Microelements are essential for the function of the innate immune system. A deficiency in zinc or copper results in an increased susceptibility to bacterial infections. Mycobacterium marinum, a close relative of Mycobacterium tuberculosis (Mtb), and its hosts. We use the professional phagocyte Dictyostelium discoideum to investigate the role of Zn2+ during M. marinum infection. We show that M. marinum senses toxic levels of Zn2+ and responds by upregulating one of its isoforms of the Zn2+ efflux transporter CtpC. Deletion of ctpC (MMAR_1271) leads to growth inhibition in broth supplemented with Zn2+ as well as reduced intracellular growth. Both phenotypes were fully rescued by constitutive ectopic expression of the Mtb CtpC orthologue demonstrating that MMAR_1271 is the functional CtpC Zn2+ efflux transporter in M. marinum. Infection leads to the accumulation of Zn2+ inside the Mycobacterium-containing vacuole (MCV), achieved by the induction and recruitment of the D. discoideum Zn2+ efflux pumps ZntA and ZntB. In cells lacking ZntA, there is further attenuation of M. marinum growth, presumably due to a compensatory efflux of Zn2+ into the MCV, carried out by ZntB, the main Zn2+ transporter in endosomes and phagosomes. Counterintuitively, bacterial growth is also impaired in zntB KO cells, in which MCVs appear to accumulate less Zn2+ than in wild-type cells, suggesting restriction by other Zn2+-mediated mechanisms. Absence of CtpC further epistatically attenuates the intracellular proliferation of M. marinum in zntA and zntB KO cells, confirming that mycobacteria face noxious levels of Zn2+.Macrophages use diverse strategies to restrict intracellular pathogens, including either depriving the bacteria of (micro)nutrients such as transition metals or intoxicating them via metal accumulation. Little is known about the chemical warfare between  Mycobacterium marinum is a pathogen that causes a tuberculosis-like infection in fish and amphibians, in which it generates granulomas very similar to those caused in humans by its close relative Mycobacterium tuberculosis (Mtb). M. marinum also opportunistically infects humans, but these infections are limited to the skin and extremities due to their lower temperature . Like Mtb, M. marinum was thought to be a vacuolar pathogen. However, accumulating evidence indicates that both bacteria also reside and proliferate in the cytosol, where access to nutrients is not limited but bacteria are exposed to cytosolic defenses  13M. mariM. marinum infection has been shown to induce inducible nitric oxide synthase (iNOS) gene expression in fish macrophages (M. marinum is targeted for digestion by the autophagy machinery of murine macrophages (D. discoideum (M. marinum avoids phagosome maturation by (i) modulating the composition of its cell wall , (ii) iM. marinum and its hosts, and especially about the manipulation of transition metals. In the case of Mtb, immune cells deprive the bacteria of essential nutrients such as iron and manganese, while they intoxicate the mycobacteria by accumulating copper and zinc inside the MCV  are altered in fish granulomas compared to resting macrophages  and 11 Zn2+ influx transporters of the ZIP family (Zpls). ZntA locates to the contractile vacuole (CV), an organelle that regulates the osmotic and metal balances in the cell, and ZntB to organelles of the endosomal pathway  the CV network, (ii) zincosomes , and (iii) phagosomes containing beads and nonpathogenic bacteria such as Escherichia coli or Mycobacterium smegmatis  appeared devoid of Zn2+, likely because it leaked out of either subtly or visibly broken MCVs  and CtpC-like (MMAR_2147). Inspection of our parallel RNA-sequencing (RNA-Seq) results of M. marinum and D. discoideum during infection  compared to M. marinum grown in broth . Colors indicate the differential expression of P-type ATPases (in logFC) in infected cells compared to bacteria that were grown in 7H9 medium: from dark red (highest expression) to dark blue (lowest expression). Download FIG S2, PDF file, 0.2 MB.Differential expression of Copyright © 2021 Hanna et al.2021Hanna et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01313-20.8TABLE S1M. marinum P-type ATPases (A), D. discoideum CV genes (B), D. discoideumzntA to zntD transporters (C), and D. discoideumzplA to zplG transporters (D) during infection. D. discoideum wt was either infected with GFP-expressing M. marinum wt or mock infected. Samples were collected at different times postinfection  and sorted for enrichment in infected cells . Shown are the logarithmic fold changes (LogFC) in expression between infected and mock-infected samples (for D. discoideum genes) or between intracellular M. marinum and bacteria grown in broth (for M. marinum genes). Data extracted from reference Table S1, DOCX file, 0.03 MB.Differential expression of Copyright © 2021 Hanna et al.2021Hanna et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the ctpC is influenced by Zn2+. To this end, the expression levels of selected P-type ATPases were determined by qRT-PCR analysis. Supplementing the growth medium with increasing concentration of ZnSO4 strongly enhanced the transcription of ctpC but not ctpC-like, ctpV, or ctpV-like  Growth of GFP- or E2-Crimson-expressing M. marinum wt, ΔctpC (GFP expressing), and ΔctpC::ctpC (E2-Crimson expressing) was assessed in broth by measuring the increase of fluorescence. Shown is the fold increase in bacterial fluorescence over time for two independent biological replicates. Error bars indicate the SD from eight technical replicates. Statistical differences of pairwise comparisons were calculated with a Fisher LSD post hoc test after two-way ANOVA . (E to G) GFP-expressing M. marinum wt and ΔctpC were plated on 7H11 or 7H11 supplemented with increasing concentrations of ZnSO4 (E), MnCl2 (F), or CuSO4 (G)  as described in Materials and Methods. Shown are the bacterial colonies obtained in one representative of three independent biological replicates, after 7 days of incubation at 32°C. Download FIG S3, PDF file, 0.9 MB.High concentrations of ZnCopyright © 2021 Hanna et al.2021Hanna et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the D. discoideum Zn2+ efflux transporters ZntA, ZntB, ZntC, and ZntD . In. InD. dimethanol  suggestsmethanol  38)..D. disco10.1128/mBio.01313-20.4FIG S4M. marinum (shown in cyan). MCVs were visualized by staining for p80 (green). Arrows point to ZntC/D-negative MCVs. Scale bars, 5 μm. (C to E) Live imaging of ZntA-mCherry-, Rab11a-RFP-, and Rab11c-RFP-expressing cells (shown in magenta) infected with GFP-expressing M. marinum (shown in cyan). Images were taken at the indicated time points. Two examples are for shown for ZntA-, Rab11a-, or Rab11c-positive MCVs at 26 or 21 hpi, respectively. Arrowheads point to MCVs decorated with ZntA-mCherry, Rab11a-RFP, or Rab11c-RFP. Scale bars, 5 μm. (F) Enrichment of D. discoideum CV proteins at the MCV and their differential expression during M. marinum infection. Table showing the presence of selected CV proteins in the MCV proteome. Data were retrieved from Table S1 of the work of Gueho et al. . Colors indicate the strength of expression (in logFC) in infected cells compared to mock infected: from dark red (highest expression) to dark blue (lowest expression). Download FIG S4, PDF file, 0.7 MB.ZntC and ZntD transporters do not localize at the MCV. (A and B) Immunofluorescence staining at different times postinfection of ZntC- or ZntD-mCherry-expressing cells (shown in magenta) infected with GFP-expressing o et al. . InformaCopyright © 2021 Hanna et al.2021Hanna et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01313-20.9TABLE S2M. marinum MCVs  D. discoideum material used for this study. (B) M. marinum material used for this study. Download Table S3, DOCX file, 0.03 MB.Copyright © 2021 Hanna et al.2021Hanna et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2+ transporter but that ZntA might also contribute to the import of Zn2+ into the MCV . In. InM. ma2+ increases in acidic zincosomes and phagosomes, while in cells lacking ZntB, zincosomes contain low levels of Zn2+  cells while the levels of Zn2+ were lower in MCVs from zntB KO cells than from wt cells  and the capacity of M. marinum to detoxify its own intracellular Zn2+  Plaque formation by Ax2(Ka) wt and zntA KO (A) or Ax4 wt and zntB KO cells (C) on GFP-expressing M. marinum wt or ΔctpC, as described in Materials and Methods. Shown is one representative experiment from three independent biological replicates. (B and D) Quantification of three independent experiments. Plaquing score was calculated as described in Materials and Methods. (E and F) Plaque formation by Ax2(Ka) wt and zntA KO (E) or Ax4 wt and zntB KO cells (F) on nonpathogenic bacteria, as described in Materials and Methods. Download FIG S5, PDF file, 0.2 MB.Growth of Copyright © 2021 Hanna et al.2021Hanna et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the znt KOs on M. marinum intracellular growth. Strikingly, absence of ZntA led to a decrease in the intracellular load of M. marinum  is strong evidence for the role of Zn2+ during infection.Next, we monitored the impact of  marinum , suggestnfection  and C. T+-levels , was decKO cells . This ep2+ inside the MCVs was lower in cells lacking ZntB  Ax2(Ka) wt or zntA KO cells (A) and Ax4 wt or zntB KO cells (B) were infected with GFP-expressing M. marinum wt and ΔctpC. The percentage of ubiquitinated bacteria was assessed by immunofluorescence using FK2 antibodies and manual counting at 26 hpi. A total of 106, 95, 95, and 68 bacteria were counted in Ax2(Ka) wt, Ax2(Ka) zntA KO, Ax4 wt, and zntB KO cells infected with M. marinum wt, respectively, and a total of 115, 91, 94, and 86 bacteria were counted in Ax2(Ka) wt, zntA KO, Ax4 wt, and zntB KO cells infected with M. marinum ΔctpC, respectively. Unpaired t tests showed no statistical differences. (C and D) Ax2(Ka) wt or zntA KO cells (C) and Ax4 wt or zntB KO cells (D) expressing mCherry-Plin were infected with GFP-expressing M. marinum wt and ΔctpC. The percentage of cells with intracellular bacteria colocalizing with Plin was assessed manually at 26 hpi. A total of 178, 136, 85, and 29 bacteria were counted in Ax2(Ka) wt, zntA KO, AX4 wt, and zntB KO cells infected with M. marinum wt, respectively, and a total of 96, 85, 65, and 19 bacteria were counted in Ax2(Ka) wt, zntA KO, Ax4 wt, and zntB KO cells infected with M. marinum ΔctpC, respectively. Error bars indicate the SEM from four (C) or three (D) independent experiments. Statistical differences are indicated with an asterisk and were calculated with an unpaired t test . Download FIG S6, PDF file, 0.06 MB.Membrane damage and phagosomal escape of Copyright © 2021 Hanna et al.2021Hanna et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2+ pools for nutritional immunity and/or metal intoxication purposes. Accordingly, infected neutrophils reduce Streptococcus pyogenes growth by releasing the Zn2+ scavenger calprotectin   and four ZnTs (ZntA to -D) . Colors indicate the strength of expression of zplA to -G (in logFC) in infected cells compared to mock infected: from dark red (highest expression) to dark blue (lowest expression). Download FIG S7, PDF file, 0.04 MB.Differential expression of the Copyright © 2021 Hanna et al.2021Hanna et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro, M. marinum senses and reacts to elevated levels of Zn2+ by specifically inducing the expression of the Zn2+ efflux pump CtpC  to the cytosol. This model is in line with a previous study  is even more attenuated for intracellular growth in a D. discoideum mutant itself affected in a Zn-related gene (zntA or zntB) is a demonstration that the two mutants interact genetically in a way that is best explainable by a dual perturbation in Zn2+ homeostasis. Second, the increased attenuation of the ΔctpC mutant in the zntA KO host is most likely due to the elevated MCV Zn2+ concentration, while the increased attenuation in the zntB KO is likely due to inefficient MCV escape and prolonged exposure to the residual lumenal Zn2+ concentration.Strikingly, the intracellular growth of  or ZntB , althouged to wt . We propD. discoideum restricts an infection with M. marinum by using one or more Zn2+-dependent host defense mechanisms, which are counteracted by the pathogen via the specific induction of its Zn2+ exporter CtpC. To our knowledge, this is the first study reporting the role of CtpC in the resistance of M. marinum to Zn2+-mediated host defense.In summary, we conclude that D. discoideum material used in this study is listed in The M. marinum strains and plasmids used in the present study are listed in M. marinum ΔctpC was generated by specialized phage transduction as previously described  from the medium-strength tetracycline promoter. The vector is then stably integrated into the genomic attB site. Mtb ctpC was amplified from genomic DNA using the primer pair oHK375/oHK376 to yield pHK124. The correct insertion was verified by sequencing using the primers oHK328, oHK340, and oHK377. Mycobacteria were cultured at 150 rpm at 32°C in Middlebrook 7H9 (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC), 0.2% glycerol, and 0.05% Tween 80. Bacterial clumping was minimized by adding 5-mm glass beads during cultivation. Mutants and plasmid carriers were grown in medium supplemented with hygromycin (100 μg/ml), kanamycin (25 μg/ml), and/or ampicillin (100 μg/ml).The escribed  19) wit witM. ma5/oHK146 . The PCRcassette  using thcassette  to constM. marinum wt and ΔctpC were grown in liquid 7H9-OADC-glycerol-Tween at 32°C in shaking until reaching an optical density at 600 nm (OD600) of approximately 0.5. For growth assays on agar, serial 10-fold dilutions were performed and 5 μl of each dilution was plated on 7H11-OADC-glycerol-Tween containing different concentrations  of ZnSO4, MnCl2, and CuSO4. Pictures were taken after 7 days of incubation at 32°C. For growth in broth, overnight cultures of green fluorescent protein (GFP)-expressing M. marinum wt and ΔctpC as well as of E2-Crimson -expressing wt and ΔctpC::ctpC were diluted to 5 × 105 bacteria/ml, centrifuged, and resuspended in 7H9 medium containing increasing ZnSO4 concentrations as indicated. Bacteria were distributed in 96-well plates at 105/well (Perkin Elmer). Growth at 32°C under shaking conditions was assessed by measuring fluorescence at 509 nm for the GFP signal and 646 nm for the E2-Crimson signal in a plate reader . The growth of the ΔctpC (GFP) and ΔctpC::ctpC (E2-Crimson) strains was normalized to the M. marinum wt strains expressing GFP and E2-Crimson, respectively.6 cells/ml in filtered HL5-C. Five micrograms per milliliter of streptomycin and 5 U/ml of penicillin were added to prevent extracellular bacterial growth. The infected cells were then incubated at 25°C at 130 rpm, and samples were taken for analysis at the indicated time points.Infections were performed as previously described , 60. Aft2+ during infection, infected cells were plated on μ-dishes (ibidi), medium was exchanged to Soerensen buffer, and intracellular Zn2+ was stained with 5 μM NBD-TPEA , 561-nm (mCherry), and 640-nm (Vybrant Ruby) laser lines, respectively. To quantify the integrated intensity of NBD-TPEA inside MCVs, cells were infected with mCherry-expressing M. marinum. At the indicated time points, samples were stained with NBD-TPEA and images were taken using the ImageXpress spinning disc confocal microscope (Molecular Devices). Here, mCherry-expressing bacteria were excited using the Texas Red laser line and NBD-TPEA-expressing bacteria were excited using the GFP laser line. The integrated intensity per MCV area was assessed using an analysis pipeline created in MetaXpress Custom Module Editor (Molecular Devices). Briefly, MCVs were defined as a sphere around mCherry-labeled bacteria that were detected using the Texas Red channel. NBD-TPEA inside MCVs was detected using the settings for the GFP channel. The normalized integrated intensity of NBD-TPEA was divided by the area of each MCV, leading to the integrated intensity per MCV area.To monitor the subcellular localization of Zno. N1040 ) for 30 Antibodies against p80  were froM. marinum expressing GFP as described above. In brief, at each time point, a homogeneous population of infected cells was obtained by GFP fluorescence-activated cell sorting (FACS). The total RNA was extracted, and the rRNA from host and bacteria was depleted, before generation of the cDNA libraries using a Bio-Rad iScript kit and sequencing. The resulting reads were mapped in parallel against the D. discoideum and M. marinum genome. Differential expression analysis of the infection time course was performed using the R package limma, by comparing on one hand infected D. discoideum cells to mock -infected cells, and on the other hand intracellular M. marinum to M. marinum grown in broth.RNA-Seq data were gathered from the work of Hanna et al. . Briefly2+ on the expression of ctpC, ctpC-like, ctpV, and ctpV-like, M. marinum strains were exposed to various concentrations of ZnSO4  for 2 h. Bacteria were harvested, RNA was extracted, and cDNA was synthesized using a Bio-Rad iScript kit. For each gene tested, the mean calculated threshold cycles (TC) were averaged and normalized to the TC of a gene with constant expression (sigA). The normalized TC was used for calculating the fold change using the ΔΔTC method. Briefly, relative levels of target mRNA, normalized with respect to an endogenous control (sigA), were expressed as 2−ΔΔTC (fold), where ΔTC = TC of the target gene − TC of the control gene (sigA), and ΔΔTC = ΔTC of the studied set of conditions − ΔTC of the calibrator conditions, as previously described  were plated on nontreated, white F96 MicroWell plates (Nunc) or black 96-well plates (Perkin Elmer) covered with a gas-permeable moisture barrier seal (BioConcept). The course of infection was monitored by measuring either luminescence or fluorescence (at 509 and 646 nm) as a proxy of bacterial growth using a Synergy Mx microplate reader (BioTek) at a constant temperature of 25°C for around 70 h with 1-h intervals. The growth of the ΔctpC (GFP) and ΔctpC::ctpC (E2-Crimson) strains was normalized to M. marinum wt strains expressing GFP and E2-Crimson, respectively.Intracellular growth of escribed . BrieflyD. discoideum cells were added to the bacterial lawn and plates were incubated at 22°C for 4 to 7 days until plaques were visible. Quantification was performed by scoring the appearance of plaques in at least three independent experiments. The logarithmic plaquing score was defined as follows: plaque formation in wells with 10 amoebae yielded a score of 1,000; in the cases where cells did not grow at lower dilutions, they obtained the corresponding lower scores of 100, 10, and 1. The ability of D. discoideum Ax2(Ka), zntA KO, Ax4, and zntB KO to form plaques on M. marinum wt and ΔctpC was assessed as described previously  were added to the bacterial lawn, and plates were incubated at 25°C for 7 days until plaques were visible. The scoring was performed as described above.Phagocytic plaque assays in the presence of nonpathogenic bacteria as food source were performed as described previously . Brieflyeviously , 65. BriM. marinum were identified by performing a protein BLAST search using NCBI PSI-BLAST and the sequence of Mtb CtpC. The results were then cross-compared to the annotations of M. marinum P-type ATPases available on Mycobrowser  using the G-INS-1 strategy and “leave gappy regions” to generate a phylogenetic tree in phylo.ilo using NJ conserved sites and the JTT substitution model (Putative P-type ATPases of obrowser . The proon model .M. marinum P-type ATPases (D. discoideum CV genes (zntA to -D transporters (zplA to -G transporters (https://doi.org/10.1101/590810 (https://doi.org/10.1101/592717 (Information on the differential expression of  ATPases  as well CV genes , zntA tosporters , and zplsporters  was extr1/590810 . Informa1/590810  was extr1/592717 .


This approach describes how the social distancing measures currently in effect can be used to re‐think higher education based on a reasonable use of technology. Taken together, the COVID‐19 crisis can be a time of major reform in higher education that will accelerate the process of digitalization in an unprecedented way.As a result of the COVID‐19 outbreak, teaching in universities needed to be quickly transitioned from regular on‐campus classes into technology‐enhanced teaching formats. In this article, we present the case study of Chemnitz University of Technology (Germany), where digital classes were introduced in a matter of weeks. By analyzing syllabus data, we found that the use of video and video conferencing is an important current development. Related to these findings, we present evidence from instructional psychology and social media research that can help in the design of teaching during this crisis. We highlight the need for multimodal learning, that is, learning settings that use multiple sensory modalities. Importantly, we present a strategy of  Although technology‐enhanced learning already played an important part in most German universities, the COVID‐19 pandemic has required to expand the existing infrastructure and highlighted areas that universities should focus on in their digitalization strategy. Furthermore, this challenge has greatly accelerated the digitalization of the German higher education sector. However, it needs to be highlighted that most schools and some universities in Germany are not adequately equipped to handle the current transformation towards fully‐digital forms of education Kerres, . Further22.1With its 10,389 students .Additional measures taken by the university include the establishment of a psychological counseling hotline and several technical changes. One of the most important new tools introduced during this time is the setup of the video conference software BigBlueButton  chats. On the other hand, 146 classes were stated to make use of video‐based teaching formats . Besides, 30 classes aim at using the newly introduced BigBlueButton video conference service of Chemnitz University of Technology. Interestingly, the video‐based formats are particularly often used within the Faculty of Electrical Engineering and Information Technology. Although the descriptions provided in the syllabus are not necessarily exhaustive and may have omitted more traditional tools such as forums and chat‐rooms despite being used in digital classes, these numbers give us an impression for the heightened demand of video‐based forms of teaching.3As described in this case study, the COVID‐19 crisis required a swift response in the transition from on‐campus classes to digital teaching formats. In a matter of days, teaching concepts needed to be made compatible with the emergency situation. Our case study revealed that there is a high interest in the usage of video technology. Several classes feature video recordings, streams, and video conferences. The newly introduced open‐source video conference system BigBlueButton already elicits a high demand.These results show that the COVID‐19 crisis has already proven to be a significant accelerator for the digitalization of teaching at this particular university. The dire situation has been understood as an opportunity for change and innovation Yan, . When co3.1hybrid campuses. This emerging strategy of combining digital and face‐to‐face teaching appears to be a promising choice for the following months and years in which additional waves of COVID‐19 and related infections may spread rapidly and unexpectedly all over the globe. Before the COVID‐19 crisis, the teaching format of blended learning was already in use  can actually induce cognitive load by itself (Makransky, Terkildsen, & Mayer, Besides, various other findings run counter to educators' intuitions. As we have seen in our case study, videos and video conferencing appear to be important new components of digital classes. However, a large‐scale investigation found that seeing the instructor's face in an instructional video does not affect learning (Kizilcec, Bailenson, & Gomez, Budgetary limits and legal hurdles can severely impact technology‐enhanced learning (see Kerres, 4While the sudden and unexpected outbreak of COVID‐19 required university lecturers and administrators to quickly adapt to a new situation, the data presented in this case study provide evidence that most university courses can be rapidly transformed into a digital format. Given the fact that there may be some heterogeneity concerning the experience with technology‐enhanced learning throughout the teaching staff, it will be interesting to assess how well less experienced users of e‐learning services have dealt with this emergency situation. In any event, this situation has greatly accelerated the digitalization of university teaching and may contribute to the permanent establishment of hybrid campuses that offer flexibility and autonomy for students as well as educators. Time will tell whether university educators who switched to online teaching formats merely consider technology‐enhanced learning as the second‐best alternative to face‐to‐face on‐campus classes or whether they use this situation to re‐invent teaching.The authors declare no potential conflict of interest.


In the central nervous system, hyperpolarization-activated, cyclic nucleotide-gated (HCN1–4) channels have been implicated in neuronal excitability and synaptic transmission. It has been reported that HCN channels are expressed in the spinal cord, but knowledge about their physiological roles, as well as their distribution profiles, appear to be limited. We generated a transgenic mouse in which the expression of HCN4 can be reversibly knocked down using a genetic tetracycline-dependent switch and conducted genetically validated immunohistochemistry for HCN4. We found that the somata of HCN4-immunoreactive (IR) cells were largely restricted to the ventral part of the inner lamina II and lamina III. Many of these cells were either parvalbumin- or protein kinase Cγ (PKCγ)-IR. By using two different mouse strains in which reporters are expressed only in inhibitory neurons, we determined that the vast majority of HCN4-IR cells were excitatory neurons. Mechanical and thermal noxious stimulation did not induce c-Fos expression in HCN4-IR cells. PKCγ-neurons in this area are known to play a pivotal role in the polysynaptic pathway between tactile afferents and nociceptive projection cells that contributes to tactile allodynia. Therefore, pharmacological and/or genetic manipulations of HCN4-expressing neurons may provide a novel therapeutic strategy for the pain relief of tactile allodynia. The HCN channel family comprises four subtypes (HCN1–4) that form heteromultimers. Among those, HCN1 and HCN4 possess the fastest and slowest activation kinetics upon hyperpolarization, respectively. The voltage-dependence of activation of HCN2 and HCN4 is highly sensitive to cyclic adenosine monophosphate (cAMP), whereas that of HCN1 and HCN3 is almost insensitive to cAMP [Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels are expressed in both the peripheral nervous system (PNS) and central nervous system (CNS), and these ion channels generate the hyperpolarization-activated, non-selective cation current . HCN4, a well-known pacemaker channel in the sinoatrial node, is expressed in a minor population of trigeminal ganglion and DRG neurons –5. In thh currents and high-frequency firing [In the spinal cord, HCN channels are reportedly expressed in both the ventral and dorsal horns –14. Whily firing . On the All animal experiments in this report were approved by the Animal Ethics Committee of Kurume University animal care and experiments (#2020–30). Animal care and experiments conformed to the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health .+/tTA_TRE mice has been described previously [+/tTA_TRE mice were identified by genomic PCR using the following primers: forward (5′-CGA ATA AGA AGG CTG GCT CTG CAC C-3′), reverse (5′-GAG CAG CCT ACA TTG TAT TGG CAT G-3′). Inhibition of HCN4 expression by the mutant allele was performed by providing food pellets containing doxycycline  ad libitum for 2 weeks before the experiments [+/Luc mice were generated as previously reported [+/Luc mice were identified by genomic PCR using the following primers: forward (5′-CGA GCT GGA CGG CGA CGT AAA CGG C-3′), reverse (5′-CAT CCT TAG GGA GAA TTT GTT GAC C-3′). The knock-in mice in which cDNA of GFP had been targeted into the locus of glutamate decarboxylase 67 (GAD67-GFP mouse) were obtained from RIKEN BRC (#03674) [− 1) and then used for the following experiments.The generation of the HCN4eviously , 18. HCNeriments . HCN4+/Lreported . HCN4+/L(#03674) . These G(#03674) . The VGA− 1; PerkinElmer, Waltham, MA, USA) was injected intraperitoneally into HCN4Luc/tTA_TRE mice. The mouse was deeply anesthetized 30 min after the injection, and the lumbar spinal cord was dissected out. Transverse sections (400 μm thickness) of the spinal cord were obtained using a microslicer . Luminescent imaging was performed using an EM-CCD camera .150 μL of XenoLight RediJect D-luciferin . Thereafter, lumbar spinal cords were dissected out and stored in the same solution. Horizontal sections of L3-L4 (600 μm thickness) were obtained using a microslicer. Total RNA was extracted from the preparation using Trizol reagent . Superscript III (Thermo Fisher Scientific) was used to synthesize single-strand cDNA. We used predesigned TaqMan Gene Expression assay probes for mouse HCN4  to perform the quantitative real-time polymerase chain reaction (qPCR). Relative levels of mRNAs were normalized to the level of 18S rRNA .iv). The lamina III was defined as the gray matter that has 80 μm thickness under lamina II, according to the Allen Brain Atlas [After deep anesthesia, the mice were perfused transcardially with 4% paraformaldehyde solution. The lumbar spinal cords were dissected out and post-fixed in the same fixative for 2 h. Then, the spinal cords were cryoprotected overnight in phosphate-buffered saline containing 30% w/v sucrose and mounted in OCT Compound . Transverse sections (30 μm thickness) were cut on a cryostat , incubated in 50% ethanol for 30 min to enhance antibody penetration, and then incubated for 72 h in a cocktail of primary antibodies , IIiv, and lamina III, referring to the immunoreactivity of the PKCγ plexus. The ventral border of lamina III was drawn according to the definition of lamina III as mentioned above. We initially marked the cell nuclei with NeuN whose sizes were largest in the 3rd to 6th sections of each z-series consisting of 8 sections. We then examined the HCN4-immunoreactivity of the marked cells. Coexpression of GAD67-GFP or VGAT-Venus fluorescence was assessed in the same way. We did not perform NeuN immunohistochemistry when we examined the colocalization of PKCγ-, PV-, or c-Fos-immunoreactivities with HCN4-immunoreactivity.To examine the proportion of neurons in laminae I–III, we scanned the dorsal horn sections sequentially with a 20× lens and produced z-series of 8 optical sections in 2-μm steps. It was necessary to scan 1–2 z-series from each section to cover the whole area of laminae I–III. We first determined the gray matter outlines and then the boundaries between the dorsal part of the inner lamina II  pinching of skin folds at 10 points by the same experimenter in the same way . Each point of the skin was pinched using Adson forceps with 1 × 2 teeth  for 3 s with equal force until the teeth of forceps completely bite into the skin: (2) immersion of the left hind paw in water at 52 °C for 15 s (n = 3 mice). Two hours after the noxious stimulation, the mice were transcardially perfused with 4% paraformaldehyde under urethane anesthesia (1.5 g kg− 1). Thereafter, the lumbar spinal cords of these mice were dissected out and used for immunohistochemistry and quantitative analysis, as described above.Six HCN4n = 3) and HCN4Luc/tTA_TRE mice which were fed with DOX-containing food pellet for 4 weeks (n = 3). These mice were placed on an elevated grid of wire and the plantar surface of hind paw was stimulated with calibrated von Frey monofilaments . Fifty percent paw withdrawal threshold was determined by Dixon’s up-down method [We carried out von Frey assay using wild type mice .For multiple comparisons in Fig. +/Luc mouse [We have previously demonstrated that HCN4-expressing loci in the heart can be visualized with chemical luminescence using the HCN4uc mouse . In this+/tTA_TRE mouse [+/tTA_TRE mouse, the HCN4 promoter drives the tetracycline transactivator (tTA) expression, which then binds to the tetracycline responsive element followed by a cytomegalovirus minimal promoter, and overexpresses the HCN4 protein in physiological expression loci .To overcome this problem, we aimed to conduct the immunohistochemical study with genetic validation. Previously, we developed the HCN4RE mouse . In the oci Fig. a. We the+/tTA_TRE mice , whereas there were only a few HCN4-IR cells in laminae I–IIid , 15.3% (14.3–17.0%), and 19.7% (17.0–22.8%) of all neurons in laminae I–IIid, IIiv, and III, respectively  and 16.9% (lamina III) were neither PV-IR nor PKCγ-IR. These results seemed to support the findings that 78–84% of PV-IR cells and 53.9% of PKCγ-IR cells in laminae II–III are reportedly HCN4-IR [PKCγ-IR cells accounted for 74.6% of HCN4-IR cells in lamina IIiv Fig. a, d and iv Fig. d. Among  HCN4-IR , 17.Reportedly, only ~ 70% of PV-IR cells in the murine SDH express VGAT mRNA. The rest of PV-IR cells (~ 30%) express mRNA of the vesicular glutamate transporter 2 (VGLUT2) . PKCγ ce+/tTA_TRE mice with VGAT-Venus mice and aimed to determine the population of inhibitory HCN4-IR neurons using the double transgenic HCN4+/tTA_TRE::VGAT-Venus mice [id , 5.2% in lamina IIiv (0–11.8%), and 2.9% (0–4.8%) in lamina III. These findings confirmed that the majority of HCN4-IR cells were not inhibitory interneurons, but excitatory interneurons. Furthermore, most of the cells that were IR for both PV and HCN4 were not Venus-positive, suggesting that HCN4 was expressed by excitatory PV-IR cells.In the SDH, inhibitory interneurons are GABAergic and/or glycinergic; GABAergic neurons tend to be concentrated in superficial areas whereas glycinergic neurons occupy deeper laminae . Becausenus mice  Fig. 4)+/tTA_TRE+/tTA_TRE::GAD67-GFP mouse in which GABAergic inhibitory neurons are visualized by GFP  of HCN4-IR cells in laminae I–IIid, 4.0% (0–8.7%) in lamina IIiv, and 4.1% (1.6–9.1%) in lamina III. We therefore concluded that the vast majority of HCN4-IR cells in laminae I–III were not inhibitory interneurons.In addition, we also carried out immunohistochemical studies in the double transgenic HCN4o  of HCN4-IR cells in laminae I–IIid. In laminae IIiv and III, c-Fos expression was not induced in HCN4-IR cells  in laminae I–IIid, 4.0 in lamina IIiv, and 5.5 in lamina III. The neurons that showed both HCN4- and c-Fos-immunoreactivity accounted for 12.5% (range 0–25%) of HCN4-IR cells in laminae I–IIid. The neurons that showed both HCN4- and c-Fos-immunoreactivity accounted for 3.8% (range 0–8.3%) of HCN4-IR cells in lamina IIiv. No HCN4-IR cell in lamina III expressed c-Fos  in laminae I–IILuc/tTA_TRE mice with DOX administration (HCN4-knockdown mice). Two weeks after starting DOX administration, we could not observe obvious alteration in general behavior of HCN4-knockdown mice. This finding was in good agreement with the previous report on brain-specific HCN4 conditional knockout mice [As shown in Fig. out mice . We thenIn this study, we described for the first time the distribution of HCN4-immunoreactivity in the mouse spinal cord by immunohistochemistry using a knockdown-verified specific antibody. We found that HCN4-immunoreactivity was concentrated in the SDH, especially near the border of laminae II and III. We also examined the colocalization of HCN4-immunoreactivity with commonly used neurochemical markers and revealed that most HCN4-IR cells expressed either PKCγ or PV. Using transgenic mice in which fluorescent reporter proteins are selectively expressed only in inhibitory neurons, we demonstrated for the first time that the vast majority of HCN4-IR cells were limited to excitatory interneurons in this area.Luc/tTA_TRE mice by DOX administration. Consistent with our previous western blotting experiments, HCN4-immunoreactivity was completely absent following DOX treatment. This result allows us to consider that HCN4-immunoreactivity faithfully reflects the locations of HCN4 proteins in the spinal cord. In other studies, HCN4-immunoreactivities were reportedly found throughout the spinal cord including the ventral horn [+/tTA_TRE mice, in which HCN4 was overexpressed at its physiological expression loci. This result also agreed with the finding that in HCN4Luc/tTA_TRE mice, the luminescence signals were restricted to the SDH. Based on these findings, we concluded that the location of HCN4-IR neurons was restricted to the SDH.In our previous report, we verified the specificity of the same antibody against HCN4 by western blotting of protein samples obtained from the same set of HCN4-transgenic mice used in this study. It is not known, however, whether an antibody whose specificity has been verified by western blotting can provide specific immunohistochemical staining of brain/spinal cord slice preparations in general. To verify whether immunoreactivities with the same antibody were specific against HCN4 in immunohistochemistry, we knocked down the expression of HCN4 in HCN4ral horn . Howeverral horn , 17. As i–III showed HCN4-immunoreactivity, whereas 64.8% of them were HCN1-IR [In this study, we confirmed that 60% of the HCN4-IR neurons were PV-IR in lamina III Fig. . In acco HCN1-IR .i–III are the source of axo-axonic inputs onto presynaptic terminals of low-threshold mechanoreceptor (LTMR) afferents and exert presynaptic inhibition. PV-terminals having contacts with LTMR afferents were clearly shown to be VGAT-positive, and the dysfunction of inhibitory PV-neurons was implicated in the development of tactile allodynia [h current.In the SDH, the population of PV-positive neurons reportedly consists of both inhibitory (70%) and excitatory (30%) interneurons , 32. It llodynia , 33, 34.h upon hyperpolarization [h in putative inhibitory neurons that expressed GFP driven by the PV promoter, they also demonstrated that Pax2 (a marker of inhibitory neurons) did not colocalize with HCN4 [Seemingly, this rule does not apply to the neurons in LTMR recipient zone (LTMR-RZ). In the LTMR-RZ, many types of neurons show tonic firing patterns. Most notably, both excitatory and inhibitory PV-neurons in the LTMR-RZ exhibit tonic firing and the voltage sag (a slowly developing depolarization during hyperpolarizing current injection), which is generated by the activation of Irization . Althougith HCN4 . Furtheriv and III was that of PKCγ-IR neurons (Fig. h was recorded in 70.8% of PKCγ-positive interneurons [h current [Another population of neurons in which we observed HCN4-immunoreactivity in laminae IIons Fig. . In accorneurons . However current . It appe current , 38. In o, which successively have monosynaptic excitatory connections with nociceptive projection neurons in lamina I [iv–III [Peripheral information is conveyed by primary afferent fibers. In the SDH, they are not only relayed to projection neurons but also processed by local circuits. Recently, genetic ablation studies of spinal interneurons have revealed the importance of such local circuits for the transmission of noxious, pruritic, and tactile sensations . It is elamina I . Besides [iv–III . Althoug+/PKCγ+ neurons may be particularly important for the development of mechanical allodynia [It has been suggested that peripheral nerve injury suppresses this feed-forward mechanism and induces mechanical allodynia . In the llodynia .iv ~ III. The PKCγ-expressing neurons reportedly receive the synaptic inputs from myelinated Aβ- and Aδ fibers, which convey tactile information, but not acute nociceptive information [As shown in the present study, HCN4-expressing cells in the SDH did not respond to the noxious stimulation. This result seems to agree with the distribution of HCN4-expressing cells revealed in this study. The HCN4-expressing neurons largely overlapped with the PKCγ-expressing neurons in lamina IIormation , 46. Howormation , 41. It ormation . These fHcn4 gene contains an enhancer element that is regulated by AP1 and MEF2 [In PKCγ-expressing neurons of the rat SDH, the phosphorylation of cAMP response element-binding protein was reportedly increased after partial sciatic nerve ligation, suggesting that the ligation increased cAMP levels in PKCγ-expressing neurons . Becauseand MEF2 , 51, theand MEF2 . For theand MEF2 , 52, 53.h in the anterior cingulate cortex reportedly reduces mechanical allodynia [h in the HCN4-expressing excitatory SDH interneurons may provide a clue to clarify the pathophysiological roles of these cells in mechanical allodynia.In the present study, global knockdown of HCN4 did not induced significant change in nocifensive behavior. However, it appears difficult to interpret the effects of global HCN4-knockdown, because HCN4 is widely expressed in many locus of the brain, and the pharmacological inhibition of Illodynia . In the Additional file 1: Table S1. Primary antibodies. Table S2. Secondary antibodies.Additional file 2: Figure S1. The majority of HCN4-IR cells do not express GAD67.Additional file 3: Figure S2. Fifty-percent paw-withdrawal threshold did not differ between wild type mice and HCN4-knockdown mice.


Host vulnerabilities associated with acute malnutrition could facilitate the ability of specific enteric pathogens to cause diarrhoea and associated mortality. Using data from the Global Enteric Multicenter Study, we assessed whether acute malnutrition modifies the association between common enteric pathogens and moderate-to-severe diarrhoea, and whether associations between enteric pathogens and death were modified by acute malnutrition.Children with moderate-to-severe diarrhoea and age-matched and community-matched controls were included in this post-hoc analysis if their mid-upper arm circumference had been measured and if they were older than 6 months of age. Acute malnutrition was defined as mid-upper arm circumference below 12·5 cm, capturing both severe acute malnutrition (<11·5 cm) and moderate acute malnutrition (≥11·5 cm and <12·5 cm). We tested whether acute malnutrition modified associations between enteric pathogens and moderate-to-severe diarrhoea in conditional logistic regression models. Among children with moderate-to-severe diarrhoea, Cox proportional hazards regression evaluated the modifying effect of acute malnutrition on the relationship between pathogens and 60-day fatality rate.Escherichia coli among children aged 6–11 months was 2·08 (95% CI 1·14–3·79) in children with acute malnutrition, and 0·97 (0·77–1·23) in children with better nutritional status, compared with healthy controls. Enterotoxigenic E coli producing heat-stable toxin among children aged 12–23 months also had a stronger association with moderate-to-severe diarrhoea in children with acute malnutrition (aOR 7·60 [2·63–21·95]) than among similarly aged children with better nutritional status (aOR 2·39 [1·76–3·25]). Results for Shigella spp, norovirus, and sapovirus suggested they had a stronger association with moderate-to-severe diarrhoea than other pathogens among children with better nutritional status, although Shigella spp remained associated with moderate-to-severe diarrhoea in both nutritional groups. 92 (64%) of 144 children with moderate-to-severe diarrhoea who died had acute malnutrition. Pathogen-specific 60-day fatality rates for all pathogens were higher among children with acute malnutrition, but no individual pathogen had a significantly larger increase in its relative association with mortality.The age, site, and co-infection adjusted odds ratios (aORs) for moderate-to-severe diarrhoea associated with typical enteropathogenic Acute malnutrition might strengthen associations between specific pathogens and moderate-to-severe diarrhoea. However, the strong link between acute malnutrition and mortality during moderate-to-severe diarrhoea in children is not limited to specific infections, and affects a broad spectrum of enteric pathogens. Interventions addressing acute malnutrition could be an effective way to lower the mortality of both childhood malnutrition and diarrhoea.The Bill & Melinda Gates Foundation. Children with acute malnutrition are three times more likely to die from diarrhoea than children with better nutritional status, and over 200 000 diarrhoeal deaths are attributed to acute malnutrition annually.Escherichia coli, enteroaggregative E coli, Shigella spp, Salmonella spp, Campylobacter spp, Entamoeba histolytica, and Cryptosporidium spp have been shown to be more common among children with acute malnutrition presenting with diarrhoea, compared with children who are better nourished.Enterotoxigenic Evidence before this studyCampylobacter spp, Entamoeba histolytica, and Cryptosporidium spp are more common among children with diarrhoea if that child has malnutrition, but these studies did not include community controls, limiting their ability to differentiate enteric pathogens associated with prolonged asymptomatic carriage from those that have an increased propensity to cause diarrhoea or death in children with acute malnutrition.Height-for-age Z-score, a marker of chronic malnutrition, was shown to be an important risk factor for death during episodes of childhood moderate-to-severe diarrhoea in the Global Enteric Multicenter Study (GEMS). However, little is known about acute malnutrition and its interactions with enteric pathogens. Previous data have suggested that members of the Enterobacteriacae family, Added value of this studyUsing cases of moderate-to-severe diarrhoea and community controls from GEMS, this analysis attempts to differentiate prolonged asymptomatic carriage of specific pathogens from increased virulence associated with malnutrition. We also compared the 60-day fatality rates of common enteric pathogens across childhood nutritional strata, to establish the contribution of individual pathogens to the high case fatality observed in children with acute malnutrition and moderate-to-severe diarrhoea.Implications of all the available evidenceThere is evidence that some pathogens are adept at capitalising on the vulnerabilities of children with acute malnutrition. However, the magnitude of these interactions does not explain the substantial increase in mortality observed in children with acute malnutrition. Children with moderate-to-severe diarrhoea who have acute malnutrition are at considerably higher risk of death than children with better nutritional status and diarrhoea, and children in the community with acute malnutrition. This increase in mortality occurs irrespective of the inciting pathogen, which suggests that pathogen-specific therapies will only lead to a modest reduction in malnutrition-associated diarrhoeal mortality. Interventions that address the broader scope of malnutrition-associated death could make an important contribution to reducing global diarrhoeal mortality.GEMS recruited children aged 0–59 months with moderate-to-severe diarrhoea and asymptomatic controls in Bangladesh, India, Kenya, Mali, Mozambique, Pakistan, and The Gambia.Detailed clinical, sociodemographic, and anthropometric data were collected at enrolment. Children received guideline-recommended treatment for diarrhoea at the discretion of the clinician, and cases and controls were visited approximately 60 days (range 50–90) after enrolment to measure vital status. Children with severe acute malnutrition were referred to local standard-of-care malnutrition management programmes, but some national guidelines do not include moderate acute malnutrition treatment.E coli, and Salmonella, Shigella, Campylobacter, Aeromonas, and Vibrio spp. Three E coli colonies were combined, and multiplex PCR was used to classify E coli pathotypes.Giardia lamblia, Entamoeba histolytica, and Cryptosporidium spp were identified with commercially available enzyme-linked immunoassays .All children provided at least 3 g of stool within 12 h of registration at the health facility, as previously described.For this post-hoc analysis, acute malnutrition was defined as mid-upper arm circumference below 12·5 cm, capturing both severe acute malnutrition (<11·5 cm) and moderate acute malnutrition (≥11·5 cm and <12·5 cm). Mid-upper arm circumference is the optimal measure for identifying acute malnutrition among children with diarrhoea, as weight is more susceptible to dehydration.E coli producing heat-stable toxin [ST-ETEC], Cryptosporidium spp, Shigella spp) to control for confounding by co-infection. Controls were age-matched, but continuous and quadratic age in months were also tested in pooled age-group models. Interactions may differ between relative (odds ratio [OR]) and absolute (risk difference) models, therefore we also calculated relative excess risk due to interaction on the absolute risk scale.Conditional logistic regression was used to estimate the risk of diarrhoea given specific enteric infections in the age strata , as per the GEMS primary analysis.E coli [EPEC], ST-ETEC, Entamoeba histolytica, Cryptosporidium spp), and clustered by site. Models including quadratic terms for age in months were run. All Cox models satisfied the proportional hazards assumption, which was tested using Schoenfeld residuals. Again, relative excess risk due to interaction models were used to calculate interactions on the absolute scale.The effect of acute malnutrition on the relationship between pathogens and mortality was assessed using 60-day follow-up data collected from moderate-to-severe diarrhoea cases. The incidence of death among moderate-to-severe diarrhoea cases with and without acute malnutrition was calculated using observed person-time and compared using a Kaplan-Meier plot and Cox proportional hazards regression. Children were censored at the date of death or at day 60 of follow-up. Children who died after 60 days post-enrolment were counted as alive at 60 days. Pathogen-specific mortality with 95% CIs were calculated separately by acute malnutrition status, assuming a Poisson distribution. The 60-day fatality rate for children in the control group with and without acute malnutrition was calculated as a reference for the main analysis. To test for interactions between acute malnutrition and specific pathogens, the hazard ratio (HR) for death associated with each pathogen, relative to children without that pathogen, among moderate-to-severe diarrhoea cases with and without acute malnutrition was calculated using Cox proportional hazards regression, and the interaction term between nutritional status and pathogen tested using the Wald statistic. To provide generalisability to current syndromic management, additional analyses were done to test an interaction between dysentery (present or absent) or acute watery diarrhoea (present or absent) and nutritional status. Dysentery was defined by the clinician or caregiver, or by laboratory-reported bloody stool. Acute watery diarrhoea was defined by the clinician when the child left the facility. There were too few deaths to stratify by age group, so Cox models were adjusted for age group as binary indicator variables and for pathogens associated with death in the original GEMS analysis . Finally, to understand if socioeconomic status or height-for-age Z-score confounds the effect of malnutrition on diarrhoeal mortality, we further adjusted the Cox proportional hazards model for height-for-age Z-score, quadratic height-for-age Z-score, caregiver education , household crowding (continuous), improved water source (binary), improved toilet type (binary), and a composite asset assessment constructed using a principal component analysis. The principal component methods are described in the The funder of the study had no role in in study design, data collection, data analysis, or data interpretation. An investigator employed by the funder (LML) participated in writing of the manuscript to provide expertise on diarrhoea aetiology and management.vs 1442 [20·6%] of 6987 children with better nutritional status and moderate-to-severe diarrhoea), more likely to be admitted to hospital , and less likely to be prescribed outpatient antibiotics  than children with better nutritional status.This post-hoc analysis included 8182 children aged 6–59 months with moderate-to-severe diarrhoea and 11 590 controls . ChildreShigella spp among children aged 12–23 months and 24–59 months, and sapovirus among children aged 24–59 months  of 2·09 (95% CI 1·33–3·30). Similarly, ST-ETEC had a stronger association with moderate-to-severe diarrhoea in children with acute malnutrition across all age groups (rOR 2·59 [1·18–5·71]). Conversely, Shigella spp had a consistently stronger association with moderate-to-severe diarrhoea among children without malnutrition in each age strata, with a pooled age-group rOR of 0·28 (0·15–0·53). The association of norovirus with moderate-to-severe diarrhoea had an inverse interaction with nutritional status across age strata; norovirus had a 28% weaker association with moderate-to-severe diarrhoea among children with acute malnutrition than among children with better nutritional status (rOR 0·72 [0·46–1·12]). The pooled age-group rOR for sapovirus was 0·55 (0·30–0·99). Rotavirus appeared to have no interaction with malnutrition in relative models, but in absolute models there was a higher risk of moderate-to-severe diarrhoea when rotavirus was detected among acutely malnourished compared with better nourished children (At least one enteric pathogen was identified in 6726 (82·2%) of 8182 cases and 8198 (70·7%) of 11 590 controls. In adjusted and unadjusted models , acute m9 months . The agechildren . There aOf 172 deaths in the study population, 144 (83·7%) were among children with moderate-to-severe diarrhoea. 92 (64%) of 144 children with moderate-to-severe diarrhoea who died had acute malnutrition . The 60-day fatality rate  for children with acute malnutrition and moderate-to-severe diarrhoea was 8·2 deaths per 100 children (95% CI 6·7–10·1), with a median time to death of 15 days , but the remaining pathogens either showed no evidence of an interaction, or were too rare for an accurate estimation.Each pathogen-specific case fatality rate was higher among children with acute malnutrition than among children with better nutritional status . The fivCryptosporidium spp, Entamoeba spp, and typical EPEC trended toward having an interaction with nutritional status that suggested these pathogens were associated with additional risk of death among children with acute malnutrition  had contradictory results in relative and absolute risk models. For example, in logistic regression Shigella spp had a stronger association with moderate-to-severe diarrhoea among children without acute malnutrition, but this interaction was not present when looking at absolute risk. Because particularly virulent pathogens can cause severe disease in otherwise healthy children whereas less virulent pathogens may only cause severe disease in children with underlying vulnerabilities, the relative importance of virulent pathogens may appear greatest among children with better nutritional status.The GEMS control group enabled this analysis to differentiate malnutrition-associated differences in diarrhoea aetiology from pathogens that are more prevalent among malnourished children due to prolonged carriage.Linear growth stunting was an important risk factor for mortality in the original GEMS analysis.Enteric dysfunction and microbiome dysbiosis are linked to acute malnutrition,Shigella spp are not commonly associated with sepsis or bacteraemia, but infection with these pathogens could indirectly facilitate Gram-negative sepsis. A study in Kenya,ClinicalTrials.gov, NCT03130114) and the First Line Antimicrobials in Complicated Severe Acute Malnutrition trial  will offer important insights into antibiotic regimens for malnourished children.Malnourished children are more likely to present with Gram-negative sepsis than children who are better nourished,We found scarce evidence of confounding by socioeconomic status, but capturing socioeconomic status across diverse settings is challenging, and the data could be subject to social desirability bias, suggesting socioeconomic vulnerability could still explain some of the relationship between malnutrition and mortality. Pathogenic bacteria could be a marker of socioeconomic status through inadequate access to water, sanitation, and hygiene facilities, and mid-upper arm circumference is also a measure of socioeconomic vulnerability. Children with both low mid-upper arm circumference and enteric bacterial pathogens could come from more disadvantaged social situations. Incorporating a holistic assessment of childhood vulnerability into management could provide a more accurate method of targeting interventions, such as an increased frequency of follow-up appointments and young infant feeding interventions.Shigella spp.This analysis has several important limitations. HIV is strongly associated with acute malnutrition, certain enteric pathogens, and death, and HIV exposure or infection can confound some of these results. However, analyses at sites with a low HIV prevalence suggest that this confounding is minimal. Misclassification of nutritional status can also affect our results, as dehydration does reduce mid-upper arm circumference and is a strong risk factor for death. However, mid-upper arm circumference is the most robust measure of nutritional status among children with diarrhoea.Acute malnutrition is a crucial predictor of death during and after an episode of moderate-to-severe diarrhoea, and clinicians should be aware that children with acute malnutrition and diarrhoea are at high risk for mortality and consider additional follow-up and close monitoring. There is a clear need for improved evidence to guide the management of children with moderate acute malnutrition, who comprise a large population at high risk for mortality, for whom no widely implemented management strategies have been defined. The increased mortality associated with malnutrition, irrespective of the infecting pathogen, suggests that interventions targeting specific pathogens would only lead to modest reductions in malnutrition-associated diarrhoeal mortality, and that more lives could be saved through interventions that address underlying malnutrition and the mechanistic pathways that contribute to malnutrition-associated vulnerability.


Larimichthys crocea). These TLRs share high sequence homology with other ichthyic TLRs, while also having their own characters; qtPCR was determined and the results found that the three genes were constitutively expressed in all examined tissues: TLR5M was highly expressed in the spleen and liver; TLR13 expression was high in the kidney, liver, and spleen. And TLRs were upregulated following stimulation with Vibrio parahaemolyticus in the liver, spleen, and kidney. Immunofluorescence staining revealed that TLR5M were localized in the cytoplasm, while TLR5S and TLR13 were in the endosome. The evolutionary analysis has shown that TLR13 was clustered with TLR11, 19, 20, 21, and 22, while TLR5 and TLR3 were classified into a group; these results suggest that TLRs are vital in the defense of L. crocea against bacterial infection and further increase our understanding of TLR function in innate immunity in teleosts.TLRs (Toll-like receptors) are essential in host defense against pathogens. There are two types of TLR5, namely, membrane form of TLR5 (TLR5M) and soluble form of TLR5 (TLR5S), both of which perform a crucial role in flagellin response. TLR13 is a TLR that localizes to endosomes and recognizes nucleic acids released by internal microorganisms, including viruses, bacteria, and fungi. Here, the full-length coding sequence (CDS), protein structure, and immune response and subcellular localization of TLR5 (TLR5S) and TLR13 were characterized in large yellow croaker ( TLRs (Toll-like receptors) were first discovered in Drosophila embryos and control the formation of the dorsal-ventral polarity of early embryos . FurtherTLRs are type I transmembrane proteins composed of extracellular, transmembrane, and intracellular signal domains: the extracellular domain has a repetitive leucine-rich repeat (LRR); the ligand-induced dimerization of TLR triggers the recruitment of adaptor proteins to the intracellular Toll/IL-1 receptor homology domain (TIR), thereby initiating signal transduction . The extThe first fish TLR gene was discovered in goldfish in 2002, and several TLRs were retrieved from the genomic data of pufferfish and zebrafish, respectively, in 2004 . So far,Sparus aurata can bind to flagellin of Vibrio anguillarum, and then, TLR5M activates the inflammatory response. But it is still controversial whether the exact function of TLR5S is to enhance the innate immune response or to avoid the excessive inflammatory response of TLR5M [TLR5-ligand interaction in mammals, TLR5, is primarily responsible for the detection of a kind of flagellin in bacterial flagella and the specific identification of constant functional domains that are relatively conserved between different bacteria . To fishof TLR5M . TherefoAs per previous studies, TLR13 is a receptor for bacterial RNA; small-interfering RNA against TLR13 reduced cytokine induction by bRNA in DCs ; additioLarimichthys crocea is one of the most economically important fish endemic in the eastern and southern coastal waters of China. It has a well-developed innate immune system [L. crocea are seriously damaged by overfishing, while the cultured L. crocea is vulnerable to various pathogens [L. crocea TLRs are published, while TLR5, which has two forms, namely, membrane form (TLR5M) and soluble form (TLR5S), was poorly understood; meantime, TLR13, one of the important receptor, and the mechanism of innate immunity of large yellow croakers need further study. Therefore, this study was focused on three genes, and the results will contribute to our understanding of the immune system in fish.e system . Howeverathogens , 25. At The examined fishes (weight 800 ± 15 g), obtained from Zhejiang Dahaiyang Technology Co. Ltd. , were clinically healthy. They were maintained at 25°C in an aerated seawater tank and fed a commercial diet for two weeks prior to the start of the experiments. The water in the tank was changed daily.L. crocea after being anaesthetized by immersion in MS222.For the basal tissue expression analysis, tissues  were isolated from six unchallenged V. parahaemolyticus  and PBS (as control) at a dose of 300 μL/200 g, respectively. Then, the liver, kidney, and spleen tissues were collected from three fishes per group, at 0 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h postinjection. All procedures were performed under the guidelines of the Regulations for the Administration of Laboratory Animals  and were approved by the Animal Ethics Committee of Zhejiang Ocean University .To investigate the transcriptional modulations of lcTLRs, two independent groups of 100 individuals were infected with Total RNA was isolated from examined tissues using the Trizol Total RNA Kit  following the manufacturer's instructions. Then, the concentration was measured by a UV-spectrophotometer , and the quality of RNA was analyzed by 1% agarose gel by visualizing the intensity of 18 and 28s ribosomal RNA. After that, cDNA was synthesized using the M-MLV RTase cDNA Synthesis Kit  following the guidelines of its manufacturer's instructions. Finally, using the cDNAs as templates, PCR amplification was conducted using a thermal cycler  under the following conditions: 50°C for 2 min, 95°C for 30 s, and then 40 cycles of 95°C for 15 s, 58°C for 45 s, and 95°C for 15 s, followed by a final extension at 72°C for 5 min.μL using the PrimeScriptTM RT reagent kit  following the manufacturer's instructions. Then, qRT-PCR was carried out in a reaction mixture of 20 μL, containing 0.8 μL primer-F (10 μmol/L), 0.8 μL primer-R (10 μmol/L), 8 μL 2x SYBR ®Premix Ex TaqTM II, 1 μL cDNA sample (100 ng/μL), 0.4 μL ROX II, and 9 μL ddH2O (the reagent concentration refers to its manufacturer's instructions) in triplicate. The reference gene β-actin was used to normalize the expressions of TLR5M, TLR5S, and TLR13. Primers for qRT-PCR  to obtain the relative mRNA expression. The mRNA expression in muscle was treated as a control for comparisons with various tissues, and the 0 h time point comprised the control for the expressions post V. parahaemolyticus infection. Two-tailed Student's t-test was performed to determine the statistical significance of differences observed between the experimental and control groups using SPSS Statistics 19 (IBM). ∗p < 0.05 and ∗∗p < 0.01 were considered statistically significant and indicated in Results.All data were analyzed using the 2We used pcDNA3.1-C-eGFP plasmids  to transform the overexpressed protein TLR5S, TLR5M, and TLR13 with green fluorescence. Transfection was using FTX and PLUS reagents . Cells were seeded in 24-well plates and cultured. DAPI was used to locate the nucleus, and Dil was used to locate the cytomembrane. The localization of protein TLR5S, TLR5M, and TLR13 was observed under a Nikon TE2000 microscope.L. crocea whole-genome data [http://genes.mit.edu/GENSCAN.html) and ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/). The theoretical molecular weight (MW) and isoelectric point (pI) were determined by Expasy-ProtParam (http://web.expasy.org/protparam/). The domains of the proteins were predicted using the online tool SMART (http://smart.embl-heidelberg.de) and pictured by IBS (http://ibs.biocuckoo.org). The secondary structure and tertiary structure were predicted using Phyre2 (http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index) [https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) and in the Gene Database (https://www.ncbi.nlm.nih.gov/gene/). Multiple sequence alignments were performed with MEGA7 using ClustalW and ESPript3 (http://espript.ibcp.fr/ESPript/ESPript/index.php). The phylogenetic tree was constructed by MEGA7, using the neighbor-joining method.The gene sequences of TLR5M, TLR5S, and TLR13 were obtained from the ome data . Their cd=index) . The homThe coding region (CDS) of the TLR5M gene was deduced to be 2658 bp in length and encodes a protein of 885 aa with a predicted MW of 101.369 kDa and a pI of 5.69.The 1926 bp length CDS of the TLR5S gene encoded a protein of 641 aa with a theoretical MW of 71.612 kDa and a pI of 8.94. The domains of TLR5M and TLR5S and their homologous genes in other species are shown in β-sheets, 14 all located in the horseshoe-shaped concave surface, while the 7 α-helices were located on the horseshoe-shaped convex surface, and the rest were irregularly curled; the intracellular region was 5 α-helix surrounded by 4 parallel β fold. TLR5S had only the extracellular region, and its concave surface had 19 β folds, and the convex surface has 7 α-helices. TLR13 was composed of an intracellular region, a transmembrane domain, and an intracellular region. The extracellular region had 19 β-sheets and 7 α-helices, and the intracellular region was also surrounded by 6 α-helices with 4 β-sheets4 , TLR1, 2, 6, 8, and 10 were clustered together (TLR1 group), and the remaining TLRs formed a large group . The large groups of 13 TLRs could be subdivided into 3 groups: TLR7, 8, and 9 clustered into one group (TLR7 subgroup); TLR3 and TLR5 clustered together (TLR3 subgroup); TLR11, 13, 19, 20, 21, and 22 clustered into a group (TLR11 subgroup). Previously, vertebrate TLRs were divided into six major TLR families  according to evolution. Our evolutionary analysis results were similar to their conclusions, but TLR3 and TLR5 were classified into one family, and the TLR11 family was complemented by TLR19 and TLR20.The correct localization of a protein in a cell is crucial to understand the function of the protein. Hence, we examined the subcellular localization of TLR5M, TLR5S, and TLR13 . As the The functional domains of TLRs were highly conserved, and the differences were mainly expressed in the N-terminal LRR domain. The LRR domain is the extracellular domain of the TLR and is involved in the recognition and binding of ligands, so its N-terminal differences should have an effect on TLR recognition and binding ligands.Recent studies demonstrate that piscine contains two types of TLR5, namely, membrane form of TLR5 (TLR5M) and soluble form of TLR5 (TLR5S), both of which perform a crucial role in immunity response .Miichthys miiuy TLR5M and two more LRRs than the human TLR5M. TLR5S had only the extracellular region, and its concave surface had 19 β folds, and the convex surface has 7 α-helices. TLR5M and TLR5S were also studied in a few fish species, such as Pelteobagrus vachelli, Epinephelus coioides, M. miiuy, Pangasianodon hypophthalmus, and Paralichthys olivaceus [It was obvious that TLR5M had 11 LRRs, 1 LRR_CT, a transmembrane domain, and a TIR domain, which was one LRR less than livaceus –31.Although the functional domain predicted structure of TLR5M did not have LRR_NT, its N-terminus contained a typical 24Cx(8)C33 cysteine cluster of LRR_NT and was conserved almost exclusively among all fish TLR5M. LRR_NT (19-50) of TLR5Ss also had a Cx(8)C cysteine cluster, which was conserved between humans and fish . LRR_CT Ctenopharyngodon idellus yielded similar result sequences of TLR5M/S, and TLR13 contain multiple conserved Cys residues, which are likely to form disulfide bonds to help maintain the stability of the extracellular structure of TLRs , but TLR13 has one LRR less than sldTLR13, while sldTLR13 had one N-terminal LRR-TYP more than TLR13 (E. coioides), the cDNA of gTLR13 is 3559 bp in length, and the ORF is 2844 bp in length, encoding a protein of 947 amino acid residues (aa). Its domain includes a signal peptide, 13 LRR, a C-terminal LRR, a transmembrane domain, and a TLR structure [The length and functional domain structure of TLR13 is similar to that of an TLR13 . LRR numtructure .L. crocea and other species was in the number, type, and distribution of N-terminal LRRs 41 [Analysis of TLR sequences revealed that all three functional domains  of TLRs, except TLR5S (without transmembrane and intracellular TIR domains) and TLR21 (transmembrane domain deletion), are available . The dif LRRs 41 .Transcriptional expression of lcTLR5/TLR13 and genes in the TLR signal pathway TLR5 is located on the cell membrane, and it is capable of detecting flagellin and specifically recognizing a relatively conserved constant domain among different bacteria. TLR13 is a TLR that localizes to endosomes and recognizes nucleic acids released by internal microorganisms, including viruses, bacteria, and fungi .C. irritans can cause an increase in TLR5M and TLR5S transcription levels in the fins and spleen of E. coioides [M. miiuy TLR5S in the liver and kidney increased significantly after infection by Vibrio harveyi [S. maximus and found that TLR5M was upregulated in fin, head, kidney, and spleen [L. crocea. As shown in Studies have shown that stimulation of coioides . The exp harveyi . Jiang ed spleen . In addid spleen . It has d spleen . Howeverd spleen . In thisFrom the phylogenetic tree, we found that all TLRs were clustered into 4 groups, and the TLR5M/S and TLR13 were clustered in one group, although the genetic distance between them is not so close as that between TLR5M/S and TLR13, which may be the reason for the similar 3D model of TLR5M. All TLRs were clustered into 4 groups, in which TLR15 and TLR4 were grouped separately (TLR15 group and TLR4 group), TLR1, 2, 6, 8, and 10 were clustered together (TLR1 group), and the remaining TLRs form a large group ; the large groups of 13 TLRs could be subdivided into 3 groups: TLR7, 8, and 9 clustered into one group(TLR7 subgroup), TLR3 and TLR5 clustered together (TLR3 subgroup), and TLR11, 13, 19, 20, 21, and 22 clustered into a group (TLR11 subgroup); previously, vertebrate TLRs were divided into six major TLR families  according to evolution. Our evolutionary analysis results were similar to their conclusions, but TLR3 and TLR 5 were classified into one family, and the TLR 11 family was complemented by TLR19 and TLR20.L. crocea, which was different from other fish; TLR13 localized in endosomes, which basically recognize nucleic acids released by intracellular microorganisms, including viruses, bacteria, and fungi. In this study, TLR13 of L. crocea protein was distributed in endosomes; its proteins were localized in the cytoplasm in a punctuate manner.As per the previous studies, there two types of TLR5 of fish, namely, membrane form of TLR5 (TLR5M) and soluble form of TLR5 (TLR5S) . Interes


N,N-dimethylacrylamide—and evaluated the MPCP’s usefulness for dry eye treatment using a rabbit model treated with N-acetylcysteine. The MPCP particle size was 50–250 nm, and the form was similar to that of micelles. The MPCP viscosity (approximately 0.95 mPa·s) was 1.17-fold that of purified water, and a decrease in the transepithelial electrical resistance value  was not observed in the immortalized human corneal epithelial cell line HCE-T cell (HCE-T cell layer). The MPCP enhanced the water maintenance on the cornea, and the instillation of MPCP increased the lacrimal fluid volume and prolonged the tear film breakup time without an increase in total mucin contents in the lacrimal fluid of the normal rabbits. The therapeutic potential of the MPCP for dry eye was evaluated using an N-acetylcysteine-treated rabbit model, and, in our investigation, we found that MPCP enhanced the volume of lacrimal fluid and promoted an improvement in the tear film breakup levels. These findings regarding the creation and characteristics of a novel MPCP will provide relevant information for designing further studies to develop a treatment for dry eyes.The polymer that includes 2-methacryloyloxy ethyl phosphorylcholine (MPC) is well-known as an effectively hydrating multifunction agent. In this study, we prepared an MPC polymer (MPCP) using radical polymerization with co-monomers—MPC/Stearyl Methacrylate/ Dry eye disease is a complex and multifactorial ocular disease, and tear film instability, visual disturbance, and discomfort are commonly observed, with potential damage to the ocular surface . It was ®  and Diquas®  in Asia; Xiidra®  and Restasis®  in North America; and Ikervis®  in Europe. In addition, CequaTM  has been used in the US since 2018. CequaTM enhances lacrimal fluid production in patients with dry eye disease. Overall, these medicines are used to stabilize the tear film and/or prevent the inflammation of the ocular surface [Current treatments for dry eye disease aim to prevent the objective signs and clinical symptoms of the disease and recover the quality of life of the patients. The main approved treatments are as follows: Mucosta surface .The polymer including 2-methacryloyloxy ethyl phosphorylcholine (MPC) is well-known for being not only an antiadhesive and antithrombogenetic agent, but also a significant hydrating multifunction agent ,9,10. ThN,N-dimethylacrylamide (MPCP), and evaluated the polymer’s usefulness for dry eye treatment using a rabbit model treated with N-acetylcysteine.In this study, we prepared a novel MPC polymer using radical polymerization with co-monomers, MPC/stearyl methacrylate/N-acetylcysteine six times per day . This protocol was performed following our previous report [Male adult rabbits (weight 2.58 ± 0.75 kg) were obtained from Shimizu Laboratory Supplies Co., Ltd. , and the protocol was approved by the Kindai University . The experiments using the rabbits were performed according to the Association for Research in Vision and Ophthalmology (ARVO) and Kindai University guidelines. Thirty microliters of 0.1% MPCP were instilled in single applications at 14:00 h. For repeat applications, 0.1% MPCP (30 µL) was instilled once a day (14:00 h) for 5 days, and the measurement of the levels of lacrimal fluid, mucin, tear film breakup time (TBUT), ocular surface, and tear film breakup began at 16:00 h (single application) and 18:00 h (repetitive application). The dry eye model using rabbits was conducted via the instillation (30 µL) of 10% TM  and rat tail collagen type 1 were purchased from Costar  and Sigma , respectively. All other chemicals used were of the highest purity commercially available.Dulbecco’s modified Eagle’s medium/Ham’s F12, penicillin, streptomycin, and fetal bovine serum were provided from GIBCO . Cell Count Reagent SF was purchased from Nacalai Tesque, Inc. , and the Tear mucin assay ELISA kit was obtained from Cosmo Bio Co., Ltd. . Transwell-ClearN,N-dimethylacrylamide, with a composition ratio of 50:5:45, and purified using the dialysis method. After polymerization, we measured the residual monomers and calculated that the conversion rate of this polymer in each monomer was >99%. We thus confirmed that the target polymer was obtained. In this study, we used MPCP diluted to a 1% aqueous solution with purified water. To confirm the MPCP concentration of the aqueous solution, we studied the residue upon drying. The structural formula of the MPCP is illustrated in MPCP was obtained via radical polymerization with co-monomers, MPC/stearyl methacrylate/A NANOSIGHT LM10  was used to measure the size and number of MPCP nanoparticles—the measurement was performed for 60 s at 405 nm. Atomic force microscopy (AFM) images were obtained via an A SPM-9700 , and AFM images were created by combining the phase and height images. The viscosity of MPCP was measured with an SV-1A at 10–40 °C  . To confThe immortalized human corneal epithelial cell line (HCE-T cell) used in this study were developed by Araki-Sasaki et al. . The HCE4 cells) in a culture medium containing 0.1% MPCP and incubating the medium for 12 h. Then, a Cell Count Reagent SF was added, and the absorbance (Abs) at 450 nm was measured according to the manufacturer’s protocol. The Cell Count Reagent SF is based on the conversion of the reagent to formazan salts according to mitochondrial activity and, as a metabolic assay, is related to the cell number and its metabolic state and efficiency. Therefore, the cell number was also measured via counting under a microscope , and the cell adhesion was evaluated using a combination of data on the cell number and Cell Count Reagent SF. The cell adhesion (%) was represented as the Abs ratio of the MPCP treatment and non-treatment [The MPCP treatment was carried out by seeding HCE-T cells (1 × 10reatment .4 cells) by changing to a culture medium with 0.1% MPCP and incubating the medium for 24 h. Then, the Cell Count Reagent SF was added, and the Abs at 450 nm was measured according to the manufacturer’s protocol. Cell proliferation (%) was recorded as the Abs ratio of the MPCP treatment and non-treatment [The cell cultures were treated with MPCP 1 d after seeding  coated with rat tail collagen type 1 (71.5 µg/cm2) and grown for seven days until the cells reached confluency. The HCE-T cells were then exposed to an air–liquid interface for two weeks with the culture medium containing the vehicle or 0.1% MPCP replaced every other day. In this process, 50 µL of the medium solution with or without MPCP was dropped onto the donor side twice a day (9:00 and 19:00 h) so that the donor would not dry completely. In this study, chopstick electrodes connected to an epithelial Volt–Ohm meter Millicell-ERS  were used to measure the transepithelial electrical resistance (TER) and followed the differentiation stages during cultivation.The cell layer models (multilayer) consisting of one cell (only HCE-T cells) were cultured by following our previous reports ,18. The 2 were used for the cell toxicity analysis. The HCE-T cell layer model was treated with the vehicle and 0.1% MPCP, and the changes in TER values were measured for 60 min [Differentiated cells with TER values greater than 350 Ω·cmr 60 min .The rabbits were euthanized by injecting a lethal dose of pentobarbital, and the corneas were carefully separated from other ocular tissues. The individual corneas were treated with the vehicle, 0.1% MPC-BMA, and 0.1% MPCP for 1 min. After that, the cornea was placed on a plastic cell, and the changes in the weight and number of MPCP particles were measured at 22 °C. In this study, the changes in weight were expressed as water retention in the cornea. Moreover, the samples  on the cornea were collected by pipette, and the number of MPCP particles in the samples was measured with NANOSIGHT LM10, as described above.The ocular surface was monitored by using a dry eye monitor DR-1 , and the TBUT and changes in the ocular surface were monitored following our previous study . Each raThe volume of lacrimal fluid in rabbits instilled with MPCP was measured using Schirmer tear test strips , and the mucin was measured using a Tear mucin assay ELISA kit according to the manufacturer’s instructions. Briefly, lacrimal fluid was collected with Schirmer tear test strips, and the Schirmer tear test strips were added into elution buffer of a tear mucin assay ELISA kit to extract the mucin. After that, the extracted mucin was measured by the Tear mucin assay ELISA kit, and a fluorescence microplate reader (Absorption/Emission = 336/383 nm) . The muct-test, and Dunnett’s multiple comparisons test was used for the statistical analysis, where p < 0.05 was considered significant. The data are expressed as the mean ± standard error (S.E.).Differences between the mean values were analyzed with an ANOVA, followed by Student’s N,N-dimethylacrylamide accelerates the polymerization between MPC and stearyl methacrylate and strengthens the structure of polymer nano-sphere in an aqueous solution. 8 particles/mL). n = 3), the contact angle using MPC-BMA was 26.2 ± 2.0° (n = 3), and the control was 29.2 ± 0.4° (n = 3). These results indicate that the MPCP was slightly hydrophilic and had a higher wettability than the MPC-BMA and the control.2 when we exposed them to an air–liquid interface for two weeks. The TER in the MPCP-treated HCE-T cells was similar to non-treated values before exposure to the air–liquid interface; however, during exposure to the air–liquid interface, the TER values in the MPCP-treated HCE-T cells were significantly higher than those found in the HCE-T cells without MPCP treatment. n = 5) and TBUT  in the rabbits instilled with MPC-BMA tended to increase, although both the lacrimal fluid volume and TBUT were significantly lower than those in the MPCP. As shown in N-acetylcysteine-treated rabbit model  MPCP. Strong tear film breakup levels were observed and still persisted five days later. Repeat treatment with MPCP attenuated the tear film breakup levels five days post-N-acetylcysteine treatment, and the therapeutic effect of MPCP was significantly higher than that in MPC-BMA .Next, we investigated the usefulness of MPCP as a therapy for dry eye using the it model . The levectively A,B. Bothectively A,B. FiguIn this study, we designed a novel MPCP that includes the zwitterionic group stearyl methacrylat, and the acryl group, and found that the MPCP has a high affinity for water and moisturizes the ocular surface in comparison with the MPC-BMA reported previously (preexisting MPC polymer) . We alsoN,N-dimethylacrylamide was used to prepare the MPCP in this study, and MPCP was purified using the dialysis method. The over-30-nm nanoparticles reported previously were not detected in the MPC-BMA [First, we designed MPCP and evaluated its characteristics. MPC/stearyl methacrylate/ MPC-BMA . In contNext, we investigated the effects of MPCP on corneal epithelial cells using HCE-T cells. We previously reported that MPC-BMA does not affect the cell adhesion and growth of HCE-T cells, with little toxicity in HCE-T cells . The MPCFurther, we investigated the changes in the ocular surface in normal rabbits instilled with MPCP . The insWe found that the number of MPCP nanoparticles decreased on the cornea after treatment C. The MPN-acetylcysteine, a reducing agent and mucolytic [N-acetylcysteine caused a decrease in the mucin layer in the conjunctiva and cornea, desquamation of conjunctival and corneal epithelial cells, elimination of microvilli [N-acetylcysteine is widely applied as a mucin-reduced model and may be useful for evaluating the therapeutic effect of the MPC polymer for dry eye. Therefore, we treated a rabbit model with N-acetylcysteine and measured the effect of MPCP on the ocular surface. First, we attempted to measure the tear film break-up time , although it remains important to assess the systemic parameters and pharmakokinetics in the rabbit model. In addition, further studies are needed to apply the MPCP as dry eye drops. In future work, we intend to measure the therapeutic effect under a combination of MPCP and commercially available eye drops, such as MucostaN,N-dimethylacrylamide (50:5:45) and demonstrated that this MPCP provides high water affinity in comparison with the preexisting MPC polymer. The instillation of the MPCP prolonged the retention of lacrimal fluid in the eye and enhanced the TBUT in the rabbits. In addition, the MPCP accelerated the normalizing of the ocular surface in the dry eye model by keeping the tear film on the cornea. Moreover, the therapeutic effect of MPCP was significantly higher than that of the MPC-BMA reported previously [In this study, we prepared a novel MPCP using MPC/stearyl methacrylate/eviously . Thus, t