Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the primary agents involved in the porcine respiratory disease complex, economically one of the most important diseases in pigs worldwide. The pathogen adheres to the ciliated epithelium of the trachea, bronchi, and bronchioles, causes damage to the mucosal clearance system, modulates the immune system and renders the animal more susceptible to other respiratory infections. The pathogenesis is very complex and not yet fully understood. Cell-mediated and likely also mucosal humoral responses are considered important for protection, although infected animals are not able to rapidly clear the pathogen from the respiratory tract. Vaccination is frequently practiced worldwide to control M. hyopneumoniae infections and the associated performance losses, animal welfare issues, and treatment costs. Commercial vaccines are mostly bacterins that are administered intramuscularly. However, the commercial vaccines provide only partial protection, they do not prevent infection and have a limited effect on transmission. Therefore, there is a need for novel vaccines that confer a better protection. The present paper gives a short overview of the pathogenesis and immune responses following M. hyopneumoniae infection, outlines the major limitations of the commercial vaccines and reviews the different experimental M. hyopneumoniae vaccines that have been developed and tested in mice and pigs. Most experimental subunit, DNA and vector vaccines are based on the P97 adhesin or other factors that are important for pathogen survival and pathogenesis. Other studies focused on bacterins combined with novel adjuvants. Very few efforts have been directed towards the development of attenuated vaccines, although such vaccines may have great potential. As cell-mediated and likely also humoral mucosal responses are important for protection, new vaccines should aim to target these arms of the immune response. The selection of proper antigens, administration route and type of adjuvant and carrier molecule is essential for success. Also practical aspects, such as cost of the vaccine, ease of production, transport and administration, and possible combination with vaccines against other porcine pathogens, are important. Possible avenues for further research to develop better vaccines and to achieve a more sustainable control of M. hyopneumoniae infections are discussed. Mycoplasma hyopneumoniae (M. hyopneumoniae) is the most important Mycoplasma sp. in swine health management. It is the primary pathogen of enzootic pneumonia, a chronic respiratory disease in pigs, and one of the primary agents involved in the porcine respiratory disease complex [M. hyopneumoniae are highly prevalent in almost all swine producing areas, and they cause significant economic losses due to increased medication use and decreased performance of the pigs [ complex . Infectithe pigs .M. hyopneumoniae lacks a cell wall. The organism is very difficult to isolate because of its slow growth and potential overgrowth with other swine mycoplasmas. The pathogen-host interactions are very complex and not fully characterized. The organism is primarily identified on the mucosal surface of the trachea, bronchi, and bronchioles [M. hyopneumoniae predisposes animals to concurrent infections with other respiratory pathogens.Similar to other mycoplasmas, nchioles . It affenchioles . TherefoM. hyopneumoniae infections in pig herds can be accomplished by optimizing management, housing and biosecurity practices [M. hyopneumoniae. Antimicrobial medication can limit the consequences of the disease and decrease the infection load [M. hyopneumoniae. Medication with antimicrobials is also discouraged because of the risk of antimicrobial resistance development [M. hyopneumoniae has been shown to be a useful tool to control M. hyopneumoniae infections. Different inactivated, whole-cell vaccines are commercially available and vaccination is frequently practiced worldwide. In infected herds, vaccination decreases clinical signs and lung lesions due to M. hyopneumoniae infections, performance losses of the animals and antimicrobial use. Although the commercial vaccines are widely used and cost-efficient in many farms, they induce only a partial protection and do not prevent infection.Control of ractices . Treatmeion load , but it elopment . VaccinaM. hyopneumoniae with the host and the limitations of the currently available commercial vaccines. Next, the different experimental vaccines that have been developed and tested in mice and pigs are reviewed. Finally, avenues for further research are provided in order to improve M. hyopneumoniae vaccination and achieve a more sustainable control of M. hyopneumoniae infections.The present paper discusses the complex interaction of M. hyopneumoniae is a small (0.2–0.4 µm) and pleomorphic organism as it lacks a shape-defining cell-wall. Studies have shown a high diversity at genomic, antigenic, and proteomic level between different strains [M. hyopneumoniae strains  were sequenced in 2004 [M. hyopneumoniae genomes are available, 11 already assembled and annotated, and 12 not fully assembled. In general, the genomes are small in size, namely 0.86–0.96 Mb, and in each of them, there are 528 to 691 protein-encoding genes [M. hyopneumoniae genes encode surface proteins, the function of many of them is not yet known [M. hyopneumoniae a complex transcriptional organization, unique intrinsic terminator stem-loop formation and individual ribonuclease P (RNase P) structure [ strains . The gen in 2004 , 10. Sintructure .M. hyopneumoniae and the limited number of secreted or surface proteins is particularly interesting for use of recombinant DNA technology. However, M. hyopneumoniae uses an unusual genetic code. The amino acid tryptophan is not encoded by TGG, but by TGA, which is a stop codon in most organisms [M. hyopneumoniae containing TGA codons in E. coli, the most attractive system used for production of recombinant proteins. However, mutations replacing TGA codons with TGG have been used successfully to solve this problem [The small genome of rganisms . This di problem .M. hyopneumoniae adheres to the ciliated epithelial cells of the trachea, bronchi and bronchioles underneath the mucous layer. Adhesion is followed by the induction of ciliostasis, loss of cilia, and eventually epithelial cell death [Upon inhalation, ll death .. hyopneumoniae proteins have been associated with cell adhesion, including several related to the P97/P102 paralog families and other surface proteins that moonlight as adhesins [M. hyopneumoniae adhesins can even be higher, considering that its surfaceome includes more than 290 proteins and that many uncharacterized surface displayed proteins may bear adhesion properties. Different adhesins may vary in abundance at the cell surface between strains. This may be due to differential transcriptional rates of the respective genes, differential translational rates of the corresponding mRNAs and/or post-translational events, including their export to the cell membrane and proteolytic processing. Proteolytic processing of adhesins can shape the bacterial surface architecture [A repertoire of at least 35 Madhesins , 18. Theitecture , generatitecture .M. hyopneumoniae and also the most intensively studied one, and therefore, it has been tested in many experimental vaccines. P97 contains two repeat regions (R1 and R2), located in the C-terminal portion. The sites that are involved in cilium binding are located in the R1 region and at least seven AAKPV/E repeats are required for functional binding. Both R1 and R2 are involved in the attachment of M. hyopneumoniae to the extracellular matrix of the respiratory tract [P97 is one of the most important adhesins of ry tract .M. hyopneumoniae [M. hyopneumoniae may mediate subsequent adherence to swine respiratory cilia. Further research is needed to investigate whether adherence to fibronectin and plasminogen may facilitate internalization of M. hyopneumoniae and facilitate its traffic via the circulatory system and penetration into host organs, such as liver, kidneys and spleen [M. hyopneumoniae infection is unclear, but it may influence the migration of inflammatory cells and stimulate the release of pro-inflammatory cytokines [Apart from the cilia-exposed glycans, some swine extracellular matrix molecules, such as fibronectin and plasminogen, also provide binding sites for surface adhesins of eumoniae . The fibd spleen . The exaytokines .M. hyopneumoniae P97 adhesin and other proteins, including lipoproteins, glycolytic enzymes, chaperones and translation factors [M. hyopneumoniae also interact with other cytoskeletal proteins, such as vimentin, keratin, tubulin, myosin, and tropomyosin [Extracellular actin is also used as a surface receptor by different proteoforms of  factors . Apart fpomyosin .Mycoplasma species. Mycoplasma hyopneumoniae can produce H2O2 in the presence of glycerol in vitro. However, this is strain dependent and the attenuated type strain J does not produce detectable amounts of H2O2 [M. hyopneumoniae remains to be investigated. Ferrarini et al. [M. hyopneumoniae is able to take up myo-inositol and use it as an alternative energy source in the absence of glucose. Since myo-inositol is freely available in the serum of pigs, it might be a suitable alternative energy source for M. hyopneumoniae residing in the highly vascularized lungs.Adhesion serves as the starting point of infection which is then assisted by other virulence factors. Classical virulence factors like toxins are generally lacking in  of H2O2 . Whetheri et al.  showed tM. hyopneumoniae, whole membrane lipoprotein fractions induced apoptosis in various cell types, including porcine peripheral blood mononuclear cells (PBMCs) [Lipid associated membrane proteins (LAMP) have also been implicated in the pathogenicity of mycoplasmas. They interact with the host immune system mainly through Toll-like receptors (TLRs), such as TLR2 . In M. h (PBMCs) . Further (PBMCs) .Mycoplasma bovis (M. bovis), MnuA was shown to degrade DNA-based neutrophil and macrophage extracellular traps , thereby enabling M. bovis to escape these traps [mnuA gene is also present in M. hyopneumoniae [M. hyopneumoniae to escape the host immune defense and using at the same time the nucleotides and protein synthesis materials as nutrients for proliferation [Mycoplasmas need to scavenge nutrients including nucleotides from their environment and therefore, they are well known for their potent membrane nucleases . A well-se traps  and not eumoniae . Therefoferation .Mycoplasma hyopneumoniae may release extracellular DNA that allows the organism to form biofilms on abiotic and host surfaces [M. hyopneumoniae biofilm formation are thus far mostly unknown.surfaces . BiofilmMycoplasma genitalium, an immunoglobulin (Ig) G binding protein, called protein M, has been identified. This protein not only fixes IgG very efficiently but also prevents subsequent antigen–antibody binding and subsequent signaling pathways of the bound antibodies [Mycoplasma mycoides subspecies capri encode a Mycoplasma Ig binding protein (MIB) and a Mycoplasma Ig protease (MIP). The complex of MIB and Ig is necessary for the proteolytic activity of MIP. The two proteins are encoded by two genes and are often detected in multiple copies in various Mycoplasma sp., including M. hyopneumoniae [M. hyopneumoniae.In tibodies . In vitreumoniae . FurtherM. hyopneumoniae [M. hyopneumoniae remains unclear.Integrative and conjugative elements (ICE) are self-transmissible mobile genetic elements involved in horizontal gene transfer, thereby providing new virulence and/or antibiotic resistance traits. Such ICE have been identified in eumoniae , but theM. hyopneumoniae [M. hyopneumoniae is a genome-reduced organism that is characterized by a limited set of biosynthetic pathways, as such it is not surprising that further loss of enzymatic functions might have a large influence on survival and growth of the microorganism. This also holds true for lipoproteins involved in nutrient acquisition [M. hyopneumoniae strains and M. flocculare provided tens of novel candidates to enzootic pneumonia determinants or virulence factors [Several large-scale comparisons on the genome, transcriptome, proteome, metabolome and secretome level have been performed in order to investigate virulence and pathogenesis of eumoniae , 36. Liuuisition . A compr factors .Mycoplasma-induced pneumonia [The interaction of the pathogen with the immune system of the host is not yet fully elucidated, and it is clear that some components of the immune system may both help and hinder the development of neumonia , 38. Infneumonia .M. hyopneumoniae by porcine alveolar macrophages [M. hyopneumoniae infection. M. hyopneumoniae was also demonstrated to strongly activate monocytes and B cells in vitro, with the B cell-activation resulting in a potent polyreactive antibody response [M. hyopneumoniae infection is still unknown. Also, it is not clear why there is a low neutrophil infiltration upon M. hyopneumoniae infection.It has been shown that Toll-like receptor 2 (TLR2) and TLR6 are important in the recognition of rophages . The actrophages , indicatresponse . NeverthM. hyopneumoniae. Among the up-regulated genes, they found several genes related to immune response and inflammation, such as C3 complement, SAA3, chemokines (CXCL2 and CCL20) and galectins. These chemokines may attract myeloid cells. The study also suggested that ciliostasis caused by this pathogen might partially be explained by the down-regulation of ciliary genes. The innate immune responses against M. hyopneumoniae and mycoplasma in general have been reviewed in more detail elsewhere [Recently, Mucha et al.  investiglsewhere .M. hyopneumoniae-specific serum IgG antibodies are detected 3–4 weeks post-infection (pi), peak after 11–12 weeks and then decrease very gradually [. Mycoplasma hyopneumoniae-specific IgM in serum can be detected as early as 9 days pi under experimental conditions. The percentage of IgM positive pigs peaks at 14 days pi and rapidly decreases afterwards [M. hyopneumoniae-specific antibodies precede specific serum antibodies following infection but decline faster [Mycoplasma hyopneumoniae-specific IgA can be detected in nasal swabs as early as 6 days pi, peak 12–16 days pi and decline steadily afterwards to reach pre-immune levels by day 84 pi.After experimental infection, radually . After bradually . Interesradually . Mycoplaterwards . When ine faster . MycoplaMycoplasma hyopneumoniae-specific IgG levels in serum induced by vaccination are not correlated with the severity of lung lesions in M. hyopneumoniae-infected pigs, suggesting that systemic antibodies play a minor role in protective immunity [M. hyopneumoniae is still unclear. Some studies demonstrated that specific antibody levels in the respiratory tract did not correlate with protection [M. hyopneumoniae-specific secretory IgA in preventing adhesion of the microorganism to the ciliated cells of the respiratory tract [M. hyopneumoniae, resulting in phagocytosis by macrophages and neutrophils [M. hyopneumoniae by alveolar macrophages in vitro.immunity . The rolotection , 49, whery tract –52. Furttrophils . Howevertrophils  recentlyM. hyopneumoniae [Mycoplasma-induced pneumonia [Mycoplasma pulmonis challenge studies using various T cell subset-depleted mice indicate that T helper 1 (Th1), Th17 and CD8+ T cell responses are responsible for protection against Mycoplasma disease. In contrast, Th2 responses are less efficient in controlling the infection and thus contribute to immunopathology [M. hyopneumoniae vaccination-challenge study resulting in a significant reduction of lung lesions in the vaccinated group, Thacker et al. [M. hyopneumoniae-vaccination studies using a M. hyopneumoniae-resistant and a non-resistant pig line, higher serum levels of IFN-γ and IL-17A, but lower levels of IL-4 and CD4+ T cells were detected in the resistant line compared to the non-resistant line after vaccination [Mycoplasma disease. Next to that, Marchioro et al. [+/CD8+ ratio, and thus a higher relative number of CD8+ cells in pigs vaccinated with a commercial M. hyopneumoniae bacterin compared to control pigs receiving a physiological saline solution. This supports the hypothesis that CD8+ T cells have a protective role in Mycoplasma infections and could partially explain the beneficial effects observed after vaccination against M. hyopneumoniae.T cell-mediated immune responses are generally considered important for protection against Mycoplasmas causing local respiratory infections such as eumoniae . T cellsneumonia . Mycoplaathology . In a M.r et al.  observedcination . As IFN-o et al.  found a Mycoplasma infections by IFN-γ-mediated activation of macrophage killing. It is now well-established that Th17 immune responses are important to protect mucosal surfaces, to promote epithelial cell regeneration, mucous and antimicrobial protein production, and the release of neutrophil recruitment [+ cells is killing infected cells [M. hyopneumoniae is able to invade porcine epithelial cells [+ cells might be relevant in the immune response against M. hyopneumoniae. Interestingly, studies performed in the M. pulmonis mouse model suggest that CD8+ T cells might dampen the pro-inflammatory Th cell responses responsible for lung damage and clinical disease [+ T cells may skew the responses towards a protective Th1 response. Another possibility could be that CD8+ T cells kill antigen presenting cells (APCs), thus reducing the possibility of Th cell activation [T helper 1 responses may contribute to protection against ruitment . Followiruitment  and by eruitment . The majed cells . Since tal cells , this ch disease . It is stivation .M. hyopneumoniae and grows easier in culture medium than recent field isolates. This strain was isolated in 1963 from a field outbreak of enzootic pneumonia in the UK. Commercial bacterin vaccines are licensed either for single or double vaccination, and combinations with porcine circovirus type 2 (PCV-2) or Glaesserella parasuis (formerly Haemophilus parasuis) are available. Most bacterin vaccines should be administered intramuscularly, but some bacterins are also licensed for intradermal administration. An inactivated vaccine based on soluble antigens of M. hyopneumoniae is also commercially available [Commercial vaccines mostly consist of adjuvanted inactivated, whole-cell preparations . Most vvailable .M. hyopneumoniae have been licensed in Mexico and in China [M. hyopneumoniae (strain LKR) that should be applied intranasally [M. hyopneumoniae adhesins , cell envelope proteins (P95), cell surface antigens (P36), and secreted proteins and chaperone protein (DnaK) [Attenuated vaccines against in China . The vacanasally . The attanasally . This fintigens P, and secn (DnaK) . Residuan (DnaK) .M. hyopneumoniae infections. However, the immune mechanisms resulting in partial protection are not fully elucidated. Several studies observed lower levels of the pro-inflammatory cytokines associated with lymphoid hyperplasia and pneumonia lesions, such as TNF-α, Il-6 and IL-1β in M. hyopneumoniae-vaccinated pigs compared to non-vaccinated ones [Commercial vaccines induce partial protection against ted ones , 64. Morted ones . Indeed,ted ones  demonstrted ones .The number of animals seroconverting after vaccination, as well as the antibody levels induced in serum and respiratory tract washings may vary depending on the vaccine composition, administration route, vaccination strategy (single or double vaccination) and the infection status of the animal . The serM. hyopneumoniae-specific IFN-γ-secreting cells in the blood and lung tissue of vaccinated animals [Several studies found an increase in  animals , 64, 68.M. hyopneumoniae infections: improvement of daily weight gain (2–8%), feed conversion ratio (2–5%) and sometimes mortality rate. Additionally, shorter time to reach slaughter weight, less variation in slaughter weight (more homogeneous carcasses), reduced clinical signs (coughing), lower prevalence and severity of lesions typically caused by M. hyopneumoniae and lower treatment costs, are observed [M. hyopneumoniae organisms in the respiratory tract [The major advantages of piglet vaccination relate to increased animal welfare and a decrease of the performance losses due to observed . The curry tract , 65, 70 ry tract .M. hyopneumoniae strains, and maternal immunity. A recent study [M. hyopneumoniae vaccine responses, although bacterial diversity indexes alone were not predictive for antibody responses among individual pigs.Different factors that may influence vaccine efficacy have been described by Maes et al. . The mosnt study  also hinM. hyopneumoniae is often incomplete and vaccination does not prevent colonization. Transmission models under experimental [M. hyopneumoniae. New vaccines and/or administration routes are therefore needed. A recent pilot study could not demonstrate transmission of M. hyopneumoniae between seeder and contact animals in case both had been vaccinated multiple times against M. hyopneumoniae [Drawbacks of the current vaccines are that the protection against clinical signs and lesions typically caused by rimental  and fielrimental , 73 alsoeumoniae , suggestM. hyopneumoniae infections is ongoing. An overview of the peer-reviewed studies (since 1995) that have investigated different experimental vaccines in mice is shown in Table M. hyopneumoniae in pigs were not found. No publications in peer-reviewed scientific journals have been found about experimental vaccines based on attenuated strains.Research on the development of novel vaccines that may offer better protection against es since 995 that M. hyopneumoniae is only causing disease in pigs, the efficacy of the vaccines was not tested in mice. All studies assessed the humoral responses in serum, many also included cell-mediated immunity (CMI), whereas the humoral response in the BAL fluid was less frequently examined.The studies in mice focused on the construction and development of the vaccines and the evaluation of the immune responses. As Salmonella Typhi in mice and Mycobacterium avium subsp. paratuberculosis in cattle [M. hyopneumoniae. P37 is a lipoprotein that belongs to the ATP-binding cassette (ABC) transporters [E. coli as expression system.In terms of antigen selection, most studies included outer membrane proteins that are immunogenic and/or considered important for adhesion such as P97, P46, P71 and P95. However, also other antigens were tested such as NrdF, P36, HSP70, P42, P37 and MnuA. NrdF is an essential enzyme for metabolic processes. It catalyzes the conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates, an essential step in DNA replication . P36 or n cattle . P42 is n cattle . It is hsporters , whereassporters . The antSalmonella, Actinobacillus and Bacillus, while two studies used adenovirus vectors. The subunit vaccines were based on single antigens or different antigens that were mixed with an adjuvant. In three of the five studies, the antigens were constructed as a chimeric protein. Subunit vaccines were used in combination with adjuvants based on oil, aluminium hydroxide, the B subunit of heat-labile enterotoxin of E. coli (LTB) or Mycobacterium tuberculosis ESAT-6 protein. Vaccination with DNA constructs encoding potential antigens might be promising, as in general, DNA constructs are stable, easy to handle and can be administered via various routes.Most vector vaccines were based on bacterial vectors namely Most vaccines in mice were applied two or three times, and were administered via the parenteral route, mainly intramuscularly, although oral (vector vaccines), intranasal and intraperitoneal administration routes were evaluated as well.The major outcome parameters of the studies in mice were humoral responses in serum and in some studies also in BAL fluid, and CMI responses. Humoral responses in serum were assessed by measuring IgG against the antigens included in the vaccine, some studies also measured isotypes of IgG. In general, IgG1 in mice is indicative of a Th2 response, whereas IgG2a is predominantly produced during a Th1-type response . CMI resSalmonella vector vaccine were poor, IgA was only found in BAL fluid in one study and IFN-γ production in another study , and five out of eight a reduction of the M. hyopneumoniae load in the BAL fluid. Three studies [M. hyopneumoniae 232 strain were expressed under in vitro culture conditions. This might be one of the reasons of the incomplete protection induced by commercial vaccines. Nonetheless, the efficacy of the experimental vaccines was even lower, suggesting that immunity induced by the antigens selected for preparing these vaccines is not sufficient for improved protection, although it is well known that these antigens like P97 have important roles in the pathogenesis of M. hyopneumoniae infections.In terms of efficacy, 11 out of 14 studies showed a significant reduction of the lung lesions caused by  studies , 52, 97  studies , or to t studies  showed tAll studies could demonstrate specific IgG responses in the serum, except for two vector vaccines , 95. In M. hyopneumoniae compared to the type strain J, in combination with different novel adjuvants. These included different Toll-like receptor ligands , the MINCLE agonist trehalose 6,6-dibehenate (TDB), and STING ligand cyclic diadenylate monophosphate (c-di-AMP). They were used in combination with different carriers namely liposomes, polylactic-co-glycolic acid (PLGA) microparticles or squalene-in-water emulsion (SWE) , and possible combination with other vaccines are important.From an immunological point of view, a major challenge is to induce a protective immunity at the mucosal surface of primary adherence and multiplication of M. hyopneumoniae to gilts under field conditions, resulted in infection of the animals with this pathogen [Administration of the antigens intranasally or via aerosols might be the most appropriate routes to accomplish mucosal protection. The number of experimental vaccines that have evaluated this route of immunization is still very limited. This is mainly due to important difficulties that are encountered by using the mucosal route such as: (1) the antigen should be present at and below the mucosal barrier in sufficient amounts allowing sufficient capturing by antigen presenting cells, (2) mucosal tolerance mechanisms should be overcome, (3) protective immune mechanisms should be activated and, (4) minimal/or no influence on mucosal functionality should occur. Mucosal adjuvants e.g. microbe-derived substances, including TLR ligands, could be crucial in reaching these goals , 110. Hepathogen . Also, Fpathogen  showed tM. hyopneumoniae vaccine was administered orally to pigs via a tube into the esophagus on three occasions. IgA was detected in nasal secretions and saliva, but IgA was not measured in BAL fluid. However, lung lesions following challenge infection were significantly reduced in vaccinated pigs, pointing to protective immunity in these animals. Placing a tube in the esophagus is labor intensive, time consuming and not animal welfare friendly. The stability of the antigen during the mouth-stomach-intestine passage might also be an issue. Therefore, further research should focus on whether M. hyopneumoniae vaccines mixed in the diet and consumed by the pigs can also provide protection. Such a method might require a higher antigen dose and also the stability of the antigen in the feed might be an issue. However, it would be animal welfare friendly and require much less time and labor than administration of the vaccine via a tube to each individual animal.Providing the antigen orally might also induce mucosal immunity in the respiratory tract via the common mucosal immune system . In the M. hyopneumoniae-specific IgA levels were observed in respiratory tract washings of parenterally vaccinated pigs compared to non-vaccinated pigs after challenge infection, indicating an anamnestic immune response. This suggests that priming of the mucosal immune system is possible after parenteral vaccine administration. Intradermal vaccination directly targets epidermal Langerhans cells and dermal dendritic cells, which are essential for efficient T and B cell priming. In this sense, intradermal vaccination against M. hyopneumoniae can be an asset, as more of these specialized APCs are present in the skin compared to muscle tissue [Most experimental vaccines were applied via the parenteral route, mostly intramuscularly. The studies using parenteral administration however could not demonstrate measurable IgA responses in the BAL fluid prior to challenge, and therefore, this route is less suitable for inducing mucosal humoral responses. However, higher e tissue . In addiBacterial genes and antigens involved in survival of the bacterium in the host and/or that render the bacterium harmful to the host could be included in vaccines or could be targets for attenuation to develop attenuated vaccines. Many experimental vaccine studies have included P97 and other proteins that are important for adherence or metabolism. However, subunit vaccines based on the most important adhesion namely P97, did not provide sufficient protection. The adhesion process is complex and involves many different adhesins, therefore, including one or only a few antigens might be insufficient. This might also explain why bacterins, which include a broad array of antigens, perform better in terms of efficacy than subunit vaccines. Further research on subunit vaccines should therefore not focus on one or a few antigens, but include different and carefully selected antigens.M. hyopneumoniae infection, adjuvants that stimulate this arm of the immune system should preferably be selected [Adjuvants are incorporated into vaccines to increase and direct the immunogenic responses to antigens. Many adjuvants activate the innate immune system through pattern-recognition receptors (PRRs) present in immune cells. Receptor-ligand interactions lead to expression of genes that encode cytokines, chemokines, and costimulatory molecules responsible for priming, expansion, and polarization of immune responses . Adjuvanselected . Commonlselected .M. hyopneumoniae vaccines is very scarce. Research on adjuvant development should therefore not only identify more inflammatory adjuvants, but also try to separate the potency of adjuvants from the adverse effects they can induce.Adjuvants not only increase the immunogenic responses, but can also lead to adverse reactions such as inflammation at the injection site, granuloma and sterile abscess formation, malaise, fever and other systemic reactions. Apart from the study of Matthijs et al. , informaM. hyopneumoniae vaccines, very few studies have focused on developing experimental attenuated M. hyopneumoniae vaccines. This might be due to the difficult isolation and cultivation characteristics of M. hyopneumoniae. In addition, reversion of virulence is always a concern in case of attenuated vaccines. One study [M. hyopneumoniae strain could be used as a potential vaccine. However, infection with this strain did not protect piglets against infection with a highly virulent M. hyopneumoniae isolate 1 month later, suggesting that low virulent strains might not be suitable as such to be used as vaccines. Current technology allows however to selectively delete specific genes important for replication or virulence in pathogenic organisms, which might be useful for the development of attenuated vaccines against M. hyopneumoniae. At this stage however, these genes are not yet fully elucidated, and therefore, further research is needed to investigate genes of M. hyopneumoniae involved in pathogenesis and virulence.Attenuated vaccines have great potential to better stimulate a mucosal immune response at the respiratory tract compared to parenterally administered vaccines. However, apart from the two current commercially available attenuated ne study  tested wM. hyopneumoniae with the respiratory tract and the immune system and the fact that naturally elicited immune responses upon infection are not able to rapidly clear the pathogen from the animal, make the search for protective immune responses and efficacious novel vaccines challenging. Different experimental vaccines against M. hyopneumoniae have been developed and tested in mice and pigs. Most of them were based on the P97 adhesin or other factors considered important in the pathogenesis, or were bacterins combined with novel adjuvants. As cell-mediated and likely also mucosal humoral responses are important for protection, new vaccines should aim to target these arms of the immune response. Very few research has been directed towards the development of attenuated vaccines, although such vaccines may have great potential.The very complex interaction of


Mortierella alpina is a safe source of polyunsaturated fatty acids (PUFA) in industrial food and feed production. Besides PUFA production, pharmaceutically relevant surface-active and antimicrobial oligopeptides were isolated from this basal fungus. Both production of fatty acids and oligopeptides rely on the biosynthesis and high turnover of branched-chain-amino acids (BCAA), especially l-leucine. However, the regulation of BCAA biosynthesis in basal fungi is largely unknown. Here, we report on the regulation of the leucine, isoleucine, and valine metabolism in M. alpina. In contrast to higher fungi, the biosynthetic genes for BCAA are hardly transcriptionally regulated, as shown by qRT-PCR analysis, which suggests a constant production of BCAAs. However, the enzymes of the leucine metabolism are tightly metabolically regulated. Three enzymes of the leucine metabolism were heterologously produced in Escherichia coli, one of which is inhibited by allosteric feedback loops: The key regulator is the α-isopropylmalate synthase LeuA1, which is strongly disabled by l-leucine, α-ketoisocaproate, and propionyl-CoA, the precursor of the odd-chain fatty acid catabolism. Its gene is not related to homologs from higher fungi, but it has been inherited from a phototrophic ancestor by horizontal gene transfer.The oleaginous fungus  It has been well-studied in the ascomycete Saccharomyces cerevisiae +), ranging from 0 to 500 µg/mL, served as reference standard.Details on the extraction procedure and chromatographical analyses are described in the method C . A calibl-glutamate (Glu) and the reaction was started with 10 µL of α-keto acids KIC, KIV, and KMV (1 mM each). After 2, 5, 10, 15, 20, and 40 min at 25 °C, the reaction was stopped by shock-freezing in liquid nitrogen. The reaction mixture was lyophilized, dissolved in 100 µL methanol, centrifuged , and the supernatant was subjected for quantification on a HILIC-UHPLC system, using a calibration curve with Glu as reference (BatA and BatC activities were determined in BCAA aminotransferase (BAT) buffer , 50 mM Tris-HCl, pH 8.0). To determine kinetic parameters and alternative substrates, a discontinuous assay was performed: 190 µL master mix (5 mM α-ketoglutarate (KG), 130 nM BatA or BatC in BAT buffer) was mixed. In a two-step approach, the reaction was initially started by adding 10 µL of pools of α-amino acids (1 mM each). Subsequently, individual α-amino acids were tested . For the BCAA synthetic direction, KG was replaced by eference . For theabs = 412 nm and λabs = 800 nm as reference wavelength at 35 °C for 8 min in a Clariostar plate reader (BMG Labtech). The continuous assay was used to determine activity, kinetic parameters of substrates and inhibitors , pH optimum, and metal ion dependencies towards divalent cations  or monovalent cations . To study the feedback inhibition of the enzyme, 0–5 mM BCAAs  or KIC were added. The inhibition by CoA esters was tested in the presence of 0–200 µM Prop-CoA or Mal-CoA. To determine the temperature optimum, a discontinuous assay was performed: the reaction mixture (100 µL) was incubated at 4–60 °C, and reaction was stopped by the addition of 50 µL ethanol after 8 min incubation. After the addition of 50 µL DTNB (200 µM), the mixture was assayed colorimetrically, as described above.LeuA1 activity was determined in α-isopropylmalate synthase (IPMS) buffer . To determine kinetic parameters and alternative substrates, a continuous assay in a 100 µL-scale was performed by detecting free thiol groups of coenzyme A (CoA) using Ellman’s reagent , DTNB): 25 µL master mix (80 nM LeuA1 in IPMS buffer) were mixed. Next, 20 µL of alternative acyl CoA substrates  and 5 µL α-keto acids , α-ketobutyrate (KB), α-ketoglutarate (KG), or pyruvate at a final concentration of 0–200 µM) were added, and the reaction was started by the addition of 50 µL DTNB . Colorimetric detection of the released yellow product (2-nitro-5-thio-benzoic acid) was carried out in a 96-well plate at λSaccharomyces cerevisiae and Pyricularia (syn. Magnaporthe) oryzae as queries (ilvB-ilvF1-ilvE1 and ilvF2-ilvE2-leuB1). Similarly, the genes for the cytosolic late steps in Leu biosynthesis are partially duplicated  but are not part of a gene cluster. In S. cerevisiae and P. oryzae, two paralogous genes encoding for a mitochondrial and cytosolic version of the α-isopropylmalate synthase (IPMS) can be found, denoted as leu4 and leu9. However, only one leu4 copy was identified in M. alpina (leuA1), but the overall similarity is comparatively low . This suggests that leuA1 is not related to genes of higher fungi and might have been introduced by HGT from another genetic source. This has been suggested for the leuA gene from the basal fungus Phycomyces blakesleeanus  and Dikarya ,28, a hoM. alpina, the fungus was cultured in minimal medium in absence or presence of 10 mM Ile, Leu, Val, or all BCAAs. Uptake of the three BCAAs was monitored by UHPLC-MS . This suggested, that malpinins are preferably produced under BCAA-limited conditions. Interestingly, according to qRT-PCR analysis, the expression of the corresponding malpinin synthase gene  is strongly regulated by the presence of BCAAs ,  and malpinin content dropped , . Since L dropped B. In sump < 0.001) all genes required for Leu biosynthesis, indicating that Ile excess is equivalent to Leu limitation in the cell. Similarly, feeding of Val significantly (p < 0.01) induced expression of genes responsible for the early Leu biosynthetic steps . In contrast, presence of Leu did not significantly alter the expression levels of any of the biosynthetic genes, suggesting that breakdown of Leu requires at least in part the same set of enzymes as for the anabolic pathway. Moreover, repression by Leu is still evident and intensified, when all three BCAA were added. The genes for the early steps in Leu biosynthesis were unaffected but two genes for the late steps were downregulated, especially leuA1  and leuA2 , suggesting that the encoded enzymes are the gatekeepers for the Leu biosynthesis. Therefore, Leu seems to be the major repressor for BCAA biosynthesis genes and overcomes the inducing effects by Ile or Val. Interestingly, the two potential BAT genes (batA and batC) are not significantly regulated by any of the supplementations, suggesting a requirement for both BCAA biosynthesis and degradation. This sharply contrasts the BCAA-dependent transcriptional regulation of BAT genes in other fungi [To monitor the expression of the Leu biosynthesis genes, fungal cultures were supplemented with individual or all BCAAs and qRT-PCR expression analyses were performed . In geneer fungi ,26.M. alpina genome encodes the complete subset of mitochondrial or cytosolic enzymes to produce BCAAs, but the genes are hardly transcriptionally regulated by Leu, contrasting observations in Dikarya. Hence, a more pronounced metabolic regulation was examined. Since basal fungi are hardly accessible for gene knock-out strategies, we aimed at the biochemical characterization of selected heterologously produced enzymes of the BCAA metabolism.In summary, the M. alpina encodes only one enzyme . In P. oryzae, a third distantly related BAT is postulated (Bat3), and a similar enzyme  is encoded in the M. alpina genome. According to Target-P [The BCAA aminotransferases (BAT) catalyse the final step in BCAA biosynthesis and the first step in BCAA degradation, i.e., the transamination of an α-keto acid to its respective BCAA and vice versa. Interestingly, genomes of ascomycetes usually encode at least two—usually inversely regulated—genes for BATs for anabolic BCAA biosynthesis and BCAA catabolism, respectively . HoweverTarget-P  and MitoTarget-P , BatA isme BatA, 5.2 kDa. Target-P .E. coli , and BatC were heterologously produced and purified from E. coli . During E. coli , which iMortierellaceae [l-Ile, l-Leu, l-Val) and their respective α-keto acids in a PLP-dependent manner (345). Some side activity could be detected with structurally similar amino acids such as l-Met and l-Phe (data not shown). In contrast, BatC was inactive, and neither converted any BCAA (l-amino acid (data not shown). These data, in combination with its low expression  LeuA1 is a cytosolic acetyl-CoA dependent Cor α-IPM . IPMS acor α-IPM . Hence, E. coli and purfied as N-terminally His6-tagged protein  known to coordinate divalent cations [ium ions . However as Ca2+ , indicatand Mn2+ C, whilstar range . LeuA1 s cations . Hence, Feedback inhibition by Leu and KIC. LeuA1 converts KIV and KB in an acetyl-CoA depended manner  are accepted as cosubstrates (Cupriavidus necator (−1 [M. alpina, Prop-CoA, but not Mal-CoA, inhibits the α-IPM synthesis by 43% at 200 µM (KΜ and decreases kcat values (M. alpina. A lack of Val in the cell is probably sensed by its accumulating catabolic product Prop-CoA, which inhibits the KIV-consuming LeuA1, resulting in sufficient intracellular KIV to regenerate Val via BatA (bstrates . This co necator  binds anator (−1 . In M. at 200 µM J. Prop-Ct values , as suggvia BatA .M. alpina, when compared to Dikarya. The absence of a common transcriptional regulator similar to Leu3 in S. cerevisiae [A. nidulans [M. alpina, a constant Leu turnover is required.Our data indicated a different mode of BCAA regulation in basal fungi dissimilar to other fungal divisions. The transcriptional regulation of BCAA biosynthesis is much less abundant in revisiae  or LeuB nidulans , suggestAspergillus terreus, or the aflatoxin biosynthesis in Aspergillus flavus, which are regulated by asparagine, methionine, or glutamine, respectively [Fusarium graminearum, the trichothecene transcriptional activator Tri6 is both a regulator for mycotoxin production and BCAA degradation, showing that primary and secondary metabolism are interwoven [Leu represses the expression of the malpinin synthetase gene and access of BCAAs impairs malpinin production. On first sight, this appears as an inverse regulation, since BCAAs are required of malpinin biosynthesis as well. However, an access of secondary metabolites may lead to a self-intoxication and is usually circumvented by a down-regulated expression, as shown for the gliotoxin gene cluster . Many asectively ,47,48. Iterwoven . Fungal terwoven . Howeverterwoven .leuA1 and leuA2, i.e., the first two genes in the late-stage phase of Leu biosynthesis. Transcriptional regulation of its homologs in ascomycetes rely on the zinc knuckle transcription factor Leu3/LeuB [leu3 homolog is missing in M. alpina, and a merely constitutive expression of Leu biosynthesis is observed.In contrast to the malpinin biosynthesis, the genes of BCAA biosynthesis are marginally regulated by altered expression. Dikarya usually rely on both a transcriptional and metabolic BCAA regulation . The BCAeu3/LeuB , which beu3/LeuB . HoweverbatA gene, encoding the sole BCAA aminotransferase, is constitutively expressed like a house-keeping gene and is required for both BCAA anabolism and catabolism. This contrasts the regulation in ascomycetes or plants, in which at least two, and up to six, individual BATs are partially inversely transcriptionally regulated and, hence, are responsible for either BCAA biosynthesis or degradation. Human and yeast BATs form homodimers [M. alpina to regulate the metabolic flow of BCAAs. Indeed, mitochondrial compartmentation of BatA circumvents a competition with the cytosolic IPMS, LeuA1, for its common substrate KIV (The modimers ,55, whilmodimers . The remmodimers  is requirate KIV .M. alpina (leuA1 gene is most likely originated by HGT from a photosynthetic ancestor such as a cyanobacterium, whilst the Dikarya may have evolved an independent IPMS version [P. blakesleeanus can—at least in part—functionally replace the IPMS in yeast [S. cerevisiae [M. tuberculosis IPMS [M. alpina LeuA1. In contrast to Leu, KIC is clearly a competitive inhibitor competing with KIV for the substrate binding pocket. The conversion of KIC to Leu by mitochondrial BatA requires a back-flow of cytosolic KIC into the mitochondria via monocarboxylate transporters [In contrast to BatA, IPMS is the major regulatory key enzyme in Leu and Val biosynthesis in . alpina . Phyloge version . Indeed, version . IPMS plin yeast ,5. An alrevisiae  and confsis IPMS . A similsporters , and KICsporters ,61. PropM. alpina may help to increase the production yields of polyunsaturated fatty acids.In contrast to higher fungi, the BCAA biosynthesis pathways in basal fungi are predominantly metabolically, rather than transcriptionally, regulated. BCAA production is a constitutive process, as it is required for protein biosynthesis, fatty acid metabolism, and secondary metabolite production as well. However, it can be quickly modulated if environmental metabolite fluxes change. The main regulatory protein is the plant-derived α-isopropyl malate synthase, whose gene has been presumably introduced by horizontal gene transfer. The intimin interaction of basal fungi with bacteria or plants is a widely described phenomenon. Interspecific gene transfer provides an efficient strategy to adapt to altered environmental conditions and may have contributed to the ubiquitous spread of basal fungi in various ecological niches. From an industrial angle, the described complex inhibition loops regulating the leucine metabolism in


Although a direct relationship between tinnitus or hearing difficulties and COVID-19 has been suggested, current literature provides inconsistent results, and no research has been undertaken in older adults.In November 2020, we conducted the LOST in Lombardia survey, a telephone-based cross-sectional study on a sample of 4,400 individuals representative of the general population aged ≥65 years from Lombardy region, Northern Italy. Individuals with diagnosed tinnitus and/or hearing loss were asked whether their conditions had improved or deteriorated in 2020 compared to 2019.n = 6) or hearing loss (n = 13) had COVID-19. The incidence of tinnitus was lower in 2020  than in previous years . There was no change in the incidence of hearing loss (p = 0.134).Overall, 8.1% of older adults reported a diagnosis of tinnitus and 10.5% of hearing loss. In 2020 compared to 2019, among individuals with tinnitus, those with increasing severity (5.0%) were similar to those decreasing it (5.3%). Among individuals with hearing loss, more people reported an increase (13.6%) than a decrease (3.2%) in their disease severity. No individual with a diagnosis in 2020 of tinnitus (In this large representative sample of older adults, on average neither COVID-19 confinement nor SARS-CoV-2 infection appeared to increase the severity or incidence of tinnitus. The increased severity of hearing difficulties may totally or partially be explained by physiologic deterioration of the condition, or by a misperception due to the use of face-masks. When the first cases of SARS-CoV-2 were diagnosed in Lombardy in February 2020 , Italy bGiven the direct relationship with mental health outcomes , 6, aggrBeukes and colleagues conducted a systematic review to understand the influence of the COVID-19 pandemic or SARS-CoV-2 infection on tinnitus . AlthougTinnitus is strongly associated with hearing loss , so an iThe purpose of this study is to assess the effects of confinement due to the COVID-19 pandemic on tinnitus and hearing loss in older adults in the Lombardy area.We used data from a telephone-based cross-sectional survey performed by Doxa, the Italian division of the Worldwide Independent Network/Gallup International Association, and coordinated by the Mario Negri Institute and other Italian universities and research institutions . The LOcParticipants were randomly selected from a list of 30,000 households, representative of the families in Lombardy in terms of province and size of municipality. A quota approach was employed to assure the representativeness of the older Lombardy population in terms of sex, age, and province of residence. The study protocol was approved by the coordinating group's ethical committee . All individuals gave their informed consent to participate in the study.Trained interviewers administered by telephone a questionnaire including information on socio-demographic characteristics, such as age and sex. SARS-CoV-2 infection by was assessed by respondents who self-reported the method of virus identification .A specific section of the questionnaire focused on chronic conditions, including tinnitus and hearing loss. Respondents were asked: (i) whether they were currently affected by tinnitus and/or hearing loss, (ii) for those affected, the year of first diagnosis by a physician, and (iii) whether their condition had worsened, improved, or did not change during the COVID-19 emergency, comparing their conditions at the time of the interview (autumn 2020) with the previous year (autumn 2019).We employed descriptive statistics and calculated incidence rates (IRs) and their corresponding 95% confidence intervals (CIs), using Fisher's exact method for tinnitus and hearing loss. We used a Chi-square test to compare incidence rates in 2020 vs. the mean incidence rate of the previous two decades (1999–2019). To analyze the relationship between sex and age with tinnitus and hearing loss, we computed odds ratios (ORs) and 95% CIs through unconditional multiple logistic regression models, after adjustment for sex, age and level of education. All analyses considered a statistical weight to ensure that the sample was representative of the general older population of the Lombardy region in terms of sex, age, and province of residence. The software SAS 9.4  was used for statistical analyses.p for trend <0.001). Hearing loss was reported less frequently by women than men  and increased with increasing age (p for trend <0.001). Among individuals reporting hearing loss, a percentage of 14.9% people reported tinnitus, while in the group of individuals without hearing loss, only 7.3% reported a perception of tinnitus.Out of 4,400 individuals, 358 (8.1%) reported a diagnosis of tinnitus and 463 (10.5%) of hearing loss . No statAmong individuals with a diagnosis of tinnitus (8% of the whole sample), 5.3% reported a decrease in symptom severity while 5.0% reported tinnitus worsened in 2020 compared to 2019 . The prop = 0.026). The IR for hearing loss was 50.1 (95% CI: 46.1–54.2) in 1999–2019 and 32.9 (95% CI: 17.5–56.3) in 2020 .The IR for tinnitus was 36.0 per 10,000 person-years (95% CI: 32.6–40.0) in 1999–2019 and 14.8 (95% CI: 5.4–32.3) in 2020 (p = 0.042 compared to no COVID-19 patients). This association resulted in a crude OR of 2.5 (95% CI 1.0–6.2).Of the 358 individuals with tinnitus, 16 (4.5%) reported a diagnosis of COVID-19. None of them showed any change in its severity . Of the None of the participants with COVID-19 reported a first diagnosis of either tinnitus or hearing loss in 2020 (data not shown in tables).In this representative sample of older adults from Northern Italy, we found that among individuals reporting a diagnosis of tinnitus (8% of the whole sample), in 5% the symptom improved and in 5% it worsened in autumn 2020  compared to 2019. Among individuals reporting a diagnosis of hearing loss (10% of the whole sample), in 3% their condition improved and in 14% it worsened.Our findings on the role of the COVID-19 pandemic on tinnitus severity contrast with current evidence suggesting a worsening of tinnitus due to the pandemic . In a coHowever, we confirm a possible role of the COVID-19 pandemic on the perception of a worsening of the severity of hearing difficulties , 13. We To our knowledge, this is the first study investigating changes in the incidence rates of tinnitus and hearing loss due to the COVID-19 pandemic . AlthougAmong tinnitus patients infected by SARS-CoV-2, none reported changes in their tinnitus status, suggesting that the infection has limited impact, if any, on tinnitus severity. For hearing loss, the proportion of individuals infected by SARS-CoV-2 reporting a worsening of their audiological impairment was higher than those with no infection, although the large majority of SARS-CoV-2 positive individuals reported no change in hearing loss severity. Moreover, none of the COVID-19 patients indicated a concurrent diagnosis of either tinnitus or hearing loss in 2020, thus suggesting that the SARS-CoV-2 infection had no substantial impact on the severity of either tinnitus or hearing loss.We acknowledge several limitations in our study, including those inherent to its cross-sectional design. We were therefore unable to demonstrate any causal relationship. Moreover, the sample size, although large enough to represent the geriatric population of the Lombardy region, was inadequate to derive robust estimates in selected subpopulations. For example, the incidence rates of tinnitus and hearing loss in 2020 were based on only 6 and 13 cases, respectively. As a telephone-based survey, we introduced an indirect selection bias because only telephone owners were included in our population. However, this was the most accurate mode of data collection in the pandemic period, where contacts had to be kept to a minimum, particularly for the elderly who are less likely to participate in online surveys.Another limitation is that both tinnitus and hearing loss diagnoses were self-reported, and validated questionnaires were not administered to assess tinnitus or hearing loss. However, to our knowledge, this is the first representative study specifically undertaken on the geriatric population during the COVID-19 pandemic.If generalized to the whole population of Lombardy, our estimates amount to more than 185 and 240 thousand older people, respectively, being diagnosed with tinnitus and hearing loss. Our findings do not appear to support the hypothesis that the COVID-19 pandemic as a societal stressor has enhanced the severity or incidence of tinnitus. The worsening of hearing difficulties between 2019 and 2020 may totally or partially be explained by a physiologic deterioration of the condition in the 1-year span, and by the fact that the patients had to cope with their illness at a time when face-masks prevented them from reading lips, and generally made it harder to hear each other's words.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by EC of Fondazione IRCCS Istituto Neurologico Carlo Besta. The patients/participants provided their written informed consent to participate in this study.Silvano Gallus, Cristina Bosetti, Carlotta Micaela Jarach, Alessandra Lugo, Chiara Stival: Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milan, ItalyGianluca Serafini, Andrea Amerio, Mario Amore: Università di Genova, Genoa, ItalyDavid Stuckler, Roberto De Sena, Simone Ghislandi, Yuxi Wang: Università Bocconi, Milan, Italy*, Marialaura Bonaccio*, Francesco Gianfagna°: Università degli Studi dell'Insubria, Varese, ItalyLicia Iacoviello*IRCCS Neuromed, Pozzilli, Italy°Mediterranea Cardiocentro, Napoli, ItalyAnna Odone, Carlo Signorelli, Giansanto Mosconi, Giacomo Vigezzi: Università di Pavia, Pavia, Italy and Università Vita-Salute San Raffaele, Milan, ItalyLuca Cavalieri d'Oro, Magda Rognoni, Luca Paroni: Agenzia per la Tutela della Salute della Brianza, Monza, ItalyAlberto Zucchi, Roberta Ciampichini: Agenzia per la Tutela della Salute di Bergamo, Bergamo, ItalyCMJ: conceptualization, methodology, formal analysis, writing–original draft, and visualization. AL: supervision and writing-review and editing. CB, AA, LC, LI, AO, DS, and AZ: writing-review and editing and funding acquisition. CS, PB, WG, CC, and WS: writing-review and editing. SG: conceptualization, methodology, supervision, writing-review and editing, and funding acquisition. LOST in Lombardia Study Investigators: funding acquisition. All authors contributed to the article and approved the submitted version.The project is funded by a research grant of the DG-Welfare of Lombardy Region  and by a grant of the AXA . The work of AL and SG is partially supported by Unification of Treatments and Interventions for Tinnitus Patients–UNITI project, which has received funding from the European Union's Horizon 2020 Research and Innovation Programme, Grant Agreement Number 848261; the work of CMJ and SG is partially supported by Tinnitus Genetic and Environmental Risks–Tiger project, which has received funding from the European Union's Horizon 2020 Research and Innovation Programme, Grant Agreement Number GNP-182.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The stria vascularis (SV) is a highly vascularized tissue lining the lateral wall of the cochlea. The SV maintains cochlear fluid homeostasis, generating the endocochlear potential that is required for sound transduction. In addition, the SV acts as an important blood-labyrinth barrier, tightly regulating the passage of molecules from the blood into the cochlea. A healthy SV is therefore vital for hearing function. Degeneration of the SV is a leading cause of age-related hearing loss, and has been associated with several hearing disorders, including Norrie disease, Meniere’s disease, Alport syndrome, Waardenburg syndrome, and Cytomegalovirus-induced hearing loss. Despite the SV’s important role in hearing, there is still much that remains to be discovered, including cell-specific function within the SV, mechanisms of SV degeneration, and potential protective or regenerative therapies. In this review, we discuss recent discoveries elucidating the molecular regulatory networks of SV function, mechanisms underlying degeneration of the SV, and otoprotective strategies for preventing drug-induced SV damage. We also highlight recent clinical developments for treating SV-related hearing loss and discuss future research trajectories in the field. This maintains the endolymph at a high K+ (∼157 mM), low sodium  state, that contrasts with the low K+ (∼4.2–6.0 mM), high Na+ (∼141–148 mM) state of perilymph (The stria vascularis (SV) is a highly vascularized tissue located in the lateral wall of the cochlea that contributes to cochlear homeostasis in two ways . First, erilymph . This coerilymph . For thierilymph .+ channels in the hair cell stereocilia. Once open, these channels allow for the influx of K+ and the depolarization of the hair cell (2+) channels are activated, allowing for the influx of Ca2+ and release of neurotransmitters, creating a signal that is sent to and processed by the brain , that tightly controls the transfer of material from the SV capillary network into the endolymph . The BLB+ from the SV into the scala media , and the Notch signaling pathway effector gene, Hes-related family BHLH transcription factor with YRPW motif (Heyl), expressed in marginal cells; the K+ inwardly-rectifying channel J13 (Kcnj13) and a VEGF receptor gene, neuropilin 2 (Nrp2), expressed in intermediate cells; and the SRY-Box transcription factor 8 (Sox8), and the steroid/thyroid hormone receptor gene, nuclear receptor 2F2 (Nr2f2), expressed in basal cells. Further evaluation of these genes will likely provide important insight into cell-specific roles within the SV. In addition, Korrapati et al. used the Pharos database (https://pharos.nih.gov/) to demonstrate that 26 of the newly identified SV genes can be targeted by 93 FDA approved drugs, opening immediate avenues for SV-specific therapeutics.While knowledge of the SV layers remains limited, recent RNA sequencing and bioinformatic analysis have been used to further elucidate cell-specific gene expression in the SV. For instance, single-cell RNA sequencing of the adult CBA/J mouse SV identified previously unknown gene expression in the SV . Newly iAnxa1) and dipeptidyl peptidase like 10 (Dpp10), which are target genes for the bric-a-brac (BTB) domain and cap‘n’collar (CNC) homolog 2 (Bach2) transcription factor—a potent immune regulator (Slc26A4), and the K+ inwardly-rectifying channel J10 (Kcnj10) in root cells suggests a role in maintaining the endocochlear potential (+ channel activity (Kcnj10) within root cells, providing further evidence that root cells contribute to K+ recycling in the cochlea , vascular integrity, and blood flow control , CD68 molecule (CD68), integrin alpha M (CD11b), macrophage/monocyte antibody (MOMA2), glutathione S-transferase alpha 4 (GSTα4), glutathione S-transferase (GST), and Kir4.1 . PVM/M’s2+ interactions  in transgenic NMRI mice that were either pigmented and expressing melanin (YRT2 mice), non-pigmented but expressing the melanin precursor L-DOPA (TyrTH mice), or albino non-transgenic NMRI littermates (NMRI), demonstrated that aging YRT2 and TyrTH mice exhibit significantly less hearing loss after noise exposure than NMRI littermates  in the SV of aged guinea pigs (1-year-old) when compared to young guinea pigs (2-week-old). The reduced TMEM16A expression corresponded with increased hearing thresholds  function . While the mechanism of effect needs to be better defined, these therapeutic advances could significantly improve the outcomes experienced by individuals with Meniere’s disease, especially when compared to gentamicin treatment. In addition, a recent study by Gu et al. used published RNA-sequencing data in adult mice and compared it to published datasets regarding Meniere’s disease in humans to localize known Meniere’s disease genes within specific cells of the SV (Esrrb) and Na+/K+ transporting ATPase 1b2 (Atp1b2) expression was identified in marginal cells, endothelin receptor type B (Ednrb) and transmembrane protein 176A (Tmem176A) in intermediate cells, and solute carrier 44a2 (Slc44a2) and collagen type 11 a2 (Col11a2) in basal cells. Future research to determine the involvement of these genes in Meniere’s disease and SV function may improve Meniere’s disease models and identify novel therapies.Currently, there is no cure for Meniere’s disease and treatments to manage Meniere’s symptoms are limited. For severe cases of vertigo, the ototoxic aminoglycoside antibiotic, gentamicin, is injected intratympanically. This approach provides relief by destroying vestibular hair cells, which subsequently removes dysfunctional balance sensation. However, vestibular hair cell death impairs normal balance and intratympanic gentamicin can also damage cochlear hair cells, causing permanent hearing loss. Therefore, the corticosteroids methylprednisolone (NCT00802529) and dexamethasone (NCT03664674) have been tested in human clinical trials as potential gentamicin replacements . Encouraf the SV . EstrogeAlport syndrome is a rare inherited type IV collagen disorder, affecting approximately 1 in 5,000 people . Alport NFKBIA), in the SV of COL4A4 knockout mice when compared to wildtype controls. This would suggest that the host’s inflammatory response damages the SV in those with Alport syndrome . This study will produce important human-based information, but more research is needed in both animal models and humans to identify SV pathology so that treatments may be developed.Mitf), paired box 3 (Pax3), SRY-box transcription factor 10 (Sox10), Ednrb, endothelin 3 (Edn3), and snail family transcription factor 2 (Snai2) , Kcnj13, and solute carrier 45A2 (Slc45a2) are downregulated  with congenital CMV infection  infection is one of the most common causes of pediatric sensorineural hearing loss. 35% of infants with symptomatic CMV and 7–10% of infants with asymptomatic CMV will develop hearing loss . The mecnfection . Notablynfection . IncreasThere is no cure for CMV-induced hearing loss. However, valganciclovir, an FDA approved, orally administered, anti-viral medication prevented CMV induced hearing loss in a phase three clinical trial (NCT00466817; The SV has a vital role for hearing. It regulates the strict ionic composition of cochlear fluids and produces the endocochlear potential required for sound transduction, while also protecting the cochlea by providing immune surveillance and maintaining the BLB. However, the BLB remains one of the most under studied aspects of the ear, despite being one of the most important targets for delivering inner ear therapeutics. Nevertheless, single-cell sequencing technologies, combined with human focused histology and functional hearing assessments have significantly improved our understanding of the SV. Novel SV cell types have been identified and known cell types are proving to have more dynamic roles than previously thought. Direct avenues for clinical intervention have been identified, such as gene networks that can be targeted by FDA approved therapeutics. Follow up studies using knockout models to evaluate the importance of genes identified by single nucleus RNA sequencing will also provide greater detail regarding cell-specific roles in the SV. Furthermore, evaluation of these SV cell-specific genes might also identify biomarkers for diagnosing or monitoring SV health. To date, only a few inner ear biomarkers have been evaluated, but none are specific to the SV .While several questions remain regarding SV development and function, significant groundwork has set the scene for burgeoning discovery. It is likely that this will subsequently facilitate the development of research tools such as organoids or organ-on-a-chip technologies, that have proven invaluable for high throughput drug screening and disease modelling in other fields. Overall, critical knowledge has been gained particularly elucidating the mechanisms of age-related SV degeneration, ototoxic outcomes, and SV-associated hearing disorders, providing exciting opportunities for preventing hearing loss in the future.


P < 0.001). The prevalence of ANLs in patients with scores 1–3 was 1.2, 13.5, and 38.9% respectfully. When controlled for other significant variables, patients with a higher qFIT score were statistically more likely to have an ANL . A negative qFIT had a high NPV for the presence of ANLs . These results strongly suggest that qFIT has validity as a prioritisation tool in patients with iron deficiency; both allowing for a more informed decision of investigation of patients with very low risk of malignancy, and in identifying higher risk patients who may benefit from more urgent endoscopy.Difficulty in providing endoscopy for patients with iron deficiency anaemia (IDA) during the COVID-19 pandemic has highlighted the requirement for a prioritisation tool. We aimed to test the validity of qFIT as a prioritisation tool in patients with iron deficiency and its ability to identify patients with advanced neoplastic lesions (ANLs). Data collected from patients referred with biochemically proven iron deficiency (ferritin ≤ 15 μg/L) and synchronous qFIT who underwent full gastrointestinal investigation within NHS Greater Glasgow and Clyde was analysed retrospectively. Patients who did not undergo full investigation, defined as gastroscopy and colonoscopy or CT colonography, were excluded. ANLs were defined as defined as upper GI cancer, colorectal adenoma ≥ 1 cm or colorectal cancer. Area under the curve (AUC) analysis was performed on qFIT results and outcome, defined as the presence of an ANL. AUC analysis guided cut-off scores for qFIT. Patients with a qFIT of <10, 10–200, >200, were allocated a score of 1, 2, and 3, respectively. A total of 575 patients met criteria for inclusion into the study. Overall, qFIT results strongly predicted the presence of ANLs (AUC 0.87, CI 0.81–0.92;  Iron deficiency anaemia (IDA) remains a prevalent condition affecting 2–5% of adult men and post-menopausal women in developed nations  and is aIn the context of a reduced endoscopy capacity and delivery of services as a result of the ongoing COVID-19 pandemic, providing timely bidirectional endoscopic investigations for patients with IDA is challenging. There is concern that limited endoscopy service provision as a result of the pandemic will result in delayed diagnosis of underlying GI neoplasia . A strat® healthcare information system and information regarding patient demographics, referrals, clinical details, investigations and outcomes were recorded. Biochemically proven iron deficiency was defined as a ferritin ≤ 15 μg/L. Patients who did not undergo full investigation, defined as gastroscopy and colonoscopy or CT colonography, were excluded from the study. All patients had investigations carried out within 6 months of qFIT testing. Outcome from radiological or endoscopic investigation was obtained from electronic clinical notes. The presence of GI symptoms and recurrent anaemia was obtained from referral letters from primary care physicians and electronic clinical records. Recurrent anaemia was defined as the presence of anaemia more than 2 years prior to investigation. A positive qFIT was defined as ≥10 μg Hb/g. We defined advanced neoplastic lesion (ANL) as upper gastrointestinal cancer, colorectal adenoma ≥1 cm or colorectal cancer (CRC). We defined other significant lesions as gastric/duodenal ulceration, reflux oesophagitis (LA classification C/D), vascular abnormalities, polyp ≥1 cm and active inflammatory bowel disease.We searched laboratory databases for patients with proven iron deficiency across Greater Glasgow and Clyde health board between January 1, 2019 and December 31, 2019 and matched these with synchronous qFIT (HM-JACKarc) results. Patients were identified by interrogation of the TrakCarehttp://www.R-project.org). All results are expressed as the mean ± standard error for continuous variables, or as a percentage for categorical variables. Student T-test, Or Welch T-Test was carried out to compare the means of continuous parametric and non-parametric variables, respectively. Chi-squared tests were used for comparison of categorical variables. All tests were two-tailed and a value whereby P < 0.05 was considered as statistical significance.All calculations were carried out using SPSS version 23  and R 4.0.0 for Mac (Area under the receiver operator curve (AUROC) was conducted to determine the predictive validity of qFIT for detecting ANL. A cut-off of <10 μg Hb/g Hb/g was used as this currently is the cut-off used by NICE to determine if a test is negative. We then calculated the furthest co-ordinate from this which maintained an acceptable Youden Index; this corresponded to a cut-off of 200 μg Hb/g Hb/g. All qFIT results were grouped 1, 2, and 3 if a result fell into a range of <10 μg Hb/g Hb/g, 10–200, μg Hb/g and >200 μg Hb/g, respectively. Variables which displayed statistical significance at univariate analysis were considered for hierarchical analysis within the multivariate logistic regression equation. Regression was used to calculate odds ratios for qFIT scores and their association with ANLs. Positive predictive values (PPV), negative predictive values (NPV), sensitivity and specificity were calculated, with 95% confidence intervals (CI) for all categorised GI pathology.P < 0.001). There were two patients who had concurrent upper and lower gastrointestinal pathology at time of endoscopy: patient 1; gastric ulcer/ileal ulcer and patient 2; gastric ulcer/proctitis.A total of 1,769 patients had proven iron deficiency with a matched ferritin and qFIT result. Within this cohort, 944 patients received at least one type of endoscopic or radiological investigation and 575 patients underwent full GI investigation as described above for inclusion into the study. Descriptive statistics for common demographic and clinical parameters are recorded in . A totalCut-off analysis aided the allocation of groupings by qFIT results of <10, 10–200, and >200 μg Hb/g. P < 0.01, The validity of qFIT as a prioritisation tool was tested in AUROC analysis which showed that qFIT significantly predicted the presence of ANL  and higher still in patients with a qFIT >200 μg . Following adjustment for qFIT group within the multivariate logistic regression the association between anticoagulation and presence of ANL was lost.During univariate analysis only qFIT result and treatment with anticoagulation were associated with a higher prevalence of ANL. When these variables were analysed together with multivariate logistic regression  the prevP = 0.05). Though patients who were receiving anticoagulant therapy were more likely to be associated with higher rates of ANL , this was not observed in patients who were receiving antiplatelet therapy .Patients who were referred with recurrent anaemia had a non-significant lower risk of ANL  has been established as an accurate test in identifying colorectal cancer (CRC) and is used for national bowel cancer screening programs . CurrentThe results from our study support a role for using qFIT to help prioritise the endoscopic investigation of patients referred with iron deficiency to gastroenterology out-patient services. The strong correlation with qFIT levels not only identifies those with an increased risk of having ANL dependent on the qFIT value but also confidently identifies those with a significantly lower risk.We have suggested cut off values for qFIT results aided by AUROC analysis which offer a pragmatic risk stratification tool for clinicians who are investigating patients with iron deficiency. We found an ANL (cancer) in 1.19% (0.3%) of those with qFIT <10 μg, in 13.5% (8%) of those with qFIT 10–200 μg and in 38.9% (28%) of those with qFIT > 200 μg . We haveOur study has demonstrated that qFIT has an excellent NPV for identifying patients with iron deficiency who do not have ANL [98.8% (97.0–99.5%)]. In a pandemic, it may be more appropriate to use a test to exclude cancer, on the presumption that significant polyps will be picked up with resumption of bowel cancer screening programmes. Using this criteria, a qFIT would have a NPV of 99.7% in our population, and applying this cut off would have avoided 550/943 (58.3%) patients undergoing investigation. Although our results are in the broader context of iron deficiency, they are in keeping with previous studies evaluating the predictive validity of qFIT in patients with iron deficiency anaemia which also demonstrate a high NPV with a negative qFIT , 13, 14.In addition, we included the diagnostic performance of qFIT for other significant GI pathology. We found other significant upper GI lesions in 7.2% and lower GI lesions in 3.5% of cases . OverallThere is little information around the utility of qFIT in detecting upper GI malignancy. In our study, qFIT appeared to detect all patients with upper GI malignancy Importantly, however the sample size within this group is low and caution should be taken with its interpretation. Currently it is not recommended that FOB testing or qFIT be used as a diagnostic tool in the investigation of IDA (BSG guidelines), and its use in the assessment of upper GI blood loss and malignancy is controversial . This isIn our group of patients age, sex and haemoglobin values were not independent risk factors for underlying ANLs as has been suggested in previous studies . It is iWithin this study, we observed a non-significant trend between a lower prevalence of ANL and the history of recurrent anaemia. These patients were noted to have been iron deficient more than 2 years prior to current referral episode. A small but significant yield in follow up or following repeat bidirectional endoscopy has previously been reported , 23. We Almost half of our study population were recorded as being symptomatic at referral or at time of investigation, however we observed that the presence of GI symptoms and iron deficiency was not associated with increased finding of ANL. Although it was out with the scope of this paper to test the significance of individual symptoms and their association with neoplasia we note that previous meta-analyses assessing the predictive value of individual symptoms have had inconsistent results –26.We recognise that our study had a number of limitations. Firstly, it was a retrospective study of clinical records of patients referred over a 12 month period. Because it is retrospective, we therefore could not account for all potential bias. A degree of selection bias is likely due to the fact that patients with IDWA are not generally referred for bidirectional endoscopy unless there are other factors influencing the decision for referral. There was a relatively short follow-up period. All patients with a documented ferritin ≤ 15 μg/L during this period were not included as they did not have matched qFIT results available. Approximately 50% of patients with matched qFIT results were either not referred for investigation or were pending investigation at the time of data collection. Data collected regarding patient symptoms were gathered from primary care referral letters, clinical letters and endoscopy reports which may have varying degrees of accuracy. Moreover, we did not collect data on the location of colorectal adenomas that were observed at endoscopy. Although all investigations were completed within 6 months, we have not specified the exact time from qFIT test to investigation, possibly not observing an interaction between delay to investigation and diagnosis of ANL. A final limitation is that data on patients with small bowel lesions was not collected; we therefore cannot comment on the utility of qFIT for this patient group. However, we note current guidance  does notIn conclusion, our study strongly supports the role of qFIT as a prioritisation tool in patients with iron deficiency. During the current global pandemic endoscopy services may benefit from being able to prioritise the investigation of patients based on the qFIT level to expedite investigation of those at highest risk. For those with a negative qFIT, investigation may take place at a lower priority, or in the context of a very low likelihood of significant pathology, allow a better informed discussion with patients around the likely benefit of undergoing investigation. Whilst such an approach is of clear benefit during time of limitation of endoscopic resources, it also likely to be beneficial in the recovery period and beyond.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.WC responsible for data analysis, literature review, and article write up. AC responsible for project design and data collection. SB and AS contributors to data analysis and literature review. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Emission rate and influencing parameters were then computed using a well‐mixed dispersion model. The emission rate from the index case was estimated at 130 quanta h−1 (interquartile (97–155 quanta h−1). Measured lactose concentrations in the room were found relatively homogenous . Air renewal was found to play an important role for event durations greater than 0.5 h and loss rate below 2–3 h−1. The estimated emission rate suggests a high viral load in the index case and/or a high SARS‐CoV‐2 infection coefficient. High probabilities of infection in similar indoor situations are related to unfavorable conditions of ventilation, emission rate, and event durations. Source emission control appears essential to reduce aerosolized infection in events lasting longer than 0.5 h.There is increasing evidence of SARS‐CoV‐2 transmission via aerosol; the number of cases of transmission via this route reported in the literature remains however limited. This study examines a case of clustering that occurred in a courtroom, in which 5 of the 10 participants were tested positive within days of the hearing. Ventilation loss rates and dispersion of fine aerosols were measured through CO This case study contributes to the growing body evidence highlighting possible SARS‐CoV‐2 transmission through aerosol.The known exposure conditions in the courtroom allow us to reasonably exclude other transmission modes.Probabilities of transmission in room of similar sizes and various event durations and air renewal were estimated.1The role of aerosols in the transmission of COVID is currently under debate. Superspreading events, in which aerosols may have been a significant factor, are regularly reported in the press. However, the cases reported in the scientific literature remain rare and we were able to identify few studies that explicitly attribute the emission to aerosols , whereas it can pass to 6 m, in the case of an air movement of 50 m s−1.µm tend to settle on the floor, offices, and neighboring surfaces.Simulations of the propagation of respiratory and aerosol droplets generated by speech over a wide range of temperatures (0–40°C) and relative humidity (0–92%) show that 95% of the droplets are deposited over a distance of less than 1.4 m.6 to 109 copies ml−1 with a maximum of up to 1011 copies ml−1.6, there is a 37% probability that a droplet of 50 μm (before dehydration) contains at least one virus.The theoretical transport capacity of a spherical droplet depends on its volume, which is proportional to the cube of its radius. It is therefore possible to estimate, for a given viral load scenario, the probability of finding the virus in a droplet or aerosol immediately after emission. Analysis of oral fluid from infected patients shows viral RNA loads ranging from 10There are numerous field studies showing the presence of SARS‐CoV‐2 or similar viruses in aerosols. These studies have mostly been conducted in hospital settings where COVID patients are concentrated.In this study, the hypothesis of aerosol contamination in a poorly ventilated space is addressed through the case of a SARS‐CoV‐2 cluster occurring in a courtroom.1.1In October 2020, Unisanté's occupational physicians were called upon by a Vaud state department following the appearance of symptoms compatible with COVID for 5 of the 10 people who participated in a hearing in the same courtroom. The situation was investigated to highlight the course of the hearing, the protective measures put in place, and the ventilation conditions in the courtroom.The hearing took place behind closed doors on the afternoon of 30.09.2020 from 2 to 5 p.m. in a courtroom belonging to the Canton of Vaud (Switzerland). As this was a formal event, the records of the hearing made it possible to retrace the positions and activities of the participants quite precisely. Nine people, identified P1 to P9, attended the entire hearing and a 10th person  attended the hearing for only 34 min.P1 arrived alone by car. The hearing started at 2 p.m. in the presence of P1, P2, P3, and P4. From 2:05 p.m., P5, P6, P7, P8, and P9 were also present in the room. The respective positions of the participants in the hearing room are shown in Figure During the hearing, the people did not have a microphone and the exchanges took place in an oratory mode. According to the chairperson, during the hearing, P2, P8, and P5 were the 3 people who spoke the longest and loudest. P1 (index case) and P4 spoke very little. P1, P2, P3, and P4 conversed during breaks, but in “normal” voice. The exchange of objects between participants was very limited. A paper document circulated between P8, P2, P1, and P4. P3 also walked up to P5 and P8 to have them sign a document during the hearing.At the time of the hearing, Switzerland was at the beginning of the second epidemic wave. The 14 days incidence of cases in the country was about 100 cases/100 000 inhabitants. General hygiene measures and the wearing of masks in closed public places and a social distance of at least 1.5 m were in force. The wearing of a face mask was mandatory inside the building, except when individuals were seated in their assigned seats in the courtroom. Participants therefore removed their masks once they were seated at least 1.5 m apart. Disinfectant was available for hand washing. The participants have, in principle, complied with the existing protective measures.Examination of the ventilation conditions during the hearing showed that the room was poorly ventilated. The building's logistics department recorded a breakdown of the ventilation system from 28.09.2020 to 6.10.2020. This failure was not known to the participants at the time of the hearing, and therefore during the 3 h of the hearing, the mechanical ventilation did not work. The courtroom was equipped with a narrow window of 42 × 205 cm (width × height). During the hearing, the door and window were always kept closed for confidentiality reasons. But the window was opened during the breaks. Based on weather data, the average daily relative humidity (outdoors) on the day of the hearing was 93.5%. The indoor relative humidity at the time was not known.P1 can be considered as the index case and that P2, P3, P4, and possibly P5 are likely secondary cases following the hearing.Except for P5, who had a contact with a positive case prior to the hearing, contact tracing did not identify other risk situations for secondary cases . In particular, P2, P3, and P4 had no contact with P1 outside of the hearing and no contact with other COVID cases during the window of contagiousness.The index case (P1) presented cough symptoms from 30.09 with a positive test on 1.10. All secondary cases were tested positive within the next week  was no longer available at the time of this evaluation. However, it was possible to retrospectively analyze the sample from a secondary case, which tested negative for S‐gene dropout. It can be concluded that the cluster is not due to the UK variant.2.2Field measurements were conducted on December 21, 2020, in the absence of any hearing in the courtroom. Compared to the studied situation, the only difference during this field measurement was the presence of plexiglas™ dividers (65 × 110 cm) between each seat.2 from a pressurized cylinder into the courtroom, until a concentration of about 2200 ppm was reached, the ventilation system being stopped to mimic the conditions at the time of the hearing. CO2 was measured using a direct reading CO2 sensor , located on a table in the middle of the room (P10). Two ventilators were in function during the measurement in order to homogenize the ambient air. Once injected, the decaying CO2 level in the room was recorded during about 30 min in absence of any person. CO2 measurements were repeated with the window open, to mimic the air renewal conditions during the breaks. The air renewal rate was obtained by plotting the natural logarithm of the CO2 levels as a function of the time and computing the slope of the linear part of the plot.The air renewal was measured by injecting CO−1) filled with a lactose solution (0.1 M in water). The generator was located in P1, mimicking the emission of the index case. Air sampling was performed at all positions (P1–P10), using sampling pumps  operating at a flow rate of 2 liter per minute and connected to a sampling open‐face cassette containing a cleaned quartz microfiber filter . Nobody was present in the room once the generation system and the pumps were started. The generation and sampling lasted about 80 min. The aerosol characterization was done by installing a direct reading aerosol spectrometer  at P1 and P3. After sampling, the filters were extracted in water and this solution was analyzed for lactose using an ICS‐5000 ion chromatography system  equipped with a DP 5000 pump, an autosampler AS‐AP, and a thermostatized compartment DC 500 with an electrochemical detector. Lactose aerosols measurement was chosen because of the submicronic size distribution as well as sensitivity and reliability of lactose determination by ion chromatography.The aerosol dissemination within the room was investigated using an ultrasonic atomizer system  is an exponential function of the number of inhaled virus quanta (n). The term quanta is used in evaluating airborne infections, conveying information on both virulence and quantity of infection material. A quanta is the dose of aerosol required to cause infection in 63% of susceptible persons in a room. The formulation of Equ.1 is based on several underlying assumptions: (a) the infectious individual emits SARS‐CoV‐2 at a constant rate, (b) the initial airborne concentration of virus is zero, (c) the latency time of the disease is longer than the time scale of the event, (d) the infectious aerosol is homogeneously distributed in the ambient air, and (e) viruses are eliminated, in a first‐order decrease, by a combination of ventilation, deposition on surfaces, and inactivation. Assumptions a, b, and c appear relatively trivial when considering the moderate variability of aerosol emission in the same activity  and the duration of the event relative to the latency time. Hypotheses d and e are less trivial, but are common assumptions in the field of aeraulic. An ideal mixing is for instance difficult to achieve in practice, since the geometry of the room or the presence of obstacles  will lead to the creation of short circuits or poorly ventilated areas (dead zones). This model is however commonly used to simplify an overly complex physical situation and obtain an estimate with reasonable resources. Moreover, experimental measurement of the air change rate allows the model to be verified and adjusted.The probability of infection =Eλ·quations  and 5),,E [q h−12.4n = 1 000 000). To facilitate the comparison of results, the parameters used are compared with those proposed by Miller for the Skagit Valley situation.The calculations have been implemented on Stata/IC 16.1 . Table A transmission probability (secondary attack rate) of 33%–44% (3/9–4/9) was used for the courtroom situation. Without considering the index case, 4 of the 9 people present were indeed confirmed SARS‐CoV‐2 positive within days of the hearing. For one of the cases (P5), however, contamination by the index case is not very likely due to the short incubation period and prior contact with a positive case at a party. The most likely value is therefore 3/9 with an upper estimate of 4/9.−1with the window closed and of 1.25 h−1 with the window open. Since it is difficult to know if the window was open during all the breaks, a uniform distribution ranging between these two values was used for λv.The air renewal measured in the room (loss rate to ventilation λv) was of 0.23 h−1.Volumetric inhalation rates were taken from the Binazzi study. The value used here is that of "reading aloud with a normal voice", which is lower than that of singing. Doremalen's study was selected to determine a plausible range of loss rate due to virus inactivation. The half‐life interval proposed by this study is 0.64–2.64 h, corresponding to a k of 0.26–1.08 h3−1 with an interquartile (25–75 percentile) of 97–155 q h−1 and a 50th percentile of 122 q h−1. To evaluate the effect of our hypothesis on the distribution of the loss rate due to ventilation, a simulation was also carried out for a closed window for the duration of the hearing (λv = 0.23). In this more conservative case, an average emission rate of 90 q h−1 is sufficient to produce the same attack rate. The emission rate remains in the same order of magnitude, but the distribution of the emission rate is significantly narrower, reflecting the smaller range of uncertainty used on the input parameters.The probable distribution of the resulting emission rate is shown in Figure n = 8, mean 336 µgm−3, SD = 39 µgm−3; interquartile range 315–344 µgm−3). Even with the presence of plexiglas™ (added after the hearing), which tends to limit horizontal airflows, these concentrations remain in the same order of magnitude, suggesting that the well‐mixed model used is adequate.The investigations conducted, by lactose emission, allowed a better understanding of the distribution of aerosols inside the room. The results of the sampling carried out are presented in Table Examination of the direct reading measurements made at locations P1 and P3 shows differences in the aerosol size distribution see . At P1, −1 and three possible event durations: 0.5, 1.5, and 3 h. The distributions used for the simulation parameters are those already described in Table v) is shown in Figure −1. The effect of the emission rate E is shown in Figure An exposure scenario was considered to illustrate the influence of environmental and emitter‐related parameters (the index case) on the contamination conditions in the room. The model of the well‐mixed room, previously described in Equations  and 5),,5), was −1. For higher emission rates and event times, this growth is attenuated, probably due to the exponential nature of the probability of contamination . For longer event times, a rapid increase is observed between 0 and 300 q h4n = 5) investigated in this study is small in size, which makes the estimation of the attack rate (probability of secondary infection) uncertain. It cannot be excluded that some contamination may have occurred outside of this situation. On the other hand, it is possible that individuals who would have already been contaminated during the first spring wave, but who were asymptomatic, were present in the courtroom and benefited from some immunity. Despite of these uncertainties, the main interest of this case is that the conditions of the courtroom hearing were well known and documented, which allows us to reasonably exclude that the contaminations observed could have been explained by other transmission modes.Existing knowledge on the aeraulic behavior of fine particles, speech and respiration‐related emissions, and experimental evidence on the viability of the virus in the air support a contribution of SARS‐CoV‐2 transmission by inhalation of aerosols. However, its identification in situ is difficult, probably because it is often not possible to distinguish the relative contribution of the different transmission routes. This study contributes to the modest but growing body of case studies highlighting this mode of transmission. Compared to other similar studies, the cluster , but also by the fact that Miller et al. made conservative assumptions. In particular, they hypothesized that inhalation of aerosols was the dominant form of contamination, whereas it is likely that some of the 53 identified cases were contaminated by contact or droplets. The values we estimated for the courtroom situation are close to the upper emission range estimated by Buonanno et al.2 q h−1 in individuals with elevated viral loads and assuming a high infectivity coefficient (ci). This value of emission rate corresponds, for example, to a viral load of 109 RNA copies ml−1 for a ci of 0.1.The emission rate distribution obtained in this study has a mean value of 1.3 × 10−1 and relatively short event durations . For these situations, the ventilation loss rate plays only a marginal role in transmission. On the other hand, high infection probabilities , which may lead to large infectious clusters, are due to the combination of several unfavorable parameters, generally requiring emission rates greater than 100 q h−1, ventilation rates <2 h−1, and event durations equal to or greater than 1.5 h. This is consistent with the cases of super propagation reported in the literature and associated with aerosols, which are surprisingly few in number and highlight situations of confinement, insufficient ventilation, and/or high emissions rates.Analysis of the influence of event duration, emission rate, and ventilation loss rate highlights the importance of these parameters in the indoor transmission of SARS‐CoV‐2. On the one hand, situations with a low theoretical infection probability are mainly associated with emission rates below 100 q h3 room and the results obtained are not readily transposable to larger or smaller volumes such as conference rooms or domestic rooms. It is interesting to note that 150 m3 is typically in the order of magnitude of the volume of classrooms, the ventilation of which is a matter of debate. Our results suggest that while room ventilation is essential, it is difficult to control the risk of contamination with this parameter alone because of the residual probability of infection at high ventilation rates, brought by the variability of the other parameters . Keeping in mind that the number of transmitters and targets also increases linearly with the number of people in the room. Wearing a community or surgical mask, which contributes to mitigate emission at source, especially during voice bursts, coughing, or sneezing, seems essential.The example given here is that of a 150 m5DV and SS planned the study. JJS, DV, and GS planned the field investigations. SS and CP investigated the case study JJS, and GS conducted field investigations. JJS, GS, and DV conducted the data analysis, DV did the modeling and wrote the first draft. All co‐authors contributed to the revision and finalization of the manuscript. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted.The author received no specific funding for this work.No conflict of interest.https://publons.com/publon/10.1111/ina.12866.The peer review history for this article is available at Supplementary MaterialClick here for additional data file.