Neurodegenerative diseases are among the leading causes of disability and death worldwide. The disease-related socioeconomic burden is expected to increase with the steadily increasing life expectancy. In spite of decades of clinical and basic research, most strategies designed to manage degenerative brain diseases are palliative. This is not surprising as neurodegeneration progresses “silently” for decades before symptoms are noticed. Importantly, conceptual models with heuristic value used to study neurodegeneration have been constructed retrospectively, based on signs and symptoms already present in affected patients; a circumstance that may confound causes and consequences. Hence, innovative, paradigm-shifting views of the etiology of these diseases are necessary to enable their timely prevention and treatment. Here, we outline four alternative views, not mutually exclusive, on different etiological paths toward neurodegeneration. First, we propose neurodegeneration as being a secondary outcome of a primary cardiovascular cause with vascular pathology disrupting the vital homeostatic interactions between the vasculature and the brain, resulting in cognitive impairment, dementia, and cerebrovascular events such as stroke. Second, we suggest that the persistence of senescent cells in neuronal circuits may favor, together with systemic metabolic diseases, neurodegeneration to occur. Third, we argue that neurodegeneration may start in response to altered body and brain trophic interactions established via the hardwire that connects peripheral targets with central neuronal structures or by means of extracellular vesicle (EV)-mediated communication. Lastly, we elaborate on how lifespan body dysbiosis may be linked to the origin of neurodegeneration. We highlight the existence of bacterial products that modulate the gut-brain axis causing neuroinflammation and neuronal dysfunction. As a concluding section, we end by recommending research avenues to investigate these etiological paths in the future. We think that this requires an integrated, interdisciplinary conceptual research approach based on the investigation of the multimodal aspects of physiology and pathophysiology. It involves utilizing proper conceptual models, experimental animal units, and identifying currently unused opportunities derived from human data. Overall, the proposed etiological paths and experimental recommendations will be important guidelines for future cross-discipline research to overcome the translational roadblock and to develop causative treatments for neurodegenerative diseases. Neurological disorders are the leading cause of disability-adjusted life-years and the second-leading cause of deaths worldwide. This burden has increased substantially over the past 25 years because of expanding population numbers and aging  and willAt intermediate and advanced stages, frequently by the time a diagnosis is made, neurodegeneration has affected higher brain areas that control cognitive, sensory, and motor functions. Because the majority of cases are seen by the age of 50 or older in both men and women, many scientists think of neurodegenerative diseases as processes that reflect abnormal aging. Likewise, since neurodegeneration involves the loss of neuronal populations brain-wide, many researchers believe that neuronal death results from altered processes intrinsic to the nervous system, such as neuroinflammation, abnormal accumulation or deteriorated clearance of toxic proteins, and reduced anti-oxidative defenses.Hence, current explicative models of neurodegenerative diseases account for signs and symptoms already seen in affected patients. In fact, the majority of these causative models have been formulated in retrospect, based on what is already present in the patient’s brain. Given the nature of these models, it is difficult to identify real causes from mere consequences and epiphenomena. This is even more puzzling given that neurodegenerative diseases are life-spanning processes that start silently at least 20 to 30 years before the pathological signs and symptoms are recognized by patients and diagnosed by physicians. In addition, accumulating information suggests an extraneural origin of neurodegeneration.Clearly, the essential premises guiding research on neurodegenerative diseases seem at least partly incorrect and somewhat misleading. This may explain why, despite researchers’ best efforts, neurodegeneration is still a growing health concern and the reason why therapeutic measures are currently palliative, while preventive approaches are often ineffective .Here, we outline four different paths as the basis for a paradigm shift toward an etiologic view on neurodegeneration: (1) the vascular origin of neurodegeneration, (2) cellular senescence as the origin of neurodegeneration, (3) body–brain trophic interactions as the origin of neurodegeneration, and (4) lifespan gut dysbiosis as the origin of neurodegeneration. We did not cover here genetic components as risk factors for neurodegeneration, a comprehensive analysis of which can be found elsewhere . We provNeurons depend on blood vessels to deliver oxygen and nutrients, for the removal of carbon dioxide and other by-products of metabolism from the brain’s interstitial space, which helps to maintain the homeostasis of the cerebral microenvironment. Therefore, to function properly, it is thought that the cerebral circulation has developed adaptive mechanisms to provide the trophic molecules and energy substrates needed for neuronal cells at different times and regions, depending on neural activity .The recognition of these mechanisms led to the concept of neurovascular coupling , which rThe main players of these dynamic adjustments in the cerebral blood flow are neurons, glia, and vascular cells. All of them come together in a functional morphological entity termed the “neurovascular unit”. This view explains why cerebrovascular diseases linked to neurodegeneration are considered as stand-alone etiopathological states . VasculaCerebral blood vessels originate from large cerebral arteries arising from the circle of Willis. As they find the way through the brain, large cerebral arteries divide into pial arteries and arterioles, which grow gradually deeper and form the penetrating arteries and arterioles that become progressively smaller until they turn into cerebral capillaries . The dev2+ and calcium-dependent enzymes that exert effects on the endothelium, astrocytes, and pericytes. Because of their close association with synapses and microvessels, astrocytes are in charge of the neurovascular transmission of signals to the capillaries and arterioles while endothelial cells exert a retrograde propagation of vasomotor responses to regulate cerebral blood flow , prostacyclins, endothelial-derived hyperpolarizing factor (EDHF), and endothelin have been proposed as the main vasoactive factors related to the endothelial response to neural activity. NO promotes the vasodilation of cerebral blood vessels by stimulating soluble guanylate cyclase in the vascular muscle, leading to an intracellular increase of cGMP and relaxation . NO is gIn the CNS, nNOS is mainly located in neurons, astrocytes, and neuronal stem cells and has been implicated in synaptic plasticity and in the central control of blood pressure. Abnormal NO signaling is likely to contribute to a variety of neurodegenerative pathologies such as excitotoxicity following stroke, multiple sclerosis (MS), Alzheimer’s disease (AD), and Parkinson’s disease (PD) .iNOS expression had initially been shown for macrophages, but it is now clear that it can be induced by cytokines in a variety of cell types in the context of inflammation . During Endothelial NOS is mostly expressed in endothelial cells, although the isozyme has been detected in certain neurons of the brain . eNOS-deIn addition to the production and release of NO, cyclooxygenase produces prostacyclin, which induces the relaxation of cerebral vessels by the activation of adenylate cyclase with the accumulation of cAMP and the activation of potassium channels. Similarly, in large cerebral arteries, the production of EDHF by the cytochrome P-450 monooxygenase metabolism of arachidonic acid causes relaxation by hyperpolarizing the underlying vascular muscle through the activation of potassium channels. The contribution of EDHF increases as the vessel size decreases, with a predominance of EDHF activity in resistance vessels and a compensatory upregulation of hyperpolarization in states characterized by reduced NO availability .Besides vasodilatory factors, under certain conditions, the cerebral endothelium produces vasoconstrictive substances such as endothelins leading to a potent and long-lasting contraction of cerebral vessels dependent on extracellular calcium and the activation of protein kinase C .The alterations of any of the cellular players involved in the neurovascular unit may impair its coupling and functional hyperemia, resulting in an alteration of homeostasis leading to brain dysfunction .According to the World Health Organization, cardiovascular diseases (CVDs) lead to one-third of all annual deaths globally, which is about 17.9 million total deaths . CVDs arBecause the factors contributing to vascular pathology are often preventable or treatable, targeting them may prove useful for preventing or delaying the onset of neurodegenerative diseases. Epidemiological studies show that treating hypertension and hyperlipidemia reduces the incidence of neurodegenerative diseases  and slowHypertension – or blood pressure at or beyond 130 mmHg/80 mmHg (systolic/diastolic blood pressure) according to the 2017 American College of Cardiology/American Heart Association guidelines  – affectHypertension alters cerebrovascular autoregulation  by impaiThe disruption of the adjustment mechanisms by a sustained increase in the blood pressure has been linked to different neurodegenerative diseases such as stroke . HyperteAtherosclerosis, a progressive disease, characterized by the accumulation of lipids and fibrous elements in the large arteries, is a major cause of heart disease and stroke .The damage leading to atherosclerosis is elicited over years and is highly influenced by lifestyle. The pathogenic sequence starts with the recruitment of circulating monocytes into the intima, where they differentiate into macrophages and internalize modified lipoproteins to become foam cells (fat-laden M2 macrophages). Then, chemokines and growth factors induce the proliferation of neighboring smooth muscle cells and the synthesis of extracellular matrix components within the intimal compartment, generating a fibromuscular plaque that undergoes progressive structural remodeling resulting in the formation of a fibrous cap, overlying a lipid-rich, necrotic core accompanied by varying degrees of matrix remodeling and calcification . If a ruEpidemiological and post-mortem studies have shown that the atherosclerosis of the arteries that supply the brain is associated with a two- to threefold increased odds of dementia . InteresCerebral small-vessel disease comprises white matter lesions (WMLs), lacunar infarcts, and cerebral microbleeds and is a frequent neuroimaging finding (best seen on magnetic resonance images) in elderly people .Factors such as arterial stiffness, also related to aging and hypertension, are considered to expose the small vessels in the brain to highly pulsatile pressure and flow . This caCerebral small-vessel disease is associated with an increased risk of cognitive decline, dementia, stroke, balance disturbances, and parkinsonism . IndividAtrial fibrillation (AF) is the most common arrhythmia affecting 1 to 2% of the general population . AF is cAF can lead to reduced cardiac output, intermittent cerebral hypoperfusion, and transient hypertension in the cerebral capillaries . Such chAF is associated with increased morbidity and mortality and has long been known as one of the main risk factors for embolic stroke . It is ndirectly involved in the pathophysiology of neurodegenerative diseases.The link between vascular risk factors and neurodegenerative disease is strengthened by the evidence showing that vascular and neurodegenerative pathophysiological changes often co-exist in patients with a diverse spectrum of neurodegenerative diseases. Several lines of evidence even suggest that vascular mechanisms are The event that triggers the onset of a stroke, such as the rupture of an atherosclerotic plaque, the embolization of a blood clot, or the damage to a vessel and subsequent bleeding, is often preceded by vascular pathophysiological changes that accumulate over time. One of the earliest changes is endothelial dysfunction induced by oxidative stress , which oThe same vascular mechanisms that lead to acute stroke can also lead to cognitive impairment . In the Vascular pathology leading to white matter damage, manifesting as WML, seems to play a particularly important role in the pathogenesis of vascular dementia . Very ofThe link between vascular pathology and neurodegeneration has also been thoroughly studied in AD. Vascular damage, initiated by vascular and genetic predisposing factors, precedes the accumulation of beta-amyloid (Aβ), which eventually leads to neurodegeneration in AD . NeuroimVascular damage may facilitate neurodegeneration by altering a major route through which substances are cleared from the interstitial space of the brain parenchyma. This route is known as the glymphatic system, which facilitates the movement of fluid from perivascular (Virchow-Robin) spaces surrounding pial arterioles to the interstitium into spaces surrounding the brain’s deep veins and then to the cervical lymph nodes .Vascular risk factors cause blockages in and around small arteries, which can manifest as an enlargement of the perivascular spaces  that areInterestingly, the spatial distribution of these enlarged perivascular spaces has been linked to different underlying pathologies. A predominant distribution in the basal ganglia indicates vascular disease caused by hypertension , while aCerebral amyloid angiopathy, which leads to intracerebral hemorrhage that aggravates the cognitive impairment , is founBlood–brain barrier dysfunction, which involves damage to pericytes and endothelial tight junction molecules, has been observed in several neurodegenerative diseases, including AD, PD, amyotrophic lateral sclerosis (ALS), and Huntington’s disease . VasculaThe presence and severity of vascular damage occurring at the cellular and molecular level, affect the clinical course of neurodegenerative diseases. Large neuropathological studies show the post-mortem presence of infarcts, lacunes, leukoencephalopathy, atherosclerosis, or hemorrhages in 80% of AD patients compared to 67% of similarly aged people without cognitive impairment .Interestingly, the effects of vascular pathology and neurodegeneration on cognitive decline seem to be synergistic, particularly in patients with moderate AD pathology . StudiesSince a main feature of aging is the accumulation of senescent cells in various tissues , cellulaCIP1/WAF1 (encoded by Cdkn1a) and/or p16INK4A (encoded by Cdkn2a), as well as by apoptosis resistance , associated with the expression of tumor suppressors p21sistance . SenesceHowever, the most important feature of senescent cells is the secretion of pro-inflammatory cytokines, growth factors, extracellular enzymes, and metalloproteases, collectively known as senescence-associated secretory phenotype (SASP) . In earlinflammaging), a major contributor to age-related dysfunctions. SASP molecules also have an autocrine role, fostering the senescent phenotype, and a paracrine role inducing senescence in surrounding cells .In a recent publication, using weighted gene co-expression analysis, in vitro exposure of astrocytes to Aβ1–42 triggers senescence, leading to the secretion of pro-inflammatory cytokines, including interleukin 6  deposition, the degeneration of hippocampal neurons, brain shrinkage, and the thinning of the dentate gyrus, thus preserving cognitive function. The transgenic mice that did not undergo a senescent transformation of microglia cells maintained their cognitive functions .Interestingly, neurogenesis and learning and memory can be impaired when blood and plasma from aged animals are transfused to young animals, showing that systemic factors can affect brain integrity. This phenomenon may be related to chemokine levels, especially the C–C Motif Chemokine Ligand 2 (CCL2), an important SASP component . It is pCellular senescence accompanying neurological conditions involves many cell types of the CNS , which mThere is no consensus whether and to what extent age influences infarct size: several studies reported larger  or smallFurthermore, the immune response to ischemic brain injury differs between aged and young mice. In a recent report, older mice displayed an increased number of brain-infiltrating and peripheral neutrophils with decreased phagocytic potential, increased levels of reactive oxygen species and extracellular matrix-degrading enzymes . Rawji aIn vitro and in vivo experiments show that aging influences the state of endothelial cells, pericytes, and tight junctions, in turn resulting in a compromised function of the BBB , which, in fact, favor the accumulation of senescent cells by facilitating the functional changes of cellular distribution and regulation of adipose tissue. As mentioned before, cellular senescence in aging may contribute to a chronic inflammatory state in the brain and peripheral tissues .Interestingly, obesity generates an accelerated state of aging, pro-oxidant factors as well as glucotoxicity . It is aFurthermore, the affected glial cells in obese mice display excessive fat accumulation, which is in agreement with the role of glial cells on the senescence-mediated induction of neurodegeneration. Since SASP contributes to inflammation, metabolic dysregulation, progeria, pulmonary fibrosis, geriatric syndromes, and the loss of resilience, the clearance of senescent cells may delay or even alleviate multiple age-related diseases .Taking into consideration the increasing body of evidence on the role of cellular senescence in neurodegenerative, cerebrovascular, and metabolic disorders, we propose to investigate senescent cell accumulation in the periphery and in the nervous system as well as the influence of SASP on processes in the CNS, such as neuroplasticity, specific cellular populations, and on the brain–body signaling. This will help to understand the normal process of aging and senescence in an integrative way.The disruption of the bidirectional interaction between neurons and the cells they innervate has not been considered as a putative etiologic mechanism of neurodegeneration. According to the trophic theory of neural connections, the body is represented several times within the nervous system along the sensory and motor pathways . Thus, cFurthermore, novel requirements of the body affect the peripheral and central connections of peripheral neurons. In early phases of metabolic disorders, adipocytes and immune cells are related to peripheral nerve remodeling of both sympathetic and sensory fibers . In addiVarious neurodegenerative diseases share common molecular and cellular mechanisms including the misfolding of proteins that undergo conformational changes leading to tissue deposition and the formation of inclusion bodies. Increasing evidence suggests that these neurodegenerative proteinopathies may originate in the peripheral nervous system  or even In PD and dementia with Lewy bodies (DLBs), the histopathological hallmark is the occurrence of neuronal α-synuclein aggregates, also known as Lewy bodies. Lewy bodies are typically found in the substantia nigra in PD. However, LBs can also be present in peripheral organs, neural or not, including the sympathetic ganglia, ENS, cardiac and pelvic plexuses, submandibular gland, adrenal medulla, and the skin . MoreoveIn AD, NFTs are observed in the spinal and sympathetic ganglia. However, it seems that they develop independently of the cerebral AD pathology . In ALS,Sensory abnormalities have been described in neurodegenerative diseases including PD and AD. In this regard, the loss of olfaction represents an important early clinical symptom frequently preceding motor and cognitive impairments typically observed in these neurodegenerative diseases . The olfGiven that the OE is directly accessible to external factors, it is vulnerable to environmental insults. Thus, external agents  may trigger neurodegeneration through the OE . Once thMoreover, xenobiotics and particles in contact with the OE can also induce the production of reactive oxygen species triggering a local inflammatory reaction in a similar way to that induced by misfolded proteins . AlternaPain abnormalities are part of the clinical features in patients with PD and the impaired nociceptive processing depends on nociceptor degeneration . Skin biThe accumulation of misfolded proteins such as α-synuclein and parkin due to a dysfunction of the ubiquitin-proteasome system may partially explain the painful nociceptor dysfunction . In AD, Autonomic dysfunction is present in several neurodegenerative diseases including PD, multiple system atrophy, AD, and other types of dementia such as DLB, PD with dementia, and frontotemporal lobar degeneration . OrthostThe presence of orthostatic hypertension may be due to the depletion of cardiac vagal sympathetic nerves as well as the α-synuclein pathology in sympathetic ganglia and adrenal gland that have been reported in Lewy body diseases, which includes PD, PD with dementia, and DLB . InteresGastrointestinal dysfunction is closely linked to the dysfunction of the ENS. Enteric dysfunction has been reported in PD, AD, ALS, and frontotemporal dementia . BiopsieLikewise, in AD patients, Aβ immunoreactivity is found in the submucosa of the intestine . In addiAn increasing body of evidence suggests that metabolic disturbances may contribute to neurodegenerative processes in the CNS. We discuss here briefly the impact of diabetes mellitus (DM) and obesity on neurodegeneration.Diabetes mellitus is one of the most common metabolic diseases and a major disorder of insulin regulation with increasing incidence . EspeciaT2D develops under a cluster of risk factors that includes high blood glucose, obesity, increased blood triacylglycerols and insulin resistance, which individually or collectively, also increase the risk for neurodegeneration/neuronal death, functional and structural brain changes, culminating in cognitive dysfunction that underlies dementia-type disorders , which may arise from a complex interplay between T2D and brain aging. Additionally, decreased brain insulin levels/signaling and glucose metabolism in both pathologies further suggests that an effective treatment strategy for one disorder maybe also beneficial in the other .Obesity is clinically identified based on measurements of body mass index, but can be generally defined as the condition in which excess body fat has accumulated to an extent that can negatively affect health. This definition is based on the dramatically enhanced risk for a myriad of disorders, including T2D, CVD, gastrointestinal and respiratory disorders, and several types of cancer . Moreover emerging evidence that dietary restriction can forestall the development of AD and PD is consistent with a major “metabolic” component to these diseases, and provides optimism that these devastating brain disorders of aging may be largely preventable .Advanced glycation end products generated by chronic hyperglycemia and their receptor RAGE provide critical links between diabetes and AD . ObesityHowever, due to increased incidence, especially in the aging population, the impact of metabolic disease on neurodegenerative processes will increase tremendously in the upcoming decades. We believe therefore that potential therapeutic approaches aiming at preventing/treating neurodegenerative diseases should include metabolic disorders.Accumulating evidence suggests that patients with neurodegenerative diseases commonly develop sensory and autonomic dysfunctions during maturity. This raises the possibility that the peripheral nervous system may be the gateway to alter brain physiology. The trophic theory of neural connections states that there is a mutual dependence between neurons and their targets . OlfactoRecently, a novel system of intercellular communication has been proposed to modulate changes in neuronal physiology and to participate in the propagation of misfolded proteins . This syApparently, all the cell types of the body can release EVs to transmit information in the form of proteins, RNAs, and lipids. EVs are classified according to their subcellular origin in exosomes when originating from multivesicular bodies and in ectosomes (or microvesicles) when formed from the plasma membrane . DespiteExtracellular vesicles are involved in several processes of brain development and normal functioning, including synapse formation, synaptic plasticity, and communication between glia and neurons. The signaling proteins of hydrophobic nature, such as the Wnt family members and other regulatory proteins, are loaded into EVs . During Drosophila neuromuscular junction, EVs containing the Wnt-binding protein Evennes Interrupt and the Wnt 1 homolog Wingless promote synaptic growth when released from the presynaptic terminal -challenged mice . Dorsal Recent findings also indicate that EV-mediated communication can be an additional mechanism operating during trauma. After nerve injury, dorsal root ganglia neuron-derived exosomes loaded with microRNA-21 increase the infiltration of inflammatory macrophages, which contributes to neuronal sensitization . Furtherin vivo and in vitro.Despite the few studies focusing on the influence of peripherally derived EVs on peripheral neurons, it is likely that peripheral terminals establish a bidirectional interaction with the cellular elements surrounding them. Both sensory and autonomic axon terminals usually end as free nerve endings in the extracellular matrix intercalated between different cell types. Peripheral neurons are responsive to EVs derived from mesenchymal stem cells and Schwann cells . The genTaken together, these findings indicate that neurons are susceptible to modify their functioning or gene expression profile as a result of incorporating EVs from neighboring cells. A challenge for future studies is to determine if EVs from any cell source can modify neuronal functioning and how these events are associated with physiological alterations that precede neurodegeneration in the brain.Microbiota is defined as a community of all microorganisms living in our body. Microbiota therefore includes all taxonomic domains such as fungi, protozoa, viruses, and bacteria. Bacterial microbiota living in the gut (further referred as GM) represent about 90% of all microbiota in the body , with anA delicate balance exists between the GM composition and its interaction with the host, while an imbalance in this composition is commonly known as dysbiosis. Gut dysbiosis has been implicated in the development of chronic diseases, such as irritable bowel syndrome , coeliacNotably, those metabolic diseases, when developed during adulthood, are considered important risk factors for AD . FurtherDuring aging, there is an increased permeability of the gut epithelium . This coRecent reports concomitantly demonstrate a gut dysbiosis in PD , ALS, anUnderstanding how GM may be implicated in brain pathologies requires a clear comparison among the data reported from different research groups. The GM is mainly dominated by four phyla: Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria . The firImportantly, variations in the experimental design or the type of samples can be observed. Moreover, differences in the animal model, chronological stage of the pathology (age), handling and diet significantly impact GM . Thus, eDespite this strong variation, we can observe a general increased abundance of pro-inflammatory bacteria in AD mice and AD patients  comparedA bidirectional communication between the gastrointestinal system and the brain has been described and has led to the concept of gut-brain axis . Recent Several groups have implicated the neurotoxin LPS present in the outer membrane of gram-negative bacteria. LPS plays key roles in the host-pathogen interactions of the innate immune system . AlteratSystemic infections have been related to a higher possibility to develop AD  as well 1–42 . SCFAs are produced by the GM after the degradation of non-digestible polysaccharides, with butyrate, acetate, and propionate being the more abundant fermentation products . About 9Some alterations in fecal SCFAs concentration have been described in AD transgenic mice compared to controls . ButyratFecal samples of children with autism spectrum disorder present an increased level of propionate . In addiAnother important pathway connecting the brain and the GM is the involvement of the immune system  studied,However, the optimal (in terms of prognosis after stroke) composition of the GM has not been identified yet and is possibly difficult to pinpoint. Some findings indicate that the complete absence (in germ-free mice) or deep antibiotic depletion of GM can be detrimental, leading to increased infarct volumes  or increWe can conclude that a predominantly pro-inflammatory GM and the release of neurotoxic substances may negatively affect brain functions, causing systemic and central inflammation over decades. This is in support of the recent hypothesis of an infectious etiology for neurodegenerative diseases, such as AD . The subAs we have described above, the neurocentric view entertained as the predominant focus of our effort in treating neurodegeneration is likely insufficient to ensure successful therapeutic intervention.Investigating the pathophysiological processes behind neurodegeneration in detail requires the use of animal models. A plethora of animal models of neurodegeneration exists, and the nuances of these models are beyond the scope of this manuscript  seen in human patients. Examples of such problems are the rodent models of stroke, particularly ones that involve surgical ligation or occlusion of blood vessels in otherwise healthy, young animals .Another important issue is the way these models are characterized. For animal models to be used to study neurodegeneration, two essential steps must be taken. First, the validity of the model for the question at hand should be thoroughly and systematically investigated. This is preferably done in a multi-centric and multi-disciplinary way to ensure the generalizability and robustness of the results. Second, the external  validity of the model  should bFor these strategies to be successful, research into and using these models must be conducted in the setting of appropriate, rigorous, and transparent experimental design and reporting . Beyond Although animal models are a powerful tool toward understanding the etiology of neurodegenerative diseases, they still present considerable limitations . As a coThe recent increase in the generation of digital data including imaging, genotypic, and phenotypic information coupled with a fast development of computational methods allows us to obtain new insights from this massive amount of data. Major efforts from consortiums such as the Alzheimer’s Disease Initiative (ADNI), the Australian Imaging, Biomarkers & Lifestyle Study of Ageing (AIBL), the Parkinson’s Progression Markers Initiative (PPMI) or more recently the United Kingdom Biobank have made available an unprecedented amount of data, which can be exploited through the use of big data algorithms.Having large amounts of information coming from heterogeneous populations and acquisition systems increases the power of statistic approaches and reduces the risk of bias. Big data approaches are inherently limited since in general they cannot be used to perform causal inference, but rather to find correlations or associations between variables. However, these correlations have proven to be an invaluable tool for hypothesis generation, drug and safety surveillance, disease and treatment heterogeneity among many other applications .Such computational models have been used for example to build a model of senescence signaling via DNA damage, insulin-TOR, FoxO3a transcription factors, oxidative stress response, mitochondrial regulation, and mitophagy using a systems biology approach . A recenRecent trends in stem cell methods have also opened new possibilities for human brain cell cultures, which can in turn be used to model neurodegeneration. Induced pluripotent stem cells and neural stem cell cultures can now be used to generate human neurons from human and sometimes patient cells . This haDisease is typically defined in juxtaposition to the state of health, which has been even more generally defined as “a state of complete physical, mental, and social well-being” . HoweverTypically, pathophysiological events of neurodegeneration are detected through behavioral observations and self-reporting and subsequently confirmed and classified using neurological examination and diagnostic imaging. The result of this diagnostic approach usually reveals typical defining features for each neurological disease , that allows the classification of the disorder. However, these defining characteristics are not necessarily the root causes of the disease but rather elements of the disease manifestation or even merely epiphenomena. In addition to the complex and usually obscure causal relationships giving rise to brain disorders, a parameter that further complicates the identification of causation is the widespread and intricate comorbidities between presumably distinct and unrelated disorders.Together, these facts limit the therapeutic potential to treat the symptoms rather than the causes. The complex causality chain hinders the early detection and treatment of the root causes of brain disorders overall and neurodegeneration more specifically. These limitations highlight the critical need for a drastic shift of perspective and the improvement of the models used to investigate and understand neurodegenerative diseases.Given the complexity of the brain and its disorders, researchers and clinicians have retreated to a highly segregated approach, focusing on describing subsets of the symptoms of each disease and trying to identify mechanistic explanations and treatments for those symptoms. However, the lack of a holistic perspective, taking into account the deviations from the default homeostatic state at an organismal level, renders this endeavor nearly impossible and doomed to fail.Importantly, characterizing the expected, default healthy brain, is a necessary precondition to being able to identify and meaningfully interpret the deviations from it. Thus, characterizing and understanding the brain function in health is of paramount importance in the attempt to understand and treat the diseased brain.In summary, there is an identifiable need for an interdisciplinary approach to explore the brain function at multiple levels, from molecular to systems, while maintaining a holistic perspective that treats the brain as an element of a complex system, rather than a separate entity. Using this approach, fundamental research can proceed hand-in-hand and maintain a constant dialogue with clinical research, to propose, improve, and revise current models of health and disease.We hope that our work inspires the opening of new research avenues among those interested in understanding and pursuing the complexities of the “neuro-degenerating” brain focusing on lifespan processes. Therefore, the proposed etiological paths and suggestions will be important guidelines for future cross-discipline research to overcome the translational roadblock and to develop causative treatments for neurological disorders.AP, GG-O, and MZ applied for funding and organized the workshop. AK, AP, AV, BG-B, MZ, NK, and XC were participants of the subgroup “The Vascular Origin of Neurodegeneration.” AV-C, JP-V, KR, KW, LR, and SC-O of the subgroup “Cellular Senescence as the Origin of Neurodegeneration.” CP-C, EM-M, GG-O, HG, and JL-N of the subgroup “Body–Brain Trophic Interactions as the Origin of Neurodegeneration.” Accordingly, the authors wrote the respective sections of this manuscript, except CP-C who together with TS wrote the section “Gut Dysbiosis as the Origin of Neurodegeneration” with contributions from KW. AP contributed to the section “Body–Brain Trophic Interactions as the Origin of Neurodegeneration.” BG-B wrote the section on “Studying Human Disease in Human Systems.” AK on “The Lack of Adequate Experimental Models to Study the Etiology.” NK wrote the conclusion. MZ wrote the remaining sections of the manuscript. AV commented on the manuscript. All authors discussed and commented on the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The infratemporal fossa (ITF) is an anatomical lateral skull base space composed by the zygoma, temporal, and the greater wing of the sphenoid bone. Due to its difficult approach, surgical intervention at the ITF has remained a heavy burden to surgeons. The aim of this article is to review basic skull base approaches and ITF structures and to avoid severe complications based on the accurate surgical knowledge.A search of the recent literature using MEDLINE (PubMed), Embase, Cochrane Library, and other online tools was executed using the following keyword combinations: infratemporal fossa, subtemporal fossa, transzygomatic approach, orbitozygomatic approach, transmaxillary approach, facial translocation approach, midface degloving, zygomatico-transmandibular approach, and lateral skull base. Aside from our Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) trial, there have been very few randomized controlled trials. The search data for this review are summarized based on the authors’ diverse clinical experiences.We divided our results based on representative skull base approaches and the anatomy of the ITF. Basic approaches to the ITF include endoscopic endonasal, transzygomatic, orbitozygomatic, zygomatico-transmandibular, transmaxillary, facial translocation, and the midfacial degloving approach. The borders and inner structures of the ITF  are summarized, and the modified zygomatico-transmandibular approach (ZTMA) is described in detail.An anatomical basic knowledge would be required for the appropriate management of the ITF pathology for diverse specialized doctors, including maxillofacial, plastic, and vascular surgeons. The ITF approach, in conjunction with the application of microsurgical techniques and improved perioperative care, has permitted significant advances and successful curative outcomes for patients having malignancy in ITF. Malignant tumors of the infratemporal fossa (ITF) usually originate from the paranasal sinuses, oral cavity, or the skull base structures. Although the incidence of ITF malignancy is less than 3% of all head and neck tumors, a surgical management is the only treatment for complete control –3. FurthThe treatment of skull base tumors is a multidisciplinary work composed of plastic and reconstructive surgeons, anesthesiologists, radiologists, rehabilitation specialist, and neurosurgeon. Additionally, medical and radiotherapeutic oncologists are also important for their chemotherapy combined with radiation therapy pre- or postoperatively.In this review article, we focus on representative skull base approaches and the complex anatomy of the ITF by summarizing its borders, inner structures, and lateral skull dissection techniques. This article also describes the modified zygomatico-transmandibular approach (ZTMA), which is presented in detail from the maxillofacial surgeon’s point of view.A search of the recent literature using MEDLINE (PubMed), Embase, Cochrane Library, and other online tools was executed with the following keyword combinations: infratemporal fossa, subtemporal fossa, transzygomatic approach (TZA), orbitozygomatic approach (OZA), transmaxillary approach (TMA), facial translocation approach (FTA), midface degloving approach (MDA), zygomatico-transmandibular approach (ZTMA), and lateral skull base. The search data were summarized based on the authors’ diverse clinical experiences. A statement of ethics approval was provided by the Department of Oral and Maxillofacial Surgery at Seoul National University Dental Hospital, with the approval of the Institutional Review Board of Seoul National University (S-D20170024).We approached to summary variations in infratemporal skull base approaches, and the clinical anatomy of the ITF.The infratemporal fossa approach (ITFA) is one of the several approaches to the skull base, including maxillary swing, midfacial degloving, facial translocation, endonasal transsphenoidal, subfrontal interhemispheric, orbitozygomatic, pterional, subtemporal, middle cranial fossa, translabyrinthine, transcochlear, retrolabyrinthine presigmoid, retrosigmoid suboccipital, far lateral transcondylar, transcervical, and transpalatal –5.Compared with extensive transcranial approaches for the sinonasal or nasopharyngeal malignancies, endoscopic endonasal approach (EEA) was developed as an alternative to the middle and posterior cerebral fossae . EEA couFor the approach to the anterior temporal dura and middle fossa, TZA could be considered by vertical sectioning of zygomatic arch and by the fronto-temporo-sphenoidal craniotomy Fig. b. TZA isOZA could be chosen for the wide exposure of lateral skull base, middle cranial fossa, basilar apex, and ITF . PterionThe ZTMA is a modified approach to lateral skull base surgery. It combines the TZA and the OZA, adding a mandibulotomy procedure Fig. d. For moTMA directly exposes the anterior and lateral skull base including ITF. Several important anatomies including mandibular branch (V3), PPF, maxillary artery (MA), lateral pterygoid muscle (LPM), foramen rotundum, and foramen ovale are exposed sequentially  in this FTA is a somewhat rare approach, but often chosen for extensive tumors involving nasopharynx, nasal cavity, posterior maxillary sinus, and ITF. This unesthetic approach could be chosen in these order, such as subtemporal, preauricular, infratemporal, maxillary swing, TMA, and finally FTA . ExcludiFrom 1992 onward, MDA has gained popularity and could be effective in removing a tumor without late complications. Closed rhinoplasty incisions with direct opening of nasal cavity, paranasal sinus, and anterior skull base structures are essential for the MDA , vessels (including MA with veins and the pterygoid plexus of veins), nerves , and the temporomandibular joint (TMJ).LPM has a superior head with infratemporal crest and surface initiation, the anterior capsule of TMJ insertion, an inferior head with a lateral surface for pterygoid plate initiation, and the pterygoid fovea below the mandibular head and disc. The main action of the LPM is protruding the jaw and opening the mouth. MPM has a deep head on the lateral pterygoid plate and a smaller superficial head. The main action of the MPM is pulling the mandible upwards, forwards, and medially and closing the mouth during chewing. The LPM and MPM contain venous plexuses that connect with facial veins.The MA, or internal maxillary artery (IMA), is composed of three parts according to its course.Branches of the first mandibular part of the MA sit between the initial mandibular part and the posterior part of LPM. They include the auricular artery deep to the external acoustic meatus, the tympanic artery anterior to the tympanic membrane, the middle meningeal artery (MMA) to the dura mater , the accessory meningeal artery to the cranial cavity, and the alveolar artery inferior to the mandibular gingiva and teeth. The second part of the MA is the pterygoid portion between LPM and PPF. It mainly serves the masticatory muscles and includes the masseteric artery, deep temporal artery, pterygoid branch, and buccal artery. The third pterygopalatine part comprises a short course in the PPF, from the sphenopalatine artery to the sphenopalatine foramen. It includes the posterior superior alveolar, infraorbital, descending palatine, sphenopalatine artery, and artery of pterygoid canal.The deep veins of the face can be divided into the maxillary vein and pterygoid plexus. These are counterpart to the superficial veins of the face . The maxillary vein courses with the first division of the MA, starting from the posterior part of the pterygoid plexus and continuous with the superficial temporal and retromandibular vein. The pterygoid plexus forms the main venous bundles in the ITF. It is located between the LPM and MPM and between the temporal muscle and LPM. The pterygoid plexus communicates with the anterior facial vein and cavernous sinus through the foramen Vesalius, foramen lacerum, and foramen ovale.Nerves within the ITF are divided into three groups: branches from the trunk , anterior branches , and posterior branches . The V3 descends through the foramen ovale into the ITF and divides into sensory and motor branches. These comprise the auriculotemporal, inferior alveolar, lingual, and buccal nerves. Branches also supply the four muscles of mastication, but not the buccinator . The chorda tympani nerve (CTN) carries taste fibers from the anterior two thirds of the tongue and parasympathetic innervation to the submandibular and sublingual glands. It originates from the facial nerve in the temporal region and passes anteriorly to enter in the middle ear, where it is separated from the tympanic membrane by the handle of malleus. It enters the ITF and descends medial to the spine of sphenoid and then to the LPM.Basic lateral skull dissection could be proceeded as follows: (1) check and confirm the origin and insertion of the masseter muscle; (2) retract the cutting zygomatic arch and inserted masseter muscle in a downward: (3) check and confirm the insertion of the temporal muscle to the coronoid process; (4) confirm the masseteric nerve and vessels at the mandibular notch; (5) carefully cut the coronoid process without causing soft tissue injury by approaching antero-inferiorly from the mandibular notch. Inspect the coronoid process with the attached temporalis and remove the temporal muscle after locating the anterior and posterior deep temporal nerve and vessels; (6) after marking the horizontal line on the external surface of the lingual nerve, cut without injuring the mandibular foramen or inferior alveolar nerve and vessels; (7) separate the ramus from the insertion of the periosteal elevator to the inside of the mandibular neck; and (8) after removal of this portion of the ramus, the contents of the ITF will be exposed. Two significant differences, such as the short distance between mandible and ITF, and the more distant location of facial nerve from the mandible, could be considered in the pediatrics , 20, 21.In spite of disagreement among some clinicians regarding the exact limits of the ITF and/or recommended surgical approaches, we have described the modified ZTMA here because of its versatility. It is suitable for tumors originating from the ITF, tumors originating from the greater wing of the anterior temporal base or sphenoid bone, and tumors spreading to the ITF. Although the safe resection of tympanic bone or the safe control of the infratemporal facial nerve (or jugular bulb) are not feasible, most ITF tumors in the oral and maxillofacial region can be removed by this conventional approach.The initial incision could be adjusted by combining a hemicoronal or submental-submandibular to the conventional parotidectomy incision for the lateral transparotid approach. The anterior facial flap elevation plane is similar to the normal facial lifting that occurs when the facial nerve trunk is not cut, and the parietomassetric fascia is dissected outward Fig. a. In theDuring the transfacial dissection approach, the dissection proceeds along the deep part of the transfacial layer so as not to damage the frontal branch. In the lower part of the preauricular region, the dissection is performed like a facial lifting along the parotidial-masseteric fascia. The incision is usually similar to the radical neck dissection incision, connecting the mastoid process and the hyoid bone. The dissection of the scalp flap extends to the superior orbital rim along the subpericranial plane. The cheek area extends to the parotid gland, and the subclavian incision is usually sufficient for the mental foramen.Full mobilization of the entire parotid gland is necessary for localization of facial nerve main trunk. At first, the parotid sheath near the zygoma is removed from the external auditory meatus. The parotid gland is separated from the tragal cartilage and bony ear canal. The mastoid-leading edge and the ear lobule are separated. Then, sharp dissection of the sternocleidomastoid (SCM) muscle, adherent fascia (attached to the parotid tail), and sternomandibular ligaments (connected to the mandibular angle) can be performed. At this point, the anterior branch of the external jugular vein and the greater auricular nerve should be cut. If possible, the posterior branch should be preserved for ear lobe sensation. If the external meatus cartilage is further dissected from the parotid capsule, the bony resistance of the styloid process can be felt. When the tail of the parotid is pulled all the way to the outside, the retromandibular vein is seen from the parotid gland. At this point, bleeding due to congestion should be avoided. The whole cleavage should be retained, as this may help with access to the facial nerve Fig. b.For identification and exposure of facial nerve main trunk, the posterior digastric muscle, stylohyoid muscle, and stylopharyngeus muscle can be seen by anterior gentle traction of the mobilized parotid gland. The pointer cartilage tip and the vaginal process of the styloid process are visible during further dissection Fig. b. In mosAfter exposing the zygomatic body, the periosteum is stripped and zygomatic cortex is removed by osteotomy at the zygomatic root for the posterior part, the zygomaticofrontal suture for the upper part, and the zygomaticomaxillary buttress. This is on the zygomaticofacial nerve level from the inside. The zygomatic arch can be separated from the masseter muscle by transection with electrocautery or can be moved downward with the masseter muscle attached, to expose the ITF Fig. a.In order to confirm the IOF, orbitozygomatic osteotomy is essential. After the periorbit is elevated from the outer or lower wall of the orbit, the tip of the reciprocating saw is positioned in the outermost part of the IOF from the lower part of the orbit. Osteotomy should be performed along the malar eminence. A beveled or V-shaped bone cut is performed using a reciprocating saw to increase the exposure and return the bone to its original position after the operation. If the tumor mass does not involve the temporal bone or the petrous portion of the internal carotid artery (ICA), this level of osteotomy is sufficient for exposure to the infratemporal skull base. However, if removal of the petrous portion of the ICA is required, the glenoid fossa should be removed together with osteotomized bony segments, and temporal craniotomy may be performed to expose the glenoid fossa. TMJ capsule can be freed from the fossa and lowered for full exposure, but the capsule and meniscus should be preserved if possible. If additional exposure is required, the condyle can be removed from the sigmoid notch. A V-shaped bone cut can be performed using a reciprocating saw to remove the outer two thirds of the glenoid fossa. By thus removing the glenoid fossa, the ICA inside the glenoid fossa can be protected.The pericranium-attached temporal muscle is elevated from the temporal fossa by the remaining 2–3 mm fascia on the muscular margin, for its relocation during closing reconstruction. Inferior observation of the temporalis is recommended until full exposure of the infratemporal crest is achieved. The soft tissues of the sigmoid notch should be protected to prevent IMA injury inside of the mandibular ramus, and the coronoid process should be removed or fractured to increase the inferior rotation arc of the temporal muscle. Wide dissection on the soft tissues of the infratemporal skull base is not recommended due to continuous venous bleeding in the pterygoid plexus. Subtemporal craniotomy can be used to confirm the neural and vascular foramina Fig. d.Although mandibulotomy can give a wide additional exposure, partial mandibulectomy should be executed (for a negative tumor margin) in the case of invasive mandibular malignancy. For preserving the mandibular condyle, mandibular cutting near the third molar should be done after exposure of the condylar head. The joint capsule and inner tissue can be protected with a curved periosteal elevator. Complete detachment of the masseter and medial pterygoid muscles from the ramus should be followed. The resected mandible can be retracted anteriorly, and ligations of inferior alveolar nerves and vessels on the lingual course performed, for full exposure of the anatomical contents of the ITF Fig. a.Further lateral dissection of the SCM muscle through the caudal limb of the initial incision line can expose the carotid sheath with the internal, common, and external carotid artery and internal jugular vein (IJV). Cranial nerves, including the vagus, spinal accessory, and hypoglossal, should be confirmed and preserved. IMA in the ITF and its related branches could be also identified Fig. e.Additional bone removal can occur with a bone rongeur after temporal craniotomy. Using the curved line of the lateral pterygoid plate, the foramen ovale, foramen spinosum, and sphenoidal spine are found posteriorly. If needed, cauterization of the MMA with bipolar electrocautery and surgical packing for the purpose of bleeding control at the venous communication on the foramen ovale should be done. If complete detachment of the petrous portion of the ICA is needed, V3 can be cut on the foramen ovale. The whole bone and soft tumor mass can be removed by the appropriate evaluation of the sphenoid sinus after resection of the bone between maxillary branch (V2) and V3 Fig. f.Exposed dura mater should be closed by water-tight suture combined with lyophilized dura or SCM muscle graft. All upper aerodigestive tract defects related to tumor resection should also be closed. If the temporalis muscle with its vascular supply is intact, trigeminal nerve transposition is beneficial for covering the ICA and for dead space closure. Repositioning of the resected orbitozygomatic bone with titanium microplates or 2–0 braided nylon sutures should be considered first, only excluding the intentional zygoma removal for avoiding any compression on flap closure. TMJ reconstruction with repositioning of the resected mandibular condyle should also be considered in the case of exposed petrous ICA Fig. g and i.ITF should be selected according to age, gender, facial growth or shape, biological characteristics of pathology, and surgeon’s experience. Any approach should strive for the complete resection of pathology and a careful consideration of postoperative consequences . Minimal but sufficient exposure for the complete resection of pathology is a high priority, along with minimizing the risk to cranial nerves .A focus on the prevention and management of postoperative complications is important, and we have summarized the following recommendations: (1) careful bony cutting is needed to avoid hearing loss due to cochlear injury. Comparing the strong marrow bone near the cochlea and labyrinth to the other marrow bone is a useful technique. (2) During dissection to the petrous bone, a close approach near the cartilaginous ear canal can prevent facial nerve injury, and no over-retraction of the greater superficial petrosal nerve should be kept. (3) A damaged ICA should be directly restored after temporary clipping. To avoid ICA injury, only a diamond drill is recommended when removing bone near the ICA. Where the ICA courses through the temporal bone, the periosteal layer surrounds and protects it. Two or three sympathetic nerves run between the ICA and periosteal layer. Therefore, it is necessary to preserve the periosteal layer as much as possible during ICA dissection. (4) For the prevention of trigeminal ganglion and root injury, excessive traction or manipulation should be avoided. (5) In the case of dura injury, CSF may leak into the skin or eustachian tube. If damage occurs, it should be restored with a fat or temporoparietal fascial flap. The cartilage portion of the eustachian tube should be carefully closed. If necessary, head elevation and spinal drainage should be considered, with prophylactic antibiotics administration.We have analyzed the following surgical approaches to ITF: incision with facial flap elevation; mobilization of the parotid gland; facial nerve exposure and main trunk transaction; orbitozygomatic osteotomy; temporal muscle dissection with coronoidectomy, mandibulotomy, and/or partial mandibulectomy; identification of the ICA, IJV, and IMA locations; mass resection with temporal or skull base craniotomy; and dura mater repair with closing reconstruction. This ZTMA is versatile and often recommended based on our previous surgical experiences. The ITF approach, in conjunction with the application of microsurgical techniques and improved perioperative care, has permitted significant advances in the successful curative outcomes of patients with extended malignant pathology of the lateral skull base.


Microorganisms can have a profound and varying effect on the chemical character of environments and, thereby, ecological health. Their capacity to consume or transform contaminants leads to contrasting outcomes, such as the dissipation of nutrient pollution via denitrification, the breakdown of spilled oil, or eutrophication via primary producer overgrowth. Microorganisms can have a profound and varying effect on the chemical character of environments and, thereby, ecological health. Their capacity to consume or transform contaminants leads to contrasting outcomes, such as the dissipation of nutrient pollution via denitrification, the breakdown of spilled oil, or eutrophication via primary producer overgrowth. Recovering the genomes of organisms directly from the environment is useful to gain insights into resource usage, interspecies collaborations (producers and consumers), and trait acquisition. Microbial data can also be considered alongside the broader biological character of an environment through the co-recovery of eukaryotic DNA. The contributions of individual microorganisms  to snapshots of ecosystem processes can be determined by integrating genomics with functional methods. This combined approach enables a detailed understanding of how microbial communities drive biogeochemical cycles, and although currently limited by scale, key attributes can be effectively extrapolated with lower-resolution methods to determine wider ecological relevance. One of the obvious attractions of environmental genomics (and recovering metagenome-assembled genomes [MAGs]) is that it provides valuable insights into the lifestyles of uncultivated microorganisms and mechanisms of trait acquisition  . The recUnderstanding the metabolic capacity of organisms can reveal the types of resources  that are important for sustaining life within an environment. For example, in addition to leaked metabolites , environComplex microbial processes or interactions can be resolved using integrated approaches, such as functional genomics , alongside host-related or geochemical data , 9. GeocCombining genomics with transcriptomics or proteomics enables metabolic activity to be linked to specific organisms within communities to determine individual contributions . This enCandidatus Udaeobacter copiosus” in grassland soils) , which limits the scale of application. This is particularly problematic in environments where large sample numbers are needed to understand trends, due to a high level of spatial heterogeneity   or tempod soils) . The homd soils) . To corrThe constraints of traditional marker gene assays, which segregate and ring-fence taxonomic groups of organisms, is beginning to fall away with untargeted sequencing of environmental DNA. The boundless quality of shotgun sequencing leads to the intentional or accidental recovery of nontarget  DNA from environmental samples. While this is overtly the case for host microbiome studies, it can be equally valid for other environments where eukaryote DNA is co-recovered, such as terrestrial soil, marine sediment, or stream biofilms . The co-Environmental genomic approaches are aptly suited to recovering the small tidy population genomes of bacteria, archaea, and viruses. Nevertheless, a natural extension of the method is to microbial eukaryotes: diverse protists, such as diatoms and ciliates , and alsMicrobial ecologists may increasingly find themselves meeting with unexpected companions in the environmental molecular space. Environmental DNA (eDNA) is gaining popularity among diverse ecologists who research groups of organisms as wide ranging as fungi, metazoans, and plants. The appeal is due to the persistence of DNA in the environment and the strong potential of recovering the DNA of larger organisms from environmental sources, such as soil or filtered water, without needing to capture or physically isolate these organisms . The sco


The biogenicity problem of geological materials is one of the most challenging ones in the field of paleo and astrobiology. As one goes deeper in time, the traces of life become feeble and ambiguous, blending with the surrounding geology. Well-preserved metasedimentary rocks from the Archaean are relatively rare, and in very few cases contain structures resembling biological traces or fossils. These putative biosignatures have been studied for decades and many biogenicity criteria have been developed, but there is still no consensus for many of the proposed structures. Synchrotron-based techniques, especially on new generation sources, have the potential for contributing to this field of research, providing high sensitivity and resolution that can be advantageous for different scientific problems. Exploring the X-ray and matter interactions on a range of geological materials can provide insights on morphology, elemental composition, oxidation states, crystalline structure, magnetic properties, and others, which can measurably contribute to the investigation of biogenicity of putative biosignatures. Here, we provide an overview of selected synchrotron-based techniques that have the potential to be applied in different types of questions on the study of biosignatures preserved in the geological record. The development of 3rd and recently 4th generation synchrotron sources will favor a deeper understanding of the earliest records of life on Earth and also bring up potential analytical approaches to be applied for the search of biosignatures in meteorites and samples returned from Mars in the near future. The Viking missions on the 1970’s found chemical traces that could be related to metabolism on the surface of Mars  are now being routinely applied on Earth sciences. Meanwhile, techniques based on synchrotron light are gaining more space as promising tools for the non-destructive investigation of paleobiological samples, giving rise to an emerging field known as paleometry . Some ofper unit area), broad, continuous energy range, and high coherence, which are unattainable by conventional light sources, such as lasers, lamps or X-ray tubes. These characteristics allow the use of multiple physical phenomena to unveil the original elemental and molecular composition, mineralogy and morphology of samples, with higher sensitivity, penetrability and resolution than conventional techniques applied for the same purposes. In the present review, we will discuss recent applications and potentials of some synchrotron-based techniques for evaluating biosignatures in the geological record.Synchrotron accelerators produce light with unique qualities, such as high brilliance  morphological features, such as preserved cells or extracellular components, as fossilized extracellular polymeric substance (EPS); (ii) biogenic minerals, such as those biologically-induced  or biologically-controlled , which sometimes present crystallographic characteristics and/or physical properties that can be distinguished for those abiotic minerals ; (iii) specific textures or biogenic fabrics in rocks originated by microbial activity, such as fenestral fabrics in microbialites; (iv) molecular vestiges linked to biological activity, such as organic ligands, lipids, organic macromolecules, etc; (v) specific chemical characteristics of bioprocessing, such as chirality in organic molecules; (vi) stable isotopes favorable to biogenic patterns and bioprocessing. Interpreting these signals as biosignatures is not always trivial, especially if these samples are from deep time context . One exaThe complexity of interpretation also increase considering the possibilities of posterior contaminations and the several changes in the original characteristics of mineralogy and geological features, the action of metamorphism and changes of temperature which can degrade or modify the organic biosignatures, besides several other alterations during the post-burial processes over time. Thus, it is important to do critical interpretations of the signs of early life in order to avoid misinterpretations and/or equivocal biosignatures. There are several examples in the literature regarding the contestation or reassessment of alleged evidence of ancient life on Earth and outside it . Some wiMorphological traces of microorganisms are important biosignatures for providing direct insights into ancient life ultrastructure, evolution, paleo-environment and also to help to understand the physico-chemical conditions that allowed their long-term preservation. High resolution microscopies applied so far, such as Scanning Electron Microscopy (SEM) or Transmission Electron Microscopy (TEM) have allowed significative advances on the understanding of the earliest fossilized microbes. Their application, however, is limited by the low penetration depth of electrons, thus requiring destructive sample preparation for exposing the structures from within the rock matrix , or by preparing ultra thin (<100 nm) sections that can sample only a small fraction of the specimen. 3D images can be achieved by using a focused ion beam (FIB) coupled to a SEM (FIB-SEM), and combining sequential milling with concurrent Energy Dispersisve Spectroscopy (EDS-SEM) imaging. Although being a destructive approach, it has allowed even some of the most ancient microfossils to be investigated at the nanoscale, providing insights into their chemistry, ultrastructure, taphonomy and investigating the biogenicity of these structures .X-rays can penetrate objects and provide information of their interior non-destructively, therefore having the potential of overcoming the limitations of electron microscopy. Conventional X-ray imaging, such as the widely known Computed Tomography (CT), is based in the absorption of X-rays, a physical interaction which relies on the densities and/or atomic numbers of the materials comprising the specimens. For mineralized structures such as some fossils, this can represent a limitation in contrast. Synchrotron sources allow other contrast modalities to be explored, such as the phase contrast. Phase-contrast μ-CT has become critical for paleobiological studies in the last decades , due to Metabolic processes of biological systems can generate traces or patterns uncommon to abiotic systems. For geobiological materials, these biosignatures can be present in the form of biominerals with different crystalline organizations or specific distributions, mineral assemblages in association with organic matter and modifications on the mineral surface . Some elIdentifying and mapping the chemical elements present in geobiological structures can provide means of supporting their biogenicity and also understanding their ecological interactions and modes of preservation. Nevertheless, identifying these elements and distinguishing patterns generated by biotic from abiotic processes require approaches with high-spatial resolution, sensitivity to a broad range of elements and to trace element concentrations. In questions about biogenicity studies, in particular for elemental distribution analyzes such as X-ray fluorescence mapping, it is important to consider the co-location of elements with morphological structures of interest, original lithology and its possible changes, the co-occurrence of biogeochemical elements of interest and also their abundance limits taking into account the geological and preservational context. The combination of as much evidence as possible that can explore the depositional history, and especially the diagenetic alterations that may have exerted any changes in the preservation of authigenic characteristics, it is important to minimize any misinterpretation, especially in deep time rocks.Synchrotron-based X-ray fluorescence (SR-XRF) allows the identification, mapping and semi-quantification of chemical elements even in concentrations of parts per billion (ppb), and has a spatial resolution primarily dependent on the size of the X-ray beam, which, in the case of the new generation synchrotrons, can reach nanometric dimensions. The range of elements that can be detected with this approach depends on the energy of the X-rays, once the fluorescence phenomenon relies on the removal of strongly-binded inner-shell electrons, creating vacancies that need to be filled by external electrons. The difference of energy between the electrons involved in this process is emitted as photons, generating a fluorescence pattern which is specific to each element. One example of case study using SR-μ-XRF was applied to discuss the biogenicity of pyrites in a microbial context in order to understand the role of bacteria in the tafonomical process of well-preserved fossilized organisms in Crato Basin, Brazil . The autStudying organic-walled microfossils, One of the main challenges in the search of chemical biosignatures associated to microfossils and microbialites is the fact that the original biochemical components of microorganisms are degraded and altered over time, while other non-biogenic elements are incorporated in the course of diagenetic processes . The metThe possibility to tune the energy of X-rays in synchrotron sources allows the application of X-ray Absorption Spectroscopy (XAS), an approach based on the analysis of the absorption profile of a given element prior to the core electrons removal with the incidence of X-rays . The different regions analyzed on the absorption spectra gives rise to two different techniques, X-ray Absorption Near Edge Structure (XANES), and Extended X-ray Absorption Fine Structure (EXAFS). These techniques are strongly sensitive to the oxidation state of the elements and can also provide information about the coordination of the absorbing ions. When associated to the small beams available at scanning X-ray microscopes, speciation maps with nanometric resolution can be achieved. These techniques have already been applied at lower concentrations and at less-than-perfect sample conditions , as the ca. 1.9 Ga Gunflint Formation. In this study, XANES provided information about the chemical complexity of kerogen, which presented aromatic carbon and oxygenated functional groups. There are several examples of the use of these techniques as a complement both in reinforcing and in questioning the biogenicity of ancient microbialites and microfossils. With the intense photon beams of 3rd and 4th generation synchrotron sources, radiation damage of the samples should also be considered. Potentially preserved organic molecules are the most fragile ones, and can suffer from photooxidation and breakup depending on the measuring condition. However, even inorganic signatures can be altered, as X-rays can change the oxidation state of elements  is a potential technique to be used in the study of mineral biosignatures due to its capability of identifying crystalline phases such as inorganic and organic ordered structures preserved and/or secreted by living organisms. This kind of technique is selective and can identify synthetic or natural minerals . Even if–4∘), considering the Bragg-Brentano geometry and the measurement in 2θ, which can be used to deconvolute very close peaks. This can be used to distinguish biotic and abiotic crystals, for example, magnetic compounds like greigite and magnetites produced by bacteria , is able to give information about the orbital magnetic moment and the spin of the material. The technique provides information about the 3d electronic states in transition metals, such as Fe, Ni, Co, etc., which is responsible for the magnetic properties of the minerals . XMCD ha2+ content in the intracellular magnetites. The exceptional reducing power of bacteria such as Shewanella putrefaciens probably explains the high concentration of Fe2+ in comparison to nanoparticles of abiotic origin.Also using XMCD, The optical activity from organic and inorganic compounds has also been proposed as a tool for the detection and characterization of biosignatures. For example, kerogen , proteinXEOL is a photon-in/photon out technique in which X-rays are used to excite core levels and getting light emitted in the range from the UV to IR . It is sThe complexity of attestations of biogenicity on geological and paleobiological materials makes it essential to explore multiple and complementary approaches in different length and sensitivity scales. For the micron- and nanoscales synchrotron-based techniques represent the forefront of the application of photons for the inspection of a wide range of materials, allowing complex and heterogeneous samples to be studied at an unprecedented level of detail. Synchrotron approaches are been consolidated as important tools for the deeper understanding of the records of ancient life on Earth and for the non-destructive investigation of extremely rare samples, such as meteorites and rocks that will be retrieved from Mars in the near-future sample return missions.The recent developments in synchrotron sources also brings good perspectives for the study of biosignatures. The novel 4th generation sources, such as MAX IV in Sweden, Sirius in Brazil, and the upgraded sources ESRF-II in France, APS-U in the United States and Spring8-II in Japan are opening up new avenues for the nanoscale investigation of different types of materials. For geobiological specimens, these machines will allow the achievement of nanometric spatial resolution for resolving preserved morphological fossils and also microbial-mineral interactions with different chemical and morphological contrast information, high energy and high spectral resolution for probing, mapping and speciating heavy Z elements and high sensitivity to elements in trace concentrations. These advances will allow complex and important questions on the early chemical and morphological biosignatures to be attacked, likely consolidating synchrotron paleometry and nanopalebiology within biogeosciences and astrobiology.All authors contributed to the literature revision and manuscript writing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tree frogs have the remarkable ability to attach to smooth, rough, dry, and wet surfaces using their versatile toe pads. Tree frog attachment involves the secretion of mucus into the pad-substrate gap, requiring adaptations towards mucus drainage and pad lubrication. Here, we present an overview of tree frog attachment, with focus on (i) the morphology and material of the toe pad; (ii) the functional demands on the toe pad arising from ecology, lifestyle, and phylogenetics; (iii) experimental data of attachment performance such as adhesion and friction forces; and (iv) potential perspectives on future developments in the field. By revisiting reported data and observations, we discuss the involved mechanisms of attachment and propose new hypotheses for further research. Among others, we address the following questions: Do capillary and hydrodynamic forces explain the strong friction of the toe pads directly, or indirectly by promoting dry attachment mechanisms? If friction primarily relies on van der Waals (vdW) forces instead, how much do these forces contribute to adhesion in the wet environment tree frogs live in and what role does the mucus play? We show that both pad morphology and measured attachment performance suggest the coaction of several attachment mechanisms  with situation-dependent relative importance. Current analytical models of capillary and hydrodynamic adhesion, caused by the secreted mucus and by environmental liquids, do not capture the contributions of these mechanisms in a comprehensive and accurate way. We argue that the soft pad material and a hierarchical surface pattern on the ventral pad surface enhance the effective contact area and facilitate gap-closure by macro- to nanoscopic drainage of interstitial liquids, which may give rise to a significant contribution of vdW interactions to tree frog attachment. Increasing the comprehension of the complex mechanism of tree frog attachment contributes to a better understanding of other biological attachment systems (e.g. in geckos and insects) and is expected to stimulate the development of a wide array of bioinspired adhesive applications.The online version of this article (10.1186/s12983-018-0273-x) contains supplementary material, which is available to authorized users. Strong, reversible, and repeatable grip to diverse substrates is a basic requirement for climbing animals . A wide With their toe pads, tree frogs attach to a wide range of substrates, from smooth glass to rough wood  in both Several attachment mechanisms have been proposed for tree frogs’ toe pads e.g. , 24, 25), 2524, 2Despite the substantial progress made in the understanding of tree frog attachment over the last centuries, several questions remain unanswered. For example, do capillary and hydrodynamic forces explain the strong friction of the toe pads directly, or indirectly by promoting dry attachment mechanisms? If friction primarily relies on vdW forces instead, how much do these forces contribute to adhesion in the wet environment tree frogs live in and what is the function of the mucus? Are there other attachment mechanisms active in the toe pads and how do these mechanisms interact? Several questions concerning the functional morphology of the attachment apparatus also remain open. Can the smooth soft toe pads of tree frogs conform closely to rough substrates to form a large area of dry contact and strong vdW forces, as described for the hairy attachment organs of geckos , 27 and We discuss these questions by revisiting evidence regarding the attachment performance of tree frogs or, when information is lacking, formulate new hypotheses for further research. First, we describe the morphology and material properties of the toe pads. Subsequently, a set of functional demands regarding adhesion and friction, which the toe pad presumably accommodates, is presented as well as the physical fundamentals of the mechanisms that have been proposed in previous research to explain tree frog attachment. Next, we discuss the observed attachment performance of tree frogs with respect to the stated questions, the functional demands, the morphological and material properties of the pad, and the physical fundamentals of attachment. Finally, we present conclusions of the reviewed knowledge available on tree frog attachment and provide perspectives for potential future developments in the field.1) down to the nanoscopic features of the toe pad epidermis . To get insight in where and how contact forces are generated, we categorise the morphological elements based on their potential functionality (e.g. attachment control and force transmission). Furthermore, we discuss the material properties of the pad and the secreted mucus. For open questions on the pad morphology (and for possible approaches to answer these), we refer to the final section.In this section, we describe the morphology of the limbs of tree frogs from the macroscopic anatomy Fig. A1 down tAp of single pads was reported by Linnenbach , Ba-Omar et al. , Mizuhira ().With respect to the effects of the variation of substrate properties on tree frog attachment, only roughness has been tested extensively. In geckos, the effects of variations in surface and subsurface energy as well as stiffness of the substrate have been analysed experimentally , 116. AnIn contrast to geckos, tree frogs secrete mucus, which presumably fulfils various functions: (i) epidermal water and oxygen uptake, (ii) capillary and hydrodynamic force generation, (iii) avoidance of pad stiffening and a reduced conformability, and (iv) lubrication and hence reduction of abrasive wear. Studying the mucus properties is central to elucidating tree frog attachment.Ra≤10–20 µm), vdW forces could be dominant and any liquid between pad and substrate would weaken this adhesion component. On rougher substrates, a liquid filled pad-substrate gap may support capillary adhesion, which then could  compensate for the expected reduction of vdW forces. Based on this hypothetical interplay, we predict a trade-off in force generation based on capillarity and vdW interactions: Liquid filling the pad-substrate gap enhances capillary forces, but too much liquid reduces the Hamaker constant and vdW interactions.Considering the effects of variations of roughness and wetting on tree frog attachment, we propose an interplay of vdW and capillary forces depending of substrate roughness: On low-roughness substrates  and consequently the importance of dry contacts: The hierarchical surface pattern on the ventral pad surface presumably reduces the effective bending stiffness of the surface and increases the wetted area of the pad. The intercellular channel network, the convex pad curvature, and macroscopic grooves on the ventral epidermis may facilitate viscosity-dominated drainage of interstitial liquids and gap-closure in the central part of the pad, and inertia-dominated drainage in the grooves and in the peripheral contact region, unaffected by the presence of epidermal surface structures . FurtheThe trajectories of the epidermal tonofilaments presumably facilitate force transmission through the epidermis, but how are forces transmitted from there to the phalanx and to other body parts? We suggest a detailed morphological analysis  of tree frogs’ toe pads, with focus on force-transmitting structures, such as cytoskeletal elements, connective tissue, and muscular tissue. Such an analysis may show (i) where exactly contact forces are generated, (ii) which types of loading are dominant in the pads, and (iii) whether shear stiffening, as observed in geckos , 117, isLittle is known about active components and attachment control in the toe pads. Can tree frogs actively modify the geometry of the epidermal channels to control, for example, hydrodynamic force generation? Do the pads facilitate energy recovery during take offs from compliant substrates ? The musNext to capillary friction and suction  could further advance the design of biomimetic adhesives. In this light, we propose a comparative examination of intraspecific differences in habitat, attachment performance, and morphology of the whole pad as well as the epidermal surface structures, as done in geckos , 122. OnAdditional file 1Symbols and abbreviations. List of symbols, List of abbreviations. Morphology and material properties of a toe pad. Geometrical model of the surface of the ventral toe pad epidermis, Fibre-matrix analogy of a toe pad, Scaling of tenacity with snout-vent-length. Attachment performance of tree frogs. The rotating platform experiment, Capillary adhesion of deformable objects, Capillary friction, Suction, Kendall peeling model, Johnson-Kendall-Roberts model. (DOCX 125 kb)


The purpose of this study is to discuss the total joint reconstruction surgery for a patient with recurrent ankylosis in bilateral temporomandibular joints (TMJs) using three-dimensional (3D) virtual surgical planning, computer-aided manufacturing (CAD/CAM)-fabricated surgical guides, and stock TMJ prostheses.A 66-year-old female patient, who had a history of multiple TMJ surgeries, complained of severe difficulty in eating and trismus. The 3D virtual surgery was performed with a virtual surgery software . After confirmation of the location of the upper margin for resection of the root of the zygoma and the lower margin for resection of the ankylosed condyle, and the position of the fossa and condyle components of stock TMJ prosthesis , the surgical guides were fabricated with CAD/CAM technology. Under general anesthesia, osteotomy and placement of the stock TMJ prosthesis (Biomet) were carried out according to the surgical planning. At 2 months after the operation, the patient was able to open her mouth up to 30 mm without complication.For a patient who has recurrent ankylosis in bilateral TMJs, total joint reconstruction surgery using 3D virtual surgical planning, CAD/CAM-fabricated surgical guides, and stock TMJ prostheses may be an effective surgical treatment option. Ankylosis of the temporomandibular joint (TMJ) is a disabling condition in mastication, speech, facial expression, pain, and oral hygiene, resulting in compromise of patient’s quality of life , 2. The There are a variety of surgical options to manage the TMJ ankylosis including gap arthroplasty, interpositional arthroplasty, and total joint reconstruction . In caseThe most frequently used alloplastic prosthetic device for total joint reconstruction is stock TMJ prosthesis and custom-made TMJ prosthesis . The stoOn the other hand, the custom-made TMJ prosthesis system only requires a single operation. Due to use of computer-aided design and computer-aided manufacturing (CAD/CAM)-fabricated surgical guide and custom-made TMJ prosthesis, the precise operation can be performed with less time-consuming due to accurate match of the custom-made TMJ prosthesis with individual patient’s anatomy , 14. AltTherefore, it is necessary to combine the advantages of the two systems: low-cost and single operation with precise outcome. We combined three-dimensional (3D) virtual surgical planning, CAD/CAM-fabricated surgical guides, and stock TMJ prostheses for total joint reconstruction. The purpose of this case report was to discuss the total joint reconstruction surgery for a patient with recurrent ankylosis in bilateral TMJs using above-mentioned technology.A 66-year-old female patient visited the Department of Oral and Maxillofacial Surgery, Seoul National University Dental Hospital, with a complaint of severe difficulty in eating due to limitation of jaw function and trismus. The patient had a history of multiple TMJ surgeries for reduction of condyle fracture of both sides and arthroplasty/coronoidectomy due to trismus. Clinically, the patient exhibited the maximum mouth opening of 8 mm and relapse of severe trismus Fig. . After CThe 3D virtual surgery was performed with a specific navigation surgery software . As the first step, both condyles and their surrounding hyperplastic bone were removed, while making it sure to avoid the mandibular foramen. The second step was virtual placement of the condyle and fossa components of the stock TMJ prosthesis. After placement of the fossa and condylar components, any areas of interference were investigated by virtually opening the jaw.After confirmation of the position of the fossa and condyle components, the surgical guides were fabricated by using the CAD/CAM technology. The surgical guides consist of the upper and lower parts. The upper part is designed to be adapted onto the root of the zygoma, which guides the location of upper margin for resection of the root of the zygoma and the position of the fossa component of the stock TMJ prosthesis. The lower part is designed to be adapted onto the mandibular angle area, which guides the location of lower margin for the resection of the ankylosed condyle and has several drill holes for fixation screws of the condyle component of the stock TMJ prosthesis Fig. .Fig. 3RUnder general anesthesia, the total joint reconstruction surgery was performed via preauricular and submandibular incisions. Osteotomy and placement of the stock TMJ prosthesis were carried out according to the 3D virtual surgical planning Fig. . At 2 moComplete bony ankylosis of the TMJ is a significant problem for patients as well as doctors. When the total joint reconstruction surgery with alloplastic materials is planned, it would be necessary to combine the advantages of stock TMJ prosthesis and custom-made TMJ prosthesis: a single-stage approach, low-cost, more precise surgical outcome, and less operation time-consuming.There are several considerations for accurate placement of the stock TMJ prosthesis. First, since the stock TMJ prosthesis does not fit perfectly to the resection site due to straight design, leading to difficulty in properly positioning the condylar component medio-laterally. As a solution for this problem, the fossa component of the stock TMJ prosthesis system was fixated, followed by fixation of the condylar component and adjustment of its medio-lateral position by controlling the depth of the fixation screws.Second, because the tissue surface of the condylar component of the stock TMJ prosthesis is flat, it is difficult to get a perfect adaptation onto the resection site. Therefore, the frontal ramus inclination of the condylar component of the stock TMJ prosthesis should be meticulously investigated. In the frontal view, if the lower margin of the condyle components of the stock TMJ prosthesis at the mandibular angle area is located medial to the glenoid fossa, the condyle head portion of the condyle components of the stock TMJ prosthesis may be positioned too laterally. In contrast, if the lower margin of the condyle components of the stock TMJ prosthesis at the mandibular angle area is located lateral to the glenoid fossa, opposite situation may arise. By controlling the insertion depth of fixation screws, such problems can be overcome. The insertion depth of fixation screws can be controlled while observing the relationship between the condyle head portion of the condyle component and the glenoid fossa. For example, if the frontal ramus inclination of the condylar component of the stock TMJ prosthesis is inclined to inward, the upper fixation screws for the condyle components can be inserted loosely, while the lower fixation screws can be inserted to their full depth.The advantage of the system we used is that the cutting condyle and positioning TMJ prostheses can be determined according to the patient’s anatomy before surgery. Therefore, the need to perform the staged operation in two times is reduced, the operation time can be shortened, and using stock prostheses is cheaper than customized TMJ prostheses. In disadvantage, it’s less accurate than customized. The three-dimensional position can be grossly positioned the same as the surgical plan, but because the adaptation of prostheses is not accurate, minor differences such as the frontal ramal angle may occur. In order to overcome this drawback, the condyle prostheses should be placed first, and then the condylar part can be positioned more accurately. Another disadvantage of this system is that when condyle cutting, prediction of medio-lateral depth is not possible, so the perception of the safety zone toward the cranial base must be present before surgery.In this case with recurrent bony TMJ ankylosis in both sides, all treatment objectives of total joint reconstruction surgery including improvement of mouth opening, correction of deformity, pain relief, and prevention of re-ankylosis were met with the use of 3D virtual surgical planning, CAD/CAM-fabricated surgical guides, and stock TMJ prostheses. However, it is necessary to increase the sample size and longitudinal long-term follow-up for evaluating possible complications.For a patient who has recurrent ankylosis in bilateral TMJs, total joint reconstruction surgery using 3D virtual surgical planning, CAD/CAM-fabricated surgical guides, and stock TMJ prostheses may be an effective surgical option to obtain precise surgical outcome with low-cost, less operation time consuming, and a single-stage approach.