Mycotoxins are secondary metabolites produced by fungus. Many mycotoxin species are highly toxic and are frequently found in cereals and feedstuffs. So, powerful detection methods are vital and effective ways to prevent feed contamination. Traditional detection methods can no longer meet the needs of massive, real-time, simple, and fast mycotoxin monitoring. Rapid detection methods based on advanced material and sensor technology are the future trend. In this review, we highlight recent progress of mycotoxin rapid detection strategies in feedstuffs and foods, especially for simultaneous multiplex mycotoxin determination. Immunoassays, biosensors, and the prominent roles of nanomaterials are introduced. The principles of different types of recognition and signal transduction are explained, and the merits and pitfalls of these methods are compared. Furthermore, limitations and challenges of existing rapid sensing strategies and perspectives of future research are discussed. Topics related to food safety have been attracting significant attention all over the world. Contaminants in cereals and animal products are related closely to human health. Mycotoxins are secondary metabolites of fungus and may be produced during growth, production, processing, and storage of cereals, grains and feedstuffs . There aTraditional analytical methods such as enzyme-linked immunoassay (ELISA), thin layer chromatography, gas chromatography or high-performance liquid chromatography (HPLC) coupled with ultraviolet detection, fluorescence detector electron capture detectors, diode array detectors, and mass spectrometry (MS) detectors have been used to detect mycotoxins for decades –10. MostRapid detection technology is a concept relative to traditional analysis methods and laboratory detection technology. It is often based on interdisciplinary subjects such as nanomaterials science, immunology, molecular biology, spectroscopy, and electrochemistry. Rapid detection is simple, cheap, easy to operate, and only requires portable instrument and a very short detection duration. It enables detection method to meet the need of the real-time on-site mycotoxins screening in the field of food safety. Herein, we review published research reports on rapid detection technology for mycotoxins from 2016 to 2021 including immunoassays and biosensors. Principles of recognition and signal transduction strategies are explained, and the pros and cons compared to existing method are discussed. Especially, we highlight studies on simultaneous detection of multiple mycotoxins. Limitations, challenges and perspectives of the future developments in this field are discussed.Specific recognition of analytes is the primary step of rapid detection, which ensures the specificity and selectivity of the analytical method. In this section, different types of recognition elements for mycotoxin detection are introduced including antibodies, aptamers, and molecularly imprinted polymers (MIPs).Antibodies are immunoglobulins produced by the immune system that specifically bind to the corresponding antigens. Based on the preparation processes used, antibodies are classified into polyclonal, monoclonal, or recombinant categories. Antibody-antigen recognition is regarded as a gold standard because of its properties of high specificity and affinity. Antibodies and antigens are the most widely commercialized recognition and capture agents applied in immunoassays and biosensors for clinical diagnosis, disease treatment, environment monitor, and food safety control –15. HoweTo overcome these limitations of antibodies, variable region of heavy chains antibody (nanobody) and phage-displayed mimotope peptides may be some utility in solving these antibody problems . NanobodAptamers are small fragments of oligonucleotide sequences (single stranded DNA or RNA), which usually contain 10 to 100 bases. Aptamers bind to their targets by folding into specific three-dimensional structures. They are selected from a combinatorial DNA library by a technology named systematic evolution of ligands exponential enrichment (SELEX) and can bind to various targets ranging from ions to cells. Aptamers are usually considered to be ideal affinity reagents alternatives to antibodies. Compared to antibodies, aptamers are inexpensive, stable, reversible, not limited by immunogenicity of targets, and do not require immunization of animals during production. Aptamers have high bioavailability and are easy to modify. As an excellent alternative to antibodies, aptamers are being deployed in biosensor, disease diagnosis, and drug delivery –24. HoweMolecular imprinting is a technology developed based on bionic science and simulation of the interactions of enzymes with their target substrates and receptors with their antibodies in nature. Molecularly imprinted polymers (MIPs) are obtained by mixing imprinted molecules (template molecules) with appropriate functional monomers  and crosslinking agents. Then polymerize them and eluting the imprinted molecules by appropriate methods. Molecularly imprinted polymers have been called “artificial antibodies”. They are preserved easily, resistant to extreme conditions such as high temperature, high pressure, acid, and alkali, and difficult to destroy by biodegradation . ConsequImmunoassays are based on immunological principles that use antibodies to recognize and capture target antigens or haptens. Enzyme-linked immunosorbent assays (ELISA) are early immunoassays that have been commercially applied in mycotoxin detection with good sensitivity and accuracy . Enzyme-Lateral flow immunoassays (LFIA), also known as immunochromatographic assays (ICA), are the most frequently used and commercialized rapid sensing platform and widely applied in disease diagnosis, point-of-care testing (POCT) and food safety monitoring because of its low cost, rapidity and simplicity , 40. TheHigh-quality antibodies and antigens are primary guarantees for the high sensitivity and specificity of LFIA. However, the recognition of limited immunogenic targets by antibodies has hindered development of LFIA in several fields of study. Secondly, there is a contradiction in the competitive detection mode itself. That is, within a certain range, the less quantity of antibodies makes the competition between free target analytes and the immobilized antigens more effective on the test zone. So, the lower the specific antibody concentration, the lower the detection limit. However, the amount of the label conjugated with antibody is reduced simultaneously with the low concentration of antibody, which decreases signal intensity of the test line and hinders successful detection of the targets.To overcome these shortcomings above, researchers developed better-performed antibodies or signal labels with high luminescent intensity and stability . BesidesGold nanoparticles (AuNPs) are the most frequently used signal labels in LFIA because they are easy to synthesize, inexpensive, and emit visible red to purple light when they aggregate. However, an obvious drawback of AuNPs-based LFIA is the instability and weak optical intensity of AuNPs resulting in low sensitivity , 43. RecUse of various fluorescent labels with higher stability and signal intensity in LFIA to enhance the sensitivity has become commonplace. Quantum dots (QDs) are semiconductor nanoparticles that possess excellent optical properties including size-tunable emission, broad adsorption, narrow photoluminescence spectra, high photostability, large Stokes’s shift, and long fluorescence lifetime . Fluores2O3 nanocubes used as LFIA label for simultaneously detecting two mycotoxins. These nanocubes have been employed to detect AFB1 and DON residues in mung bean, millet and corn, with the visual LOD for AFB1 and DON of 0.01 and 0.18 ng/mL, respectively [Time-resolved fluorescent microspheres (TRFMs) are recently employed as signal labels frequently. Lanthanides such as Eu (III), Tb (III) have much longer fluorescence lifetime than the previously mentioned fluorescent materials so that they can eliminate the background fluorescence interference from the matrix, thus improving the sensitivity of LFIA –53. For ectively .Fig. 1SResearchers have compared various signal labels to provide a reference for selection of appropriate labels. For instance, colloidal gold (CG) and QDs as labels for multi-mycotoxin detection in cereal matrices have been compared. The QD-LFIA was more sensitive and economical than CG-LFIA . In anotGold nanoparticles and sliver nanoparticles can provide another detectable signal termed Surface-Enhanced Raman Spectroscopy (SERS). Surface-Enhanced Raman Spectroscopy effect is the phenomenon that in the excitation region of some specially prepared metal good conductor surface or colloidal sol. The Raman scattering signal of adsorbed molecules is greatly enhanced compared with the ordinary Raman scattering signal because the enhanced electromagnetic field on the sample surface. When SERS nanotags  are exposed to a laser light source, the incident field is obviously enhanced at active sites (electromagnetic “hot spots”) due to localized surface plasmon resonance effects , 59. ComIn addition to competitive assays, researchers have explored LFIA based on fluorescence quenching principle to improve sensitivity. Competitive assays need many target analytes to compete with Immobilized antigen then make the signal of test lines “turn off”. The signal is an indirect signal, which leads to relative low sensitivity. While the fluorescence quenching LFIA can provide a “turn on” signal, which responses to the amount of target analytes directly , 62. InnBased on existing studies, fluorescence quenching LFIAs exhibited a better performance than traditional AgNPs-based LFIAs but there have been hardly any researches on multiple mycotoxin detection using fluorescence quenching LFIA. Most of traditional LFIAs and fluorescence quenching LFIA can only render qualitative or semi-quantitative results at present. False positive and false negative results are still a problem. Selected studies of LFIAs for mycotoxin detection published in recent years are listed and compared in Table Biosensors are detection devices that can convert the information measured into electrical signals or other information output of the required form. The biosensor system mainly consists of a biorecognition (biosensing) element and a transducer element. Biorecognition elements such as antibodies, antigens, nucleic acids, and enzymes are used to identify and sense target analytes. Transducer elements convert the physical quantity signal of generated by the recognition elements into detectable signals. According to the principle of the signal transduction, biosensors can be classified into electrochemical, optical, mass-sensitive, and thermal sensors. They have become vital and powerful analytical tools widely applied in various fields including medicine, food, agriculture, environment, and industry. Biosensor systems were introduced to enhance food safety in the 1980s and provide great commercial potential currently , 72–74. Optical sensors rely on variation in optical signals generated by transducer from molecular recognition events on sensing element. This approach excels in superiorities of simplicity, speed of detection, sensitivity and visualization . On the Colorimetric biosensors are based on color changes induced by target analytes that can be easily distinguished by the naked eye. Gold or silver nanoparticles are most frequently applied as indicators to utilize their surface plasmon resonance (SPR) properties. The general principle is that recognized targets induce aggregation of AuNPs or AgNPs from well-dispersed state resulting in color change from red to blue.3O4/GO based platform and a Fe3O4@Au based platform for AFB1 and OTA sensing. Quantitative detection of OTA and AFB1 was respectively carried out by release of thymolphthalein and 3,3′,5,5′-tetramethylbenzidine catalyzed by gold nanoparticles, respectively. In this work, OTA and AFB1 were easily and visibly detected in one system with the linear ranges of 0.5–80 and 5–250 ng/mL and did not interfere with each other because of different color reaction conditions.A colorimetric aptasensor for simultaneously detecting ochratoxin A and aflatoxin B1 in peanut was reported . ResearcIn another attempt, a colorimetric sensor based on array of gold and silver nanoparticles was fabricated for simultaneous detection of AFB1, AFG1, AFM1, OTA and ZEN . MycotoxAt present, fluorescent biosensors are the most popular optical sensors because of their high sensitivity, stable signal, and fast response. Various nano-materials such as magnetic nanoparticles (MNPs), quantum dots (QDs), carbon nanoparticles (CNPs), gold nanoparticles (AuNPs), fluorescent nanobeads, and graphene oxide (GO) have been widely applied in construction of fluorescent sensors –80. LantFigure 2 nanosheet and dual-color gold nanoclusters (AuNCs) for simultaneous detection of AFB1 and ZEN in maize [2 were connected by AFB1/ZEN-aptamer-BSA resulting in fluorescent quenching of AuNCs. Fluorescence turned on when target analytes were present , as a subclass of chemiluminescence or electrochemistry, is regarded as the reverse of photoelectron chemistry. Voltage or current serves as the excitation source and the generated chemiluminescence is used as the detectable signal. With ECL, the electro-active species generated in the vicinity of the electrode surface are in excited states emitting light that arises from the high-energy electron-transfer reactions of these species. Luminol and Ru (bpy)y of ECL , 89. Eley of ECL . HoweverA nicking endonuclease-powered DNA walking machine was proposed to fabricate an ECL aptasensor for OTA detection . QuantumSurface plasmon resonance (SPR) sensors are optical sensors based on changes in the refractive index due to the mass changes that occur when molecules bind to the sensor surface. These changes in refractive index can provide direct recognition information between a target and a probe on the sensor surface. Therefore, SPR sensor enables monitoring the real-time interaction among molecules on a tiny chip which can be a powerful analytical tool for purpose of medical diagnostics, food safety analysis and environmental monitoring , 93.A competitive-type SPR sensor for DON and OTA detection by imaging nanoplasmonics was developed . A portaAnother study described a SPR-based aptasensor in a direct assay format for AFB1 detection. The aptamers were modified on the commercial sensor chip surface, and the SPR signals increased when AFB1 bound to aptamers to achieve quantification with the LOD of 0.4 nmol/L (124.8 pg/mL) . A labelBy comparing these studies, we can infer that direct SPR-sensors are simpler and more sensitive than indirect SPR-sensors. However, because the small molecule analytes (such as mycotoxins) cannot cause obvious mass concentration changes at the sensor surface, the direct SPR-sensor’s chip surface must be modified with noble metal NPs for signal amplification. More advanced portable SPR instruments are required for practical application.Optical biosensor platforms that recently reported for mycotoxin determinations are listed in Table Electrochemical biosensors are based on changes in outputted electrical signals that produced by the chemical reactions between electrode-immobilized recognition elements and target analytes. According to the types of detectable electrical signals, electrochemical sensors can be categorized into amperometric, potentiometric, conductometric, impedimetric and voltammetric methods. Besides, the electrode system  is vital to the sensors because identification of target analytes needs to be finished on the electrode. The electrode is also used to conduct electrical signals , 109.2, and porous metal organic framework) to increase their surface area [2-Thi). Thin-layer MoS2 is an important graphene analog that is used frequently as a supporting substrate for stabilizing nanoparticles [2-Thi composites to obtain synergistic signal amplification. Therefore, this immunosensor was easy to operate as well as offered a higher sensitivity compared with the original ZEN monoclonal antibodies.Traditional electrochemical immunosensors using general electrodes have some defects of low sensitivity, low stability, and inability to detect multiple targets simultaneously. To address these issues, improving performance of working electrodes and achieving signal amplification have become necessary. Commonly used working electrodes such as glass carbon, gold, and silver electrodes have recently been modified with various nanomaterials  have attracted more and more attention recently. Aptasensors exhibit greater diversity and universality than immunosensors. They are easier to construct for multiple analytes detection systems and sensitivity of aptasensor can be greatly improved by a ratiometric mode. Fig. Fc), and increased current intensity of THI (ITHI) [Another dual-target electrochemical aptasensor was developed based on co-reduced molybdenum disulfide and gold nanoparticles (rMoS2-Au) modified electrodes . AptamerI (ITHI) . It is wMolecularly imprinted polymers (MIPs) can serve as the recognition elements in electrochemical sensors which are favorable alternatives to antibodies. MIPs are mechanical and chemical stable and provide thickness-controlled MIP films on electrode surfaces, which permit direct communication between the films and the transducer. A novel MIP-sensor with two functionalization methods for AFB2 detection in milk was developed. Researches compared ZnO-NPs/chitosan (CS)/AFB2 and ZnO-NPs/Cs/polypyrrole (PPy)/AFB2 composite that electrodeposited on the screen-printed electrode . They shIn general, electrochemical biosensors show reliable sensitivity and selectivity as well as great potential for miniaturization and portability. But the modification steps involved in the electrochemical biosensors’ preparation are complicated. Reproducibility and interference resistance of traditional electrodes in complex matrices still is problematic. Compared with optical detection methods, there are no huge advantages for development of multiple target detection systems. Table Photoelectrochemical (PEC) sensors are emerging as an analytical technology that combines features of both optics and electrochemistry. In PEC sensors, light source, electrochemical workstation, and signal acquisition system are main components. Light is used as the excitation source and the generated photocurrent is used as the detection signal, which significantly reduces background interference . PhotoacIn recent years, a novel material has been be applied in fluorescent and electrochemical sensing, termed metal−organic frameworks (MOFs). Metal−organic frameworks are porous coordination polymers with huge surface area, that exhibit excellent optical, electrochemical and catalytic properties , 141. LuVarious mycotoxin contamination occurs frequently and unavoidably in feedstuffs and cereals, which poses enormous risks to public health and leads to economic losses. This paper introduces advanced analytical methods and nanomaterials for mycotoxin detection in recent years including immunoassays and biosensors, especially for multiple mycotoxin detections. Target recognition, signal transduction, and nanoparticles are keys to rapid detection techniques. Analytical methods introduced in this paper are divided into many categories based on these three aspects.Colorimetric immunoassays are simple, visible, and low-cost methods without the requirement of expensive instruments but the instability, background interference, and poor sensitivity are considerable drawbacks. Therefore, many fluorescent nanoparticles have been introduced to rapid detection and greatly enhance sensitivity and stability. However, some fluorescent signal labels are susceptible to fluorescent bleaching, autofluorescence or environmental interference and require complex labeling steps. Electrochemical biosensors are classic and powerful sensing strategy because of their high sensitivity and ability to be miniaturized, but they are hard to resist interferences, complex electrode modifications are required and electrode fouling and degradation still need to be solved. Photoelectrochemical and electrochemiluminescence sensors are regarded as combining the merits of optical and electrochemical strategies, which show low background, fast response, and high sensitivity. However, the absence of advanced PEC and ECL instrumentation limits practical application. And signal stability and ability to analyze multiple targets of PEC and ECL sensors is not sufficient. Label-free SPR biosensors simplify preparation and detection procedures. They have excellent analytical performance and offers direct and real-time detection platforms by using tiny chips, which make them have great potential for commercial detection of mycotoxins. But SPR sensors are not sensitive to binding events of low molecular weight molecules so that some modifications on chips and sophisticated instruments are still required. Both advantages and disadvantages exist in each kind of detection strategy. We need to choose the appropriate approach according to the conditions and requirements.In spite of numerous successes with laboratory-based mycotoxin detection, there are still many limitations and challenges to conquer in achieving practical applications.Firstly, nanomaterials are widely applied almost in all sensing strategies but the function of nanomaterials currently available are not perfect. There are many studies improving material properties and biosensor performances by using hybrid nanostructured materials, but they are not convenient for practical application and mass manufacturing. Developing promising nanomaterials that have excellent optical or electrochemical properties, but also are inexpensive, easy to prepare and environmentally friendly is still a focus of future research. This is also one of the core driving forces behind development of biosensor and immunoassay-based analytical methods.Secondly, most of the existing sensing strategies are based on single signal output, which are susceptible to instrument conditions and environmental interferences resulting in poor reproducibility and stability. To overcome this drawback, ratiometric sensor with dual signals is an ideal solution. Detection results are based on the ratio of two signals enable self-built-in corrections thus greatly improving sensitivity and accuracy of sensing method. Two different luminescent particles connected together to form a FRET system in optical sensors or two signal labels served as response/reference system in electrochemical sensors are mainstream of ratiometric sensor design. But there are few reports on the detection of mycotoxins by ratiometric biosensors at present.Lastly, simplicity, sensitivity, and high throughput have not really been implemented simultaneously in a rapid detection method. High-sensitive methods such as electrochemical biosensors often require complex modification operations, while simple and portable LFIA tend to produce qualitative or semi-quantitative detection results. Nowadays, with the development of micro-electro-mechanical systems (MEMS), sensor devices can be miniaturized at a tiny size. There are more and more reports on smartphone-based microfluidic biosensor systems which provide the portable, rapid, and sensitive sensing platforms. This is also a future trend that makes biosensors to better serve humans. Moreover, faced with a wide variety of mycotoxins, there have been many reports on the LFIA strip with multiplex test lines, multiplex SPR biochips, biosensors with multiple labels and integrating biosensors with microarrays and microfluidic systems. Most examples discussed in this paper are appropriate for up to three to six mycotoxins and involved complex designs. But achieving high-throughput quantitative detection of a dozen to dozens of analytes under the premise of guaranteed sensitivity still needs hard work.Despite many challenges, rapid detection methods based on cross-discipline play an increasingly important role in food safety, industrial manufacture, environmental monitoring, and clinical diagnoses. Traditional determination technology has not been adapted to the demands of rapid, on-site, and large-scale screening. Fast, simple, sensitive, low-cost, and high-throughput analytical methods with portable instrument are growing need. With development of nanomaterials and biosensor technology, rapid detection will receive much more attention in the future.


Fungi represent one of the most diverse and abundant eukaryotes on earth. The interplay between mold exposure and the host immune system is still not fully elucidated. Literature research focusing on up-to-date publications is providing a heterogenous picture of evidence and opinions regarding the role of mold and mycotoxins in the development of immune diseases. While the induction of allergic immune responses by molds is generally acknowledged, other direct health effects like the toxic mold syndrome are controversially discussed. However, recent observations indicate a particular importance of mold/mycotoxin exposure in individuals with pre-existing dysregulation of the immune system, due to exacerbation of underlying pathophysiology including allergic and non-allergic chronic inflammatory diseases, autoimmune disorders, and even human immunodeficiency virus (HIV) disease progression. In this review, we focus on the impact of mycotoxins regarding their impact on disease progression in pre-existing immune dysregulation. This is complemented by experimental in vivo and in vitro findings to present cellular and molecular modes of action. Furthermore, we discuss hypothetical mechanisms of action, where evidence is missing since much remains to be discovered. Humans are exposed to multiple fungi. Many fungi live in harmony with humans, rarely causing diseases. Possible health effects associated with fungi generally fall into the category infections (mycosis), allergic reactions, or toxic effects . Fungi cMolds are fungi, which grow in form of multicellular filaments called hyphae and are found in distinct environments: air, soil, plants, animals, and human hosts ,6. It isSignificant exposure to mycotoxins has also been found in developed countries , even inIn the past decade, mycotoxins have attracted considerable attention due to their potential of strong toxicity. Harmful effects caused by mycotoxins include acute poisoning after consumption of high dosages, which may even result in death . AF, as The immune system plays a key role in host–pathogen interaction and is essential for protecting living organisms against pathogens like molds and mycotoxins. Intact barrier functions, innate, and adaptive immune responses are essential for effective and successful elimination of infectious pathogens and toxic metabolites. These mechanisms might be disturbed in response to fungal cell wall antigens, or immunomodulatory and immunotoxic actions of mycotoxins, and therefore are critical for the development or exacerbation of immune system-related diseases.The present review aims to indicate that, next to direct effects of mold and mycotoxin exposure in immunocompetent individuals, exposure in the presence of pre-existing immune dysregulation is of particular concern. First, we briefly describe how mycotoxins directly exert immunomodulatory effects. Next, we illustrate how mycotoxins might trigger the onset or exacerbation of chronic inflammatory diseases, autoimmune disorders, and progression of HIV disease ,27,28,29The toxic mold syndrome is described as a symptom complex including various vague characteristics like cognitive impairment, emotional disturbance, respiratory complaints but also symptoms like muscle ache. It is thought to be a direct effect of indoor “black mold” and its mycotoxins . NeverthAlternaria spp., Cladosporium spp., Aspergillus spp. and Penicillium spp. belong to the phylum Ascomycota for which currently 88 allergens are described . Allergens include enolase, heat shock proteins, cyclophilins, proteases, redoxins, and disulphide isomerases .,70.69,70Since AF and OTA exposure, as well as HIV-infection, are known to cause immune modulation ,71,72, aThe immune system plays a central role in mold-host interaction. Several organ-specific barrier functions are impaired by mycotoxins ,74,75. AHerein, we summarize the immune response to mold and mycotoxins under different impaired immune system conditions. Therefore, we focus on in vivo and in vitro models to sum up and partly discuss the current knowledge of cellular and molecular modes of action.C. albicans and the mold A. fumigatus but the relative contribution of C-type lectin receptors in the development of immune responses against nonpathogenic molds as for instance Penicillium spp., Cladosporium spp. or A. alternata is lacking and the mechanisms involved are not formally identified yet.Mold species and its components including mold spores and hyphal fragments have been recognized to be involved in inflammation and as allergens that can cause different diseases . The immA. fumigatus . T. T54]. TThe role of chronic inflammation in the pathogenesis of inflammatory arthritis, for example, RA and spondylarthritis, is well established in that immune cytokines attributed to genetic and environmental factors drive systemic inflammation ,135. OTAelevated .In the past years, several human studies  supporteSince both, mycotoxins and HIV cause immunosuppression, chronic exposure to mycotoxins in HIV-positive people could lead to higher levels of viral burden and disease progression. Mycotoxins like OTA , AF 41]41], and + T cells [Past studies showed significantly lower percentages of CD4 T cells ,143 and + T cell numbers were still in normal range [+ T cell activation was increased, which is important in regard to HIV replication [However, recent human studies showing increased mycotoxin levels and elevated HIV burden, while CD4al range . In anotlication . Moreovelication .Regarding these findings, a modulation of the immune response by mycotoxins resulting in increased HIV burden and HIV disease progression c is curr+ and CD8+ T cells resulting in immune suppression and disease progression [In addition, stimulation of proinflammatory pathways due to impaired oxidative stress response might be crucial ,145. Progression .According to available data, it can be hypothesized that mycotoxin exposure in HIV-positive individuals exacerbate HIV burden in acute phase—at least in part via ROS, enhancing oxidative stress and inflammatory response, while chronification of inflammation during the chronic phase might then enhance immunosuppression and disease progression.Mold and mycotoxin exposure results mainly from contaminated food and inhalation of spores. Mold spores range in their size from 3 to 40 µm allowing them to flow in the air and being inhaled by animals and humans. Spores serve as vectors for mycotoxins and are pathogenic due to allergenic and infectious properties. Irrespective of exposure route, mycotoxins are systematically bioavailable affecting different system organ classes. In immunocompetent individuals, direct allergenic, and immune modulatory effects of mycotoxins are well described and accepted. In contrast, from a clinical point of view the concept of toxic mold syndrome that might be directly caused by mold or mycotoxins is discussed controversially. Moreover, according to critical opinions there is no sufficient evidence that mold/mycotoxin exposure might directly induce autoimmune diseases.The situation appears different considering the role of mold and in particular of mycotoxins as risk factors in the onset and severity of various diseases in individuals with an already impaired immune system. Exacerbation of asthma has already been shown in well-designed human cohort studies, meta-analyses, and animal models. Although there seems to be an association between mold/mycotoxin exposure and exacerbation of other dysregulated immune conditions like inflammatory bowel diseases, autoimmune diseases or disease progression in HIV-positive patients, no causality could be demonstrated to date.Epidemiological studies are important for the estimation of risk factors regarding diseases. Future human cohort studies regarding immune system disorders including chronic inflammatory disease, autoimmune diseases, and HIV disease should consider environmental chemicals like mycotoxins to a greater extend to evaluate the actual relevance for human disease progression.(1)Study objectives should include investigation of the adjuvant activity of mycotoxins of the addressed immune system disorders. For better understanding of the underlying pathological mechanism, future research should investigate the key modes of action including the alteration of barrier functions, exacerbation of inflammation and T-cell differentiation. Moreover, the measurement of relevant disease parameters like the influence on gastrointestinal microbiota in IBD, or axonal demyelination in MS is strongly recommended.(2)Using appropriate disease models for a valuable translation to human exposure and clinical research is suggested. Therefore, it is necessary to determine state of the art disease models and use disease-specific biomarkers as readout. Real-world human mycotoxin exposure scenarios should be mimicked in model organisms to be able to make estimations about critical threshold concentration for specific vulnerable populations.(3)Investigations to develop prevention or treatment strategies to face the exacerbation of pre-existing immune system disorders due to mycotoxin contamination should be addressed as well. It should be questioned if the harmful effects are reversible after elimination of the mycotoxin or by interfering with the mycotoxin modes of action. E.g., the development of sensitive and specific detection assays for multiplex mycotoxin detection could be established for comprehensive diagnostics.(4)Next to mycotoxin exposure, also invasion mechanisms of mold might be of interest depending on the disease model. Under this scope, antifungal treatment options should be investigated as well.However, to determine a causal relationship randomized controlled double blind interventional human studies are needed, which are unethical and therefore justifiably not allowed. Therefore, well-conducted in vivo and in vitro research studies with plausible explanations of pathological mechanism should be performed. Additionally, the invasion of mold under such conditions should be addressed as well. Although emerging evidence outlined in the current review clearly indicates that mycotoxins have an exacerbating effect on pre-existing immunological disorders, the key elements have yet to be determined. To do so, we encourage future research to consider the following points:These different research approaches are needed to complement the current clinical knowledge and link the dots until well-studied interventions are available to properly assess the combination of concern—mold, mycotoxins and a dysregulated immune system.


Gram-negative bacteria include a number of pathogens that cause disease in humans and animals. Although antibiotics are still effective in treating a considerable range of infections caused by Gram-negative bacteria, the alarming increase of antimicrobial resistance (AMR) induced by excessive use of antibiotics has raised global concerns. Therefore, alternative strategies must be developed to prevent and treat bacterial infections and prevent the advent of a postantibiotic era. Vaccines, one of the greatest achievements in the history of medical science, hold extraordinary potential to prevent bacterial infections and thereby reduce the need for antibiotics. Novel bacterial vaccines are urgently needed, however, and outer membrane vesicles (OMVs), naturally produced by Gram-negative bacteria, represent a promising and versatile tool that can be employed as adjuvants, antigens, and delivery platforms in the development of vaccines against Gram-negative bacteria. Here, we provide an overview of the many roles OMVs can play in vaccine development and the mechanisms behind these applications. Methods to improve OMV yields and a comparison of different strategies for OMV isolation aiming at cost-effective production of OMV-based vaccines are also reviewed. Human beings have greatly benefited from the discovery and application of antibiotics to fight bacterial infections and significantly improve health and life span as well as enable efficient animal husbandry and food production , 2. For Streptococcus pneumoniae and Haemophilus influenzae type b, which dramatically reduced antibiotic use in humans , for which the effectiveness has been limited by the antigenic variation of outer membrane proteins used as protective antigens in the vaccine formulation employed  are spherical particles secreted by all Gram-negative bacteria investigated to date . AlthougThe host innate immune system can recognize OMVs efficiently, facilitating induction of inflammatory response and antigen presentation and subsequently leading to a specific immune response to OMVs. The initiation of this process is largely attributed to the presence of pathogen-associated molecular patterns (PAMPs) on the OMVs. PAMPs are molecules, such as LPS or PG, that are widely conserved among microorganisms. PAMPs present on the OMVs can be detected by the pattern recognition receptors (PRRs) ubiquitously expressed by innate immune cells that, in turn, initiate signal cascades, leading to the priming of the host immune response . This caThe interplay between OMV-associated PAMPs and innate immune cells plays an important role through induction of proinflammatory cytokine secretion by different cell types –33. Amon–+ T cell activation.The interactions between PAMPs and innate PRRs promote activation and maturation of antigen-presenting cells (APCs) in the transition toward adaptive immune responses. Activated APCs can express a variety of surface molecules that are needed for presentation of protein antigens to T cells. As shown in + T cells, it is worth noting that OMVs, as a mixture of exogenous antigens, are capable of programming APCs for antigen presentation to CD8+ T cells (referred to as cross-presentation). Lee et al. showed that OMVs could enhance cross-priming of CD8+ T cells when coimmunized with antigens as adjuvants, indicated by increased gamma interferon (IFN-γ) secretion of mice splenocytes restimulated with MHC-I-restricted peptides .OMVs can be viewed as nonreplicative representations of the parental bacterial cell by possessing antigenicity similar to and a higher safety profile than intact yet attenuated bacterial cells. In addition to PAMPs, OMVs also carry a combination of pathogen-specific antigens that can stimulate long-lasting adaptive immunity and immune memory, both of which are crucial in conferring protection against future infections. Furthermore, the inactivation procedure during production of conventional bacterial vaccines or subunit vaccines typically denatures proteins and other components, potentially reducing the antigenic diversity in these vaccine types. In comparison, OMV-based vaccines offer the potential of providing a broader and more effective protection by presenting a repertoire of antigens in their native conformation. Thus, OMVs are regarded as promising vaccine candidates against the bacteria from which they are derived. A good example is the recent OMV-based vaccine against the meningococcus MenB . Several46–10.1128/mBio.01707-21.1TABLE S1TABLE S1, DOCX file, 0.04 MB.Examples of experimental OMV-based vaccines. Download Copyright © 2021 Zhu et al.2021Zhu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro and in vivo, not all Gram-negative bacteria are equally suitable for OMV isolation. This can be due to strict growth requirements, low growth rate, low vesiculation rate, or a combination of these factors, which render the OMV yield too low . Hbp consists of a cleavable β-domain that anchors on the outer membrane and a passenger domain that can be replaced with heterologous antigens of interest. Normally, Hbp synthesized in bacteria is translocated across the cell envelope and secreted into the extracellular milieu by the enzymatic lysis of the link between passenger and β-domain. To achieve the purpose of surface display, a noncleavable mutation was introduced into Hbp to keep heterologous passengers retained on the outer membrane. Based on this principle, the feasibility of employing Hbp for heterologous protein display in OMVs has been demonstrated and applied in experimental vaccine research –67. Alte–Escherichia coli and Salmonella spp. The potential of alternative carrier proteins expressed by less well-characterized bacterial species still remains to be investigated. Some examples of alternative carrier systems are the proteins fHbp and ApfA, adapted for membrane enrichment of antigens in N. meningitidis and Actinobacillus pleuropneumoniae, respectively  signal sequence or to periplasmic proteins such as OmpA , 73. HowS. pneumoniae and Campylobacter jejuni , displayed by E. coli OMVs able to elicit PNAG-specific antibodies. However, antibodies against PNAG in its native acetylated conformation were shown to be ineffective in preventing infection from PNAG-positive bacteria  and are regarded as a practical source of membrane antigens in vaccine manufacturing , 89.N. meningitidis, shifting the OMV production mode from batch to continuous process resulted in an estimated 9-fold increase of OMV production under a specific dilution rate  is the most widely used method for OMV isolation. This technique consists of multiple steps of low-speed centrifugation followed by a final ultracentrifugation step, which ultimately leads to the production of an OMV pellet. Although simplicity of execution and relatively low technical requirements characterize DC as one of the most approachable methods for OMV isolation, contaminants such as pili, flagella, and soluble components, which cannot be separated by centrifugation alone, are still present in the isolated OMV pellet . TherefoSize-exclusion chromatography (SEC) isolates OMVs by trapping them into nano-sized pores in porous resin particles to delay their elution time to achieve purification. SEC has frequently been employed as a secondary step to enhance the purity of crude OMV extractions in early research. Recently, however, it has been adopted for the direct isolation of OMVs from cell-free supernatants. SEC possesses the advantage of simplicity of execution and can lead to OMV isolations with high purity rates from cell-free supernatants in one step , 94. SECin vivo immunization trials. An additional advantage of HFD is that it offers a considerably consistent reproducibility from batch to batch, a characteristic rather crucial for vaccine development  was originally developed by Musante et al. as a way to quickly identify bacterial urinary tract infections by selectively enriching urine samples . The met96–elopment . As HFD E. coli strain to express a recombinant OmpA protein fused with a repeated histidine sequence tag (His tag), thereby labeling the resulting OMVs secreted in culture. OMV-containing cultures were subsequently subjected to immobilized metal affinity chromatography (IMAC) to selectively isolate His-tag-marked OMVs while avoiding contamination by unlabeled wild-type OMVs (Another technique described for the isolation of OMVs is affinity purification. This method relies on the affinity adsorption between a tag incorporated into OMVs and the ligands that specifically bind the tag. As a proof of concept, Alves et al. engineered an ype OMVs . From thVaccination plays a complementary role to antibiotic treatment in reducing incidence and morbidity of bacterial infections. During the initial course of a bacterial infection, antibiotics represent an indispensable tool due to their fast mode of action and broad spectrum, which often enables clinicians to treat sudden outbreaks even when the causative agent is still uncertain . Infectein silico screening of antigens according to their subcellular location, conservation, and hydrophobicity to predict those that are most likely to be vaccine candidates, which greatly simplifies the process of antigen selection (First-generation vaccines represented a big step forward in both human and animal health over the past century. These vaccines are based on relatively crude formulations according to modern standards, mostly relying on the administration of inactivated or attenuated pathogenic microorganisms in order to generate immunity in healthy individuals. Despite their effectiveness, first-generation vaccines presented several safety risks, mostly due to the risk of incomplete inactivation or reversion of virulence of the pathogens included in the formulations . Subsequelection . More reelection . Fourth-election . Exampleelection . Nucleicelection . Artificelection .Due to their nonreplicative nature, antigenic properties, and adjuvanticity, OMVs as immunogens do not really fit in any of the previously mentioned vaccine categories. As such, OMV-based vaccines constitute a category of their own.The pluripotency of OMVs as adjuvants, antigens, and delivery platforms is being extensively investigated and has been proven by the development of several OMV-based pharmaceutical prototypes in the last 2 decades, which have been well summarized in other reviews , 112. NoDespite the universal nature of OMV secretion among Gram-negative bacterial species, secreted OMVs are often a rather heterogeneous mix of particles possessing significantly different attributes and functions. As a consequence of this intrinsic variability, OMVs isolated from different bacterial species have been shown to present very different protein and, consequently, immunological profiles. For this reason, very limited assumptions can be made at the beginning of any research endeavor focusing on previously undescribed OMVs. Any novel OMV-producing strains need to be individually characterized and often genetically engineered to increase OMV yields, a lengthy and time-consuming process. Further insights into the relationship between OMV composition and their resulting immunological profile would also be beneficial for accelerating the development of novel OMV products.In conclusion, the immunological properties of OMVs characterize them as a promising new avenue for the development of novel vaccine formulations and will almost certainly represent a useful tool at our disposal in combating bacterial infectious diseases and tackling the global challenge posed by AMR.


Haemophilus influenzae (NTHi) is an ubiquitous commensal-turned-pathogen that colonises the respiratory mucosa in airways diseases including Chronic Obstructive Pulmonary Disease (COPD). COPD is a progressive inflammatory syndrome of the lungs, encompassing chronic bronchitis that is characterised by mucus hypersecretion and impaired mucociliary clearance and creates a static, protective, humid, and nutrient-rich environment, with dysregulated mucosal immunity; a favourable environment for NTHi colonisation. Several recent large COPD cohort studies have reported NTHi as a significant and recurrent aetiological pathogen in acute exacerbations of COPD. NTHi proliferation has been associated with increased hospitalisation, disease severity, morbidity and significant lung microbiome shifts. However, some cohorts with patients at different severities of COPD do not report that NTHi is a significant aetiological pathogen in their COPD patients, indicating other obligate pathogens including Moraxella catarrhalis, Streptococcus pneumoniae and Pseudomonas aeruginosa as the cause. NTHi is an ubiquitous organism across healthy non-smokers, healthy smokers and COPD patients from childhood to adulthood, but it currently remains unclear why NTHi becomes pathogenic in only some cohorts of COPD patients, and what behaviours, interactions and adaptations are driving this susceptibility. There is emerging evidence that biofilm-phase NTHi may play a significant role in COPD. NTHi displays many hallmarks of the biofilm lifestyle and expresses key biofilm formation-promoting genes. These include the autoinducer-mediated quorum sensing system, epithelial- and mucus-binding adhesins and expression of a protective, self-produced polymeric substance matrix. These NTHi biofilms exhibit extreme tolerance to antimicrobial treatments and the immune system as well as expressing synergistic interspecific interactions with other lung pathogens including S. pneumoniae and M. catarrhalis. Whilst the majority of our understanding surrounding NTHi as a biofilm arises from otitis media or in-vitro bacterial monoculture models, the role of NTHi biofilms in the COPD lung is now being studied. This review explores the evidence for the existence of NTHi biofilms and their impact in the COPD lung. Understanding the nature of chronic and recurrent NTHi infections in acute exacerbations of COPD could have important implications for clinical treatment and identification of novel bactericidal targets.Non-typeable  COPD is currently the third leading cause of global mortality affecting an estimated 174 – 384 million people worldwide and is responsible for 3.2 million deaths annually . COPD isChronic bronchitis is particularly important with respect to the role of bacteria and chronic infections. An immunological cascade in response to inhaled particles and gases drives airway structural remodelling that leads to hypertrophy and hyperplasia of sub-mucosal bronchial glands and mucus-producing goblet cells , formingTogether these conditions may lead to significant microbiome shifts and favour the proliferation of fastidious, facultatively anaerobic pathogens, and drive a biofilm lifestyle.Contrary to historical views, the lung is not a sterile environment but has a rich and diverse microflora. The role of the lung microbiome and microbial dysbiosis in respiratory diseases is becoming increasingly understood through the advent of 16S rRNA sequencing of sputum, deep lung brushes and biopsy samples , where tPseudomonas, Streptococcus, Prevotella, Fusobacterium, Veillonella Porphyromonas and Haemophilus spp.  is responsible for a large subset of AECOPD hospitalisations  vaccine and prescription of COPD maintenance drugs  is the density-dependent coordination of gene expression  across bomologue  and thesomologue , leadingin-vitro  strains grown in-vitro  or the cin-vitro . Scanninsistence . Disruptsistence  showing m matrix , with gsLuxS mutants. Instead, QSeB/C mutants show significant biofilm biomass and surface coverage in continuous flow models over 24 – 48 hours   webs of One of the lung’s primary defences against bacteria is activation of complement that opsonises invading pathogens for phagocytosis by neutrophils and macrophages . HoweverP. aeruginosa, the aetiological pathogen in chronic cystic fibrosis (CF) infections, NTHi biofilms have a limited carbohydrate or exopolysaccharide component , a strong and flexible transmembrane filament that has diverse functional roles in pathogenicity. Other interactions include the sharing of β-lactamases and eDNA   through P. aeruginosa isolates and improving the efficacy of antibiotics  awarded for JW’s doctoral studies.KS reports grants from AstraZeneca, outside the conduct of the study; TW reports grants and personal fees from AstraZeneca, outside the conduct of the study; personal fees and other from MMH, grants and personal fees from GSK, grants and personal fees from AZ, personal fees from BI, grants and personal fees from Synairgen, outside the submitted work.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.


Sepsis survivors experience a persistent myopathy characterized by skeletal muscle weakness, atrophy, and an inability to repair/regenerate damaged or dysfunctional myofibers. The origins and mechanisms of this persistent sepsis-induced myopathy are likely complex and multifactorial. Nevertheless, the pathobiology is thought to be triggered by the interaction between circulating pathogens and impaired muscle metabolic status. In addition, while in the hospital, septic patients often experience prolonged periods of physical inactivity due to bed rest, which may exacerbate the myopathy. Physical rehabilitation emerges as a potential tool to prevent the decline in physical function in septic patients. Currently, there is no consensus regarding effective rehabilitation strategies for sepsis-induced myopathy. The optimal timing to initiate the rehabilitation intervention currently lacks consensus as well. In this review, we summarize the evidence on the fundamental pathobiological mechanisms of sepsis-induced myopathy and discuss the recent evidence on in-hospital and post-discharge rehabilitation as well as other potential interventions that may prevent physical disability and death of sepsis survivors. Sepsis is a debilitating disease with medical costs exceeding $17 billion per year and is associated with long-term poor physical outcomes as well as increased all-cause mortality . Higher One of the main characteristics of sepsis is the acute derangement of pro-inflammatory and anti-inflammatory (immunosuppression) responses in all patient age groups, with older adults not returning to hemostasis after surviving sepsis. The improved implementation of evidence-based intensive care unit (ICU) clinical management has resulted in decreased early hospital mortality. However, many survivors develop chronic critical illness (CCI), with persistent inflammation from which they may never fully recover . SpecifiSepsis survivors who return home frequently face exacerbation of comorbidities and develop chronic physical disability due to discontinued hospital physical therapy (PT) or occupational therapy (OT) . PersistThough the primary biological mechanisms for skeletal muscle abnormalities in this population are still unknown, several candidates have been identified as contributing factors. For example, the negative outcomes in muscle function are presumably exacerbated by high levels of physical inactivity due to bed rest during prolonged ICU admissions. Contributing to this vicious cycle, weak and atrophied muscles become more susceptible to injury . Sepsis Given the biological evidence of sepsis-induced myopathy and failure to recover health status and physical function over at least 1 year in older sepsis survivors, there is an urgent need for practical solutions to improve functional outcomes in this home-bound population at high risk for disability and death. In this review, we will summarize the existing evidence on the fundamental pathobiological mechanisms of sepsis-induced myopathy, recent evidence on in-hospital and post-discharge rehabilitation, and other potential interventions that may prevent physical disability and death of sepsis survivors.The most accepted model for the origin of sepsis-induced myopathy is that it is triggered by circulating pathogens and cytokines that signal skeletal muscle pathways associated with halted protein synthesis (due to overproduction of reactive oxygen species) and accelerated protein degradation (due to enhanced proteasome proteolytic degradation and autophagy pathways) . The actMuscles sense and respond to circulating pathogens and signals arriving from damaged cells via receptors, particularly toll-like receptors (TLRs) on the sarcolemma. All membrane-bound TLRs, such as TLR4 and interleukin (IL)-1β signal through an adapter protein, myeloid differentiation primary response 88 (MyD88), which eventually activates downstream nuclear factor κB (NF-κB) signaling networks. NFκB is a central player in intracellular inflammatory responses and is a critical link between inflammation and most forms of muscle atrophy and myopathy ,27. The Another factor that determines the fate of muscle function in sepsis is mitochondrial status. In whole muscle, mitochondrial dysfunction can drive persistent intrinsic tissue inflammation, loss of muscle mass, and loss of force production after sepsis . The breSCs are unipotent muscle-resident myogenic stem cells that participate in skeletal muscle regeneration and repair during recovery from injury. They and their immediate surrounding cells and microenvironment are referred to as the SC niche. SCs are characterized by the expression of transcription factor Pax7, which is required for their self-renewal ,38. ThesImpaired skeletal muscle regrowth and decreased SC content have been demonstrated in human septic patients displaying sustained and progressive atrophy and weakness following hospitalization  14,41].,41.14,41Despite the metabolic and regenerative barriers in skeletal muscle in response to sepsis, early rehabilitation has been attempted in critically ill patients. We will discuss the main outcomes of these studies in the following section.A number of systematic and narrative reviews of randomized clinical trials (RCTs) over the past 8 years have assessed the available published evidence for effective early rehabilitation efforts in reducing the severity of muscle weakness, mobility, and quality of life after critical illness ,52,53,54In evaluating the results of rehab in all conditions of post-intensive care syndrome, the outcomes of early rehabilitation are mixed but can be roughly divided into three categories: (1) no improvements in long or short-term physical outcomes  appear to include very early initiation of rehab and (2) careful execution of programmed mobility and range of motion exercises by experienced therapists. In addition, (3) there appears to be no evidence, at this time, that alternative approaches such as extensive resistance exercise, ergometry, or EMS provide a consistent advantage over the careful application of physiotherapy.Early rehabilitation is critically important for recovery from a critical illness ,62; howeStudies have shown that a 12-week multi-modal home-based intervention including cognitive and physical rehabilitation in middle-aged (41–59 years old) critical illness survivors (25% sepsis patients) was feasible and effective in improving physical and cognitive outcomes . They alWhile structured facility-based physical activity is a powerful tool to improve overall health in many health conditions and diseases, most structured physical activity training programs would not be feasible in older sepsis survivors for a few reasons. Firstly, most patients are unlikely to be capable of regularly traveling to participate in structured physical activity programs with multiple visits to intervention sites due to poor health status and distant living locations. Secondly, group-based interventions may be difficult to customize to each individual patient’s level of function. Thirdly, participants may feel less safe in public places, particularly in the era of COVID-19. For these reasons, programs are needed to overcome these limitations and be tailored to individuals in their home environment.In sedentary women (55–70 years old), 12 weeks of home-based physical-activity consulting and monitoring of step-count goals increased their daily step count and decreased sedentary time . FurtherWhile sepsis mortality continues to decline through advances in critical care support, long-term outcomes after sepsis remain poor; contributing to these poor post-sepsis outcomes is sepsis-induced myopathy. It is thought that this myopathy is triggered by circulating mediators that signal skeletal muscle pathways associated with decreased protein synthesis and accelerated protein degradation . These p


Translating the human microbiome in health and disease requires detailed information about each microbial member—from individual annotated genomes to community functional dynamics to relationships with the host. Toward this goal, phylogenetic descriptions of the microbiome are evolving into mechanistic analyses. Recent years have seen a dramatic rise in metagenomic and metabolomic studies using microbial-driven pathologies as models to understand host–microbiome interactions. These studies are charting community-wide ecological maps that provide insight into microbial determinants of health and disease, layering in impacts of the microbiome at different life stages. Although much is known about early microbiome development, trajectories during aging and later in life require further characterization.The microbiome impacts every organ system and aspect of physiology, often through as-yet-unknown mechanisms. Moreover, the influence of the microbiome extends beyond the local environment: neuroactive molecules produced by gut commensals reach the brain to mediate mood, depression, and mental illness . UnderstGenome Medicine highlights the advances made in understanding strain diversity, microbial biochemistry, disease risk, and clinical outcomes. In this Editorial, I highlight key areas and recent progress that has been made in the field toward translating the microbiome and defining the many pathways that modulate host biology.This special issue of Metagenomic sequences provide a framework to generate testable mechanistic hypotheses; however, functional validation is limited by the availability of culturable isolates. Strain collections are being assembled, but those aiming to capture the breadth of diversity within the microbiome underrepresent strain-specific functional diversity. This obstacle is being circumvented in part by computational advancements, such as comparative genomics platforms that can analyze a single bacterial class, map genetic diversity to phenotypes, and identify potential competitive fitness genes through the quantification of highly related strains . This apRecently, we have learnt that subsets of microbiome-reactive T cells are characteristic of health and various diseases, but our knowledge of microbial antigens is incomplete . ComputaAdaptive immunity is additionally affected by microbial metabolites, and recent work revisited the effects of bacterially modified bile acids on T cell biology. Lithocholic acid (LCA) derivatives were found to regulate the differentiation of naive T cells into Treg and Th17 cells, the balance of which plays a critical role in inflammatory bowel diseases (IBD) . A netwoDietary fibers modulate the composition and function of the microbiome, shaping host–microbiome interactions. Fiber-rich diets maintain a healthy, diverse microbiome and lead to beneficial metabolites such as short-chain fatty acids (SCFAs), which promote mucus and antimicrobial peptides production as well as maintain intestinal barrier integrity and robust immunity. Fiber-poor diets reduce diversity and alter microbial functions, impairing host physiology and increasing susceptibility to infection and chronic inflammation. Interventional increases in the dietary glycans, fructooligosaccharides and polydextrose, have been shown to cause quantifiable and consistent microbiome responses . ImportaPrevotella copri, which has been correlated with improved glucose and insulin tolerance when subjects consume a high fiber diet [Prevotella copri and presented metagenomic analyses defining four carbohydrate-metabolizing clades highly prevalent in non-industrialized populations, which typically have high-fiber diets. Functional analyses confirmed that isolates from each clade utilize distinct sets of plant-derived polysaccharides. Thus, there is increasing interest in restoration of the healthy state potentially through dietary interventions or by utilizing microbial carbohydrate-active enzymes, CAZymes, to replace metabolic activity lost through diet-dependent microbiome changes.Specific microbial commensals have been linked to beneficial health effects that are dependent on diet. An example is ber diet , 12. TwoBacteroides highly abundant in their microbiomes. Infants born in Russian Karelia, where autoimmunity is less prevalent, were exposed to a structurally distinct LPS from Escherichia coli. The study demonstrated that Bacteroides LPS inhibits innate immune activation by E. coli LPS, providing evidence that early life colonization by immune-silencing microbiota may hinder immune education and enhance susceptibility to autoimmune disease.Discoveries in microbiome-associated diseases point toward mechanisms underlying pathophysiology. Addressing concurrent increases in autoimmune and allergic disease rates, the hygiene hypothesis posits that early childhood exposure to certain microbes contributes to immune development and protects against these diseases. To test this, a longitudinal study followed children genetically predisposed to autoimmunity from birth to age three in neighboring countries . InfantsGain- and loss-of-function mutations in C9orf72 contribute to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Microbiomes of ALS patients differ from unaffected individuals, and recent evidence suggests that interactions between C9orf72 and the microbiome influence inflammatory responses within the nervous system. The latter stemmed from the observation that, despite identical genetic backgrounds, C9orf72-deficient mice reared in separate facilities harbored distinct microbiota and displayed marked differences in disease severity. A reduction in immune-stimulating bacteria protected C9orf72-deficient mice from neuroinflammation and early mortality, even after initial onset . In contThe microbiome influences cancer onset, progression, and response to therapy. While effects can be exerted indirectly through release of bioactive molecules into the circulation, it is also possible for the tumor microenvironment to support its own microbiome. Bacteria have long been detected in tumors, but the small biomass of the microenvironment made it challenging to exclude the possibility of contamination. A recent analysis surveyed bacteria in primary tumors and adjacent normal tissue across seven cancer types from body sites both exposed to and protected from microbes . Each tuCurrent efforts are building extensive biobanks of human gut isolates with corresponding multi-omic data, not only to advance mechanistic research but to preserve and expand our understanding of microbiome biodiversity across communities worldwide. Research to date largely centers on industrialized populations, providing little insight into the microbiomes of non-industrialized populations that are often more diverse. Moreover, therapies based on or targeted for industrialized microbiomes might be ineffectual or even detrimental for other populations. Longitudinal microbiome analyses of the Hadza, an indigenous hunter-gatherer society, illuminated important roles played by gut microbes at risk of extinction from industrialization . The HadHuman cohort studies will continue to be integral for translating the microbiome in health and disease. Treatment-naive cohorts and longitudinal studies revealed important alterations in microbiome community structure and function during IBD  and servMulti-omic data exposed extensive “microbial dark matter” within the microbiome. Much about these unculturable microbes and their metabolic capabilities remains unknown, but new computational and biochemical approaches are enabling the characterization of unannotated molecules. The next steps will be to resolve the human metabolome into host, microbial, or co- metabolites  and build chemical libraries that can be tested in functional assays. A complete annotation of the microbes, genes, proteins, and metabolites within the human microbiome will be essential to hypothesis-based functional studies that reveal biological mechanisms at work in health and disease.While a promising route to the improvement of human health, microbiome-based therapeutics face several current challenges. One is the need to study immunomodulatory molecules and other bioactives derived from microbes at physiological concentrations. Then, deep mechanistic understandings need to be developed with particular emphasis on potential pleiotropic effects. For example, a single microbial metabolite may bind multiple host receptors, each with a distinct, often cell type-specific biological outcome. Stimulation thresholds determined by signal strength, duration, or both might also dictate host responses. Synergistic or antagonistic effects that are not observed in reductionist models could appear in more complex contexts and emphasize the critical importance of conducting functional studies in simple systems, such as mono- and co-cultures, as well as sophisticated models that more closely resemble tissues. Lastly, pharmacokinetic processes of administration, absorption, distribution, metabolism, and excretion need to be considered when evaluating metabolites or microbes themselves as therapeutics. The future of microbiome-based precision medicine will rely on extensive hypothesis-based functional work, as well as full consideration of the complexities between microbes and their hosts, in order to improve human health.


This Special Issue concerns the theme of how parents may influence child and adolescent weight-related and obesity developments. The ten articles in this collection cover a wide range of research designs and span a wide developmental age range of studies focusing on eating and weight-related outcomes in (very young) children or adolescents. All studies have one main central message, that is, parents, parenting and the home environment play an important role in children’s eating and weight development.To date, two prospective studies examining eating behaviour and weight development among adolescents both suggest that parents affect later adolescents’ eating behaviour ,2. The sIn this regard, it is also important to understand contextual and individual moderators that might determine whether and how parental stress, parenting, and/or child attachment may affect children’s weight-related outcomes. For instance, Beijers et al.  have proMoreover, Quick et al.  found thNotably, some parental stress eliciting mechanisms may already operate at a very early stage, prenatal and during lactation. Larsen and Bode  reviewedMoreover, the potential importance of an automatic lifestyle may also be reflected in the data of more than four thousand six year-old children from the Generation R Study by Yang-Huang et al.  on the cAnother way to avoid practical barriers and reach vulnerable families may be through eHealth, offering a way to reach families with tailored health information. In this regard, Chau, and colleagues  found thTo conclude, the articles in this Special Issue support the idea that parents and the home environment play an important role in children’s weight-related development and that parental interventions to prevent childhood obesity should avoid practical barriers and facilitate the reach of vulnerable parents. Some studies refer to (clustering of) lifestyle habits, in specific home contexts , among specific vulnerable groups , possibly reflecting underlying automatic (parenting) processes. Other studies support the idea that parental stress, parenting, and related parent–child attachment and child emotion regulation are important factors in determining child weight-related outcomes. As parental stress may elicit a more automatic unhealthy processes within families, more attention should be paid to parental stress in childhood obesity science. Future high-quality research is needed to understand when, how and for whom parental stress begets obesity in children. Longer-term prospective designs should include more vulnerable subgroups  and investigate the effects of different forms of stress among diverse child age groups, while examining potential interrelated or additive mechanisms . Experience sampling methods may provide specific insight into more momentary, and potentially more automatic, mechanisms, through which parental stress may increase child obesity risk. Moreover, intervention studies targeting stress among parents may further provide causal evidence of the parental stress–child obesity link and the underlying mechanisms involved. These areas would be fruitful pursuits for childhood obesity prevention science.


This commentary encourages the regular archiving of nucleic-acid-stabilized serial samples of wastewaters and/or sewage. Stabilized samples would facilitate retrospective reconstitution of built environments’ biological fluids. Biological time capsules would allow retrospective searches for nucleic acids from viruses such as SARS-CoV-2. Current resources for testing need not be diverted if samples are saved in case they become important in the future. Systematic storage would facilitate investigation into the origin and prevalence of viruses and other agents. Comparison of prevalence data from individual and clinical samplings with community wastewater would allow valuable comparison, contrast and correlation among different testing modalities. Current interest is focused on SARS-CoV-2, but archived samples could become valuable in many contexts including surveys for other infectious and chemical agents whose identity is not currently known. Archived time series of wastewater will take their place alongside other biological repositories and records including those from medical facilities, museums, eDNA, living cell and tissue collections. Together these will prove invaluable records of the evolving Anthropocene. The purpose of this commentary is to encourage the regular archiving of nucleic-acid-stabilized serial samples of untreated waste water and sewage from multiple systems and subsystems of our built environment. There is an important literature on preserving wastewater samples , 2 for cThere is a significant and rapidly growing literature on SARS-CoV-2 virus detection in sewage, including tracing the virus to specific buildings; surely more use of sewage for “real time” analysis will come –11. It sBiological diaries, archives, and culture collections are invaluable in contexts including medical repositories, museums, collections of living and revivable tissues and cells in biobanks, seed banks of biodiversity, and many other contexts , 14. BioHistorical samples have proven important in tracing the origin and spread of infectious diseases in previous instances. However, in the cases of which we are aware, biological materials were originally collected for other purposes. Molecular methods of analysis used, to study the origin of HIV, for example, were not even dreamed of at the time samples were taken . MoleculThere are many contexts for which a properly preserved time series of biological samples of built environments are potentially of inestimable value. Archeologists and physical anthropologists make good use of the information obtained from ancient latrines, valuable biological time capsules that were not deliberately created –22. MuseEnvironmental DNA (eDNA) is increasingly recognized and used for species assays in aquatic systems to supplement or partially replace trawl or other macroscopic methods of census , 24. MetLeonardo Da Vinci conceived of Earth and also architectural entities such as cities as akin to living organisms . The conAnalytic methods are likely to improve over the coming years and to increase the information that can be obtained from archived samples. The most exciting developments might be unanticipated, but some possibilities for innovation are outlined below.Retroactive analysis of wastewater samples has the potential to inform epidemiology, kinetics in closed and semi-closed systems, and the understanding of microbial evolution in built environments. Sewage samples archived for other purposes and stored at -20 °C have been retroactively assayed for SARS-CoV-2 and found positive . When diConsider the controversy concerning the origin of SARS-CoV-2 –38. ArchThe number of positive cases determined from individual tests has several uncertainties. How are people to be tested chosen? The kinetics of viral appearance and disappearance in individuals are uncertain variables. The shared fluid of built environments is a complementary sampling strategy that pools all the individuals, and with statistical qualifications has been found to agree reasonably well with individual sampling from the community . DependiPCR and rtPCR based methods for virus detection now largely depend on having the target sequence in hand. Non-targeted detection methods are becoming more sophisticated, allowing detection of genetic elements and agents that have not been previously defined. Potential nucleic-acid targets include viruses, bacteria, protists, parasites, and antibiotic-resistance genes on plasmids. More speculatively, and dependent upon engineering beyond today’s norms, there is a possibility that viral nucleic acid in sewage is present inside host cells. Single cell isolation might allow identification of infected individuals directly from wastewater. This future technical capability raises questions concerning conflicts of privacy and public health .The storage of samples for future nucleic acid analysis is currently not as widespread as its potential suggest it should be. What nudges  might enCurrent sewage analysis includes some pharmaceuticals, cosmetics, illegal drugs, viruses, pesticides and other chemicals of concern –47. The


As skilled nursing in the United States moves to a value-based model, malnutrition care remains a critical gap area that is associated with multiple poor health outcomes, including hospital readmissions and declines in functional status, psychosocial well-being, and quality of life. Malnutrition is often undiagnosed/untreated, even though it impacts up to half of skilled nursing facility (SNF) residents, and COVID-19 infections/related symptoms have likely further increased this risk. In acute care, malnutrition quality measures have been both developed/tested, and nutrition-focused quality improvement programs (QIPs) have been shown to reduce costs and effectively improve care processes and patient outcomes. Less is known about such quality initiatives in SNF care. This perspective paper reviewed malnutrition-related quality measures and nutrition-focused QIPs in SNFs and nursing home care. It identified that although the Centers for Medicare & Medicaid Services (CMS) has had a nursing home Quality Assurance and Performance Improvement (QAPI) program for 10 years and has had SNF quality measures for nearly 20 years, there are no malnutrition-specific quality measures for SNFs and very few published nutrition-focused QIPs in SNFs. This represents an important care gap that should be addressed to improve resident health outcomes as SNFs more fully move to a value-based care model. Skilled nursing facilities (SNFs) continue to account for the majority of spending for post-acute care services in the United States (US) . PopulatIn skilled nursing care—as in all other areas of US healthcare—federal Medicare continues its progress toward value-based payment and away from traditional fee-for-service models. A significant shift occurred in October 2019, when the US Centers for Medicare & Medicaid Services (CMS) moved from the Resource Utilization Groups, Version IV (RUG-IV), to the Patient Driven Payment Model (PDPM). PDPM focuses on quality of care and patient outcomes, as opposed to quantity of provided services. Specifically, PDPM assigns each resident a case-mix classification based on five different components addressing acuity of illness and management needs, which then drives the daily reimbursement rate for that individual. CMS has commented that while the RUG-IV system reduced resident care to a single, typically volume-driven, case-mix group, PDPM focuses on the individual needs/goals of each resident .One critical area of individualized resident need is nutrition, and the Federal Nursing Home Reform Act legally requires that nursing home meals meet the daily nutrition and special dietary needs of each resident . Speech In addition to impacting individual resident needs, nutrition can also impact SNF business models. Tavakoli et al.  suggest SNFs may be challenged by an aging population, as more people need supportive care but may have poorer health outcomes because of unrecognized, undiagnosed, and untreated malnutrition. Quality measures and quality improvement programs (QIPs) can help address these challenges and improve care processes to benefit both residents and SNFs. In acute care, malnutrition quality measures have been developed/tested and nutrition QIPs have been shown to be effective in identifying and intervening for malnutrition . HoweverThis perspective paper reviews malnutrition-related quality measures and nutrition-focused QIPs in SNFs and nursing home care. To provide context, it initially summarizes the problem of malnutrition in SNFs and outlines the CMS quality framework for nursing home care. It then summarizes our review and findings of SNF quality measures and nutrition-focused QIPs. The paper characterizes the gap in skilled nursing care at the intersection of value-based healthcare models and resident malnutrition and makes the case for how quality measures and nutrition-focused QIPs can help address this gap to benefit resident and SNF outcomes. Age-related changes—appetite loss, limited ability to chew/swallow, polypharmacy, and cognitive/functional declines—commonly affect diet and nutrition. The most common form of malnutrition in older adults is protein-calorie malnutrition, which is associated with multiple poor health outcomes including decreased immunity, increased infection rates, delayed wound healing, and decreased respiratory and cardiac function, as well as increased healthcare costs . MalnutrIn addition to poorer health outcomes, malnutrition in nursing homes is associated with declines in functional status and psychosocial well-being and can negatively influence quality of life . It is nNutrition intervention has been associated with improved health status and related cost savings across healthcare systems, including residents in nursing homes ,21,22,23Ideally, malnutrition is identified in other institutional or community settings even before individuals enter skilled nursing care. Yet this often does not occur. An estimated 20–50% of patients admitted to the hospital are malnourished  but lessUnder the PDPM, every resident admitted to the SNF requires an appropriate nutrition assessment within 5 days of the admission to identify all SLP and NTA comorbidities. Data from the Minimum Data Set (MDS) gathered on admission can help inform the nutrition assessment, but when malnutrition is identified, it must be documented as a diagnosis. If a correct malnutrition diagnosis is not recorded within the 5-day timeframe, treatment delays resulting in poorer patient outcomes can occur, and resources to effectively address nutrition needs may be impacted. Thus, more needs to be done to better identify as well as document malnutrition diagnoses in SNFs, including leveraging the SNF quality framework. CMS has a long-established framework for improving the quality of nursing home care, including a national Nursing Home Quality Initiative (NHQI) launched nearly two decades ago. The NHQI was built on a set of validated, publicly reported quality measures aimed at helping consumers and their families make more informed decisions in selecting a nursing home. Another NHQI goal has been making quality information publicly available to proactively motivate providers to address/improve their quality of care .The key features of SNF value-based payments under CMS are (1) evaluation according to performance on a hospital readmission quality measure; (2) scoring on both improvement and achievement; (3) confidential quarterly feedback report on performance quality; and (4) earned incentive payments based on performance . Rates oDesign and Scope;Governance and Leadership;Feedback, Data Systems, and Monitoring;Performance Improvement Projects (PIPs);Systematic Analysis and Systematic Action .A different part of CMS’ quality framework is the Quality Assurance and Performance Improvement (QAPI) management system, which was established in response to provisions under the 2010 Affordable Care Act (ACA). The ACA requires nursing care facilities to adopt systems to continuously identify and correct deficiencies and promote and sustain performance improvement. CMS’ approach for executing this through the nursing home QAPI program is based on five key elements of effective quality management and implementation science:CMS defines a PIP as a “concentrated effort on a particular problem in one area of the facility or facility wide; it involves gathering information systematically to clarify issues or problems and intervening for improvements” . As malnOur review of malnutrition quality improvement in SNFs had two components: SNF quality measurement, and published, nutrition-focused QIPs in SNFs.CMS annually adopts and removes measures from its SNF quality reporting program . In reviWe next reviewed English language literature from the last 10 years (2011–2021) to identify malnutrition and nutrition-focused QIPs and quality effectiveness process initiatives in SNFs/nursing home care. Key search terms included the following: SNFs, long-term care, assisted living, senior care, nursing homes, and nutrition-focused quality improvement/effectiveness  as well We found 6 publications on nutrition-focused QIPs in skilled nursing facilities. The majority was published abstracts and most concentrated on efficacy/effectiveness versus clinical practice changes/process improvements .We identified very few published nutrition-focused QIPs in SNFs, and the majority of the studies did not focus on US SNF populations. Among our findings were that Elia et al.  undertooBecause of the limited literature base specific to nutrition-focused QIPs in SNFs, lessons from nutrition-focused QIPs in other care settings may be relevant, as poor nutrition status predisposes residents to falls and fractures, impaired skin integrity (pressure ulcers/injuries), and limited mobility . One exaNutrition-focused QIP models have been proven effective when they include malnutrition risk screening at admission, prompt initiation of ONS for at-risk patients, and nutrition education and follow-up. Specifically, a nutrition-focused QIP was associated with an overall cost-savings of over $4.8 million in the hospital setting  and a coDrawing on such literature, nutrition-focused QIPs in SNFs could impact multiple components of the nutrition care process to improve timeliness, effectiveness, and outcomes. We suggest several potential QIP areas for process improvement in SNFs .However, adjustment for systematic differences in SNFs that could impact uptake and embedding of new practices may be needed. For example, because many SNF residents have other comorbidities related to malnutrition, such as disability  and obesIn addition, interventions that may be suitable in other care settings, such as patient nutrition education, may have less of a role and other considerations such as addressing medications that can impact appetite, and avoiding needlessly restrictive diets may become primary areas of focus. Indeed, it is the position of the Academy of Nutrition and Dietetics that in post-acute care settings, older adults who prioritize quality of life/personal choice receive the least restrictive diet appropriate to meet their nutrition needs . In addiAlthough CMS has had a nursing home QAPI program for 10 years and has had SNF quality measures for nearly 20 years, our review identified that there are no malnutrition-specific quality measures for SNFs and very few published nutrition-focused QIPs in SNFs. Yet a case can be made that both are needed to better identify and intervene for malnutrition and benefit health outcomes as SNFs more fully move to a value-based care model and policymakers, healthcare organizations, and interdisciplinary teams all have a role.The Improving Medicare Post-Acute Care Transformation Act of 2014 (IMPACT Act) requires development of quality measures . ScreeniThere are several reasons why SNF nutrition-focused QIPs are needed. First, when malnutrition and other nutrition-related conditions are identified early, they can be part of the PDPM case-mix adjustment components, which identify complexity of care and resource needs. In addition, malnutrition impacts multiple, existing quality measures, including readmission, which is a key performance measure for SNF value-based care reimbursement.Second, effective malnutrition care is an interdisciplinary process in SNFs. The medical complexity of residents in post-acute settings such as SNFs is increasing; for example, the percent of residents receiving total parenteral nutrition (TPN) is greater in SNFs today . HoweverNearly a decade ago, Dyck and Schumacher identified nurse leaders as playing pivotal roles in evidence-based clinical interventions for unintentional weight loss among older adults in long-term care . Today, Third, because the PDPM model requires resident conditions be fully documented within 5 days, optimization of the malnutrition care process has become more important than ever, which is where nutrition-focused QIPs play a critical role. QIPs can help identify specific areas where the malnutrition care process or team communications may be breaking down and then test models for improvement. In acute care, improvement models include nursing screening and intervening for malnutrition, under the supervision of an RDN or physician, and using well-established electronic health record-cued algorithms . Such moThe next steps for developing SNF malnutrition quality measures and nutrition-focused QIPs align with the ongoing progress in nursing home care reform. Nutrition status was mentioned in the 2022 SNF QRP final rule as a condition that CMS may consider in future measure development efforts . More reIn addition, nursing homes are routinely surveyed to assure they are in compliance with federal requirements, and CMS’ Requirements of Participation help clarify the intent of federal Medicare regulations . While aAs the US population grows older, more people with age-associated diseases and disabilities will increase the demand for skilled nursing care. Further, older age is associated with greater likelihood of worsening nutrition status, due to the merging influences of advancing age, poor dietary intake, and acute/chronic disease. In acute care, malnutrition quality measures have been both developed/tested, and nutrition-focused QIPs have been shown to reduce costs and effectively improve care processes and patient outcomes. Our review identified that even though there has long been a SNF quality improvement framework, there still are no malnutrition-specific quality measures and very few published nutrition-focused QIPs in SNFs. Reasons for this are unknown and represent an important area for future research. More importantly, the gap in malnutrition quality care continues to impact the health, independence, and quality of life for older people. Thus, policymakers should include malnutrition-focused quality measures as part of SNF quality reporting programs, and care providers should routinely include malnutrition care in facility QAPI programs. Taking these steps can harness the benefits of improved nutrition status as a pathway for reducing readmissions, increasing quality of life, and better supporting SNF value-based care.