Primary human brain microvascular endothelial cells (BMECs) were productively infected by all studied ZIKV strains at MOI 0.01, and were analyzed by plaque assay, immunofluorescence for NS1 protein, and qRT-PCR at 2 and 6 days post-infection (dpi). Compared to mock-infected controls, expression level of ZO-1 was significantly upregulated in ZIKV-H-infected BMECs, while occludin and claudin-5 levels were significantly downregulated in BMECs infected by all three studied viral strains. Interestingly, BMEC permeability was not disturbed by ZIKV infection, even in the presence of a very high viral load (MOI 10). All studied ZIKV strains productively infected wild-type C57BL/J mice after intravenous infection with 107 PFU. Viral load was detected in the plasma, spleen, and brain from 1 to 8 dpi. Peak brain infection was observed at 2 dpi; therefore, TJ protein expression was assessed at this time point. Claudin-5 was significantly downregulated in ZIKV-U-infected animals and the BBB integrity was significantly disturbed in ZIKV-H-infected animals. Our results suggest that ZIKV penetrates the brain parenchyma early after infection with concurrent alterations of TJ protein expression and disruption of the BBB permeability in a strain-dependent manner.The blood–brain barrier (BBB) selectively regulates the cellular exchange of macromolecules between the circulation and the central nervous system (CNS). Here, we hypothesize that Zika virus (ZIKV) infects the brain via a disrupted BBB and altered expression of tight junction (TJ) proteins, which are structural components of the BBB. To assess this hypothesis,  Flaviviridae family of viruses. These viruses have significant neuroinvasive characteristics and are regarded as neurotropic  disrupt the complex structural and functional architecture of the central nervous system (CNS). In addition, several neurological disorders are often associated with autoimmune mechanisms initiated by a viral infection, such as Guillain-Barré syndrome . Neurotrrotropic .Zika virus is an enveloped, mosquito-borne flavivirus, containing a single-stranded positive-sense RNA genome . Two maiin vivo and in vitro models of ZIKV-infection.In recent years, ZIKV has been recognized as the cause of severe neurological disorders, such as Guillain-Barre syndrome in adults  and micrAedes albopictus clone, mosquito cells) and Vero cells  were obtained from the American Type Culture Collection (ATCC). C6/36 cells  were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing GlutaMAX (Thermo Fisher Scientific) and supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) at 28°C with 5% CO2. Vero cells (ATCC CCL-81) were cultured in DMEM containing GlutaMAX (Thermo Fisher Scientific) and supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific) at 37°C with 5% CO2. Primary human BMECs  were cultured in pre-coated plates with Attachment Factor (Cell Systems) and with Cell Systems Medium, pre-formulated with 10% serum and supplemented with CultureBoost containing animal derived growth factors (Cell Systems), at 37°C with 5% CO2.C6/36 cells . Virus was tittered by plaque assay using Vero cells with a 0.7% agarose overlay. Foci of plaques were detected at 3 dpi, following fixation with 10% paraformaldehyde solution and staining with 1% crystal violet.Zika virus strains R103451 , PRVABC9 , and MR 766  were obtained from ATCC and propagated in C3/36 mosquito cells at a multiplicity of infection (MOI) of 0.01. Supernatants were collected three to four days post-infection (dpi), clarified by centrifugation at 1,000 × 7 plaques forming units (PFU) of ZIKV-H, ZIKV-PR, ZIKV-U, or vehicle . After animals were euthanized, whole blood was collected via cardiac puncture, followed by perfusion with normal saline. Brain and spleen tissue were harvested, snap frozen in liquid nitrogen, and stored at –80°C. Whole blood samples were centrifuged at 1,000 × g for 10 min to obtain plasma and stored at –80°C.All animal procedures were approved by the University of Miami Institutional Care and Use Committee (IACUC) in accordance with the National Institutes of Health (NIH) guidelines. Male and female C57BL/6J mice (aged 10–12 weeks) were purchased from Jackson Laboratory and randomly assigned to various ZIKV infection groups. Mice were anesthetized intraperitoneally with a mixture of ketamine (100 mg/kg body weight) and xylazine (5 mg/kg body weight), then injected intravenously via retro-orbital venous sinus with 100 μL of 108 to 101 viral copies) and the mouse housekeeping gene hemoglobin beta chain complex (HBB) (108 to 101 gene copies), as previously described by our group . Total RNA was also isolated from 400 μL of tissue homogenates  or 100 μL of cell culture lysates, using RNeasy tissue kit (Qiagen). RT-qPCR of plasma and tissue total RNA samples was performed using primers specific for all ZIKV strains evaluated in the present study (5′-CCG CTG CCC AAC ACA AG-3′ and 5′-CCA CTA ACG TTC TTT TGC AGA CAT-3′), probe (5′-6FAM-AGC CTA CCT TGA CAA GCA GTC AGA CAC TCA A-IABkFQ-3′) (Integrated DNA Technologies), and the qScript XLT 1-Step RT-qPCR ToughMix (Quantabio) reaction mix in a 7500 Real-Time PCR System (Thermo Fisher Scientific). RNA absolute quantification per cell was achieved by fitting to serially diluted standard curves of plasmids containing the target region of ZIKV  with freshly added protease inhibitors (Thermo Fisher Scientific) and filtered through a 300 μm nitrocellulose mesh filter (EMDMilipore). Then, 26% dextran  in isolation buffer was added to the filtered brain homogenate, mixed, and centrifuged at 5,800 × g for 20 min at 4°C. The supernatant was removed, pellets were resuspended in isolation buffer, and filtered through a 120 μm nitrocellulose mesh filter (EMDMilipore). Filtered homogenates were then re-pelleted by centrifugation  and re-resuspended either in 200 μL of radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific) supplemented with protease inhibitors for immunoblotting, or in PBS for RNA sequencing.Mouse brain microvessels were isolated as previously described . BrieflyProtein fractions from cell culture lysates and isolated brain microvessels were extracted with RIPA buffer supplemented with protease inhibitors and 1% Triton X-100 to inactivate ZIKV. Protein concentrations were measured by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). The proteins (20 μg/well) were loaded on sodium dodecyl sulfate (SDS) polyacrylamide 4–20% ready gels (BioRad) and electrotransferred to a nitrocellulose membrane using a transfer pack system (BioRad). The blots were probed overnight at 4°C with the following primary antibodies: rabbit anti-ZIKV NS-1 , rabbit anti-Claudin-5 , rabbit anti-ZO-1 , and rabbit anti-Occludin   in 5% BSA in TBS-T, overnight at 4°C. Then, the samples were incubated with conjugated secondary anti-rabbit antibody (Licor)  in 5% BSA in TBS-T, for 1 h at room temperature. The blots were washed three times in TBS-T for 5 min after each incubation step. Membranes were imaged in the Licor CLX imaging system and signal quantification was performed using ImageStudio 4.0 software. The GAPDH housekeeping gene was used as a reference.Immunostaining was performed on primary human BMECs cultured on 8-well chamber slides (Thermo Fisher Scientific). Upon 80% confluency, cells were infected with different strains of ZIKV  at MOI 0.01 for 48 h. After the incubation period, cells were carefully washed twice with PBS and fixed with 4% paraformaldehyde solution. Fixed cell monolayers were permeabilized with 0.1% Triton-X solution in PBS, followed by blocking with 4% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Chamber slides were incubated overnight at 4°C with rabbit antibodies against the ZIKV NS-1 protein  (diluted 1:100 in 4% BSA in PBS). Anti-rabbit Alexa-594 secondary antibody (diluted 1:500 in 1% BSA in PBS) (Thermo Fisher Scientific) and Hoechst  (Thermo Fisher Scientific) were used for detection of ZIKV NS-1 protein and cell nuclei, respectively. Imaging was performed on an Olympus FluoView 1200 confocal microscope with a 60× oil immersion lens and analyzed using ImageJ software.In vitro endothelial permeability assays were performed in 6-well 0.4 μm pore transwell plates (Corning). Primary human BMECs were seeded at 2 × 105 cells per insert in appropriate media, which was changed every 48 h, and transendothelial electrical resistance (TEER) measurements were acquired daily (data not shown). Five days after seeding, cells were inoculated with different strains of ZIKV at MOI 10, diluted in the appropriate media, in quadruplicates. At 2 dpi, media was replaced with Hanks balanced salt solution in both chambers, and fluorescently tagged dextran  of 10 kDa or 20 kDa was added to the upper chamber at a final concentration of 0.5 mg/mL. Fluorescent marker translocation was analyzed after 90 min of incubation by transferring 100 μL aliquots from the lower chamber to a 96-well plate and reading fluorescence at 485 nm (Ex) and 525 nm (Em).In vivo BBB permeability assays were performed using sodium fluorescein (NaF) as described before . Proteins were precipitated with 100% trichloroacetic acid  overnight at 4°C. After the incubation period, samples were centrifuged at 10,000 × g, 15 min, at 4°C and supernatants were harvested and mixed with 0.05 M sodium tetraborate buffer. A NaF standard curve was prepared by serially diluting 0.01% NaF in saline at a 1:2 ratio, and a total of 12 NaF concentrations were used. NaF fluorescence was measured in a fluorescent plate reader . Protein levels were measured by the Pierce BCA Protein Assay Kit for normalization of NaF fluorescence.d before , 2016. BA total of 32 animals were infected with different strains of ZIKV  or vehicle to characterize the impact of infection on transcriptome of brain microvessels. Each group was composed of 8 animals, 4 males and 4 females. Animals were sacrificed at 2 dpi and whole brain tissue was submitted for microvessel isolation, as described above and elsewhere . Total Rt test with significance value at p < 0.05.Experimental treatments were compared pairwise with control treatments using two-way ANOVA, followed by Dunnett’s multiple comparison test of Student’s 8 ZIKV RNA copies/mL were detected in both supernatants and cell lysates. The viral burden reached a peak at 4 dpi (approximately 1010 ZIKV RNA copies/mL in the supernatants and cell lysates), and at 6 dpi viral load decreased to the levels found at 2 dpi, the effect that was accompanied by cell detachment as assessed by microscopy, suggesting loss of cell viability (data not shown).We hypothesized that ZIKV, as other neuropathogenic viruses, invades the CNS by disrupting the BBB. Therefore, our first approach was to verify the infectivity of different strains of ZIKV in BMECs, the main component of the BBB. Primary human BMECs were cultured on 6-well plates, and when 80% confluency was reached, cells were infected with ZIKV-H, ZIKV-PR, ZIKV-U at MOI of 0.01, in quadruplicates, or exposed to media only (mock-infected control). The inoculum was left in contact with the cells for 2 h to allow viral adsorption, carefully washed with PBS, and then replaced with fresh uninfected media. Cell culture supernatants and cell lysates were collected at 2, 4, and 6 dpi, and submitted to RNA extraction. ZIKV levels were assessed by RT-qPCR in cell culture supernatants and BMEC lysates  were performed with BMECs infected with ZIKV-H at MOI 0.01 and 1; however, no alteration of BMEC barrier integrity was detected; therefore, the subsequent experiments were performed at MOI 10 to ensure that all cultured cells were infected. Control cultures were exposed to medium only. Then, media in the apical compartment of the transwell system was replaced with medium containing fluorescently tagged dextran of either 20 kDa or 10 kDa  at MOI 0.01 and lysed in RIPA buffer at 2, 4, and 6 dpi . The expin vitro analyses, we next evaluated the impact of ZIKV infection on BBB permeability and TJ protein expression in vivo. In order to assess the kinetics of ZIKV infection, immunocompetent C57BL/6J male mice were infected with 107 PFU of ZIKV-PR or vehicle and euthanized at 1 dpi (n = 4), 2 dpi (n = 4), 4 dpi (n = 4), 6 dpi (n = 4), and 8 dpi (n = 4). Systemic infection was verified by the presence of ZIKV RNA in spleen tissue homogenates and plasma samples by ZIKV-specific RT-qPCR. In brain tissue homogenates, ZIKV RNA levels peaked at 2 dpi  per group  were infected intravenously with 103 to 1.2 × 105 copies/mL in ZIKV-H-infected animals, from 8.3 × 103 to 0.8 × 105 copies/mL in ZIKV-PR-infected animals, and from 0.3 × 103 to 3.7 × 105 copies/mL in ZIKV-U-infected animals  compared to ZIKV-PR (1.6 × 104 copies/106 cells) and ZIKV-U (1.5 × 103 copies/106 cells); however, the differences were not significant . These data suggest that immunocompetent mice are a feasible model to study ZIKV infection, including neuroinfection.Brain infection was detected in 3 out of 6 mice infected with ZIKV-H or with ZIKV-PR, and in 5 out of 6 mice infected with ZIKV-U. Mean levels of ZIKV RNA showed tendency to be slightly lower in ZIKV-H-infected animals  per group were infected with ZIKV-H, ZIKV-PR, and ZIKV-U for 48 h. Mock-infected animals were included as negative controls. As illustrated in p-values, between mock-infected and ZIKV-infected animals. In addition, p = 2.4 × 10–26). The Xist gene ensures X-chromosome inactivation in female placental mammals, and therefore its expression is expected to be low in male animals. Gene expression related to BBB function was not significantly changed in microvessels isolated from ZIKV-infected animals as compared to mock-infected controls. However, the analysis revealed upregulation of genes involved in immune responses against viral infection, such as Ddx60 (p = 1.2 × 10–24), Dhx58 (p = 8.8 × 10–45) and Nlrc5 (p = 5.4 × 10–45). In addition, IFN-related immune response genes, such as Zbp1 (p = 6.8 × 10–44) and genes of the Oas , H2-Q , Irf , and Stat  families were upregulated in microvessels of ZIKV-infected animals. Interestingly, the gene Rnf213 was significantly upregulated in ZIKV-infected animals (p = 3.9 × 10–46). Rnf213 encodes a type Zn-finger protein involved in mediating protein-protein interactions and has been reported as a susceptibility gene for Moyamoya disease, a vascular disorder of intracranial arteries (p = 3.0 × 10–28). The expression of Lgals3bp is upregulated in the context of HIV-1 and HCV infections, and its expression is associated with upregulation of IL-2, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and TNF-α, contributing to an antiviral state . Interestingly, one gene related to neurogenesis signaling pathway and one gene related to cell differentiation signaling pathway  were significantly downregulated in ZIKV-infected animals, as compared to mock-infected animals .Regarding the mostly downregulated genes, ribosomal protein encoding genes were significantly downregulated in ZIKV-infected animals, compared to mock-infected animals   . These s (PE243) , i.e., a (PE243) .in vivo were shown to be transient and regulated by the interaction of the host with viral factors, such as pathogen-associated molecular patterns   occurred only among animals infected with the Honduras strain. In addition, a clear trend between the presence of the neuroinfection and increased BBB permeability was observed in mice infected with ZIKV-H but not ZIKV-U or ZIKV-PR, indicating strain-specific effects  . Recentl effects . The par process .Interestingly, when we evaluated the expression of TJ proteins after ZIKV infection, significant changes starting at 2 dpi, and at a physiologically relevant dose (MOI 0.01) were observed . Expressin vivo analyses of TJ protein expression in ZIKV-infected brains agree with in vitro data. In ZIKV-H-infected animals, all TJ proteins analyzed tended to be upregulated. In ZIKV-PR infected animals, a tendency in the upregulation of ZO-1 and occludin expression was evident in mock-infected brains; however, a reverse trend was observed in infected brains. Regarding infection with ZIKV-U stains, TJ proteins were downregulated in infected brains, achieving statistical significance only for claudin-5. Overall, the pattern of the observed changes suggests specific responses from the BBB during neuroinfection by individual strains of ZIKV. However, the kinetics of TJ alterations may depend not only on different viral strains but be individually tuned for individual TJ proteins. It also should be noted that modulation of the BBB permeability is frequently regulated by transient phosphorylation/dephosphorylation of TJ proteins and not their overall expression levels  in accordance with the National Institutes of Health (NIH) guidelines.AL designed and performed all the experiments, analyzed the data, and wrote the manuscript. IA and LB contributed to generation of the data. NE-H and MN conceived and designed the studies. MT conceived and designed the studies, and wrote the manuscript.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.


It has been known for more than a century that, in adult vertebrates, the maintenance of taste buds depends on their afferent nerves. However, the initial formation of taste buds is proposed to be nerve-independent in amphibians, and evidence to the contrary in mammals has been endlessly debated, mostly due to indirect and incomplete means to impede innervation during the protracted perinatal period of taste bud differentiation. Here, by genetically ablating, in mice, all somatic (i.e. touch) or visceral (i.e. taste) neurons for the oral cavity, we show that the latter but not the former are absolutely required for the proper formation of their target organs, the taste buds. Taste buds are onion-shaped clusters of 60–100 taste receptors and support cells, embedded in epidermal papillae and distributed in a punctate pattern in the tongue and soft palate epithelia. They sense nutrients in the oral cavity and transmit taste information to the termini of sensory neurons, through conventional  and non-Neurog1 and Neurog2. As early as day 9.5 of embryonic development (E9.5), the knockout of Neurog1 blocks neuronal differentiation in the trigeminal, superior and jugular ganglia  from, presumably, visceral or somatic sensory fibers, respectively (which thus navigate to their target independently of each other)  of the anterior tongue and soft palate are innervated by somatic sensory neurons (for touch and pain) located in the trigeminal ganglion and visceral sensory neurons (for taste) located in the geniculate ganglion . The cir ganglia  which ha neurons . In eachh other) . In doubthe head , all nerthe head .sonic hedgehog (Shh), after a diffuse phase throughout the oral epithelium, resolves around E12.5 into a punctate pattern corresponding to taste placodes, which also start expressing Prox1 and high levels of Sox2  —  in both wild type and Neurog1/2 KO  — , and theand Hes6  were swiand Hes6 . All thee palate . Expressnockouts . Betweenog1/2 KO . Thus, fog1/2 KO . The sinog1/2 KO . Howeverexplants  and see  stalled , corrobonockouts . A similurog2 KO , which lurog2 KO . Therefosenchyme , also diCK8, spanning the height of the oral epithelium, to different extents in different locations. From this developmental stage on, we will designate these taste bud anlagen, irrespective of their size and degree of maturation, as ‘CK8-positive (CK8+) cell clusters’. In the palate, they almost reach their mature size and structure by E18.5  in the palate  and their cross-sectional area  so that the overall area occupied by CK8+ cells was 80% smaller than in wild types  for the normal pregnancy of C57BL/6 mice). The degree of atrophy was even more pronounced at E20.5 than at E18.5, due to a decrease in the number of CK8+ cell clusters (now 89% fewer than in wild type) and a stagnation of their average size (while their wild type counterparts had enlarged), so that the total area occupied by CK8+ cells in the soft palate was smaller by 96% relative to wild type , we devised a second genetic strategy to prevent visceral innervation of the soft palate. We found that ablating the transcription factor Foxg1, expressed in epibranchial placodes , but for the constitution, maintenance or proliferation of the pool of progenitors required for bud formation, possibly reminiscent of the role of parasympathetic nerves in the organogenesis of salivary glands , a hallmark of taste cell progenitors  (At E20.5 (P2), many palatal taste buds in wild types are fully mature and probably functional . In lineel Kcnq1   , togethes Sox2+) . We coul+ cell clusters of aneural oral epithelia resembled, by their small size yet expression of terminal differentiation markers, those induced by ectopic expression of Shh  or 4% paraformaldehyde (PFA)-perfused pups (at P0 (E18.5) and P2 (E20.5)) were fixed in 4% PFA overnight at 4°C, and the tongue or palate was dissected out. For cryostat sections, tissues were embedded in 7% gelatine/15% sucrose and sectioned at 20 μm. For vibratome sections, tissues were embedded in 3% agarose (in PBS) and cut at 100 μm.Immunohistochemical reactions were done with the Vectastain Elite ABC Kits  and color revealed by DAB .For anti-Pkd2l1 and anti-Sox2 immunoreactions, light fixation was required with 4% PFA at 4°C for 3 hr.Hes6 (obtained from Ryoichiro Kageyama), Ascl1 (obtained from François Guillemot), Shh (obtained from Andrew McMahon) and Sox2 (obtained from Dr. Robin Lovell-Badge).Antisense RNA Probes used were Foxg1Cre (BF-1) KO (Cre in the Foxg1 locus. RRID:MGI:5806112BF-1) KO : a knockNeurog1 KO  solution (Delvosteron: NaCl (0.9%)=1:1) was injected subcutaneously in each pregnant mouse. Pups were surgically delivered on day 20.5 of embryonic development.Proligestone (Delvosteron) was used to prolong the gestation of + cell clusters, one cryosection out of two through the entire soft palate was analyzed in Neurog1 and Neurog2 KO embryos, wild types serving as controls. This raw number was used as an estimate of the number of CK8+ cell clusters in the palate, without multiplying by two and performing the Abercrombie correction. Indeed, all the clusters we counted in the wild type spanned the whole height of the epithelium and/or had a visible pore, de facto excluding objects that would be tangentially sectioned. This amounts to a systematic loss of ‘caps’, an important source of errors in the Abercrombie correction (For the quantification of CK8rrection . Our metrrection  would be+ clusters (if there were less than 10), or up to ten (if there were more) were imaged, for the wild type in 2 to 3 sections around the midline of the soft palate (where taste buds were the densest), for the mutants, in one out of two sections throughout the palate. Confocal imaging was performed on a Leica SP5 microscope or a LSM 880 Airyscan Zeiss microscope. The surface occupied by each CK8+ cluster was automatically outlined and measured with the Fiji software, and the mean cross-sectional area was calculated for each genotype. To calculate the entire surface occupied by CK8+ cell clusters on alternate sections of one half of soft palate, the mean value of the calculated surfaces of CK8+ cell clusters was multiplied by the raw, uncorrected number of CK8+ clusters. Statistical analysis was performed using unpaired two-tailed t-test. Results are expressed as mean ± SD. All graphs were performed with GraphPad Prism software. For the reason that we did not count ‘caps’ of wild type CK8+ clusters (see above), the decrease in total area is likely underestimated.For each genotype, all CK8 In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by Marianne Bronner as the Senior Editor, a Reviewing Editor, and three reviewers. The following individual involved in review of your submission has agreed to reveal their identity: Igor Adameyko (Reviewer #3).Thank you for submitting your article "Taste bud formation depends on taste nerves" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.As you will see, all of the reviewers were impressed with the significance and novelty of your work, but each reviewer also had specific and useful comments for improving the manuscript. Specifically, there are suggestions relate to the quantification of the data (reviewers 1 and 2), the over-simplification of the Title and Abstract (reviewers 2 and 3), and the Discussion section .I am including the three reviews at the end of this letter. I appreciate that the reviewers' comments cover a range of suggestions for improving the manuscript. We trust that most of these can be accommodated in a reasonable period of time. We look forward to receiving your revised manuscript.Reviewer #1:This is a nifty short report on genetic ablation of either somatosensory or visceral sensory neurons and the effects on development of taste buds. The work is generally clear and well-written and offers a modicum of new information on the neural dependency of the sensory end organs for taste.A major obstacle to clarity in this work is the lack of a clear definition of how the authors use the term "Taste buds". As the MS describes, development in this system proceeds from non-innervated placodes through some intermediate stages culminating in a mature taste bud. The terminology used in this work seems inconsistent, sometimes referring to the intermediate stages as "taste anlage", but never clearly defining the term. Does a taste anlagen contain elongate, differentiated taste cells? When does a small collection of elongate taste cells become a "taste bud"? For example, Figure 2 shows elongate differentiated taste cells in Neurog2 KO animals, but the caption suggests that taste buds do not form in this KO line. Since the bar graphs of this figure report on number of taste buds observed, it is essential that the authors define what structure was counted as a taste bud. If in fact the authors are counting as taste buds these small collections of differentiated elongate cells, then they should be correcting for the sampling bias comparing larger objects to smaller objects in histological sections, e.g. by Abercrombie correction. If not, this is essential. Crucial for this calculation is the section thickness and the spacing between sections in relation to the size of the objects being studied. For example, if the section thickness is 15um and the object is 50um in depth, then each object is guaranteed to be counted twice if alternate sections are measured. Conversely, in the same set of sections a 10um object will be counted only once giving the impression that there are twice as many large objects as small objects when in fact, there would be equal numbers.Also missing from this report is any mention of the development of taste bud primordia (placodes?) in vitro - a situation entirely devoid of innervation. This would seem highly relevant to this paper. Relevant references:Mbiene, Maccallum and Mistretta, 1997.Mistretta et al., 2003.Hall, Bell and Finger, 2003.Ozdener et al., 2006.Reviewer #2:Understanding the role of innervation in taste bud development is complicated by the normal occurrence of taste buds in the palate and different parts of the tongue, with taste cells maturing at different rates in each of these locations. Fan et al. exploited their genetic models to dissect the involvement of nerves in taste bud formation at different stages and in distinct locations.Developmental defects in these mouse mutants include the absence of neurons that normally innervate the taste buds, and these unique neuronal knockouts therefore can be used to assess whether nerves are required in taste bud development. The authors showed that at early stages when nerves are just about to establish contacts with their peripheral targets, the circumvallate placode at the posterior of the tongue is unique in its nerve dependence for expression of certain early genes and for taste bud formation. Expression of some of the same genes during development of non-taste palatal placodes or lingual placodes giving rise to taste buds of the fungiform papillae in contrast remains largely unaffected. Nerves thus participate in taste bud formation at specific developmental windows that vary by location. This variation in nerve dependence for formation of distinct taste bud populations raises questions as to the choice of title, which understates the complexity of taste bud dependence on innervation.Sox2 associated with progenitor cells. However, sample numbers and quantification of these observations were not provided in the current manuscript. A revision of the manuscript is recommended before acceptance for publication. Please see below for specific questions and suggestions.It would be helpful if the authors could provide an overview describing taste bud development and maturation at each location and at distinct developmental stages. Using the Neurog2 knockout alone in palatal taste bud development, a previously unappreciated early involvement of nerves in taste bud maintenance was noted. The authors showed morphological defects in the number of K8+ cells and reduced proliferation and reduced expression of 1) The Abstract and Title do not include any reference to the complexity of nerve involvement in formation of taste buds at different locations and developmental stages.2) Does circumvallate atrophy occur in the Neurog2 single knockout, as was noted for the Neurog1/2 double KO?3) Please report the number of mutant animals examined in Figure 1.Sox2Sox2+ progenitor cells and proliferative status as indicated by Ki67+. Additional time points at E16.5 and E17.5 would also be informative.4) One of the most interesting aspects of the paper is mutant effects on progenitor cells, however, more samples are needed to quantify Reviewer #3:This focused and strong study answers an important biological question addressing the importance of innervation in initial development of taste buds and putting the regeneration of taste buds into an ontogeny-like framework. The authors provide a clear and straightforward answer in the form of a short report.I have very few comments:1) I think that the abstract slightly over-exaggerates the real conclusions: not all taste buds depend on the nerve presence in their development.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4197660/2) It should be very briefly discussed why different taste buds demonstrate differential preferences for the specific visceral sensory nerves during their development. Why the embryonic environment is permissive for some buds that are not innervated at all in case of a double Neurog1/2 KO? Can it be connected to the extraordinary high levels of Shh in developing rugi of the palate? This would fit the results described in the paper from Linda Barlow lab: 3) In Figure 2—figure supplement 1: In case of Neurog2 KO, Tuj1+ green fibers are very proximal to the CK8 staining and the location of forming taste bud. Are those nerves the remaining touch afferents? They stay in place in Neurog2 KO, although they do not support the development of fungiform papillae. It will be good if the authors clarify this in a figure legend for pedagogical purposes.4) Can it be that nerve-associated cells (SCPs and Schwann cells) play a key role in signaling to the developing taste buds, and not the neurons, which support them ? Worth discussing in one or two phrases.Overall, this is a good report that requires only some tuning of the text. Reviewer #1:This is a nifty short report on genetic ablation of either somatosensory or visceral sensory neurons and the effects on development of taste buds. The work is generally clear and well-written and offers a modicum of new information on the neural dependency of the sensory end organs for taste.A major obstacle to clarity in this work is the lack of a clear definition of how the authors use the term "Taste buds". As the MS describes, development in this system proceeds from non-innervated placodes through some intermediate stages culminating in a mature taste bud. The terminology used in this work seems inconsistent, sometimes referring to the intermediate stages as "taste anlage", but never clearly defining the term. Does a taste anlagen contain elongate, differentiated taste cells? When does a small collection of elongate taste cells become a "taste bud"?Shh or CK8 spans 3 and 2 of these phases, respectively, and their restriction to future taste bud cells is progressive. As for terminal differentiation markers , the onset of their expression is not precisely known. So, to the question “When does a small collection of elongate taste cells become a "taste bud"?” there is, in our opinion, no rigorous answer.The referee rightly points to the lack of clear-cut transition during taste bud formation. Indeed, we don’t know of any morphological criterion or global gene expression event that sharply demarcates the three canonical stages of taste bud formation: “placode”, “papilla” , and “taste organ” (i.e. a papilla equipped with a mature taste bud). The expression of + cells at E18.5 or later, “CK8+ cells clusters” whether they are mature taste buds, immature taste buds , or taste buds which are atrophic in the mutants, to any extent. This common term reflects the fact that all such formations are not distinguished by dichotomous morphological or gene expression criteria, but distributed on a gradient of sizes (and we quantify the sizes).This said, to homogenize the nomenclature as requested, we now call any group of elongated CK8+ cells between E16.5 and 18.5, before they are clearly elongated or grouped in onion shaped structures, all the more so, since we show that some of these cells  never become taste cells.This leaves undefined the nature of CK8Nevertheless, we have kept the word “taste bud” in the title of the paper and figures and in the Discussion section, because a large majority of CK8+ clusters have vanished in the mutants and the remaining ones are abnormally small, so that, despite the lack of definition of the exact moment where a collection of CK8+ cells qualifies as a “taste bud”, we feel justified in saying that “taste bud formation requires taste nerves” [see responses to criticisms of the title below].For example, Figure 2 shows elongate differentiated taste cells in Neurog2 KO animals, but the caption suggests that taste buds do not form in this KO line. Since the bar graphs of this figure report on number of taste buds observed, it is essential that the authors define what structure was counted as a taste bud.+ cell clusters” in the legend and in the figure. And we counted all CK8+ cell clusters (down to single cells when that occurred in the mutants).See explanation above. We have now replaced “taste buds” by “CK8If in fact the authors are counting as taste buds these small collections of differentiated elongate cells, then they should be correcting for the sampling bias comparing larger objects to smaller objects in histological sections, e.g. by Abercrombie correction. If not, this is essential. Crucial for this calculation is the section thickness and the spacing between sections in relation to the size of the objects being studied. For example, if the section thickness is 15um and the object is 50um in depth, then each object is guaranteed to be counted twice if alternate sections are measured. Conversely, in the same set of sections a 10um object will be counted only once giving the impression that there are twice as many large objects as small objects when in fact, there would be equal numbers.We do not think that the Abercrombie correction, which was devised to count cell nuclei in a volume of tissue, is appropriate for the comparison of very different objects between a wild type and mutant condition, particularly because of the indirect estimate of H (the height of the object perpendicular to the plane of section) and the “lost caps” effect We now make this reasoning explicit in the Material and methods section: in the following way:+ cell clusters in the palate, without multiplying by two and performing the Abercrombie correction. Indeed, all the clusters we counted in the wild type spanned the whole height of the epithelium and/or had a visible pore, de facto excluding objects that would be tangentially sectioned. This amounts to a systematic loss of “caps”, an important source of errors in the Abercrombie correction . Our method makes it next to impossible that clusters (with an average width of 28μm(E18.5) and 38μm (E20.5)) would be counted more than once on alternate 20μm sections. On the other hand, a few clusters were probably lost, if they were centered on one of the uncounted section. The accuracy of our method is verified by the fact that we found the same number of clusters at E20.5 and E18.5 despite the 36% increase in average diameter, and also that we found (not shown) the same number of clusters on alternate 30μm sections (as opposed to 20μm).”“This raw number was used as an estimate of the number of CK8In any case, this raw number is the only one relevant to the calculation of the total volume occupied by CK8+ cells, that we estimate through the proxy of the total surface occupied by CK8+ cells on the sections we count (one 20μm section out of two). The total surface occupied by taste cells is the total number of patches of CK8+ cells seen on sections, multiplied by the average size of the patches. If two patches on two sections happened to correspond to the same “CK8+ cell cluster”, it would be a realistic reflection of the fact that this cluster is big and occupies a large volume . This is biologically the most important figure. If the number of CK8+ clusters was unchanged in the mutants, but their size decreased on average by 98%, the conclusion of our paper, i.e. that “taste bud formation depends on taste nerves” would remain unchanged.Also missing from this report is any mention of the development of taste bud primordia (placodes?) in vitro - a situation entirely devoid of innervation. This would seem highly relevant to this paper. Relevant references:Mbiene, Maccallum and Mistretta, 1997.Mistretta et al., 2003.Hall, Bell and Finger, 2003.Ozdener et al., 2006.We did cite the first paper, historically, to show that fungiform papillae develop in tongue explants. As requested, we have now added the following citation:, Maccallum and Mistretta, 1997 and Hall, Bell and Finger, 2003. And we added Mistretta et al., 2003 on the topic of the CV papilla, making explicit that we find, like her, that Shh is switched on in the CV placode, but leaving implicit the contradiction of her claim that CV papilla formation is nerve independent . On the other hand, it is not easy to see how the taste cell culture system described by Ozdener et al., 2006 informs about nerve dependency in vivo.Reviewer #2:[…] It would be helpful if the authors could provide an overview describing taste bud development and maturation at each location and at distinct developmental stages.The circumvallate placode is unique in its dependency on nerves for subsequent papilla formation, but not for taste bud formation, which are just as dependent as in other papillae. To help conceptually disentangle the two issues, we now show, the absence of taste buds in the neonatal circumvallate organ (as expected since the organ itself does not form) .Using the Neurog2 knockout alone in palatal taste bud development, a previously unappreciated early involvement of nerves in taste bud maintenance was noted. The authors showed morphological defects in the number of K8+ cells and reduced proliferation and reduced expression of Sox2 associated with progenitor cells. However, sample numbers and quantification of these observations were not provided in the current manuscript. A revision of the manuscript is recommended before acceptance for publication.We do not document “maintenance” but appearance.Please see below for specific questions and suggestions.1) The Abstract and Title do not include any reference to the complexity of nerve involvement in formation of taste buds at different locations and developmental stages.The “complexity” (which is rather an asynchrony) is in the development itself, not in the requirement for nerves in that development. The development of taste buds is not synchronous at every place  and their demise at different stages in the absence of nerves simply reflects that.The only true “complexity”, not encapsulated by the Title or the Abstract, is that the CV organ depends on nerves, not only for its resident taste buds but also for the papilla itself. In other words, it depends on the nerves even more than other taste organs. The title does not include this additional feature, but remains perfectly true, concerning taste buds.We think that one virtue of our title (and of the paper), is precisely to simplify a field — which has become replete, over decades, with appearances of complexities and special cases, quantification of partial phenotypes, at different phases, in different locations, etc. — by encapsulating the fact that no complete, mature taste bud forms in the absence of nerves. This most important message would be jeopardized by the addition of what is basically a bonus, the idiosyncrasy of the circumvallate papilla .2) Does circumvallate atrophy occur in the Neurog2 single knockout, as was noted for the Neurog1/2 double KO?Neurog2 KO looks exactly like the double Neurog2/Neurog1 knock out. This is now stated in the text as: “A similar phenotype was obtained in single Neurog2 KO , which lack a petrosal ganglion .As requested, we now show in a new supplementary figure that the 3) Please report the number of mutant animals examined in Figure 1.Two animals were used for each probe. This is now stated in the legend.+ progenitor cells and proliferative status as indicated by Ki67+. Additional time points at E16.5 and E17.5 would also be informative.4) One of the most interesting aspects of the paper is mutant effects on progenitor cells, however, more samples are needed to quantify Sox2Sox2Sox2+ cells and Ki67 cells in mutants and wild type ad E18.5, and add this data to Figure 4. On a pilot experiment (that we do not show), we could not see any difference at E16.5.As requested, we now counted Reviewer #3:This focused and strong study answers an important biological question addressing the importance of innervation in initial development of taste buds and putting the regeneration of taste buds into an ontogeny-like framework. The authors provide a clear and straightforward answer in the form of a short report.I have very few comments:1) I think that the abstract slightly over-exaggerates the real conclusions: not all taste buds depend on the nerve presence in their development.+ cell clusters in the mutants are the same, from animal to animal. We believe that our short and clear abstract reflects the findings that we describe, without exaggeration, and that the field will benefit from that clear message [see response to referee #2]. Nevertheless, we softened the abstract as requested, it now reads “we show that the latter but not the former are absolutely required for the proper formation of their target organs, the taste buds”, leaving room for the formation of very rudimentary, atrophic organs.It is not clear to us what are the taste buds that the referee has in mind that would not depend on nerves. Unless the referee means that our statement would be justified only if the disappearance of taste cells was by 100% as opposed to 96%? Also, we have no indication that the atrophic residual CK82) It should be very briefly discussed why different taste buds demonstrate differential preferences for the specific visceral sensory nerves during their development.papilla shows a specific dependency that other papillae don’t.Taste buds per se don’t demonstrate differential preferences (see response to referee#2). Only the circumvallate https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4197660/Why the embryonic environment is permissive for some buds that are not innervated at all in case of a double Neurog1/2 KO? Can it be connected to the extraordinary high levels of Shh in developing rugi of the palate? This would fit the results described in the paper from Linda Barlow lab: We do not see true “taste buds” (i.e. CK8+ clusters which would not be atrophic) in the mutants. The relationship of the atrophic residual taste buds  to the ectopic ones in the Barlow paper is made explicit in the text. .3) In Figure 2—figure supplement 1: In case of Neurog2 KO, Tuj1+ green fibers are very proximal to the CK8 staining and the location of forming taste bud. Are those nerves the remaining touch afferents? They stay in place in Neurog2 KO, although they do not support the development of fungiform papillae. It will be good if the authors clarify this in a figure legend for pedagogical purposes.Neurog1 and Neurog2 knockout at E16.5, the epithelium of the tongue and of the soft palate retained nerve fascicles at regularly spaced locations (corresponding to the taste organs) from, presumably, visceral or somatic sensory fibers, respectively (which thus navigate to their target independently of each other)”. As requested, we have now added the following sentence in the legend of Figure 1—figure supplement 1: “Since Neurog2 KO have lost the geniculate, but keep the trigeminal ganglion, the residual innervation of taste organs in these mutants correspond to the somatic (touch and pain) fibers”.In the original main text we wrote: “In each single 4) Can it be that nerve-associated cells (SCPs and Schwann cells) play a key role in signaling to the developing taste buds, and not the neurons, which support them ? Worth discussing in one or two phrases.As requested we now mention this possibility in the text as follows: “This specificity [of nerve fiber type] makes less likely the possibility, that we cannot exclude however, that the nerve associated cells (Schwann cell precursors and Schwann cells) have the inducing role, rather than the nerve fibers themselves.”


Public health guidelines suggest that physical activity can be accumulated in multiple short bouts dispersed through the day. A synthesis of the evidence for this approach is lacking.Our objective was to undertake a systematic review and meta-analysis to examine if exercise interventions consisting of a single bout of exercise compared with interventions comprising the same total duration, mode, and intensity of exercise accumulated over the course of the day have different effects on health outcomes in adults.Six electronic databases were searched (Jan 1970–29 August 2018). Two authors identified studies that evaluated the effects of a single bout of exercise compared with the same intensity, total duration, and mode of exercise accumulated in multiple bouts over the course of a day, in community-dwelling adults. Risk of bias was assessed using the Cochrane Collaboration tool. Pooled effects were reported as standardised mean differences (MDs) and 95% confidence intervals (CIs) using a random effects model.I2 = 0%; five studies, 211 participants). In subgroup analyses, accumulating > 150 min of weekly exercise in multiple bouts per day resulted in small effects on body fat percentage  compared with 150 min of exercise amassed via single continuous bouts per day. There was a decrease in low-density lipoprotein (LDL) cholesterol with accumulated versus continuous exercise . No differences were observed for any other blood biomarker .A total of 19 studies involving 1080 participants met the inclusion criteria. There were no differences between accumulated and continuous groups for any cardiorespiratory fitness or blood pressure outcomes. A difference was found in body mass changes from baseline to post-intervention in favour of accumulated exercise compared with continuous .The online version of this article (10.1007/s40279-019-01145-2) contains supplementary material, which is available to authorized users. Globally, approximately one quarter of adults 23.3%) are failing to meet current recommendations for physical activity . Since 13.3% are Acute responses to physical activity have been observed during and in the hours following a single bout of physical activity . Reductin = 7). Furthermore, the majority of included studies relied on self-reported measures of exercise , which may have impacted the reliability of comparisons ; (2) body fatness (e.g. body fat percentage); and (3) cardiovascular risk factors (e.g. blood pressure and blood lipids) measured using standard techniques. Secondary outcomes included psychological/psychosocial parameters , other anthropometric measures (e.g. lean mass and waist-to-hip ratio), and objectively measured physical activity and sedentary behaviour derived from accelerometers. We also recorded adverse events and adherence to exercise programmes.Our primary outcomes included (1) cardiorespiratory fitness , with contrast analysis revealing statistical increases in the control group, but not in the two exercise conditions. Eguchi et al. [p < 0.05) in both accumulated and continuous exercise groups compared with control, but no differences between exercise groups. In the same study, no statistical differences in 2-h insulin were observed between exercise groups or control.Additional blood biomarker outcomes not included in the meta-analysis were as follows: One study  showed mer study  found noer study  observedi et al.  found noi et al.  found stI2 = 27% and 0.68, 95% CI 0.03–1.32, I2 = 0%). However, no statistical differences in vigour subscale scores were found. Only one of these studies included a control group, so accumulated exercise versus control comparisons were not possible. Similarly, both studies prescribed an exercise dose of 150 min (both 5 days of 3 × 10-min bouts), so no subgroup analysis by exercise dose was possible. Only Osei-Tutu and Campagna [In meta-analyses involving only 41 participants in two studies , 37 thatCampagna  reportedp = 0.034) at mid-intervention, and greater improvements in self-efficacy among accumulated bout exercisers compared with control (p = 0.001) at the end of the 12-week intervention. However, the effect on self-efficacy was similar between the two exercise patterns. Murphy et al. [p < 0.05) only. The third trial [d = 0.40 in both cases).Three trials , 33, 38 rd trial  reportedp = 0.046), whereas no differences were found within or between accumulated and continuous exercise bout groups. In the second trial, Asikainen et al. [p = 0.021).Two trials , 38 compn et al.  measuredn et al.  also obsNo statistical differences were found for daily energy intake  or percentage of daily energy intake from fat between accumulated and continuous exercise, accumulated exercise and control, or any subgroup analysis by exercise dose. In one study  that didp = 0.008). In the same trial, however, no between-group differences were observed in the proportion of participants reaching the maximum score on the one-leg standing balance test. Finally, walking time on the UKK 2-km Walk Test increased statistically in both exercise groups when compared with the control group (p < 0.001). Similarly, Shiau et al. [p < 0.05) in maximal strength (via 1 RM bench press), anaerobic performance (via 30-s Wingate test), and blood lactate response to anaerobic exercise (30-s Wingate) after a 12 week accumulated (three bouts of one set of each exercise per session for 3 days/week) or continuous (three sets of each resistance exercise per session for 3 days/week) resistance training intervention, but no statistical between-group differences. DeBusk et al. [Asikainen et al.  reportedu et al.  reportedk et al.  reportedk et al.  reportedThis is the first meta-analysis considering the effects of splitting a continuous bout of exercise into shorter bouts of the same intensity and overall duration dispersed throughout the day. The majority of the studies included (16 of 19) were small (< 30 participants), and therefore, probably did not have sufficient power to detect changes in some outcomes. Pooling the weighted data in this analysis increases the power to detect such changes. The findings suggest that accumulating exercise in short bouts (at least 10 min) over the course of the day produces similar effects on a range of health-related outcomes, including cardiorespiratory fitness, blood pressure, lipids, and glucose metabolism, to performing the same exercise in one continuous bout. This strengthens the evidence base for current physical activity guidelines which suggest that short bouts are equivalent to longer continuous bouts.Within our analysis, there is evidence from a small number of studies that accumulated bouts of exercise produce slightly more favourable changes in body mass and LDL cholesterol than continuous bouts of the same intensity and total duration. The mechanisms underlying potential differences in these effects on body mass have not been well elucidated. It is plausible, however, that the acute increase in metabolic rate induced by exercise  results Accumulated exercise has often been promoted as a more palatable way of meeting physical activity recommendations. This suggestion is intuitively appealing given that time is often cited as a barrier to achieving sufficient daily physical activity . Howevern = 12/19) of the studies comparing the effects of continuous and accumulated exercise. This may reflect both the accessibility of walking in terms of cost, skill requirement and acceptability among participants but it is also likely to be because unlike many other forms of exercise, walking can be easily incorporated into daily life. It is unlikely that someone choosing swimming or another facility-based exercise or one which requires a change of clothing would choose to split a continuous daily bout into multiple shorter bouts. This underscores the utility of walking as a flexible mode of exercise eminently suitable for helping people meet current physical activity guidelines. Of note is the predominance of female participants in the trials included in this study. This contrasts with the trends towards male participation in other exercise trials [Walking was the mode of activity employed in most  definition) . Many ofOur analysis suggests that splitting a continuous bout of exercise into shorter bouts of equivalent total duration dispersed over the course of a day does not alter its potential to evoke physiological effects likely to provide health benefit. Moreover, for weight loss, the fractionalisation of a single exercise bout into multiple bouts spread across the day may provide greater benefit. These findings provide further evidence that bout length, at least when bout duration is greater than 10 min, is not a determinant of the health effects associated with regular exercise.Supplementary material 1 (DOCX 13 kb)Supplementary material 2 (DOCX 18 kb)Supplementary material 3 (DOCX 27 kb)Below is the link to the electronic supplementary material.


Neurospora crassa. Here we provide evidence indicating that czt-1 is allelic to acr-3, a previously described locus that we now found to harbor a point mutation in its coding sequence. This nonsynonymous amino acid substitution in a low complexity region of CZT-1/ACR-3 caused a robust gain-of-function that led to reduced sensitivity to acriflavine and staurosporine, and increased expression of the drug efflux pump abc-3. Thus, accumulating evidence shows that CZT-1 is an important broad regulator of the cellular response to various antifungal compounds that appear to share common molecular targets.Fungal infections have far-reaching implications that range from severe human disease to a panoply of disruptive agricultural and ecological effects, making it imperative to identify and understand the molecular pathways governing the response to antifungal compounds. In this context, CZT-1 (cell death-activated zinc cluster transcription factor) functions as a master regulator of cell death and drug susceptibility in  Human fungal infections have been, until recently, a largely underestimated public health problem with dramatic worldwide ramifications. Superficial skin infections occur in approximately a quarter of the world’s population, and invasive fungal infections, while less incident, are associated with high mortality rates—about 1.5 million people every year ,3. In adNeurospora crassa, are of great importance due to the wide availability of genetic tools, such as targeted gene deletion strain collections and the accumulation of strains obtained over the years by classical forward genetics methodologies [Theoretically, most human fungal infections are relatively easy to treat provided that access to antifungal drugs is assured. However, a restricted number of drug options against fungal infections are available, including polyene (amphotericin B), echinocandin and azole drugs ,5. The sdologies .N. crassa response to staurosporine involves well-defined alterations in the levels of cytosolic calcium, a process mediated by a putative transient receptor potential (TRP) channel at the plasma membrane and regulated by phospholipase C [N. crassa response to staurosporine is largely controlled by the Zn2Cys6 binuclear cluster transcription factor CZT-1 [czt-1 is allelic to acr-3, a locus that had been described in a mutant N. crassa strain obtained decades ago by random mutagenesis and that exhibits increased resistance to the fungal growth inhibitors acriflavine and malachite green [czt-1 results in gain-of-function and is the likely cause for the enhanced tolerance to acriflavine of the acr-3 strain. This report expands the importance of CZT-1 as an important regulator of susceptibility to antifungal compounds.Staurosporine is a natural bacterial alkaloid, protein kinase inhibitor and prototypical anticancer drug that has been customarily used as a cell death inducer in various organisms, including fungi . The N. lipase C , as welllipase C . At the or CZT-1 . Here wete green . We showN. crassa was performed on Vogel’s minimal medium with 2% sucrose (plus 1.5% agar for solid medium). Asexual spores were obtained by growing strains in glass tubes with slanted medium for one week until full sporulation was evident. Mutant strains were obtained from the Fungal Genetics Stock Center [acr-3 mutant was obtained using a routine sequencing methodology [6 cells/mL were grown at 26 °C in liquid VMM for 7 h; RNA was then isolated using the ZR Fungal/Bacterial RNA MicroPrep kit (Zymo Research), used for cDNA preparation using the SuperScript First-Strand Synthesis System kit (Life Technologies) and the relative expression of abc-3 in different strains using actin (NCU04173) as the reference gene was obtained using the 2Ct−ΔΔ calculation method from mixes containing previously described primers [Routine cultivation of k Center . The genhodology . Radial hodology . Stauros primers  and SYBRN. crassa has resulted in a collection of strains displaying morphological and developmental phenotypes that has been complemented with a genetic map of more than 1000 phenotypic markers and hundreds of other features like telomeres, centromeres, nucleolus organizer region, translocations, inversions and duplications [N. crassa mutant FGSC1215 (acr-3 mat-a) was generated by UV light-based random mutagenesis and exhibits increased resistance to acriflavine [acr-3 (acriflavine resistant-3) is one of seven loci in N. crassa associated with altered tolerance to this drug [The application of classical forward genetics methodologies in ications . More reications . The N. iflavine ; acr-3 , FGSC1209 (acr-3 mat-A) or FGSC875 (acr-1 mat-a) [acr-3 mutant was czt-1; czt-1 is also located in the genomic region previously identified to contain the acr-3 locus. In fact, czt-1 and abc-3 are adjacent to each other and CZT-1 is a transcriptional regulator of abc-3 [czt-1 (NCU09974) in FGSC1215 (acr-3) revealed a C>T non-synonymous mutation in its coding sequence  . Thus, wof abc-3 ,13. Targsequence A. Two tr domain” . It willacr-3 mutant grew slightly slower than the wild type  were almost ineffective against both mating type strains of acr-3  [2Cys6 proteins are fungal specific, constitute the largest family of transcription factors in N. crassa [In summary, we report here that the CU05733) . Zn2Cys6. crassa  and have. crassa ,21,23. A. crassa  and acri. crassa  and prot. crassa . Further


Glycine max L.) has been used as a traditional medicine because its seed coat contains various natural phenolic compounds such as anthocyanins. The objective of this study was to reveal the genetic variation in the agricultural traits, phytochemicals, and antioxidant activity of 172 Korean black soybean landraces (KBSLs) and establish a relationship among them. The evaluation of three agricultural traits , six phytochemicals , and four antioxidant activities , 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS), ferric-reducing antioxidant power (FRAP), and the total polyphenol content (TPC) of 172 KBSLs were analyzed in 2012 and 2015. The agricultural traits, phytochemicals, and antioxidant activities of the 172 KBSLs showed wide variation among the accessions and years. In correlation analysis, the agricultural traits and phytochemicals showed positive and negative correlations with phytochemicals and antioxidant activity, respectively. The principal component analyses result indicated that phytochemicals accounted for most of the variability in the KBSLs. In clustering analysis, the 172 KBSLs were classified into four clusters. These results could lead to expanding the knowledge of the agricultural traits, phytochemicals, and antioxidant activity of the KBSLs, which are valuable materials for the development of new soybean varieties.Black soybean ( Glycine max L. Merr.) is widely cultivated and consumed throughout the world as grains, tofu, and soy milk [Soybean  were evaluated. The aim of this study was to reveal the genetic variation in 172 KBSLs to provide information on KBSLs which are valuable as a functional food crop and/or a new dietary ingredient.A total of 172 KBSLs were obtained from the National Agrobiodiversity Center of the Rural Development Administration (RDA), Republic of Korea. Each accession was sown with 20 seeds on May 25, 2012, at Suwon  and on May 27, 2015, at Jeonju . Standard RDA soybean management practices were applied for the cultivation. Days to 50% flowering  and days to maturity  of each KBSL were observed during cultivation, and 100-seed weight (SW) was measured after harvest. The harvested seeds were refrigerated at −20 °C until they were retrieved for analyses.In order to analyze the anthocyanin content in each KBSL, the hand-peeled seed coats (100 mg) of 172 KBSLs were mixed with 15 mL of 1% HCl in 99% MeOH for 24 h at 4 °C in the dark. After centrifugation at 13000 rpm for 10 min, each specimen was filtered through a 0.45 µm syringe filter and analyzed with an Agilent 1260 Infinity HPLC system . The analysis was performed using a Waters XSelect HSS Cyano XP system . The HPLC conditions were as follows: solvent A, 0.1% TFA/H2O; solvent B, 0.1% TFA/CH3CN; gradient, 5% (B) for 0.3 min, 20% (B) for 6.0 min, 95% (B) for 8.0 min, and 5% (B) for 10 min; column temperature, 40 °C; and flow rate, 0.5 mL/min. The filter detector was set at 520 nm .To analyze the isoflavone content in each KBSL, 100 mg of each sample (whole seeds) was added to 2 mL of 80% MeOH and incubated with sonication for 1 h. The sample in each tube was hydrolyzed using 150 µL of 2N NaOH. After mixing for 10 min, the solutions were neutralized with 50 µl of glacial acetic acid. The sample was centrifuged for 5 min at 3000 rpm, and the collected supernatant was then filtered using a 0.45 µm syringe filter prior to analysis with an Agilent 1260 Infinity HPLC system (Agilent Technology). The analysis was performed using a Proshell 120 SB-C15 . The HPLC conditions were as follows: solvent A, 0.1% TFA/H2O; solvent B, 0.1%TFA/CH3CN; gradient, 10% (B) for 0.35 min, 10%–30% (B) in 3.96 min, held at 30% (B) for 0.36 min, and re-equilibrated at 10% (B) for 1.8 min; column temperature, 30 °C; and flow rate, 0.58 mL/min. The filter detector was set at 254 nm .To compare antioxidant activities in 172 KBSLs, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric-reducing antioxidant power (FRAP), and total polyphenol content (TPC) assays were performed using previous methods described by Lee et al. . To anal50 were converted to 1/IC50 before clustering analysis. Integration of the antioxidant capacity results derived from different chemical methods was used to calculate the relative antioxidant capacity index (RACI) [All the data collected from three replicate experiments were expressed as the mean ± standard deviation. Duncan’s multiple-range test and correlation analyses were used to determine the differences between the 172 KBSLs using SPSS Statistics 20 . The DPPH results expressed as ICx (RACI) . Hierarcx (RACI) . PAST3 sx (RACI) . p < 0.001) and experimental years (p < 0.001), whereas the DM was only significantly different between the accessions (p < 0.001) (There were variations in the 172 KBSLs in days to 50% flowering (DF), days to maturity (DM), and 100-seed weight (SW) in 2012 and 2015 . The DF < 0.001) .p < 0.001), the experimental years (p < 0.001), and the interaction between year and accessions (p < 0.001), whereas the C3G content was different between black soybean accessions (p < 0.001) and the interaction between year and accessions (p < 0.001) (The content of three anthocyanins (delphinidin-3-O-b-D-glucoside (D3G), cyanidin-3-O-b-D-glucoside (C3G), and petunidin-3-O-b-D-glucoside (Pt3G)) and isoflavone aglycones  was measured in the 172 KBSLs . Among a< 0.001) .p < 0.001), experimental years (p < 0.001), and the interaction between year and accessions (p < 0.001). In the three isoflavone aglycones, the contents of daidzin, glycitin, and genestin were 14.1 to 108.7, 0.8 to 43.2, and 15.6 to 115.6 mg/100 g dried seeds in 2012 and 4.6 to 81.0, 0.4 to 19.9, and 5.2 to 56.7 mg/100 g dried seeds in 2015, respectively. The three isoflavone contents were significantly different between the black soybean accessions (50) in 2012 and 16.4 to 154.6 (IC50) in 2015, with average values of 90.3 and 59.5 (IC50), respectively, among the accessions evaluated , experimental years (p < 0.001), and the interactions between years and accessions (p < 0.01), respectively, whereas the ABTS activity was significantly different between black soybean accessions (p < 0.01) and the interaction between year and accessions (p < 0.01). In the results of RACI, IT178047 was the highest (2.21), followed by IT274457 (1.64), IT178132 (1.52), and IT177807 with the lowest value (−1.49)  .p < 0.001); D3G and Pt3G, r = 0.267 (p < 0.001); C3G and Pt3G, r = 0.287 (p < 00.01)) and isoflavones ; daidzin and genestin, r = 0.554 (p < 0.001); glycitin and genestin, r = 0.362 (p < 0.0001)). Between the anthocyanins and antioxidants, C3G showed correlations with DPPH , ABTS , TPC , and FRAP . D3G showed correlations with DPPH , ABTS , and FRAP , whereas Pt3G was not corrected with antioxidant activities. Among the isoflavones, only genestin showed a positive correlation with ABTS activities . Among the agricultural traits, the DF and DM showed correlations with anthocyanins, isoflavones, and antioxidant activity, whereas the SW was only correlated with isoflavones. The correlations between agricultural traits, phytochemicals, and antioxidant activities are shown in PCA using the agricultural traits, phytochemicals, and antioxidant activities of 172 KBSLs indicated that five principal components (PCs) with eigenvalues >1 could explain 73.94% of the total variance . The firThe first two PCs are plotted in The 172 KBSL were classified into four clusters according to their agricultural traits, phytochemicals, and antioxidant activities . ClusterIn this study, the agricultural traits, phytochemicals, and antioxidant activity of 172 KBSLs were evaluated to identify their potential as new breeding materials. The results showed that the 172 KBSLs had wide variations in agricultural traits, phytochemicals, and antioxidant activity. Hoisington et al.  suggesteMany methods measuring antioxidant activity have been developed and used to determine the antioxidant capacity of plant extracts because they use relatively standard equipment and can deliver fast and reproducible results . In thisThe DF was positively correlated with anthocyanins, genestin, and antioxidant activities. Phommalath et al.  reportedThe black soybeans exhibited high levels of antioxidant activity because of their high concentrations of phenolic compounds . In addiGenetic variability is important to the success of a breeding program . Therefo


Staphylococcus aureus (SA) based on specific aptamer and catalysis of dsDNA-SYBR Green I (SG I) complex. SA specific aptamer was immobilized on a 96-well plate by hybridization with the capture probe anchored on the plate surface through streptavidin-biotin binding. In presence of SA, the aptamer was dissociated from the capture probe-aptamer duplex due to the stronger interaction between the aptamer and SA. The consequent single-strand capture probe could be hybridized with a three-way junction (TWJ) probe. With the presence of SG I, the dsDNA-SG I complex catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) under photo-irradiation, producing sensitive photo-catalyzed colorimetric response to SA. Under the optimal conditions, the proposed method could directly detect SA with the limit of detection (LOD) at 81 CFU mL−1 in PBS buffer in 5.5 hours, which demonstrated the sensitive and fast quantification of target pathogenic bacteria. The method showed weak colorimetric signal to Escherichia coli and Pseudomonas aeruginosa, indicating the high specificity for SA. In addition, the method can simultaneously detect 96 samples which can be used for high throughput analysis. The designed method may become a powerful tool for pathogenic microorganisms screening in clinical diagnostics, food safety and environmental monitoring.To develop a high throughput colorimetric biosensor for detection of  Staphylococcus aureus (SA) is the most common pathogen that causes a wide range of human infections. It is the major cause of bacteremia, infective endocarditis as well as skin and soft tissue infection and device-related infections1. Rapid identification of SA in the early stages of infection is important for reducing high mortality. However, the conventional culture method, known as “gold standard” for bacterial detection, usually requires 3–5 days incubation. It also needs at least 12 hours of growth on solid media to complete the identification2. Time consuming and insensitive are common problems with these methods.3, polymerase chain reaction (PCR)4, surface plasmon resonance biosensor5, electrochemical biosensor6 and so on. Despite improvements, these methods still require sophisticated equipment, complex sample preparation and long-term blood culture, which limit their use in clinical applications7. Besides, false positive results often occur in PCR detection8. Therefore, the development of a new platform that can distinguish SA in a short time is highly desired.Various methods which can shorten the detection time and increase the sensitivity have been used for bacterial detection, including enzyme-linked immunosorbent assay (ELISA)et al.2 has strong binding affinity to the bacteria. The TWJ DNA nanostructure used in the assay for signal amplification is another high light of the study. In recent years, as an enzyme-free amplification method, more and more attention has been paid to the self-assembly of nanostructures, which are molecules spontaneously combine into stable, well-defined aggregates under equilibrium conditions9. Among them, TWJ composed of three complementary oligonucleotide branches has become an extremely important building block for constructing DNA structure and dynamic assembly. Meanwhile, the TWJ strategy has obvious advantages, including simple probe design, economical biosensor manufacturing and excellent signal amplification. Therefore, it opens broad prospects for applications in biosensing and bioanalysis11.In this study, an aptamer-based high throughput colorimetric biosensor for detection of SA was devised. The SA specific aptamer, reported by Y.S. Xu 12. Compared with G-quadruplex-based DNAzyme14, peroxidase mimicking nanozyme15, and HRP17, which also have similar catalytic activity for TMB oxidation, the dsDNA-SG I proposed here offers several distinct advantages, such as simple and universal, label-free, visual sensing, suitable for convenient biosensor designs, etc.X. F. Zhang etal reported a new discovery that the dsDNA-SG I complex possessed photocatalytic activity, which could catalyze the oxidation of oxidase substrates TMB with dissolved oxygen under photo-irradiationIn this study, for the first time, we designed an aptamer-based photo-irradiation colorimetric biosensor for detection of SA.By using this method, a new platform which can distinguish SA in a short time with selectivity and sensitivity has been achieved. The TWJ DNA nanostructure exhibited excellent signal amplification effect through self-assembly, and the dsDNA-SG I complex possessed photocatalytic activity, which made the method more specific and simpler. In addition, this analysis is a high throughput analysis which can be used to detect 96 samples at once. Compared with other reported methods, our biosensor has such advantages, include fast speed and more sensitive and specific. So, it may be a powerful tool for SA screening in clinical diagnostics.DNA oligonucleotides were synthesized and purified by Sangon Inc. . Their sequences are listed in Table −1. The concentration was estimated by calculating the average number of CFU.The strains of SA were cultured with Luria-Bertani medium at 37 °C for 24 hours, and then we diluted the SA suspension using 0.1 M PBS buffer to obtain an appropriate optical density value of about 0.5 at 600 nm. After that, the bacterial solution was serially diluted and inoculated onto the solid medium for 24 hours at 37 °C to quantify the CFU mL2HPO4, 50 mM citric acid, pH 5.0) to the final concentration 5.0 µg mL−1. Then add 200 µL of the coating solution to each well. The plates were closed in a humidified box and incubated at 35 °C overnight.Streptavidin was diluted in the coating buffer  were added to combine with the capture probe. After incubated at 37 °C for 90 minutes, 30 μL SYBR Green I (1:100) was embed in the dsDNA for 30 minutes, and then washed with PBS buffer. At last, 50 μL TMB (200 mg/L) and 20 μL 10% HAn overview of the high throughput colorimetric biosensor for detection of SA based on TWJ DNA nanostructure and the catalysis of dsDNA-SG was illustrated in Fig. 10.Compared to a single oligonucleotide branch, the TWJ nanostructure composed of three mutually complementary oligonucleotide branches  can carry more SG I. Then the dsDNA-SG I complex catalyze oxidation of TMB under LED photo-irradiation. The catalytic color will further increase and the sensitivity will be improved. Meanwhile, the TWJ strategy also simplified probe design and biosensor fabrication, as well as excellent signal amplification. Thus, it opens a promising avenue for applications in biosensing and bioanalysis4 CFU mL−1. With the increasing incubation time, the absorbance increased and tended to a steady value at 120 minutes. Prolonged incubation time did not increase the signal response obviously. So, 120 minutes was identified as the optimal incubation time. The effect of TWJ hybridization time for capture probe and P1/P2/P3 probe was also studied in the time range from 30 to 120 minutes.  and Pseudomonas aeruginosa (PA) were tested under the same experimental conditions as those for SA. The concentrations of the test bacteria were 104 CFU mL−1. To further prove the identification ability of this method, mixtures of microorganisms  were also tested in the meantime. Each bacterium and the mixtures of microorganisms were tested for three times. The results were shown in Fig. E. coli) were only as low as the blank, but the SA and microorganisms’ mixtures had obvious absorbance responses, indicating that the colorimetric biosensor had a good specificity and identification ability for the detection of SA.To prove the specificity of the biosensor, two bacterias, 7 CFU mL−1. Each concentration was tested for three times. The absorbance responses of the biosensor to different SA concentrations were shown in Fig. −1 in real milk samples. In addition, the method can complete the detection in 5.5 hours. Compared with traditional culture methods (3–5 days), this method is simple, fast, with higher sensitivity and specificity, showing the potential as a pragmatic tool for SA detection in real samples.In order to prove the proposed colorimetric biosensor can detect SA in actual samples sensitively and specifically, the cultured SA were inoculated into milk at a concentration of 0 to 10Staphylococcus aureus has been successfully developed. The biosensor shows wide linear range, low detection limit, high specificity, and could be used for detection of SA in real samples. Importantly, the application of TWJ DNA nanostructure and photo catalytic activity of dsDNA–SG I complex not only amplifying the detection signal, but also shortening the detection time. The method provided a direct sensing platform for detection of SA and the whole analytical process can be finished in 5.5 hours. The bioassay strategy could be used to develop other assay method for pathogenic bacteria and would become a powerful tool for pathogenic microorganism screening in clinical diagnostics, food safety, biothreat detection and environmental monitoring.Aptamer-based high throughput colorimetric biosensor for direct detection of


Correction to: Implement Sci Commun (2021) 2:6https://doi.org/10.1186/s43058-020-00105-6Following publication of the original article , the autThis affiliation has been added to Rinad S. Beidas in the author list and the original article  has been


Thus, a variety of mechanisms have been proposed for the selective catalytic oxidation of various hydrocarbons including light alkanes, olefins, and simple aromatics by the biological metalloproteins and their biomimetics either in their homogeneous or heterogeneous platforms. Most studies involving these metalloproteins are Fe or Cu monooxygenases. The pathways carried out by these metalloenzymes in the oxidation of C–H bonds invoke either radical reaction mechanisms including Fenton's chemistry and hydrogen atom transfer followed by radical rebound reaction mechanism or electrophilic oxygenation/O-atom transfer by metal-oxygen species. In this review, we discuss the metal oxide nano-catalysts obtained from metal salts/molecular precursors  that can easily form in situ through the oxidation of substrates using H2O2(aq) in CH3CN, and be facilely separated from the reaction mixtures as well as recycled for several times with comparable catalytic efficiency for the highly selective conversion from hydrocarbons including aromatics to oxygenates. The mechanistic insights revealed from the oxy-functionalization of simple aromatics mediated by the novel biomimetic metal oxide materials can pave the way toward developing facile, cost-effective, and highly efficient nano-catalysts for the selective partial oxidation of simple aromatics.The process of selective oxy-functionalization of hydrocarbons using peroxide, O To further examine the production of benzyl hydroperoxide, the treatment of PPh3 can further assist for its quantification . However, with slow addition of H2O2(aq), ~45% sp3 C–H bond oxidation products were observed, and the ratios for benzyl hydroperoxide, benzaldehyde, and benzyl alcohol were identified to be 18, 25, and 2%, respectively .Molecular iron complexes including polycyclic cage-like compounds, biomimetic model complexes, and encapsulated within zeolite cages have been shown with high catalytic activity in the oxidation of hydrocarbons including alkanes and aromatics   and rela(2O2(aq) . These p(2O2(aq) , and ele(2O2(aq)  study. Iectively . Neverth2H0, 1]toluene for its oxidation catalyzed by Fe(ClO4)2 catalyst using H2O2 in CH3CN exhibited high NIH-shift ratios of 83–86%  in CH3CN  using Cu(ClO4)2 , and better selectivity for p-BQ (77%), than other volumes of water  using two structural models of copper oxide and trinuclear copper cluster model (Cu2.8O4.6), respectively, can both give reasonable fits of Rfit = 0.027 and 0.15%, respectively -peroxo)Cu2+ (Cu–Cu distance ~3.6 Å) that can potentially reach an equilibrium of a Cu3+(μ-O)2Cu3+ structure 2Cu3+ can exhibit much lower activation energy for a facile oxo-transfer toward the inert C–H bond  C–H bonds of toluene via the formation of arene oxide intermediate followed by an NIH rearrangement process.The EPR spectrum of copper oxide nanoparticles at 298 K indicated an isotropic signal, reminiscent of a previous study of pMMO with trinuclear copper cluster feature , and acetic acid were used as co-catalysts to prompt the oxidation reactions. We recently reported a unique vanadium nanorod (Vnr) catalyst  assists in accumulating more redox active centers ) and commercial V2O5  as an oxidant displayed 68% 18O-enriched PhOH; however, there was negligible enrichment using 2 and 18O2. The results implicate H2O2(aq) as the essential oxidant for OAT mediated by the vanadium oxygenated species.ectively . In addi2 adsorption–desorption isotherms and XRD studies reveal the porous structure and crystalline feature of these catalysts that formed from the core metal oxide with the organic linker as a framework. These three catalysts similarly possessed mesoporous structure in a pore size range of 1.5–30 nm and BET surface area of each was in the range of 20–30 m2/g that can be essential for catalytic activation of the small aromatic compounds. The mesopore structure of the catalysts has a type of slit-like pore formed by the effect of substrate (benzene/toluene) acting as template while preparing in situ  with additional H2O proportions in CH3CN  orientation that can be crucial for the related additional activities  to the Fe/Cu perchlorate and VCl3 in CH3CN for the selective oxidation of simple aromatics  or benzyl alcohol/benzaldehyde (sp3 C–H bond oxidation). This indicates that, in the presence of organic residues including CH3CN, benzene, and/or toluene, the oxidant of H2O2(aq) can not only accumulate HO·/HOO· but is also essential for the self-assembly of the heterogeneous metal-oxide hybrid catalyst formation with active metal oxygenated species.Overall, the study provides an efficient strategy that accumulates active Fe, Cu, and V oxide species of organic–inorganic hybrid nano-catalysts, respectively, through the addition of 35% H3CN is a polar solvent that can be miscible with polar H2O2 and H2O as well as the non-polar aromatic substrates, leading to oxygenate formation. When the metal salts dissolved in the reaction mixtures of aromatics, the free Cu/Fe/V ions presumably coordinate with CH3CN serving as a ligand to form metal complexes  in CH3CN without any additional substrates  can occur and are essential to assist the metal oxide polymerization/formation.CH2O2(aq) is the cause of further oxidation of initially formed phenols and the appearance of tar  in CH3CN catalyzed by the metal oxide nano-catalysts can give rise to reactivity from the specific FeIV = O species on poly-iron oxo clusters (Enami et al., 4+/V5+ redox sites stabilized by PCA (Wanna et al., via the arene oxide intermediates (Guroff et al., In addition, it has already been known that catalytic aromatic hydroxylation with H2O2/O2, in a heterogeneous platform, can drive selective oxidation of simple aromatics such as benzene and toluene. Furthermore, these unique catalysts effectively tuned sp2vs. sp3 C–H bond hydroxylation of toluene and achieved selective double oxidation of benzene to p-BQ. In many cases, the reaction mechanism proceeds significantly through OAT process via the formation of the arene oxide intermediate, although the free radicals that initiated oxidation of C–H bond also participate. These nano-catalysts prepared in situ and composed of organic–inorganic hybrid exhibited recyclable properties, controlled selectivity, higher activity, and were greener than their bulk oxide counterparts, which make them potentially promising for large-scale application.In summary, highly dispersed active Fe, Cu, and V nano-catalysts with HWW, DJ, NT, and RR conducted the research of catalytic oxidative conversion of simple aromatics. WW, DJ, NT, RR, and Y-FT carried out the identification and characterization of the metal oxide materials. SY organized the research. WW, DJ, NT, and SY wrote the paper. 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.


In terms of the absolute number, the SWRO feed water after DAF–UF supports 1.5 × 106 cells/mL, which is 1.25 times higher than after DMF–CF. This corresponds to the higher cleaning-in-place (CIP) frequency of SWRO with DAF–UF compared to DMF–CF as pre-treatment, indicating that the BGP method has an added value in monitoring the biofouling potential in SWRO systems.Measuring the bacterial growth potential of seawater reverse osmosis (SWRO) feed water is an issue that is receiving growing attention. This study developed and demonstrated the applicability of the flow-cytometry (FCM)-based bacterial growth potential (BGP) method to assess the biofouling potential in SWRO systems using natural microbial consortium. This method is relatively fast (2–3 days) compared to conventional bioassays. The effect of the potential introduction of nutrients during measurement has been studied thoroughly to achieve the lowest measure value of about 45,000 cells/mL, which is equivalent to about (10 µg-C glucose/L). The BGP method was applied in two full-scale SWRO plants that included (i) dissolved air flotation (DAF) and ultra-filtration (UF); (ii) dual-media filtration (DMF) and cartridge filter (CF), which were compared with the cleaning frequency of the plants. A significant reduction (54%) in BGP was observed through DAF–UF as pre-treatment (with 0.5 mg Fe Biofouling in seawater reverse osmosis (SWRO) systems remains the major challenge for its cost-effective operation ,2,3,4,5.Pseudomonas fluorescens Pl 7 bacteria for a period of 14 days [Spirillum sp. NOX together with Pl 7 [(Flavobacteriumjohnsoniaestrain A3) was introduced to utilize polysaccharides and proteins [A promising online detection of biofouling was attempted using a membrane fouling simulator (MFS)  and biof 14 days  and lateith Pl 7 ,12. Lateproteins , but stiproteins . Ross etproteins  showed tVibrio fischeri and Vibrio harveyi, respectively. These methods are very fast—1 h for the Weinrich method and 1–3 days for the Jeong method. The use of a single bacterial strain allows normalization of the yield based on a carbon source, enabling conversion of bacterial growth to a carbon concentration. However, a challenge of using the single strain is it may not reflect broader substrate utilization. These methods also cannot use natural bacteria as not all indigenous bacteria have the property of bioluminescence. Therefore, it is of great importance to develop a BGP method using natural consortium bacteria, which provide reliable information regarding biofouling potential in SWRO systems. In seawater, Weinrich et al.  and Jeondirect method and ATP filtration method are 0.3 ng-ATP/L, 0.06 ng-ATP/L, respectively. ® Green I  [Dixon et al.  and QuekMO, USA) ,25.® Green I and Propidium Iodide (PI). The dye PI penetrates bacterial cells with disrupted membranes while SYBR® Green I can bind the nucleic acid of both live and dead bacterial cells [7 cells/mL) and, if exceeded, the sample needs to be diluted. Moreover, the effect of salinity on bacterial count using FCM needs to be studied, otherwise the result of bacterial shock while diluting the samples may underestimate the BGP results. FCM can distinguished the live and dead bacterial cells by staining with SYBRal cells . Recentlal cells  and Farhal cells  publisheal cells ,27. MoreVerify the reproducibility and effect of salinity while enumerating marine bacterial cells using FCM;Verify the effect of the introduction of nutrients that might originate from chemicals and/or bottles during BGP method;Develop a calibration curve and the LOD of the measurement using both artificial and natural seawater using glucose as substrate;Measure bacterial growth potential along the pre-treatment train of an SWRO desalination plant using an indigenous bacterial consortium.The objective of this article is to further develop and demonstrate the applicability of using the FCM-based BGP method to assess the biofouling potential in the pre-treatment and feed of SWRO systems using natural consortium bacteria. The following aspects have been investigated and are described in this article: All the glassware/vials/caps were first washed with lab detergent , rinsed three times with Milli-Q water, soaked overnight in 0.2 M HCl solution and again rinsed with Milli-Q water, and were air-dried. Finally, all the glassware/vials were heated in a muffle furnace at 550 °C for 6 h to remove all traces of organic material while the caps were bathed in 100 g/L sodium persulfate solution at 60 °C for 1 h and then rinsed with Milli-Q water and air-dried.2CO3, NaHCO3, CaCl2.2H2O, KCl, Na2SO4, MgCl2.6H2O, NaCl) in Milli-Q water  of collected seawater from the North Sea  was added to the vial containing 20 mL of sample. Samples were then incubated at a temperature of 30 °C [0) from the maximum bacterial count (Nmax) during the incubation period. The net bacterial growth was considered as an indicator for BGP. The protocol involves filtration of a sample (V > 60 mL) through 0.22 µm PVDF filters to remove large particles and bacteria from seawater samples. Before the sample filtration, a 0.22 µm filter was flushed with Milli-Q water to remove the released carbon from the filter. The choice of the 0.22 µm filtration approach for the BGP test was based on the comparative study made with different approaches. The 0.22 µm filtered seawater sample (20 mL) was transferred into clean vials (in triplicate). A volume of 200 µL  using artificial seawater . The ASWThe possible contamination that might originate from the bottles and chemicals used during the preparation of blank  and its contribution to the BGP measurement was demonstrated considering the following three scenarios. For each scenario, the BGP was measured and compared. Scenario 1: ASW  was prepared using salts NaCl, MgCl2.6H2O, Na2SO4, CaCl2.2H2O, KCl, NaHCO3, Na2CO3 and tested for the condition below to foresee the effect of bottle and chemical contamination on BGP.-No heating of bottle and chemicals;-Heating of bottle  and no heating of chemical;-Heating of bottle and chemical (NaCl only) at 550 °C, 6 h.Scenario 2: ASW  was prepared using salts, NaCl only, to minimize the effect of chemical contamination from other salts. In this case, both the bottle and NaCl were heated at 550 °C, 6 h.Scenario 3: ASW  was prepared using salts, NaCl and NaHCO3. The chemical NaHCO3 was added to maintain the buffer capacity of the ASW. In this case, both the bottle and NaCl were heated at 550 °C, 6 h.In all cases, heating of NaCl was only considered due to its higher melting point > 550 °C. To demonstrate the effect of salinity while enumerating seawater bacteria using FCM, the ASW  and 100 µg/L of P (NaH2PO4). The BGP was measured using the protocol as described in The BGP method was calibrated using glucose as substrate in both ASW and natural seawater samples. A glucose concentration that ranged from 0 to 2000 µg-C glucose/L was added in both water samples. All the samples were then spiked with a fixed concentration of 500 µg /L of The BGP method was applied to monitor the bacterial growth potential along the treatment process trains of full-scale desalination plants located in the Middle East. The raw seawater of both of the SWRO plants comes from open intakes and has similar characteristics to raw seawater properties. Some basic water quality parameters were salinity (69–71 mS/cm), TDS (~50 g/L), turbidity (4–10 NTU), water temperature (22–30 °C).2SO4 in both plants. The coagulant (FeCl3) was continuously dosed in both plants at a concentration of 0.5 ppm of FeCl3 before DAF and 0.8 ppm of FeCl3 before DMF. The de-chlorination was performed before the SWRO unit by dosing Na2S2O5 (sodium metabisulfite). The dosing pump for Na2S2O5 was controlled based on the oxidation-reduction potential (ORP) value, which was set to a level of 250 mV. The general scheme of the plants included: (i) dissolved air flotation/ultrafiltration/reverse osmosis (DAF–UF–RO) and (ii) dual media filtration/cartridge filter/reverse osmosis (DMF–CF–RO) as shown in Brief specifications and operating conditions of the two plants are presented in As illustrated in 2 = 0.99) between the percentage of seawater and live bacterial cell concentrations was observed, with a percentage deviation that ranged from 0.6% to 9.1%. Furthermore, the effect of salinity when seawater bacteria were stained with fluorescence staining dye SYBR® Green I (SG) and Propidium Iodide (PI) and enumerated using FCM was performed. As illustrated in 2.6H2O, Na2SO4, CaCl2.2H2O, KCl, NaHCO3, and Na2CO3.3, where NaCl and bottle were heated at 550 °C for 6 h) showed the live net bacterial growth approximately to the level of 43,000 ± 12,000 cells/mL, which is approximately 92% lower than in sample A. While compared to the net bacterial growth in sample C and D, we observed no substantial difference despite the fact that in sample D no chemicals that constitute calcium and magnesium were added. Previous studies suggested that potassium, magnesium, and calcium are also essential elements required for the growth of marine bacteria [3, where chemical (NaCl) and bottle were heated at 550 °C for 6 h, substantially reduced the effect of nutrients that originate from chemicals and bottles during BGP measurement.The consortium of bacteria proliferated to approximately 600,000 ± 65,000 cells/mL in sample A . The higher growth in this sample was attributed to the introduction of nutrients originated from chemicals and bottles used during the preparation of ASW samples. To prove this, another test was performed with sample B , which revealed that the live net bacterial growth was approximately 16% lower compared to that measured in sample A. Furthermore, a test performed with sample C  showed substantial reduction (90%) in live net bacterial growth. The result illustrates that heating of the major salt (NaCl) and bottle in a muffle furnace at 550 °C for 6 h substantially reduced the effect of nutrients originated from bottle and chemicals added to prepare ASW on BGP measurements. As shown in bacteria ,31. As wglucose/L is as shown in 2 > 0.95 for substrate concentration ranging from 0 to 2000 µg-Cglucose/L in both ASW and natural seawater. While for lower range (0–100 µg-Cglucose/L), R2 = 0.88 was observed  and Spirillum sp. NOX (1.2 × 107 CFU/µg acetate-C) [The result of BGP calibration performed with ASW and natural seawater and fortified with 0–2000 µg-Cobserved . The speetate-C) . glucose/L. The measured LMV for the BGP method was slightly lower than 10 µg-C acetate/L, a threshold value beyond which the biofouling is expected in freshwater [glucose/L. The higher measured ECC could be attributed to the occurrence of algal blooms in the seawater where the sample was collected. Using the calculated specific bacterial yield and the net bacterial growth, the equivalent carbon concentration (ECC) was calculated according to Equation (1) in both ASW and natural seawater . AccordiBiofouling potential was measured and compared over the treatment process trains of two desalination plants, which included DAF–UF–RO and DMF–CF–RO using the FCM-based BGP method. As illustrated in 3 coagulant and followed by ultrafiltration. Moreover, in terms of an absolute number of bacterial growth, the SWRO feed water after DAF and UF supports 1.5 × 106 cells/mL, which is 1.25 times higher than in SWRO feed water of DMF–CF, as illustrated in Furthermore, the pre-treatment option DAF–UF showed a reduction of 54% in net bacterial growth, while it was only 40% by DMF–CF. This could be due to the higher removal of the biodegradable organic matter in dissolved air flotation operated with 0.5 mg/L of FeClNevertheless, other water quality parameters measured for SWRO feed water of the DAF–UF–RO and DMF–CF–RO schemes during the time of the study showed very low fouling potential, as shown in As illustrated in -An FCM-based BGP method for seawater using natural microbial consortium as inoculum was developed and applied in full-scale SWRO plants. The developed method was relatively fast (2–3 days) to monitor the biofouling potential of pre-treatment and SWRO feed water.-The percentage deviation on the reproducibility of the FCM measurement was below 10% and the variation in the FCM-based BGP method was approximately <5% and <20% when the method was applied for ASW and natural seawater, respectively.-3, where bottles and NaCl were heated at 550 °C for 6 h. With this approach, the lowest measured value of the FCM-based BGP method was approximately 10 µg-Cglucose/L.The effect of nutrients on the BGP method that originated from the bottle and chemicals was substantially reduced by 92% when blank (ASW) was prepared using NaCl and NaHCO-2 ~ 0.9) between carbon concentration (0–2000 µg-Cglucose/L) and live net bacterial growth, in both artificial and natural seawater.The FCM-based BGP method showed good linear correlation (R-3+/L), while it was 40% with DMF–CF (with 0.8 mg Fe3+/L).The method was applied to measure the bacterial growth potential through pre-treatment trains of two SWRO desalination plants in the Middle East. A significant reduction (54%) in bacterial growth potential was noticed through DAF–UF as pre-treatment (with 0.5 mg Fe-The absolute number of bacterial growth supported by the SWRO feed water after DAF–UF was approximately 1.25 times higher than SWRO feed water after DMF–CF. This corresponds to the higher CIP frequency of SWRO with DAF–UF as pre-treatment, suggesting that the FCM-based BGP method is a promising tool for measuring the biofouling potential in SWRO feed water. However, more experiments are required to develop a sound relationship between the BGP and the pressure drop increase in SWRO plants.