Cytogenetic approaches play an essential role as a quick evaluation of the first genetic effects after mutagenic treatment. Although labor-intensive and time-consuming, they are essential for the analyses of cytotoxic and genotoxic effects in mutagenesis and environmental monitoring. Over the years, conventional cytogenetic analyses were a part of routine laboratory testing in plant genotoxicity. Among the methods that are used to study genotoxicity in plants, the micronucleus test particularly represents a significant force. Currently, cytogenetic techniques go beyond the simple detection of chromosome aberrations. The intensive development of molecular biology and the significantly improved microscopic visualization and evaluation methods constituted significant support to traditional cytogenetics. Over the past years, distinct approaches have allowed an understanding the mechanisms of formation, structure, and genetic activity of the micronuclei. Although there are many studies on this topic in humans and animals, knowledge in plants is significantly limited. This article provides a comprehensive overview of the current knowledge on micronuclei characteristics in plants. We pay particular attention to how the recent contemporary achievements have influenced the understanding of micronuclei in plant cells. Together with the current progress, we present the latest applications of the micronucleus test in mutagenesis and assess the state of the environment.  Cytogenetics is the branch of genetics, cytology, and cell biology that analyses the nuclear genomes at the chromosome level. Cytogenetics makes the chromosome a substantial target in elementary plant cell biology and other fields such as mutagenesis and genotoxicity studies. Standard cytogenetic methods were, and are still, commonly used. Modern cytogenetic technologies involving advanced microscopy and imaging methods, that progress in the analyses on epigenetic DNA and histone modifications as well as DNA damage by using fluorescent antibodies benefit plant genome structure, dynamics, and evolution. They have also served the comprehensive evaluation of the effects of various mutagens on the plant genome that are observed as chromosome aberrations, including micronuclei (MN). Mutagens affect the structure of DNA and cause double-strand breaks (DSBs) leading to MN formation. The elimination of MN causes DNA loss. Micronuclei are induced by many mutagenic factors, both physical and chemical, as well as those of an environmental nature. The analysis of their frequency is the basis of the commonly used micronucleus test. We provide a comprehensive overview of the current knowledge on MN characteristics in plants. This paper focuses on critical scientific problems: Is the distribution of DNA damage that led to micronuclei formation random? What is the origin of plant micronuclei? Are epigenetic processes involved in micronuclei formation? How could there be a role of the genetic activity of chromatin in the formation of micronuclei?Allium, Nicotiana, and Vicia [Micronuclei (MN) are structural chromosome aberrations that are detected in non-dividing cells during interphase. Among numerous genotoxicity assays, the micronucleus (MN) test is especially recommended to evaluate the genotoxic effects of chemical and physical agents, as well as mixtures of substances. Since 1959 when the MN assay was first applied in kidney beans, followed the treatment with gamma-ray ,3. Currend Vicia ,11,12,13nd Vicia ,15,16,17Compared to the chromosomal aberrations (CA) assays that are applied to mitotically divided cells, the MN test is less time-consuming and easier to perform. Most mutagens decrease mitotic activity, thus making the chromosome aberrations analyses in dividing cells, especially in metaphases, challenging and often impossible. Although the knowledge on different aspects of the origin, structure, genetic activity, and micronuclei in plants has been explored in recent years, there is still much less that is known than in humans and animals. There are many reviews of MN in humans and animals, also from the last few years ,25,26,27Micronuclei (MN) are small, extranuclear bodies that are located next to the parental nucleus in the cytoplasm. Micronuclei are detected in the meristematic interphase cells of the shoots or roots, in the next cell cycle, followed by treatment with mutagen. MN can originate in two ways. They can arise from acentric fragments resulting from double-strand breaks (DSBs) which are not repaired or repaired improperly. The micronuclei could also occur from the entire chromosome(s) that does not attach to the mitotic spindle at metaphase. Likewise, micronuclei that have arisen from entire chromosomes could result from kinetochore damage, failure of the cell cycle control system, or centromeric DNA hypomethylation. Thus, the knowledge on the origin of micronuclei allows for assessing the mutagen’s mechanism of action as clastogenic or aneugenic. Changes in the structure of the chromosomes, such as chromosome fragments and delayed chromosomes, can also be detected during mitosis; however, their detection is not as straightforward as during interphase. Some micronuclei might also be derived from the breakage of anaphase bridges that are formed from dicentric chromosomes, concatenated ring chromosomes, the union of sister chromatids, unresolved sister chromatid connections, or chromosomes that have merged by telomere fusion. The number of micronuclei in a single cell is most often one, but sometimes cells with a higher MN number are observed, depending on the number of chromosome fragments or delayed chromosomes. It still needs to be emphasized that, taking into account the mechanism of micronucleus formation, the frequency of dividing cells after the mutagenic treatment influences the frequency of micronuclei. Cells need to divide so that chromosome fragments can be removed outside the newly formed daughter nuclei and create an MN.From the data that are available for animals and humans, micronuclei can be lost from the cells and incorporated into the nucleus . There aThe conventional cytogenetic is recognized as the approach for the detection and basic description of the MN after mutagenic treatment. Changes in chromosome morphology are usually detected using the basic chromosome-staining techniques, such as the Feulgen method, acetoorcein, and Giemsa stainings. Among these staining methods, the Feulgen technique  is charaNowadays, fluorescent methods, e.g., DAPI   or acridThe fluorescence methods are quick and precise, and even small micronuclei can be detected . Still, The early and current achievements of molecular cytogenetics have led to progress in the detection and detailed characterization of micronuclei (MN) ,33. ModeA breakthrough in the analyses of the localization of DNA damage at the chromosomal level in plants came with applying the fluorescence in situ hybridization (FISH). It provides information on the possible ‘hot spots’ in plant genomes for DNA damage after the action of mutagens. Also, it gives information on the mechanisms of the biological effect of the individual agents that induce DNA damage. This knowledge is particularly crucial in plant mutagenesis, as the use of the chemical and physical mutagens is the most common way to obtain mutants. This technique could detect even extremely small aberrations in dividing and non-dividing cells. Arabidopsis thaliana (Arabidopsis)-type (TTTAGGG)n telomeric sequences, and ribosomal DNA (rDNA), which give strong and easily observed FISH signals, have found application in the detailed characterization of MN. These DNA sequences’ advantages are evolutionary conservation and location at a specific chromosome region. Repetitive dispersed DNA sequences are not a good source for probe pool for fluorescence in situ hybridization to study the origin of micronuclei.There is only one morphological type of micronuclei that may differ in size . The sizArabidopsis thaliana. Applying the rDNA as probes showed rules regarding gamma-ray–induced MN formation in barley (Hordeum vulgare)  . Brachypodium distachyon [Lolium multiflorum were not correlated with rDNA sites [5S rDNA-bearing chromosomes are shown to be more often involved in MN formation than NOR chromosomes in barley ,42. Simistachyon . The hotNA sites .The use of the centromere and telomere-specific DNA sequences for FISH also provided some rules regarding the origin of MN. It confirmed that the gamma ray-induced MN may originate from acentric fragments or whole lagging chromosomes. Thus, this approach allows the distinguishing of the micronuclei being a clastogenic and aneugenic effect of mutagens. However, most MN had only telomeric DNA signals, indicating that terminal deletion is the primary type of chromosome aberration leading to their formation . Comparing the contribution of particular chromosome fragments in MN that are induced by different chemical clastogens, the maleic acid hydrazide (MH) and nitroso-N-methyl-urea (MNU) have shown the difference in the size of the chromosome fragments that are involved in the MN. Most MH-induced MN originated from large acentric fragments, whereas MNU-induced MN is from small terminal chromosome fragments ,42. Secale cereale L.) [FISH provides much more information about MN formation with DNA probes that are dedicated to different chromosomes or particular chromosomes. Standard A- and B-chromosome-specific probes were successfully used in the rye gamma-irradiated cells   for the Brachypodium distachyon root-tip cells that were subjected to a chemical mutagen [One of the FISH approaches that is used to detect and characterize micronuclei in plants is multicolor FISH (mcFISH). It is based on the two consecutive FISH experiments that use a pair or pairs of probe sets that are removed after each experiment and include the reprobing step. Combining more than two differently labeled DNA probes on the same nuclei slide makes this technique more informative . For the mutagen . Brachypodium distachyon (The most advanced FISH-based approach in plants is chromosome painting (CP), which permits the selective visualization of entire chromosomes or their specific segments during mitosis as well the interphase ,52,53,54stachyon . BAC-FISH-based chromosome painting provides new information on the composition, origin, and mechanisms of micronuclei formation that is induced by MH-treatment and X-radiation in Brachypodium by showing the ‘fragile spots’ of DNA breaks . Site-spTo summarize, FISH provides new insights into the localization of DNA breaks on plant chromosomes, proving the non-random distributions of chromosome aberrations. The reasons for this non-random distribution may be the spatial organization of the nucleus at the interphase, the diverse transcriptional activity of specific chromosome regions, and chromosome size. Single BAC-FISH-based chromosome barcoding and ‘chromosome painting’ approaches have proven to be effective in analyzing the mechanism of micronuclei formation in plants after mutagenic treatment. The advantages of the FISH technique in terms of accuracy and quality of quantitative analyses make the technique one that is likely to become more widespread in DNA damage studies in plants.The nucleolus, whose primary function is ribosomal RNA (rRNA) synthesis and ribosome biogenesis, plays a crucial role in the response to biotic and abiotic stress. This aspect has not been extensively studied in plants . VariousThe p53 transcription factor plays a significant role in the DNA damage response (DDR) in mammalian cells to maintain genome stability . Plants Crepis capillaris [In plants, cytogenetic studies of the activity of rRNA genes in MN seem to be particularly important as the frequent involvement of the rRNA genes in their formation was shown for a few species: barley , Brachyppillaris ,43. Vicia faba, with a nucleolar organizer, could synthesize protein and replicate DNA [The transcriptional activity of 35S rRNA genes that are present in MN that were analyzed using silver-staining is always maintained in barley . MN in Vcate DNA . Studiescate DNA ,69. The cate DNA . Many studies on the transcriptional activity in micronuclei were performed in humans . It depePrecise genome replication is crucial in maintaining the stability of genomes and any replication errors are critical for living cells. The studies on the genetic activity of chromatin in MN also includes the ability to replicate DNA. The early studies on the micronuclear chromatin replication indicate the heterogenous behavior of MN in animal cells . DNA synNowadays, there has been progress in detecting S-phase nuclei and DNA replication in the MN. BrdU, with many disadvantages, such as a denaturation step and low specify that is correlated with the size of antibody signals, has been replaced by modern labeling higher resolutions techniques—“click” reaction with 5-ethynyl-2′-deoxyuridine (EdU) ,75 Figu.Distinct rules have been observed in plant cells when the replication ability of micronuclei was analyzed using the pulse EdU labeling method . The preThe DNA damage response (DDR) plays a role in maintaining the genome integrity in response to abiotic and biotic stresses ,79,80,81Many methods have been developed to detect and localize DNA damage in a genome, quantify the repair processes, and thus provide better insight into the mutagenesis process in various organisms . DNA breEpigenetic modifications of chromatin, which are defined as being mitotically- and meiotically-heritable changes in the gene expression patterns that arise independent of the changes in DNA sequence, are essential for many biological processes, including growth and reproduction. Post-translational modifications of histones and DNA methylation are the main epigenetic modifications that have a causal role in establishing different chromatin states. Chromatin is a dynamic complex of DNA and proteins. The two main chromatin states can be distinguished: compacted and repressed, the so-called heterochromatin, or the less condensed and gene-rich euchromatin. Earlier studies indicated that the heterochromatic regions represent ‘hot spots’ for the aberrations that are induced by S-phase-dependent mutagens. DNA methylation is one of the epigenetic modifications that has been studied in plants most intensively. At the chemical level, this process involves the covalent addition of a methyl group to the 5th position of cytosine in a pyrimidine ring. It is catalyzed by the methyltransferase enzymes using S-adenosyl methionine as the methyl group donor. In plants, the heterochromatin domains are determined by the methylation of cytosines (5mC), and there is a close link between DNA and histone methylation. DNA methylation is highly concentrated in the heterochromatin domains, mainly in the centromeric regions and repetitive sequences. Cytologically, heterochromatin, which has a high level of methylated DNA, can be defined as intensively DAPI-stained chromocenters during the interphase.Additionally, the same specific patterns of 5mC can be found along the metaphase chromosomes . Also, DB. distachyon genome was studied. DNA methylation might respond to mutagenic treatments [However, similar levels of histone H3 methylation, histone H4 acetylation, and 5mC in the MN and its parental nucleus were observed more often. Rarely, the differences in the level of epigenetic modification between the MN and its parental nuclei were observed . The evaeatments . It demoFor the MN test, the microscope is obligatorily used for the visual detection of micronuclei and their manual counting based on observing a significant number of cells. Despite the many advantages of MN, their analysis by picking out and manually counting with microscopy is time-consuming, requires the proper skills, and is prone to subjectivity. On the other hand, the visual confirmation of MN and visualization of cytoplasm to associate MN to a particular cell is an advantage of this method. The use of high definition fluorescence microscopy that is equipped with a high-sensitivity camera allows the precise detection and quantification of micronuclei and automatically captures images. The development of microscopic and bioimaging techniques enables the rapid and versatile assessment of MN. These approaches improved the statistical power of this method and the robustness of the MN assay. Previously, laser scanning cytometry (LSC) and conventional flow cytometry methods were successfully applied to identify and enumerate MN ,88. ThisIn conclusion, we have highlighted the possibilities of the detection and detailed analysis of MN in plants, emphasizing the research directions using modern molecular cytogenetic approaches. These collected approaches provide future directions to study MN in plants. The most important advancement from the development of the molecular cytogenetic techniques for the MN analysis is based on fluorescence in situ hybridization (FISH) and its variants. With the application of FISH, many obstacles that are connected to the small size and uniformity of chromosomes were overcome. Thus micronuclei, which are very small, could be analyzed even in species that are characterized by a small genome. The other cytogenetic advances, e.g., in chromosome preparation, such as extended fiber-FISH, are in no way needed for the study of MN, although they are very helpful in the analysis of plant genome structure. The imaging and signal amplification technologies have improved the ability to detect small gene-sized probes in micronuclei. Recently, the main driver of plant cytogenetics are next generation sequencing (NGS) platforms, as well as bioinformatics that enables analyses of DNA sequences. Up to date, based on the whole genome sequencing achievements for many species, the linking DNA sequence to the physical chromosomes enable the development of new areas of plant genomics, epigenetics, and evolution. The integration of the big data and next-/third-generation sequencing, with the cytogenetics offers possibilities for new insight into micronuclei structure in the future.Although several advances have recently been made in the studies of MN in plants , a numbeThere is no knowledge on the possible cell lethal events and their risk to the organism. These new approaches may help to clarify whether micronuclei and genomic instability are related to other cellular mechanisms that have not been described so far.


Nutritional manipulation of livestock diets has long been considered asa tool for the control of infectious diseases. The objective of this literature review is to provide a comprehensive and critical view of the studies that have investigated the interactions between synthetic amino acids supplementation and immune response against infectious diseases in livestock. We performed a literature search in two main databases, PubMed and Web of Science. Based on our criteria for eligibility of the research articles, we selected 58 studies. Most of the studies selected focus on poultry and three amino acids that are all associated with a significant improvement in host response: methionine, threonine and arginine. The most described immune mechanisms associated with synthetic amino acids supplementation were directed against intracellular pathogens. We highlight the need for more analytical studies using these three amino acids, particularly in livestock other than poultry, and their use with other types of pathogens.Infectious diseases represent one of the most critical threats to animal production worldwide. Due to the rise of pathogen resistance and consumer concern about chemical-free and environmentally friendly productions, the use of antimicrobials drugs is no longer desirable. The close relationship between nutrition and infection has led to numerous studies about livestock. The impact of feeding strategies, including synthetic amino acid supplementation, on host response to various infections has been investigated in different livestock animals. This systematic review provides a synthesis of the experimental studies on the interactions between synthetic amino acid supplementation and immune response to infectious diseases in livestock. Following PRISMA guidelines, quantitative research was conducted using two literature databases, PubMed and Web of Science. The eligibility criteria for the research articles were: (1) the host is a livestock animal; (2) the supplementation with at least one synthetic amino acid; (3) at least one mediator of immunity is measured; (4) at least one production trait is measured. Data were extracted from 58 selected studies. Articles on poultry were the most numerous; few contained experiments using ruminants and pigs. Most of the authors hypothesized that synthetic amino acid supplementation would particularly improve the animals’ immune response against intracellular pathogens. An increase in T and natural killer lymphocytes and macrophages activation, intracellular redox state, lymphocytes proliferation and antibodies production were the most described immune mechanisms associated with synthetic amino acid supplementation. Most of the selected studies focused on three amino acids , all of which are associated with a significant improvement of the host immune response. The use of synthetic amino acid supplementation appears as an encouraging perspective for livestock infectious disease management, and research must concentrate on more analytical studies using these three amino acids.  Infectious diseases are one of the most significant threats to livestock farming worldwide. These diseases have important economic impacts and can lead to food insecurity by increasing morbidity and mortality, reducing market value and productivity ,2. The pA vaccination strategy to improve livestock immunization against specific pathogens is a vital tool for the efficient prevention of infectious diseases. However, vaccination is not available for all infectious diseases of importance. Some vaccines have a number of shortcomings with regard to safety, efficacy and/or user-friendliness that limit their effectiveness . The genIn recent decades, knowledge regarding livestock nutrition has considerably evolved in terms of improvements in feed efficiency, whatever the production considered. Most of the time, these nutritional strategies to maximize production performances were developed in healthy, un-challenged animals. However, it has long been shown that optimal immune function is dependent on an adequate supply of protein, energy and micronutrients , thus highlighting the close relationship between host nutrition and the immune system .In animal production, the key role of dietary protein and amino acids, which are the building blocks of proteins, on different functions of the immune system, has been investigated ,14,15. IThe study methodology was based on the guideline of “Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement” . The lit*The use of truncation (*) allows a query to be made only on the root of the word. Furthermore, the reference lists from selected articles provide additional articles. Studies were closely evaluated and selected for inclusion if they were performed using livestock to measure the effect of dietary supplementation . They had to have at least one particular synthetic amino acid on the expression of at least one mediator of the immune response following an immune challenge, and measure at least one production trait. Information extracted from each study included livestock types, immune challenge , immune mediators and the impact on their expression , dietary synthetic amino acid supplementation (quantity), basal diets, number of animals per group and production traits . A totalA total of 2322 results were provided by the Web of Science and PubMed databases . After eThe main objective of this systematic review was to synthesize the knowledge on the effect of dietary supplementation with synthetic amino acids on the immune response to infectious diseases in livestock. The bibliographical research was focused on all livestock, but only three types were found: poultry, ruminant and pig. The research articles on poultry were the most numerous compared to pigs and ruminants (48 out of 58 articles), and chicken was the main poultry studied. One of the major inclusion criteria was monitoring an immune challenge, including live bacteria, virus, parasite and their associated molecular pattern. The studies using non-specific immune stimulators  were excluded. Among the 20 proteinogenic amino acids, only 9 were found in the selected research articles. The most studied amino acids were methionine (24 articles), followed by arginine (19 articles) and threonine (12). The results of the qualitative evaluation of the risk of bias are presented in The fine-tuning of nutritional strategies to support optimal function of the immune system in livestock is of great interest for the development of sustainable management in animal health. The immune response against invading pathogens is expensive in terms of proteins and calories because of the energy and protein requirements of immune cells for the synthesis of immune mediators and repairing damaged tissue . MoreoveMethionine and cysteine are the two principal sulphur-containing amino acids used as a substrate for proteins biosynthesis . These tEscherichia coli LPS (lipopolysaccharide) injection, notably by the production of several cytokines  by macrophages [Eimeria IgA in broilers [In turkeys and broilers, it has been shown that higher dietary methionine levels  increased the level of methionine in the peripheral blood, and was correlated with higher levels of peripheral leucocytes and IgG after vaccine challenges ,31,32. Irophages . The supbroilers .Haemonchus contortus and Trichostrongylus colubriformis was studied. Cysteine was reported to increase blood eosinophil counts and globule leucocytes in the abomasal mucosa following experimental infection [Few studies have investigated the role of cysteine in the improvement in immune responses. The bibliographic analysis performed here showed only one article in which the impact of cysteine supplementation on the immune response of sheep against nfection . FurtherEimeria and E. coli [The interaction between dietary glutamine and immunity has been well studied in mice . Glutami E. coli . Similar E. coli , this hiThe basic amino acids, lysine and arginine, play important roles in membrane protein activity and the actions of antimicrobial, toxin and cell-penetrating peptides. In poultry, due to the absence of a functional urea cycle, arginine is an essential amino acid . Many stEscherichia coli LPS (lipopolysaccharide), arginine supplementation (0.5% above the NRC recommendation) increased the infiltration of ileal mucosa by IgA-secreting cells, CD8+ and CD4+ T cells [Escherichia coli K88 [Arginine metabolism has been described as a key regulator of innate and adaptive immunity. Cells of the myeloid lineage, such as macrophages and dendritic cells, could modulate the immune response by regulating the expression of two enzymes: NOS (nitric oxide synthase) and arginase . NOS use T cells . Many pi T cells . This pocoli K88 . An averBranched-chain amino acids (BCAA), which include leucine, isoleucine and valine, contribute notably to the synthesis of glutamine in the skeletal muscle and are essential for lymphocytes’ proliferation in response to immune stimulation . Thus, dE. coli K88 (ETEC) [Threonine is implicated in many biological functions, such as, body protein synthesis, collagen, antibody and uric production or pancreatic enzymes . In broi8 (ETEC) . In broi8 (ETEC) . The ant8 (ETEC) . Indeed,Most of the selected studies focused on three amino acids: methionine, threonine and arginine, which were all associated with a positive impact on the host immune response. The mechanisms of immunity improved by dietary amino acids in livestock animals involved both Th1 and Th2 responses, and Th17 to a lesser extent. We have summarized the effects of the different amino acids studied in the selected research articles in a schematic representation of the immune response . The most important effects of amino acids on immunity were mostly observed at a high level of supplementation. The majority of the studies were on chicken, and few studies investigated the effect of amino acid supplementation on ruminant immune response. This may be due to the fact that ruminant physiology limits amino acid availability. To overcome this limitation, the utilization of rumen-protected amino acids is of interest. Therefore, there is a need for more studies in pigs and ruminants with live pathogens to increase our knowledge on the effect of dietary supplementation with synthetic amino acids on the immune response against infection.


Background: Enterobiasis or oxyuriasis from Enterobius vermicularis is an infection usually localized in the large bowel and cecum. Generally, the symptoms are characterized by anal itching, and intestinal or nervous disorders. Rarely, it is responsible for death. Methods: A forensic autopsy of a 52-year-old white male inmate who died 5 days after hospitalization was performed. Histological and toxicological analyses were also performed. Results: The death occurred by localization of Enterobius vermicularis in the duodenum and in the proximal ileum, with intestinal haemorrhage, inflammation, and peritonitis documented by histological examination. Conclusion: This is a common infectious disease, and can rarely occur with a fatal outcome, even in advanced populations. The lack of knowledge related to the rarity of death from enterobiasis disease can determine a dangerous concern. Gastrointestinal infections are a less common cause of sudden death compared to other conditions such as cardiovascular diseases, but they are equally important ,2. AmongWe report a case of death with forensic autopsy, in which EV in the duodenum and in the proximal ileum was found histologically. Such a death is rare, but this report shows that it can occur.Case History: A 52-year-old white male inmate with a history of non-steroidal anti-inflammatory drugs (NSAIDs) therapy and enalapril therapy for hypertension was admitted to the emergency room for repeated lipothymia in the absence of sweating, with hematemesis from the previous evening and melaena from three days before. The patient was hemodynamically unstable with acute anemia. The hemoglobin value upon admission was 6g/dL, while the procalcitonin in the blood was not evaluated. Therefore, a computed tomography (CT) scan of the abdomen was performed, which revealed a narrow lumen of the second portion of the duodenum; furthermore, the esophagus-gastro-duodenoscopy (EGDS) examination revealed multiple sub-centimeter lymph node formations in the stomach with normodistended walls due to insufflation, and fundus and gastric bodies occupied by food residues and clots; at the level of the first duodenum, there was an ulcerated lesion covered by a large clot. After a worsening of the condition, the patient was transferred to Intensive Care, was intubated and underwent therapy to restore hemodynamic balance. On the fifth day, the hemodynamics were unstable, and the anemia persisted. An emergency gastroscopy was performed in resuscitation, which revealed the absence of blood in the esophagus, stomach, and duodenum, and ulcerative lesion of the duodenal bulb with circumferential extension to the intestinal wall. Conditions precipitated due to common complications of hypovolemia. Hemorrhagic shock and peritonitis due to enterobiasis were assessed as causes of death. After 72 h, an autopsy was performed in accordance with the recommendations on the harmonization of forensic autopsy rules of the Committee of Ministers of the Council of Europe (1999) and according to the commonly accepted criteria for sudden cardiac death (SCD). Femoral blood was analyzed for alcohol (ethanol) and volatiles by head-space gas chromatography coupled with a flame ionization detector (GC/HS-FID). All post-mortem specimens were screened for the presence of the main different classes of drugs , using immunological or chromatographic methods as appropriate. A systematic toxicological analysis (STA) was performed by the LC-MS/MS system (API 3200 triple quadrupole ABI-SCIEX) in multiple reaction monitoring (MRM) mode.The autopsy showed some thin blood-stained fluid in the peritoneal cavity between the intestinal loops at the duodenum and the proximal ileum in the absence of macroscopic evident perforations A. We didThe toxicological analysis showed no evidence of drugs or alcohol.For each organ, sampling was carried out. After fixation in 10% neutral buffered formalin and paraffin embedding, sections for hematoxylin−eosin (E&E) staining for morphological diagnosis were performed. The histology of the duodenum and proximal ileum tract showed diffuse necrosis with multiple areas where trans-mural hemorrhage, granulocytic inflammation, and EV were detected . In partEnterobiasis or oxyuriasis is an intestinal parasitosis that mainly affects children, with predominantly anal itching and intestinal symptoms. After the ingestion of infective eggs, larvae hatch in the duodenum and descend into the crypts of the duodenum and the ileum where they develop and mutate into adult worms. In the beginning, larvae adhere to the mucosal surfaces of the small intestine, where they develop to sexually mature adult form and establish a chronic infection causing mucosal inflammation and ulceration. Then, the larvae can also penetrate the deeper layers of the intestinal wall where they establish chronic inflammation, destroying the tissue layers. Commonly, adult individuals settle in the colon from where pregnant females migrate to the perianal area at night, while the host is sleeping, to lay eggs. In fact, a diagnosis of enterobiasis in the living can be performed by applying a transparent adhesive tape in the perianal area immediately after waking up, followed by microscopic analysis. A reliable diagnosis of enterobiasis is also based on a correct intestinal sampling. Multiple samples of the intestinal wall must be taken at random in order to find the pinworms inside. In histological samples, the thick cuticles and internal organs of the worms can be easily recognizable within the intestinal wall, from the mucosal epithelium to the serosa, according to the transverse and longitudinal sections of paraffin inclusions .Sometimes, although very rarely, EV can also migrate into the other tracts, such as the urinary bladder, peritoneum, kidneys, liver, lungs, and other organs. Generally, it is considered that EV is a non-invasive parasite, which cannot penetrate a healthy intestinal mucous membrane. However, there is increasing evidence that EV infection may be the cause of ileal inflammation. By causing mucosal inflammation and ulceration, pinworms damage the integrity of the bowel mucosa and can then penetrate the deeper layers of the bowel wall or lead to intestinal perforation and hemorrhage ,12,13,14This case is interesting because it offers a reflection in the framing of an inmate with a history of drugs associated with repeated events of lipothymia, hematemesis, and melaena caused by intestinal parasitosis. Unfortunately, there was not enough time to diagnose the EV infections due to the rapid worsening of the patient’s condition. The instrumental investigations were able to identify the ulcerative complications that may be common to different diseases, such as the side effects of drug medications. A CT of the abdomen, EGDS, and gastroscopy showed the absence of blood in the oesophagus, stomach, and duodenum, along with ulcerative lesions of the duodenal bulb with circumferential extension to the intestinal wall. However, the diagnosis of EV infections is generally made on the identification of the eggs in the anal region or of the pinworm after intestinal biopsy. Therefore, intestinal parasitosis should be considered in the differential diagnosis of clinical syndromes like gastrointestinal haemorrhages.This case study presents the worst consequences of enterobiasis in a young adult. A large number of human pinworms were observed histologically in the duodenum and ileum wall, surrounded by inflammatory cell infiltration, bowel hemorrhage, and peritonitis. Therefore, a correct differential diagnosis between enterobiasis and gastrointestinal ulcer hemorrhages from other causes can be useful for correct treatment of patients.


The World Health Organization predicts that infertility will be the third major health threat after cancer and cardiovascular disease, and will become a hot topic in medical research. Studies have shown that epigenetic changes are an important component of gametogenesis and related reproductive diseases. Epigenetic regulation of noncoding RNA (ncRNA) is appropriate and is a research hotspot in the biomedical field; these include long noncoding RNA (lncRNA), microRNA (miRNA), and PIWI-interacting RNA (piRNA). As vital members of the intracellular gene regulatory network, they affect various life activities of cells. LncRNA functions as a molecular bait, molecular signal and molecular scaffold in the body through molecular guidance. miRNAs are critical regulators of gene expression; they mainly control the stability or translation of their target mRNA after transcription. piRNA functions mainly through silencing genomic transposable elements and the post-transcriptional regulation of mRNAs in animal germ cells. Current studies have shown that these ncRNAs also play significant roles in the reproductive system and are involved in the regulation of essential cellular events in spermatogenesis and follicular development. The abnormal expression of ncRNA is closely linked to testicular germ cell tumors, poly cystic ovary syndrome and other diseases. This paper briefly presents the research on the reproductive process and reproductive diseases involving ncRNAs. With the development of genome-wide sequencing technology and high-throughput sequencing technology, it has been found that about 93% of the DNA sequences in the human genome can be transcribed into RNA, but only about 2% of the DNA sequences eventually encode proteins, with 90% of the DNA sequences being transcribed into ncRNAs . AccordiSpermatogenesis is a process through which male animals produce male gametes continuously. That is, spermatogonia stem cells (SSCs) undergo a series of strictly regulated physiological progress to constitute spermatozoa. Spermatogenesis consists of the following stages: stem-cell mitosis produces spermatocytes which undergo two meiosis to produce haploid round sperm cells, which eventually become mature sperm . Each stThe ovary is the female reproductive organ. Its main functions consist of secreting sex hormones and producing mature eggs. The follicles, the fundamental unit of the ovary, are comprised of the oocytes located in the middle, and the surrounding granuloma cells and membrane cells. According to the different stages of development, follicles can be divided into primordial follicles, primary follicles, secondary follicles, astral follicles and preparatory follicles. In fact, only a few follicles can develop to the ovulation stage and ovulate, and most follicles move towards atresia at different stages of development. Maturation or atresia of an egg during follicular development is formally regulated by a variety of factors, including paracrine and autocrine factors in the endocrine and ovary, and cellular communication between oocytes and granulosa cells, and between granuloma cells and granulosa cells . TherefoIn recent years, abundant hallmark studies in various organisms show that non-coding RNA directly and indirectly regulate the reproductive system. For example, it interacts with proteins and regulates their functions , regulatMicroRNAs (miRNAs) are a class of noncoding RNAs with regulatory functions discovered in eukaryotes in recent years. They are primarily involved in the regulation of genes at the post-transcriptional level. The regulatory functions of miRNAs are indispensable, and they have recently been found to play an important regulatory role in immune diseases, cancer and reproductive diseases . miRNAs The biosynthesis of miRNAs in animals  has been preliminarily interpreted. First, the primary transcripts of miRNA genes (pri-miRNAs) are converted into precursor miRNAs (pre-miRNAs) in the nucleus by the RNase, Drosha. After the initial splicing, pre-miRNAs are transferred from the nucleus to the cytoplasm under the action of the transporter Exportin-5, and then further cleaved by another RNase, Dicer, to produce mature miRNAs. These mature miRNAs, together with other proteins, form the RNA-induced silencing complex (RISC), which causes the degradation of target mRNA or the inhibition of translation . This prhttps://www.mirbase.org/) revealed 2654 mature miRNAs in the human genome and 1978 miRNAs in the mouse genome , GDF9 and BMP15 . In addiMammalian transcriptome analysis revealed that most lncRNAs are transcribed by RNA polymerase II, which is similar to protein-coding mRNA, with a 5 ′methylation cap and a polyadenylate tail but without a distinct open reading frame . AlthougA large number of lncRNAs have been detected in the testes of humans, rats and mice during specific developmental stages; some of the functions of lncRNAs have been characterized and annotated, for instance, Mrhl (meiotic recombination hot spot locus) , Tsx te, Dmr Dm and HongSSCs are a uniquely male germ line stem cell, which can support spermatogenesis and maintain male fertility. Liang et al. found that 241 specific lncRNAs can play an essential role in maintaining SSC survival and self-renewal through protein coding genes and miRNA . Weng etIn view of the fact that a large number of lncRNAs have been found to be expressed in testicular development and male germ cells, but only a few have studied their functions during the development of male germ cells, the regulatory mechanism of lncRNA in spermatogenesis is worth further in-depth investigation.Cumulus cells (CCs) are closer to oocytes than parietal granule cells, participate in the coordination of late follicular development and oocyte maturation, promote amino acid transport, sterol biosynthesis and glycolysis, and provide energy substrates for oocyte meiosis recovery . Xu et aIn ovarian granule cells of patients with PCOS, the lncRNA HCG26 (HLA complex group 26) is highly expressed and its expression level is linked to the number of ovarian antral follicles. The down-regulation of HCG26 expression in the human ovarian granule cell line KGN can inhibit cell proliferation and cell cycle progression, and also affect the expression of the aromatase gene and the production of 17β-estradiol . It is s, are essential factors for stem cell regeneration and male germ cell development in vertebrates  of target mRNA by a base incomplete pairing approach similar to miRNA. Inducing deadenylation and degradation of target mRNA, and depending on the different sequences of piRNA in millions, pi-RISC mediates degradation of thousands of different mRNA during late sperm cell development . In addiIn recent years, researchers have conducted in-depth studies and found that piRNA is not only specifically expressed in testis, but also abundant piRNAs are detected in ovary . It has Currently, infertile couples account for about 15% of married couples worldwide, and male infertility accounts for 40–50% . The etiA study using miRNA chip technology to screen the semen of infertile and healthy men found that there were 52 differentially expressed miRNAs, of which 21 were highly expressed miRNAs and 31 showed low expression in infertile patients . Using tAt the gene level, lncRNAs are not separated from DNA methylation. However, this model is commonly seen in the combination of lncRNA and methylated transferase , and the localization of this enzyme to the promoter (CpG island) and methylation of the gene, thus inhibiting gene transcription . Peng etPCOS (Polycystic ovary syndrome) is the principal element of female infertility, and is often characterized by hyperandrogenemia, oligomenorrhea and polycystic ovary syndrome, affecting 6–10% of women worldwide . In addiWith the development of gene sequencing technology, countless researchers have carried out the study of lncRNA sequencing in PCOS. The researchers completed microarrays or deep sequencing of different ovarian cells from PCOS patients or a rat model . These rIn addition, many lncRNAs are involved in the occurrence and development of PCOS, lnc-MAP3K13-7:1 was highly expressed and accompanied by DNA hypommethylation in granulosa cells of PCOS patients. The study demonstrated that Lnc-MAP3K13-7:1 bound DNMT1 as a recruitment agent and mediated its ubiquitination degradation. The CDKN1A promoter was demethylated and its expression level increased, finally, GCs proliferation was inhibited . CTBP1-AResearch into the role of lncRNAs in PCOS has just begun. Although the sequencing results show that there are numerous differentially expressed lncRNAs, research into these differentially expressed lncRNAs is not prolific enough, and only a few studies have discussed the function and mechanism of lncRNAs. Current studies mainly focus on the expression and mechanism of lncRNAs in peripheral blood leukocytes, cumulus cells, granulosa cells and follicular fluid. According to the current research, lncRNAs may participate in the production of steroids, enhance the activity of steroid receptors, obesity and IR and other metabolic processes, affect the development, proliferation and apoptosis of ovarian cells, and then lead to the development of PCOS . The stuIn recent years, more and more studies have investigated miRNA expression in women with PCOS . It has Ovarian cancer constitutes one of the most common cancers of the female reproductive system. However, the molecular mechanism of ovarian cancer remained unclear, so it is necessary to further study the biological behavior of ovarian cancer at present. NcRNAs provided a suitable cut-off point in the diagnosis and treatment of ovarian cancer. Previous studies have revealed that microRNA-224 inhibits the expression of cyclin A by targeting KLLN, thereby promoting the proliferation of epithelial ovarian cancer cells . In addiNon-coding RNAs are important regulatory molecules in the human body and are essential for the development of a pathophysiological state. The formation of gametes is extremely composite, and spermatogenesis can be simply summarized as the proliferation and meiosis of primitive germ cells, with the formation of mature sperm occurring due to the deformation of sperm cells. The development of follicles requires the passage of primordial follicles, primary follicles, secondary follicles, and mature follicles, and the eventual discharge of the cumulus oocyte complex. These processes are strictly and precisely regulated, and the failure of any one of them can intervene in the continuation of life. Non-coding RNAs are an important element in these processes.Despite the fact that current high-throughput experimental techniques and open ncRNA database resources have given rise to the discovery of many ncRNAs, the function of these ncRNAs in gametogenesis and reproductive disease remains unclear. Therefore, it can be inferred that there are still innumerable reproductive ncRNAs with important functions to be studied. This is also suggested that we have great potential to study the regulatory mechanism of ncRNAs in gametogenesis and reproductive disease. MiRNA binding to mRNAs is reversible, enabling the regulation of gene expression. Competitive endogenous RNA (ceRNA) competitively binds miRNA to regulate gene expression. LncRNAs can act as a sponge of miRNAs, adsorbing miRNAs and interacting with each other to constitute a huge and complex ceRNA network which is able to regulate the function of target genes.Research into the role and mechanism of ncRNAs in the generative process is a difficult and persistent task. Understanding the role of post-transcriptional gene regulation in reproduction will help to clarify the causes of reproductive failure and hopefully provide methods and targets for the treatment of infertility, as well as additional contraceptive methods. Scientists need to continue to clarify the functions of lncRNA in spermatogenesis and oogenesis, and strive for the diagnosis and treatment of infertility.


The purpose of this study was to evaluate the efficacy and safety of photobiomodulation as an adjuvant treatment for primary headache. A systematic review of randomized clinical trials was performed. For such, electronic searches were performed in the MEDLINE, Embase, Cochrane Library, LILACS, PEDro, PsycInfo, Clinicaltrials.gov., and WHO/ICTRP databases, with no restrictions imposed regarding language or year of publication. We included studies that assessed any photobiomodulation therapy as an adjuvant treatment for primary headache compared to sham treatment, no treatment, or another intervention. The methodological assessment was conducted using the Cochrane Risk of Bias tool. The certainty of the evidence was classified using the GRADE approach. Four randomized clinical trials were included. Most of the included studies had an overall high risk of bias. Compared to sham treatment, photobiomodulation had a clinically important effect on pain in individuals with primary headache. Despite the benefits reported for other outcomes, the estimates were imprecise, and the certainty of the evidence was graded as low. These findings are considered insufficient to support the use of photobiomodulation in the treatment of primary headache. Randomized clinical trials, with higher methodological quality, are needed to enhance the reliability of the estimated effects. Headache is a common form of pain throughout the world. In most cases, the diagnosis of headaches is based entirely on patient history, as an isolated physical examination rarely provides adequate diagnostic information ,2. AccorThe International Headache Society published a classification system and operational diagnostic criteria for headache based on clinical consensus, classifying headache into primary  and secondary . The conventional treatment for primary headache is pharmacological . However, medication overuse can also be a cause of headache. In such cases, withdrawal should be counselled, so that the prophylactic treatment is effective .Photobiomodulation with a low-level laser or light-emitting diode (LED) has been used for different therapeutic purposes since the end of the 1960s, when there was a considerable advance in the production of equipment and applications in the field of medicine ,6,7. LigPhotobiomodulation is a recent adjuvant treatment for numerous neurological and psychological conditions. It seems to increase the metabolic capacity of neurons, and stimulate anti-inflammatory, anti-apoptotic, and antioxidant responses as neurogenesis and synaptogenesis . The nonTherefore, this systematic review was performed to evaluate the efficacy and safety of photobiomodulation as an adjuvant treatment for primary headache.This systematic review followed the methodological recommendations of the Cochrane Handbook for Systematic Reviews of Interventions  and the We only considered randomized clinical trials (RCT) with parallel or crossover designs, independently of the publication status (complete article or abstract).We included RCTs involving adults (18 years of age or older) diagnosed with primary headache.We included RCTs that assessed any photobiomodulation therapy as an adjuvant treatment for primary headache. The following comparators were considered: placebo (sham), no intervention, or different interventions, such as botulinum toxin type A, acupuncture, among others.Pain intensity during a headache episode, measured by any validated scale or questionnaire, such as visual analogue scale (VAS), numeric rating scale, among others.Severity, duration, and frequency of episodes of headache.Serious adverse events during or after treatment that could lead to hospitalization or death.Minor adverse events, measured as the proportion of participants with at least one adverse event during or after treatment .Analgesics needed.Quality of life .Patient acceptability.The evaluation time of the outcomes was considered according to the follow-up analyzed in the included RCTs. In addition, the following intervals were considered for possible grouping in a meta-analysis: immediately after treatment, short term (up to three months after treatment), and long term (more than three months after treatment).Comprehensive and sensitive search strategies were conducted in the following six electronic databases on 14 February 2021, with no restrictions imposed regarding language or publication date: MEDLINE (via PubMed), EMBASE (via Elsevier), The Cochrane Central Register of Controlled Trials (CENTRAL) (via Wiley), Physiotherapy Evidence Database (PEDro), Literatura Latino Americana e do Caribeem Ciências da Saúde  via Biblioteca Virtual emSaúde ), and PsycInfo (via APA).www.clinicaltrials.gov, accessed on 24 December 2021) and International Clinical Trials Registry Platform (ICTPR) of the World Health Organization . In addition, the grey literature was searched in the OpenGrey database . The search strategies are presented in Ongoing clinical trials were searched in the following clinical trial registry databases: ClinicalTrials.gov , aspects of the intervention and comparator groups, analyzed outcomes, follow-up time and results. When necessary, the authors of the trials were contacted to provide additional information or test data.Two authors independently extracted the following information on included RCTs using a form previously prepared in Microsoft Office ExcelThe methodological quality of the included studies was assessed by two independent reviewers using the Cochrane Risk of Bias (RoB) table . This to2 statistic, where greater than 50% is considered substantial heterogeneity : −2.43 to 3.19; p < 0.00001).One month after treatment: a significant reduction in the frequency of episodes was found in the LLLT group compared to the acupuncture group .Four months after treatment: an increase in the number of episodes was found in the LLLT group, and a significant reduction was found in the acupuncture group ;Immediately after treatment: ;One month after treatment (short term) .Four months after treatment (long term) , and the optimization of these parameters was not discussed.One study , with 50In comparing the effects of LLLT and BTX-A on the frequency of episodes of headache,  reportedAnother study  comparedIn comparing infrared LLLT and TENS , no signBesides the inherent differences of each study, regarding the population, dosimetric parameters, and groups studied, divergences were found regarding the behaviour of the treatments over time. Moreover, the results should be interpreted with caution, due to the low quality of evidence.One study  reportedThe strong points of the present review are the use of a sensitive search strategy in six databases and hand searches of manuscript reference lists, with no restrictions imposed regarding the language or date of publication. Moreover, two independent reviewers screened the trials, data extraction, and appraisal of methodological quality, as recommended by the Cochrane Handbook for Systematic Reviews of Intervention, and the certainty of the evidence was carefully appraised using the GRADE approach .Future studies need to be based on the Consolidated Standards of Reporting Trials (CONSORT statement) . MoreoveThe current evidence does not support the use of photobiomodulation to reduce pain and the frequency of episodes of primary headache. Further randomized clinical trials, with adequate sample sizes and methodological quality, are needed to investigate the efficacy and safety of photobiomodulation as an adjuvant treatment for primary headache.


Antioxidants are an heterogeneous group of compounds able to counteract cell oxidation by acting as reducing agents, as free radical scavengers, and quenchers of radical species and other pro-oxidants, such as metals . Plants Owing to their antioxidant and antimicrobial characteristics, many efforts are devoted to increase their content in plants in the field , to prevThis Special Issue aimed to collect the most recent outcomes of plant antioxidants research, with particular regards to all aspects of antioxidant application and the production of healthier and safer foods.Food processing and storage appear to be two important fields of intervention to maintain high levels of phenols, flavonoids, and tannins, and promote the generation of antioxidant peptides. Thermal treatments are usually used to ensure microbial safety and to inactivate undesired enzymatic activities . At highThe current trends of research explore novel biotechnological protocols to reduce the loss of antioxidants, and investigate the conventional methods to assess their impact on their content and bioaccessibility. In addition, the effects of added-antioxidants on thermal treatments and the storage of different food products is another important topic. Predictive modelling of antioxidant and protein oxidation, with the support of artificial neural networks, is also emerging as a powerful tool to monitor quality changes.Three different studies evaluated the impact of novel and conventional processing techniques on strawberry , coffee Stübler and colleagues  investigHaile and colleagues  comparedCannabis sativa L.). Hemp bioprocessing consisted of a combined enzymatic treatment with commercial food grade enzymes and ad hoc selected lactic acid bacteria. The antioxidant potential of hemp was significantly improved through intense proteolysis followed by release of antioxidants peptides and bound phenolics. The antioxidant properties increased when tested in vitro on the human keratinocyte cell line. Due to proteolysis, hemp-food protein quality available for digestion and assimilation was also improved.Pontonio and colleagues  studied The effect of added antioxidants to reduce food oxidation and maintain high quality during the shelf life is an important matter of research. Three different studies focused on this topic, analyzing lipid and protein oxidation in meat, namely beef , pork 1, and lamKaczmarek and colleagues  developeMuzolf-Panek and colleagues  evaluate−1) in lamb diet increased the antioxidant capacity of postmortem meat and reduced drip loss during storage, likely due to the regulation of the expression of oxidases, metabolic enzymes, calcium channels, and structural proteins. The lipid oxidative stability was also promoted.Wang and colleagues  evaluateFlores and colleagues  studied Supplementation of antioxidants can be performed in various foods, not only to promote its oxidative stability, but also to obtain healthier and functional foods. Indeed, the study of Cedeño-Pinos and colleagues  focused The changes in food quality induced by the application of natural preservatives, like plant antioxidants, that were the focus of these studies can be investigated with different technological approaches. One is spectroscopy, reviewed in depth by Hassaun and colleagues . SpectroAntioxidants can be also used as part of a packaging material in polymer and biopolymer formulations. They represent a cost effective and green alternative to synthetic antioxidants, with lower safety and environmental issues. This is the case of the study of Rojas-Lema and colleagues  who studAmong the novel technologies used to improve the antioxidant profile of fruits and vegetables, light emitting diodes (LEDs) is one of the most promising. LEDs show unique spectra wavelengths, high luminous efficacy, lower operational cost, a lack of radiant heat, and longer lifespan compared to the traditional light systems.As reviewed by Loi and colleagues , light qIn the research of Palmitessa and colleagues , the effThe interest in specific antioxidants is still high in the scientific community. Two reviews focused on two different antioxidants, namely hydroxytyrosol and anthocyanins.Hydroxytyrosol is one of the main olive antioxidants, recognized by EFSA for its role in reducing the risk of cardiovascular diseases . Silva aYamuangmorn and colleagues  reviewedAronia melanocarpa, Cornus mas, and Chaenomeles superba leaf extracts, which were previously used to extend the shelf life and improve the health benefits of meat products. The anti-cancer activity of these extracts was assessed in Caco-2 cells. The authors measured the antiproliferative activity and the morphological changes induced by the extracts. When low concentrations were used, the antioxidant properties stimulated DNA repair in Caco-2 cells, whereas at higher concentrations the extracts acted as pro-oxidant, leading to DNA damage. Further research is still required; nonetheless this preliminary study shed light into the possible use of these extracts as anticancer agents.These reviews highlighted that there is increasing interest on the effects of plant antioxidants on human health and, in particular, on the characterization of the biological activity of novel antioxidants or plant extracts. Efenberger-Szmechtyk and colleagues  studied The papers published both in forms of original contribution and up-to-date literature reviews provide a comprehensive and interesting overview of the most recent and innovative trends of research in the field of plant antioxidants for food safety and quality. Nonetheless, further research is still needed to continue exploring the immense potential of plant antioxidants.


This paper presents an enhanced PDR-BLE compensation mechanism for improving indoor localization, which is considerably resilient against variant uncertainties. The proposed method of ePDR-BLE compensation mechanism (EPBCM) takes advantage of the non-requirement of linearization of the system around its current state in an unscented Kalman filter (UKF) and Kalman filter (KF) in smoothing of received signal strength indicator (RSSI) values. In this paper, a fusion of conflicting information and the activity detection approach of an object in an indoor environment contemplates varying magnitude of accelerometer values based on the hidden Markov model (HMM). On the estimated orientation, the proposed approach remunerates the inadvertent body acceleration and magnetic distortion sensor data. Moreover, EPBCM can precisely calculate the velocity and position by reducing the position drift, which gives rise to a fault in zero-velocity and heading error. The developed EPBCM localization algorithm using Bluetooth low energy beacons (BLE) was applied and analyzed in an indoor environment. The experiments conducted in an indoor scenario shows the results of various activities performed by the object and achieves better orientation estimation, zero velocity measurements, and high position accuracy than other methods in the literature. Indoor location-based services (LBS) have always been of great importance because people live and work in indoor environments most of their lives. Massive wireless networks are built according to the IEEE 802.11 wireless Ethernet standard . GNSS anA large variety of reconfigured wireless devices (RWD) are already in use, namely Bluetooth low energy (BLE) beacons, smartphones, Wi-Fi, ultra-wideband (UWB) beacons, and digital cameras for indoor positioning ,12. For Pedestrian dead reckoning (PDR) is an independent approach, and its primary principle is to calculate the step length, determine walking direction, and step size by using inertial sensors (IS)  to calculate the moving target information and positions . BluetooIn this study, EPBCM was developed to track the position of an object in an indoor environment. Quaternions and their conversion to Euler angles are explained. Smartphone-based inertial measurement unit (IMU) sensors were used for the data collection. A complementary filter was used to integrate the information of pitch, roll, and angular velocity to obtain orientation tracking. The state vector used for the estimation of orientation in UKF contains the prior gyros bias errors and Euler angle errors. The HMM-based activity detection approach was developed to recognize the various activities  performed by the smartphone user. The state transitions probability matrix and observation probability matrix were calculated based on the changing magnitudes of the accelerometer values. The compensation mechanism increases the position accuracy by reducing the drift error. KF was used for smoothing RSSI measurements obtained from the BLE beacon. The average weighted centroid localization algorithm (AWCLA) was used for the proximity calculation between the smartphone and fixed BLE beacon position. Various performance analyses were used to evaluate the significance of the proposed EPBCM based on HMM and AWCLA, such as clustering of raw data to know about the activities performed, comparison of position accuracy of PDR, BLE, and EPBCM, indoor localization visualization using IMU sensor data, and orientation estimation based on AHRS and UKF.Developed mechanism based on complementary filter to integrate roll, pitch, and angular velocity information to obtain orientation tracking information.Developed HMM-based activity detection approach to recognize the various performed activities.Smoothing of RSSI measurements by passing through the Kalman filter to remove noise and enhance the accuracy of distance calculation between the beacon and mobile phone user.Weight assignment based on the power of RSSI measurements and use of AWCLA for the proximity calculation between the BLE beacon and smartphone.Furthermore, different evaluation metrics were utilized to evaluate the effectiveness of the proposed EPBCM based on HMM and AWCLA, such as a comparison, in terms of position accuracy, confirmation of activity detection by clustering the sensor data to visualize the performed activities and compare with HMM-based activity detection approach, and comparison of the orientation estimation approach based on AHRS and UKF.The notable contributions of the proposed study is as follows:The rest of the paper is divided into the following sections. The internet of things is an evolving field, attracting much attention from the research community ,41,42,43A microelectromechanical systems (MEMS) are used to offer beacon-free solutions, consisting of a gyrometer, an accelerometer, and magnetometer. Due to the lightweight, smaller size, and lower cost of MEMS, sensors are often employed for the PDR system. Conventionally, the indirect estimation of walking speed and walking direction is conducted by using the PDR approach . PDR canTo identify and track objects automatically, various technologies related to indoor positioning include Wi-Fi, ultra wide band (UWB), radio frequency identification (RFID), and BLE. To achieve better positioning accuracy and to reduce the positioning system cost based on RFID, the authors in  presenteTo predict and measure the motion of the body based on IMU wearable devices, many machine learning algorithms are used. To reduce the error in an indoor environment, a deep neural network fingerprinting based indoor positioning approach is used . In 70]70], the To the best of the authors′ knowledge, all of the indoor positioning systems are developed based on single or hybrid localization algorithms that have problems of high position errors and more computational costs, particularly for hybrid localization algorithms. Moreover, all of these only determine the location of an object in an indoor environment. In some cases, sensors mounted on the foot, hand, arm, and chest are considered to track the orientation and detect the activity of an object up to the binary level. Furthermore, all of these existing indoor positioning systems face different issues, such as high localization errors, high computational costs, and high hardware costs, to name of few, as mentioned in In this section, an EPBCM based on HMM and AWCLA was implemented to decrease drift and error in the position caused by navigation algorithms. The details of the proposed EPBCM are shown in q and k are basis elements and The orientation between the inertial navigational frame Q. Quaternions The amount of the rotation that should be performed about the vector part specifies by the element The rotation can be reversed by simply inverting the attitude quaternion The inertial frame is an unmoving Earth-fixed set of axes reference. In the inertial frame, x-axes point north, y-axes point east, and z-axes point down. This configuration of axes in the inertial frame reference is called north–east Down (NED). The sequence of rotations is used to represent a given orientation, which is the first yaw, then pitch, and finally roll. YAW rotation results in the new coordinate frame as illustrated in the Similarly, if the rotation around pitch represents Performing rotation around the newf2 x-axis, SBF is obtained. The SBF contains yaw, pitch, and roll, and the rotation matrix for moving from the newf2 to SBF is given by:To complete the rotation matrix for moving from the inertial frame to the body frame—it is shown in Equation (7)QinfsTo get the accurate angular rates in the proper frames, the x-axis IMU output must be rotated into the newf2 frame, the y-axis IMU output must be rotated into the newf1 frame, and the z-axis IMU output must be turned into the INF. The resulting transformation matrix for converting body-frame angular rates to Euler angular rates is given byLet Gimbal lock becomes a problem when using Euler angles and the above operation illustrates mathematically. When the pitch angle The quaternions data can be converted to Euler angles on the receiving end, and the exact equations for converting from quaternions to Euler angles depend on the order of rotations. The sensors move from the inertial navigational frame to the sensor body frame using the first yaw, then pitch, and finally roll. The Euler angles can be obtained from the quaternions, and this results in the following conversion equation:To tackle the problem that arose due to the arctan and ained in .In this study, the detailed explanation of orientation estimation based on UKF is presented as shown in e errors .(11)δΨ=lication . The staThe state equation for attitude estimation system isThe process model is adopted as The measurement equation is represented by, The variance of measurement noise is ethod in .x contains the orientation information q and the gyro bias K and l, ith column of the matrix represent i. The prior estimates of covariance For calculating the posterior first and second order statistics of a random variable, which undergoes the unscented transformation. By using a minimal set of deterministically chosen weighted sample points, the state distribution is again represented by a Gaussian random variable (GRV). Covariance and true mean completely capture the prior random variable by using the weighted sample points called sigma-points. In this case, the error state Equation , and traIn the above two equations To determine posterior covariance, e zero ρ . The valned from .Magnetic measurement and gravity vector are not good choices for the reckoning of the horizontal component of the state vector. Moreover, yaw correction is not possible by using a gravity vector. Due to the described issues and to ensure gravity correction, it does not act on the yaw estimate, and as magnetic anomalies only influence the pitch estimates and roll, we divide the measurement update into two steps. For the case of gravity vector measurement update:K the We set the third part of R, diagonal covariance inflation (CI) approach in [To adaptively adjust the measurement covariance roach in  is impleThe determination of the error covariance k activities performed The observation model uses the inertial measurement (IMU) acceleration data received from the different activities performed by the person containing the smartphone. By performing various activities while holding a smartphone in a user′s hand, fingerprints are collected of each activity in an offline phase. Let In previous research, the observation model′s role was to assign a weight To each PA in each zone he model .(27)KPrecThe path loss is expressed in dB and it takes place exponentially with distance as shown in Equation (49)PlosThe relation between distance and RSSI in  can be dThe above equation can be rewritten as:A and B, and it is among the control unit, current state, and previous state. At time step t, the state of interest is T, and noise measurement is The relationship is modeled through matrices Likewise, the steps update and prediction based on the Kalman filter are as followsWe define RSSI signals as the state of interest t, and Kalman Gain (KG).The RSSI estimation is computed in three steps. Step 1: g. To estimate the unknown mobile position, the weighted centroid localization algorithm uses Equations (59)wij=1Here Equation (62)wij=Calculations of smartphone locations in indoor surroundings based on KF, which integrates the smoothed RSSI measurements based on the beacon weight. In the proposed algorithm based on KF, RSSI measurements are pre-processed and integrated. The proposed beacon-based localization eliminates noise and smooth RSSI values. By using the estimated filtered RSSI values, the distance between the deployed beacons and smartphones is calculated. The strength of the received RSSI signal is directly proportional to the power delivered to the beacon. The calculation of the actual distance between the deployed beacons and smartphones by using centroid points of beacons.The error in distance is calculated using equation Experiments were conducted on a Windows PC with 12GB RAM. A front end (desktop application) was developed using Java, and the clustering techniques were applied in python. Well-know python libraries, including NumPy, SkLearn, and Scipy, were used for clustering experiments. In addition, NCSS was used for the visualization of data in PC. Furthermore, the simulation time for acquiring data for every instance was one minute (60 s). The required software and hardware components are listed in To validate our enhanced PDR-BLE compensation mechanism based on HMM and AWCLA for improving indoor localization, the person moves in an indoor environment with a Bluetooth enabled smartphone. The mobile was tested in seven different locations. Actual smartphone coordinates were compared with the estimated positions. Seven Bluetooth low energy beacons with known coordinates named By using the Kalman filter in the proposed algorithm, the collected RSSI values were then smoothed, and for position estimation of the smartphone, the weight of each BLE-beacon was used. The figure shows various smartphone and deployed BLE beacon positions.To validate our KF approach mentioned in The estimated mobile positions using EPBCM algorithm, the BLE-beacon and PDR is given in To validate the EPBCM localization algorithm, six mobile positions were chosen and compared with other estimated positions using different approaches. The measurements of accelerometer, magnetometer, and gyroscope were used to track the position and orientation of an object moving in an indoor environment. Walking down two flights of stairs, trajectory is shown in The purpose of showing this result is to evaluate the performance of UKF-based orientation tracking and data collection from smartphone-based IMU sensors. The starting point is the mobile computing lab (MCL) situated on the fourth floor of the JNU, and the ending point is the end of two flights of stairs. However, the exact ground truth cannot be given, but the person′s orientation and activity can be determined. Compared to the flat ground, the error in stair walking is more significant. This is due to the fact that the calculations of vertical acceleration in stair walking has to be calculated for this scenario and it must include gravitational acceleration.In this experiment, several pedestrian walking experiments were conducted in the engineering building of JNU, and the final results are shown in In this study, an EPBCM based localization algorithm was developed to estimate the position of an object in an indoor environment. The proposed system is a combination of two localization algorithms—PDR and BLE-beacons. In the proposed position estimation module, orientation is estimated using the unscented Kalman filter and HMM-based activity detection by considering the varying measurements of the accelerometer sensor. In this paper, the BLE-beacon-based localization algorithm is used as a compensation algorithm. In this compensation model, the path-loss model is used for distance measurement. The Kalman filter is used for noise elimination, smoothing RSSI measurements, and integrates raw BLE-beacons measurements to reduce the positioning error. The position estimation combinator is designed to combine the position coordinates of the two localization algorithms and get the enhanced position, containing less error. For comparative analysis, we compare the HMM-based activity detection approach results with the K-mean clustering technique. We also compare the three localization algorithm results, showing the proposed indoor positioning system containing the least error. It also shows that the proposed algorithm deviation from the actual location is the minimum. The walking scenario experimental results show that the proposed EPBCM based on HMM and AWCLA is feasible for the indoor positioning system. At some points, the positioning error is tiny, although to maintain a high accuracy level, more beacon deployment must be required to get more compensation in error at different mobile locations.


Cerebellar ataxias (CAs) manifest with a combination of motor incoordination, cognitive, affective and recently identified social symptoms. Novel therapies aim to stop the progression of the subgroup of the degenerative ataxias, or even to cure the disease with a functional and anatomical restoration of the cerebellar circuitry in the near future. The goal of stopping the progression of the disease is particularly relevant if applied at a very early stage of the disease, when the cerebellar reserve is only slightly impaired. Therefore, the search of the prodromal phase or pre-ataxic stage of CAs represents a very important challenge for the scientific community. The identification of pre-manifest individuals and the recruitment of individuals at risk has become a key-challenge to address neuroprotective therapies. The feasibility is high due to the recent progress in the biological and morphological biomarkers of CAs. Cerebellar ataxias (CAs) represent a highly heterogeneous group of neurological disorders with a core group of clinical manifestations embracing motor incoordination, cognitive and affective symptoms, and social deficits . In the The recent development of molecular therapies opens the door leading to the reversal of the irremediable course and transforms debilitating incurable diseases into treatable conditions . The fieRNA depletion or DNA addition are emerging techniques. siRNA, miRNA and CRISPR-based techniques are being validated in animal models of CAs. Antisense oligonucleotides (ASOs) represent an example of a very promising approach to revert the course of the disease even before the occurrence of symptoms ,5. ExperThe identification of early predictors is an essential step to identify disease onset, monitor the early progression, improve our understanding of the natural history and establish novel therapies . QuantitFluid biomarkers  are used to predict the onset of the disease and to monitor the progression of ataxia. In polyglutaminopathies, the abnormal protein itself can be assessed both in blood and CSF . The useAmongst the imaging biomarkers, several important findings have appeared in the literature. Not only do brain volumetry and ROI (region of interest)-based studies allow the early identification of structural lesions, but the involvement of the white matter tracts can be demonstrated at the level of the cerebellum and cerebellar peduncles by DTI techniques ,22,23,24The identification of the prodromal stage is currently highly challenging. The neuronal loss leads to a decrease in the cerebellar reserve. Clinical symptoms become manifest once a threshold of manifestations is reached .Cerebellum represents that main stock of neurons in the brain (about 60%), and therefore the question of how to preserve this “natural neuroanatomical neuronal bank” is straightforward. Cerebellar reserve is defined as the capacity for tissue compensation and restoration following pathological injury to the circuitry . The anaAs a function of the injury, two types of cerebellar compensation and restoration can evolve: (a) structural cerebellar reserve: when the cause elicits transient structural damage in a limited area , the lost cerebellar functions are compensated for by other cerebellar areas, which are anatomically preserved and are extra-cerebellar regions within the cerebro–cerebellar networks and brainstem-cerebellum networks; (b) functional cerebellar reserve: the neuronal loss is progressive and diffuse in this case, e.g., immune-mediated, metabolic, and degenerative CAs. Compensatory mechanisms to account for vanishing cerebellar functions are embedded in the circuitry itself and, due to the numerous inflow–outflow tracts of the cerebellum, also involve specific tracts such as the olivocerebellar pathway (climbing fibers), the ponto-cerebellar tracts (mossy fibers), the spino-cerebellar tracts (mossy fibers) and/or the aminergic/cholinergic afferent tracts. In spite of their self-recovery capacities, some patients do not show recovery from CAs. In such instances, it is assumed that the pathology has moved from a treatable condition to an untreatable state . This trIt is critical to promptly identify and address alarming symptoms that, if untreated, may lead to permanent cerebellar dysfunction. The concept gives rise to the term “brainstem attack”. The brainstem attack is a reversible deficit due to the transient loss of motor control and temporary motor, ocular motor, and vestibular deficits. A brainstem attack, classically seen in vascular compromise, i.e., the posterior circulation of transient ischemic attacks, can have an important differential in impending autoimmune disease that, if untreated, can lead to permanent damage, as is the case with its vascular etiology. The vascular brainstem attack symptoms should be promptly addressed with aggressive antiplatelet therapy, which is the adequate perfusion pressure maintenance, and, if needed, the addition of antilipid agents. Immune etiologies, on the contrary, are addressed with immunomodulators, steroids, or intravenous immunoglobulin therapy. There are distinct differences between vascular and immune brainstem attacks; vascular attacks lead to episodes of several hours to days in length, while immune etiology is generally associated with much prolonged, days- to week-long deficits. If untreated, the impairment in the immune etiology leads to a neurodegenerative disorder, while vascular etiology may result in a stroke. Typically occurring in a stroke, the brain stem attack can also present with a neurodegenerative or autoimmune disease. The brainstem attack is thought to be secondary to the brainstem or cerebellar Purkinje neuron dysfunction. These neurons provide critical feedback to a number of motor circuits, including facilitating saccades, pursuit, gaze holding, and VOR.In terms of cerebellar reserve, the process of neuronal death resulting in atrophy helps us to better understand how neuronal loss manifests clinically and why some patients develop cerebellar symptoms quickly despite a small apparent atrophy. In a subgroup of patients, the atrophy of the cerebellum is silent whereas in others the symptomatology remains unchanged. This reflects the collapse of the dynamic capacity for compensation and restoration, mainly due to advanced cell death . ReserveNovel tools are now available to restore cerebellar functions in a non-invasive way, especially by means of neuromodulation techniques. The cerebellum represents an ideal target for this approach for several reasons, including the anatomical location of the cerebellum immediately below the skull, the very high concentration of neurons in the cerebellar cortex, its organization into modules linking the cerebellar cortex/nuclei/the inferior olive, the multiple forms of plasticity between parallel fibers/Purkinje neurons, the remote effects of the cerebellum upon the brainstem nuclei/basal ganglia/cerebral cortex, the multiple roles of the cerebellum in oculomotor and motor controls, cognitive operations, and social/affective regulation . The neuOne of the challenges of the cerebellum community is to identify the prodromal stages of cerebellar disorders, so that early therapeutic strategies can be administered at a time when the cerebellar reserve is still in a position to respond. It is however, encouraging that currently irreversible and incurable cerebellar degenerative disorders will soon be addressable. The ongoing cutting-edge research in neurotherapeutics, neuromodulation, and physiological and biological marker development will bring us closer to a much more effective treatment early on in the disease course.The identification of pre-manifest individuals and the recruitment of individuals at risk has become a major challenge to address neuroprotective strategies. Due to the rarity of CAs, their clinical heterogeneity, and the variability in penetrance and clinical progression, multi-center studies are required. There is space for improvements in clinical rating scales, because the currently used scales show both floor and ceiling effects, which create difficulties in handling patients with very mild deficits, in addition to the subjective effect inherent to clinical scores. A combination of clinical scales with quality-of-life measures, wearable sensors allowing free-living motor activity monitoring, neuroimaging biomarkers  and fluid biomarkers  is likely the best approach, not only to identify conversions from the asymptomatic stage to symptomatic condition, but also for the general monitoring of ataxias ,38. Pure


Antimicrobial peptides (AMPs) are short cationic amphipathic peptides with a wide range of antimicrobial properties and play an important role in the maintenance of immune homeostasis by modulating immune responses in the reproductive tract. As intra-amniotic infection and microbial dysbiosis emerge as common causes of preterm births (PTBs), a better understanding of the AMPs involved in the development of PTB is essential. The altered expression of AMPs has been reported in PTB-related clinical presentations, such as preterm labor, intra-amniotic infection/inflammation, premature rupture of membranes, and cervical insufficiency. Moreover, it was previously reported that dysregulation of AMPs may affect the pregnancy prognosis. This review aims to describe the expression of AMPs associated with PTBs and to provide new perspectives on the role of AMPs in PTB. Preterm birth (PTB) refers to a multi-etiological condition that occurs in more than one out of ten child births, and approximately 1.1 million neonates die from prematurity-related complications each year ,3,4,5,6.The cellular structure of the reproductive tract and various immunological functions form a barrier against microbial pathogens during pregnancy. Uterine, vaginal, and cervical epithelial cells build a physical barrier by expressing intercellular junctions, including tight or occluding junctions and adherent junctions ,19,20,21Considering that intrauterine infection or inflammation and vaginal dysbiosis are some of the key triggers of PTB, AMPs may have an array of effects on PTB. Therefore, in this review, we highlight the recent findings regarding the altered expression of PTB-associated AMPs and their potential influences on the development of PTB.AMPs are evolutionarily conserved molecules that are produced in all multicellular organisms from prokaryotes to humans, and they are the first-line defense against microbial pathogens ,42,43,44AMPs have direct and rapid antimicrobial activities by destroying the physical integrity of microbial membranes or by acting on intracellular targets via membrane translocation . BacteriRecent studies have demonstrated that AMPs perform a broad range of immunomodulatory functions beyond their antimicrobial activity . The molThere is mounting recent evidence supporting that the commensal microbiota in the body plays a pivotal role in host defense through colonization resistance and development of the mucosal immune system; thus, the balance of commensal microbiota has an impact on health and the state of disease. AMPs have both antimicrobial and immunomodulatory properties, which directly and indirectly affect the composition of commensal microbiota. A recent report showed that intestinal commensal species increase resistance to antimicrobial activities of AMPs by up to four times by reducing the overall negative charge on cell surfaces via LPS modification . MoreoveAMPs, which are distributed throughout the female reproductive tract during pregnancy, play a role in preventing infection through their antimicrobial activities and modulating immune responses. HNP 1–3 were reported to be expressed in the vernix caseosa, chorion, placental trophoblasts, and amniotic fluid ,101,102.Lactobacillus-dominant commensal bacteria, which prevent the attachment and invasion of microbial pathogens  and mannose-binding lectin [MBL2]) that were more frequent in neonates born to pregnancies complicated by PPROM ,129. MorSeveral studies on significant alterations in the expression of AMPs in PTB-associated clinical presentations and correlation of the level of AMP expression with pregnancy prognosis suggest that AMPs play an important role in the development of PTB.In view of the complex host defense properties of AMPs, such as antimicrobial and immunomodulatory properties and shaping of the composition of the commensal microbiota, many AMP-based therapeutic agents have recently been investigated, not only as new anti-infectives that can overcome the limitations of antibiotics related to drug-resistant pathogens, but also as immunomodulatory agents in a variety of indications, such as chronic inflammatory disorders and wound healing. As infections and microbial dysbiosis emerge as common causes of PTB, further research on functions and mechanisms of action of AMPs may enhance our understanding of the pathogenesis of PTB and provide promising treatment options.


Today, a wider perspective of gastrocardiac syndrome has emerged. First, the cardiovascular risk factor chronic systemic inflammation may reflect gastroenterological inflammatory conditions, such as inflammatory bowel disease and gastrointestinal infections, in particular, chronic Helicobacter pylori infection. Furthermore, since contemporary treatment of cardiovascular disease commonly includes potent antithrombotic medications, the cardiovascular benefit in terms of a decrease in the incidence of recurrent ischemic events and death needs to be carefully balanced with an increased risk of gastrointestinal bleeding. Several strategies to target chronic gastrointestinal inflammation and to diagnose and treat Helicobacter pylori to reduce the risk of cardiovascular events and gastrointestinal bleeding are available but residual controversy remains and large-scale gastro-cardiology trials are needed to determine the optimal treatment approaches. In perspective, the centennial gastrocardiac syndrome is more relevant than ever in a contemporary gastroenterology and cardiology setting. A collaborative subspecialty, namely Gastro-cardiology, would introduce novel unique means to study, diagnose and treat gastrocardiac conditions with the aim to reduce the risk of cardiovascular and bleeding events to improve the prognosis for gastro-cardiology patients.The gastrocardiac syndrome was coined originally at the beginning of the 19 The gastrocardiac syndrome was coined by Ludwig von Roemheld in 1913 to describe an alleged gastric-cardiopathy (Helicobacter pylori (H. pylori) infection as an additional ventriculo-duodenal causal factor for atherosclerosis progression and cardiovascular events (H. pylori has been established over the last two decades (H. pylori and atherosclerosis could relate to two pathways: (1) chronic inflammation caused by direct colonization of the vascular wall enhancing and disrupting atherosclerotic lesions, and (2) a systemic inflammatory response in reaction to the colonization of the gastric mucosa    and non-invasive  methods are established  by UBT   to decrehol use) . Howeverhol use) . Further PPI use , is reco PPI use , 26. Conablished . As inva) by UBT , current) by UBT . No contpatients , in agred at 11% . Importaecessary . Thus, gH. pylori eradication on subsequent ulcer bleeding in infected individuals taking aspirin daily was completed enrolling 30,000 patients . Patient enrollment is expected to start in November 2021.To date, the residual controversy on how to handle the risk of UGIB in patients with cardiovascular disease urges the need for randomized gastro-cardiology trials. Recently, the Helicobacter eradication aspirin trial (HEAT), a double-blind, placebo-controlled, randomized trial of the effects of patients  and otheIn perspective, the centennial gastrocardiac syndrome is more relevant than ever in a contemporary gastroenterology and cardiology setting. A collaborative subspecialty to improve diagnosis and treatment of gastrocardiac conditions and to reduce the cardiovascular risk and complications of ischemic-, arrhythmic- and valvular heart diseases would introduce novel unique means to improve the outcomes of gastro-cardiology patients.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.RH and MB: concept and design, critical revision of the manuscript for important intellectual content, and drafting of the manuscript. All authors contributed to the article and approved the submitted version.RH was supported by the Swedish Heart-Lung Foundation ; the Swedish Research Council ; and the Region Stockholm  . The funding organizations had no role in the preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.