Central nervous system (CNS) diseases are the leading causes of death and disabilities in the world. It is quite challenging to treat CNS diseases efficiently because of the blood–brain barrier (BBB). It is a physical barrier with tight junction proteins and high selectivity to limit the substance transportation between the blood and neural tissues. Thus, it is important to understand BBB transport mechanisms for developing novel drug carriers to overcome the BBB. This paper introduces the structure of the BBB and its physiological transport mechanisms. Meanwhile, different strategies for crossing the BBB by using nanomaterial-based drug carriers are reviewed, including carrier-mediated, adsorptive-mediated, and receptor-mediated transcytosis. Since the viral-induced CNS diseases are associated with BBB breakdown, various neurotropic viruses and their mechanisms on BBB disruption are reviewed and discussed, which are considered as an alternative solution to overcome the BBB. Therefore, most recent studies on virus-mimicking nanocarriers for drug delivery to cross the BBB are also reviewed and discussed. On the other hand, the routes of administration of drug-loaded nanocarriers to the CNS have been reviewed. In sum, this paper reviews and discusses various strategies and routes of nano-formulated drug delivery systems across the BBB to the brain, which will contribute to the advanced diagnosis and treatment of CNS diseases. Central nervous system (CNS) diseases, such as dementia, Alzheimer’s disease and gliomas, are the leading causes of disability and death ,2,3,4. HThus, it is critically important and an urgent demand to understand the BBB transportation and have efficient strategies to deliver drugs through crossing the BBB. Recently, nanomaterial-based drug delivery systems have been studied by exploiting physiological BBB transport (transcytosis mechanism). Inorganic and organic nanostructures with surface modification have been designed as drug carriers to cross the BBB. In addition, during the period of the COVID-19 pandemic, the studies on neurotropic viruses, their BBB disruption mechanisms and their invasion methods to the CNS are vital to fight the global crisis. Meanwhile, their BBB disruption and/or circumvention mechanisms may give an inspiration on developing innovative drug delivery systems for the treatment of these CNS diseases.In this paper, the structure of the BBB and its physiological transport mechanisms are introduced, followed by the introduction of the viral disruption mechanism. Nanomaterial-based drug carriers with enhanced capability for BBB transportation are reviewed based on different transport mechanisms . Furthermore, nanocarriers by exploiting viral-induced BBB disruption mechanisms are discussed, which is considered an alternative method to cross the BBB and could be the next-generation drug vehicles. Finally, we discuss various delivery routes of nano-formulated drugs to the brain.The BBB is one of the most extensive and exclusive physical barriers that maintain homeostasis within the CNS by protecting it from toxins and metabolic fluctuations . The BBBMajor physiological BBB transport mechanisms are passive diffusion, carrier-mediated transcytosis (CMT), adsorptive-mediated transcytosis (AMT) and receptor-mediated transcytosis (RMT). However, nanomaterials, with the advantages of a high drug-loading capacity, good stability, biodegradability, low toxicity and versatility, make good drug transportation systems across the BBB and make the delivery of the loaded drugs into the CNS possible ,14,15,16At the same time, some therapy strategies by using mechanical, ultrasound and chemical agents were able to disrupt BBB and aid the transportation of drugs across the BBB. However, such disruption was reported to cause severe vasculopathy, chronic neuropathologic changes and seizures in animal models . It is wSubstances cross the BBB by following one of the four transport mechanisms: passive diffusion, carrier-mediated transport, adsorptive-mediated transcytosis, and receptor-mediated transport . PassiveCarrier-mediated transport is one of the most common transport mechanisms. Substances enter the endothelial cells via their corresponding transmembrane proteins on the cell membrane. An example would be glucose transportation via glucose transporter type 1 (GLUT1). GLUT1 is able to recognize glucose, mannose and galactose and actively transport these substances through the BBB . AnotherAdsorptive-mediated transcytosis (AMT) results from the electrostatic interaction between the positively charged ligands and the negatively charged cell membrane. It is mediated by clathrin-dependent endocytosis and is unidirectional from blood to brain . ReceptoIn addition to the physiological transport mechanisms, many viruses, such as Japanese encephalitis virus (JEV), human immunodeficiency virus-1 (HIV-1) and rabies virus, are found to be able to infect the CNS by other mechanisms and may cause severe neurologic syndromes. Flavivirus, coronavirus and other neurotropic viruses are briefly introduced, and their possible disruption mechanisms are also reviewed  and discFlaviviruses are major emerging human pathogens, and some of the flaviviruses, such as West Nile virus (WNV), Japanese encephalitis virus (JEV), Dengu virus and Zika virus, are regarded to invade the CNS by various mechanisms ,41. DuriIn addition, HCoV-OC43 and HCoV-229E are two wild-spread coronaviruses and are proved to be neuro-invasive . The HCoSARS-CoV-2 is a SARS-like single-stranded RNA coronavirus with 29,903 bp , and hasThe other coronavirus with neuro-invasive potential is SARS-CoV, which caused the SARS pandemic. It has been clear that SARS-CoV is able to infect monocytes-macrophages  and dendOther viruses such as rabies virus and herpes simplex type 1 virus (HSV-1) are both regarded as neurotropic viruses and have a big effect on the CNS. Rabies virus is a highly neurotropic RNA virus, and the glycoprotein of that may have high specific affinity to the nicotinic acetylcholine receptor (nAchR) on neuronal cells , the neuThe infection mechanisms of those viruses can be classified as passive diffusion ; endothelial cell infection ; virus transcytosis ; cell-associated virus transport  . These pIn the past decades, various studies on investigating the ability and efficiency of nanomaterials used as drug carriers to cross the BBB have been reported. Inorganic nanomaterials, such as silica NPs , gold NPThe blood–brain barrier, formed by brain capillary endothelial cells, is a dynamic interface that takes control of the influx and efflux of numerous molecules between the brain and blood. Not only could this barrier exclude most of the drug molecules but it also possesses several transporter systems, which actively and selectively allows for the passage of desired molecules, including endogenous substances and nutrients, such as peptide, amino acid and glucose ,72,73,74Glucose is the main energy source for the metabolism of the mammalian brain and this molecule can be transported through the BBB via GLUT, since it cannot be synthesized by brain neurons. GLUT1 is highly expressed as a glycosylated form in the endothelial cells of the BBB . The cap glucose .System L, a sodium ion-independent bidirectional transporter, plays a key role in amino acid homeostasis in the brain ,91. LAT1The initial progress of transcytosis is the uptake of NPs by endocytosis . EndocytPoly(propylene imine) (PPI) and poly(ethylenimine) (PEI) are two examples of cationic polymers that are able to condense nucleic acid by ionic interaction and also efficiently deliver genes by escaping from endosomes using proton buffering capacity . The endIn addition to the cationic polymeric core, some polysaccharide can also be used as positively charged materials to enhance the AMT process and more efficiently facilitate the penetrability of the BBB. Maltodextrin NPs have been found to be capable of binding to anionic sites of the cell membrane at an early stage of endocytosis and succeed in penetrating such a barrier via a cholesterol-dependent exocytosis process . AnotherFurthermore, the zeta potential of the drug delivery system should not be the only factor taken into account when developing a carrier for crossing the BBB; the lipid solubility of the drug carrier is also a vital factor for enhanced AMT. This is because lipid membranes of the endothelium have an innate property to offer an effective diffusive route for lipid-soluble agents ,111. BasHowever, a positively charged surface is not always good for drug delivery systems designed for crossing the BBB. The cationic surface may represent a higher cytotoxic effect than the neutral counterparts ,113,114.In contrast to AMT, the binding interaction in RMT has a specific target and has much higher binding affinity between the ligands and the receptors. RMT is initiated by a ligand binding onto its receptor, followed by receptor-mediated endocytosis resulting in the movement of ligand-conjugated drug vehicles across or inside cells. In receptor-mediated drug delivery carriers, the surface of the delivery system is not required to be positively charged but can also be neutral or negatively charged, reducing the potential for increased cytotoxicity. vβ3 and αvβ5 integrins [3O4 nanoparticles across the BBB via the Lf-receptor-meditated pathway. On the other hand, insulin regulates glucose metabolism in the brain and its receptors can be found on the surface of vascular endothelial cells in the brain [Depending on the types of ligands and specific application area, there are various receptors involved in RMT, such as the transferrin (Tf) receptor ,118,123,ntegrins ,125. Trantegrins . It is entegrins . Based ontegrins ,118) arentegrins , pegylatntegrins , polylacntegrins ,118 , by a host-response-induced BBB disruption, and/or by a Trojan horse mechanism via monocytes ,42,62. TBased on the neurotropic property of a specific virus and the related mechanisms mentioned above, several non-viral drug carriers and viral vectors have been developed to enhance the penetration of the BBB and transport drug molecules to the CNS. The rabies virus glycoprotein peptide (RVG29) is a peptide with 29 amino acids derived from the rabies virus glycoprotein and has an innate property to specifically bind to nAchR. Because of its high brain penetration capability, several RVG-conjugated drug delivery systems, such as PEI , chitosaAnimal studies show that the current forms of the administration of nanoparticle-carried drugs to the central nervous system are: 1. systemic  administration, 2. direct brain/intrathecal administration, and 3. transnasal administration. The most convenient route for nano-drug administration is systemic delivery . However, the percentage of administered drug reaching the brain via this way is usually below 1–4%, due to the low permeability and the poor BBB selectivity, which means that the remaining 96–99% of the drug is off-target and would be potentially responsible for the systemic side effects, mainly phagocytosed by monocytes and macrophages and accumulated in the liver and spleen ,138.The direct delivery of nano-drugs to the CNS, bypassing the BBB and brain–spinal cord barrier (BSCB), is possible by intrathecal injection with direct drug delivery into the cerebrospinal fluid. This route of delivery reveals several advantages over systemic, peripheral administration. It leads to an immediate high concentration of the drug in the cerebrospinal fluid; thus, smaller doses of nano-drugs could be used, thereby minimizing any potential side effects. Importantly, the tightness of the BBB prevents the systemic spread of the nano-drug and significantly limits its penetration from the brain to the general circulation, which effectively reduces its side effects and toxicity. Compared to the freely administered molecules, intrathecal administered nanomedicines are well retained within the central nervous system, and encapsulated payloads experience slower clearance and mixing within the cerebrospinal fluid, which can enhance tissue exposure . The encThe intranasal route of drug delivery offers a unique opportunity for the delivery of pharmaceutically active ingredients (APIs) to the central nervous system. It is the less invasive route of drug delivery compared to the intrathecal administration method, and it already has been used successfully in clinical trials, showing improved cognition after intranasal insulin application in Alzheimer’s disease patients ,141,142.Notwithstanding the significant advantages of transnasal drug delivery routes, the proper formulation of drugs prepared for administration remains an important challenge, especially for drugs with adverse physicochemical and biopharmaceutical properties, such as rapid chemical or enzymatic degradation, poor moisture solubility, and low permeability. It requires a formulation capable of increasing drug transport to the brain, without interfering with the structure and physiology of the nasal epithelium. Pharmaceutical nanotechnologies are of strategic importance for developing the formulation of these substances for transnasal drug delivery to the brain. Nanomedicines could further contribute to making nose-to-brain delivery a reality.The potential limitations of the method are the small volume of the nasal cavity, which limits the amount of formulation that can be administered this way, poor access to the olfactory region using conventional nasal application devices, the short residence time of the drug in the nasal cavity before it is removed outside, low hydrophilic bioavailability, and possible mucosal irritations . In the Comparison of the brain targeting of olanzapine loaded PLGA NPs with a free drug in a solution, administered both intravenously and intranasally to sheep, showed that the uptake of NPs into the brain was 6.35 and 10.86 times higher, respectively. Noteworthy is the significant advantage of the nasal route of nanomedicine administration in increasing olanzapine transport to brain tissue . NimodypQuantitative data analysis in 73 publications from the last 30 years on the transnasal route of drug administration revealed such large differences in work results that it was impossible to establish a correlation between the physicochemical properties of drugs and their formulas and the effectiveness of targeting in the brain. The only regularity that can be directly compared was the effect of the drug form on its transport from the nose to the brain. The percentage of drugs reaching the brain from various formulations shows that drugs encapsulated in particles reach the brain in a greater amount (60%) than the free drug administered intranasally in solution (36.6%), and it should be noted that 24 of the 32 compared particulate drug formulations had a size of 50–200 nm .Undoubtedly, the transnasal route of administering the drug’s nano-formulation directly to the brain tissue still requires a lot of research, especially the standardization of nanomedicine preparation methods, the functionalization of NPs, arming them with directional particles that improve transport to the brain, etc. This is a prerequisite for obtaining more reproducible, more comparable results of experiments in vitro and in vivo on laboratory animals . On the Regardless of the path that nanomedicines have used to reach the brain, it is necessary to better understand the effects of the nano-formulation building elements themselves on neurons as well as on glia cells. Specifically, chronic toxicity to the central nervous system, as well as the immunogenicity of nanocarrier components, must be evaluated in detail, especially for medications anticipated for long-lasting treatment ,138,146.The complex structure of the BBB allows for the extensive filtration of materials for the protection of the brain and CNS. Although such filtration is necessary, it leads to difficulties in delivering drugs to treat various CNS diseases. The investigation of the physiological transport and viral-induced BBB breakdown mechanisms has provided innovative strategies for overcoming the BBB. The nanomaterial-based drug delivery systems functionalized with endogenous substances and essential nutrients for the brain, as well as their derivatives, are able to penetrate the BBB and deliver drugs to the brain via CMT. However, the molecules conjugated on their surface, which can be transported by specific carriers, must be well designed. Free endogenous substances and nutrients may have a competitive effect with such a drug carrier system and lower the drug transport efficiency. In addition, drug nano-carriers with cationic molecules and ligand modified on their surface both illustrate higher BBB permeation and brain accumulation via AMT and RMT, respectively. The binding affinity between the ligand and receptor pair for RMT is stronger than it is for AMT, and the binding sites are specific, while AMT drug delivery carriers have a higher binding capacity and the inhibition by saturation seldom happens. Furthermore, drug transport system coupling dual functional molecules (manipulating AMT and RMT together) were found to have a synergistic effect on crossing the BBB. In addition to the drug carriers based on the physiological BBB transporting mechanism, the studies on exploiting viruses with nanomaterials as drug carriers are under investigation and still at an early stage. However, drug carriers combining VLP and the functional proteins on the viruses are proved to be effective for the BBB transportation.To conclude, nanomaterials have proven their ability to cross the BBB and their potential in drug delivery systems involving the BBB. Further studies investigating the synergistic effects of CMT, AMT and RMT in BBB drug delivery and the identification of compatible nanomaterial–drug pairs would allow for a wider range of applications in the diagnostic imaging of CNS diseases and effective treatment through drug delivery systems. The further rapid development of pharmaceutical nanotechnology and the improvement of direct drug delivery to the brain that bypasses brain barriers promise a breakthrough in the near future in the treatment of many nervous system diseases, including inflammatory, autoimmune and mental diseases, as well as neurodegeneration and brain cancer.


A search was conducted in the Medical Literature Analysis and Retrieval System Online/PubMed and Virtual Health Library databases using the descriptors provided by Medical Subject Headings (MeSH) and Health Science Descriptors (DeCS).Bacteroides, Bifidobacteria, and Lactobacillus in the first days of life and more significant microbial variability in the following weeks. The microbiome of infants born via C-section is similar to the maternal skin and the hospital setting and less diverse, mainly composed of Staphylococcus, Streptococcus, and Clostridium.infants born vaginally presented a greater concentration of the maternal vaginal microbiota provides newborns with a greater variety of colonizing microorganisms responsible for boosting and preparing the immune system. Vaginal birth is the ideal birth route, and C-sections should only be performed when there are medical indications. The Brazilian obstetrical context is of concern and considered a public health problem due to various factors, including the C-section epidemic. According to the World Health Organization (WHO), the rate of C-sections should not exceed 15%--There is a higher risk of bleeding in the intra- and post-operative of C-sections-The route of birth delivery may also influence a newborn’s health. Children born via C-section are at increased risk of developing asthma, systemic connective tissue disorders, juvenile arthritis, inflammatory bowel disease, immune deficiencies, and leukemiaStudies suggest that the immune system of a newborn is widely stimulated when first exposed to microorganisms during neonatal lifeTherefore, we propose that the route of birth delivery influences the colonization of microorganisms in newborns. Nonetheless, studies seldom address the mechanisms involved in this adaptation according to the type of birth. This study’s findings are expected to support the choice of the type of birth, decreasing unnecessary C-sections without a medical indication. Fetal morbidity and mortality caused by C-sections and inappropriate adaptation of the neonatal immunological system are also expected to decrease.Therefore, this study’s objective was to analyze scientific evidence concerning the relationship between the route of birth delivery and the newborns’ acquisition of microbiota.This is an integrative review, which can be defined as a method capable of synthesizing scientific knowledge regarding a given problemThis study strictly followed the proposed stagesPICO, which stands for Patient, Intervention, Comparison, and Outcomes, was the strategy used to establish the guiding question. These four components are essential to establish the guiding question and, consequently, seek scientific evidenceTherefore, the topic chosen was the acquisition of microbiota among newborns according to the route of birth delivery. The guiding question was: What is the production of knowledge concerning the association between the acquisition of microbiota among newborns and the route of birth delivery? The first element of the strategy (P) refers to newborns; the second (I) to C-section; the third refers to vaginal delivery (C); and the fourth element (O) refers to newborns’ acquired microbiota.Inclusion criteria were: papers published in Portuguese, English, or Spanish; answering the guiding question; with up to ten years since publication. Exclusion criteria were letters, editorials, experts’ opinions, reviews, and studies addressing premature infants, or not comparing the route of birth nor addressing newborns.Índice Bibliográfico Español en Ciencias de la Salud (IBECS) , on August 10th, 2020. The following descriptors were used in the search strategy: ((“MICROBIOTA”) AND  OR (“CESAREAN SECTION”) OR (“PARTURITION”)) NOT ) in the PubMed and (tw:(MICROBIOTA)) AND (tw:(HUMANS)) AND ) AND NOT ) in the VHL.An advanced search was performed in the following electronic databases to select the studies for this review: Medical Literature Analysis and Retrieval System Online (MEDLINE) (via PubMed), Latin American and Caribbean Health Sciences Literature (LILACS), and The equivalent descriptors in Portuguese and Spanish were used in the VHL to identify Latin American studies, namely: (tw:(MICROBIOTA)) AND (tw:(HUMANOS)) AND (tw:(PARTO OR CESÁREA OR PARTO OBSTÉTRICO)) AND NOT (tw:(LEITE HUMANO OR LECHE HUMANA)).The descriptors used in the PubMed were selected from the Medical Subject Headings (MeSH terms), used for the efficient indexing of publication in the topicThe Preferred Reporting Items for Systematic Reviews and Meta-Analyses - PRISMA was adapted to guide this review’s reportAfterward, the full texts of the studies selected were considered in a new screening process. The researchers independently examined all the publications to choose the studies that met the inclusion criteria previously mentioned. Conflicts were resolved in a consensual meeting.The studies were then analyzed using an instrument to collect dataThe studies were also analyzed according to the level of evidence. In this study, the level of evidence was organized into seven levels: Level I - systematic review or meta-analysis of randomized controlled clinical trials; Level II - evidence obtained from at least one well-designed randomized controlled clinical trial; Level III - well-designed controlled trial without randomization; Level IV - evidence from well-designed case-control or cohort studies; Level V - systematic reviews of descriptive and qualitative studies; Level VI - evidence form a single descriptive or qualitative study; Level VII - evidence from the opinion of authorities and/or reports of expert committeesThe studies were tabulated and rigorously analyzed, interpreted, and synthesized. Two thematic categories were chosen according to the topics addressed in the studies to interpret the results.This study’s sample was composed of 25 studies. The entire process of searching, excluding, and selecting studies is described in detail in a flowchart .The year of publication among the studies selected ranged from 2010 and 2019: 32% of the studies were published in 2016, followed by 12% of the publications published in 2018, 2017, and 2015. Regarding the country of origin, the United States presented the highest number of studies (20%), followed by Sweden and China, with 16% each. All the papers were written in English, and the periodical with the highest number of publications was PloS One with three papers. Nature Medicine, Nat Commun, EBioMedicine, Journal of Allergy and Clinical Immunology, Journal of Pediatric Gastroenterology and Nutrition, and Scientific Reports published two papers each.Analysis of the studies revealed similar results and discussions. Hence, two analytical categories emerged from the studies’ analysis: microbiota and immune system and route of birth delivery and neonatal microbiota. Both are presented in the discussion section.,-A rise in the C-section rates, diet changes, the indiscriminate use of antibiotics, and antimicrobial agents have altered the natural microbial composition of the human body. These may disturb the balance of commensal organisms and alter their metabolic network, favoring the growth of potentially pathogenic constituents, negatively reflecting on the population’s healthTherefore, it is essential to understand the importance of the microbiota for the immune system and its relationship with the route of birth delivery and a newborn’s microbiota composition.,,,,-Studies show that microbiota is a crucial and active inducer of regulatory responses of the immune system,The neonatal period is considered a critical window in the development of the immunological system. Appropriate microbial stimulation is essential for the appropriate maturation of TReg cells response-,Additionally, the human organism’s microbiota limits the colonization of pathogens based on a competition for metabolites, a process known as “colonization resistance”Bifidobacterium and Lactobacillus are considered the ideal bacteria for the human body because they are commensal bacteria,,,Bifidobacterium between one week and three months of age was associated with a lower risk of developing eczema-Bifidobacterium and Lactobacillus seem to be related to the emergence of allergies,,Bifidobacterium are found in the long run among individuals with asthma, compared to recently diagnosed individualsBacteroides. Some of them help regulate gut immunity, and a deficiency of these bacteria was identified among infants born via C-section. This situation causes the rupture of tolerogenic response, contributing to inflammation and obesity,,Another component found in the microbiota of newborns is ,,These claims are explained based on the Old Friends theory. Commensal microorganisms train the immunological system to develop tolerance mechanisms as a strategy for their own survival. Hence, the immune system recognizes and eliminates harmful bacteria but does not react against useful species or the organism itself-,,Regarding the route of birth delivery and neonatal microbiota, the first influences the acquisition and colonization of bacteria in newborns’ bodies,-,-,,,,-,-,-,,,,,-,-,,Many of an infant’s body sites come into contact with the environment’s microbiota, and colonization occurs. The studies analyzed the neonatal microbial colonization in various body sites, and the gut was the site most frequently studied using fecal samplesBacteroides, Bifidobacterium, and Lactobacillus in the first days of life, and a greater microbial variability occurs in the following weeks,,,,-,,,Lactobacillus, Bifidobacterium, and Streptococcus-Lactobacillus target species. Hence, the composition of a pregnant woman’s vaginal microbiota is more stable, conferring a protective role against ascending infectionsInfants born vaginally have a higher concentration of ,,-Staphylococcus, Streptococcus, and Clostridium. Although, a longitudinal studyInfants born via C-section present a microbiome similar to the maternal skin and the hospital environmentStaphylococcus, Clostridium, Klebsiella, Enterobacter, and Enterococcus species were more frequently found among infants born via C-section. These bacteria are resistant to various antibiotics and are endemic in the hospital setting-,-,,The On the other hand, the effects of elective and emergency C-sections differ,,,The microbiota of healthy adults is stable and diversifiedDespite the systematized search and selection of studies, it is worth mentioning that it is impossible to exhaust the entire literature addressing this topic. Therefore, this study’s limitations include the search method, which was restricted to only two databases, the 10-year timeframe, and the fact that only studies written in Portuguese, English, or Spanish (the languages the authors are proficient) were selected.Notwithstanding its limitations, this study presents evidence of 25 studies, most of which were clinical trials and cohort studies. It clearly shows that the route of birth influences an infant’s microbiota, and it is assumed that it may compromise infants’ health, influencing the development of allergies, immunological/metabolic diseases, and obesity. Thus, this study contributes to the advancement of scientific knowledge and reinforces the importance of policies and practices intended to discourage C-sections worldwide, but especially in Brazil.Microbial colonization during the fetal period, intrapartum, and after birth is crucial in the host-microbial mutualism; the primary function is the maturation and development of newborns’ immune systems. The maternal vaginal microbiota provides newborns with a greater variety of colonizing microorganisms, responsible for training and adapting the infants’ immune system. Therefore, it is clear that vaginal delivery is ideal, and only in the presence of medical indications should a C-section be endeavored.Currently, the Brazilian obstetrical service presents high rates of C-sections. The implications of using this route of birth delivery involve increased fetal, neonatal, and maternal morbidity and mortality. In this sense, this study supports the choice of the route of birth delivery and, consequently, favors a decrease in unnecessary C-sections. It is crucial to provide information to pregnant women and/or couples and health workers to make informed and educated decisions.Research addressing the role of microbiota in human health and its association with the route of birth tends to increase, and further studies are crucial to understanding the complex interaction between one’s microbiota and the immune system while the route of birth delivery influences both. Thus, there are gaps concerning the mechanisms involved in the training and maturation of newborns’ immune systems and its implications in adulthood.


Heart rhythm disturbances caused by different etiologies may affect pediatric and adult patients with life-threatening consequences. When pharmacological therapy is ineffective in treating the disturbances, the implantation of electronic devices to control and/or restore normal heart pacing is a unique clinical management option. Although these artificial devices are life-saving, they display many limitations; not least, they do not have any capability to adapt to somatic growth or respond to neuroautonomic physiological changes. A biological pacemaker could offer a new clinical solution for restoring heart rhythms in the conditions of disorder in the cardiac conduction system. Several experimental approaches, such as cell-based, gene-based approaches, and the combination of both, for the generation of biological pacemakers are currently established and widely studied. Pacemaker bioengineering is also emerging as a technology to regenerate nodal tissues. This review analyzes and summarizes the strategies applied so far for the development of biological pacemakers, and discusses current translational challenges toward the first-in-human clinical application. All these membrane ion channels concur to the physiological automaticity of SAN, but a long debate is still going on the predominant one in controlling the spontaneous diastolic depolarization (DD)  and INaCa (intracellular Ca2+ clock) in the normal total pacemaker clock , and other cytotypes, i.e., the transitional cells. Each PC displays a clear cytoplasmic zone around a large, centrally located nucleus with little glycogen amount and randomly oriented myofibrils and small mitochondria. Proximity to and arrangement in clusters or grapes are mainly observed among PCs, but the interconnection is poor with the absence of intercalated disks and tight junctions James, . During sinus venosus as other cell types composing the node, i.e., fibroblasts and nerve cells.Around PC grape-like clusters, transitional cells distribute in the outer half part of SAN. Despite a similar elongated aspect to working myocardial cells, transitional elements are smaller in size, very interweaved, abundant in glycogen, less rich in myofibrils and mitochondria, distributed in an arranged fashion. Transitional cells are much higher in number than PCs and are responsible for transmitting the sinus impulse to the ordinary myocardium. These SAN cells have an embryonic origin in the An important component of SAN is its main artery, prevalently originating from the right coronary artery as its first branch. The SAN artery crosses the node centrally and is widely interconnected with the right and circumflex coronary tree, thus supplying blood to the atrium and the same node. A copious net of capillaries, arterioles, and venules supports the vascularization of the whole SAN.Due to its proximity and arterial connection to the aorta, SAN also exerts a sensor role for monitoring central aortic pressure and pulse, a servomechanism stabilizing pulse and impulse. Moreover, adrenergic and cholinergic nerves operate as further functional stabilizers in the beat-to-beat frame and in the longest time. In particular, the balanced autonomic innervation contributes to the stable performance of SAN in its postnatal maturity James, .AVN lies at the conjunction of the right atrium and the right ventricle. For this purpose, Tawara  describevia the right coronary artery in the right heart dominance case  pathway, and the route via the terminal crest connects to the slow pathway ; thus, the potential action transmission is highly controlled, and the atrial and ventricular excitation–contraction cycle is very coordinated also in the case of atrial fibrillation or tachycardia. AVN-generated automaticity is only subordinate to SAN and might impose only with the dysfunctional pacemaking of the latter , i.e., embryonic stem cells (ESCs) and induced PSCs (iPSCs), are considered the most promising stem cell types regarding their ability to differentiate into a virtually unlimited number of body cell types, including those derived from the cardiac lineage  are widely utilized in stem cell-based methodologies due to their ability to differentiate into spontaneously beating cardiomyocytes, which functionally express HCN channels  are finding larger applications for the generation of PCs-like cardiomyocytes. Modern protocols currently allow to effectively generate hiPSCs-derived cardiomyocytes with the typical cardiac physiological features: a variety of ion channels, specific contracting apparatus, excitation, propagation, etc. /bone morphogenetic proteins (BMP) signaling modulation, they observed that eGFP-negative cells were expectedly NKX2.5-negative but also expressed ISL1 and the posterior heart field marker podoplanin (PDPN) at high levels. This pattern of expression in these cardiac progenitors is typical of the native SAN during development. After further differentiation, these cells also showed a strong induction of TBX3, SHOX2, TBX18, and HCN4 and displayed the electrophysiological features of PCs. So-selected eGFP-negative, ISL1-positive, and NKX2.5-negative cardiac progenitors were demonstrated to be clonogenic and multipotent , chronotropic responses, and the ability to fire pacemaker action potentials and pace ventricular cardiomyocytes in vitro. After the transfer into the apex of rat hearts submitted to AV block, these SAN-like cardiomyocytes demonstrated pacing activity in the host tissue and were able to function as a biological pacemaker  and distinctive parameters  for the prospective of clinical transplantation  to prevent the automaticity suppression in guinea pig ventricular myocytes. Reduction in the number of inward rectifier potassium ion channels  in the myocardium by overexpressing a KIR2.1-dominant-negative construct (KIR2.1AAA) was adopted. The suppression of IK1 induced ventricular cardiomyocytes to depolarize spontaneously, thus producing pacemaker activity . Thus, the regulation of these specific miRs deserves more attention for possible pharmacological targeting in therapeutic strategies preventing the irreversible dysfunction of SAN.A locally enhanced chronotropic activity might potentially be achieved through the HCN overexpression in the subsidiary atrial pacemaker tissue, which is physiologically bradycardic but shares several characteristics with SAN, including the differential expression of TBX3, HCN1, Nav1.5, and Cx43 with respect to the right atrium. It was shown, in fact, that pacing could be accelerated by the localized HCN2 or HCN212 overexpression  or adenylyl cyclase (AC) genes , was recently demonstrated in bradycardia models. In particular, the lentiviral transduction of HCN2 and SkM1 was more efficient than their nucleofection-mediated gene transfer. Moreover, virally transduced cells survived better in vivo  were proved to express functional cardiac pacemaker HCN2 channels and induce spontaneous pacemaker activity, triggering the contraction of ventricle cardiomyocytes f current were also confirmed , is upregulated in the developing epicardium and injury-activated epicardial stem cells, giving rise, through the epithelial-mesenchymal transition, to the fibroblasts and smooth muscle cells of the coronary arteries  and induce the differentiation of SAN myocardium  and reliance on non-specialized components . Zhang et al. , representing less attractive options for the future clinical application in humans .Delivery constructs that target a specific cell type of the heart are not yet available, and vehiculated genes are, therefore, introduced at the site of injection where they are randomly inserted. It has been speculated that a non-targeted gene delivery might have a causative role in the unsustained activity at the AV level whether native, functional AVN PCs are the objects of the forced expression of channel proteins , and optical mapping of the epicardial electrical activity in perfused, excided hearts are often employed to analyze biopacing activity  or associated health risks . With a considerable level of technology implemented in these approaches, the manufacturing costs are particularly high, representing a critical barrier for distribution in both industrialized and non-industrialized countries. As for other advanced therapeutic medicinal hypotheses, the balancing between benefits and risks associated to any biopacemaking approach must be critically valued in healthcare management programs to prevent any harmful exposure of patients affected by the disturbances of the conduction system. Indeed, the possibility of clinically testing these hypotheses depends on the local sanitary legislation, as well as on government/federal funding allocated, and, finally, on the consent of the patient. In the cases where no available treatment options are suitable to (re)establish rhythm functionality as for fetal subjects with congenital AV block or adult patients with device-related infections  and tissue engineering strategies will also require additional investigation before reaching the clinical application: the acceptable level of reproducibility and long-term functioning is not yet achieved. Although the biological pacing created by gene transfer showed hitherto to be the most successful biopacemaking strategy in short-term preclinical evaluations, the application of tissue engineering principles is expected to keep its promises to overcome all the limitations shown by the other approaches to clinically replace electronic pacemakers.At the present moment, the best strategy for the first clinical testing of a biological pacemaker appears to be its combination with electronic devices  differentiate them. Nevertheless, each of them potentially could be safe and effective and more promising compared to artificial electronic devices, so far clinically applied. In summary, biological pacemakers are expected to improve and expand the spectrum of the therapeutic strategies for the treatment of disorders in the cardiac conduction system.We overviewed a significant progress in the field of biological pacemaker development in the last few years. We have every reason to believe that modern biological pacing technology will demonstrate its validity in preclinical investigation LI conceived the manuscript. NN and LI contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


The cause of the loss of basal forebrain cholinergic neurons (BFCNs) and their terminal synapses in the cerebral cortex and hippocampus in Alzheimer’s disease (AD) has provoked a decades-long controversy. The cholinergic phenotype of this neuronal system, involved in numerous cognitive mechanisms, is tightly dependent on the target-derived nerve growth factor (NGF). Consequently, the loss of BFCNs cholinergic phenotype in AD was initially suspected to be due to an NGF trophic failure. However, in AD there is a normal NGF synthesis and abundance of the NGF precursor (proNGF), therefore the NGF trophic failure hypothesis for the atrophy of BCNs was abandoned. In this review, we discuss the history of NGF-dependency of BFCNs and the atrophy of these neurons in Alzheimer’s disease (AD). Further to it, we propose that trophic factor failure explains the BFCNs atrophy in AD. We discuss evidence of the occurrence of a brain NGF metabolic pathway, the dysregulation of which, in AD explains the severe deficiency of NGF trophic support for the maintenance of BFCNs cholinergic phenotype. Finally, we revise recent evidence that the NGF metabolic dysregulation in AD pathology starts at preclinical stages. We also propose that the alteration of NGF metabolism-related markers in body fluids might assist in the AD preclinical diagnosis. This review deals with some general historical aspects regarding the fascinating discovery of nerve growth factor (NGF). It tells how this trophic factor was first found to be significant for the survival of basal forebrain cholinergic neurons (BFCNs) during developmental stages. It also relates how NGF was later established as responsible for the maintenance of their neuronal phenotype in the adult and fully differentiated central nervous system (CNS).For decades, it was assumed that mature NGF (mNGF) was generated intracellularly from its precursor molecule (proNGF) and released in an activity-dependent manner as the mature form. This assumption stemmed from studies performed in vitro and applying ELISA methods unable to distinguish mNGF from proNGF. However, investigations made ex vivo with cerebral cortex slices stimulated with carbachol, glutamate, or potassium chloride revealed only the activity-dependent release of proNGF (not mNGF) along with the release of a cluster of zymogens, convertases, and endogenous regulators composing a metabolic cascade responsible for the extracellular maturation and degradation of NGF. This metabolic pathway, discovered by us and referred to as the NGF metabolic cascade, is described in detail in this review along with its marked dysregulation in the Alzheimer’s disease (AD) pathology.We discuss here how this metabolic dysregulation results in reduced mNGF generation and its exacerbated degradation provoking a trophic void for the maintenance of highly NGF-dependent BFCNs. We also discuss the evidence of an early, preclinical, NGF metabolic dysregulation in the continuum of the AD pathology in sporadic cases as well as in Down syndrome (DS) with preclinical AD pathology.Finally, this review discusses the possible causative effect of CNS amyloid burden and inflammation for this AD-pathology-related NGF metabolic dysregulation. It also points to the potential value of studying levels of members of this metabolic cascade in body fluids  and plasma) to assist in the early detection of preclinical AD pathology.For decades, embryologists wondered if the growth of limbs was controlled by the nervous system or the reverse. Viktor Hamburger’s finding that the extirpation of the wing bud in chick embryos prevented the development of the CNS led him to advance the idea that “The wing stimulates the central nervous system by way of a reflex arc and that only its sensory fields are the acting parts. They send nervous excitations centripetally to the spinal ganglia” . This paThe purification of NGF in large amounts then allowed for the derivation of antibodies against NGF. With these tools in hand, Levi-Montalcini demonstrated that NGF is necessary for the development, growth, and maintenance of the sympathetic and sensory systems, thus establishing NGF as the first neurotrophic factor to be identified ,12,13, aIn the 1980s, it was demonstrated that NGF also acts on the development of cholinergic neurons of the rat CNS where it increases the activity of choline acetyltransferase (ChAT), the enzyme responsible for the generation of acetylcholine, both in vitro and in vivo ,26,27,28Importantly, the application of NGF and of anti-NGF antibodies in vitro indicated that the embryonic survival and development of BFCNs are highly dependent on the expression of both NGF and its receptors tropomyosin receptor kinase A (TrkA) and p75NTR ,38, furtThat NGF is critical for the survival and maintenance of the phenotype of specific subsets of peripheral neurons and basal forebrain cholinergic nuclei during development and maturation is quite clear. The idea that NGF is essential for the survival of BFCNs is largely based on the in vitro studies cited above.adult CNS. The selective uptake and retrograde transport of NGF from the dorsal hippocampus to the adult forebrain cholinergic system was first shown by Schwab and collaborators [There is also strong evidence showing that NGF acts as a neurotrophic factor for cholinergic neurons of the borators . Howeverborators ,41,42.The concept that NGF remains as a “survival” factor in the adult brain emerged from the protective effects of exogenously administered NGF in models of axotomy of the fimbria fornix which disconnects the cholinergic neurons of the medial septum from the hippocampus, where the target-derived neurotrophin is largely produced. Such axonal transection results in the disappearance of ChAT-immunoreactive neurons of the medial septum. This neuronal loss can be prevented by the intraventricular application of exogenous mature NGF protein and was therefore interpreted as an NGF-dependent survival mechanism ,44,45. TThe above sequence of investigations strongly supports the concept that diminished NGF trophic support does not provoke cholinergic neuronal cell death but rather progressive retrograde atrophy of these neurons ,48,49. TAs discussed above, NGF supply appears to have a definitive effect on the survival of BFCNs in in vitro conditions and during development. A strong argument for the critical role of NGF in the developmental survival of these neurons has been provided by Chen and collaborators  as the lexogenous NGF in AD, a subject of continued interest and discussed in authoritative reviews [endogenous NGF should be a more promising avenue. The existence of a CNS metabolic pathway regulating the available mature NGF, as discussed below, might offer hitherto unknown therapeutic possibilities. This objective is also substantiated by the evidence that the trophic support offered by minute amounts of endogenous mature NGF is sufficient to regulate the day-to-day number of NGF-sensitive cholinergic synapses in the cerebral cortex [The efficacy of exogenous NGF in recovering lesion-induced atrophy of BFCNs has been strongly demonstrated by many laboratories, as reviewed in ,71,72,73 reviews ,75,76. Ol cortex . A findil cortex ; fundameMuch of the interest in the neurobiology of trophic maintenance of BFCNs has been provoked by the well-established notion that this system plays a fundamental role in higher CNS functions and memory , and subAltogether, the above studies led to the development and application of anticholinesterases in AD, which for decades have remained the most widely applied strategy for symptomatic treatment ,85,86. HA new investigative paradigm regarding the NGF trophic support of BFCNs is presently available with the discovery of a metabolic pathway controlling the availability of mNGF as well as its extracellular degradation  Figure . The disIn brief, mNGF is produced transiently in the synaptic cleft via an NGF metabolic cascade in which proNGF, zymogens, convertases, and endogenous regulators are co-secreted from BFCN-target neurons in an activity-dependent manner . Upon thOur lab has validated this pathway pharmacologically, demonstrating that applying neuroserpin or blocking plasmin activity compromises the conversion of proNGF to mature NGF leading (as in the AD pathology) to increased proNGF brain levels, atrophy of BFCNs, and loss of cholinergic synapses ,102,103.That hypothesis is supported by the finding that an NGF metabolic dysregulation is indeed present in the AD pathology even at preclinical stages as demonstrated in the analysis of post-mortem brain tissue, plasma, and CSF ,105,106.We have shown that the postmortem frontal cortex tissue from AD patients has notably increased protein levels of neuroserpin, the endogenous tPA inhibitor, leading to decreased tPA protein levels and activity resulting in lower plasmin brain content ,106,109.The AD NGF metabolic dysregulation is a double-edged sword, as not only is NGF maturation compromised, but also the degradation of the already diminished mNGF is intensified, due to a substantial elevation of MMP-9 and MMP-3 activity ,104,110.https://www.nia.nih.gov/research/abc-ds, accessed on 2 December 2021) [https://santpaumemoryunit.com/alzheimer-down-unit/, accessed on 2 December 2021) and in longitudinal clinical studies [A similar imbalance of the NGF pathway has been evidenced in the brains of individuals with DS at preclinical AD stages ,113,114.er 2021)  and the  studies ,124.In DS brains, proNGF accumulation is accompanied by increases in neuroserpin protein levels, TIMP1 transcripts and MMP-9 activity as well as by decreases in plasminogen protein levels and in tPA transcripts ,113,114.The NGF metabolic dysregulation has also been replicated in the brain of transgenic rodent models of the human AD-like amyloid pathology ,129,130 In consequence, we hypothesize that the NGF metabolic dysregulation in the continuum of the AD pathology, resulting in the abrogation of trophic support to the NGF-dependent BFCNs, is ultimately the cause of their well-established atrophy in this neurodegenerative condition. We further postulate that the NGF trophic failure leading to a progressive degeneration of BFCNs makes them vulnerable to tau pathology ,140. TheThe consequences of a compromised NGF metabolic pathway on BFCNs in the AD pathology are schematically represented in The NGF dysmetabolism in the AD pathology is likely to be provoked by progressive Aβ burden and other pathological factors of which CNS inflammation is an important candidate . The tigThe widescale acknowledgment that the neuropathological progression of AD begins decades before clinical symptom onset, and that the neurological damage present at this point is likely irreversible ,160,161,As high levels of proNGF and neuroserpin have been found in both CSF and plasma, reflecting the brain’s NGF metabolic dysfunction ,162,163,In this review, we highlight the existence of an entire CNS metabolic pathway explaining the activity-dependent release of proNGF along with a cluster of molecules which, in a highly coordinated fashion, provoke the conversion of the NGF precursor molecule to its mature and trophic active form in the extracellular space, in proximity to their cognate receptors located in cholinergic synaptic terminals. The mNGF rapidly binds to these receptors to be internalized and retrogradely transported to the neuronal soma to fulfill its trophic functions. The function of the remnant unbound mNGF in the extracellular space is terminated by the proteolytic degradation effected by matrix metalloproteases.The AD pathology impairs proNGF to mNGF conversion and facilitates mNGF degradation. This compromised NGF trophic support to BFCNs explains the well-known atrophy of these neurons in AD and it is already present at AD preclinical stages. An aspect in line with the remarkable advances in brain imaging revealing that the atrophy of the BFCN area starts at preclinical stages of the continuum of this neurodegenerative condition.We also pose the possible contribution of alterations of body fluids biomarkers of the NGF metabolic dysregulation to signal preclinical stages of AD.Finally, it is conceivable that pharmacological corrections of the NGF metabolic pathway could assist in preserving the trophic support of BFCNS in silent AD stages and thus prevent the atrophy of these neurons and losses of cortical and hippocampal cholinergic synapses, therefore improving cognitive outcomes.


The risk factors affecting workers' unsafe acts were comprehensively identified by Human Factors Analysis and Classification System (HFACS) and grounded theory based on interview data and accident reports from deep coal mines. Firstly, we collected accident case and field interview data from deep coal mines issued by authoritative institutions. Then, the data were coded according to grounded theory to obtain relevant concepts and types. The HFACS model was used to classify the concepts and categories. Finally, the relationship between core and secondary categories was sorted out by applying a story plot. The results show that risk factors of unsafe acts of deep coal mine workers include environmental factors, organizational influence, unsafe supervision and unsafe state of miners, and the main manifestations of unsafe acts are errors and violations. Among them, the unsafe state of miners is the intermediate variable, and other factors indirectly affect risky actions of coal miners through unsafe sates. Resource management, organizational processes and failure to correct problems are the top three risk factors that occur more frequently in unsafe acts. The three most common types of unsafe act are unreasonable labor organization, failure to enforce rules, and inadequate technical specifications. By combining grounded theory and the HFACS framework to analyze data, risk factors for deep coal miners can be quickly identified, and more precise and comprehensive conceptual models of risk factors in unsafe acts of deep coal miners can be obtained. As one of the most important global energy sources, coal plays a vital role in the world's energy structure. Still, the frequent occurrence of coal mining accidents dramatically threatens the safety of coal production and workers' lives. Studies have shown that more than 90% of coal mine accidents are caused by human factors. These factors often manifest in various unsafe behaviors of people , 2. At tRecently, scholars have carried out studies on the influencing factors of coal miners' unsafe acts. For example, Kapp et al.  studied Previous research has been enlightening that the study on the risk factors of deep coal mine workers' unsafe act needs to be carried out from many aspects, such as individual workers, organization, management, and environment. The risk factors affecting the unsafe act of deep coal miners are complex and diverse, and few scholars systematically analyze them from multiple levels. Although some scholars have used the HFAC model to analyze the risk factors of the unsafe act of miners, the risk factors are not comprehensive, and there is a lack of analysis of the unsafe state of workers , 12. TheBased on the above analysis, the purpose of this study is to use grounded theory and the HFACS model to analyze the interview data of deep coal mine workers and coal mine accident reports to identify the risk factors affecting workers' unsafe acts. The HFACS model combined with grounded theory can accurately identify the human factors in coal mine accidents and find out the causes of hazardous behaviors of coal miners. It is hoped that this study can improve the depth and breadth of accident analysis methods of deep coal mines, clarify the causes of unsafe acts of deep coal miners, and provide a theoretical basis for formulating intervention strategies. The Improved HFACS-CM model constructed in this study can point out various risk factors of unsafe acts of deep coal miners and point out the interaction of factors at different levels. It can also point out how various factors of human factors ultimately lead to coal mine accidents. The research results of this paper enrich the research methods of unsafe behavior of deep coal miners and lay a particular theoretical foundation for future scholars in this field. Since the research of this paper is based on the accident report of deep coal mines and the interview of deep coal mine workers, the research results of this paper have more practical guiding value for the prevention and control of unsafe acts of deep coal mine workers. Based on the Improved HFACS-CM model constructed in this study, coal mine safety managers can deeply understand the causes of workers' unsafe acts and how human factors lead to coal mine accidents. Based on this, they can establish more effective measures to prevent and control workers' unsafe acts to reduce the occurrence of workers' unsafe acts and coal mine accidents.Shappell and Wiegmann established the HFACS model based on the Swiss cheese model . This moIn the original HFACS model, human error is divided into four levels . From hiThe first layer is organizational influence, which refers to the weak organizational framework, defects, lack of team culture, and other factors, laying a foundation for accidents. This level is divided into three subcategories: resource management, organizational climate, and organizational processes. Resource management refers to misguided decisions made by an enterprise in terms of workforce, equipment, and investment, such as improper staffing, lack of education and training, and insufficient equipment and investment. Organizational climate refers to the working environment of an organization, which is defined as the internal factors that affect individual performance, such as teamwork and communication. Finally, organizational processes include internal administrative factors such as formal processes, methods, and oversight, for example, work commitments, reward and punishment systems, and safety procedures.The second layer is unsafe supervision, which refers to poor operation habits, worker process mistakes, and behaviors that violate rules set by the appropriate supervision and management system. This layer primarily includes inadequate supervision, planned inappropriate operations, failed to correct problems, and supervision violations. Inadequate supervision refers to the failure of work and performance supervision of workers. Planned inappropriate operations are defects in the pre-set operation plan, such as unreasonable labor organization and ineffective implementation of risk response measures. Failure to correct problems refers to management personnel's negligence of effective preventive measures against the improper operation of workers, equipment defects, management errors, and other issues. Finally, supervision violations refer to workers' behaviors who disregard the supervision and management systems, for example, allowing unqualified people to work in high-risk mines.The third layer is the precondition for unsafe acts, which describes the psychological and physiological states and operational skills that cause hazardous actions. This level includes three sub-categories: environmental factors, conditions of operators, and personnel factors. Environmental factors refer to the factors that adversely affect worker productivity, including the physical and technical environments. Physical environment refers to physical factors such as temperature, noise, and lighting. Technical environment refers to worker performance factors, including equipment, mining design, safety monitoring systems, among others. Operator conditions refer to psychological and physiological states that negatively affect the individual performance of workers. Finally, personnel factors are bad decisions due to a lack of coordination among team members and inadequate personal preparation, for example, a lack of safety equipment.The fourth layer is unsafe acts, which refers to personally risky actions. There are two types of unsafe acts: errors and violations. Errors refer to unintentional actions, divided into three types: decision, skill, and perceptual errors. Violations refer to intentional disregard of rules and regulations, divided into regular and exceptional types. Regular or routine violations are those tolerated by managers, such as eliminating work steps, risky operations, and disregarding rules and regulations. Exceptional violations refer to those that occur under particular circumstances. These violations are unusual because they occur outside regulations and laws, such as the production organization during the COVID-19 (Corona Virus Disease 2019) pandemic.Although the original HFACS model factors are an excellent fit, some of them do not fully apply to components of risk associated with unsafe acts of deep coal miners. The model should be improved according to the characteristics of the mining industry and the specific accident site situation , 28. TheIn deep coal mining, physical conditions, such as geological structure, temperature, humidity, and other environmental characteristics, significantly influence workers' unsafe behaviors. In addition, with the continuous progress of mining technology and the rapid development of society, coal miners' behaviors are not only affected by internal factors and the external environment, such as information technology, economy, and politics. Therefore, in this study, the environmental factors included in the preconditions of unsafe acts in the original model are moved to the first layer. The new level (Level 1) consists of the physical, technical, and policy environments, which are summarized as environmental factors. In real-world coal mine production, the premise of unsafe acts in the original model is changed to the unsafe state of miners, including workers' mental and physiological states and business ability. Achieving coal mine production objectives requires cooperation and communication between team members. Therefore, this study adds teamwork and communication to the original HFACS model and classifies them within the organizational climate. The improved HFACS-CM model is shown in In the improved HFACS-CM model, the causes of accidents in deep coal mines are divided into five levels, including environmental factors, organizational influence, unsafe supervision, unsafe state of miners, and the unsafe acts of miners. Compared with the traditional HFACS model, the new model takes environmental factors as the first level and changes the precondition for the unsafe acts to the unsafe state of miners according to the real-world situation. According to the types of hazardous acts in the improved HFACS-CM model  and the Grounded theory is a bottom-up analytical method based on experience and materials, which focuses on the treatment of problem situations and can produce solutions to problems , 29. ThiThe analytical steps of grounded theory mainly include data acquisition, open, axial, and selective coding and model saturation testing, as shown in As shown in HFACS model and grounded theory are used together in this study to construct a conceptual model of risk factors for the unsafe acts of deep coal miners. First, based on grounded theory, we fully extracted coal miner workers' risk factors and types of hazardous actions of coal mine workers from coal mine accident reports and workers' interviews. Next, the factors were mapped according to the improved HFACS-CM model. Finally, the conceptual model of the risk factors of unsafe acts of deep coal mine workers was formed. In the process of extracting as many risk factors and types of hazardous acts of workers as possible, this study also analyzes the action path of these factors on unsafe acts and coal mine accidents.The range of data in grounded theory is so broad that everything is data . In otheBased on previous studies, this study designed an interview outline for risk factors of unsafe acts of deep coal mine workers from three aspects: influencing factors, unsafe behaviors, and their results, as shown in The sociodemographic characteristics of respondents in this study are shown in In grounded theory, original data is crucial for establishing new theoretical systems, and the information released by authoritative institutions is more reliable and accurate. Therefore, the coal mine accident report issued by China Coal Mine Safety Production Network is selected as the original data. This website has accumulated a large number of coal mine accident cases. These data contain detailed process descriptions and cause analysis of accidents, which apply to grounded theory analysis. Furthermore, the accident investigation report can reveal the cause of the accident in detail, the fault in the risk management process of the coal mine enterprise, and the defects of various constructive documents. Therefore, this paper uses accident investigation reports as the original data of grounded theory. Only the deep coal mine accident report with a mining depth over 600 m is selected in selecting coal mine accident reports. After screening, 40 accident reports from 2016 to 2021 are selected. The accident types mainly include ten major accident types, including rock-burst accidents, coal seam explosion accidents, water disasters, collapse accidents, mechanical and electrical accidents, coal and gas outburst accidents, roof accidents, and so on, as shown in Open coding requires researchers to conduct an in-depth analysis of the original data with an open mind and code the data according to the state of the data. Open coding is divided into two steps: conceptual analysis and classification analysis. The first step of conceptual analysis is to conceptualize the original data by disassembling the data and using more refined sentences to express the meaning of the original sentence. In this step, similar and identical statements in the original material are combined, some direct and vague statements are deleted, and only the statements related to the topic are retained. The second step of classification analysis is based on the core theme of risk factors of unsafe acts of deep coal mine workers, classifying and analyzing the types and risk factors of unsafe acts caused by interview data and accident reports sentence by sentence. This study combines the method of expert consultation with the technique of personal coding, reducing the number of personal subjective opinions. With the help of the expertise and experience of experts in the field, the subjectivity of coding is reduced. Nvivo 11 software is used to improve the scientific nature of the coding process. The coding materials were derived from 32 accident reports and 20 interview records. Through conceptual analysis and repeated arrangement and adjustment of original data, 327 concepts and 52 categories were abstracted. Then the similar categories of the 52 categories were combined to get 32 categories. This study obtained 52 secondary and 32 primary categories after open coding of the original data, as shown in After open coding, we identified the main concept categories from 32 unsafe acts categories. The correlations between the main concept categories were established. These are the key content of axial coding. The process of axial coding further analyzed causal relationships and logical connections between categories obtained in open coding, and concepts that cannot be grouped with other concepts are deleted. Through the repeated comparisons and systematic clustering of 32 categories, 15 main categories were obtained, as shown in the third column of Selective coding sorts out the relationships among the main categories, abstracts the core categories that summarize all categories and clarifies the relations between the core categories and sub-categories using a storyline. The core category is the thread of a fishnet, connecting all other categories. It serves as an outline. Through selective coding, five core categories of environmental factors, organizational influence, unsafe supervision, unsafe state of miners, and unsafe acts of miners were obtained, as shown in the fifth column of The storyline categorizes the risk factors influencing the unsafe actions of deep coal miners into four core categories: organizational influence, unsafe supervision, unsafe state, and environmental factors, which significantly impact the unsafe actions of miners. After analysis, the following storyline was be obtained. On the one hand, with the recent depletion of shallow coal mines, the depth of deep coal mines is increasing. The complex and changeable mine environment makes the mining of coal more difficult. It also makes supervision and administration of management departments difficult. Inadequate management of miners occurs due to the lack of oversight provided by management departments, weak risk awareness, imperfect supervision systems, and other reasons. For example, due to the unreasonable staffing of the management department, appropriate department leaders are frequently transferred to other positions. As a result, leaders are not focused and unwilling to manage the front-line miners, which may increase the likelihood of errors, violations, and other unsafe outcomes. On the other hand, the complex deep mine environment requires coal mining enterprises to increase investment in industrial processes, such as introducing advanced equipment, advanced technology, and hiring high-end talent. However, due to the lack of safety investment, the miners' skills and psychological quality will be significantly tested, greatly impacting worker performance and safety-related behaviors. Environmental factors affect organizational factors, organizational factors affect unsafe supervision, and unsafe supervision affects the worker conditions, thus affecting the workers' unsafe actions. The five core categories above dominate the other categories. They are ultimately summed up as a storyline that describes the risk formation process of unsafe behaviors of deep coal miners.A conceptual model of risk factors for unsafe acts of deep coal mine workers is constructed by summarizing the coding results of accident reports and interview records, including five core categories and 32 sub-categories of factors, as shown in The improved HFACS-CM model has similarities with many published HFACS models, but there are also some differences. First of all, the classification and description of the original HFACS model are relatively general. In contrast, the improved HFACS-CM model describes the risk factors of workers' unsafe acts at different levels according to the characteristics of the mining industry. Secondly, the hierarchy of human factors in the original HFACS model is divided. Still, the primary and secondary factors and the causal relationship between them cannot be identified. The improved HFACS-CM model elucidated the primary and secondary factors and the causal connection. Finally, the enhanced HFACS model analyzes human errors and violations at all system levels. This study has obtained a conceptual model of the risk factors of the unsafe act of workers in the deep coal mine, which has a more apparent conceptual relationship and hierarchical relationship.A saturation test is needed to ensure the reliability and integrity of the conceptual model. If no new categories and logical relations appear in the original conceptual model by adding new data, the initial conceptual model is considered theoretically saturated. Otherwise, data collection must continue. The study's saturation test was performed 2 weeks after we formed the theoretical model. The saturation test was carried out by another member of the research group to eliminate the subjective influence of the researchers. The test data were eight previously prepared deep-coal mine accident reports. The test results showed that there were no new concepts. Furthermore, logical relations appear after the tests of the three coding processes of the grounded theory, indicating that the theoretical model previously constructed was saturated and there was no need to add data for analysis.Through statistical analysis of accident reports in deep coal mines, we obtained the frequency of risk factors of workers' unsafe acts, as shown in According to the improved HFACS-CM model, the unsafe acts of deep coal mine workers are classified. These unsafe acts can be divided into five categories. They are Decision errors, Skill-based errors, Perceptual errors, Routine violations, and Exceptional violations. Decision errors include Adventure Work; Skill-based errors include Imperfect Technical Specifications and Lack of Protection; Perceptual errors include Lack of Safety Awareness, Lack of Concentration, Fluke Mind, and Weak Sense of Responsibility. Routine violations include Unreasonable Staffing, Unreasonable Labor Organization, and Failure to Enforce Rules; Exceptional violations include Poor Risk Identification and Assessment Ability and Poor Risk Management ability. Through statistical analysis of accident reports in deep coal mines, the frequency of various unsafe acts is shown in Based on the HFACS model and grounded theory, we established the improved HFACS-CM model. It was mapped by analyzing deep coal mine workers' accident reports and interview records. Finally, the conceptual model of risk factors for the unsafe acts of deep coal mine workers was established. Based on grounded theory, we concluded that risk factors affecting the unsafe actions of deep coal miners are mainly environmental factors, organizational influence, unsafe supervision, and unsafe state of miners. Among these factors, workers' unsafe state is direct, all others are indirect. The technique used in this study identified risk factors and systematically analyzed them.The contributions of this study to coal mining safety include the following:This research object of the study is deep coal mine workers. Our findings guide effectively addressing their unsafe behaviors. The unsafe acts of deep coal miners is the direct factor leading to coal mine accidents. However, previous research focused more on the miners' errors , 33, whiIn this study, the HFACS model and grounded theory were used comprehensively, enriching the identification methods of risk factors for unsafe acts of miners and revealing the formation mechanisms of hazardous behaviors. The factors influencing coal mine workers' unsafe acts are complex and varied. Previous studies focused on unilateral factors such as fatigue, physical condition, and organizational defects –5, 24. IThis study provides a more profound and broader analysis of deep coal mine accidents. Although previous studies also statistically analyzed causes of accidents in coal mines from a human perspective , 36, theThe results provide valuable information for coal mine safety management practices.Coal mine safety managers should pay attention to the complex factors contributing to workers' unsafe acts. They should work to reduce the occurrence of coal mine workers' unsafe behaviors from a systems perspective. The administration department of coal mine safety should take measures to intervene in the organizational factors that influence workers' behavior and in supervisory factors that impact working conditions. At the same time, managers should also consider the influence of these factors on workers' unsafe actions through their unsafe state. The conceptual model of risk factors constructed in this study shows that environmental factors, organizational factors, supervision factors, and workers' unsafe state interact, ultimately resulting in workers' unsafe actions. The coal mine safety managers can create safer conditions for workers and reduce the possibility of risky behaviors by improving the underground working environment, strengthening the organizational management, and enhancing supervision inside the mine.Resource management for deep coal miners should be the critical content of unsafe acts intervention. It includes safety education and training, personnel allocation, and safety investment. Safety education and training often appeared among the risk factors of unsafe acts. For example, when education and training are incomplete, workers lack safety awareness and knowledge, which will affect workers' ability to identify and respond to risks , 8. TherSupervision and preventative management of unsafe actions of coal miners also need to be strengthened. Based on the conceptual model of risk factors, Managers of coal mines can take the following measures to improve the supervision of workers' unsafe acts. First, strengthen the education and training of managers to let them timely guide miners' work and correct their unsafe acts. Second, improve legal standards and operating procedures to help workers reduce errors and violations. More detailed and comprehensive operational practices can help workers reduce the number of unsafe acts. More comprehensive rules and regulations would make it easier for managers to regulate miners' acts and reduce workers' violations. Finally, strengthen safety education and training for front-line miners, especially safety and professional knowledge. Rich professional knowledge and safety knowledge can enhance miners' safety awareness and professional skills and reduce the occurrence of safety acts.Unreasonable labor organization and failure to enforce rules are the core risk factors of unsafe acts of deep coal mine workers. In the process of intervening in unsafe acts of miners, the manager should emphasize controlling these two factors. Reasonable labor organization is the result of making good operation plans. Therefore, coal mining enterprises need to introduce professional management personnel. Experienced managers can develop scientific, reasonable work plans but efficiently find loopholes in the management process, effectively preventing unsafe worker behavior. Managers of coal mines can adopt intelligent devices to monitor compliance on the job for those who fail to enforce rules. For example, Managers can use wearable devices to monitor workers' physical and psychological indicators to judge their condition.In this study, the HFACS model and grounded theory were used to analyze interview records and case reports of deep coal mine accidents to identify risk factors and manifestations of workers' unsafe actions. This comprehensive risk identification method can accurately find the factors implicit in the text data, which can investigate and analyze the risky behaviors of deep coal miners. The risk factor conceptual model constructed accurately describes the causes of unsafe acts of miners and provides a theoretical basis for safety improvements. Scholars can also apply the research methods in this paper to other fields to identify risk factors for workers' unsafe actions, such as crews and pilots.There may be some limitations to this study. First, the conceptual model of risk factors constructed in this study was obtained by qualitative methods, and the influence of each factor on the unsafe act was not quantitatively analyzed. Based on this conceptual model, subsequent studies can quantitatively analyze the impact of various factors on workers' unsafe acts. In addition, the analysis process of grounded theory used in this paper was carried out manually with the help of Nvivo software. However, the amount of data collected and summarized manually is limited. Conducting manual analysis on large amounts of data is difficult. In the future, it should be considered to introduce data mining technology into the grounded theory to achieve rapid acquisition of crucial information from massive data and combine it with research content for coding.This study provides a valuable basis and enlightenment for coal mine enterprises to supervise and manage unsafe behaviors of employees in practice and has practical guiding significance. Combined with occupational health-related laws and regulations and ISO 45001:2018, this paper puts forward some suggestions on the supervision and management of coal mine workers' unsafe behaviors from two aspects of control measures and supervision and management.In terms of measures to control workers' unsafe behaviors, the risk factor identification model of coal mine workers' unsafe behaviors constructed in this paper can help managers of coal mine enterprises better understand the factors leading to coal mine workers' unsafe behaviors. According to the risk identification model, managers of coal mine enterprises can take measures to improve the unsafe situation of workers from three aspects: environmental factors, organizational influence, and organizational supervision, to reduce the occurrence of unsafe behavior. Improve the working environment for workers. Managers constantly improve and optimize the operating environment to eliminate and reduce workers' adverse psychological and physiological reactions caused by the negative environment. A good environment can make the operator work happily to avoid unsafe behavior. Strengthen safety education and training and enrich workers with safety knowledge. Safety education and training can continuously improve workers' safety awareness and skill quality to improve the overall quality of workers and reduce the occurrence of unsafe behaviors. Strengthen safety culture and create an excellent safe atmosphere to infect staff. Good safety culture and atmosphere can help workers establish safety concepts, make workers take the initiative to abide by rules and regulations and laws and regulations from the ideological point of view, and reduce unsafe behavior. Enterprises strengthen communication within the organization. Good communication within the organization can help employees solve various problems encountered in the work process to maintain a stable working mood and avoid unsafe behavior. Strengthen supervision and strictly restrict workers' behavior. Adequate on-site safety supervision is an essential means to standardize workers' safety behavior and ensure the implementation of safety systems and measures. Strict supervision and management of workers' safety behavior is the most direct way of effectively eliminating all kinds of unsafe behavior. Enterprises strengthen reward and punishment mechanisms. A perfect safety reward and punishment mechanism can timely punish workers' unsafe behaviors and effectively reward workers' outstanding performance, promoting workers to take the initiative to reduce the occurrence of unsafe behaviors.The managers of coal mining enterprises can take the following measures to supervise and control the unsafe acts of coal miners. Establish and improve the mechanism for discovering, reporting, and appealing to workers' unsafe behaviors. Improve the education and evaluation system for workers with unsafe behaviors, and carry out professional intervention for workers with unsafe behaviors. Establish a system of returning and visiting workers who have performed unsafe behaviors to avoid the recurrence of unsafe behaviors. Conduct statistical analysis on various unsafe behaviors of workers to analyze the causes of unsafe behaviors and formulate effective control measures. Perfect the accountability system of workers' unsafe behavior to reasonably punish workers who have shown unsafe behavior.102455019@qq.com.The data analyzed in this study is subject to the following licenses/restrictions: Raw data can only be used by journal editors and reviewers, not publicly by others. Requests to access these datasets should be directed to Xue Wang, XW designed and conceptualized the study, and wrote the manuscript. LY supervised the project and obtained funding. XW, JZ, and ZQ downloaded the related papers and also obtained funding. All authors participated in screening the articles and provided critical feedback, significantly contributed to the study, and approved the final manuscript.This study was supported by the National Natural Science Foundation of China under the grant (No. 71971003), the Major of National Social Science Foundation of China (No. 20ZDA084), and the Graduate Innovation Fund Project (No. 2020CX1009).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.