The increasing global burden of cardiometabolic diseases highlights the urgent clinical need for better personalized prediction and intervention strategies. Early diagnosis and prevention could greatly reduce the enormous socio-economic burden posed by these states. Plasma lipids including total cholesterol, triglycerides, HDL-C, and LDL-C have been at the center stage of the prediction and prevention strategies for cardiovascular disease; however, the bulk of cardiovascular disease events cannot be explained sufficiently by these lipid parameters. The shift from traditional serum lipid measurements that are poorly descriptive of the total serum lipidomic profile to comprehensive lipid profiling is an urgent need, since a wealth of metabolic information is currently underutilized in the clinical setting. The tremendous advances in the field of lipidomics in the last two decades has facilitated the research efforts to unravel the lipid dysregulation in cardiometabolic diseases, enabling the understanding of the underlying pathophysiological mechanisms and identification of predictive biomarkers beyond traditional lipids. This review presents an overview of the application of lipidomics in the study of serum lipoproteins in cardiometabolic diseases. Integrating the emerging multiomics with lipidomics holds great potential in moving toward this goal. Human plasma is estimated to consist of thousands of functionally and chemically diverse molecular lipid species , which aA cluster of metabolic diseases is triggered by abnormalities in lipid metabolism such as dyslipidemia and obesity, culminating in the metabolic syndrome (MetS) and, furthermore, to prevalent chronic diseases including insulin-resistance states, type 2 diabetes (T2D), atherosclerosis, and cardiovascular disease (CVD). Both genetic and environmental factors  are the cause of these lipid-related cardiometabolic diseases and they may exist long before becoming clinically apparent. Dysregulated lipid metabolism is recognized as an established risk factor in CVD. However, it is still largely unknown how and which lipid molecular species affect the risk of CVD and potentially improve CVD prediction in addition to the traditional serum lipid biomarkers [Comprehensive lipid profiling, or lipidomics, defines the chemical lipid phenotype of biological matrices; as such, it has a unique potential to unravel and elucidate the underlying aberrant molecular lipid-related pathways associated with the incidence, severity, and progression of cardiometabolic diseases ,4. AccorIn this review, the first section focuses on the existing conventional serum lipid biomarkers used for the diagnosis of lipid disorders, monitoring the response of patients to lipid-lowering therapy and also for the assessment of CVD risk and the need for the shift to the comprehensive lipid profiling. This is followed by the detailed description of the steps comprising the lipidomics workflow, and finally, the last part of the review emphasizes the disturbances occurring in lipid composition of serum lipoproteins in cardiometabolic diseases.Disturbances in lipid metabolism play a central role in the onset and progression of cardiometabolic diseases ,6,7. In Since the landmark publications from the Framingham study , the afoSince human plasma consists of thousands of functionally and chemically diverse molecular lipid species , the afowww.lipidmaps.org, accessed on 1 December 2022). The International Lipid Classification and Nomenclature Committee under the sponsorship of the LIPID MAPS Consortium established a “Comprehensive Classification System for Lipids” based on well-defined chemical and biochemical principles together with ontology [www.lipidmaps.org).Lipidomics is considered to be a branch of metabolomics, to which it is very closely related and aims to the full characterization of lipid molecular species and of their biological roles with respect to expression of proteins involved in lipid metabolism and function, including gene regulation. The term lipidomics was used for the first time by Kishimoto et al. in 2001 . Two yeaontology ,13. Thisontology   and multivariate analysis, either in an unsupervised ) or supervised ) manner. The last step of the lipidomics workflow is the data interpretation, the identification of lipid biomarkers and aberrant lipid-related metabolic pathways. For that, available software tools and databases including the Kyoto Encyclopedia of Genes and Genomes (KEGG), LIPID MAPS, and MetaboAnalyst, which further enable pathway and enrichment analysis, lipid mapping, and visualization, are widely used in applications of lipidomics [Regardless of the analytical approach chosen for the detection and characterization of lipid molecules, a broad range of statistical analyses can be performed to determine potential differences between “healthy” and diseased groups or between groups at different disease stages, including univariate  or highly complex biofluids  and are collected from healthy individuals and/or patients . Each ofThe optimal protocol for the collection and storage of samples depends on multiple parameters, including the type of sample , the physicochemical properties of the compounds of interest, etc. For example, the preparation procedure methods for solid samples are more labor intensive than those for biofluids because additional steps, such as sample homogenization, prior to lipid extraction are required. In contrast, those for biofluids tend to be more straightforward. The most clinical lipidomics studies are based on blood-derived samples (plasma/serum), which can be collected with low invasiveness and minimal health risk. Blood-derived samples contain lipids in the form of lipoproteins, as well as metabolites secreted by different tissues and cells in response to various physiological or pathological stimuli. As a consequence, serum and plasma can provide important information at a systemic level that is representative of the overall metabolic status of the human organism , which iBlood collection: traditionally, blood is collected from a peripheral vein after an overnight fast (12–14 h). Serum is obtained after clotting by centrifugation, which allows the removal of fibrin clots, blood cells, and related coagulation factors, whereas plasma is obtained by adding anticoagulants  before removal of blood cells by centrifugation. Then, it is directly divided into prelabeled aliquots to limit freeze–thaw cycles and stored as fast as possible at −80 °C until the lipid extraction procedure is carried out. Blood collection tubes are a critical step in the preanalytical phase; however, only few data exist in the literature concerning their effect on the lipid components [mponents . Becausemponents . In addimponents , as wellmponents , should Blood-derived samples storage: the storage conditions of blood-derived samples strongly affect the quality and quantity of lipidome. Many lipid components undergo chemical and enzymatic degradation [radation . For exaradation . Since tradation . In addiWhen the lipids carried on lipoprotein particles are within our interest, then serum lipoproteins including very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), LDL, and HDL should be separated. Analytical methods such as ultracentrifugation, column chromatography, and gel electrophoresis can be used for their separation ,26,27. HLipids are embedded in a biological matrix instead of being in a free form. Thus, prior to their detection, the structurally diverse lipid species must be extracted from their native state into suitable single-organic solvents or solvent mixtures. Lipid extraction is without a doubt the major limiting step to analyzing the complete set of lipids in biological systems in the lipidomics approach. For their extraction, we should use solvents that dissolve lipids readily and also overcome the interactions between lipids and components such as protein and polysaccharides via hydrophobic or van der Waals forces. If the biological matrices are tissues or cells, an additional homogenization step is required before the lipid extraction procedure. For the extraction of lipids, there are two significant challenges to overcome: extraction efficiency and complete removal of non-lipid constituents. Both challenges are influenced by parameters such as temperature, sample/solvent ratio, mixing times, and extraction under inert gasses, which in turn may be responsible for artifacts appeared in the identification of lipid molecules and the inconsistencies in their quantification.A variety of well-established lipid-extraction techniques have been reported in the literature including monophasic  and biphasic approaches  and solid-phase extraction (SPE)) ,30,31. Mv/v), whereas in Bligh and Dyer, the chloroform:methanol ratio is 1:2 (v/v).The aforementioned techniques are reasonably efficient in terms of conferring a general lipidome representative of the vast diversity of endogenous lipids; however, the choice of a particular lipid-extraction protocol and organic solvent depends predominantly on the lipids of interest, the range of concentrations at which they exist, and the samples being analyzed. According to the scope of the present review article, we will focus on the methods used for extraction of all classes of detectable lipids from serum lipoproteins with minimal contamination of non-lipid molecules such as proteins and carbohydrates. LLE chloroform/methanol-based protocols, such as Folch  or BlighFollowing lipid extraction, the stability of the extracted lipids is also an important consideration, particularly if lipid molecules contain unsaturated double bonds that can be subjected to oxidation in the presence of oxygen in air. To minimize this effect, the removal of air from the headspace of sample vials containing the lipid extracts, by passing inert gases, such as nitrogen, over the mouths of the vials prior to sealing them, can also help minimize the occurrence of lipid oxidation reactions.After the lipid extraction procedure, the next key step in the lipidomic workflow is the qualitative and quantitative analysis of lipid classes extracted from biological samples. Because lipids are highly complex and structurally diverse with heterogeneous physiochemical properties their comprehensive analysis in a direct and quantitative manner presents considerable analytical challenges.Historically, the earliest attempts for the study of lipid composition of cells, body fluids, and tissues were performed by conventional chromatographic separation-based techniques such as high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), gas chromatography (GC), liquid chromatography (LC), or electrophoretic techniques such as capillary electrophoresis (CE) ,39,40. A1H NMR or 31P NMR) in the high micromolar to millimolar range compared with MS-based techniques and the low natural abundance of 13C for 13C NMR. Its sensitivity can be improved by using very strong superconducting magnets and cryoprobes that operate the receiver circuitry at low temperatures to reduce electronic noise. In addition, NMR has been successfully hyphenated to LC as liquid chromatography-NMR (LC-NMR) for accurate lipid separation and subsequent structural elucidation tackling the problem of signal overlaps.The two most commonly used analytical tools for lipid analyses are the NMR and MS techniques. MS is a powerful analytical technique for the detection of various classes, subclasses, and individual molecular species of lipids in a biological sample with high sensitivity and high throughput ,44,45. AA multidimensional NMR technique could obtain a better resolution of the signals attributed to lipids without sacrificing the sensitivity, as it allows their dispersion into two dimensions, providing an information-rich two-dimensional lipid map in which key unique peaks can be resolved and used for the identification and quantification of specific lipid molecules . The devIn the field of serum lipoprotein research, NMR has been proven to be a main tool in the study of the structure of lipoprotein particles. The first publication on lipoprotein structure was entitled “Structure of human serum lipoproteins: nuclear magnetic resonance supports a micellar model”, by Steim et al., involved a magnetic field frequency of 60 MHz and was dated from 1968 . Since t1H NMR spectroscopy in the detailed analysis of lipids extracted from cell membranes was pioneered by Sparling et al. in 1989 [Historically, the first application of  in 1989 . Then, s in 1989 ,59,60,61In this part of the review, the application of the NMR on the comprehensive analysis of lipids extracted from serum lipoproteins will be discussed. It is well-known that the lipids located on the core and surface of serum lipoproteins are qualitatively similar but quantitatively different. For example, triglycerides are the predominant core lipid in VLDL, while cholesterol esters predominate in the cores of HDL and LDL particles. Compared to LDL, HDL contains higher amounts of phospholipids, particularly phosphatidylcholine. Regardless of the lipoprotein class, the lipids will appear in the NMR spectrum with more than one signal, and their intensity is linearly related to their concentrations. For example, the signal attributed to triglycerides will appear in the NMR spectra of both LDL and HDL lipid extracts but with higher intensity in the NMR spectrum of LDL lipid extracts compared to that of HDL because of the higher content of triglycerides in the LDL particle’s core.1H NMR spectrum of the total lipid extract of HDL lipoprotein particles is shown in A typical Regardless of the analytical technique used for the lipidomic analysis, the study and interpretation of the resulting lipidomic data can be accomplished using either a targeted approach or an untargeted approach .In a targeted approach, the physicochemical properties of a pre-defined set of lipid constituents of interest are a priori known. This allows for a more selective biological sample preparation optimized for the set of lipids analyzed, thus providing their absolute concentrations. As opposed to the targeted strategy, the untargeted lipidomic approach aims to confer a general overview and the reproducible measurement of as many lipid molecules as possible in the total lipid extract of a given biological sample. Thus, a global lipidome is yielded where alterations in lipid patterns occurring under biological perturbation or stimulation can be detected, or aberrant lipid compounds, whose structure and chemical formulas are unknown, can be reliably and unambiguously identified. With this approach, peak areas are reported for lipid compositional characteristics instead of absolute concentrations, resulting in semi-quantitative lipidomic data.Lipoprotein metabolism is an extremely complex process during which lipoprotein particles exchange their proteins and lipids and undergo extensive remodeling by lipid transfer proteins, lipoprotein receptors, and lipophilic enzymes. Imbalances in lipid metabolism are linked to various cardiometabolic diseases such as obesity, diabetes, chronic renal failure, or cardiovascular disease. To understand the pathogenesis of these diseases and subsequently develop successful treatment, it is necessary to be able to track the aberrant lipid-related metabolic pathways. These states may contribute to the vulnerability of serum lipoproteins to compositional and structural modifications, causing disproportionation in two or more of their components beyond cholesterol and triglycerides, which are routinely determined.Before investigating the alterations occurring in serum lipoproteins in cardiometabolic diseases, it is important to define the lipid composition among healthy individuals. Studies have shown that the total lipid content differs across lipoproteins ,65,66. VHDL or atheroprotective lipoproteins, apart from the well-recognized antiatherogenic role of HDLs in reverse cholesterol transport (RCT), have been postulated to exert numerous functions including antioxidant, anti-inflammatory, vasodilatory, antithrombotic, immunomodulatory, and endothelial repair properties that may contribute to the protection from atherosclerosis. Structure–function studies suggest that the ability of HDLs to exert these functional properties is inextricably related to the overall structure and composition (lipidome or proteome) , rather The influence of disproportions in lipid components on the HDL structure has been investigated in reconstituted discoidal and spherical HDL particles. Changes in core neutral lipids (cholesterol esters and triglycerides) have distinct effects on an HDL particle’s stability and induce conformational and structural changes on the primary apoprotein, apoAI. An increase in triglyceride content has a negative effect on a particle’s stability, whereas the stability is enhanced by the presence of cholesterol ester molecules . The conPhospholipids play an important role in regulating the structure and function of HDLs. Rye KA et al. in 2000 showed that phospholipids depletion not only had a major effect on the size of HDLs but also influenced the cholesterol ester transfer protein (CETP)-mediated remodeling and dissociation of apoAI . The fluAs mentioned above, disturbances in different HDL lipid molecules beyond cholesterol could lead to the formation of dysfunctional or even pro-atherogenic particles. The lipid composition of HDLs appears to be compromised by acute and chronic inflammatory conditions, as well as by metabolic decompensation, including obesity, metabolic syndrome, insulin-resistance states, nonalcoholic fatty liver disease (NAFLD), chronic renal failure, cardiovascular disease, etc. This section reviews the impact of cardiometabolic diseases on the modification of HDL lipidome that compromise the functionality of HDL.The prevalence of overweight and obesity is increasing and represents a primary health concern because of the increased risk for development of T2D and CVD. The specific underlying mechanisms linking the expansion of adipose tissue, a characteristic of the obese state, to cardiometabolic comorbidities associated with obesity are still poorly understood. Serum HDL-C levels and cholesterol efflux capacity are often decreased, especially when obesity is associated with metabolic syndrome (MetS). The latter includes a clustering of clinical findings of abdominal obesity, hyperglycemia, hypertriglyceridemia, hypertension, and low HDL-C levels. MetS is tightly associated with lipid disorder, which relies on the ectopic accumulation of lipids (lipotoxicity), as well as on mitochondrial dysfunction leading to an increased CVD risk. In addition, it is characterized by several metabolite changes in plasma, reflecting abnormalities in several metabolic pathways. However, the specific underlying pathophysiologic mechanisms pertaining to how altered lipid metabolism can potentially result in disease onset, have largely remained unaccounted for.Central obesity is associated with modifications of the atherogenic nature to lipoprotein distribution and composition including increased synthesis of larger VLDL particles and a shift to small, dense LDLs that have been positively associated with increased CVD risk . BesidesHDL isolated from patients with MetS display a reduced ability to activate eNOS and, consequently, to promote the production of nitric oxide mainly because of depletion of HDLs in a sphingolipid named sphingosine-1-phosphate (S1P) ,82. The According to a WHO report, in 1980–2014, the number of people suffering from diabetes increased from 108 to 422 million. The World Diabetes Federation predicts that the incidence will reach 700 million by 2045. Disturbances in glucose homeostasis and lipid metabolism, both well-recognized predisposing factors for CVD, are highly interrelated at multiple metabolic crossroads. Thus, aberrations in pathways involved in lipoprotein metabolism could at least partially explain the risk for the development of atherosclerosis in individuals with carbohydrate-metabolism derangement.SAA) [In insulin-resistance states, HDLs undergo several structural and compositional disturbances that are beyond their cholesterol content. As a result, functionally deficient HDL particles are formed by several proposed mechanisms . For exaSAA)  because SAA) .Meanwhile, the functional status of HDLs is strictly related to alterations occurring in lipid components. Increased triglyceride content in HDL core caused by insulin resistance and elevated CETP activity ,92, abnoPrediabetes is a clinically silent, insulin-resistant state that often remains undiagnosed and is associated with an increased risk for the development of T2D  and CVD In addition to adipose tissue, the liver is another key organ early associated with insulin resistance and diabetes. NAFLD is one of the most common forms of chronic liver diseases in the Western countries, affecting approximately 25% of the general population, and is a major risk factor leading to MetS, T2D, CVD, chronic liver disease, and liver failure . It is cAlthough there has been remarkable progress in the elucidation of NAFLD pathogenesis, the pathophysiological pathways underlying lipotoxicity and the transition of simple steatosis to NASH are still incompletely understood . Liver bChronic renal failure is associated with an increased risk of CVD as these patients develop accelerated atherosclerosis . Renal dStudies have shown that oxidized LDL-C levels increase as kidney function declines . The chrCVD is mostly associated with alterations of blood levels for one or more lipid classes. The cholesterol content of HDLs is not a causal marker because many functional properties of HDL are exerted either by entire particles or specific constituents (lipid or protein) other than cholesterol. It has been shown that overt CVD dramatically alters the composition and overall structure of HDLs and endows the particles with abnormal biological and physico-chemical properties, and finally alters quality and impairs functional status. Indeed, the inflammation burden of CVD induces changes in the protein or lipid components of HDLs, beyond cholesterol content, resulting in the formation of dysfunctional particles . HoweverHDLs lose their atheroprotective properties and become gradually proatherogenic. Cholesterol efflux capacity is decreased , while nThe first studies conducted in HDLs isolated from CVD patients revealed alterations in individual lipid molecules and specifically in phospholipids, whose content was lower in these patients ,118,119.The increasing global burden of cardiometabolic diseases highlights the urgent clinical need for better personalized prediction and intervention strategies for the right individual at the right time (at an early stage). The knowledge of the alterations in lipid metabolic pathways in a broad spectrum of CVD etiologies could offer us valuable information on how to control lipid metabolism. To this aim, the advent of lipidomics approaches and other omics approaches have led to tremendous progress in the cardiometabolic diseases research field in last two decades. Integrating the emerging multiomics with lipidomics holds a great potential in moving toward this goal.


Editorial on the Research TopicHighlights in abdominal and pelvic pain 2021/2022Highlights in Abdominal and Pelvic Pain 2021/22. This Research Topic has a selection of high-impact articles authored by four groups of excellent international leaders in the field of pain research and has shared varied theories and/or experiences in the clinical treatment and therapy of abdominal and pelvic pain research. The work presented here highlights the broad diversity of research carried out in the abdominal and pelvic pain section, not specifically limited to years, and aims to highlight the main areas of interest among many clinical departments: anesthesiology and pain medicine, neurology, obstetrics and gynecology, urology, gastroenterology, immunology and allergy, surgery and orthopedics, and general medicine. Of course, state-of-the-art concepts, such as the brain-gut axis, or vice versa, also exist in some research collection papers  such as irritable bowel syndrome (IBS). To date, pharmacological treatments for patients with chronic visceral pain generally lack efficacy and are fraught with unwanted side effects. Cognitive behavioral therapy has emerged as a psychotherapy that shows efficacy in ameliorating stress-induced chronic visceral pain. It is interesting to read these articles on acupuncture mechanism disputes for disorders of the digestive system and the potential cellular mechanism . It is well known that IBS is more common in children than in adults and that it affects the quality of human life. Once considered a disorder caused by abnormalities in gut smooth muscle, visceral hypersensitivity, and/or central nervous system hypervigilance, a change in focus has been observed to include both central and peripheral mechanisms in pathophysiology in the past decade. Related peripheral mechanisms include abnormal colonic transit and rectal evacuation, intraluminal intestinal irritants, altered gut microbiome, and enteroendocrine cell dysfunction. The United States Food and Drug Administration approved the authors to conduct a thorough safety study of VSL#3 in adults with IBS before its use in a randomized study in children with IBS. The researchers used this opportunity to investigate adult patients with IBS with matched fecal omics data and the effects of two dosing durations of VSL#3 on gut permeability, recovery of probiotic organisms, and changes in gut microbiome composition determined by shotgun metagenomic sequencing, as well as alterations in metabolome profile (bile acid). The results of the RCT suggested that the probiotic organisms identified in the stool were significantly correlated with improved colonic permeability and clinical symptoms.The second article, a wonderfully novel article about probiotic organisms identified in stool that were significantly correlated with improvement in colonic permeability and clinical symptoms, led future studies to investigate the mechanistic roles of VSL#3 and colonic permeability in the pathophysiology of IBS in a larger randomized controlled trial  IBS subjects reported episodic memories of the onset of IBS symptoms, often GI infections/enteritis (35.7%). Episodic memories were associated with greater IBS symptom severity/bother, higher anxiety/depression, and poorer health-related quality of life (HRQOL). However, the subtest memory specificity was not different based on episodic memory. Patients with IBS often reported episodic memory associated with the onset of symptoms, and this recall appears to be associated with more severe symptoms. To date, the much discussed concept of intestinal microflora has been described for the special scientific mechanism; more recently, the main flow mechanism of the nervous and mental axis plays a substantially key role (The fourth article, wonderfully related to the psychological gut brain axis, is by


The complications of prematurity are the leading cause of neonatal mortality worldwide, with the highest burden in the low- and middle-income countries of South Asia and Sub-Saharan Africa. A major driver of this prematurity-related neonatal mortality is respiratory distress syndrome due to immature lungs and surfactant deficiency. The World Health Organization's Every Newborn Action Plan target is for 80% of districts to have resources available to care for small and sick newborns, including premature infants with respiratory distress syndrome. Evidence-based interventions for respiratory distress syndrome management exist for the peripartum, delivery and neonatal intensive care period- however, cost, resources, and infrastructure limit their availability in low- and middle-income countries. Existing research and implementation gaps include the safe use of antenatal corticosteroid in non-tertiary settings, establishing emergency transportation services from low to high level care facilities, optimized delivery room resuscitation, provision of affordable caffeine and surfactant as well as implementing non-traditional methods of surfactant administration. There is also a need to optimize affordable continuous positive airway pressure devices able to blend oxygen, provide humidity and deliver reliable pressure. If the high prematurity-related neonatal mortality experienced in low- and middle-income countries is to be mitigated, a concerted effort by researchers, implementers and policy developers is required to address these key modalities. Childhood mortality is predominantly driven by deaths in the neonatal period (the first 28 days of life) . In the Between and within-country variation in premature birth rates and prematurity-related mortality exist, with low- and middle-income countries (LMIC) carrying the highest burden . Over 90The drivers of prematurity related mortality are multifaceted and related to the immature organ-systems of the preterm newborn. However, respiratory distress syndrome (RDS), which results from lung immaturity and surfactant deficiency, contributes about 45% of case-fatality due to prematurity in LMICs . Risk ofThe development of RDS begins with impaired or delayed surfactant synthesis and secretion in the immature lung. Between 24 to 28 weeks of gestation, type II alveolar epithelial cells begin production of surfactant, however, this innate surfactant production is often insufficient for extra-uterine life until after 35 weeks . The priThe incidence of RDS is inversely related to gestational age. It occurs in 98% of preterm infants between 22 and 24 weeks gestation but only 25% of those with birth weight between 1,251 to 1,500 grams , 16. TheIn HICs, RDS is diagnosed in preterm infants who have signs including supraclavicular, intercostal, and subcostal retraction, grunting and flaring of the nares, requirement for supplemental oxygen as dictated by hypoxia from pulse oximetry, and have chest radiographic findings of diffuse haziness and air bronchograms . AdditioHowever, the use of chest radiographs, pulse oximetry and blood gas analysis are resource intensive and not commonly available in LRS. Hence, the most feasible assessments for RDS in LRS include objective criteria for assessing work of breathing, such as scoring systems that are simple, non-invasive, inexpensive and have shown both prognostic value and good inter-rater-reliability. Most commonly in use are the Downes score and the Silverman Andersen Respiratory Severity Score , 20. In In the 1960s, animal studies showed corticosteroids accelerated lung maturation . In subsInformed by models and strong evidence from both high, and upper middle-income countries that suggested ACS may prevent prematurity-related neonatal mortality –38, the After this study, the WHO issued guarded recommendations for ACS to be used in health facilities capable of assessing gestational age, diagnosing maternal infection, and providing emergent obstetric and preterm neonatal care, including resuscitation, respiratory, thermal, and nutritional support . SubsequTaken together, these trials on ACS among pregnant women in LMICs at risk of preterm delivery indicate that with close monitoring and the provision of neonatal interventions for preterm newborns, ACS has the potential to substantially reduce prematurity-related mortality. However, a significant proportion of deliveries in LMICs still occur in resource limited settings where the quality of care required to reap the benefits of ACS may be unavailable. Research on how to effectively monitor women at risk of preterm delivery, optimize neonatal resuscitation and increase availability of level two newborn care including respiratory support with limited resources as recommended by WHO is required. Also, investments and research into optimizing the referral and safe transportation systems to higher-level facilities for the at-risk mother, would increase access to ACS.in utero transfer of pregnancies at risk for preterm delivery to a center capable of providing high-quality obstetric and neonatal care is not always feasible  .The Neonatal Resuscitation Program recommends the use of CPAP when respiratory distress persists after initial resuscitation despite establishment of a normal heart rate and spontaneous respiration . This usvia a nasal interface enhances lung inflation, decreases work of breathing and is associated with decreased mortality due to RDS (The primary consequence of RDS (surfactant deficiency) is alveolar collapse and the loss of functional residual capacity. In spontaneously breathing infants with RDS, CPAP provides continuous distending pressure to the airway and lungs , 60. Thie to RDS . CPAP ale to RDS .CPAP devices can be grouped into two broad categories based on the method of pressure generation. Devices that use an adaptive flip valve located at the nasal interface to generate CPAP are termed variable flow devices . DevicesEvidence from HIC suggest that compared to supplemental oxygen, the use of CPAP is an effective treatment for preterm infants with RDS . A 2020 The cost of CPAP devices, unavailability of consumables and spare parts, maintenance needs, and dependence on electricity have limited the availability and use of commercialized CPAP devices in LMICs , 69. To 2O, provide humidification, blend oxygen, and have flow capacity of 0–10 L/min. In 2020, UNICEF published a target product profile to align CPAP innovators and other stakeholders to the most important performance and operational characteristics, as well as target pricing to aid in the development of effective and safe CPAP devices that can be scaled . Excerptvia the Pumani device (25 subjects) or oxygen (23 subjects). The study found that survival to hospital discharge in the CPAP and oxygen groups respectively was not statistically significant . However, in the per-protocol analysis a more significant number of subjects in the CPAP group survived to hospital discharge 77.2 vs. 47.8% . The study reported no pneumothoraxes, but that bleeding from the nose was more common in the CPAP group  (p = 0.036)  patients in the CPAP group, and 4%  patients in the control group died   of NICUs capable of providing surfactant replacement therapy did so with the thin catheter method . There a(35/54)] . FurtherOxygen therapy with CPAP for RDS is the most prevalent form of respiratory support in LMIC , 100. HoThe cost of MV and lack of capacity to use MV are deterrents to its use . MV is nDuring the COVID-19 pandemic, the lack of, and need for MV generated awareness of its necessity in LMICs among patient populations outside of the neonatal period –158. TheAdequate nutrition is critical for the continued growth and development of the premature respiratory system. Premature infants are at risk of insensitive water losses and nutritional failure by virtue of their dermal and intestinal immaturity. Liberal or excessive fluid administration is associated with poor respiratory outcomes in preterm infants , and optTo achieve the sustainable developmental goals targets, small and sick newborns need high-quality inpatient care at the right time and in the right place . Transfohow women at risk of preterm labor in rural communities can be treated safely with ACS and have the desired pregnancy outcome?how can low-cost CPAP devices be optimized to be as effective and safe as the gold standard devices?how can providers learn and maintain direct laryngoscopy skills and the complexities of MV?how can the cost of essential medications like surfactant, caffeine, and devices like CPAP and MV be made more affordable, so they are accessible to providers in LMICs?To bridge this knowledge gap, thoughtful research designs are required. Research needs to focus equally on implementation as well as evidence generation, taking into context the resource limitations of the clinical setting . These tMost global recommendations and predictive models of interventions to improve prematurity-related mortality in LMICs indicate that the greatest benefit will only be achieved with a comprehensive approach to implementing these evidence-based interventions , 154. CrRespiratory distress syndrome is a major driver of prematurity related neonatal mortality. High quality trials conducted in HRS have shown interventions to be effective in reducing the RDS specific neonatal mortality. However, these standard of care interventions in HRS have sparse and sporadic coverage in LRS. When tested in LRS these interventions have not consistently had the same beneficial effect, likely due to the lack of critical ancillary services and core neonatal care practices. To achieve a neonatal mortality rate as low as 12 per 1,000 live births in year 2030 , guided OE, MB, and AH conceptualized the review. Initial draft by OE, IO, and AH with revisions by MB. All authors 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. The handling Editor declared a shared committee with one of the authors MB.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.


Trypanosomatid parasites are kinetoplastid microorganisms, some of which cycle between the gut of insect vectors and the tissues of vertebrate hosts . In the The diseases caused by trypanosomatids are among the so-called neglected tropical diseases (NTDs), which affect millions of individuals mostly in low and middle-income countries from tropical and subtropical regions. In addition to their close relationship with poverty, these diseases permanently deform and disable a large number of poor people, who are thus trapped in their poverty situation. Sadly, these diseases are often forgotten by governments and the pharmaceutical industry, due to the lack of political voice of the affected populations and the reduced economic revenue from this low-income market , 5.Although chemotherapy is critically important in reducing the parasite burden, the variable efficacy of the drugs against different clinical stages of the infections, their frequently associated adverse effects, and our poor understanding of drug-resistant parasite phenotypes remain major concerns. To date, there are no licensed human vaccines against trypanosomatid infections and the majority of the immunization studies have been conducted only at the pre-clinical level . Thus, tTrypanosomatid parasites drive major modifications of the immune system and frequently the immune response evoked does not result in protection . Even woIn recent years, in addition to the identification of vaccine antigens, further efforts have focused on the development of efficient antigen delivery systems, adjuvants and vaccination regimens to enhance the protective responses to defined immunogens , 11.Trypanosoma cruzi infection. However, this inflammatory profile is also frequently linked to tissue damage and considered partially responsible for the pathology of Chagas disease. Silva et al. address both aspects of the immune response by pre-treating animals with alpha-tocopherol, an isomer of the E-vitamin, prior to the infection. Alpha-tocopherol has previously been described as a potential adjuvant to enhance immune responses to vaccines in several models. They showed that a pre-treatment with several 100 mg/kg doses of alpha-tocopherol induced an increase in the number of IFN-gamma-producing immune cells and effector memory T cells, resulting in better control of parasite levels in the blood. Interestingly, this pre-treatment also resulted in reduced tissue damage associated with infection and a higher survival rate. This pathology protective effect may be due to the development of more IL-10-producing CD8+ T cells in the treated animals, which would counterbalance the inflammatory response while preventing an excessive destruction of the tissues. These complementary characteristics modulating the immune response make alpha-tocopherol treatment an interesting component to be tested as a vaccine adjuvant. As noted by the authors, although its use as a prophylactic measure before infection may not be practical, this treatment might become important in the context of a therapeutic vaccine, where the goal is to potentiate an already present immune response against the chronic infection while preventing further pathology. If this dual effect is also confirmed in the chronic model, treatment with alpha-tocopherol could give researchers a new tool to enhance the host response to promote complete T. cruzi clearance of chronic infections.A strong inflammatory immune response with IFN-gamma as a key cytokine is considered to be crucial in the control of Leishmania infect humans and other mammals causing a wide spectrum of diseases, ranging from cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), diffuse cutaneous leishmaniasis (DCL), and visceral leishmaniasis (VL), depending on parasite virulence factors and the immune response established by the host  in infected dogs compared to healthy controls. Through weighted gene co-expression network analysis, they identified four main co-expression modules associated with cell cycle processes, endoplasmic reticulum stress, regulation of the immune response, and regulation of the B cell apoptotic process. Interestingly, these co-expression modules, were correlated with the monocyte concentration in blood and the clinical stage of the sick dogs. As expected for infected animals, some of the genes displaying the highest differential expression as well as two of the four co-expression modules, were involved in the immune response. Notably, the analysis also identified 21 differentially expressed lncRNAs and one module associated with chromatin organization in sick dogs, from which the authors suggest a possible role for epigenetic regulation processes in the immunopathogenesis of canine leishmaniasis.A delicate balance between inflammatory and regulatory responses is required to achieve immune control of infantum . Furtherinfantum , 16. To L. donovani cases with unusual cutaneous manifestations have been described in Sri Lanka, Nepal, and India  through TLR2 and TLR4 receptors on the surface of neutrophils. This JNK phosphorylation triggers the release of DNA and the burst of ROS by neutrophils resulting in the formation of the NETs. As such, this study identifies a T. brucei–LPG-induced activation pathway for NET formation with features that are shared by other trypanosomatid infections.The host innate immune system plays a pivotal role in the recognition of kinetoplastid infections. Polymorphonuclear neutrophils react against protozoan parasites by different effector mechanisms, which include the release of immunomodulatory molecules , 20, phat manner , 22. To In conclusion, this Research Topic provides new knowledge regarding the host- pathogen relationships of kinetoplastid parasites and the description of potential targets and approaches that could augment protective immune responses. Some of the mechanisms that parasites can orchestrate to counteract the host defenses and establish a functional infection are also considered. Nevertheless, our understanding of the immune system interaction with these highly complex pathogens is far from complete, and many challenges remain in our fight against these parasites.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The editors want to acknowledge the authors for their contributions to this Research Topic. We would also like to thank to the staff of Frontiers in Major Tropical Diseases Immunology for making it possible for us to conduct this Research Topic.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.


Septic patients were commonly affected by coagulation disorders; thus, they are at high risk of thrombotic complications. In the last decades, novel knowledge has emerged about the interconnected and reciprocal influence of immune and coagulation systems. This phenomenon is called immunothrombosis, and it indicates an effective response whereby immune cells and the coagulation cascade cooperate to limit pathogen invasion and endothelial damage. When this network becomes dysregulated due to a systemic inflammatory activation, as occurs during sepsis, it can result in pathological thrombosis. Endothelium, platelets and neutrophils are the main characters involved in this process, together with the TF and coagulation cascade, playing a critical role in both the host defense and in thrombogenesis. A deeper understanding of this relationship may allow us to answer the growing need for clinical instruments to establish the thrombotic risk and treatments that consider more the connection between coagulation and inflammation. Heparin remains the principal therapeutical response to this phenomenon, although not sufficiently effective. To date, no other significant alternatives have been found yet. In this review, we discuss the role of sepsis-related inflammation in the development and resolution of venous thromboembolism and its clinical implications, from bench to bedside. Thrombosis is the pathophysiological mechanism underlying the three main cardiovascular disorders , and of Sepsis is a life-threatening syndrome characterized by multi-organ dysfunction, consequent to a dysregulated immune, endocrine and metabolic response to infection ,9 and clHistorically, Virchow’s triad was the cornerstone of the knowledge about pathophysiological processes leading to thrombosis; it consists of three elements strictly interconnected with each other: blood flow alterations, vascular wall damage or dysfunction and hypercoagulability ,15. DespDuring sepsis, several pathophysiological processes occur simultaneously , and amoIn this context, many actors are involved, starting from endothelium, platelets and coagulation factors, cytokines and immune cells to their complex interactions, that appear to be at the basis of not only the microvascular phenomena but also of deep vein thrombosis, depending on the site, depth and extent of vascular wall damage and, consequently, on the specific activated pathway ,20. InteTherefore, the purpose of this review is to collect and summarize the current knowledge on the subject by investigating the molecular and immunologic pathways of venous thrombosis and underlining the role of sepsis-related inflammation in the development and resolution of VTE and their clinical implications.First of all, we identified some key words to explore the association between sepsis and venous thromboembolism, such as sepsis, thromboembolism, venous thrombosis, deep vein thrombosis, thromboinflammation and coagulopathy. Then, we performed a literature search in the PubMed and Medline databases from 1 January 2012 to 31 January 2022, examining in depth the various characters of the process and the relationship between them. We favored the inclusion of articles from the last 10 years to give up-to-date information, although we did not exclude older reports when highly referenced and when more recent evidence was unavailable. Moreover, we also included some studies related to COVID-19 infection, which presents a particular elevated incidence of VTE, although the pathophysiological mechanisms are not fully understood yet. We selected the relevant references identified by the search strategy, and we excluded some of them if the title and/or abstract were not appropriate for the aim of the review. Full texts were obtained for relevant references and when the significance of an article could not be excluded from the abstract. We chose to consider this lapse of time and all types of articles to obtain a comprehensive overview of this topic, initially without any language restriction. Moreover, we supplemented our search by manually reviewing the reference lists of all the retrieved articles, not to exclude any relevant source of information.As it is well-known, the main purpose of hemostasis is to stop bleeding by repairing the damaged vascular wall after injury, with a series of highly regulated steps leading to the development of thrombin and fibrin. Furthermore, hemostasis has another protective function, phylogenetically ancient and evolutionarily preserved , aimed aThe endothelium is an important regulator of vascular homeostasis with several physiological functions. It is a regulator of the vasomotor tone, a carrier for cells, nutrients and inflammatory/anti-inflammatory signaling; it has natural anticoagulant properties through its glycocalyx made of heparan sulfates and chondroitin sulfate and through transmembrane molecules such as TM (thrombomodulin), EPCR  and TFPI (tissue factor pathway inhibitor) ; it inflDuring sepsis, it undergoes various phenomena, including damage, activation and dysfunction, and acquires pro-adhesive, procoagulant and antifibrinolytic phenotypes, acting as a bridge between the immune system response and coagulation cascade. This phenotypic shift can be triggered by hypoxia, tissue injury, cytokine signals, hemodynamic changes, endotoxemia or pathogen recognition ,26.The first element of connection between the endothelium, immune cells and platelets during sepsis is related to “endothelial activation”, which leads to an increased expression and release of adhesion molecules with a pro-adhesive behavior. It is mediated by selectins expressed on ECs (E-selectin), on leucocytes (L-selectin) and platelets (P-selectin), by immunoglobulin-like receptors and receptors from the integrin family that form three heterodimers of β2-integrin collectively named the CD11/CD18 complex and, third, by intracellular adhesion molecules (ICAMs) that mediate the cross-talk with activated leukocytes . In earlThe regulation of expression, binding avidity and modulation of these adhesion molecules are operated by biological mediators such as circulating pathogen-associated molecular pattern (PAMPs) and damage-associated molecular pattern (DAMPs) molecules; endothelial-derived cytokines ); local synthesis of the platelet activating factor (PAF); triggering intracellular signaling cascades that activate the transcription factors NF-κB (nuclear factor kappa light-chain enhancer of activated B cells)  and promFurthermore, the endothelium can assume a procoagulant phenotype when it is damaged during sepsis. Vessel injury consists of cellular alteration or the interruption of the endothelial line, and it produces hypoxia and inflammation, provoking a sort of endotheliopathy with impairment of the membrane anticoagulant components, such as endothelial glycocalyx, tissue factor pathway inhibitor, thrombomodulin (TM) and protein C, with the induction of a hypercoagulability  14,35].,35.14,35This impairment assumes systemic characteristics because of the nature of disseminated sepsis that leads to generalized endothelial involvement  due to aThere are many mechanisms involved in endothelial damage during sepsis, as shown in The endothelium of the venous system is more easily exposed to insults and more predisposed to the formation of macrothrombi in correspondence with the vein valve sinus, because in these areas, there is a condition of hypoxia and slowing of the blood flow . It mainIn this procoagulant state begun from vascular wall injury, we can identify two main coagulation pathways that lead to thrombosis, triggered by a different depth of damage . On the one hand, if the injury due to complement activation is confined to the endothelium, mainly the von Willebrand factor (vWF) path is stimulated ,47. The However, consequently to the isolated endothelial damage, an increased expression of surface adhesion molecules such as P-selectin and of vWF facilitates the subsequent binding of circulating platelets and leukocytes ,53, leadOn the other hand, in systemic inflammatory syndromes such as sepsis; chemical or physical vascular damage sustained by infectious pathogens; inflammatory cytokines ; reactive oxygen species (ROS) or other injurious agents, can interrupt the integrity of the endothelial barrier with the exposure of collagen and tissue factor (TF) to the bloodstream, with TF rapid upregulation on perivascular cells and monocytes . Tissue For this reason, many authors consider TF expression the initiating event in the coagulopathy of acute sepsis because of its central role in the activation of the coagulation cascade and proinflammatory pathways. In fact, through the protease-activated receptor (PAR) signaling, the TF VIIa complex induces the release of cytokines, chemokines and grow factors , and in The other TF effects are carried out by a second mediator mentioned above: thrombin. It is a serine protease that it is detectable within a few hours in sepsis experimental models obtained after the infusion of tumor necrosis factor and endotoxins ,63,64. TSepsis inflammation directly stimulates the release of platelet-activating factor (PAF), which accelerates platelets activation . After PIdeally, the endothelium can oppose thrombin production when a vessel is repaired, balancing procoagulant and anticoagulant pathways after injury. However, when the local damage becomes systemic and sustained, as in sepsis, there is an upregulation of procoagulant mechanisms with a shift towards this state ,75, and As mentioned above, an increasing body of evidence supports an important role of immune cells not only in the response to pathogens but also in the genesis of thrombotic processes that occur during sepsis. A prominent role is played by monocyte for the exposure and release of TF . HoweverEverything starts with the adhesion of monocytes and neutrophils to the venous wall through two main interactions: with the binding of PSGL-1 on the leukocyte surface to P-selectin exposed on activated endothelial cells and platelets  and withNeutrophils participate in the sepsis procoagulant phenotype; first, with the degradation of natural anticoagulants through the direct TM cleavage by neutrophil elastase, MPO and serine proteases  and, secExtracellular trap (ET) production has also been described in other leukocytes such as monocytes, eosinophils and basophils. Monocyte ET release depends on oxidative burst, and also, in this case, it has been demonstrated as a procoagulant activity, but at the present state of knowledge, the main trap producers in venous thrombosis remain neutrophils . The role of neutrophils in thrombus formation is further confirmed by numerous proof: their depletion and their inability of producing NETs due to deficiency in peptidyl-arginine deiminase 4 (PAD 4), and NET degradation by the administration of DNase or deficiency in P-selectin prevents DVT in murine models ,67,93,94Other observations suggest that NETs and their degradation products can also counterbalance procoagulant stimuli and promote thrombolysis, probably following temporal kinetics, depending on changes of the inflammatory setting during sepsis. For example, in vitro, extracellular DNAs downregulate prothrombotic functions of neutrophils and monocytes binding TLR9 receptors , cathepsConsidering the above, neutrophils appear as an interesting target for therapeutic interventions, but their systemic modulation or depletion would compromise the entire host defense system, so it is not a sustainable therapeutic option. Therefore, the attention is shifting on multiple strategies to inhibit NET effects as potential treatments for both uncontrolled inflammatory and thrombotic diseases .As we learned from the last few years of a pandemic, the Coronavirus disease of 2019 (COVID-19) presents a significantly increased risk for micro- and macrovascular thrombosis, both in venous and arterial systems , with thIn COVID-19, the process seems to start from the downregulation of angiotensin converting enzyme 2 (ACE-2) activity due to its use by SARS-CoV-2 to enter human cells . This enIt is an excessive ROS production—in particular, from mitochondria—that triggers endothelial dysfunction. The resulting oxidative stress generates a positive feedback loop, which perpetuates the overproduction of ROS, mitochondrial dysfunction, endothelial damage and, consequently, a dysregulated inflammatory response, with chronic implications . Upon enThese mechanisms show also in COVID-19 the close interaction between an exaggerated immune response (sometimes autoimmune) and the activation of the entire coagulation system, but many aspects about the extent of the response, the thrombotic events and the impact of interindividual differences remain still unknown.Coagulation disorders have a huge impact on patients’ prognosis, because they are a major cause of death during sepsis. They range from a slight decrease in platelet count to more dangerous clinical phenotypes very different from each other, such as VTE or DIC. Usually, coagulation dysfunctions are evaluated with some laboratory tests, of which the first four listed in Therefore, there is a growing need for clinical instruments that more consider the connection between coagulation and inflammation. On this line of thought, the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Hemostasis (ISTH) defined both the ISTH overt DIC and sepsis-induced coagulopathy (SIC) scoring systems . The staThe diagnostic approach of VTE in septic patients starts from the evaluation of the usual clinical prediction rules, such as Wells and Geneva scores. However, it is not clear whether these scores show a reduced performance or not in septic patients who may present clinical characteristics similar to VTE, such as tachycardia, hypoxia and prolonged immobilization, to name a few. In the future, the diagnostic performance of the usual clinical prediction rules could be improved by adding the dosage of blood elements related to the intertwining of coagulation and immune systems. For example, soluble P-selectin associated with a Wells score has shown a specificity of 96% and a PPV of 100% in detecting VTE . As saidMoreover, there is a lack in predictive instruments that consider also the influence of inflammatory processes—in particular, during sepsis. In general, for medical inpatients is important to be able to predict the thromboembolic risk in order to set the appropriate prophylaxis. Two scores are usually used for this purpose: the PADUA Prediction Score and the IMPROVE-DD  VTE risk score. In septic patients, the first one is not so valuable; in fact, Vardi et al. found that the PADUA Prediction Score was insufficient to detect subjects likely to develop venous thromboembolism and ineffective in predicting anticoagulant administration. Nevertheless, it was capable of identifying the risk of poor outcome and mortality of these patients, working as a comorbid index rather than a specific VTE predictor . InsteadAdditionally, laboratory tests show analogue imprecision in septic patients, because they lose their diagnostic power according to their low specificity so that the results are altered due to the infection itself. A new element of interest could be the direct or indirect measurement of elements connected with the thromboinflammatory process, such as cell adhesion molecules or neutrophils products. For example, NETs or some of its components, such as MPO-DNA complexes, cfDNA, citrullinated histones H3 or neutrophil elastase (NE), contribute both to pathogens elimination and to thrombogenesis. In fact, on the one hand, sepsis is the predominant ICU condition associated with NET formation, and the levels of NETs are significantly related to disease severity . On the From the therapeutic point of view, the latest guidelines of Surviving Sepsis Compaign (SCC) recommend only VTE prophylaxis and the balance of overall effects favored low molecular weight heparin (LMWH) over unfractionated heparin (UFH) . These drugs are administered for their anticoagulant properties, and it is not currently known if they have some influence on the inflammatory response caused by sepsis. In fact, the use of heparin and other anticoagulant agents do not take into account the reciprocal and close correlation between immune system and coagulation factors, acting mainly on these second ones. Nevertheless, some evidence has shown that post-treatment circulating levels of cfNDA, NE and the MPO–NDA complex in patients with early administration of heparin (within 6 hours) are significantly lower than those patients with late administration , suggestDespite receiving a drug with anticoagulant properties, nearly 5–10% of people with sepsis and organ failure suffer of VTE ,144, andThe major gap of VTE prophylaxis and therapy in septic patients is that the drugs commonly available are used mainly for their anticoagulant properties, without taking into account the pathophysiological mechanisms of immunothrombosis. The ideal therapy to prevent or treat VTE will suppress excess inflammation and coagulation without disrupting haemostasis or triggering immune deficiency . CurrentA deeper and clearer understanding of the crosstalk between inflammation and coagulation can stimulate the discover of new therapeutic targets or help the insightful use of prior inflammatory targets for the prevention and treatment of venous thromboembolism.


Single-cell sequencing technology can reflect cell population heterogeneity at the single-cell level, leading to a better understanding of the role of individual cells in the microenvironment. Over the past few years, single-cell sequencing technology has not only made more new discoveries in the study of cellular heterogeneity of other rare cells such as stem cells, but has also become the most powerful research method for embryonic development, organ differentiation, cancer occurrence, and cell mapping. In this review, we outline the use of scRNA-seq in hair follicles. In particular, by focusing on landmark studies and the recent discovery of novel subpopulations of hair follicles, we summarize the phenotypic diversity of hair follicle cells and their links to hair follicle morphogenesis. Enhancing our understanding of the progress of hair follicle research will help to elucidate the regulatory mechanisms that determine the fate of different types of cells in the hair follicle, thereby guiding hair loss treatment and hair-producing economic animal breeding research.Single-cell sequencing technology can fully reflect the heterogeneity of cell populations at the single cell level, making it possible for us to re-recognize various tissues and organs. At present, the sequencing study of hair follicles is transiting from the traditional ordinary transcriptome level to the single cell level, which will provide diverse insights into the function of hair follicle cells. This review focuses on research advances in the hair follicle microenvironment obtained from scRNA-seq studies of major cell types in hair follicle development, with a special emphasis on the discovery of new subpopulations of hair follicles by single-cell techniques. We also discuss the problems and current solutions in scRNA-seq observation and look forward to its prospects. As a micro-organ embedded in animal skin tissue, the hair follicle plays a key role in the physiological processes of sensing external contact, maintaining body temperature, protecting internal tissues, and preventing foreign body invasion. As a derivative of skin, hair follicles directly affect hair growth, shedding, and regeneration. The hair follicle consists of epidermis and dermis. The epidermis of the mature hair follicle finally differentiates into three enclosed epithelial cylinders, the central most cylinder forms the shaft, the outermost cylinder forms the outer root sheath (ORS) that separates the whole structure from the dermis, and the middle cylinder forms an inner root sheath (IRS) to guide the shaft outward . The haiThe morphogenesis of hair follicles involves the specialization of various types of cells, among which the specialization of different cell types directly affects the structural composition of hair follicles. The hair follicle consists of hair follicle stem cells (HFSCs), dermal papilla (DP), and IRS, ORS, matrix, hair germ (HG), and other cells. These different types of cell interactions form complex intercellular communication networks, which guide the formation of hair follicle morphology and structure. Therefore, in order to further understand the morphogenesis of hair follicles, it is necessary to enhance the detailed elucidation of this molecular mechanism at the cellular level. Although the hair follicle process is very complex and composed of different types of cells, single cell sequencing technology that can analyze cell heterogeneity has become a popular means of hair follicle research.Due to the limitations of previous technologies, transcriptome sequencing analysis usually homogenizes the entire organs or tissues of animals, and the sequencing results are the average of all cells, ignoring the heterogeneity of gene expression between single cells, which is challenging for the detailed analysis of the characteristics of rare cells and single cells. The progress of single cell sequencing technology can provide new opportunities for exploring these hidden features ,3. At prHair follicles are composed of a variety of cell lines . Hair foHair matrix cells are located in the hair bulb at the lower end of the hair follicle. They are the receiver of the signal of the papilla cells and the precursor cells for the formation of various hair cells. They can differentiate into several different cell types. When the dermal matrix cells were signaled by the DP cells, they began to proliferate and differentiate, and finally differentiated into the IRS and hair shaft. The proliferation and differentiation of hair matrix cells directly affect the integrity and growth of hair structure. Panteleyev et al.  proposedThe DP provides the guiding signals needed to activate epithelial progenitor cells and initiate hair follicle regeneration. The DP cells play an important role in all stages of hair follicle development and life and are regarded as the ‘command center’ of hair follicles. They determine the thickness, length, and morphogenesis of hair follicles by secreting signaling molecules, such as growth factors and cytokines, and may even determine the periodic cycle of the hair follicle. There are complex and orderly signaling molecules exchanges between hair matrix cells and DP cells, including Wnt, BMP, Shh, and other signaling pathways. These signaling pathways accurately regulate the development of hair follicles through mutual exchange and dialogue . Shh is At the beginning of the anagen, DP activates the stem cells in the secondary hair germ, causing follicles to regrow downward, however, at the catagen, the epithelial cells at the base of the hair follicle undergo apoptosis, while DP remains intact and is pulled or migrates upwards until it stays next to the stem cells in the hair follicle bulge . The DP Botchkarev et al.  discoverMammalian hair coat color mainly comes from pigments synthesized by mature melanocytes in the hair matrix of the hair bulb. Melanocytes are mainly concentrated in hair follicles and the epidermis during embryonic development in vertebrates. Hair follicles and sweat glands are called the repository of melanocyte stem cells (MSCs). During the hair follicle growth cycle, MSCs migrate downward to the dermis, proliferate and differentiate into melanocyte cells (MCs) located above the papilla . MelanocThe IRS is located between the hair stem and the ORS, consisting of Henle’s layer, Huxley’s layer, and the IRS cuticle, which is the cell group that determines the formation of hair fibers . In HenlThe expression of Dlx3, FOXN-1, and HOXC-13 transcription factor plays an important role in regulating the periodic changes of hair follicles and the control of the hair matrix keratinocyte differentiation toward the hair shaft and IRS ,36. HumaThe DS is a layer of connective tissue sheath between the ORS and the dermal layer, which is the skin cell group derived from the mesenchymal source between the DS and the ORS. Numerous studies have identified DS as one of the main candidates for cell-based therapies to reverse hair loss . SurrounNicholas et al. have demonstrated that DS is a kind of smooth muscle, which can provide power for the regression movements of hair follicles in catagen, and the force pulls DP through tethered-like epithelial cells and wrapped in DP in the form of a hollow sleeve, which plays an important role in the generation of new hair shafts in the next cycle . The chaThe scRNA-seq technology is a sequencing method that can perform single-cell sequencing of nearly 10,000 cells and can identify the transcription characteristics of various cell types in biological tissues and comprehensively reveal the heterogeneity of gene expression between cells . James ehttp://xteam.xbio.top/CellMarker/) (23 August 2022) and SingleR (https://github.com/dviraran/SingleR) (23 August 2022), etc. Development is driven and controlled by temporal and spatial changes in gene transcription, followed by translation of the resulting messenger RNAs (mRNA) into proteins . AlthougIn recent years, the number of articles using scRNA-seq to analyze the process of hair follicle differentiation has also increased. Khusali et al. used scRNA-seq to distinguish different transcription states in embryonic skin, deduced the transcription state sequence passed by dermal condensates (DC) cells, and found the inference path of molecular state leading to DC cell differentiation, and it revealed that the maturation of DC-related transcription required the conduction of Wnt/β-catenin signal, and clarified that DC cells were descendants of DC progenitor cells highly propagated at telogen . Mok et Rie et al. used scRNA-seq to identify the unique cell types from follicular-enriched scalp grafts in human hair follicles, and 23 primary cell clusters were obtained, and association of specific cell subsets with known molecular characteristics of common skin diseases was explored; they confirmed previous murine and human studies and provided new insights into the differentiation and pathogenesis of the epidermis and hair follicles . The scRChae et al. revealed by scRNA-seq that activation of the Sonic hedgehog pathway regenerates a renewable dermal niche called the dermal papilla, explaining its necessity and adequacy for new hair follicles, and revealing that activation of Shh signaling in Wnt active cells promotes the fate of the dermal papilla in scar wounds . Ahlers The scRNA-seq technology plays an important role in discovering new cell types, using transcriptome differences between different cells to discover new cell types that have not been previously discovered. Especially in the case of a small number of cells, scRNA-seq has great advantages in analyzing potential small or rare cell groups. Transcriptome analysis of a single cell greatly promotes the dissection of gene expression networks in rare cell types, and, more importantly, helps to identify new cells in these cell groups . MacoskoBefore the advent of scRNA-seq technology, the specialization process of DC was only generally divided into a stage; using scRNA-seq technology, a precursor stage of DC cells, namely pre-DC cell stage, was newly discovered . VorstanObviously, with the development of the sequencing technology, scRNA-seq technology has made significant progress in the past decade and has been applied to many fields. With the continuous improvement of the methodology, our understanding of cell interaction in hair follicle development has been enhanced, and impressive progress has been made. scRNA-seq enters a seemingly mature stage, but there are also some limitations. For example, scRNA-seq requires the preparation of single cells in cell suspension and the construction of a single cell library by single cell separation technology. Therefore, in this process, the spatial location information of cells will be lost, and spatial heterogeneity is the key feature of organ function. The location information of cells is very important for the study of the cell fate regulation mechanism and the cell lineage generation process. Therefore, although the scRNA-seq dataset tells cells what happened and the molecular relationship between them, we do not know whether the cells obtained by sequencing are closely linked or far apart in the original sample, or whether organ development is related to specific tissue structure or two specific cell distances . Althoug2 capture area, which can contain 1007 spots, the diameter of each site is 100 um, and the spacing is 200 μm [2 capture area, each defined by a fiducial frame + capture area is 8 × 8 mm2, with a diameter of 55 μm per spot [In order to simultaneously obtain the transcriptional heterogeneity and spatial location information of cells, spatial transcriptomics (ST) technology emerged. At present, there is a wide range of commercial space transcriptomics technology, such as ST technology of 10×Genomics company. This technology provides high quality transcriptome data and their complete two-dimensional location information by locating frozen tissue sections on special carrier chips, arranged with reverse transcription primers and a unique positioning bar code array. At present, ST technology has been used to study the spatial consistency of gingival tissue , heart ts 200 μm . Wu et aper spot .Space transcriptomics will become an important supplement to scRNA-seq in the future . TherefoThe scRNA-seq technology can identify cell genetic information from the single cell level, which provides a powerful tool for identifying the transcriptome characteristics of various cell types in heterogeneous populations. With the continuous progress of single-cell sequencing research, we are unveiling the veil of cell fate selection and life occurrence. Analysis of cell genomes or transcriptomes at the single cell level has enabled biological research to reach unprecedented levels of resolution and scale, which is transformative in extremely complex hair follicle systems. Indeed, scRNA-seq can provide new perspectives and methods for life science research. The continuous development of scRNA-seq and its combination with multi-domain technologies and algorithms will bring a new revolution to next-generation genome sequencing. Together this will become an important tool for studying the type and state of hair follicle cells and help to develop reasonable methods for the treatment of future hair loss diseases, as well as to analyze the mechanism of hair follicle morphogenesis.


Southeast Asia is considered a global hotspot of emerging zoonotic diseases. There, wildlife is commonly traded under poor sanitary conditions in open markets; these markets have been considered ‘the perfect storm’ for zoonotic disease transmission. We assessed the potential of wildlife trade in spreading viral diseases by quantifying the number of wild animals of four mammalian orders  on sale in 14 Indonesian wildlife markets and identifying zoonotic viruses potentially hosted by these animals. We constructed a network analysis to visualize the animals that are traded alongside each other that may carry similar viruses. We recorded 6725 wild animals of at least 15 species on sale. Cities and markets with larger human population and number of stalls, respectively, offered more individuals for sale. Eight out of 15 animal taxa recorded are hosts of 17 zoonotic virus species, nine of which can infect more than one species as a host. The network analysis showed that long-tailed macaque has the greatest potential for spreading viral diseases, since it is simultaneously the most traded species, sold in 13/14 markets, and a potential host for nine viruses. It is traded alongside pig-tailed macaques in three markets, with which it shares six viruses in common . Short-nosed fruit bats and large flying foxes are potential hosts of Nipah virus and are also sold in large quantities in 10/14 markets. This study highlights the need for better surveillance and sanitary conditions to avoid the negative health impacts of unregulated wildlife markets. Infectious diseases are responsible for more than 7 million deaths annually, causing negative impacts on global health and substantial economic losses ,2. SevenGlobal trade and commerce, including wildlife trade, are recognized as key factors to the increase in emerging viral infectious diseases ,7,8. SpeIn recent decades, increased global human population and recently established domestic and international travel networks have escalated the commercialization of wildlife, creating a situation in which the extent and velocity of zoonotic pathogen movement are historically unmatched ,18. MuchPaguma larvata) for sale in wildlife markets but were not present in farmed civets . Informal conversations with sellers indicate that most of the animals must have been collected within Indonesia, mostly on Java, Bali and Sumatra, but also Borneo and possibly Sulawesi. None, or very few may have come from abroad.We recorded 6,725 wild animals of at least 15 species within the order Rodentia, Chiroptera, Carnivora and Primates for sale in the 14 wildlife markets ; Table 2p-value = 0.004) and the number of market stalls selling wildlife   or number of stalls in the market  with taxa richness on sale.The mean number of individuals offered for sale showed a positive trend related with the human population in the respective city  . ConversAt least eight (53%) out of 15 animal taxa recorded for sale are hosts of virus species listed in the database consulted for known zoonotic diseases . We recoTrachypithecus langurs are a host of Dengue virus. No virus was reported for the two recorded squirrels (Prevost’s squirrel and plantain squirrel) and two civets  on trade, neither for Asiatic small-clawed otter (Aonyx cinereus), Javan ferret badger  and slow lorises.The taxa with the highest number of zoonotic viruses recorded are both macaques; the long-tailed macaque, a potential host of 9 viruses , including Cowpox, Dengue, Hepatitis E, Herpes B, Monkeypox, Reston, Ebola, Simian Foamy, Simian retrovirus type D and Vesicular stomatitis viruses; and southern pig-tailed macaque, with 6 (35%) virus species, including Cowpox, Dengue, Herpes B, Simian Foamy, Simian retrovirus type D and St. Louis encephalitis viruses. Short-nosed fruit bats also stand out by being potentially infected by 5 (29%) of the viruses recorded, but different species from those infecting macaques, which include Influenza A, Issyk-Kul, Japanese encephalitis, Kyasanur forest disease, and Nipah viruses. Of the remaining mammal taxa sold, masked palm civet is a host of SARS-CoV (and SARS-CoV related) virus, Javan leopard cat is a host of Influenza A virus, Javan mongoose is a host of Hepatitis E virus and Rabies, large flying fox is a host of Nipah virus, and The Sankey network diagram shows that long-tailed macaque is the species with the greatest potential for spreading diseases , since iIn addition, long-tailed macaques are traded simultaneously with pig-tailed macaques in three markets, with the potential to spread six diseases in common . Indonesian short-nosed fruit bats and the large flying fox are also sold in large quantities and traded together in 10 markets, increasing the potential for the spread of Nipah virus. Furthermore, short-nosed fruit bats and the Javan leopard cat are traded in four markets in common and are potential hosts of Influenza A.Using the widespread and open trade in live wild mammals in markets on the two main islands of Indonesia as our case study, we show that live wildlife markets may provide optimal conditions for the spill over and spread of viral diseases. More populated cities and larger markets had larger quantities of animals being sold, and those same markets had on sale several mammal species that potentially share viruses in common. In addition, some of the most recorded species, such as long-tailed macaque and short-nosed fruit bats, are hosts of several virus species and are traded in large quantities across almost all markets, posing a risk of disease outbreaks to millions of people inhabiting those highly populated islands. These findings are of great concern in terms of public health, because there is a higher likelihood for an infected animal to end up in the market of a city where (i) there is a wider range of species, increasing the chance of a spill over, and (ii) that is more populated by humans, increasing the chance of fast pathogen spread and consequently for outbreaks and epidemics to occur. The number of zoonotic viruses potentially hosted by the traded species (17) in this study is very similar to the number (16) identified in wildlife traded as wild meat in Malaysia ; of thesIt is of great concern that the recorded virus species are potentially circulating in wildlife markets in Southeast Asia, since among them are some viruses responsible for causing serious and deadly diseases to humans. Nipah virus, in particular, is a bat-borne virus that has caused severe disease outbreaks in Asia, with mortality rates reaching over 90% in some cases, consisting of one of the deadliest viruses affecting humans . This viTrachypithecus langurs. In Indonesia trade in species that are not legally protected is regulated through a harvest quota system [A number of species we encountered in the markets are included on Indonesia’s list of protected species and no wild-caught individuals are allowed to enter the trade. These include the Javan leopard cat, slow lorises and a system . For mamWhile the illegal trade certainly aggravates the potential of wildlife markets in spreading diseases, the legal trade, if not properly monitored, also poses a similar threat to humans. Reducing this threat is not a simple task, once ownership, consumption and trade of wild species are usually part of the local culture, play important roles in local economy and occur more frequently in developing, but megadiverse, countries where surveillance is often insufficient ,45. It iSanitary measures should focus on the most affected species and largest markets and cities. For instance, the most commercialized species in our study, the long-tailed macaque, a potential host of nine virus species, has been recently classified by IUCN as Endangered , especiaIn 2021, the World Health Organisation (WHO), the World Organisation for Animal Health (OIE) and the United Nations Environment Programme (UNEP) have issued guidance to help reduce public health risks associated with the sale of live wild mammals , prompteIn addition to wildlife hunting and trade, the modification of environments by deforestation, intensification of agricultural production and urbanization increases the possibility of interspecies transmission due to higher probability of contact between humans and animals ,55. In fIn this study we highlight the need for better surveillance and sanitary conditions to avoid the negative health impacts of unregulated wildlife markets. More than half of the species traded in wildlife markets in Indonesia are in fact hosts of zoonotic virus species. This study could be used in the development of public health strategies in Southeast Asia, such as implementing sanitary measures and standards in wildlife markets. This information is also useful to develop awareness campaigns to educate people about the numerous health risks from trading or buying wildlife highlighted, encouraging them to buy wildlife that are legally sourced and surveilled for pathogens, or the consumption of alternative foods whenever possible. Such initiatives could have additional benefits for the conservation of threatened species by helping reduce the illegal domestic and international trade of species in and from Southeast Asia .