Cancer is a leading cause of death worldwide and therefore one of the most important public health concerns. In this contribution, we discuss recent key enabling technological innovations , including biomarker-based technologies, that potentially allow for decentralization  with the increasing availability of point-of-care technologies in the near future. These technological innovations are moving the field one step closer toward personalized oncology. Cancer is a leading cause of death worldwide and has been characterized as an heterogeneous disease composed of different cancer cell subtypes, including the mesenchymal cells, endovascular cells, and immune cells . Recent Traditionally, cancer diagnosis relies on the expensive and low resolution in vivo clinical imaging modalities, such as X-ray, computed tomography, magnetic resonance imaging, ultrasound, and optical imaging, in combination with (often) invasive surgical techniques required for tumor biopsy analysis. In this perspective article, we discuss the emergence of recent key enabling technological innovations , including biomarker-based technologies, that potentially allow decentralized diagnostics in the near future. A biomarker is defined as a measurable biological parameter  that is indicative of the disease state of the patient and should be highly sensitive, specific, and reproducible . BiomarkIn recent years, significant advances in the semiconductor industry enabled the emergence of a low cost, portable, micro nuclear magnetic resonance (micro NMR) spectrometer using a single chipset for medical point-of-care diagnostics . These dThe new paradigm of lowered engineering barriers   also opeFurthermore, the existence of tumor heterogeneities and cellular subpopulations have highlighted the strong limitations on the predictive power and diagnostic potential of different imaging modalities, and the issue represents a huge challenge for the development of new diagnostic technologies. As the scientific community learns more about the mechanisms of cancer, new diagnostic tools are being developed to access the vast Omics database, which includes the genomic, transcriptomic, proteomic, and metabolomic landscapes and evolvability of different cancers. However, the possibility of identifying a large number of genetic, protein, or metabolic alterations also has some contraindications, since only some of these alterations will contribute to tumor development. Nevertheless, the improved understanding of the underlying cellular and molecular signatures of cancer has made it possible to develop minimally or noninvasive and cost-effective diagnostic tests potentially applicable in decentralized clinical settings.The liquid biopsy of periphery blood offers valuable information , circulating DNA (ctDNA), and exosomes), and scientists are already accumulating evidence suggesting its clinical utilities as a novel biomarker in close association with tumor initiation and progression . One of Various microfluidic technologies had been developed to ‘sieve’ the CTCs  from the complex of blood matrixes . These mInterestingly, a recent report by Ghazani et al. compared the diagnosis outcome of bulk tumors and CTCs in peripheral blood samples using the micro NMR system, and only a weak correlation was found between each paired sample, suggesting that using CTCs as liquid biopsies and proxies to metastatic solid lesions could be misleading . In a seOn one hand, technologies, such as the digital polymerase chain reaction (ddPCR) and next generation sequencing (NGS), used to unravel the genetic profile of cancer cells are quickly emerging, with several advantages in comparison to conventional technologies  to the epigenome . This will require the development of machine learning in the form of mathematical models that are capable of making a reliable prediction  for disease diagnostics and management  in a personalized manner. Recently, artificial intelligence in the form of machine learning models  have been trained to interpret images of skin and breast cancer in cancer diagnosis [2-weighted, diffusion weighted, and the dynamic contrast-enhanced image.The paradigm shift of personalized oncology medicine sees the marriage between novel technologies and the discovery of novel biomarkers. In the near future, we will witness the identification of an increasing number of biomarkers from the genome  focusing on the rapid molecular phenotyping of biofluids ,46, whic


Tissue-engineered skin (TES), as an analogue of native skin, is promising for wound repair and regeneration. However, a major drawback of TES products is a lack of skin appendages and nerves to enhance skin healing, structural integrity and skin vitality. Skin appendages and nerves are important constituents for fully functional skin. To date, many studies have yielded remarkable results in the field of skin appendages reconstruction and nerve regeneration. However, patients often complain about a loss of skin sensation and even cutaneous chronic pain. Restoration of pain, temperature, and touch perceptions should now be a major challenge to solve in order to improve patients’ quality of life. Current strategies to create skin appendages and sensory nerve regeneration are mainly based on different types of seeding cells, scaffold materials, bioactive factors and involved signaling pathways. This article provides a comprehensive overview of different strategies for, and advances in, skin appendages and sensory nerve regeneration, which is an important issue in the field of tissue engineering and regenerative medicine. The skin is composed of the epidermis, dermis and subcutaneous tissue, and is the largest organ of the human body Fig. . The funTissue-engineered skin is a new type of tissue graft that can be used to directly treat wounds, induce cell migration, promote vascularisation and epithelisation, and accelerate wound healing . TES is Skin appendages include hair follicles, sweat glands and sebaceous glands. The recovery of skin sensory function is an important indicator of cutaneous regeneration. All of the skin appendages and nerves play important roles in the physical, chemical and biological functions of normal skin. Regeneration of appendages and nerves requires the use of stem cells, biomaterial scaffolds, bioactive factors and other growth-stimulating factors Fig. . Among tBiomaterial scaffolds provide seed cells with an environment for adhesion, growth, proliferation and metabolism, which are also important for skin appendages reconstruction and nerve regeneration. A number of biomaterials have been developed for possible skin-replacement therapies. Initially, pure biological materials were extracted and treated to act as scaffolds for cultured cells, and later, various synthetic and semi-synthetic materials were used to either support existing tissues and protect them, or replace them altogether . BiomateBioactive factors, including various cytokines and growth factors, are also important and play vital roles in tissue reconstruction and regeneration. A variety of bioactive factors have been shown to promote cell proliferation and differentiation, accelerate vascularisation, regulate wound inflammation, and promote appendage regeneration and nerve repair; they also have potential applicability in wound repair and tissue regeneration. Some bioactive factors, such as recombinant bovine basic fibroblast growth factor (rb-bFGF), granulocyte-macrophage colony stimulating factor (GM-CSF) and human granulocyte colony stimulating factor (hG-CSF), have already been developed commercially and gradually adopted in clinical applications –25.Tissue-engineered skin is an inspiring alternative for skin appendages and sensory nerve regeneration. Cells including stem cells, biomolecules, and biomaterials have been widely used to establish a functional vascular and nerve network and induce in situ wound healing, tissue regeneration. However, patients often complain about a loss of skin sensation and cutaneous chronic pain. Restoration of pain, temperature, and touch perceptions should be a major challenge to solve in order to improve patients’ quality of life. Optimisation of existing TES constructs or development of novel TES products, especially those involving skin appendages and sensory nerve regeneration, is a major trend in the field. Herein, we introduce the recent advances and key aspects of different strategies for skin appendages and sensory nerve regeneration, followed by descriptions of current challenges, and future directions.Hair follicles reside in the dermal layer of the skin and are made up of hair papillae, hair matrix, root sheath and hair bulges. The papilla is a large structure at the base of the hair follicle, which is composed mainly of connective tissue and a capillary loop. The papilla is surrounded by the hair matrix. The root sheath is composed of an external and internal root sheath. Finally, the bulge is located in the outer root sheath at the insertion point of the arrector pili muscle. It houses several types of stem cells, which supply the entire hair follicle with new cells, and takes part in healing of the injured epidermis , 27. HaiTo date, at least three main signalling pathways have been confirmed to participate in hair follicle development, i.e. the Wnt, Sonic hedgehog (Shh) and bone morphogenetic protein (BMP) signalling pathways. Activation of the Wnt signalling pathway is required to form hair follicles , 30. InhHair follicle regeneration can be achieved by applying a number of different types of stem cells . Stem ceThe implantation of porcine embryonic skin precursors (PESPs) to generate hair follicles and other skin appendages has been reported. Huang et al.  first reRecent advances regarding the utilisation of BMSCs have been reported , 45. GreThe dermal papilla is widely recognised as the key signalling centre for hair follicle regeneration, which is responsible for maintaining hair growth and controlling the complex hair follicle cycling system . Oliver In recent years, methods of hair follicle regeneration based on tissue engineering have been developed Table . Lee et The prerequisite for the application of hair follicle regeneration in clinical treatment is that the cells need to be of ethnic origin. However, most of the hair papilla cells studied for hair follicle regeneration are of mouse origin. Few studies have shown that human hair papilla cells cultured in vitro can regenerate intact hair follicles. The main reason is that human hair papilla cells quickly lose their induction ability in vitro culture conditions. How to maintain the induction ability of dermal papilla in vitro culture is a major problem. The induction function of hair papilla cells to hair follicles and the expression of characteristic genes of dermal papilla are highly dependent on their internal microenvironment. After in vitro culture, the expression of many characteristic genes of hair papilla cells, such as , decreased rapidly, accompanied by a decline of hair follicle induction ability. In summary, the greatest challenge in hair follicle regeneration is how to improve the conditions of in vitro three-dimensional (3D) culture, which in turn would truly simulate the growth environment of hair follicles in vivo, and maintain the biological characteristics of dermal papilla cells and HFSCs to realise reconstruction of mature hair follicles in vitro.At present, few researches on biological 3D printing to regenerate hair follicles. Abaci et al.  simulateSebaceous glands are important accessory organs of the skin. Most of the sebaceous glands are located between the hair follicle and the arrector pili muscle, and consist of one or more vesicles as well as a common short duct. The catheter consists of stratified epithelium, most of which opens in the upper part of the hair follicle, although some directly opens on the skin surface. There is a layer of immature cells with abundant organelles and the ability to generate new acinar cells around the acinus through active cell division. Sebaceous glands are distributed over the entire skin surface, except on the palms of the hands and soles of the feet. They show the highest densities on the scalp and face. The main functions of sebaceous glands are to secrete sebum and lubricate the skin. The sebaceous glands also form part of the body’s integumentary system and serve to protect the body against microorganisms. Sebaceous glands secrete acids that form the acid mantle, which is a very slightly acidic film on the surface of the skin that acts as a barrier to bacteria, viruses, and other potential contaminants that may penetrate the skin. Sebaceous glands also have antioxidant effects. Vitamin E, which is transported and secreted by the sebaceous glands, is the main component of the skin’s antioxidant system . The funIn contrast to the process of hair follicle morphogenesis, the cellular and molecular mechanisms that control morphogenesis during sebaceous gland organogenesis are still unknown . For manHair follicle bulge cells with self-renewal capacity and slow periodicity do not only differentiate into the epidermis, but also into skin appendages, including hair follicles, sweat glands, sebaceous glands, etc. Panteleyev et al.  examinedIn addition to HFSCs, other cells were also reported to reproduce sebaceous glands. GFP-positive bone marrow cells were transplanted in a mixture of embryonic mouse skin cells onto murine skin defects, and GFP-positive cells differentiated into sebaceous gland cells within 3 weeks . In anotThe bulge area located at the level of insertion of the arrector pili muscle is considered to be a site of pluripotent epithelial stem cells . This hyThe co-localisation of cells positive for the above specific factors with the developing sebaceous glands was not sufficient to confirm that they were sebaceous progenitors . FurtherSweat glands play important roles in regulating body temperature and maintaining fluid balance. Heat dissipation methods, such as conduction, convection and radiation, cease to function in the human body at environmental temperatures above 30 °C, and the secretion of sweat by sweat gland cells (SGCs) becomes the only means of heat dissipation . The sweMultiple genes and signalling pathways have been confirmed to play roles in sweat gland morphogenesis. A variety of cytokines and signal transduction pathways participate in the process of sweat gland regeneration and play important regulatory roles in the process of sweat gland development .The extracellular signal-regulated kinases (ERKs) are widely expressed protein kinases that act as intracellular signalling molecules in the regulation of various physiological processes, such as cell growth, development, division and death . The corHypohidrotic ectodermal dysplasia (HED) characterised by developmental defects of sweat glands, hair and teeth, is caused by ectodysplasin-A (EDA) gene mutation, indicating that EDA signals play indispensable roles in the development of sweat glands. EDA-A1 and EDA-A2 are two functional molecules encoded by the EDA gene. The receptors for these molecules are EDAR and XEDAR, respectively. EDA-A1 is directly related to HED and can release functional domains through hydrolysis. The functional domain connects with EDAR to activate a series of downstream signals, such as receptor adapter EDARADD (EDAR-related death domain) and nuclear factor kappa light chain enhancer of activated B cells (NF-κB), thus promoting the occurrence and development of skin appendages . The NF-In addition, Lei et al.  reportedPatients with large-area burns often suffer from loss of sweat glands, resulting in severe impairment of thermoregulatory function and a significant reduction in quality of life . TherefoEpidermal stem cells, as the specific stem cells of skin tissues, are key for skin tissue regeneration, wound repair and remodelling . TherefoMesenchymal stem cells have great potential for sweat gland regeneration. Li et al.  BMSCs diIt has recently become possible to achieve 3D reconstruction of the eccrine gland structure in vitro . Hu et aSweat gland regeneration is a complex process involving multiple genes and signaling pathways. And the regeneration of sweat gland requires more in-depth research on the mechanism that regulate stem cells reprogramming into sweat gland-like cells. Further explore the function of sweat gland development-related factors, find key genes involved in sweat gland tissue formation and their regulatory characteristics, and provide new technical methods for the reconstruction of sweat gland function in skin wound repair.The neural network in the skin is widely distributed and has a complex structure. Cutaneous innervation involves sensory and motor nerves; the former play a major role in a number of skin functions. There are three main types of sensory nerve fibres distributed on the skin, i.e. unmyelinated C group fibres responsible for touch, temperature and pain sensation, A-δ fibres involved in the perception of mechanical stimuli, temperature, and rapid-onset pain, and A-α fibres that respond to light tactile pressure and proprioception . In addiThe motor nerves of the skin are mainly sympathetic nerves, although there are a few parasympathetic nerves in the face. Although there are few skin motoneurons, they also play an important role in normal skin. The skin motoneurons, which are mostly distributed in the dermis, mainly secrete neurotransmitters, neuromodulators and neuropeptides to regulate important functions, such as sweat gland secretion, vasoconstriction and temperature balance adjustment .The skin can produce and secrete neurotrophic factors (NTFs). When the skin is injured, NTF synthesis is increased and their activities are enhanced, thus protecting the damaged neurons and promoting the repair of peripheral nerves , 113. ThThe first growth factor to be identified, NGF, was shown to play important roles in the nervous system, angiogenesis and wound healing . NGF canGlial cell line-derived neurotrophic factor was first isolated from the conditioned medium of the B49 mouse glial cell line in 1993 . GDNF waNeurotrophin-3 is a protein growth factor that exerts effects on certain neurons in the peripheral and central nervous systems; it helps to support the survival and differentiation of existing neurons, and encourages the growth and differentiation of new neurons and synapses. NT-3 was the third NTF to be characterised, after NGF and BDNF . Mice boThe efficacy of multiple factors in repairing skin nerve injury is superior to that of single factors. As the levels of various NTFs and their receptors change after nerve injury, and where they participate in nerve repair activities in concert, combining the effects of factors in vivo is more concordant with the actual state of the body , 129. ThSkin nerve repair and regeneration occur in two ways, i.e. via local stem cell proliferation and differentiation, and the extension of healthy axons. Therefore, mobilisation of stem cells to differentiate into neural tissue may promote nerve regeneration. Skin-derived precursor stem cells can be obtained by simple skin tissue biopsy, and have good multidirectional differentiation potential. They can proliferate and differentiate into nerve cells and glial cells in vitro, and can effectively induce regeneration of skin sensory nerves , 135. ThSchwann cells are known to play supporting roles in nerve regeneration . When thFollowing a deep skin defect caused by severe burns, large numbers of Fbs proliferate and accumulate around the wound and nerve endings. Although Fbs are vital for wound healing and tissue repair, they are not conducive to the regeneration of blood vessels and nerves . TherefoDuring the process of TES-induced neo-tissue formation, nerve regeneration is highly dependent on newly formed blood vessels. With lack of blood supply, nerve regeneration is almost completely inhibited. While the vascular embryo is growing, a large number of new nerve fibres surrounding the blood vessels gradually regenerate along with the extension of blood vessels, eventually reaching the epithelial layer to form FNEs or connect with receptors . There aThe regeneration of skin appendages is still one of the greatest challenges in the fields of tissue engineering and regenerative medicine. Numerous approaches have been developed over the past several decades to overcome those problems, and some encouraging results have been reported. However, further research is needed to better understand and promote the regeneration of skin appendages. The most important aspect is optimising the design, i.e. skin construction and functional positioning, of TES. The construction of scaffolds is often considered to be a key factor in TES, as scaffolds are not only used as DRTs to induce tissue regeneration, but also as carriers for inoculation of seeding cells and structures modified by growth factors. In the field of biological materials, many materials have been shown to have potential as TES scaffolds, but few TES products have been developed in the laboratory for clinical applications. In addition, as cell components and growth factors are introduced into the scaffold to create active tissue, the interactions between cells, matrix material and growth factors become more complex. Therefore, there is a great deal of research on optimising biological materials and structures. Appendage regeneration, rapid vascularisation and nerve repair of the scaffold have also become important indices of the effectiveness of scaffold construction . The dirNerve regeneration is a challenging but intriguing topic, which also requires more research attention. Firstly, nerve regeneration is not equivalent to normal functional recovery. Most reports published to date have generally regarded the number of nerves regenerated as an “observation index”, and seldom considered the recovery of sensory function after nerve regeneration . Therefore, future studies should examine how to promote neural integrity and regeneration. Secondly, in cases of severe skin injury, such as deep burns, the receptors of various nerve fibres are severely damaged. In particular, the receptors of A-β nerve fibres located deep within the skin show very poor regeneration after being completely destroyed. An effective method could promote the growth of nerve fibres into new tissues, but most of the tactile functions cannot be recovered due to the lack of receptors, while the retention of peripheral receptors largely depends on the depth of the skin defect. Although some haptics can be regenerated by reconstructing the follicular structure, the effectiveness of this approach remains to be clinically tested. Further research is required regarding the reconstruction of receptors to achieve the ultimate goal of restoring sensory function of the skin.The ultimate goal of TES is the development of a TES equivalent that combines living cells, biomaterials and bioactive factors to mimic all of the functions of human skin. Currently, although many mature TES products are commercially available, most of these products are only structurally similar to human skin, and provide only barrier function. Due to their lack of skin appendages and nerves, these tissues do not have the complete function of the intact skin and thus do not represent true regeneration thereof. The rapidly advancing progress of research on cells and materials brings the goal of achieving advanced TES containing whole skin appendages, blood vessels and nerves closer to fruition. Further steps from bench-to-bed of the research results can be anticipated in the forthcoming decades.


Sepsis is a leading cause of morbidity and mortality worldwide. It is defined as the presence of a Systemic Inflammatory Response Syndrome, and it represents a significant burden for the healthcare system. This is particularly true when it is diagnosed in the setting of nosocomial infections, which are usually a matter of concern with regard to medical liability being correlated with increasing economic costs and people’s loss of trust in healthcare. Hence, the Italian governance promotes the clinical risk management with the aim of improving the quality and safety of healthcare services. In this context, the role of medico-legal experts working in a hospital setting is fundamental for performing autopsy to diagnose sepsis and link it with possible nosocomial infections. On the other hand, medico-legal experts are party to the clinical risk management assessment, and deal with malpractice cases and therefore contribute to formulating clinical guidelines and procedures for improving patient safety and healthcare providers’ work practices. Due to this scenario, the authors here discuss the role of medico-legal experts in Italy, focusing on sepsis and nosocomial infections. Sepsis is a leading cause of morbidity and mortality worldwide. It refers to a syndrome of physiologic, pathological, and biochemical abnormalities induced by infection. Despite the efforts of the scientific community to reevaluate and revise clinical, imaging, and laboratory features, the diagnosis of sepsis is still challenging . In thisNosocomial infections (NI) are defined as infections acquired in hospitals by patients admitted for a reason other than that infection. The infection is not present or incubating at the time of admission and can appear after discharge. The most frequent NI are those resulting from surgical wounds, urinary tract infections, and lower respiratory tract infections . HospitaIn recent decades, the Italian governance has promoted Clinical Risk Management (CRM) with the aim of improving the quality and safety of healthcare services. On the other hand, a new normative scenario has been established, whereby inpatient institutions have the possibility of directly compensating damage claims arising from alleged medical negligence, thus obviating the cost of liability insurance. In this setting, a fundamental role is reserved for medico-legal experts. The four years training in forensic  medicine offered by Italian universities concentrates on general practices in forensic pathology, including autopsy and the scientific methods applied in crime investigation. In addition, physicians are trained on evaluation of damages in personal injury cases, including those linked with medical malpractice .Hence, hospital medico-legal experts perform medical autopsy and are on the staff of the Risk Management Unit (RMU) that evaluate the malpractice claims, compensate damages, and lay down elaborate care pathways for preventing errors.The association between sepsis and medical autopsy is not new in medical literature, as evidenced by the Latin axiom mortui vivos docent (‘The dead teach the living’). Ignaz Philipp Semmelweis was considered the father and pioneer of improved NI prevention, thanks to his job in supervised medical autopsy . The diaDiagnosis of sepsis deserves more attention in hospital settings when hypothesis of malpractice arises after the death—often unexpected—of inpatients. Sepsis has mortality rates as high as 30–90% , and it Did the patient suffer from a septic condition prior to death?Did sepsis cause or contribute to death?What was the specific portal of entry of microorganisms?Have clinical diagnosis and treatment been prompt and accurate?Could the patient’s death have been avoided with different management?When the forensic pathologist manages a sepsis fatality, the questions should be:The aim of autopsy and the following investigations is to understand if other underlying diseases could have been overlooked, contributing to sepsis and fatal outcome in sepsis-related fatalities of inpatients. The postmortem diagnosis of sepsis is by far the most difficult to establish in forensic pathology. Sepsis is characterized by life-threatening organ dysfunctions due to dysregulation of the host’s immune and metabolic response to infection . Innate At present, a forensic protocol or algorithm of action for postmortem diagnosis of infection-related fatalities has not been developed, even though the research about markers for sepsis has given promising results . The first issue is examination of the circumstances of death and the medical history, especially the results from antemortem diagnostic procedures such as laboratory and microbiological tests . SometimDuring autopsy, blood samples for the determination of biochemical sepsis markers should be collected by aspiration with a sterile needle and syringe from the femoral vein, after the dissection of the upper thigh. At least two postmortem blood samples at different time points are recommended, with the aim of following the concentration of the parameters from the time of death . AlternaSeveral biomarkers have been evaluated for diagnosis of sepsis-related deaths, including biochemical molecules involved in the pathogenesis of sepsis cascade ,8.C reactive protein (CRP) is an inflammation marker of acute-phase response in infections , inflammatory diseases, necrosis , trauma, and neoplasms ,8. PostmProcalcitonin (PCT) has an important role in the response to infections microbial infections ,15. PostLipopolysaccharide-binding protein (LPB) is released into blood as a type I acute-phase reactant after bacteremia or endotoxemia; an increase in postmortem serum LBP levels has been observed in sepsis groups, with a marked decrease correlated with interval after death ,20.Soluble interleukin-2 receptor (sIL-2R) is widely expressed by leukocytes as activated B cells, monocytes, eosinophil granulocytes, and natural killer cells. Its release in blood is an indirect marker of activation of T-cells which secrete IL-2 during sepsis ,8,21. Interleukin-6 (IL-6) is a proinflammatory cytokine expressed by leukocytes, fibroblasts, and endothelial cells in the early phase of inflammation ,2. TsokoPostmortem course of Interleukin-1β (IL-1β), sIL-2R, and LBP has been evaluated in septic and non-septic fatalities, showing that sIL-2R and LBP can be useful in diagnosis of sepsis in forensic autopsy practice .Endocan  is a marker of endothelial activation by proinflammatory cytokines and proangiocenic molecules in septic cases; an increase of both endocan and PCT serum levels was found in septic cases .Soluble CD14 subtype (sCD14-ST) presepsin is a type II acute-phase reactant and high-affinity receptor for LPS–LBP complexes in inflammation . IncreasThe levels of copeptin were found to be significantly higher in sepsis cases, and the concentrations correlated with PCT, CRP, and IL-6 values . Soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) is an immunoglobulin expressed on monocyte and macrophage surfaces and upregulated in biological fluids and tissues infected by gram-positive and gram-negative bacteria . IncreasPostmortem serum levels of Troponin I, Troponin T, and NT-proBNP increased in sepsis related deaths in absence of any relevant cardiovascular disease .Neopterin is a marker of T-cell activation and seems to be associated with SIRS; postmortem serum neopterin was investigated in bacterial and viral infection-related cases as well as in delayed deaths due to trauma ,28. A coGrowth arrest-specific 6 (Gas6) is expressed in leukocytes, platelets, endothelial cells, and monocytes, and increased serum levels have been described in patients with sepsis and septic shock; it is considered a general marker of inflammation rather than a specific biomarker of sepsis .According to the literature, the most useful postmortem marker of sepsis-related deaths may be serum PCT, CRP, sTREM-1, and sIL-2R . Serial The tracing of immunohistochemical antigens has been evaluated in the lungs and the liver as a sign of sepsis ,31. The Moreover, there is a lack of immunohistochemical studies in sepsis, some of them having been conducted only on animals, and therefore it is difficult to use these methods in forensic practice at present.The absence of pathognomonic alteration of organs in sepsis-related deaths is an obstacle for the pathologist . PostmorExternal examination of the cadaver may reveal signs of infection such as inflammatory infiltrations, abscesses, infected wounds (burns), hemorrhagic lesions from DICS , viral/bacterial/fungal–related skin lesions, medical devices as a potential source of infection, and the degree of putrefaction despite the refrigeration of the body ,9,11. Hemorrhagic lesions of pleural, pericardial, and peritoneal membranes as well as of the internal organs;Blood extravasations into the adrenal glands in Waterhouse–Friderichsen syndrome typical in meningococcal septicemia ;Alteration of the lungs which are heavy, congested, edematous, producing frothy/bloody/purulent discharge;The heart with signs of endocarditis, necrosis, parietal thrombi, subepicardial hemorrhages, subendocardial hemorrhages (“Sheehan hemorrhages”), dilated and flaccid left ventricle for severe left ventricular dysfunction;Enlarged kidneys for cortical edema, congested medulla, septicopyemic abscesses, pyelonephritis, or ischemic necrosis of the renal cortex;Enlarged liver with a tense Glisson’s capsule and rounded edges because of accumulation of leukocytes and interstitial edema, signs of cholangitis (cholestatic jaundice);Enlarged and swollen spleen with septic thrombophlebitis or septicopyemic abscesses; the term acute splenitis (“septic spleen”) refers to a soft consistency of the pulp draining from cut sections;Cerebral infarction, distributed, round to conelike hemorrhages;Gastrointestinal shock lesions such as subserous petechial hemorrhages, erosions, and acute ulcers.The internal examination may reveal ,11,13,32Histopathological lesions may vary considerably, and inflammatory infiltration may demonstrate different degrees of severity. Involvement of the lungs as “shock lung” , of the heart , of the liver , of the spleen (acute hyperplasia of the red pulp and infiltrations of neutrophils and macrophages), of the brain , of the gastrointestinal tract, and of the adrenals should be evaluated ,9,13,29.In conclusion, several factors such as nutritional status, immunodeficiency, immunosuppression, and preexisting pathology should be taken into account when the pathologist faces a potential sepsis-related death. The course of the infectious disease may be influenced by these factors. Therefore, all of the findings at external examination of a deceased, as well as at autopsy, histology, biomarkers analysis, and immunohistochemistry must be interpreted with considerable caution . A number of deaths of inpatients are investigated to establish the causal relationship between exogenous noxae and infection, and eventually death, because litigation for medical malpractice can arise. Despite advances in infection control measures, the diffusion of antibiotic resistance, the often delayed detection of microorganisms, and the difficult management of septicemia contribute to sepsis-related fatalities in hospital facilities. For example, infection is a leading cause of morbidity and mortality in burn patients . ThermalPatient safety and preventing medical liability are two of the most important issues in healthcare system politics in Italy. Dealing with the European Union recommendations and the initiatives conducted by several countries worldwide with the aim of decreasing the risk of harm associated with healthcare, the Italian government has promoted a program to develop activities related to patient safety and CRM since 2007 . If to eIn accordance with the evidence that errors are caused by system failures and not by bad doctors, the Italian Ministry of Health has generated a National Sentinel Event Reporting System, instituted hospital RMU, and implemented recommendations to prevent error and try to develop through training, education, and proactive processes a deep cultural change to improve the safety of the patient and operators ,43,44. TIn this situation, the Law n° 24 of March 8, 2017, represents a cornerstone that states the need to integrate patient safety and risk management from clinical, economic, and legal standpoints ,47. NextOn the basis of this complex law, the role of legal medicine experts has been confirmed in hospital clinical RMU, even in evaluation of economic costs of medical malpractice victims. In Italy, university medico-legal training offers students the opportunity to become experts in forensic pathology as well as in evaluation of damages in personal injury cases. Insurance companies generally pay medico-legal consultants for estimating damage claims as a consequence of medical malpractice. Hospital RMU have a multidisciplinary competence; they are directly connected with the local Legal Office, and one of their fundamental objectives is to prevent and to compensate organizations and healthcare providers in case of adverse events and errors . In ItalThe aim of this medico-legal analysis is twofold. Firstly, by examining the causes of the adverse event/error it is possible to map and validate the care pathway processes, and implementation of all the tools and proactive measures to prevent it. In this phase, it is possible to assess if and what kind of guidelines and evidence-based practices were used. If an avoidable error was identified, the RMU communicates to the Legal Office the need to compensate the injury and the value of the patient’s damage. A Claims Management Committee (CMC) nominated by the hospital governance provides that the damages be compensated directly by the hospital, removing the cost of liability insurance, and guaranteeing greater diligence and efficiency with lower economic outlay . AccordiSepsis related to HAI is one of the serious adverse events in healthcare. The most common types of infections may be caused by bacteria, viruses, fungi, and parasites and develop from surgical procedures, catheters in the urinary tract, blood vessels, or material inhaled into the lungs. HAI cause a significant burden for hospitalized patients in terms of length of mortality, morbidity, length of stay, and costs . From a Prevention of NI is the responsibility of all individuals and services providing healthcare. Therefore, hospital administrations appoint an Infection Control Committee (CIO) who is responsible for establishing and maintaining infection prevention and control, its monitoring, surveillance, reporting, research, and education. The CIO includes wide representation from all relevant disciplines and departments. The hospital pharmacists together with microbiologists and infectious disease experts play a fundamental role in management of NI. They are responsible not only for storing and dispensing the best evidenced anti-infectious drugs but also in development of pathways/practices for everyday sterilization and disinfection in hospital wards . It has Adhering to very stringent and standardized HAI preventive measures to minimize infections has a fundamental defensive value in management of medical liability claims. Conversely, failure to follow safe practices can amount to medical negligence ,55. In sActually, the medico-legal expert evaluates all the medical records dealing with the patients’ diseases as well as the certified steps of hospital ward sterility and infection control measures. It is also important to disclose information to patients and family members. Many times, infections and sepsis are not preventable, and even if well treated, they can progress until death; it depends on the assessment of the patient’s disease severity. Hence, there is a fundamental role of the information given to the patient and relatives during hospitalization, regarding the likelihood of having an infection strictly not linked to negligence in prevention and control of hospital microorganisms but only with the severity of the disease. With regard to NI, medico-legal analysis of the complaint or claim for compensation allows to highlight the need to introduce or improve best-practices to prevent infections. This means also reporting negligent acts of providers or the organizational lack of tools or measures to preserve patient safety. Moreover, a medico-legal report in such a case is useful for arriving at the CMC verdict for compensating the plaintiff or not. In an era in which public hospitals have a great economic burden for sustaining average/high standards of care, it is sometimes difficult to prove that every HAI control and preventive measure has been carried out. It could be enough to demonstrate the absence of qualified procedures for hand hygiene or surgery room sterilization to sustain a claim of negligence on the part of the hospital administrators or staff. Therefore, the medico-legal expert’s task in such cases is multidisciplinary, but regarding malpractice claims, the expert has to evaluate: (i) The correct diagnosis of NI; (ii) the diligent or negligent management by the organization/providers; (iii) whether the damage claimed by the patient is due to infections or to severity of patient’s pre-existing disease. In cases where an error has occurred or there is lack of evidence of best-practice use, the policy is to compensate it out of court. Generally speaking, the awards after a lawsuit on average could be triple or fourfold the size of compensation made directly by the hospital. Compensation and apology for an error is a way also to restore the trust among patients that represents the cornerstone of the doctor‒patient relationship and the core of the people’s perception of the institution .Despite the fact that sepsis remains a leading cause of morbidity and mortality, affecting over 30 million people worldwide each year, public awareness is poor and associated NI are still difficult to diagnose ,60,61,62


Cardiovascular diseases are the leading cause of death around the world. Despite the larger number of genes and loci identified, the precise mechanisms by which these genes influence risk of cardiovascular disease is not well understood. Recent advances in the development and optimization of high-throughput technologies for the generation of “omics data” have provided a deeper understanding of the processes and dynamic interactions involved in human diseases. However, the integrative analysis of “omics” data is not straightforward and represents several logistic and computational challenges. In spite of these difficulties, several studies have successfully applied integrative genomics approaches for the investigation of novel mechanisms and plasma biomarkers involved in cardiovascular diseases. In this review, we summarized recent studies aimed to understand the molecular framework of these diseases using multi-omics data from mice and humans. We discuss examples of omics studies for cardiovascular diseases focused on the integration of genomics, epigenomics, transcriptomics, and proteomics. This review also describes current gaps in the study of complex diseases using systems genetics approaches as well as potential limitations and future directions of this emerging field. Coronary artery disease (CAD) is the most common cause of cardiovascular death . Studiescis or trans regulatory elements that bind transcription factors, enhancers or promoters , CDKN2B (encoding cyclin p15), MTAP (encoding methylthioadenosine phosphorylase), and the long non-coding RNA ANRIL. Integration of genetic and transcriptomic data led to the identification of ANRIL as the top candidate causal gene for CAD at the 9p21 region (ANRIL (linear or circular isoforms), where linear transcripts were associated with atherosclerosis and circular transcripts were protective against atherosclerosis. This process is mediated through the expression of multiple genes regulated in both, cis and trans (ANRIL (DQ485454) is involved in endothelial cells functions important to the development of CAD including monocyte adhesion to endothelial cells, trans-endothelial monocyte migration, and endothelial cell migration  levels and cardiovascular risk  particle levels by modulating hepatic VLDL secretion, suggesting an important role of SORT1 in CAD , including, microRNA, genomics, proteomics and metabolomics, has recently been analyzed to identified novel drivers for CVD. In an exercise to demonstrate the utility of the C/VD database, integrative analysis of this “omics” studies showed enrichment of lipid metabolism, extracellular matrix remodeling, inflammation, and cardiac hypertrophy pathways. In addition, regulatory mechanisms mediated through miRNAs associated with the development of CAD were reported . Th. ThSORT1The identification of causal genes is a critical step toward the translation of genetic loci into biologic processes. The integration of “omic” strategies will accelerate the identification, in a more precise way, of novel molecular mechanisms implicated in CVD. This may eventually result in the characterization of novel pathways and drug targets. Although multi-omics approaches have been successfully applied for the investigation of cardiovascular diseases, the number of studies using this approach is still limited. These studies have been primarily focused on the integration of genomics, transcriptomics, epigenomics, and proteomics. Given the potential of metabolomics, metatranscriptomics, and metagenomics as tools for the identification of biomarkers with potential clinical applicability, the integration of such data will increase the understanding of cardiovascular diseases and accelerate the identification of new diagnostics or therapeutic targets . FinallyPL-M, JW, and AH-V drafted and edited 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 health of piglets is an important issue in pig production. Nutritional support for intestinal development is a significant component of piglet care, and amino acids are essential for intestinal growth and development. For suckling piglets, the sows’ milk and the maternal environment shape the structure and support the function of the intestinal tract. The composition of milk affects intestinal morphology and the digestive, absorption and barrier function. After weaning, the optimal nutritional strategies of their diet are necessary to guarantee the piglets’ intestinal development and growth performance. Amino acids are the most important ingredient in piglet diets. The aim of this review is to collect and analyze the relationship between amino acid nutrition and intestinal development of piglets, and elucidate the impacts on piglet health.The amino acids and other components of diet provide nourishment for piglet intestinal development and maturation. However, early-weaned piglets struggle with tremendous stress, impairing normal intestinal health and leading to intestinal dysfunction and even death. The high prevalence worldwide of post-weaning diarrhoea syndrome (PWDS) in piglets has led to much interest in understanding the important role of nutrients in the establishment and maintenance of a functional intestinal tract. In particular, the impacts of amino acids on these functions must be considered. Amino acid levels greatly influence intestinal development in weaning piglets. The lack of amino acids can cause marked structural and functional changes in the intestine. Therefore, a comprehensive understanding of the functions of amino acids is necessary to optimize amino acid requirements of the developing intestinal tract to maximize piglet health and growth performance. This review summarizes the role of specific amino acids , and branched-chain amino acids (BCAAs)) that have been proven to be beneficial for the intestinal health of weaned piglets. Intestinal development disorder is one of the most important causes of piglet morbidity and mortality during the immediate post-weaning period . At weanAdaptation to the dry diets containing complex components is the first challenging factor faced by the piglets after weaning, because most nutrients are supplied via the sows’ milk before weaning ,11. NutrThe piglet intestinal tract requires nutritional support for its growth and development as well as the function of digestion, absorption and the mucosal barrier ,23. In rDietary amino acids elicit a variety of nutritional and metabolic functions. Nutritionally, amino acids are classified as essential or nonessential for animals based on their traditional role in protein synthesis. However, the critical regulatory roles for amino acids in metabolism have long been ignored. In fact, amino acids and their metabolites are regulators of cellular signal transduction, gene expression and the protein post-translational modification, especially in the intestine ,27. The The abundance of arginine in pig tissues is very high, which plays an important role in regulating gene expression and cell signaling . Adding Current researches have recognized that arginine is a conditionally essential amino acid for pigs at all stages of pig production ,33. AddiGlutamate is considered to be a non-essential amino acid, but it plays an important role in maintaining the growth and health of piglets ,40. GlutGlutamate is one of the most abundant amino acids in food and animal tissues, accounting for 5% to 15% of dietary protein . Due to In pig production, low protein diets not only reduce farming costs, but also reduce environmental pollution. Replacing crude protein with crystalline amino acids can reduce dietary protein levels without compromising the growth performance of pigs ,52. ThreMany previous studies have focused on the requirement, efficacy and metabolism of threonine . RecentlTryptophan is one of the essential amino acids in pigs, and is also the main protein component of animals. Tryptophan has many physiological and biochemical functions during the growth and development of pigs. Although researches on the functions and applications of tryptophan have been ongoing, it is currently not widely used in livestock production due to the high cost of synthesis. Moreover, there are many studies on the applications of tryptophan to livestock and poultry farming, but it is difficult to exert the potential application value of tryptophan because of the lack of systemicity.As an essential amino acid in pigs, tryptophan plays an important role in immune regulation, digestion regulation, anti-stress and protein synthesis regulation. In the production of pigs, the addition of tryptophan can improve the growth performance of growing-finishing pigs and ameliorate the health of weaning piglets. Weaning of piglets may result in decreased daily feed intake, growth performance, gastrointestinal health and immune function, which may lead to susceptible diseases. Studies have shown that feeding the weaned piglets with tryptophan diets significantly reduced the frequency and duration of struggles between the piglets, and improved the integrity of the intestinal morphology . MoreoveThe metabolism of sulphur-containing amino acids (SCAAs) plays a key role in ensuring cell functions. In addition to their role in protein biosynthesis, SCAAs are important biologically active molecules. SCAAs can affect cellular metabolism and function by participating in the regulation of methylation processes and redox states . The impMore importantly, the SCAAs in the diet are critical to the normal functions of the intestinal tract . The intThe branched-chain amino acids (BCAAs) are essential substrates for protein biosynthesis. In addition, recent studies have shown that BCAAs are involved in intestinal growth and development . DietaryAs functional amino acids, BCAAs participate in and regulate key metabolic pathways to improve intestinal development and systemic growth in weaned piglets. BCAAs have a protective effect on intestinal redox status and related gene abundance in weaned piglets with growth retardation . DietaryUnderstanding the dependence of intestinal development on nutrients can help nutritionists to provide optimal nutritional support for weaned piglet health. Amino acids are important functional nutrients involved in the maintenance of the morphology and functions of intestine. Researches on the nutritional and physiological functions of essential amino acids have been very in-depth, and there are suitable recommended additions for piglets. Among them, the recommended level of lysine is 1.45%, arginine is 0.68%, threonine is 0.88%, tryptophan is 0.25%, SCAAs are 0.79% and BCAAs are 1.32% . Intesti


According to the World Health Organization, an unhealthy diet and insufficient physical activity are the leading global risks to health. Dietary behavior is a modifiable factor in cardiovascular disease (CVD) prevention. Furthermore, the fact that cardiovascular events and stress-related emotional disorders share a common epidemiology may indicate the existence of pathways linking these two diseases . Psychos Cardiovascular disease (CVD), is the leading cause of mortality and disability worldwide. According to the Global Burden of Disease study, ischemic heart disease and stroke accounted for 25% of total deaths worldwide in 2013 .The current epidemic of CVD is largely explained by several modifiable risk factors associated with lifestyle, feasible to modify. In this sense, an unbalanced diet, excessive alcohol and tobacco consumption, hypercholesterolemia, diabetes mellitus, high blood pressure, visceral obesity, physical inactivity and psychosocial stress increase the risk of future CVD events and are responsible for an estimated 90% of the population-attributable risk fraction of ischemic heart disease and stroke worldwide . Even moChronic or acute exposure to stress (defined as an ongoing or anticipated threat to homeostasis or well-being) favors to the destabilization of the dynamic balance of the organism, and its response promotes the release of chemical mediators that affect the metabolic and behavioral state in humans. Stress-induced activation of the neuroendocrine hypothalamic-pituitary-adrenal axis (HPA), upon exposure to a stressor cortisol is expected to exert widespread metabolic effects, which is mostly necessary to maintain or restore homeostasis. Stress, perhaps, is the trigger in the cascade of neuroendocrine effects that drive the development of the visceral distribution of adipose tissue, resistance to insulin and the consequent hyperinsulinemia, thus leading to the accumulation of cardiovascular risk factors . Since gThe aim of this mini-review is to summarize the findings in the literature on how stress influences the molecular pathways affecting behavior, dietary patterns (DP) and their impact on health, and the synergistic effects of this cycle.The authors conducted a literature search of available sources describing issues relating to stress, DP and behavior. Research studies were selected based on research topics found in globally acknowledged databases such as Web of Science, PubMed, Springer, and Scopus, from 2010 up to the present time and classified according to their relevancy. The information provided in the selected studies was carefully evaluated and is described and discussed in the following sections.Diet is one of the most studied factors in the pathogenesis of CVD since it affects diverse cardiometabolic risk factors such as obesity, lipid blood profile, blood pressure, glucose-insulin homeostasis, endothelial health, adipocyte metabolism, cardiac function, metabolic expenditure, weight regulation, visceral adiposity, and the microbiome .trans fat  may shut down appetite by corticotropin-releasing hormone action and epinephrine liberation. However, if the stressor agent persists, the increased cortisol increases appetite and the motivation to eat . Experimnd sugar . Researcnd sugar .14 resident microorganisms, among which bacteria are the most well-studied group, predominated by gram positive Firmicutes and gram negative Bacteroidetes  . Specifin humans . A lowerdiabetes . Dysbiosclerosis .Bifidobacterium and Eubacterium species, whereas Mediterranean and Prudent DP have been linked to increases in Lactobacillus, Bifidobacterium, and Prevotella, and decreases in Clostridium . However, dietary manipulation to modify gut microbiome is still at the incipient stage It has been detected that dietary fiber including prebiotics has a beneficial effect on gut microbiota and host health, improving insulin sensitivity, low-grade chronic inflammation, and lipid metabolism , the autonomic nervous system, the enteric nervous system and the HPA .In an organism exposed to stress, the hypothalamus stimulates the pituitary by releasing corticotropin-releasing factor (CRF). In the anterior pituitary, CRF causes the release of the adrenocorticotropic hormone (ACTH), which in turn stimulates the adrenals to cause an increased rate of synthesis and release of cortisol (cortisol in humans and corticosterone in rodents) . CortisoChronically elevated cortisol levels can disturb brain function, affecting cognition, emotion and motivation, short and long-term memory; sustained HPA axis activation also impairs the immune system response, as has been demonstrated particularly in early life stress . CortisoStress-related changes could be also mediated by neuroendocrine hormones, such as norepinephrine (NE) and dopamine (DA); it has long been known that these catecholamines can increase the growth rate of Gram-negative bacteria .In turn, changes in gut microbiome composition has been already related to cognitive disorders and mental illness. There is strong evidence linking major depression and microbiome, and preclinical evidence related to anxiety disorders .Further research is required to elucidate the exact mechanisms and mediators involved in brain-microbiota communication. A central issue that remains unresolved is which mediators the microbiota uses to communicate with the brain and how this network ultimately influences behavior and health.There are three major systems along with their mediators playing a role in inter-talk between the microbiota and the brain: neuronal communication, endocrine signaling mediators and the immune system mediators. Together, these systems create an integrated molecular network that impact both in gut and brain function.The gut communicates with the brain through a systemic, central route. The vagus nerve sends information to the brain, where it is processed in the nucleus tractus solitarius; this nucleus then projects to the parabrachial nucleus, which in turns projects to the prefrontal cortex (PFC) and amygdala. These latter are key loci that control anxiety and fear responses. It has been reported that both regions display irregularities in germ free animals (GF), including hypermyelination in the PFC.N-methyl-D-aspartate (NMDA) receptor subunit (NMDArec2B) has reduced expression in the amygdala of GF animals ] exerts its function in the brain and in the ENS. Most of 5-HT is synthetized by gut mucosal enterochromaffin cells and ENS neurons. Peripherally, 5-HT is involved in gastrointestinal function, smooth muscle contraction and relaxation, and in pain perception. In the brain it is involved in regulating mood and cognition . SerotonTogether these findings reinforce the concept that DP can have either a positive or a negative impact on the CNS by regulating critical neurotransmitters implicated in psychiatric disorders, such as depression .Finally, the immune system plays an important intermediary role between the brain and the gut. Gut-associated lymphoid tissue is the main component of mucosal-associated lymphoid tissue and corresponds to almost 70% of the entire immune system . DysbiosSeveral studies related to preclinical models of bacterial infections, probiotics treatment and analysis of germ-free (GF) animals suggest that the microbiota can influence the CNS and consequently behavior. Stress-related disorders such as anxiety and depression are among the main psychiatric conditions associated with microbiome changes .The treatment of multidimensional diseases such as CVD require multidimensional approaches. Nutritional psychiatry is a growing field of research seeking to provide clinically relevant interventions for multifactorial diseases. This emerging line of inquiry has assembled data on biological pathways such as gastrointestinal microbiota and inflammation, on DP and on how these are modulated by the environment. It has become clear that this intertwined network of environmental factors (such as stress and DP) interacting with the above-mentioned pathways combines to modulate behavior.Some trending foods and  diets have brought lasting changes to traditional diets, however, in most cases neither these nor dietary supplements have been subjected to adequate clinical trials to test their safety and efficacy. A better understanding of the interplay between diet, stress, neuronal signaling, phenotypes, and the microbiome will provide important insights into the utility of scientific evidence-based nutrition. Future studies are imperative to proactively avoid cardiovascular events in patient population.All authors equally contributed to the drafting, analyses the final version of the manuscript, and read and approved 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.


Amyotrophic lateral sclerosis (ALS) represents the major adult-onset motor neuron disease. Both human and animal studies reveal the critical implication of muscle and neuromuscular junctions (NMJs) in the initial phase of this disease. Despite the common efforts, ALS diagnosis remains particularly challenging since many other disorders can overlap yielding similar clinical phenotypic features. A combination of further research on the NMJ parameters that are specific for this disease and laboratory tests are crucial for the early determination of specific changes in the muscle, as well as in motor neuron and the prediction of ALS progression. Also, it could provide a powerful tool in the discrimination of particular ALS and ALS-mimic cases and increase the efficacy of therapeutic treatments. Amyotrophic Lateral Sclerosis (ALS) is a disease characterized by a progressive degeneration of upper motor neurons (MNs) in the motor cortex and lower motor neurons in the brainstem and the spinal cord. The death of these neurons leads to spasticity, weakness, and atrophy of the muscles, progressing to paralysis. The incidence of ALS in Europe is 2–16 per 100,000 each year , though Although the majority of ALS cases occur sporadically (sALS), there is a Mendelian inheritance in about 10% of the cases , mainly in an autosomal, dominant fashion . The twodying forward process that proposes an anterograde degeneration of motor neurons by glutamate excitotoxicity from the cortex, or a dying back phenomenon in which motor neuron degeneration starts distally at the nerve terminal or at the neuromuscular junction (NMJ) and progresses toward the cell body , is continually increasing. Their advantages lie in their fast development allowing quick generation of lines, their availability and the ease in manipulating gene expression and in drug screening. In drosophila, studies showed locomotor defects, reduced life span, and anatomical defects at the NMJ, causing impairments in synaptic transmission, in loss and gain of function models of TARDBP  are classified in four main groups in which ALS represents the most common form . AlthougStandard diagnostic criteria for ALS have been established in 1991 [El Escorial criteria [EEC] ] and werIn 2008, electrodiagnostic studies, known as the Awaji criteria , were inAlthough the main ALS evaluation remains the clinical one, laboratory testing, based on advanced techniques of electrodiagnosis, neuroimaging, immunobiochemistry, and neurogenetics, is required for accurate ALS diagnosis.Tests to rule out other neuromuscular conditions may include:The needle EMG is the most important study in determining diagnostic certainty of ALS . During This test measures how fast an electrical impulse moves through the nerve . During This technique is able to produce detailed images of the brain and spinal cord, the latter with the advantage of simultaneously investigating the upper and lower motor neurons. During several years, its application was related to the exclusion of other disorders, as tumors or hernias that can display certain of the ALS-mimic symptoms . The evoTesting hematological factors is helpful to exclude diseases that are capable of mimicking ALS symptoms. Recently, a population-based study, proposed serum albumin, creatinine levels, and lymphocyte count as markers for ALS, indicating muscle waste and inflammation respectively . Other mUsing this particular test, a small amount of cerebrospinal fluid (CSF) is taken from the lower back of the patient for laboratory tests. Thanks to its proximity to the central nervous system, the CSF is considered one preferred tissue to search for ALS biomarkers [reviewed in ]. SeveraWith this technique, a small portion of muscle is removed by needle biopsy and sent to a laboratory for histopathological analysis. Rarely performed because of its painful and invasive nature, this tool is useful when ALS diagnosis is in doubt. Generally, ALS muscles present signs of active denervation/reinnervation and an increased number of atrophic fibers .People with familial ALS (fALS) background can get an efficient diagnosis through genetic testing , 69. ThiSpinal muscular atrophy (SMA) is an inherited MND that prevalently affects children. Its incidence is 1 per 11,000 live births  Kennedy's disease] is a X-linked hereditary lower motor neuron disease, where the expanded trinucleotide repeat (CAG > 37) in the androgen receptor gene (AR) causes its nuclear inclusions and impairment of its function  overlaps ALS syndrome. The annual incidence ranges from 3 to 30 per 1,000,000 people    is the mis (IBM) . It is cis (IBM) , 97. In is (IBM) .Amyotrophic Lateral Sclerosis (ALS) and MNDs are not yet curable. However, accurate diagnosis is crucial to provide adequate counseling and information about the prognosis and disease course, and to avoid inappropriate therapy. Moreover, a good diagnosis could furnish a more equal stratification of cases and be important in the choice of additional medical support, as for example nutritional intervention strategies or physical therapy.Currently, there is not a common consensus in the use of laboratory analysis for ALS diagnosis. Basically, clinicians decide for the application of certain techniques based on their experience, expertise and hospital practice. Progress in molecular genetics and identification of specific biomarkers is ongoing, which will translate to a refined diagnostic certitude. Therefore, there is the emerging need to establish a widely accepted protocol for laboratory tests to discriminate the majority of cases that present clinical features resembling ALS.Increasing human and animal evidence proposed NMJ impairments as possible biomarkers for detection and discrimination of ALS and mimic diseases in an early, preclinical stage. However, further studies are needed to understand how these impairments could be monitored and specifically treated.M-LC and A-RB contributed to conception and drafting. EK provided useful comments and reviewed 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 phenotype of neurons and their connections depend on complex genetic and epigenetic processes that regulate the expression of genes in the nucleus during development and throughout life. Here we examined the distribution of nuclear chromatin patters in relation to the epigenetic landscape, phenotype and connections of neurons with a focus on the primate cerebral cortex. We show that nuclear patterns of chromatin in cortical neurons are related to neuron size and cortical connections. Moreover, we point to evidence that reveals an orderly sequence of events during development, linking chromatin and gene expression patterns, neuron morphology, function, and connections across cortical areas and layers. Based on this synthesis, we posit that systematic studies of changes in chromatin patterns and epigenetic marks across cortical areas will provide novel insights on the development and evolution of cortical networks, and their disruption in connectivity disorders of developmental origin, like autism. Achieving this requires embedding and interpreting genetic, transcriptional, and epigenetic studies within a framework that takes into consideration distinct types of neurons, local circuit interactions, and interareal pathways. These features vary systematically across cortical areas in parallel with laminar structure and are differentially affected in disorders. Finally, based on evidence that autism-associated genetic polymorphisms are especially prominent in excitatory neurons and connectivity disruption affects mostly limbic cortices, we employ this systematic approach to propose novel, targeted studies of projection neurons in limbic areas to elucidate the emergence and time-course of developmental disruptions in autism. Neurons are highly specialized cells that perform a wide variety of functions. Functional specialization depends on morphological, biochemical and physiological features of neurons. Accordingly, neurons can be classified based on a multitude of features including: (1) the number of dendritic trunks, which distinguish unipolar, bipolar, and multipolar neurons; (2) the length of the axon, which separates long axon (projection) from short axon  neurons; (3) the specialization of the dendrites, which distinguishes spiny and non-spiny neurons; and (4) the neurotransmitter, which differentiates excitatory , inhibitory (GABAergic or glycinergic), or modulatory  neurons. The combination of morphological and biochemical features exhibited by a given neuron underlies its function. For instance, projection neurons in the cerebral cortex are long axon-spiny-excitatory neurons and most are pyramidal but some are fusiform (von Economo cells). In contrast, the recipients of thalamic projections in cortical layer IV are local circuit-spiny-excitatory neurons with stellate cell body, but some are small pyramids. Inhibitory neurons across layers in the cerebral cortex are local circuit neurons with non-spiny or sparsely spiny dendrites and their body shape can be stellate, fusiform or triangular .The development and maintenance of structural and functional features of cells depend on the expression of genes in the nucleus. The pattern of gene expression is characteristic of each cell class as shown for glial cells and distinct subclasses of excitatory and inhibitory neurons in the cerebral cortex . Genes wHere we review the patterns of nuclear chromatin in relation with morphological, biochemical, and connectional features of neurons with a focus on the cerebral cortex in the rodent and primate brain. We conclude that the nuclear pattern of chromatin and the epigenetic landscape in neurons across areas in the adult cortex is related to cortical connections, a relationship that can be traced to development. Importantly, the sequence of events in the development of chromatin patterns, gene expression, neuron morphology, and connections across cortical areas and layers are likely disrupted in disorders of neurodevelopmental connectivity like autism spectrum disorders (ASD). Accordingly, the epigenetic marks of specific cortical neurons in individuals with ASD will reflect the developmental disruption of pathways of these disorders.pars fibrosa of neuronal nucleoli  in the cytoplasm, the more concentrated and simplified was the nuclear chromatin . Cajal anucleoli , 2017 thnucleoli ,b that enucleoli . Accordinucleoli .Figures 1A,B). Large neurons in other parts of the brain, like Purkinje cells in the cerebellum which are GABAergic  or cholinergic interneurons in the striatum , also have a large nucleolus with scant heterochromatin. The largest amount of heterochromatin and the smallest nucleolus is found in the nuclei of the smallest neurons, like glutamatergic granule cells of the cerebellum .We recently developed an algorithm that is highly effective in training experienced or untrained individuals alike to identify and distinguish neurons and glial cells stained for Nissl with high inter-observer reliability . AccordiTable 1). For instance, projection neurons in the olfactory bulb and in the cerebral cortex are excitatory and show comparable nuclear chromatin patterns, but the former are non-spiny and the latter are spiny . These large neurons of the cortex are projection neurons; they have extensive and complex dendritic trees and long axons. The largest neurons in the human and rhesus macaque cortex are the Betz cells in the primary motor cortex, which have long axons, like other layer V neurons . In contrast to long-axon neurons, local-circuit neurons of the cerebral cortex, like small cells of layer IV, have short axons, short dendritic trees, small nuclei with smaller nucleoli, more heterochromatin clumps, and lightly stained euchromatin . These nuclear features can be used to distinguish local circuit neurons in layer IV of different cortical areas . Interestingly, small neurons do not differ in size and heterochromatin distribution pattern across these species. By contrast, large neurons in primates are considerably larger than in rats, a feature that likely reflects the increase in the length of the axon of projection neurons in larger brains. In contrast with neurons, the nuclei of glial cell types in the cortex show comparable sizes and chromatin patterns across species .Here we surveyed and compared neurons in Nissl stained sections of cortical areas of humans, rhesus macaques, and rats systematically for the first time. Our analysis showed that chromatin patterns in cortical neurons are graded along body size, with progressively fewer heterochromatin grains and clumps in larger neurons . Cerebellar granule cells stand apart from cortical excitatory and inhibitory neurons . In contrast, glial cell types seem to have comparable open chromatin regions across cortical and subcortical regions .The findings of recent studies of epigenetic states that measure chromatin accessibility in thousands of single cells can be best understood in the light of chromatin patterns of neurons and glial cell types. These studies show higher variability of open chromatin regions in neurons across cortical areas , as pred neurons , as pred regions , as predFigures 1, 2). In line with this, excitatory neurons in the mouse and in the human frontal cortex have more hypo-methylated DNA regions than inhibitory interneurons . Different staining patterns with basic dyes, like those used in Nissl, suggest different chemical properties of euchromatin across neurons that could be attributed to a higher degree of decondensation of chromatin. The most decondensed chromatin is composed of 10 nm basic chromatin fibers in which histone cores and linker-histones are more exposed compared to 30 nm fibers , or have a rudimentary layer IV (dysgranular) and overall have poor laminar differentiation. In contrast, eulaminate areas have well developed layer IV and better laminar distinction , while they are larger in layer III in eulaminate cortices . In rats, the largest pyramidal neurons across cortical areas are found in layer V , as shown in classical  show that pyramids in primary sensory areas , in the primary motor area, and in extrastriate areas of the occipital lobe have the smallest basal dendritic fields across cortical areas of the macaque. In contrast, pyramids in limbic areas in the cingulate cortex have the largest dendritic fields in the macaque cerebral cortex, ten times larger than in the primary visual area. We have suggested that dendrite complexity of pyramidal neurons decreases in parallel with progressive laminar elaboration from limbic to eulaminate cortices (Table 2). Studies on the dendritic structure of pyramidal neurons in rodents are scarce and less systematic than in primates, but overall they show smaller basal dendritic trees than in macaques . A quick survey of the human cortex supports this observation for most areas and systems and allows formulation of a general principle: Layer III projection neurons in eulaminate cortices are among the largest in the cortex with the most open chromatin (euchromatin). These neurons connect the most distant cortical areas through long-range pathways. This applies both to long-range ipsilateral cortico-cortical connections . The neurofibrillary frame of the cytoskeleton develops postnatally .Classical studies with specific stains for DNA (Feulgen stain) and electron microscopy show that nuclear chromatin patterns in cortical neurons develop in parallel with morphological and cytoplasmic differentiation. The nuclei of migrating neuroblasts in the embryonic cerebral cortex of rodents have several clumps of heterochromatin (chromocenters), most of which are located under the nuclear envelope. As neuroblasts migrate and reach their position in the cortical plate, nucleolar substance gradually develops inside some chromocenters. Once in the cortical plate, nuclear heterochromatin disperses progressively and is redistributed around the developing nucleolus or nucleoli as Nissl substance appears in the cytoplasm . Interestingly, neurons are an exception to this general pattern of chromatin differentiation and show different chromatin patterns depending on the size of their body and the type of connections they make .The sequence of decision points that accompany neuron and glial cell differentiation during development is the result of changes in the transcriptome  that depThe parallel development of chromatin patterns, neuron morphology, and pathways in the cerebral cortex summarized above suggests that specific epigenetic modifications in distinct types of cortical neurons underlie typical development of the cerebral cortex and its connections. This sequence of events in cortical neurons at the nuclear, cytological and connectional level could be disrupted in neurodevelopmental disorders like ASD. Specifically, changes in post-translational modifications of histone proteins have been reported in the brains of individuals with ASD, including a widespread increase of histone 3 (H3K4) methylation at gene promoters . ModificBox 1| Principles, generalizations, and hypothesis.1.The nuclear pattern of chromatin and the size of the nucleolus of neurons is related to cell body size  of the largest neurons is reflected in the adult through a structural counterpart (doppelganger) in the form of some small neuron that never progressed beyond that stage of maturity .3.Terminally differentiated neurons show different nuclear chromatin patterns. By contrast, glial cells show comparable chromatin patterns by class .4.We hypothesize that changes in chromatin pattern and epigenetic landscape, neuron size and connections vary systematically across cortical areas in parallel to the systematic variation of laminar structure of the cortex.5.We hypothesize that analysis of epigenetic modifications in projection neurons in limbic areas of individuals with autism will reveal traces of developmental disruption.Which are the cortical neurons most likely involved in ASD? Genetic studies have described a large variety of polymorphisms in individuals with ASD see , which aBox 1 leads to the testable hypothesis that the epigenetic landscape of neurons will vary systematically across cortical areas in parallel with laminar differentiation and connections. In the future, a wide array of techniques that allow sampling of thousands of cells in human postmortem brain tissue .MG-C, HB, and BZ conceived and wrote the manuscript. HB and BZ secured funding.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.


Dyslipidemia, an increase in pro-atherogenic low-density lipoproteins (LDL) and its oxidative modifications, oxLDL, is well known to be a crucial factor in endothelial damage, a key early step and a predictor of the development of atherosclerosis. It is also well-known that atherosclerotic plaques develop in regions of low and non-unidirectional disturbed blood flow as is found by branching points, bifurcations, and in the inner curvature of the aortic arch. Our studies demonstrate a novel paradigm for dyslipidemia-induced endothelial damage, an increase in endothelial stiffness induced by the uptake of oxidized lipids.via activation of the contractile RhoA/ROCK cascade, whereas enriching the cells with cholesterol has a rescue effect [Our previous studies showed that exposure to oxLDL in vitro or to plasma dyslipidemia in vivo dramatically increases endothelial stiffness –3. Furthe effect , known te effect . Most ree effect . Specifie effect .stiffness of the vascular wall, dominated by extracellular matrix and layers of smooth muscle cells, and stiffness of the endothelial monolayer. Previous studies established that dyslipidemia, aging and cardiovascular disease are associated with stiffening of the arterial wall of major arteries, attributed to remodeling of the extracellular matrix and vascular smooth muscles (VSMCs). However, while vascular stiffening is well recognized as an important factor in aging and development of the cardiovascular disease (CVD) [In terms of the functional significance, there is a critical distinction between the se (CVD) , the rolvia a different mechanism. Specifically, we found that endothelial stiffening but not the stiffening of the vascular wall could be abrogated by the genetic deletion of CD36 or blocking of oxLDL uptake indicating the leading mechanism of endothelial stiffening in aged vessels is accumulating uptake of oxidized lipids. These findings provide a model to discriminate between the roles of endothelial and sub-endothelial stiffness in age-related vascular dysfunction.Finally, we found that endothelial stiffness increases with age independently from the stiffening of the vascular wall . As was In summary, the novel concept of these studies is in discovering the impact of oxidized lipids/plasma dyslipidemia on endothelial stiffness and a new paradigm for synergism between pro-atherogenic flow and dyslipidemia in inducing EC dysfunction: the stiffening of aortic endothelium, mediated by increased expression of CD36 and enhanced oxLDL uptake. Dyslipedimia/aging-induced stiffening of aortic endothelial cells is proposed to constitute an important confounding factor in endothelial dysfunction independent of vascular stiffening.


Rheumatoid arthritis (RA) is a highly heterogeneous syndrome in terms of clinical presentation, progression, and response to therapy. In such a complicated context, the identification of disease-related biomarkers would be undoubtedly helpful in assisting tailored approaches for every patient. Despite remarkable efforts, however, progress in new biomarker development and validation is dramatically slow. At present, none of the candidate genetic, cellular, or molecular biomarker has yet surpassed the clinical value of RA-specific autoantibodies, including rheumatoid factor (RF) and anti-citrullinated protein autoantibodies (ACPA). Rather, recent years have witnessed significant advancements in our understanding of the multiple roles that RF and ACPA play in RA pathophysiology. This has helped clarifying the mechanistic basis of the clinical associations of autoantibodies in RA. In this short review, we will briefly summarize the effector functions of RF and ACPA, and analyse how autoantibodies may help subclassifying RA patients in terms of clinical presentation and response to therapy. Our knowledge on rheumatoid arthritis (RA) has dramatically increased over the past 2 decades. Recognition that RA starts early and key processes of damage even develop before clinically apparent arthritis have fuelled the processes of early diagnosis and prompt treatment institution within the window of opportunity , 2. In pThe past 10 years of research have shed increasing and exciting light on the multiple roles of autoantibodies (both RF and ACPA) in RA pathology, briefly summarized below.All immunoglobulins (Ig), and in particular those of the IgG class, have effector functions mediated by the interaction of their Fc tail with specific activating or inhibitory receptors (FcR) present on the surface of various cell types. ACPA detected by second-generation assays are mostly of the IgG class, with predominance of the IgG1 subclass , which min vitro effects fit with clinical evidences linking the presence of IgA RF to worse disease prognosis  and IgMs (complement activation). However, recent evidence has also highlighted important effector functions of antibodies of the IgA class. Indeed, IgA potently activate neutrophils , and incrognosis , 24, andrognosis .Importantly, the unique effectors functions of autoantibodies are not only mediated by their diversification in classes and subclasses, but also by some specific biochemical characteristics including the sugar moiety attached to the Fc tail . In partAltogether, the afore described mechanisms underline the complexity of the inflammatory function of autoantibodies in RA, which depends on intricate and not completely disentangled characteristics including classes, subclasses and biochemical properties. Better understanding of ACPA and RF diversity across the different phases of the disease will hopefully allow important advancements in the biological and clinical understanding of how inflammation develops, evolves and eventually resolves in RA.The epidemiological association between ACPA and the severity of radiographic damage in RA has recently found causal explanation with the identification of the direct osteoclastogenic role of ACPA , 35. In in vivo remains to be fully proven, clinical evidences showing that ACPA-positive subjects may suffer from joint pain in the absence of overt synovitis , which in turn promotes nociceptive mechanisms . Althougynovitis  offer inIt is now well-established that autoantibodies precede the onset of clinical symptoms by several years in a proportion of patients with RA , 46. Thein vitro  linked to the presence of autoantibodies is extra-articular involvement, including cardiovascular (CV) comorbidity. Several studies have reported higher rates of CV mortality and events in autoantibody-positive patients , 63. ThiIt is however important to note here that the aforementioned associations between autoantibody diversity and local and systemic inflammation refer to patients fulfilling the 1987 classification criteria for RA. There is ample evidence now indicating that the introduction of the 2010 classification criteria has profoundly changed the clinical picture of RA . In thisDisease remission is increasingly achievable in patients with RA provided that diagnosis is established early and treatment instituted effectively . HoweverIn historical RA cohorts, RF has been shown to be inversely associated with the achievement of point remission in several studies. In the Utrecht RA cohort established in 1990, 36% of the patients achieved at least one period of remission; among other predictors, RF negativity was associated with 1.63 hazards of achieving remission . IgM RF RF positivity not only appears associated with lower rates of point remission, but also with reduced chances of maintaining the state of disease remission over time. In a population of 650 RA patients followed for a mean of 10.3 years, Raheel et al.  were ablIf ACPA thus appear rather ininfluent in the achievement and maintenance of remission on treatment, their role in predicting disease relapse after drug suspension is undoubtedly established. In the Leiden Early Arthritis Clinic and the British Early Rheumatoid Arthritis Study, sustained DMARD-free remission was achieved far less commonly by ACPA-positive patients . SimilarCollectively, data from the literature thus indicate that both RF and ACPA are important factors related to remission outcomes. RF would mostly predict lower chances of achieving and maintaining remission on treatment, whilst ACPA would represent the major obstacle for safe drug suspension.Despite being unanimously considered as important prognostic markers associated with more rapid evolution of joint damage, RA-specific autoantibodies, and in particular ACPA, have never been recommended as a guidance to assist the choice of first-line treatment in patients with early RA. Indeed, both the European and the American recommendations push for MTX as the anchor drug in all patients irrespective of autoantibody positivity , 92. ThiIf MTX efficacy appears overall similar between autoantibody-positive and –negative patients, still some differences in the magnitude of the response according to the serological status might exist. In patients with undifferentiated arthritis according to the 1987 criteria, MTX was effective at delaying progression to RA only in ACPA-positive patients, many of whom could be reclassified as having had RA according to the 2010 criteria at baseline . SimilarBased on the above described evidences, it would be tempting to speculate that, by interfering with both the innate and the adaptive branches of the immune system , MTX at If the differential response to csDMARDs according to the autoantibody profile remains questionable, the impact of the serological status on response to biological (b) DMARDs targeting different immune pathways is, at least for certain mechanisms of actions, more defined.post-hoc analysis of the AMPLE trial in 2016 initially showed that very high levels of ACPA at baseline were associated with better clinical response to abatacept but not to adalimumamb (It can be easily appreciated that bDMARDs selectively affecting the process of autoantibody production, including B cell depletion or inhibition of T cell costimulation, are particularly effective in autoantibody-positive patients. A meta-analysis of four randomized placebo-controlled trials from the rituximab clinical programme confirmed the additional treatment benefit in autoantibody-positive RA, particularly in patients for whom at least one TNF inhibitor had failed, and without significant differences between RF and ACPA . Similarlimumamb . Such aslimumamb . Data frlimumamb , 105. Aglimumamb . It is alimumamb . Althouglimumamb .post-hoc analyses on the RISING study, in which the dose of infliximab was progressively escalated (Studies on the prognostic value of autoantibodies for response to therapy to bDMARDs targeting different mechanisms of action are less conclusive. The assessment of response to TNF inhibitors according to RF and/or ACPA status has provided conflicting results. A systematic literature review of 14 studies involving more than 5,000 patients treated with infliximab, etanercept, adalimumab, and golimumab demonstrated that neither RF nor ACPA were associated with differential response . Howeverscalated , 112. Inscalated , 114. Inscalated . Howeverscalated . It is tscalated .Given the complex pathophysiology and the clinical heterogeneity of RA, biomarkers capable of guiding personalized treatment approaches are urgently needed. RA would indeed benefit from personalized approaches across all the phases of disease development and evolution Figure . Prompt ✓ The dichotomic classification of patients with RA into autoantibody-positive and -negative solely based on RF and/or ACPA appears too simplistic.✓ The autoantibody system in RA is likely to expand rapidly with the identification of new antigenic specificities.✓ The pathogenic properties of autoantibodies depend on several characteristics including levels, avidity, classes, subclasses, and biochemical properties.✓ All of these properties are likely to be modulated in different patients during different phases of the disease.✓ Further studies are needed to test whether the different characteristics of autoantibodies, alone or in combination, may serve as biomarkers for:prediction of disease development in at risk individualsprediction of response to MTXchoice of the second-line drug in MTX-failuresidentification of patients in whom remission is more likely to persist even after drug tapering/discontinuationSB and AM prepared a first draft of the manuscript. RC and CM critically reviewed the manuscript. All authors conceived the idea of this review article and approved the final version.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.


A case study was undertaken during wet and postwet seasons to improve the perennial and alternate submerged saucer-shaped ponded lands  in the coasts and northeastern plains of the Indian subtropics through pisciculture and cultivation of starch- and protein-rich aquatic food crops like water chestnut (Trapa bispinosa Roxb.) and makhana or fox nut . The study revealed that the physico-chemical properties of soils  as well as quality of water , growing fish, makhana, and water chestnut was remarkably influenced by different moisture regimes and exhibited a significant improvement of soil health. The amount of organic C, available N, P, and K content were found significantly highest in the treatment where makhana was grown under alternate flooding and drying situation with a depth >2 m as compared to other treatments. Such enrichment of soil fertility, particularly in available N and P content, might be due to the accumulation of considerable amounts of biomass and fish excreta and their subsequent decomposition in situ in the soils. Therefore, the present study suggests that the N-enriched soil may effectively be utilized further for growing subsequent arable crops surroundings during summer season, which not only saves the amount of applied N fertilizer but also increases the apparent N efficiency with simultaneous increase in yield, and would benefit the farmers in this region.