Non-arteritic anterior ischemic optic neuropathy (NAION) causes a sudden loss of vision and lacks effective treatment. Granulocyte colony-stimulating factor (G-CSF) provides neuroprotection against the experimental optic nerve injuries but also induce leukocytosis upon typical administration. We found synergetic neuroprotective effects of meloxicam and low dose G-CSF without leukocytosis in a rat model of anterior ischemic optic neuropathy (rAION). The WBC counts in the low-dose G-CSF-plus meloxicam-treated group were similar to the sham-operated group. Combination treatment of low-dose G-CSF plus meloxicam preserved RGCs survival and visual function, reduced RGC apoptosis and the macrophages infiltration, and promote more M2 phenotype of macrophage/microglial transition than the low-dose GCSF treatment or the meloxicam treatment. Moreover, the combination treatment induced higher serine/threonine kinase 1 (Akt1) expression. The combination treatment of low-dose G-CSF plus meloxicam lessened the leukocytotic side effect and provided neuroprotective effects via Akt1 activation in the rAION model. This approach provides crucial preclinical information for the development of alternative therapy in AION. The debilitating consequences for patients with NAION include severe vision impairment and visual field defect. The primarily proposed pathogenesis of NAION is the transient nonperfusion or hypoperfusion of the optic nerve head and loss of retinal ganglion cells (RGCs) following ischemic insults. The histologic evidence in an early clinical case of human NAION demonstrated the presence of apoptotic cells in the RGC layer by 30 days after the event. This reaction eventually results in RGC death and vision loss4. Currently, no definite treatment for NAION is available. Halting the injury and loss of RGCs may, therefore, rescue the visual deterioration and be a potential treatment strategy.Nonarteritic anterior ischemic optic neuropathy (NAION) is the most common acute optic neuropathy in people older than 50 years. The estimated mean annual incidence rates of NAION are 2.3–10.3 per 100,000 in the United States and 3.72 per 100,000 in Taiwan5. G-CSF can induce the mobilization of CD34 + hematopoietic stem cells from the bone marrow into the peripheral blood. G-CSF is commonly used in clinics for stem cell mobilization and bone marrow reconstitution8. Our previous studies have demonstrated that G-CSF exerts neuroprotective effects on a rat model of anterior ischemic optic neuropathy (rAION). The protective effects of G-CSF on the rAION model are achieved through dual mechanisms of anti-inflammation and antiapoptosis10. The antiapoptotic effect of G-CSF occurs through the activation of a variety of intracellular signaling pathways and is mainly dependent on phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) activation12. Early intervention with G-CSF can induce M2 microglia/macrophage polarization, reduce the expression of proinflammatory cytokines, and stabilize the blood–optic nerve barrier (BOB) to reduce macrophage infiltration in the rAION model10. However, systemic treatment with G-CSF may result in leukocytosis and some side effects, such as arthralgia, bone pain, headache, fatigue, nausea, fever, chills, and myalgia13. Our previous study also demonstrated that subcutaneous administration of G-CSF (100 μg/kg/day) for five consecutive days could result in leukocytosis in rats11. Thus, developing a new approach to reducing the side effects of G-CSF treatment and maintaining the therapeutic effects in the rAION model may provide benefits.Granulocyte colony-stimulating factor (G-CSF), a member of the cytokine family of growth factors, is a 19.6-kDa glycoprotein. It is commonly applied clinically to treat neutropenia14. The possible mechanism of meloxicam is inhibition of migration of leukocytes and blocking cyclooxygenase (COX), the enzyme responsible for the first step in the synthesis of prostaglandins15. COX can convert arachidonic acid into prostaglandin H2, which is a mediator of inflammation. Meloxicam has been demonstrated to primarily inhibit COX-2, the cyclooxygenase isozyme, mainly in inflamed tissues with long-lasting anti-inflammatory and analgesic effects16. Preferential inhibition of COX-2 leads to high anti-inflammatory potency with lower ulcerogenicity in the stomach and higher tolerability than non-selective NSAIDs. Moreover, the metabolites of meloxicam are inactive16. Therefore, it has the potential to reduce inflammation in ischemic optic neuropathy. However, whether meloxicam can enhance the therapeutic effects of G-CSF treatment for AION remains unclear.Meloxicam belongs to the enolic acid group of nonsteroidal anti-inflammatory drugs (NSAIDs) and is clinically prescribed for treating certain types of arthritis, such as rheumatoid arthritis, for reducing inflammation and pain17. Activation of M2 phenotype macrophages, reduction in proinflammatory cytokine expression, and stabilization of the BOB are supposed to be three vital approaches to optic nerve protection in the rAION model18. Enhancement of anti-inflammatory actions through different mechanisms may serve as a potential strategy in the treatment of ischemic optic nerve injury. The purpose of this study was to investigate whether treatment with low-dose G-CSF plus meloxicam results in a synergistic effect of neuroprotection and reduction of the side effect of leukocytosis in the rAION model.Macrophage/microglia has been shown to be polarizable and classifiable into M1 and M2 phenotype by expressed cytokine/chemokine profiles, surface markers, and biological functions. M1 phenotype is responsible for immunostimulation, including inflammation trigger, cell proliferation inhibition, which may cause tissue damage; M2 phenotype promotes immunosuppression and inhibits inflammation, which may improve cell proliferation and facilitate tissue repair in nervous tissuesIn this study, adult male Wistar rats weighing 150–180 g were used. The rats were purchased from the breeding colony of BioLASCO Co., Taiwan. Animal care and experimental procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The Institutional Animal Care and Use Committee of Tzu Chi Medical Center approved all animal experiments.In the drug-induced leukocytosis study, 18 rats were equally divided into six groups including the sham-operated, the PBS-treated, the meloxicam-treated , the low-dose G-CSF-treated , high-dose G-CSF-treated , and low-dose G-CSF (50 μg/kg/day) plus meloxicam (0.125 mg/kg/day)-treated group. The meloxicam was administered via the oral route and the G-CSF via subcutaneous injection.In the therapeutic evaluation, after successful induction of AION in the rats, 120 rats were equally divided into four groups. Treatment with subcutaneous injection of low-dose G-CSF only once daily, meloxicam only, low-dose G-CSF plus meloxicam, and phosphate-buffered saline  alone immediately after the rAION procedure for a total of 5 consecutive days. Another 30 rats received sham laser treatment without a photosensitizing agent to serve as normal controls. The number of rats used in this study is summarized in Fig. After the drug administration, 2–3 mL of circulating blood was drawn by cardiac puncture from the rats one week after rAION induction. WBC counts were obtained.10. Before general anesthesia, the rats were treated with Alcaine and Mydrin-P eye drops for topical anesthesia and pupil dilation, respectively. The rat was administrated by intramuscular injections of a mixture of ketamine (40 mg/kg body weight) and xylazine  for general anesthesia. Subsequently, 2.5 mM rose bengal in PBS  was intravenously administered. After rose bengal injection, the optic disc was immediately exposed to an argon green laser system  for 12 1-s pulses20. A laser fundus lens (Ocular instruments Inc.) was used to focus the laser on the optic disc. .The detailed methods and protocol of FluoroGold labeling have been described in our previous reports10. For electrode placement, the sagittal region of the skull was opened in the rats. The 4 mm screw implants were pass through the skull approximate 1.5 mm and were placed at the frontal cortex and the primary visual cortex region of both hemispheres using stereotaxic coordinates. A visual electrodiagnostic system  was used to measure the FVEP. The number of sweeps per average was 64 for each rat. A comparison of the amplitude of the P1-N2 wave in each group was made to evaluate visual function (n = 12 rats per group).The detailed FVEP recording method has been described in our previous studiesThe rats were euthanized after four weeks; the eyes were enucleated along with the optic nerve (approximately 5 mm in length). The samples were fixed in 4% paraformaldehyde and were cryoprotected by 30% sucrose; the samples were stored at 4 °C until they settled at the bottom of the tubes. Sections of 20 μm were obtained using a cryostat.To ensure the use of equivalent fields for comparison, all the paraffin or frozen sections of the retina were prepared with retinas at 1 to 2 mm distance from the ON head. TUNEL assay was used to detect apoptotic cells in the ganglion cell layer (GCL). This assay was performed according to the manufacturer’s procedure . The TdT-dUTP terminal nick-end labeling-positive cells in the GCL of each sample were calculated in 10 high-powered fields , and an average from three sections per retina was used for further comparison (n = 6 rats per group).ED-1 is a marker for phagocytic macrophages and microglia. The longitudinal section of the optic nerve was blocked with 5% FBS for one hour at room temperature. The section was labeled with an ED1 primary antibody diluted in the dilution buffer , and 0.3% Triton X-100; 1: 200) overnight at 4 °C. The section was incubated with Goat anti-mouse Alexa 488 (0.3% Triton X-100 and 1 × PBS (pH 7.2); 1: 500) for one hour at room temperature and counterstained with DAPI (0.3% Triton X-100 and 1 × PBS (pH 7.2); 1: 500). The image was acquired by using appropriate filter sets in a fluorescence microscope at × 100 magnification. ED-1+ cell counting was conducted by ImageMaster 2 Platinum software.18. Tissue RNA was extracted from optic nerve lysates obtained using the sonication method  with a Qiagen RNeasy Mini Kit . All the RNA samples were reverse transcribed at 42 °C for 30 minutes by using a high-capacity cDNA reverse transcription kit . qRT-PCR was then conducted on an AB PRISM 7300 Sequence Detection System (Applied Biosystems) with the QuantiTect SYBR green qRT-PCR kit (Qiagen). Expression levels of CypA mRNA were used for normalization to measure the expression levels of each target gene. The calculated data are presented as the mean relative expression levels ± standard deviation (SD). The primers used in this study for target gene amplification were listed in Table The markers of M2 macrophages  were evaluatedThe optic nerve samples were collected on day seven after rAION induction in each group. The protein extracts of the optic nerve were separated by using a 4–12% NuPAGE Bis-Tris gel . The separated proteins were transferred onto polyvinylidene difluoride membranes. The membranes were blocked by using 5% nonfat milk in Tris buffer saline/Tween-20 solution containing 20 mM Tris-HCl (pH 7.5), 0.5 M NaCl, and 0.5% Tween-20. Subsequently, the membranes were blotted with mouse anti-p-Akt1 antibody  followed by goat anti-mouse HRP. The developing reaction was performed by using an enhanced chemiluminescent substrate , and the relative intensities of the bands were measured by using an image analysis system .All data are represented as Mean ± Standard deviation. We performed statistical analysis with SPSS commercial software . Mann–Whitney U test was used to evaluate the differences between each group individually. Statistical significance was set at p < 0.05. All measurements in this study were performed in a masked manner.After 5-day treatment with high-dose G-CSF, low-dose G-CSF, meloxicam, and low-dose G-CSF plus meloxicam, the WBC counts were 6562 ± 662/μL, 6889 ± 693/μL, 6613 ± 557/μL, 19,633 ± 531/μL, 8897 ± 712/μL, and 9415 ± 657/μL in the sham, PBS-treated, meloxicam-treated, high-dose G-CSF-treated, low-dose G-CSF-treated, and low-dose G-CSF-plus-meloxicam-treated groups, respectively. The WBC counts increased by 2.99-fold in the high-dose G-CSF-treated group compared with the sham group (p = 0.0011), which indicates the leukocytosis effects of high-dose G-CSF. Treatments with meloxicam, low-dose G-CSF, and low-dose G-CSF plus meloxicam did not induce leukocytosis in the animals  and low-dose G-CSF-treated group (p = 0.021), respectively. Apoptotic cells (TUNEL + cells) in RGC layer in sham, PBS-, meloxicam-, low-dose G-CSF-, and the combination-treated group was 1.2 ± 0.8/HPF, 21.3 ± 2.4/HPF, 14.7 ± 3.2/HPF, 10.1 ± 4.6/HPF and, 4.1 ± 2.9/HPF, respectively  compared with treatment with meloxicam and low-dose G-CSF.In contrast to treatment with PBS, both the low-dose G-CSF- and meloxicam-treatment groups preserved a higher density of RGCs in the central retinas Fig. . Four weely Fig. . The numely Fig. . Treatmes in the combination-treated group compared with the meloxicam-treated (p = 0.021) and low-dose G-CSF-treated group (p = 0.032), respectively. Further, the qRT-PCR analysis demonstrated that the mRNA levels of Arg 1, CD206, and Fizz1 (M2 phenotypic markers) increased after treatment with meloxicam, low-dose G-CSF, and low-dose G-CSF plus meloxicam after rAION induction compared with PBS-treated group. In addition, the combination treatment exerted synergistic effects on the increased expression of Arg1, CD206, Fizz; (p = 0.005) in the rAION model  injection for five days commonly triggered leukocytosis in rats, but this undesired effect was absent with low-dose G-CSF (50 μg/kg/day) treatment. The administration of low-dose G-CSF alone or meloxicam alone yielded preservation of visual function, better RGCs survival, reduction of macrophage infiltration, and more M2 macrophage/microglia polarization after rAION insults. Notably, the neuroprotective effects of the combination treatment worked synergistically. In addition, the combination treatment induced more p-Akt1 expression than meloxicam or low-dose G-CSF treatment alone in the current model.12. We also found that leukocytosis was induced by this therapy in the rat ONC model11. Moreover, this therapy has detrimental effects on patients. Commonly encountered side effects after G-CSF use comprise arthralgia, bone pain, fatigue, nausea, fever, chills, headache, and myalgia13. Thus, for clinical applications, reducing the undesired effects induced by G-CSF therapy is essential for ophthalmic diseases. In this study, we found that consecutively treating normal Wistar rats with high-dose G-CSF (100 μg/kg/day) via subcutaneous injection would induce leukocytosis. By contrast, neither low-dose G-CSF (50 μg/kg/day) treatment nor meloxicam treatment would lead to the undesired effect. As expected, the combination treatment did not induce leukocytosis compared with normal rats. Furthermore, the addition of meloxicam to G-CSF treatment may reduce the discomfort caused by the side effects of G-CSF because meloxicam is a strong pain reliever. From the literature review, meloxicam possesses low toxicity to gastrointestinal tract mucosa and trustworthy efficacy against inflammation and pain24. Therefore, treatment with a combination of G-CSF and meloxicam is a reasonable approach for rAION.Based on our past work, we concluded that subcutaneous injection of high-dose G-CSF provided neuroprotection in both rAION and optic nerve crush (ONC) modelsin vitro26. A recent study reported that meloxicam’s neuroprotective effects maintained cell survival via the upregulation of the PI3K/Akt pathway instead of COX-2 inhibition26. In this study, we further disclosed that meloxicam induced Akt1 activation on day seven post-infarct. Couples of previous studies have indicated that the upregulation of the PI3K/AKT signaling pathway benefits the survival of injured RGCs29. In our previous study, we also demonstrated that the apoptosis of RGCs following optic nerve crush could be antagonized by G-CSF via PI3K/AKT-signaling pathway12. Taken together, we consider that meloxicam can maintain Akt1 activation to prevent RGC death after optic nerve damage. Remarkably, treatment with a combination of meloxicam plus low-dose G-CSF induced higher Ak1 activation than treatment with meloxicam only or low-dose G-CSF only in the rAION model. Therefore, we believe that combination treatment with meloxicam and low-dose G-CSF affords better protection of RGCs in the rAION model via antiapoptosis of RGCs and activation of the Akt1 signaling pathway.The neuroprotective role of meloxicam on the rAION model was first disclosed in this study. Surprisingly, meloxicam revealed comparable neuroprotective effects to low-dose G-CSF regarding antiapoptosis, inhibition of macrophage infiltration, and macrophage M2 polarization in this model. Meloxicam breaks the inflammatory cascade via inhibition of MAPK and p53 signaling pathway and owns antiapoptotic effects evidenced by alleviating oxidative stress, avoiding mitochondrial dysfunction, and reducing endoplasmic reticulum stress response 30. We found that ED1 + macrophage/microglial buildup at the optic nerve lesion site was alleviated in the meloxicam-treated and low-dose G-CSF-treated groups. The effect of attenuation was enhanced when the combination treatment of low-dose G-CSF plus meloxicam was used. Our recent study, therefore, implies that the rAION-injured optic nerves may be protected by the anti-inflammatory effects of immediate administration of meloxicam or G-CSF, and combination treatment with G-CSF plus meloxicam exerts the synergistic effect of reduction in macrophage recruitment. Some studies have demonstrated that treatment with G-CSF is able to reduce the breakdown of blood-brain barrier (BBB), brain edema, and macrophage infiltration in experimental brain injury models33. Also, our previous report revealed that protection from BOB disruption reduces macrophage infiltration into the optic nerve lesion site10. We believe that the possible mechanism of BOB disruption is associated with the modulation of the Akt signaling pathway in the rAION model. Our study also noted that Akt1 was activated by treatment with meloxicam or G-CSF alone in the rAION model. Therefore, we suggest that Akt1 activation may play a crucial role in preventing BOB breakdown after optic nerve infarct.After rAION induction, ED1 + phagocytes, including monocytes/macrophages of hematopoietic origin and microglia, are recruited into the optic nerves. The hematogenous ED1 + cells found at the optic nerve following rAION induction implicates disrupted BOB34. We ever reported that phenotype switch of macrophages from pro-inflammatory (M1) to anti-inflammatory (M2) prevent the cytokine-induced optic nerve injuries by reducing the pro-inflammatory cytokine expression and free radicals  during the inflammatory responses of optic nerve injury18. Intriguingly, our findings showed that meloxicam, a COX-2 inhibitor, alone was able to activate M2 polarization on the injured optic nerve, and the protective phenomenon was further magnified by simultaneous G-CSF and meloxicam administration.Macrophage/microglia has plasticity, and one phenotype can convert into another phenotype when driven by cytokines in different microenvironmentset al. reported that meloxicam has hematopoiesis-modulating action and can increase serum G-CSF levels in sublethally gamma-irradiated animals35. Enhancement of neuroprotective effects and M2 polarization from the combination treatment may be due to the increase in endogenous G-CSF after meloxicam administration. Also, we found that treatment with meloxicam alone or G-CSF alone activated Akt1 7 days after rAION induction. Since Akt downregulation will abolish the upregulation of M2 genes, Akt activation plays an important role in M2 macrophage differentiation36. Liu et al. demonstrated that TIPE2 activates the PI3K-AKT signaling pathway and subsequently promotes M2 polarization37. Furthermore, recent studies have reported that Akt1 and Akt2 kinase isoforms are critical to modulation of macrophage polarization and Akt1 ablation shifts macrophage towards M1 status38. Taken together, we conclude that Akt1 upregulation may be crucial to promote M2 polarization in the rAION model.Hofer In conclusion, addressing anti-inflammatory therapy through different mechanisms may provide a potential approach to the management of ischemic optic nerve injury. Combination therapy of G-CSF plus meloxicam can reduce the leukocytosis side effects, prevent postinjury RGCs apoptosis, preserve the visual function, halt the recruitment of macrophages to the optic nerve, and promote the M2 phenotype transition in macrophages. The neuroprotective effects of G-CSF and meloxicam worked synergistically. However, additional works are mandatory to more clearly elucidate the underlying mechanisms of the interaction between G-CSF and meloxicam.Supplementary information


K768 was located between α15 and α16 (termed the cis-loop). Cellular assays showed that K768 did not interfere with other toxin functions, including cell binding, intracellular trafficking, and pore formation. The equivalent K768 is conserved among the clostridial neurotoxin family of proteins as a conserved structural motif. The cis-loop appears to contribute to LC translocation.How protein toxins translocate their catalytic domain across a cell membrane is the least understood step in toxin action. This study utilized a reporter, β-lactamase, that was genetically fused to full-length, nontoxic tetanus toxin (βlac-TT) in discovery-based live-cell assays to study LC translocation. Directed mutagenesis identified a role for K soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]), while HC contains an N-terminal translocation domain (HCN) and a C-terminal receptor binding domain (HCC). HCN-mediated LC translocation is the least understood function of CNT action. Here, β-lactamase (βlac) was used as a reporter in discovery-based live-cell assays to characterize TT-mediated LC translocation. Directed mutagenesis identified a role for a charged loop (767DKE769) connecting α15 and α16 (cis-loop) within HCN in LC translocation; aliphatic substitution inhibited LC translocation but not other toxin functions such as cell binding, intracellular trafficking, or HCN-mediated pore formation. K768 was conserved among the CNTs. In molecular simulations of the HCN with a membrane, the cis-loop did not bind with the cell membrane. Taken together, the results of these studies implicate the cis-loop in LC translocation, independently of pore formation.The clostridial neurotoxins (CNTs) comprise tetanus toxin (TT) and botulinum neurotoxin (BoNT [BT]) serotypes (A to G and X) and several recently identified CNT-like proteins, including BT/En and the mosquito BoNT-like toxin Pmp1. CNTs are produced as single proteins cleaved to a light chain (LC) and a heavy chain (HC) connected by an interchain disulfide bond. LC is a zinc metalloprotease  in discovery-based live-cell assays to study LC translocation. Directed mutagenesis identified a role for K768 in LC translocation. K768 was located between α15 and α16 (termed the cis-loop). Cellular assays showed that K768 did not interfere with other toxin functions, including cell binding, intracellular trafficking, and pore formation. The equivalent K768 is conserved among the clostridial neurotoxin family of proteins as a conserved structural motif. The cis-loop appears to contribute to LC translocation. In contr humans , 5. The (SNAREs) , 7. ClosCC]/HCR) . CNTs shCC]/HCR) –10. CNT C]/HCR) –. Defininfluorescence resonance energy transfer (FRET)-based substrate, CCF2, resulting in loss of FRET detected as an unquenched emission of blue-wavelength fluorescence. The βlac-CCF2 reporter system is well established in detecting and characterizing both toxins and effectors , a β-lactamase tetanus toxin [TT(RY)]  fusion protein (βlac-TT) was developed as a reporter for a cell-based model to study LC translocation (trans-loop region (α18-loop–α18) comprising a CNT-conserved phenylalanine  is located away from the LC-HCN interchain disulfide, the cis-loop region (α15-loop–α16) comprising a CNT-conserved lysine  located near the LC-HCN interchain disulfide  required for LC translocation, independently of pore formation.Here, we used βlac-TT, a discovery-based, live-cell imaging reporter system in cells to map regions of HCN that contribute to LC translocation and identified a helix-loop-helix within HCN (cis-loop)   were tar612A, W615A, F623A) and βlac-TT] had a number of βlac translocation events similar to the number seen with βlac-TT (767AAA769), a cis-loop mutation, did not cleave cytosolic CCF2 (767RKK769) mediated numbers of βlac translocation events similar to the number seen with βlac-TT, showing that the acidic residues were not required for βlac translocation. βlac-TT(767DAE769) and βlac-TT(767AKA769) did not cleave cytosolic CCF2, indicating that the presence of 768K was necessary but not sufficient for reporter translocation  variants were subjected to trypsin digestion. βlac-TT and βlac-TT(767AAA769) were assembled with the interchain disulfide and yielded similar tryptic cleavage patterns (see 767AAA769) (767DKE769) variants bound neurons as efficiently as βlac-TT   and vari βlac-TT , indicat10.1128/mSphere.00244-20.1FIG S1cis-loop variants are stably expressed in E. coli and nick with trypsin as in the case of βlac-TT. (A) A 2-μg volume of purified βlac-TT wild type and cis-loop variants was resolved in reducing (βME) buffer by SDS-PAGE. (B) βlac-TT wild type and loop variants were incubated without (-) or with trypsin at the indicated concentrations , boiled, and resolved by SDS-PAGE under oxidizing and reducing conditions. βlac-TT and βlac-TT(AAA) digestions are shown as a representative sample. Download FIG S1, DOCX file, 0.1 MB.Copyright © 2020 Zuverink et al.2020Zuverink et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00244-20.2FIG S2cis-loop variants (40 nM) were incubated with primary rat cortical neurons in precooled low-K+ buffer for 30 min on ice as described previously  and βlac-TT(767RKK769) trafficked to the perinuclear vesicles void of synaptic vesicle 2C (SV2C), a synaptic vesicle marker protein, while HCC/A2 trafficked to SV2C-enriched perinuclear vesicles (cis-loop) variants were not defective in intracellular trafficking.TT and BT traffic to unique perinuclear vesicles of Neuro-2a cells . Like βlvesicles  12). HC. HC767AAvesicles  Thus, βlcis-loop variants mediated acidification-dependent trypan blue uptake, while βlac-HCC/T, which lacks pore-forming ability, mediated background levels of trypan blue uptake (cis-loop variants were not defective in pore formation.Trypan blue diffusion into the cytosol of Neuro-2a cells was used to measure pore formation by cell-bound βlac-TT . Tetanole uptake  26). Th. Thcis-lcis-loop predicted protonation of acidic residues D767 and E769 at low pH. Although βlac-TT(767AAA769) could not be directly assessed, pretreatment of primary neurons with bafilomycin inhibited βlac translocation by βlac-TT(767RKK769) and βlac-TT (767RKK769)  . Togethe10.1128/mSphere.00244-20.3FIG S3+ cells containing cleaved CCF2 divided by the total number of NeuN+ cells is indicated. (B) Three independent replicates are shown with standard errors of the means (SEM). **, P < 0.01; *, P < 0.05 (Student’s two-tailed t test). Download FIG S3, DOCX file, 0.05 MB.βlac-TT(RKK) cleavage of cytosolic CCF2 requires low pH and TrxR. Primary rat cortical neurons were preincubated with either 400 nM bafilomycin A1 (A) or 500 nM auranofin (B) for 30 min. Neurobasal media containing the concentrations of inhibitors indicated above with 40 nM single-chain βlac-TT (WT), βlac-TT(AAA), or βlac-TT(RKK) was incubated for 30 min at 37°C before loading with CCF2 was performed. The percentage corresponding to the number of NeuNCopyright © 2020 Zuverink et al.2020Zuverink et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the trans-loop (α18-loop–α18 (838F in TT), distanced from the LC-HCN interchain disulfide, and a cis-loop (α15-loop–α16) (767DKE769 in TT) near the LC-HCN interchain disulfide (5N0B) contained an analogous BoNT-switch like BT/A (PDB: 6DKK)  , implicaB: 6DKK)  showed tmembrane . After t767DKE769, within the α-helix15-loop–α-helix16 junction (cis-loop) was required for LC translocation but not intracellular trafficking or pore formation. Consistent with a role in orientating the unfolded LC for translocation were molecular simulations that located the cis-loop on the membrane surface  also failed to translocate βlac-LC. 768K was the only conserved amino acid within the cis-loop of the respective CNTs, supporting the idea of the cis-loop being a structural motif.LC translocation is the least understood step in CNT intoxication. This study characterized the role of conserved regions of the HCN required for LC translocation in TT, assuming that the mechanism of LC translocation is conserved among CNTs. In neurons, deletion of conserved loops and short helices of HCN in C-terminal regions relative to α12 reduced LC translocation efficiency, while deletion of N-terminal loop regions relative to α12 was dispensable for LC translocation (unpublished data). In addition, a short stretch of amino acids,  surface . These scis-loop structure is conserved in all BT serotypes, and the DKE sequence was homologous in BT/C and BT/D, but varied within the cis-loop of the other BT serotypes . Since α-TH5–α-TH6 has some membrane interaction properties, the cis-loop could contribute in membrane stability  that was previously shown to be needed for LC translocation  and amino acids that define the structural components of the cis-loop may provide additional details for LC translocation.Continued assessment of α-helix15-loop–α-helix16 (cis-loop (767DKE769 > 767AAA769 and 767DKE769 > 767RKK769) per kit instructions before transformation into Escherichia coli TG1. Primers for site-directed mutagenesis of the other βlac-TT cis-loop variants [residues K768A (DKE > DAE) and D767A and E769A (DKE > AKA)] and βlac-TT α12 mutations  were designed using Agilent or NEB Web-based tools. Briefly, templates and primers were mixed and amplified per the specifications in the Q5 kit instructions (New England Biolabs). Amplified product was incubated with KLD  before transformation of the entire reaction into E. coli TG1.βlac-TT(RY) and βlac-HCC/T were previously engineered , 41. DNAcis-loop variants were transformed into E. coli BL21(DE3) for expression and purified as previously described  at 1:1,000 to 1:250 (wt/wt) trypsin to variant in 20 mM sodium phosphate (pH 7.9)–50 mM NaCl for 1 h at 37°C to assess stability. Samples were inhibited every 10 min with 5 molar excess soybean trypsin inhibitor (Sigma-Aldrich). Proteins were subjected to 8% acrylamide SDS-PAGE (with beta-mercaptoethanol [βME]) and stained with Coomassie blue. βlac activity was assayed using fluorocillin green microplate assay (Thermo Fisher) (βlac-TT and  Fisher) .Mus musculus neuroblastoma, were cultured as described previously (d-lysine (Sigma-Aldrich) followed by assay 1 day after plating at ∼70% confluence. E18 rat cortices from Sprague Dawley rats  were triturated to single cells as described by the supplier and plated in NBActiv4   on glass-bottom total internal reflection (TIRF) plates (MatTek). TIRF plates were precoated with 20 μg/ml poly-d-lysine (Sigma-Aldrich) overnight, followed by 3 μg/ml mouse laminin for 3 h, and equilibrated with neurobasal medium for 30 min before plating cells in NBActiv4. Neurons were cultured for 7 to 12 days with a half-fresh media change, using NBActiv1  on days 4 and 7 postplating.Neuro-2a cells (ATCC CCL-131), from a eviously  with the+ buffer  and incubated on ice for 10 min. βlac-TT or scanning deletion variants or cis-loop variants (40 or 80 nM) were suspended in cooled low-K+ buffer and incubated with cells for 20 min on ice, buffer was replaced with prewarmed low-K+ buffer to reach pH 5.5 with 5 mM sodium citrate for 15 min, and the reaction mixture was incubated at 37°C . Cells were located as SV2C-positive stained cells, and Z-stacks were taken at 0.4-μm steps followed by cropping individual cells and blind deconvolution to estimate the point spread function (PSF) with 15 iterations.Plated Neuro-2a cells were loaded with 10 μg/well of sonicated ganglioside GT1b (Matreya) in minimal essential medium (MEM) containing 0.5% fetal bovine serum (FBS) for 4 h at 37°C. Cells were washed with prewarmed Dulbecco’s phosphate-buffered saline (DPBS) and incubated with 40 nM βlac-TT or variants in low-K+ buffer for 30 min on ice as described previously (βlac-TT and variants were incubated (10 or 40 nM) with precooled primary rat cortical neurons in low-Keviously . Cells w2+ and Mg2+ (HBSS−/−) (Life Technologies) and cooled to room temperature (RT) for 10 min followed by loading of media with 2 μM CCF2-AM (Life Technologies) and 1 mM probenecid (Life Technologies), an anion transport inhibitor, in HBSS−/− for 30 min. Neurons were washed and processed for immunofluorescence. The inhibitor-treated translocation assay was performed as described above, with the following preincubation modifications. Bafilomycin A1 (400 nM), a vesicular ATPase inhibitor (Sigma-Aldrich), was preincubated with neurons in neurobasal media for 30 min at 37°C before aspiration and application of 40 nM single-chain βlac-TT in neurobasal media containing inhibitor at the indicated concentration. Then, auranofin (500 nM), a thioredoxin reductase inhibitor (Sigma), was preincubated with neurons for 30 min at 37°C before its removal and addition of 40 nM single-chain βlac-TT in neurobasal media containing inhibitor at the indicated concentration. After being maintained 30 min with βlac-TT at 37°C, cells were washed and loaded with CCF2 as described above followed by fixation.Rat cortical neurons were incubated in equilibrated neurobasal media containing (10 or 40 nM) single-chain βlac-TT or variants for 30 min at 37°C. Neurons were washed with Hanks’ balanced salt solution lacking CaPrimary cortical neurons and Neuro-2a cells were fixed and permeabilized . Primarycis-loop variants, micrographs were acquired at ×20 magnification and neurons containing cleaved CCF2 were scored as positive/negative for βlac-LC translocation. At least 10 fields (containing 20 to 30 neurons) were analyzed per experiment, and at least 3 independent experiments were performed. For entry of cis-loop variants into Neuro-2a cells, Z-stacks were acquired and deconvolved. Pearson’s correlation coefficient was obtained using Nikon AR analysis software as described previously , and the structure was truncated to residues 564 to 870 [TT(HCN)], using PyMol. A coarse-grain structure with an elastic network was generated using the martinize.py script (version 2.2) with secondary-structure assignment provided by the Define Secondary Structure of Proteins (DSSP) program (version 2.0.4). Lipid and amino acid parameters were taken from Martini Lipidome and Martini (version 2.2). Simulations were performed as follows. The force field used for generating the coarse-grain structure was martini22, with an elastic bond force constant (-ef) of 500, an elastic bond upper distance bound (-eu) of 0.9, and an elastic bond lower bound (-el) of 0.5. The lipid bilayer system was generated using the insane.py script with a 20-, 20-, 30-nm box (xyz) with 48% phosphatidylcholine, 31% phosphatidylethanolamine, 9% phosphatidylserine, 5% sphingomyelin, and 3% phosphatidylinositol to mimic the mixtures of lipids found in neuronal vesicles. Using GROMACS 2018 (released 10 January 2018), the energy of the system was minimized for 2,000 steps with a 0.02-ps step size (40 ps) using the steep integrator. The system then underwent temperature equilibration from 0 to 300 K using v-rescale temperature coupling for 10,000 steps with a 0.01-ps step size (100 ps) and the md (leap-frog) integrator. Then, the system was equilibrated to 1 bar using the Berendsen barostat with semi-isotropic pressure coupling for 50,000 steps with a 0.01-ps step size (500ps) and the md (leap-frog) integrator. The system was finally equilibrated to 1 bar using the Parrinello-Rahman barostat with semi-isotropic pressure coupling for 50,000 steps with a 0.01-ps step size (500 ps) and the md (leap-frog) integrator. Simulations were carried out on MCW Research Computing Cluster Tesla using Tesla K40 and K80 graphics processing units (GPUs). Trajectories were converted and analyzed using VMD (version 1.9.3).Gangliosides were removed from the crystal structure file of TT (PDB: t test was utilized to determine if two data sets were significantly different where appropriate.Student’s unpaired, two-tailed


K and ICa,L in H9c2 cells treated with and without tt-DDE were measured using the whole-cell patch clamp method. Expressions of Kv2.1 and Cav1.2 in H9c2 cells treated with and without tt-DDE were measured by western blot analysis. After the H9c2 cells had been exposed to tt-DDE, the IK and ICa,L were significantly decreased. The expression of Kv2.1, unlike that of Cav1.2, was also significantly decreased in these cells. These changes in IK and ICa,L that were induced by tt-DDE may help to explain the association between cardiac arrhythmogenesis and cooking-oil fumes.Household air pollution has adverse effects on cardiovascular health. One of the major sources of household air pollutants is the combustion of cooking oils during cooking. Trans, trans-2,4-decadienal (tt-DDE) is a type of dienaldehyde that is present in a wide range of food and food products. It is a byproduct of the peroxidation of linoleic acid following the heating of oil during cooking. The mechanisms of the associations between household air pollution and cardiac arrhythmias are currently unclear. The purpose of this study was to determine effects of tt-DDE on the ion currents in H9c2 cells. The I Exposure to air pollution could increase the risks of ischemic heart disease, heart failure, cerebrovascular disease, and cardiac arrhythmias4. In the Air Pollution and Cardiac Risk and its Time Course (APACR) study, 60 minutes of exposure to PM2.5 (particles with a diameter of ≤2.5 μm) was associated with increased premature ventricular contractures5. Another study demonstrated that brief exposure to air pollution could trigger atrial fibrillation6. The mechanisms of the associations between air pollution and cardiovascular diseases are currently unclear. It has been proposed that increased systemic inflammatory responses, systemic oxidative stress, systemic and pulmonary artery blood pressure, and risks of atherosclerosis, as well as changes in autonomic function indicate increased cardiovascular morbidity and mortality caused by air pollution9.Epidemiological studies have revealed that exposure to air pollutants, especially particulate matter (PM), is associated with an increased incidence of respiratory diseases, as well as of cardiovascular morbidity and mortality10. Indoor exposures to PM2.5 is associated with alternation of heart rate variability that could in turn increase vulnerability to cardiac arrhythmias11. One of the major sources of household air pollutants is the combustion of cooking oils during cooking. A longitudinal study conducted by Chinese military cooks showed that exposure to compounds derived from cooking-oil fumes could cause oxidative DNA damage and lipid peroxidation12. However, the effects of the contents of cooking-oil fumes on vulnerability to cardiac arrhythmias is unknown. Trans, trans-2,4-decadienal (tt-DDE) is a type of dienaldehyde that is present in a wide range of food and food products. It is a byproduct of the peroxidation of linoleic acid following the heating of oil during cooking13. The alteration of ions currents in cardiomyocytes is one of the mechanisms involved in arrhythmogenesis. The purpose of this study was to determine these effects of tt-DDE on the ions currents in cardiomyocytes.As comprehensively demonstrated in previous studies, household air pollution also has adverse effects on cardiovascular healthK) and L-type calcium channel current , H9c2 cells were co-cultured with tt-DDE at a concentration of 1 µM.Figure K were evoked in the H9c2 cells treated with tt-DDE-free media for 18 hours (control group) with 300 ms of depolarizing step pulses from −70 to +50 mV in 10 mV increments at a holding potential of −60 mV. As described in a previous report14, IK first appeared significantly at the membrane potential of −20 mV and the currents amplitude increased with more positive membrane potentials until it reached the at membrane potentials of 50 mV   Fig. .Figure 2K were evoked in the H9c2 cells with 300 ms of depolarizing step pulses from −70 to +50 mV in 10 mV increments at a holding potential of −60 mV. Compared with the activation pattern of the IK in the control group, the IK amplitudes in the cells of this group were significantly smaller  currents and membrane potentials in the control and tt-DDE groups are analyzed in Fig. K in the tt-DDE group were significantly lower than those in the control group from 0 to 50 mV , Iler Fig. . The aveCa,L current in the control group. The ICa,L current was recorded, and it was significantly suppressed by verapamil  channels and L-type calcium (Ca2+) channels composed mainly of Kv2.1 α-subunits and Cav1.2 subunits, respectively15. Compared with the control group, the expressions of Kv2.1 in the tt-DDE group was significantly decreased   Fig. . The exp03) Fig. .Figure 4K and ICa,L were significantly decreased. The expression of Kv2.1, a component of the K+ channel, unlike that of Cav1.2, a component of the Ca2+ channel, was also significantly decreased in these cells. These changes in IK and ICa,L that were induced by tt-DDE may help to explain the association between cardiac arrhythmogenesis and cooking-oil fumes.After the H9c2 cells had been exposed to tt-DDE, the I7. Gold et al. investigated the association between the level of ambient pollution and cardiovascular function3. They found that exposure to PM2.5 and ozone could decrease vagal tone, resulting in reduced heart rate variability. As previous studies, air pollution is associated with changes in cardiac ventricular repolarization, regardless of the duration of exposure16. The APACR study showed that increases of 10 µg/m3 in the average PM2.5 concentration were associated with 8% and 3% increases in the average premature ventricular contraction count in the same and subsequent 30 min periods, respectively5. Link et al. studied the association between air pollution and cardiac arrhythmias in patients with implantable cardioverter defibrillators. They found that PM2.5 exposure was associated with an increased risk of atrial fibrillation within the subsequent hours in patients with known cardiac disease6. Indoor air pollution has also contributed to changes in heart rate variability and electrocardiography. A study of housewives in Taiwan showed that indoor exposure to PM2.5, especially during stir-frying, cleaning with detergents, and burning incense, was associated with changes in the heart rate variability index11. Collectively, these results suggest that indoor air pollution, especially in the kitchen, may contribute to cardiac arrhythmogenesis. However, the mechanisms by which arrhythmogenesis is caused by air pollution have not been well studied.Many studies have demonstrated an association between air pollution and cardiac arrhythmias13. Also, tt-DDE could induce the generation of reactive oxygen species and cell death in lung cells17. However, the cytotoxicity of tt-DDE to cardiomyocytes is seldom discussed.Cooking-oil fumes are major air pollutants in the kitchen. There is abundant tt-DDE in cooking-oil fumes, and exposure to cooking-oil fumes containing tt-DDE could induce oxidative stress and genotoxicity in human lung cellsK and ICa,L in H9c2 cells were significantly decreased. This effect may prolong the duration of the action potential of cardiomyocytes, which could increase vulnerability to cardiac arrhythmogenesis.Triggered activity, abnormal automaticity, and re-entry are major mechanisms of cardiac arrhythmogenesis. Alterations of function in ion channels and ion currents in cardiomyocytes could result in triggered activity and cardiac arrhythmias. In the current study, we found that after 18 hours of exposure to tt-DDE, the IK in H9c2 cells treated with tt-DDE. In contrast, the expression of Cav1.2 in the H9c2 cells was not significantly affected by treatment with tt-DDE. This suggests that the toxic effect of tt-DDE on the ICa,L in cardiomyocytes may not directly affect the L-type Ca2+ channel. The effects of tt-DDE on the IK and ICa,L in cardiomyocytes should be further investigated.This study also showed that the expression of Kv2.1 was significantly decreased in H9c2 cells treated with tt-DDE. This decrease in the expression of Kv2.1 in H9c2 cells could be one of the mechanisms of decreasing the IK and ICa,L of H9c2 cells were determined using only one concentration of tt-DDE (1 µM). We did not analyze effects of tt-DDE on IK and ICa,L with different concentrations of tt-DDE. Results of this study can not conclude the concentration-related effects of tt-DDE on the Ik and ICa,L of H9c2 cells. However, results of this study suggested that tt-DDE could affect the Ik and ICa,L of H9c2 cells. Second, although decrease of the IK and ICa,,L of H9c2 cells by exposure to 1 µM tt-DDE demonstrated in vitro in this study, how much and how long exposure to cooking-oil fume could achieve similar effects by exposure of 1 µM tt-DDE in vivo were unknown. Further studies in vivo are needed.There are some study limitations in this study. First, the effects of tt-DDE on the IK and ICa,L of cardiomyocytes. This decrease in the IK may be related to decreased expression of the Kv2.1. Decreases in both IK and ICa,L of cardiomyocytes could prolong the action potential of cardiomyocytes and contribute toward vulnerability to arrhythmogenesis. These may be the mechanisms involved in the development of cardiac arrhythmias associated with exposure to cooking-oil fumes.Exposure to tt-DDE has adverse effects leading to decrease in the Iin vitro model of cardiomyocytes biology, since they show similar ions currents responses to primary adult and neonatal cardiomyocytes. The cells were plated onto collagen-coated culture dishes and cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2 and 95% air at 37 °C18. Cells were used under the 20th passage.We used H9c2 cells  derived from rat embryonic myoblasts as an 19. The CCK-8 assay was performed according to the kit’s manual protocol. Briefly, the H9c2 cells were seeded in 96-well plates and incubated either with 0.1% DMSO or one of several concentrations  of tt-DDE for 24 hours. The CCK-8 solution was added before the cells were incubated for 3 hours at 37 °C. The absorbance of each well was measured at 450 nm using a Microplate Reader . Each measurement was performed in triplicate.The viability of H9c2 cells in tt-DDE-containing media was determined in an assay using the Cell Counting Kit-8 . The CCK-8 assay was based on the conversion of a water-soluble tetrazolium salt, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium, monosodium salt, to a water-soluble formazan dye upon reduction by dehydrogenases in the presence of an electron carrierK were measured using the whole-cell patch clamp method previously described in detail20. In brief, H9c2 cells treated with (tt-DDE group) or without (control group) tt-DDE-containing media for 18 hours were detached with 0.25% trypsin-0.02% EDTA solution, the supernatant was removed by centrifugation, and the pellets were resuspended in 1 ml of bath solution containing (in mM): 60 NaCl, 80 Na-gluconate, 0.1 CaCl2, 1 MgCl2, 5 KCl, 10 HEPES, and 10 glucose . A recording electrode was pulled from borosilicate glass (resistance: 4–7 MΩ), and the pipette was coated with sticky wax close to the tip to reduce capacitance, backfilled with pipette solution containing (in mM): 0.5 MgCl2, 30 KCl, 110 K-gluconate, 10 EGTA, 5 HEPES, 5 Na2ATP, and 1 GTP-tris . and gently lowered onto an H9c2 cell. Negative pressure was briefly applied to rupture the membrane, and a gigaohm seal was obtained. Cells were subsequently voltage clamped. The IK were recorded using an Axopatch 700 A amplifier , filtered at 1 kHz using a low-pass Bessel filter, digitized at 5 kHz, and stored on a computer for subsequent analysis with Clampfit 10.2. A 1 M NaCl-agar salt bridge between the bath and the Ag-AgCl reference electrode was used to minimize offset potentials. All electrical recordings were performed at room temperature.The ICa,L through L-type Ca2+ channels, perforated whole-cell patch clamp electrophysiology was used in H9c2 cells treated with (tt-DDE group) or without (control group) tt-DDE-containing media (1 µM) for 18 hours. This technique was previously described in detail18. In brief, H9c2 cells were placed in a recording dish and perfused with a bath solution containing (in mM): 135 tetraethylammonium (TEA)-Cl, 1.8 CaCl2, 2 MgCl2, 10 glucose, and 10 HEPES  using a fire polished glass pipette. To minimize outward potassium currents, Cs+ rather than K+ was used in the pipette solution. A recording electrode was pulled from borosilicate glass (resistance: 3–5 MΩ), and the pipette was coated with sticky wax close to the tip to reduce capacitance and backfilled with pipette solution containing (in mM): 140 CsCl, 1 EGTA, 1 MgCl2, 5 Na2ATP, and 5 HEPES . Membrane currents were recorded using the MultiClamp 700 A amplifier, filtered at 1 kHz using a low-pass Bessel filter, digitized at 5 kHz and stored on a computer for subsequent analysis with Clampfit 10.2. A 1 M NaCl-agar salt bridge between the bath and the Ag-AgCl reference electrode was used to minimize offset potentials. All electrical recordings were performed at room temperature.To measure the I18. In brief, total protein content was extracted using a Bio-Rad Protein Assay  and subsequently separated using a 10% denaturing acrylamide gel. The proteins were transferred to Immobilon PVDF membranes  and incubated with rabbit polyclonal antibodies for Kv2.1  and Cav1.2  for 1 hour at room temperature. Antibodies were diluted in TBS-Tween 1:500 for anti-Kv2.1 and anti-Cav1.2. Membranes were incubated with a secondary antibody  conjugated with horseradish peroxidase. Antigen-antibody complexes were detected using enhanced chemiluminescence . Densitometric analysis was conducted using LabWorks 4.5 ImageAcquisition and Analysis software . Each measurement was performed in triplicate.The protein levels of Kv2.1 and Cav1.2 in the H9c2 cells were analyzed using western blotting. The methods used in western blotting have previously been described in detailU test). The P value less than 0.05 was considered significant. All statistical analyses were performed using SPSS software .All data were expressed as mean ± standard deviation, with n indicating the number of cells. Continuous variables in the two groups (control and tt-DDE groups) were compared using non-parametric tests for two independent samples (Mann-Whitney


In situ hybridization (ISH), indirect immunofluorescence (IFA), and viral gene sequencing. Electron microscopy revealed large numbers of parvovirus particles in the organs of diseased fish, including kidney, spleen, liver, heart, brain, gill, intestine, etc. The virions were spherical in shape, non-enveloped and approximately 30nm in diameter. The TiPV was isolated and propagated in tilapia brain cells (TiB) and induced a typical cytopathic effect (CPE) after 3 days post-infection (dpi). This virus was used to experimentally infect adult tilapia and clinical disease symptoms similar to those observed naturally were replicated. Additionally, the results of ISH and IFA showed positive signals in kidney and spleen tissues from TiPV-infected fish. To identify TiPV-specific sequences, the near complete genome of TiPV was obtained and determined to be 4269 bp in size. Phylogenetic analysis of the NS1 sequence revealed that TiPV is a novel parvovirus, forms a separate branch in proposed genus Chapparvovirus of Parvoviridae. Results presented here confirm that TiPV is a novel parvovirus pathogen that can cause massive mortality in adult tilapia. This provides a basis for the further studies to define the epidemiology, pathology, diagnosis, prevention and treatment of this emerging viral disease.Tilapia is one of the most important economic and fastest-growing species in aquaculture worldwide. In 2015, an epidemic associated with severe mortality occurred in adult tilapia in Hubei, China. The causative pathogen was identified as Tilapia parvovirus (TiPV) by virus isolation, electron microscopy, experimental challenge,  Parvoviridae shown to infect a teleost host. We found that a nucleotide sequence similarity search by BLASTX had no significant matches with other viruses, while amino acid sequence comparison indicated approximately 34.6% ~ 50.0% amino acids (aa) homology with other parvoviruses. Similarities between the genomes of parvoviruses infecting hosts in different phyla or divisions indicate a need to update previously suggested hypotheses on the origins of parvovirus. Our findings may represent new avenues to explain viral evolution and suggest a need to further study parvovirus pathogenesis.A novel parvovirus isolated from adult tilapia causes substantial morbidity and mortality. Using a SISPA-PCR and RACE, we identified and characterized 4269 nucleotides of this parvovirus. Tentatively named Tilapia parvovirus (TiPV), this is to our knowledge the first putative member of the family  Oreochromis niloticus) are native to Africa, belonging to the family Cichlidae of the order Perciformes, and are the third-ranking aquaculture species after finfish species within the Cyprinidae and Salmonidae families. Annual global production equals approximately 5.88 million metric tons with a current value in excess of $11 billion U.S. dollars (USD) [Tilapia (rs (USD) . Asia leDactylogyrus and Streptococcus agalactiae are commonly found in tilapia, but they are not associated with the clinical features observed in this study, and moreover, attempts to identify it as a known viral agent, such as the herpes-like tilapia larvae encephalitis virus (TLEV) [A wide range of bacteria, fungi, and parasite diseases have been described for tilapia aquaculture in China –5. Howevs (TLEV) , the virs (TLEV) –9, iridos (TLEV)  and the s (TLEV) –12 were In situ hybridization (ISH), indirect immunofluorescence (IFA), PCR, and gene sequence analysis. This parvovirus pathogen (named TiPV) was isolated and determined to be the causative agent of an epidemic in tilapia in China.The objective of this study was to fully characterize a parvovirus pathogen isolated from tilapia in China. Here we report results from virus isolation, electron microscopy, animal experiments, Initial disease outbreaks were reported in the cage-cultured adult tilapia from August to September 2015, resulting in high mortality . Water tDactylogyrus sp. Bacterial isolation was attempted from the liver and kidney of moribund fish on BHI agar plates, and showed that the bacterial pathogen Streptococcus agalactiae was present.Microscopic examination of fresh preparations from the gills revealed a minor infestation of The histologic lesions of spleen, kidney, heart, brain and gill tissues were noted and summarized below:Spleen: The necrotic splenic white pulp of diseased fish was infiltrated by many lymphocytes and macrophages . More meBrain: Enlarged blood vessels were detected in the brain of diseased fish and were associated with infiltration of a large number of lymphocytes. The neurons were disordered and vacuolated, and had marginated nuclear chromatin in moderate and severe diseased fish . SeriousKidney: Edema of the renal glomerulus was observed in affected kidney tissues. Lymphocyte infiltration and melano-macrophage centers were observed in and adjacent to the areas of diseased glomeruli. Hematopoietic tissues were also infiltrated with a large number of lymphocytes . No melaLiver: Infiltration of lymphocytes and macrophages was showed in enlarged hepatic sinusoids. The infected hepatocytes showed a condensed and marginated nucleus . In healHeart: The infected heart showed disorder and disruption of muscular trabeculae, and widespread vacuolation of myocardial cells showed marginated nuclear chromatin . The heaGill: Primary lamellae of the infected gills were swollen and had widespread infiltration of inflammatory cells. The secondary lamellae showed congestion with a large number of blood cells. Meanwhile, swelling and necrosis of the diseased branchial epithelial cells was detected . The hea50 assays showed that virus titres reached 104.2±0.38TCID50/ml at the 3rd cell culture passage.On the initial isolation, tissue homogenates were inoculated onto TiB cells . Cell moStreptococcus agalactiae)  .The virus particles in the TiB cells were dispersedly located in the nuclei or clustered in the cytoplasm near the cell nucleus or were observed releasing at the cell membrane . Both emIn situ hybridization (ISH) and shown in The localization of viral mRNAs of TiPV in infected tilapia was investigated by The expression of viral protein was identified using indirect immunofluorescence (IFA) to determine the localization of TiPV in infected tilapia. As results show in Parvoviridae NS1 listed in the GenBank database. The genome sequence of NS1 revealed a novel parvovirus identified from diseased tilapia, which was subsequently named TiPV.Amplification results showed that no PCR or RT-PCR positive samples were found for TLEV, VNN, iridovirus, or TiLV in naturally or artificially infected tilapia. However, PCR results showed several fragments ranging from 500 to 1000 base pair (bp) in length following amplification of the DNA template, but no fragments were obtained from the RNA template. Nucleotide sequence similarity searches using BLASTX found no significant matches with any other virus, while at the amino acid levels, sequence comparison did show a certain degree of similarity with Following amplification, a nearly complete genome (4269 bp) of TiPV was obtained, including 5' untranslated region (UTR) (208 bp), ORF1 (396bp), the complete NS1 sequence (1857 bp) and NS2 (504 bp) sequences, ORF2 (216 bp), the complete VP1 (1665 bp), and a partial 3' UTR (46 bp) (GenBank accession no. MT393593). The 5′end of assembled TiPV genome appears to be complete, however, genome assembly at the 3' end is lacking .The genome contained two major open reading frames (ORFs), with the left and right ORFs encoding NS1 and VP1, respectively. The TiPV NS1 (618 aa) contains a conserved replication initiator (endonuclease) motif as well as a helicase domain similar to other parvoviruses –15 Fig . The HuHParvoviridae (~700 aa). The complete of TiPV VP1 protein shares the highest amino acid sequence identity of 37.0% with Parvoviridae sp. (GenBank accession no. AUW34315) and the phospholipase A2 (PLA2) motif was not found in the N terminus of VP1 in TiPV. In addition, two ORFs (named ORF1 and ORF2) were identified within the left-hand ORF and between the NS1 and VP1, encoded a predicted 131-aa protein and 71 aa, respectively. Both of ORF1 and ORF2 were found that there were no significant matches with other parvoviruses by using BLASTP  and VP1-specific primers (F3912), respectively  fragment from samples of naturally infected tilapia and infected TiB cells were considered to be positive for parvovirus. The exact size of the cloned PCR fragment from TiPV was 534bp .7.32± 0.21/ mg) and spleen (4.2x107.12± 0.35/ mg) were higher than those from the intestine (4.3x106.37± 0.36/ mg), heart (5.1x106.25± 0.27/ mg), or brain (1.8x106.09± 0.24/ mg). In samples of gill, liver and eye, genome copies were lower than above tissues and showed 1.8x104.32± 0.17/ mg, 3.5x104.15± 0.26/ mg and 2.8x103.78± 0.32/ mg, respectively. No significant viral quantities were found in muscle samples or and known negative tissue samples  and 3.5x103.18± 0.30/ mg (spleen) and then continued to increase to 2.6x107.67± 0.34/ mg and 3.4x107.28± 0.18/ mg by 5 dpi in the TiPV challenged fish. The concentration of virus in heart, intestine, and brain tissues from 1 to 3 dpi were lower than spleen and kidney tissues, but again showed a steady increase reaching maximum values  at 5dpi. The amount of TiPV was lower in gill, liver and eye tissue until 3 dpi, but maximum values  were recorded at 5 dpi  sampled at various time-points out to 5 days post-TiPV challenge. Results showed that the viral DNA could be quickly detected in the examined tissues of kidney and spleen at 1dpi with values of 3.2x10at 5 dpi .Tissue samples from 610 tilapia were tested for TiPV by PCR targeting the NS1 gene regions. The fish were collected at water temperatures from 22°C to 32°C. Results showed that samples from six cities in three provinces were positive for TiPV. Of 82 samples collected in Hubei province, samples from Wuhan , Jingmen  and Shayang  were shown to be positive for TiPV. In Guangdong, the positive rates of TiPV were 23.1% (9/39) in Guangzhou and were 22.6% (12/53) in Zhuhai. Of 27 tilapia samples from Haikou of Hainan, 10 of these (37.0%) were positive for TiPV . MultiplParvoviridae  protein amino acid sequences of 55 representative viruses in the family oviridae . The dedoviridae .Healthy tilapia were challenged with purified virus derived from cell culture and exhibited ocular lesions and hemorrhagic symptoms that were similar to those found in naturally diseased fish at approximately 4–6 days post-infection (dpi). The mortality reached approximately 90% within 11 dpi . The samin vitro in their reports. In our study, the virus was observed in cells of infected tilapia and TiB cells, which provides a basis for future study of the morphology of parvoviruses in the genus of Chapparvovirus.Tilapia is one of the most important and fastest-growing aquaculture species worldwide. Due to the intensive farming techniques and routine movement of tilapia, emerging diseases have been documented –12. One Parvoviridae has been classified into two subfamilies: Parvovirinae, infecting vertebrates, and Densovirinae, infecting insects and other arthropods [Parvoviruses, which frequently infect animals through the oral-fecal-route, are small, non-enveloped icosahedral viruses with a capsid of 20-30nm in diameter and linear ssDNA genomes of 5kb containing two open reading frames that code for a non-structural protein (NS) and a capsid protein (VP) . Based othropods . Parvovithropods –26. Parvthropods .Streptococcus agalactiae was confirmed in tissues from naturally infected fish as a co-infection with TiPV and may have contributed at some level to mortality, experimental challenges with the isolated virus demonstrated that TiPV is highly pathogenic without bacterial involvement. On the other hand, cohabitation infection may present a more natural way of transmission, a cohabitation challenge test should be needed for confirmation of the pathogenic agent in future investigations.Many viral pathogens have been described as potential challenges to tilapia, including herpes-like tilapia larvae encephalitis virus (TLEV) , viral nIn addition to the outbreak and findings presented here, farmed tilapia suffering with similar clinical signs were sampled from Guangdong and Hainan provinces in China and were found to be positive for TiPV by PCR and sequencing . This surep RNA is required for expression of the NP protein [Based on this work, TiPV appears to be a newly emerging pathogen for tilapia, and considering the extensive commercial production of tilapia worldwide , 2, it w protein . In addiParvoviridae usually have tightly controlled host ranges and tissue tropisms [Viruses of the family tropisms . This isIn conclusion, the causative agent of a new emerging disease associated with massive mortality in farmed tilapia, in China was identified in this study as a novel parvovirus. This virus is tentatively named tilapia parvovirus (TiPV) based on morphology, phylogenetic analysis, clinical symptoms, pathology, and pathogen challenge experiments. TiPV appears to be an emerging viral pathogen in tilapia with implications for culture of this species in China and elsewhere. Further work is needed to confirm host restriction to tilapia, expand screening efforts, and develop methods to prevent or control TiPV disease.Moribund adult tilapia, 25–30 cm in length and 500–600 g in body weight, were collected from a tilapia cage-culture farm in Jinmen county of Hubei province in August 2015. The diseased fish were kept on ice or in oxygenated bags and then transferred to laboratory for diagnosis and pathogen determination. For animal experiment, healthy tilapia (18-25cm in length) were obtained from the breeding station of Yangtze River Fisheries Research Institute, and no history of this disease was recorded. The healthy fish were acclimated in recirculation rearing systems (2 m x 1.5 m x 1.5 m) with aerated water at a temperature of 28°C and were fed with commercial foods regularly for 1 week before experimental infection.The exterior mucus, gills and viscera of clinically diseased tilapia above were sampled for parasitic examination through light microscope. For bacteria isolation, liver and kidney tissue from moribund fish were inoculated onto brain heart infusion  agar plates and incubated at 25°C for 10 days. Any colonies grown were identified using the Biolog Automatic Microbial Identification System .Tissue samples of liver, spleen, kidney, digestive tract, gill, heart and brain from diseased tilapia were fixed in 4% paraformaldehyde (PFA) for 24 hours at 4°C, and washed with Dulbecco’s phosphate-buffered saline , dehydrated with 30% sucrose/DPBS, and embedded in optimum cutting temperature compound (OCT). Samples were cut (8 μm thick) at -20°C with cryostat , stained with Hematoxylin-Eosin (HE) and examined by light microscopy with CCD picture system  , 30.Oreochromis niloticus) and developed as previously described [2 flask . The same volume of cell culture medium DMEM was used for a mock infection in TiB cells as negative control. After adsorption of the material for 1 h at 28°C, 5 ml medium supplemented with 2% FBS was added to the flasks and cell cultures were incubated at 28°C. The cells were examined daily under an inverted phase contrast microscope . When 90% cytopathic effect (CPE) was observed, the culture supernatant was harvested following a 3 times freeze-thaw cycle and centrifugation at 2880g for 20min to remove cell debris. Subculture of the isolated virus followed methods described above, and supernatant from TiB cells was stored at -80°C.Virus titer was determined using the standard 50% tissue culture infective dose (TCID50) method in a 96-well plate [Tilapia brain cell line (TiB) was used to culture potential pathogens from diseased tilapia. TiB is a fibroblast cell line derived from the brain of Nile Tilapia -labelled probe for the TiPV NS1 gene sequence (GenBank Accession no. MT393593), that was synthesized as previously described [To investigate mRNA expression of TiPV in the infected tilapia, escribed . The priPolyclonal mouse antibody raised against non-structural protein (NS1) of TiPV was prepared by ABclonal  and used as the primary antibody. IFA was performed according to the protocol described by Ma et al.  with sliTo determine if this viral pathogen matched known viruses, all samples were screened using specific primers  for herpRandom-PCR and sequence analysis were used to identify the unknown virus using sequence independent single primer amplification (SISPA-PCR) . Superna2 at 37°C. Five micrograms of pTiPV was transfected into HEK-293T cells cultured on 60-mm dishes using Lipofectamine 2000 reagent , according to manufacturer's instructions. Total RNA was extracted at 48 h post transfection using TRIzol regent .HEK-293T cells were maintained in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (FBS) in 5% COTotal RNA (30 μg) extracted from the pTiPV transfected HEK- 293T cells was subjected to electrophoresis in 1% agarose gel at constant voltage 28V for 12h. After that, the RNA was transferred onto a Hybond-N+ membrane  and UV cross-linked. The blots were hybridized by using a DIG High Prime DNA Labeling and detection Starter kitⅡ, according to the manufacturer's instruction. Signals were detected by Gel Doc XR+ system .Total DNA was extracted by DNAzol Reagent  according to the manufacturer’s recommended instructions from the purified virus obtained from infected TiB cells and tissue samples as described above. These were used as the templates for PCR and primer pairs were designed based on the non-structural protein (NS1) of parvoviruses in GenBank (GenBank accession no. MT393593) and the sequences for TiPV . Primersnd passage with a titre of 104.0 TCID50/ml). All the tissues were homogenized in lysis buffer, and virus genomic DNA was extracted using DNA viral kits  according to the manufacturer’s instructions. Real-time PCR reactions and quantitative analyses were performed primarily based on the previously described methods [2O 8.5 μl and the program was 94°C for 2 min, followed by 40 cycles of 94°C for 10 s, 60°C for 30 s. All samples from the infected group and the control group were tested three times by qPCR analysis. To determine the copy numbers of the TiPV DNA, a standard curve was generated with serial dilutions of synthesized DNA fragments of the TiPV NS1 gene. The virus genome copies per 1 mg tissues were calculated according to the Ct values following the manufacturer’s procedures .A real-time PCR assay was used for the detection and quantification of TiPV in infected tilapia tissue samples. Samples were collected from the 8±0.05 mg liver, spleen, kidney, heart, intestine, brain, eye, muscles and gill tissues of tilapia during 1–5 days post-infection with TiPV , Henan (Zhengzhou), Hainan (Haikou), Guangdong  and Guangxi (Nanning), China during 2015~2019. Tilapia were euthanized using MS-222 , and kidney and spleen were sampled and analyzed by PCR for TiPV detection. Fish were collected during different seasons, water temperatures and from ponds stocked with different sizes of tilapia. , Fig 7C.http://www.ncbi.nlm.nih.gov/blast). The deduced amino acid sequences were analyzed with the Expert Protein Analysis System (http://www.expasy.org). Multiple alignments of the deduced amino acid sequences were performed using Muscle package with default parameters. The phylogenetic tree containing the non-structural protein (NS1) sequences from other members of the family Parvoviridae was constructed with MEGA 6.0 using the Maximum-likelihood method with the substitution model of Poisson and rates of Gamma Distributed, while the support for the node was obtained with 500 bootstrap iterations.Sequences were compared for similarity using BLAST  were randomly collected and processed for PCR to show presence or absence of the pathogen.Pathogen challenge experiments were performed in triplicate using healthy tilapia; 30 fish in test group 1 were challenged by intraperitoneal (IP) injection with 0.5 ml viral supernatant of the 2t-test  for determination of statistically significant differences which are indicated in graphs with asterisk symbols .Results were expressed as means ± standard deviation (SD) and all statistical analysis were done by using Student’s The study was performed in strict accordance with the Guide for the Care and Use of Laboratory Animals Monitoring Committee of Hubei Province, China, and the protocols  were approved by the Committee on the Ethics of Animal Experiments at the Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences. The tilapia was euthanized for 20–30 min in 1 mg/ml of MS-222  before tissue collection.S1 Fig(A) Healthy kidney; (B) Diseased kidney, edematous renal glomerulus (red arrow), melano-macrophage centers (asterisk) and lymphocytes (white arrow) in affected kidney; (C) Healthy liver; (D) Diseased liver; lymphocytes (white arrow) and macrophages (black arrow) in diseased hepatic sinusoids; (E) Healthy heart; (F) Diseased heart, vacuolated mocardial cell (red arrow); (G) Healthy gill; (H) Diseased gill, inflammatory cells in primary lamellae (white arrow) and necrotic secondary branchial epithelial cells (red arrow). HE staining. Bar = 20um , 50um .(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Tau protein levels in cerebrospinal fluid are a biomarker of Alzheimer’s disease. We correlated MMSE severity to CSF tau levels in a large memory clinic sample. We retrospectively analyzed data from patients attending a memory clinic in the south shore of Boston from 2010 to 2020, and had a lumbar puncture to obtain CSF Tau levels. We compiled cognitive screen data from MMSE scores. Univariate analyses used Spearman correlation as data were non-normal. A multivariate model was created including covariates of age, sex, and race. 965 patients attended the memory clinic from 2010 to 2020. 711 had available MMSE scores. 129 subjects had lumbar punctures and available CSF tau levels. Univariate analyses showed that cognition as measured by MMSE total was not correlated to total tau levels in the CSF , but caucasian race was inversely associated with CSF tau levels . In a multivariate model, tau levels in the CSF were not associated with MMSE, race, gender, or age. In a large memory clinic sample, CSF tau levels did not correlate to MMSE scores, age, race or gender.


In this study, we aimed to examine if patterns of CSF inflammatory markers are correlated with global cognition, episodic memory, hippocampal volume, and CSF AD-related pathologies among non-demented older people.We included 217 non-demented older individuals, including 87 subjects with normal cognition (NC) and 130 subjects with mild cognitive impairment (MCI) from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) study. Hierarchical cluster analysis including nine inflammatory markers in CSF  was conducted.t-tau and p-tau levels in CSF.We identified two clusters among non-demented older people based on nine inflammatory markers in CSF. Compared to the first cluster, the second cluster showed significantly higher levels of CSF inflammatory markers . Further, the second cluster was also associated with higher levels of t-tau and p-tau levels in CSF.We observed a subgroup of non-demented older people characterized by increased levels of inflammatory markers in CSF. Further, this subgroup showed higher levels of  Accumulating evidence has suggested that neuroinflammation plays a pivotal role in the pathogenesis of Alzheimer’s disease (AD) . SeveralIn the present study, among non-demented subjects, cluster analysis was conducted to identify subgroups based on levels of nine inflammatory markers in CSF, including TNF-α, TNF-R1, TNF-R2, transforming growth factor-β1 (TGF-β1), TGF-β2, TGF-β3, IL-21, IL-6, and IL-7. Further, we compared several AD-related markers  between subgroups.1. The ADNI study was launched in 2003 as a public-private partnership, led by Principal Investigator Michael W. Weiner, MD. The ADNI study aims to examine whether clinical and neuropsychological assessment, serial magnetic resonance imaging (MRI), positron emission tomography (PET), and other biological markers can be combined to measure the progression of MCI and early AD. In the present study, we included 217 non-demented older individuals , including 87 subjects with NC and 130 subjects with MCI. At each ADNI center across the United States and Canada, local institutional review board approved the ADNI study, and each subject provided written informed consent.Data utilized in this work were extracted from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) databaseThe study participants underwent clinical and neuropsychological assessments in each ADNI center. In the present study, there were two cognitive outcomes: Mini-Mental State Examination (MMSE)  and Rey 2. In order to control the effect of sex difference in head size, adjusted hippocampal volumes were used in the present analysis. The formula is as following: Hippocampal/intracranial volumes ratio (HpVR) = hippocampal/intracranial volumes × 103.In an effort to examine neurodegeneration we utilized hippocampal volumes. The hippocampal volumes data were extracted from the ADNI file “ADNIMERGE.csv” (accessed August 2019). The neuroimaging techniques utilized by the ADNI have been detailed previously . Furthert-tau, and p-tau proteins) were analyzed by the multiplex xMAP Luminex platform and Innogenetics/Fujirebio AlzBio3 immunoassay reagents, details of which have been described previously  were examined by William Hu and J. Christina Howell, Department of Neurology, Emory University. All these markers were examined by commercially available multiplex immunoassays . CSF inflammatory markers of the ADNI samples were examined in duplicate. Values are given in pg/ml.x-mean)/sd]. Cluster analysis could categorize participants so that participants are as similar to each other as possible within a cluster. Ward’s clustering linkage method , t-tests were used. The significant variables (p < 0.1) identified in the t-tests were then entered in linear regression models with the adjustment of the potential effects of age, gender, educational years, clinical status, and APOE4 genotype. In addition, to examine whether gender modifies the relationship between cluster status and AD-related outcomes , the clusters∗gender interaction term was added in our regression models.R statistical software  (versione method  was utile method . As a ren = 161), the second cluster (n = 56) showed significantly higher levels of CSF TNF-R1, TNF-R2, TGF-β1, TGF-β3, and IL-6 .As shown in t-tau (p = 0.03). Further, the differences in CSF p-tau levels between two clusters were marginally significant  after controlling for other potential covariates.To further investigate the relationship between cluster status and CSF tau pathologies, several linear regression models were performed with the adjustment of several potential covariates. As shown in ∗gender interaction term was added in our regression models with adjustment of other covariates . However, this interaction term was not significant for any AD-related outcome . This indicated that gender did not modify the relationship between cluster status and AD-related outcomes.In addition, we further tested whether gender modifies the relationship between cluster status between each AD-related outcome  in our regression models. The cluster statust-tau and p-tau levels in CSF.In the present study, we observed a subgroup of non-demented older people characterized by increased levels of inflammatory markers in CSF . To our knowledge, this is the first study to show a subgroup of non-demented older people based on levels of nine inflammatory markers in CSF using cluster analysis. In addition, this cluster showed higher levels of Some data-driven methods, such as cluster analysis, can categorize participants into several subgroups based on heterogeneity within a group of biomarkers. Previously, several studies have been conducted using this approach among healthy controls and patients with cognitive impairment . Howevert-tau and p-tau in CSF compared to the first cluster, indicating that neuroinflammation may play a crucial role in the pathogenesis of AD. A recent meta-analysis involving 170 studies showed that levels of multiple inflammatory markers were substantially altered in comparison between AD, MCI and healthy control, also suggesting an important role of inflammation in the development of AD . Further, this subgroup showed higher levels of adni.loni.usc.edu). Requests to access the datasets should be directed to adni.loni.usc.edu.The datasets presented in this article are not readily available because we downloaded data from the ADNI study, which is open to the public (The studies involving human participants were reviewed and approved by at each ADNI center across the United States and Canada, local institutional review board approved the ADNI study, and each subject provided written informed consent. The patients/participants provided their written informed consent to participate in this study.ZS and YP conceived and designed the study. YP, BC, LC, and QZ performed the research and analyzed the data. YP wrote the manuscript. All authors approved the final version of 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.