Illicit drug use is growing among young people, which is one of the major problems in today’s society that can be associated with many medical issues, including infertility. Amphetamines, cocaine, opioids, and marijuana are the most common and the most used illicit drugs worldwide. The purpose of this review was to collect as much literature as possible about the impact of illicit drugs on male fertility and summarize their valuable data. Original studies and reviews were collected by searching the keywords “illicit drugs  and male infertility”. The obtained information was also categorized based on the content of the “Infertility in the Male” book. Almost all studies suggested that taking all kinds of illicit drugs with the effects on different parts of the male reproductive system can result in subfertility or complete infertility in the consumers. Although the data in this field are not decisive and there are some confounding factors in human studies, it can be inferred that the use of any illicit drug with an effect on male sexual health reduces fertility potency. Therefore, it is recommended that couples, who are planning to conceive, avoid taking any illicit drugs before and during treatment. Infertility is defined as failure to achieve a pregnancy after a year of unprotected intercourse. Considering the growing number of infertile couples, and psychological, social, and economic problems stemming from it, this social-medical condition has intrigued researchers in recent years. According to the latest reports, more than 50 million couples worldwide suffer from infertility. World statistics suggest male infertility is 2.5–12 percent, which accounts for 20–70 percent of couples , erectioFor writing of this manuscript, we made a list of relevant keywords and phrases and then started searching studies in PubMed, Scopus, and Web of Science databases. The keywords that we used were: Illicit drugs, Male infertility, Male reproductive system, Amphetamines, Cocaine, Opioids, and Marijuana. In this review study, the effects of various illicit drugs on each of the infertility factors were elaborated separately.Primary testicular disease, the most common cause of male infertilityIn contrast to women, in men, gametogenesis defects are generally due to damage to the testicular tissue and are rarely associated with pituitary dysfunction. Primary testicular dysfunction results in reduction or complete stopping of spermatogenesis . DecreasIt should be borne in mind that the disease is incurable and there is no known treatment to increase sperm cell numbers or change these conditions. Another important feature of the disease is the progressive process of decreasing quantity and quality of spermatozoa; azoospermia may occur in cases of severe oligospermia due to primary testicular dysfunction . TherefoDeficiency in Leydig cell functionThe alterations in Leydig cell activity is one of the issues discussed concerning the primary testicular disorder.Amphetamines The Ras signaling pathway, which is the most important hormone transport pathway, is blocked by protein kinase A. Increased cyclic adenosine monophosphate (cAMP), which acts through this protein kinase in the testis of the amphetamine-treated rats, suggested the hypothesis of inhibition of testosterone transduction and release from Leydig cells in this group of rats .Opioidset al. (1999) with the expression of healthy Leydig cells in the testis tissue of young (30–33 days old) Wistar rats treated with morphine showed that the decrease in testosterone was due to impairment of the Hypothalamus-Hypophyseal-Testicular (HPG) axis, and testosterone is reduced through the brain’s opioid system  included a report on the normal structure of seminiferous tubules  and spermatogenesis . The curCocaineRats treated with cocaine were observed with deficient spermatogenesis, abnormal seminiferous tubes, cellular destruction, cell sloughing, and abnormal cellular structure . In humaMarijuanaA 2 mg/kg injection of delta-9-tetrahydrocannabinol (THC) to rats caused reduction in enzymatic functions of Sertoli and interstitial cells. The fact that testicular functions are deranged due to THC-induced decrease of gonadotropin level is supported by gonadotropin treatment which leads to improved enzyme functions of testicles . InjectiOpioidset al. (2013) reported disturbing gametogenesis in male opioid addicts  system and some other systems . AccordiOpioidset al. (2013) determined reductions in testosterone, free testosterone, and LH, plus a constant level of FSH measured in opioid-addicted men  HPcted men . Opioidscted men . Althougcted men .The diversity among results could be attributed to the age, lifestyle  , clinicaAccording to these factors, it could be concluded that despite the prominent adjusting role of gonadocorticoids on sexual activities, the variable depends on uncontrollable factors and its alteration will not provide a convenient sexual health measurement. Abnormalities found in other organs of the male reproductive systemThe male reproductive system consists of testicles, an associated duct system, accessory sex glands, and the penis .Abnormalities of accessory sex glandsThe accessory glands of the male reproductive system include seminal vesicles, prostate, and bulbourethral glands . Few stuMarijuanaTHC reduces the semen-production-related enzymatic actions of the prostate in a dose-dependent manner . Erection and ejaculation disordersAccording to the reports, ejaculation disturbances are found in 20–30% of men, which is common among infertile men . This diAlthough it’s most likely that hormonal suppression is the main reason behind the change in sexual behaviors of the addicted men, erection disorders could play the main role in reduced sexual behavior . ErectioMarijuanaAdverse effects of cannabinoids on primates include reduced sexual arousal and erection disorders which remain even after withdrawal. In human studies some authors have demonstrated that cannabis was related to premature ejaculation , reducedOpioidsIncreased intromission was observed in morphine-treated rats as an inhibitor effect of opioids on sexual reflexes . In humaMicrobiology of semen and infections of the male reproductive systemInfections in the male reproductive system may cause damage to male fertility directly or to the testis, the vas deferens, and accessory sex glands . ClinicaOpioidsFew reports have been presented microbiological assessments on addicted men  plus leuSemen analysisA semen analysis or seminogram studies the most important characteristics of semen and sperm which include sperm motility, concentration, and morphology standardized by WHO. The analytical results are affected by sampling methods and measurement accuracies . The impMarijuanain vitro exposure to THC indicated decreased progressive motility and spontaneous acrosome reaction in human sperm  will increase the number of immotile sperm cells, while naloxone (antagonist of µ receptors) induces an increase in motile sperm cells . Heroin Enkephalin degrading enzymes Two significant enzyme pathways that hydrolyze enkephalin consist of glycine-phenylalanine hydrolase performed by enkephalinase and cleavage of the tyrosine-glycine bond by N aminopeptidase. The activity of these two enzymes is much higher in the semen compared with the rest of the tissues. The presence of aminopeptidases N in the head, neck, and along the tail of the sperm is identified as well as in different fractions of semen. However, enkephalinase is found in a very restricted area of a few sperm cells and fractions of semen. The inhibition of the activity of the enzymes will reduce sperm motility .OpioidsA study reported reduced transcript and protein of aminopeptidase N and reduced mRNA expression level of enkephalinase in mature sperm cells of heroin-addicted men. The reduction of aminopeptidase N was found to be relevant to the duration of addiction . The limMembrane channelsIonic channels, calcium channels, in particular, have a significant role in various stages of mammalian fertilization including sperm capacitation, acrosome reaction, and sperm motility . An examOpioidsDecreased expression of CatSper 1-4 mRNAs and relation with the dose-dependent motility of mice treated with Iranian Kerack was demonstrated . AccordiSperm nucleusAccurate chromatin packaging can affect morphology and motility of sperm cells. Compared with the somatic cell nucleus, the sperm nucleus is tenfold compressed, mediated by histone protein replacement by protamines. Factors affecting the procedure include histone deacetylase enzymes (HDCA) which are included in the decondensation of chromatin , along wOpioidset al. have studied the male infertility factors at the molecular level as pioneers, considering the health of sperm heads (the sperm nucleus) and protamine gene expression and proteins, particularly. Reduced gene expression and protamine-2 protein in sperm cells indicate heroin affecting protamine production. Similarly, increased expression level of HDAC1, decreased HDAC11 transcripts, and increased DFI% in male heroin addicts, indicate that alterations in the HDAC1 expression level will induce abnormal decondensation of the sperm chromatin in heroin consumers, thus increasing the risk of DNA damage (DFI). The increasing DFI% being directly related to the number of HDAC1 transcripts has confirmed the suggested hypothesis. There are few reports regarding the deacetylase functions of HDAC11. The ability of this enzyme to deacetylase histone directly has not been yet confirmed  are considered one kind of the most important non-coding small RNAs present in the RNA profile of semen which regulate the gene expression in spermatogenesis, taking part in successful fertility with transport to the oocyte during fertilization . RecentlOpioids The study conducted on male heroin addicts indicated an increased miR-122 expression level and its relation to the decreased protamine-2 transcripts, along with a decreased miR-125b-5p expression level and its relation with increased HDAC1 transcripts. The results of these studies revealed that heroin can affect the Ribonucleic acid content of semen; also miRNAs have a regulating role in gene expression as an epigenetic factor.Narcotic drug use in childhood, pre-adolescent, juvenile, and adulthood of men may lead to male infertility. While clinical studies are limited by legal and ethical restrictions and multiple drug consumption, structural and functional impairment of testis, endocrinal disorders, changes in sexual behavior and semen analysis, as well as molecular alterations of semen and spermatozoa have been reported in human and animal studies  and 5. T


The distinct molecular and biological properties of exosomes, together with their abundance and stability, make them an ideal target in liquid biopsies for early diagnosis and disease monitoring. On the other hand, in recent years, nanomaterial-based optical biosensors have been extensively investigated as novel, rapid and sensitive tools for exosome detection and discrimination. The scope of this review is to summarize and coherently discussed the diverse applications, challenges and limitations of nanosensors based on surface-enhanced Raman spectroscopy (SERS) as the optosensing technique.Exosomes are emerging as one of the most intriguing cancer biomarkers in modern oncology for early cancer diagnosis, prognosis and treatment monitoring. Concurrently, several nanoplasmonic methods have been applied and developed to tackle the challenging task of enabling the rapid, sensitive, affordable analysis of exosomes. In this review, we specifically focus our attention on the application of plasmonic devices exploiting surface-enhanced Raman spectroscopy (SERS) as the optosensing technique for the structural interrogation and characterization of the heterogeneous nature of exosomes. We summarized the current state-of-art of this field while illustrating the main strategic approaches and discuss their advantages and limitations. In the impending decades, cancer is set to become a major cause of morbidity and mortality across all regions of the globe , with an3, which approximatively contain 109 cells .,20.19,20The pronounced molecular and size heterogeneity of exosomes, even for vesicles originating from the same parental cells, confers a central role to isolation methods in (i) separating exosomes from potentially interfering protein aggregates, lipoparticles, viruses and cell debris in cell culture supernatants or bodily fluids, and (ii) discriminating different exosome subpopulations that could be related with different pathological states and stages of disease progression . The gol10–1011 [d, from the metallic surface (the decay is ∼1/(a + d)12 for a nanosphere of radius a) [In recent years, nanomaterial-based optical biosensors have been extensively investigated as novel, rapid and sensitive tools for exosome detection and discrimination ,40,41. W10–1011 . Thus, S10–1011 ,51,52 foadius a)  synthesiadius a) , the laradius a) ,56 and, adius a) . This laadius a) . NotablyMost studies aimed at correlating the intercellular signalling and pathological responses of exosomes with their composition focused on analyzing their respective nucleic acid cargoes which often mirror the phenotypes of their parental cells ,28,60,61Broadly speaking, SERS detection approaches can be classified into two main configurations: direct and indirect schemes. In direct SERS, the signal read-out is provided by the acquisition of the intrinsic SERS spectrum of the analyte, which contains a wealth of structural information representative of the molecular structure and composition. This label-free analysis can be carried out with simple and inexpensive plasmonic materials. However, it is usually restricted to the interrogation of relatively pure samples to prevent competing co-adsorptions of other molecular species that could undermine the correct interpretation and reliability of the final SERS spectrum. Moreover, direct SERS characterization of large biomolecules or supramolecular structures such as exosome poses important challenges in terms of understanding and interpretation of complex and often highly similar vibrational patterns. On the other hand, indirect approaches are designed to monitor the extrinsic SERS signal of molecular labels for detection and quantification of the target species. The most common indirect strategy relies on the use of SERS-encoded nanoparticles (or SERS tags) combined with surface ligands for selective recognition , as optical probes performing similar functions as fluorescent labels . AlthougThe acquisition of intense, well-defined and reproducible vibrational spectra is key to use direct SERS for sensing purposes. Overall, several factors determine the final intensification and ultimate spectral profile  of the vibrational fingerprint. Among those, we can identify inherent features of each element of the SERS analysis, such as the optical properties of the plasmonic material, the Raman cross-section of the target analyte and the experimental set-up  . On the In the liquid phase, the close contact between the analyte and the plasmonic surface is commonly achieved by exploiting the intrinsic chemical affinity of the molecule for gold or silver surfaces, mainly via the formation of metal-O, metal-N, and metal-S bonds  or via electrostatic interactions. Alternatively, molecular adhesion can be forced via physical evaporation of the sample solution onto the plasmonic substrate. In this regard, it is worth stressing that for large biomolecules, such as the exosome components  and even further to micro-entities such as cells, a transition from a hydrated to a dried state often results in major structural alterations that usually increase the intra-sample spectral variability ,104. AllIn the specific case of exosomes analysis, several additional features further increase the complexity of common direct SERS analysis. Firstly, the complex composition of exosomes, which mostly includes a large fraction of biomolecules with typically weak spontaneous Raman scattering , intrinsically generates an intricate vibrational spectral pattern comprising overlapping and often broad features. As a representative example, in Secondly, the size range itself of exosomes (ca. 30–150 nm) poses additional challenges. In fact, exosomes are large enough to prevent their optimum trapping into nanometric plasmonic gaps (hot spots) capable of concentrating extremely high intense EM fields in the whole analyte volume. Thus, the design and choice of the plasmonic substrate and experimental set-up analysis face two contrasting needs: (i) the necessity to expose exosomes to high electromagnetic enhancements to improve the amplification of their relatively weak Raman scattering; and (ii) the importance to immerse the vesicle in a relatively uniform electromagnetic field so that to minimize heterogeneous enhancements of random portions of exosomes due to different spatial arrangements onto the metallic surface. Indeed, such uneven exposures may lead to high spectral variability even within the same population of exosomes, an outcome that can be further aggravated by the heterogeneous distribution of the diverse molecular components on the exosome surface. Clearly, the reproducibility issue becomes particularly relevant when the SERS spectra are acquired from single/few exosomes rather than large ensembles of particles . For instance, Russo et al.  observedExosome sizes are, on the other hand, typically too small to facilitate single-particle Raman analysis. This explains why, for instance, single-cell Raman spectroscopy is a well-established and relatively straightforward tool for in vitro and in vivo interrogation of individual living cells while Raman characterization of individual/few exosomes is very limited and requires complex technologies such as exosome trapping via optical tweezers ,106,107.Finally, as the electromagnetic enhancement commonly declines very rapidly within few nanometers from the metallic surface, direct SERS analysis of whole exosomes yield spectra that are mostly dominated by the vibrational features of the molecular components of the outer membrane (mainly sugars and proteins). Thus, SERS spectra substantially disregard the lumen content as compared to normal Raman scattering of whole EVs, where nucleic acids contributions are distinguishable in the vibrational pattern . While t8 particles/mL). Subsequently, they identified 13 bands that correlated well with the relative exosome content and which were used as Raman markers for non-small-cell lung carcinoma (NSCLC) derived exosomes  with high spectral resolutions to maximize the collection of intense signals is feasible but is typically a rather time-consuming process unless state-of-the-art techniques  are used . A conve2 macroporous inverse opal structure  ,117,118 A large variety of SERS-encoded particles (SEPs), or also referred to as SERS tags, with different structural and chemical features has been reported in the literature ,120,121.−1 for MBA, DTNB and TFMBA, respectively) indirectly inform about the relative expression levels of surface protein biomarkers. The phenotype signature was expressed by normalizing the corresponding Raman signals (IEVs) and SEPs (Inanotags) using the same concentration of conditioned exosomes. The study was also repeated with C3 (bladder cancer) and SW480 -derived exosomes. 6 particles/mL in PBS, which is below the average exosome concentration in most body fluids (ca. 108 EVs/mL and above), thereby meeting the clinical requirements.In the indirect SERS analysis of exosomes, SEPs are conjugated with recognition elements that promote their selective accumulation at the surfaces of the vesicles. To facilitate the SERS interrogation, capturing substrates are also integrated into the sensing system to enable the separation and accumulation of the SEPs-decorated exosomes into a small area for ultrasensitive detection. Typically, capturing substrates consist of magnetic beads ,97,122 o2-coated magnetic particles, which allowed the indiscriminate, rapid and highly efficient removal of the exosomes from serum by exploiting the affinity of TiO2 for binding the hydrophilic phosphate head of the exosomal phospholipids. In their study, the programmed death ligand 1 (PD-L1) protein biomarker on the exosomal membrane was subsequently targeted with SEPs modified with an anti-PD-L1 antibody  and NSCLC patients of early (7) and advanced stages (10). The use of immunoaffinity magnetic beads for separation/enrichments of exosomes from biofluids is, however, often affected by issues related to limited reproducibility, long incubation time and low exosome yields (<50%) . On the antibody A. ExosomAlternatively, Trau and co-workers  tackled Increasing the density of biologically functioning antibodies at the capturing surface is also a central factor for improving the efficiency and sensitivity of the immunoassay. In this regard, Li et al.  reportedIn this review, we summarized and coherently discussed the diverse applications of SERS in the analysis of exosomes, with a special focus on the more recent and promising advances. We have also progressively highlighted current key challenges and limitations, which can be broadly associated with either the general application of SERS in biosensing and clinical diagnostic or the specific nature of exosomes as the biological target. In the first case, the translation of SERS-based analytical tools into competitive, commercial devices still faces important practical obstacles such as the production of cost-effective, robust and efficient plasmonic substrates at a large scale. Similarly, the fabrication of affordable and portable Raman spectrometers for fast data acquisition is critical for lowering the cost while providing manageable equipment for routine analysis in the clinical setting. In this regard, the integration of SERS substrates and Raman components into multifunctional platforms  is also pivotal for automatization and efficient standardization of the measuring procedures. Furthermore, as also stressed in the review, the efficient implementation of the most advanced chemometric tools appears to be the way to fully access the multidimensional information contained in large SERS data set. On the other hand, the intrinsic nature of these vesicles makes exosome-based diagnostics a difficult task, mainly due to their pronounced molecular heterogeneity and the requirement of determining the presence and relative distribution of different sub-populations, especially in complex biofluids. As pointed out, both improvement and standardization of the isolation procedures are needed to reproducibly supply exosomes with high purity in good yields, while identification of a much broader set of potential biomarkers is mandatory for enabling clinical applications.


Heterocyclic aromatic amines (HAAs) are potent carcinogenic compounds induced by the Maillard reaction in well-done cooked meats. Free amino acids, protein, creatinine, reducing sugars and nucleosides are major precursors involved in the production of polar and non-polar HAAs. The variety and yield of HAAs are linked with various factors such as meat type, heating time and temperature, cooking method and equipment, fresh meat storage time, raw material and additives, precursor’s presence, water activity, and pH level. For the isolation and identification of HAAs, advanced chromatography and spectroscopy techniques have been employed. These potent mutagens are the etiology of several types of human cancers at the ng/g level and are 100- to 2000-fold stronger than that of aflatoxins and benzopyrene, respectively. This review summarizes previous studies on the formation and types of potent mutagenic and/or carcinogenic HAAs in cooked meats. Furthermore, occurrence, risk assessment, and factors affecting HAA formation are discussed in detail. Additionally, sample extraction procedure and quantification techniques to determine these compounds are analyzed and described. Finally, an overview is presented on the promising strategy to mitigate the risk of HAAs by natural compounds and the effect of plant extracts containing antioxidants to reduce or inhibit the formation of these carcinogenic substances in cooked meats. Our diet contains meat as a significant food that contributes valuable constituents and nutrients such as all essential amino acids, high-quality proteins, vitamin B6, niacin, iron, vitamin B12, phosphorus, zinc, and selenium, as well as other biologically active materials, including carnosine, taurine, carnitine, glutathione, creatine, and ubiquinone, that are considered beneficial for human health ,4. Meat Heterocyclic aromatic amines (HAAs) can be defined as a main class of compounds that are potentially carcinogenic and extracted from proteinaceous foods during flavor forming and processing using heat . ProfessNevertheless, numerous epidemiological studies have demonstrated the relationship between the consumption of meat and an elevated risk of occurrence of different types of cancers  due to tStudies have demonstrated that regular intake of grilled meat with HAAs may enhance the risk of cancer in different organs of humans . It has Products from natural sources have been used for centuries ,27. PlanThe existence of HAAs in sufficiently heated food matrices and the monitoring of the negative effects of these toxic compounds on human health and the inhibition of their existence in cooked food is an obligatory requirement. This review demonstrates the delivery of detailed information about HAAs that is based on the knowledge from proceedings to advanced studies. Numerous pretreatments and methods are applied in different samples of food and clarify the different techniques for the analysis, determination, and isolation of HAAs. Finally, an overview is presented on a promising strategy to mitigate the risk of HAAs by natural compounds and the effect of plant extracts containing antioxidants to reduce or inhibit the formation of these carcinogenic substances in cooked meats.The identification of HAAs in various foods, such as cooked fish and meats, products of meat, extracts of meat , coffee Quantities of HAAs vary in the case of meat from different animal species, for example, fish, poultry, pork, and beef. For example, a great occurrence of PhIP  and IFP  in poultry has been observed while more MeIQx  is observed in pork, fish, and beef . It is wGibis and Weiss  conducteThe amines of heterocyclic compounds are divided into groups of carcinogenic and mutagenic compounds that are produced on the surface of foods rich in protein, such as fish and animal meats , and resReducing sugars, creatinine, and phenylalanine can be distinguished in the formation of PhIP. In these studies, scientists have identified the reaction, which leads to the formation of PhIP, by following steps through a model experiment. Firstly, the formation of phenylacetaldehyde occurs from phenylalanine through the degradation of the Strecker reaction. Then, the formation of phenylacetaldehyde is complete during a reaction with aldol and creatinine to develop an additional aldol product. PhIP is formed from the condensation of aldol by a subsequent condensation reaction . Figure Reducing sugars, such as fructose and glucose, are produced directly by sucrose hydrolysis of free amino acids and creatinine, available naturally in muscular mammalian tissue and referred to as the potential precursors, accountable for HAA formation. Moreover, various precursors such as creatinine, free amino acids, nitrogenous bases, sugars, protein, and nucleosides are produced at different levels of raw material processing . The varHAAs are not affected by the variations in pH in the actual sense. HAA precursors are sensitive to pH changes by which the formation of HAAs can ultimately be affected. Enhanced pH levels can modify the formation pathways of HAA precursors and lead to minimum HAA production. HAAs are sensitive to the contents and oxidation of lipids, and their formation is also affected by the availability of antioxidants .Polak et al.  conducteIn addition, the quantity of meat fat has a noticeable impact on the formation of HAAs, and it is believed that HAA concentration is lower in fat-rich meat than other kinds of meat. The transportation of high heat is required in fat-rich meat to enhance the time of cooking compared to lean meat . The infIt is hypothesized that the higher concentrations of free amino acids and total creatine are linked to the development of HAAs in more quantity during the treatment of thermal heat; a correlation that was quite strong was noted between these features. Despite the detailed fact that the concentration of HAAs somehow originates from reducing sugars, only frail interdependence was attained from these parameters . It was Skog  observedHAAs are considered analytes that are hazardous, and, therefore, they are categorized into two major groups according to their reactions of formation. They can be grouped into pyrolytic and thermic HAAs. Thermic HAAs can be produced by free amino acid reactions containing hexoses and creatinine at temperature ranges between 100–300 °C. They are also recognized as aminoimidazoazarenes and the IQ type. Pyrolytic HAAs are produced at temperatures above 300 °C, and pyrolytic reactions are applied between proteins and amino acids for the formation of hazardous compounds ,68.In another survey, HAA formation was detected diversely. The study described that HAAs are divided into two major groups known as polar HAAs (IQ type) and non-polar HAAs (non-IQ type), depending on their structure, temperature, and formation pathways . The groFive-membered amines of a heterocyclic nature;Six-membered amines of a heterocyclic nature.In another study by Sahar et al. , HAAs arFive-membered amines of heterocyclic compounds include five ring atoms. The structure of these cyclic compounds is comprised of four carbon atoms and one nitrogen atom. The main examples of these compounds are pyrrole and nicotine. The six-membered structure of amines of heterocyclic compounds show more similarity towards benzene; the difference is where nitrogen substitutes the carbon atom in the structural ring .Abbreviations, names of compounds, structure, molecular mass, chemical formulas, and the non-polar and polar presence of HAAs in different varieties of foods are mentioned in Progressive extraction procedures and instrumental methods have been applied for the detection and separation of HAAs. According to the kind of preparation of the sample, the experiment’s sensitivity, and the choice of systematic equipment, laboratory, and accessible facilities will be dissimilar .Colorimetric procedures have been established for the measurement of amines, including biogenic compounds and copper ions. This experiment includes a detection limit of about 5 mg/kg and enumerates the amines of biogenic compounds by 496 nm value of absorbance with the assistance of a spectrophotometer. The procedure has the benefit of considering only 45 min of analysis. Furthermore, the method is also determined as a method of cost-effectiveness and does not need specialized laboratory equipment .Fluorescence synchronous spectroscopy has been utilized to enumerate the quantity of numerous HAAs in meat samples that were heated by the measured condition . The mosYan et al.  anticipaIn recent studies, nine retrievals for HAAs were more than 50% extra (51–68%), although 1-mehyl-3-amino 5H-indole pyrido, 3-amino 1,4-5Hdimethyl- indole pyrido, PhIP, 2-amino-9H-pyrido-indole, and (MeAαC) 2-methyl-amino-3-9H-indole-pyrido had the range of 23%–32% . In additional recent studies, 15 targeted recoveries of HAAs were ranged between 35%-70%, commonly consumed in pan-fried beefsteak. Among the 15 HAAs targeted, the rates of recovery for 4,8-trimethyl-2-amino-3 quinoxaline imidazo  and (Norharman) 9H-pyrido-indole reached 42.7% and 35.9% correspondingly . SubsequThe salmonella or Ames test demonstrates that maximum HAAs are extremely mutagenic , and HAAAccording to the inspection description of the National US Program of Toxicology, HAA uptake ranges above 17 ng/kg of body weight show a strong correlation with the intake of meat-based cooked products. The continuing survey also commented that individuals who consume products of meat are mostly accountable for HAA accumulation in the body . The exp−1 [The Europe Council advises that the quantity of HAA consumption must have a lower quantity than 1 µg per day . Althoug−1 . Frequently, cases of colon cancer are reported to be about 7–9% due to red meat consumption with HAAs . RohrmanVarious risks are linked with the intake of HAAs; decreasing the exposure of HAA development by diminishing proteinaceous foods, cooking with reduced cooking time and temperatures , and a rNumerous studies have exposed the effects of inhibition with synthetic antioxidants (butylated hydroxytoluene (BHT), butyl tertiary hydroquinone , butylatPomegranate seed extracts , olives The reduction in the degree of HAAs seems to be based on the concentration and potency of the plant extracts as well as extractions of HAA concentration in the samples of meat. The results show that except for the determination of the property of antioxidants, which is an important feature in the reduction of HAA formation, other features in the ingredients of marinades have a major contribution to the extraction of precursors from the HAAs at different concentrations, including amino acids and glucose that also influence the reduction or formation of HAAs . The inhTengilimoglu-Metin et al.  describeVitaglione and Fogliano  exposed Meat products consumed, including processed meat and red meat, may result in the incorporation of possible carcinogenic substances developed by lipid oxidation in the components of meat during cooking. Several studies have confirmed that natural products can cause a reduction in the occurrence of carcinogenic substances developed during meat lipid peroxidation. Natural products that are utilized to reduce the degradation or oxidation of meat components can inhibit the formation of carcinogenic substances after or during cooking, such as the free radicals involved in HAA formation . Vitaglione and Fogliano  describeAn antioxidant can protect against the toxicity of iron that chelates ferrous iron and inhibit its reaction with oxygen, peroxides, and chelated iron and trap already developed free radicals. Phenols available in olives rapidly decreased heme-induced peroxidation of lipids and scavenged radicals of  DPPH. Vitamin E can inhibit the damage induced by iron, and vitamin C can cause the reduction of free iron into ferrous iron, stimulating the propagation and initiation of free radical reactions . Gobert Several inhibitors have been found in the studies of the Maillard reaction in specific foods . The MaiBeta vulgaris) effectively reduced the Maillard reaction’s substance formation in meat and milk protein. The spices’ role, particularly garlic and onion, as HAA formation inhibitors, has been described [During treatments of heat, the Maillard reaction can decrease HAA formation. Račkauskienė et al.  discusseescribed . Garlic escribed . Schoch escribed  suggesteescribed . Maillarescribed . PossiblIn conclusion, heterocyclic aromatic amines are potential carcinogens that can lead to a number of different cancer types. Cancer risk is associated with a high intake of well-done cooked meats due to the induction of heterocyclic aromatic amines. Hence, it is necessary to inhibit or minimize the formation of these potent mutagenic and/or carcinogenic HAAs. There are a number of possible means to inhibit the heterocyclic aromatic amines, but, among the different mitigation techniques, the utilization of natural plant extracts containing antioxidants has shown promising inhibitory effects against HAA formation. The studies included in


Microbiological analysis is an integral part of food safety quality control and monitoring systems. Recent outbreaks, associated with consumption of a variety of contaminated foods, have highlighted the difficulty in monitoring critical points of contamination along the food chain. The rapid and precise detection (and/or quantification) of foodborne pathogens, used appropriately along the food supply chain, represents a powerful tool to prevent outbreaks of foodborne disease. With this purpose in mind, and particularly for short shelf-life foods, it is necessary to increase the arsenal of available methods and develop innovative and time-effective assays which are able to complete the analytical process in as short time as possible. In this context, the major benefit of molecular methods, as alternatives to classical culture-based methods, is the significant reduction of the time taken for analysis. However, basic molecular approaches have also some critical weaknesses, such as an inability to discriminate between viable and dead cells (or infectious and non-infectious virus particles), and susceptibility to inhibitory influences.In recent years, there have been several advances in molecular-based subtyping methods, particularly regarding the application of sequence-based techniques. Different molecular methods, including more recently next- generation-based approaches, are replacing traditional methods in identifying the source of outbreaks, improving trace-back studies, and elucidating the evolution of foodborne pathogens. However, all these techniques need further validation before being adopted internationally.This Research Topic encompasses different aspects regarding the application of molecular methods for the detection, quantification, and identification of foodborne microorganisms, including the proposition of innovative solutions. The Special Issue comprises 12 original articles, contributed by 111 authors from 14 different countries.The major challenge of molecular methods is their inability to differentiate between dead/infectious and viable cells/non-infectious microorganisms  over 6 weeks.Xu et al., which utilizes pathogen interaction with host cells to detect only live microorganisms. They exploited the ability of viable enteric pathogens to adhere to intestinal cells, harnessing an antibody-based assay for specific detection of the adhered target pathogens. The Authors also proposed an innovative approach to prolong the shelf-life of cells (for at least 14 weeks) for possible point-of-need deployment and multi-sample testing on a single plate.A very different approach is proposed by A new direct lysis method for the extraction of viral RNA from dates was developed and optimized to detect the virus, quantify the contamination level, and sequence the strain(s) detected.Detection of enteric viruses in food products differs from detection of most foodborne bacteria, as viruses cannot be enriched in a food sample. The pre-PCR processing step, including sample preparation and nucleic acid extraction, is a critical step in foodborne virus detection. The quality of the extracted nucleic acids can strongly affect the efficiency of detection by a molecular method. In 2018 an outbreak of hepatitis A, caused by contaminated dates, was identified in Denmark by Rajiuddin et al. The length of the enrichment time is a bottleneck for development of culture-based methods able to detect bacterial pathogens in the same day  for rapid identification of Listeria monocytogenes and Listeria ivanovii, and non-pathogenic Listeria in edible mushroom (Flammulina velutipes) samples after a short enrichment culture step (4–12 h).Real time PCR or qPCR has been widely used for the quantification of nucleic acids with high precision, but it can be inaccurate when targets are at very low concentrations. Droplet digital polymerase chain reaction (ddPCR), a relatively new and promising technology, has many advantages over qPCR, as it can provide greater sensitivity and accuracy. It can be used for ultrasensitive and absolute nucleic acid quantification without a standard curve  emerging and foodborne outbreaks in Europe], Uelze et al. The Authors report the results of a ring trial performed in private and public laboratories, sequencing DNA samples of three bacterial species , according to their routine in-house sequencing protocols. Four different types of Illumina and one Ion Torrent sequencing platform in ten different laboratories were involved in the study. The results show differences in data quality after assembling short reads into genome assemblies.The consistency, accuracy and reproducibility of next-generation short read sequencing, between different laboratories and platforms, has been evaluated by Wu et al. Five isolates of Salmonella were analyzed, and the results demonstrated the reduction of the cost of ONT sequencing of a single isolate per flow cell, the achievement of high coverage of the genome, and a high accuracy of prediction within 1 day. This study provides food industries, food authorities, and regulators with the alternative of short-read sequencing platforms for serotype prediction for pathogen surveillance.The bioinformatics pipelines of whole genome sequencing (WGS) data, generated by the long-read sequencing platform Oxford Nanopore Technologies (ONT), have been evaluated by Vibrio species in seafood.The major economic threat to the marine aquaculture industry, with regard to consumer health, is the presence of Vibrio species, using highly specific unique outer membrane proteins (OMPs), is described by Wang et al. The Authors have applied a bioinformatic tools for screening OMPs candidates, to minimize target numbers in “in vivo” tests.Development of an accurate immunodiagnostic assay of pathogenic The Research Topic also includes two extensive surveillance studies in which the Authors apply molecular methods for characterizing the isolated strains. Very importantly, both papers have a particular focus on antibiotic-resistant bacteria  in slaughterhouses and meat shops in Pakistan has been presented by Sadiq et al. The authors collected a total of 300 samples. Fifty per cent of the analyzed samples were positive for S. aureus by phenotypic identification, and 96 isolates (63%) showed resistance to the antibiotic cefoxitin, known as a potential marker for detecting MRSA. All 150 isolates showed complete resistance to four antibiotics–neomycin, methicillin, ciprofloxacin, and tetracycline.Finally, an extensive investigation of the prevalence of methicillin-resistant All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cycling is a popular global sport and method of transportation and a significant contributor to admissions to hospital emergency units following an injury. Mountain biking events present additional challenges with remote venues and isolated courses, for which on-site medical care is often provided, for both injury and illness occurring during races. National health data may not represent these unique events, and specific data on incidence of injury and illness in mountain biking events are essential. Therefore, the aim of this study was to review the available injury and illness literature, reporting methods and risk factors in cross-country mountain biking.Search engines PubMed, Scopus, CINAHL (EBSCOhost), Scopus, PEDro and the Cochrane Library were systematically searched, and a grey literature search was performed. Narrative analyses of the types, severity and area of injuries and illness type and severity were performed as pooling of data was impossible due to insufficient high-quality studies with the same injury and illness definitions.Seven studies comprising 28,021 participants were included for analysis. Four to 71% of participants sustained an injury in a cross-country mountain bike event. Injuries to the skin were the most common, followed by bony injuries and concussion. Five to 47% of cyclists reported the onset of gastrointestinal symptoms post-event. The prevalence of illness during events ranged from 0.5 to 23.0%.The injury and illness definitions were varied and prevented clear comparisons between studies. Injury and illness present a concern in cross-country marathon mountain biking and should be investigated further to provide the true burden of these during race events.Registration: This protocol has been registered with PROSPERO International prospective register of systematic reviews (No: CRD42019134586). Illness and injury present significant concern in cross-country marathon mountain biking for both elite and amateur cyclists.The true burden of these injuries and illnesses is difficult to determine due to varied reporting methods and definitions.Further research in injury and illness epidemiology, using standardised and current definitions, is required in cross-country marathon mountain biking.Cycling is an increasingly popular sport and is used as both a recreational activity and a form of transport globally . CyclingAs participation in cycling has increased, conflicting evidence on the incidence of injury has emerged , 5. In tMost data on cycling injuries report cases presenting to emergency departments. These data include both commuter and recreational/sports cyclists, and rarely differentiate the cycling categories in their records . Many raIllness in mountain biking is poorly reported. Most of the data are from a limited number of race events , 11. GasThe reporting of injury and illness in events varies depending on the definitions used by the researchers. The International Olympic Committee uses a medical attention definition for both injury and illness, and includes all occurrences of injury or illness reported to the medical teams regardless of the effect on the athlete’s ability to continue training or to compete . SeveritInjury and illness prevention programmes require knowledge of the aetiology and magnitude of the injury or illness problem within the context of the sport , 15. TheThe methods for this systematic review followed the principles outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Statement (PRISMA) . This stObservational, cohort, epidemiological studies assessing the incidence and prevalence of injury and illness in single day and multi-stage mountain marathon cycling races were included in the study, provided they met the criteria of the UCI cross-country marathon category . ProspecThe primary outcome measures were the incidence or prevalence of injury and illness sustained during the event. Secondary outcome measures included the severity and location of the reported injuries, type of injury, rider’s ability to continue in the event, prevalence of injury preceding the event, pre-event training distances and the experience/expertise of the riders . Illness diagnosis, severity and the rider’s ability to continue in the event were additional secondary outcome measures. The definition of injury, illness and severity (of both) used in the study was noted.The following databases were searched: PubMed, CINAHL (EbscoHost), Scopus, PEDRO and the Cochrane library. Congress abstracts from cycling specific and clinical sports congresses (for example Cycling Science Conference and Winter Cycling Congress) held in the past ten years and available online were reviewed to identify unpublished studies. A grey literature search in Google Scholar was performed following the database searches. The reference lists of eligible articles, identified during the search, were manually searched.Databases were searched using the following keywords: (Mountain OR off-road OR cross-country OR races OR racing OR stage race) AND (Bicycling [MeSH]) OR cycling OR biking OR bikers OR cyclists OR bicycl*) AND  AND . The database and grey literature searches took place on 5 January 2021 and included all relevant publications up to this date.www.covidence.org).Following the keywords search, all abstracts and titles were downloaded to Covidence  screened the title and abstracts for eligibility based on the above-mentioned criteria. The two reviewers were given independent access to the platform and rated each abstract and title for inclusion or exclusion. Upon disagreement on the inclusion or exclusion of an article, the reviewers discussed and reached consensus on the article’s eligibility.Following title and abstract screening, both reviewers independently reviewed the full text articles for final eligibility and inclusion into the review. The reviewers discussed and reached consensus on the inclusion of specific studies if there was disagreement between the two reviewers. Once the articles were included, the reviewers extracted the appropriate data from the text.Data were extracted by the reviewers independently, on participants (age and sex), cycling event , injury , illness , study design, and risk of bias using a pre-designed data extraction form.The AXIS tool for critical appraisal of cross-sectional studies was used to assess the reporting quality and risk of bias . The tooRisk of bias was assessed based on the selection of participants, respondents and non-respondents (and the reason for non-responses) and the internal consistency of the studies. Risk of bias was reported as ‘unclear’ if the required information was not provided by the authors.Reporting quality assessment included questions on whether the aims, population and methods were clearly reported in the article. Results and limitations need to be adequately described and discussed. Studies were recorded as ‘unclear reporting quality’ if the content of these questions was not reported in the study.The quality of design was evaluated on its appropriateness for the aims of the study and the justification of the sample size and frame. Conflicts of interests and ethical approval were assessed in this section.Descriptive tables of all data are presented. All injury and illness data were reported as an incidence (per time period) or as prevalence (percentage). Descriptive summary tables were populated with information from each study, including study design, participants, context  and outcomes .A narrative analysis of the types, severity and area of injuries and illness type and severity was performed. A quantitative analysis would have been performed if there were three or more studies with sufficient data reported in the same format or with the same definitions of injury and illness, as either incidence or prevalence. Following risk of bias assessment, the same two reviewers decided on the exclusion of the studies in a meta-analysis based on the above criteria. Sub-group analyses of age, sex, type of cycling and experience were not conducted due to the insufficient information available in the studies, and differences in study design and definitions.The electronic search returned 3263 references for evaluation Fig. . Forty-tSeven studies were included, representing 28,021 participants. Each study had a different design, from retrospective to prospective and surveys, cohort and record audits  , 20, defThe majority of injuries reported in these studies were skin-related , 19, 20.minor , moderate  and serious/life-threatening . There are additional categories for event-related sudden cardiac arrest, sudden cardiac death and sudden death [The severity of injuries was only reported in a single study on the 2017 Swiss Epic event . Severiten death . These dOnly two studies assessed factors affecting injury , 20. NonDifferences between race settings (including terrain and temperature), length of race, availability of onsite care, and experience of cyclists may contribute to the vastly different findings between the studies. Over the 10-year period of the included studies, cycling technology has also resulted in equipment changes to bicycles, shoes, helmets and clothing. The protective effects of this equipment specifically within this category of cycling remain unknown. Without regular and ongoing epidemiological data collection using standardised injury definitions, the true burden of injuries will remain uncertain.. [. [Three of the studies investigating illness exclusively assessed gastrointestinal-related illnesses –23. Othe.  and Stua. [.  aimed toThe onset of illnesses in mountain biking may be delayed due to the incubation nature of some viral and bacterial infections . SymptomAll studies included in our review had different study designs and injury/illness definitions while purporting to have the same goal. The findings of this systematic review show that further epidemiological studies are needed in cross-country mountain biking, with standardised injury and illness definitions and recording methods. There is sufficient evidence that injury in mountain biking is an area of concern, but consistent reporting will allow for greater comparison between studies and a better global view of the injury burden. The epidemiology of illness in cross-country mountain biking is unclear and warrants further investigation. Epidemiological data are essential for risk assessment and understanding injury with a view to reducing injury and illness in these events.


The intracellular delivery of emerging biomacromolecular therapeutics, such as genes, peptides, and proteins, remains a great challenge. Unlike small hydrophobic drugs, these biotherapeutics are impermeable to the cell membrane, thus relying on the endocytic pathways for cell entry. After endocytosis, they are entrapped in the endosomes and finally degraded in lysosomes. To overcome these barriers, many carriers have been developed to facilitate the endosomal escape of these biomacromolecules. This mini-review focuses on the development of anionic pH-responsive amphiphilic carboxylate polymers for endosomal escape applications, including the design and synthesis of these polymers, the mechanistic insights of their endosomal escape capability, the challenges in the field, and future opportunities. Most successfully developed biotherapeutics up to date only target extracellular receptors, because the intracellular delivery of biomacromolecules remains a key challenge , poly(amino ester), poly(2-(dimethylamino)ethyl methacrylate), and polyamidoamine] can buffer the endosomal acidification by protonation, and cause osmotic swelling of endosomes, finally leading to endosomal rupture. The polycation induced “proton sponge” effect has long been studied, and reviewed recently , without disrupting the membrane at physiological pH  lipids in the 1980s  has been developed in the late 1990s  methacrylamide) (HPMA), polyethylene glycol , or poly (Jeffamine). The PEG block could enhance polymer solubility by forming micelles and increase the resistance to serum proteins  in the polymerization with PAA monomer generated hyperbranched PPAA, which showed lower hemolytic activity than linear PPAA at endosomal pH conditions  with hydrophobic methacrylates have the coil-to-globule conformational transition, when the pH decreases from neutral to acidic ranges  (PLP), also confirm that pH-dependent membrane-permeabilizing capability could be adjusted by grafting amino acids with different hydrophobicity or alkyl chains  polymerization (Fairbanks et al., Meanwhile, the mechanism of polymer-mediated endosomal escape has been intensively explored, along with the advances in the basic understanding of the endocytosis process itself (Skowyra et al., Notwithstanding the great achievements in both polymer synthesis tools and endosomal escape mechanism investigation, many fundamental issues remain to be addressed in this field. For example, the fate of the amphiphilic carboxylate polymers within the cells after the endosomal escape is rarely covered in the previous publications. It is not known whether the polymer carrier itself undergoes degradation in the cell, or gets expelled from the cell somehow. This issue is critical for biomedical applications, because of the long-term biosafety concerns. Further investigations are expected to result in an improved understanding of the degradative pathways of these polymers within the cells.in vivo environment.In summary, carboxylated amphiphilic polymers with pH-responsive endosomolytic activities demonstrate promising potentials for the intracellular delivery of macromolecules. From a retrospective view, this field has continuously progressed with the application of new synthetic techniques, mechanistic understanding of endocytic trafficking, and better methods for endosomal escape characterization. Up to date, various designs have been made to adapt these polymers for biomedical applications, i.e., the delivery of antigens, genes, and therapeutic peptides. Nevertheless, it is still early to expect clinical translations, due to the lack of biodegradability and long-term biosafety concerns. It would require joint efforts from polymer chemists, biologists, and pharmaceutical scientists to understand how polymers interact with endosomal membranes at the molecular level; how the endosomal escape happens at the cellular level; and finally the delivery in the complicated SW constructed the idea, designed the article, drafted and revised the manuscript. The author has agreed to publish the content of the work.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.


Culture results and clinical improvement will guide de-escalation and length of treatment. New diagnostic methods can help improve the prescription of adequate treatment. Prevention of sepsis includes vaccination and prevention of healthcare-associated infections. More research and education for awareness of sepsis is needed to improve care.Pediatric sepsis is a relevant cause of morbidity and mortality in this age group. Children are affected differently in high and low-income countries. Antibiotics are crucial for the treatment of sepsis, but indiscriminate use can increase resistance worldwide. The choice of a correct empiric therapy takes into consideration the site of infection, local epidemiology, host comorbidities and recent antibiotic exposure. Antibiotics should be administered in the first hour for patients with septic shock, and always intravenously or  Severe sepsis and septic shock are responsible for 10–25% of Pediatric Intensive Care Unit (PICU) admissions worldwide   , 9.Empiric therapy should be carefully prescribed, as it will be the first therapy the patient will receive when presenting with sepsis and septic shock, and selection of an appropriate drug can save lives. We suggest evaluating these principles when choosing the empiric therapy: identification of likely agent according to infection site and knowing the local susceptibility profile, evaluating the risk of having a bacterial infection, the severity of the disease, the host comorbidities and recent admissions and usage of antibiotics , 15 Tab.Streptococcus pneumoniae resistance to beta lactams, for instance. Local guidelines are available in most countries. For healthcare-associated infections, information can be obtained with Infection Control Committees within each hospital . Recent use of antibiotics can increase risk of resistance, varying between 30 and 90 days according do different literature . In these settings, prescription of vancomycin, linezolid, daptomycin or ceftaroline is recommended for unstable patients with suspected staphylococcal infections, such as skin and soft tissue infections  infections in 37% of 62 patients with rectal colonization. The risk factors for developing infection among carriers were being admitted to the ICU, receiving a CVC and being in a coma. Interestingly, no pediatric patients developed infections . Specific bottles for pediatric patients require 1–3 ml of blood and are sent only to aerobic culture. Data shows that the smaller the volume of blood, the higher the chance of growing contaminant agents instead of the real noncontaminant pathogen of the infection. Time to detection also correlates with volume of blood obtained. There is no consensus on the adequate volume of blood to collect for the pediatric patient, and this must be decided according to age and weight of each patient. For patients with CVC, paired cultures are indicated. Time to positivity and number of colonies are important information to diagnose catheter-related infections , 29.via intraosseous access. Intramuscular absorption of drugs is erratic and should be avoided in critically ill patients. The oral route is not indicated in patients with sepsis due to low intestinal perfusion and consequently low absorption  and pharmacodynamic (PD) bactericidal mechanisms require that the time the free drug remains above the minimum inhibitory concentration (MIC) is more than 40–70% of time , 32. ProStaphylococcus aureus, there is a higher risk of therapeutic failure with regular vancomycin doses . The main actions to prevent HAI are hand hygiene, adhesion to standard and transmission-based precautions, surface cleaning and disinfection and institution of bundles to prevent catheter-related infection, catheter-related urinary tract infection and ventilator associated pneumonia .Streptococcus pneumoniae, Haemophilus influenzae type B and Neisseria meningitidis had a huge impact on preventing meningitis and other invasive bacterial infections in children. The fall of vaccine coverage, due to shortage, vaccine hesitancy, anti-vaccine movement or during the Covid-19 pandemic represents a great risk of reemergence of vaccine-preventable diseases  and polymerase chain reaction (PCR) can decrease time for pathogen identification and susceptibility results , 45.in situ hybridization (FISH) or MALDI-TOF. Time to positivity is shorter with MALDI-TOF (60 min) and costs are lower, but best results are available when testing purified cultures samples, such as bacterial pellets or subcultures. Automated microbial system cards are used to perform antibiotic susceptibility tests with few discrepancies from traditional phenotypic methods (Commercial kits that use whole blood for pathogen identification are available. Those are PCR kits than can identify a certain number of bacteria, viruses, fungi, and the presence of resistance genes, such as mecA, vanA/B and blaKPC. MALDI-TOF can also be utilized with whole blood, but the rate of contamination and false-positive results is higher. Testing blood directly from the positive culture is possible using PCR, microarray, fluorescence  methods , 46.Staphylococcus aureus and non-penicillin or ampicillin prescription for Streptococcus A and B and Enterococcus faecalis (A study showed that time to optimal therapy was reduced from 73 to 48 h and time to pathogen identification from 55 to 29 h after implementation MALDI-TOF and PCR. There was no impact in mortality, but a reduction was noted on antibiotic prescription for patients with blood culture contaminants, vancomycin prescription for methicillin susceptible faecalis .Elevated costs are still an important barrier to implement these new technologies in low- and middle-income countries.In the 21st century, sepsis remains a major cause of morbidity and mortality in childhood and adolescence. Prevention and timely and adequate therapy are major goals to improve clinical results. Antibiotics are mandatory to treat sepsis, but indiscriminate use and resistance development can lead to higher medical costs, drug side effects and increased mortality.The choice of empiric therapy must be assertive, and take into consideration the site of infection, local epidemiology, severity of the disease, host comorbidities and recent exposure to antibiotics. Performing blood and other site cultures is fundamental for pathogen identification and will help defining the best drug, de-escalation, and duration of treatment.Sepsis prevention is the best strategy to reduce mortality. For community-acquired sepsis, the main intervention is increasing vaccine coverage. For HAI, preventing sepsis means reducing risk of contamination by using bundles, stimulating adequate hand-hygiene and surface cleaning, as well as following isolation precautions. Education of patients and families for sepsis awareness can help healthcare professionals to recognize and timely treat sepsis.Future Perspectives for the treatment of sepsis are rapid diagnostic methods for pathogen identification and antibiotic susceptibility tests. Research gaps in pediatric sepsis comprise the difficulty of developing of new drugs for this age group, the small number of patients in each study and comparing methodologies in different studies, the lack of resources destinated to research in low- and middle-income countries and the difficulty in obtaining and communicating local epidemiology data .LM and VB: substantial contributions to the conception and design of the work, drafting the work, and revising it critically for important intellectual content. 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 affiliation with one of the author VB at time of review.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.


In this review, we introduce microbially-mediated soil processes, players, their functional traits, and their links to processes at biogeochemical interfaces . A conceptual view emphasizes the central role of the rhizosphere in interactions with other biogeochemical interfaces, considering biotic and abiotic dynamic drivers. We discuss the applicability of three groups of traits based on microbial physiology, activity state, and genomic functional traits to reflect microbial growth in soil. The sensitivity and credibility of modern molecular approaches to estimate microbial-specific growth rates require further development. A link between functional traits determined by physiological  and genomic  approaches is strongly affected by environmental conditions such as carbon, nutrient availability, and ecosystem type. Therefore, we address the role of soil physico-chemical conditions and trophic interactions as drivers of microbially-mediated soil processes at relevant scales for process localization. The strengths and weaknesses of current approaches  for assessing process localization and the corresponding estimates of process rates are linked to the challenges for modeling microbially-mediated processes in heterogeneous soil microhabitats. Finally, we introduce a conceptual self-regulatory mechanism based on the flexible structure of active microbial communities. Microbial taxa best suited to each successional stage of substrate decomposition become dominant and alter the community structure. The rates of decomposition of organic compounds, therefore, are dependent on the functional traits of dominant taxa and microbial strategies, which are selected and driven by the local environment. In terrestrial ecosystems, the most critical biochemical processes are performed by soil microorganisms , and a bprimary input organic substances in soil are microbially transformed into cell constituents or excreted by cells as labile metabolic products  emissions. The direction and intensity of the consequences of microbial metabolic activity are dependent on the functional traits of the organisms performing the ecologically-relevant processes below-ground and the abiotic and biotic conditions these organisms encounter in their habitats.Microorganisms decompose organic substrates to maintain their metabolic requirements and enable their growth. Microbial growth and anabolic reactions require not only C and energy, but also a general stoichiometric composition of nutrients , which microorganisms have to mobilize from a multiphase  soil environment . If theiactive fraction of a predominantly dormant microbial community can be small in nutrient-poor or stressed environments  and the time required for dormant microorganisms to switch to active growth . The second group represents intrinsic functional traits of the microbial population, such as maximal specific growth rate (μm), generation time (Tg), and affinity of extracellular enzyme systems (Km) to soil organic substrates used for microbial growth. The third group refers to phenotypic traits at the level of functional genes, for example, those related to internal microbial metabolism, extracellular resource acquisition, or stress tolerance.Soil organic matter transformation processes mainly rely on the production of extracellular enzymes that facilitate the oxidation or hydrolysis of diverse and complex SOM compounds . The decm), and by enzyme affinities for a substrate (Km), experimentally determined under in situ soil conditions  encoding certain functions with microbial ability to grow under in situ soil conditions. At the physiological level, growth rate is dependent on a balance between two fractions of the total proteome . Thus, rhizodeposition processes overlap with leaf litter and dead root tissue degradation processes, forming a rhizosphere–detritusphere interface. Because of the spatial and chemical heterogeneity of rhizodeposits and rhizo-detritus in soil, the microbial communities and activities in these two spheres as well as in the overlapping sphere are specific . It has ulk soil . Essenticrobiota , indicatcrobiota . Moreoveood webs . Therefoood webs .Differences in the quality of organic matter input induce contrasting competition situations within the rhizosphere and detritusphere. In the rhizosphere, the majority of easily available organic C, such as sugars, amino acids, and carboxylic acids, are released through living roots . In the 2 levels  that cohere to each other more strongly than surrounding non-cohesive particles and are considered as soil structural building units . A hieraaggregatosphere interacts with the detritusphere, because root detritus  provide a substrate for microbial metabolism. Both, microbial metabolic products and rhizodeposits form sticky polymeric substances  is generally determined at the macro-scale, for example, on the landscape or soil profile level. Such approaches are important for global budget estimates. Understanding the mechanisms and spatial distribution of these processes requires, however, more precise mesocosm studies, while a shift to the micro- and even to the nanoscale is necessary to identify links between rates and distinct local processes in soil microhabitats.two-dimensional distribution of soil properties, such as pH or oxygen concentration (2 and hydrolytic enzyme activities). Zymography coupled with X-ray CT is very promising for the 3D reconstruction of enzymatic processes within soil pore spaces, in particular, if the integration of 2D chemical imaging (in this case zymography) and 3D (μ)X-ray CT is further coupled with the modeling of pore-scale processes  was achieved using novel microsensor techniques and soil zymography. These approaches enable the monitoring of the ntration  and the rocesses . For exarocesses  that coudestructive sampling disturbing soil microcosms, (ii) non-destructive imaging in situ techniques, and (iii) prediction by modeling.Existing and newly developed methods for the determination of localized process rates in soil can be differentiated into three groups based on (i) Techniques to identify microorganisms are currently mostly based on DNA sequencing approaches, as DNA has high information content regarding taxonomy . MetagenE. coli in pure culture ; in particular, modern viewpoints are mostly presented for the rhizosphere . For insrocesses . These a, pH, O2 , (ii) se, pH, O2 , (iii) z, pH, O2 , (iv) DG, pH, O2 , and (v) O2 , essential methodological uncertainties occur related to the diffusion of targeted colored or fluorescent molecules (substrates or products of reactions) between the soil and membrane as well as within the membrane . PossiblHence, a quantitative estimation of process rates and the magnitude of changes in pools and fluxes is necessary at interfaces such as the rhizosphere to clarify, for instance, how inoculants modulate the resident microbiome, how pathogenic attack affects the activity of the complex microbiota of hotspots, how grazing activities by protists, nematodes, or bacteriophages control the extinction of species, and how the rhizosphere microbiome responds to abiotic stresses . This remains a challenging task, considering the diversity of C compounds in the rhizosphere and the challenges regarding their analysis .2, P, Mn content, and enzyme activity) are satisfactorily visualized in 2D by application of sensor membranes to the root–soil interface , scanning transmission X-ray microscopy (STXM), X-ray absorption spectroscopy, micro-fluorescence spectroscopy, and nanoSIMS, as well as combined STXM-NanoSIMS , appliedTo conclude, this review suggests a conceptual view emphasizing the central role of the rhizosphere in interactions with other biogeochemical interfaces. The main drivers of plant–microbial interactions, such as substrate input through exudation and rhizodeposition, and physico-chemical conditions  are already subject to intensive research. In contrast, the driving role of trophic interactions within and between interfaces, including competition for nutrients and successional dynamics, requires more specific studies involving both higher and lower trophic levels . According to our concept, microorganisms are not the drivers, but they are the most abundant and powerful players in the soil interfaces because of the great diversity and specificity of genes encoding similar functions. The combination of phylogenic specificity and functional redundancy ensures the sustainability of soil microbial communities by the use of functional traits  as a tool to develop a microbial life strategy, which in turn affects the rates of transformation of organic compounds in soil. Thus, taxa with life strategies best adapted to the environment become dominant and alter the structure of the active microbial community. This self-regulatory mechanism maintains metabolic activity by the microbial community during the successional decomposition of organic substrates entering the soil. However, the rates of substrate decomposition are dependent on the functional traits of dominant taxa and microbial life strategy, which in turn are selected according to substrate quality and local environmental constraints, for example, water and nutrient availability. The rapid development of instrumental and molecular techniques has fueled attempts to reconsider the concepts of microbial life strategies with the goal of specifying functional groups according to their ecological relevance. This requires the identification and estimation of intrinsic traits by microbial physiology or phenotypic traits at the functional gene level. The quantitative definition of functional traits based on genetic and isotopic approaches is very promising, but demands further development with caution regarding the relevant resolution time and type of biomarker. Additional technique development is needed for ground-truth measurements of microbial growth in soil, linking physiological and molecular approaches. Therefore, the current challenge in modern ecology is the further development of cutting-edge methodologies for precise localization of biochemical processes, considering interactions within and between soil interfaces, as well as identifying and linking functional traits of plants and microbial populations that contribute to the rates of soil processes relevant at the ecosystem level.EB, SS, and BR developed the concept and all the co-authors contributed to the writing and structured the manuscript. All authors contributed to the article and approved the submitted version.RK was employed by the company Forschungszentrum Jülich GmbH. The remaining 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.


Tumor-draining lymph nodes play a paradoxical role in cancer. Surgeons often resect these sentinel lymph nodes to determine metastatic spread, thereby enabling prognosis and treatment. However, lymph nodes are vital organs for the orchestration of immune responses, due to the close encounters of dedicated immune cells. In view of the success of immunotherapy, the removal of tumor-draining lymph nodes needs to be re-evaluated and viewed in a different light. Recently, an important role for tumor-draining lymph nodes has been proposed in the immunotherapy of cancer. This new insight can change the use of immune checkpoint therapy, particularly with respect to the use in neoadjuvant settings in which lymph nodes are still operational. Immunotherapy with PD-1/PD-L1 blocking antibodies has reached a new phase and now moves towards application in early stage patients. For patients enrolled in therapy early after diagnosis, the administration of immune checkpoint blockers in adjuvant or neo-adjuvant settings appears very relevant. Adjuvant therapy is defined as the administration of therapeutics after the surgical resection of a tumor and/or sentinel lymph nodes (or tumor-draining lymph node TDLN). Neo-adjuvant therapy means therapeutic interception before scheduled tumor and/or TDLN resection. These two different settings may have crucial implications in immunotherapy, since immunotherapy will activate immune cells within the tumor microenvironment (TME). Therefore, the presence of the tumor will add to the potential outcome of the therapy. Moreover, the TDLN is an important organ in orchestrating immune responses. Although this is an accepted fact within the scientific tumor immunology community ,3,4,5 haIn this review, we will discuss the potential of TDLN in cancer immunology and, in particular, immune checkpoint therapy, emphasizing that there is still a gap in knowledge on the immunological role of the sentinel lymph node and that it is more than a route for disseminating metastases. We argue that its role in cancer immune surveillance should be seriously reconsidered and stimulate a detailed evaluation of our systemic lymphoid draining system in the immunotherapy of cancer.The TDLN has quite extensively been investigated. However, the majority of reported studies focus on lymph angiogenesis and metastases. Clinical standards on the need for the resection of the entire lymph drainage basin of the tumor compared to single sentinel nodes have changed drastically over the last years, especially in breast cancer and melanoma, with overall survival as the main parameter . HoweverUp until the breakthrough of the clinical success of the immune checkpoint blockade, TDLN were studied regularly for anti-tumor immune response factors. Since the mechanistic effects of the immune checkpoint blockade are generally assumed to rely on the reactivation of anti-tumor T cells, all focus has shifted to the TME, while the TDLN received less attention. The relevant insights and analyses made on the differences between TDLN and non-draining LN (NDLN) from before checkpoint therapy are expertly reviewed by Cochran et al. . The speBesides serving as a potential source for adoptive transfer, the targets for checkpoint blockade therapy, the most potent and successful immunotherapy at the moment, can demonstrably be detected within TDLN. Both in pre-clinical mouse models of cancer and patient samples, PD-L1 and PD-1 have been found to be expressed in TDLN more extensively than in NDLN ,3,11. AnLocal inflammation leads to lymph node swelling , which is a well-known reaction to immune activation, and has been described to be dependent on S1P1, TNF, IFN α/β and CD69 ,14,15,16Surgical resection may abruptly deplete an important source of tumor-eradicating immune cells counteracting the intended positive effects of the immunotherapy. Recent findings have indicated that immunotherapy efficacy is based on the influx of newly primed T cells into the tumor microenvironment, suggesting a non-stop process, otherwise known as the cancer immunity cycle (a term coined by Chen and Mellman ), in theAlthough the influence of TDLN on the anti-tumor immune response and, in particular, the efficacy of immunotherapy of cancer have been generally underestimated, there is a great body of research on the lymph drainage of tumors, its role in metastases dissemination and edema problems after resection ,21.Besides metastasizing tumor-cells, many other materials such as tumor debris and secreted extracellular vesicles are drained from the tumor to the lymph node. In addition, soluble factors such as chemokines and cytokines have been reported to play a role in creating an immunosuppressive environment as well as changing the stromal cells of the lymph node ,19,21,22VEGF-C, which is known to increase lymph angiogenesis and increase the risk of metastasis, also strongly increased the anti-tumor effect of immunotherapy with PD-1 checkpoint blockade therapy. Furthermore, it was shown that presence of lymphatic endothelial cells (LEC) within tumors made them more responsive to PD-1 therapy, indicating a clear link between lymph system and checkpoint blockade therapy.Two recent studies showed the importance of lymphatic drainage for the immunotherapy of cancer ,25. The Studies with the so-called “photoconversion” mice, in which the immune cells in tumors under the skin are fluorescently labelled in vivo by UV irradiation, demonstrated immune cell migration from the tumor to TDLN ,27. ThesThe success of checkpoint therapy is undisputed, but still, only a minority of patients have lasting benefits from the treatment. Therefore, combining the checkpoint blockade with other therapy modalities in an attempt to increase clinical benefit is tantamount. The most likely candidates for combinatorial treatments are the already used standard treatments, such as radiotherapy and chemotherapy. In order to implement these combinatorial strategies into daily clinical practice, it is vital to understand the effect these therapy modalities have on the immune function in space and time in order to design rational strategies. Currently, knowledge on the effects radiotherapy and chemotherapy have on the immunological function of the TDLN is limited .Radiotherapy can have a profound effect on tumor cells but can also influence healthy surrounding tissues, including TDLN. Specific doses of radiotherapy will lead to the immunogenic cell death of tumor cells, i.e., the combined release of antigens and damage-associated molecular patterns (DAMPs), which can subsequently lead to DC activation, enhanced tumor antigen uptake and presentation and increased systemic anti-tumor T cell responses. The development of potent anti-tumor T cell responses is dependent on TDLN being able to perform their immunological function, as this is where the tumor antigen would drain to and where uptake and presentation will occur. Since TDLN are under the direct influence of radiotherapy due to their location, the influence of radiotherapy could be crucial to the induction of anti-tumor immune responses, and knowledge about this effect is important for future clinical study designs.Most studies into the immunological effects of radiotherapy focus on the TME or on subsequent peripheral blood responses ,37. BattBoth of these studies point at the importance of understanding the effect radiotherapy has on TDLN in order to make rational combinations with immunotherapy.Similarly, there is little knowledge on the effects of chemotherapy on the immunological function of TDLN. Most chemotherapy agents cause lymphodepletion, as extensively shown in preclinical models and patients. However, this information is based largely on the analysis of peripheral blood. It is likely that similar processes are occurring inside lymph nodes—particularly in TDLN. However, this remains to be studied. Additionally, high doses of glucocorticoids, a common agent given to chemotherapy-treated patients to attenuate nausea and vomiting, severely hampers the antigen presentation by DC and suppresses the secretion of inflammatory cytokines such as IFNα/β and IL-1α/β, as in vitro and in vivo studies have shown . Specialhigh cells within tertiary lymphoid structures (TLS) to be implicated with good prognosis after PD-1 therapy in lung cancer patients [high cells commonly reside within lymphoid structures—even lymphoid structures organized within the tumor tissue.At present, the majority of studies into the mechanism of checkpoint blocker success are aimed at the TME. There is a wealth of knowledge on the cells and molecules involved in the mechanisms behind the success of checkpoint blocker therapy ,41. At ppatients . The preIn recent years, the number of publications on neo-adjuvant immunotherapy trials has increased exponentially. It is clear that administering checkpoint blockade in earlier stages of disease, before the resection of tumor and/or TDLN, is considered by many as a potentially efficacious treatment option that warrants extensive evaluation. In the many papers describing, the outcomes and translational research performed on patients undergoing neo-adjuvant immunotherapy, the immunological changes in TME and peripheral blood were meticulously analyzed and described. However, the immune contexture of TDLN was not studied or discussed ,60,61,62Not only is the neo-adjuvant administration of immunotherapy an excellent way to exploit the full potential of the immune cells within TME and TDLN but it also forms a great source of material for the scientific advancement of the knowledge of TDLN in terms of tumor immunology. Few of the neo-adjuvant studies published at present mention the TDLN. We want to encourage all scientists and clinicians involved in neo-adjuvant trials of immunotherapy to include the TDLN in their immune-monitoring schemes and amplify the current knowledge on the potential of the TDLN for antitumor immunity. Important issues, outstanding at present, are highlighted in Tumor draining lymph nodes play a role in cancer that goes far beyond the dissemination of metastases. The contribution of TDLN to the immunotherapy of cancer, particularly the treatment with checkpoint blockers, is slowly becoming apparent. At present, there are 154 clinical trials reported for the neo-adjuvant therapy of immunotherapy, clearly marking the next big step in the implementation of these antibodies. The role of TDLN as the orchestrator of anti-tumor immune responses is becoming more and more apparent, especially in PD-1/PD_L1 checkpoint blockade therapy.Based on recent scientific findings, as reviewed in this manuscript, we propose a serious re-evaluation of the role of the tumor-draining lymph node in relation to the modes of action of the different immune checkpoint antibodies that are now in the clinic and the possibility to make efficacious treatment combinations. We advocate the meticulous study of all aspects of TDLN immunology, thereby optimally exploiting the anti-tumor immunity present in these specialized lymphoid organs.