Progressive multifocal leukoencephalopathy is a severe demyelinating disease caused by the polyoma JC virus in patients with reduced immunocompetence. A few cases of progressive multifocal leukoencephalopathy have been reported in patients treated with fumaric acid esters.A 53-year-old Caucasian woman reported to our clinic with a first focal epileptic seizure and mild cognitive impairment. Since 1.5 years, she was treated with fumaderm for her psoriasis. During that time, her lymphocyte counts ranged between 450 and 700/μl. Cerebral magnet resonance imaging showed multifocal subcortical T2 hyperintense lesions with partial gadolinium enhancement. She did not have antibodies against human immunodeficiency virus 1 and 2 and cerebrospinal fluid-polymerase chain reaction for viral infections including a sensitive JC-virus polymerase chain reaction were negative. The diagnosis of progressive multifocal leukoencephalopathy was established by histological analysis and detection of JC-virus desoxyribonucleic acid in brain biopsy specimens. Dimethyl fumarate was stopped and Mirtazapin and Mefloquin were initiated. Neurological examination and imaging remained stable.Progressive multifocal leukoencephalopathy can occur in patients with lymphocyte counts between 450 and 700/μl, produce only faint symptoms and is not excluded by negative JC-virus-polymerase chain reaction in cerebrospinal fluid. The incidence of progressive multifocal leukoencephalopathy may thus be underestimated and a more careful surveillance of patients would be necessary. Progressive multifocal leukoencephalopathy (PML) is a severe demyelinating disease of the central nervous system (CNS) caused by the polyoma JC virus in patients with reduced immunocompetence within the CNS. PML may develop in patients treated with immunomodulatory treatment such as natalizumab, rituximab, efalizumab, infliximab or mycophenolat mofetil . RecentlA 53-year-old Caucasian woman reported to our clinic in September 2014 with a first and acute episode of transient confusion and sensory aphasia and a two days history of headache. She was on medication for arterial hypertension and hypothyroidism. Since May 2013 she received dimethyl fumarate (Fumaderm®) for the treatment of psoriasis. The patient only received topical treatment prior to dimethyl fumarate. Her clinical examination on admission was unremarkable. Cerebral magnet resonance imaging (MRI) showed several subcortical T2 hyperintense and partially contrast-enhancing lesions in the right frontal and left parietal lobes, which were increased in size on follow-up MRI two weeks later  protein was slightly elevated (54 mg/l), cell count and IgG-Index were normal and she had identical oligoclonal bands in serum and CSF. She did not have antibodies against (Human immunodeficiency virus) HIV 1 and 2 and polymerase chain reaction (PCR) for JC-virus desoxyribonucleic acid (DNA) (University Tübingen (sensitivity of JC-virus DNA: > 1000 copies) and University Düsseldorf (sensitivity of JC-virus DNA: > 4 copies/assay ), GermanTreatment with dimethyl fumarate was stopped and she was put on 250 mg Mefloquin per week and 45 mg Mirtazapin per day. The patients peripheral lymphocyte counts during treatment with dimethyl fumarate were retrospectively evaluated Fig.  and rangA few cases of PML in patients treated with fumarates have been reported , 10, 11.The incidence of PML in patients with long term immunomodulating or immunosuppressive therapy is thus possibly currently underestimated and a more careful surveillance of these patients would be necessary.Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.


Mycobacterium tuberculosis (Mtb). The only known functional fructose 1,6-bisphosphatase (FBPase) in Mtb is encoded by the glpX gene and belongs to the Class II sub-family of FBPase. We describe herein the generation of a ΔglpX strain using homologous recombination. Although the growth profile of ΔglpX is comparable to that of wild type Mtb when grown on the standard enrichment media, its growth is dysgonic with individual gluconeogenic substrates such as oleic acid, glycerol and acetate. In mice lung CFU titers of ΔglpX were 2–3 log10 lower than the wild-type Mtb strain. The results indicate that glpX gene encodes a functional FBPase and is essential for both in vitro and in vivo growth and survival of Mtb. Loss of glpX results in significant reduction of FBPase activity but not complete abolition. These findings verify that the glpX encoded FBPase II in Mtb can be a potential target for drug discovery.Several enzymes involved in central carbon metabolism and gluconeogenesisplay a critical role in survival and pathogenesis of  Mtb grows on a variety of substrates in vitro but mounting evidence indicates that during infection most of its energy is derived from fatty acids [pckA is up-regulated by acetate or palmitate, but down-regulated by glucose. Deletion of the pckA gene of Mycobacterium bovis BCG led to a reduction in the capacity of the bacteria to infect and survive in macrophages [Mtb, as it is essential for growth and survival of this pathogen during infections in mice and Mtb relies primarily on gluconeogenic substrates for in vivo growth and persistence [Mtb pathogenesis has remained largely unaddressed. Therefore, understanding the key structural and functional aspects of enzymes in the gluconeogenic pathway becomes important. Using genetic and biochemical methods, Movahedzadeh et al. have identified Rv1099c gene as a glpX gene that encodes a class II FBPase [Escherichia coli. An additional class II FBPase is encoded by the glpX gene in E. coli, which is part of the glycerol 3-phosphate regulon [Mtb allowed the identification of genes that were predicted to encode enzymes for most central metabolic pathways [Rv1099c gene of Mtb encodes the missing mycobacterial FBPase (II) [Mtb glpX (Rv1099c) gene is Similar to other class II FBPase from E. coli (GlpX) (42% identity) and Corynebacterium glutamicum FBPase II (65% identity) [glpX gene of Mtb to be essential for survival in a mice infection model. Although the method is high throughput and genome wide, it does not characterize individual transposon mutants. To investigate the role of this enzyme in mycobacterial pathogenesis, we generated an unmarked glpX gene deletion knock out (KO) in Mtb and investigated its effect on in vitro and in vivo growth and survival behavior.ty acids , 2. Whenty acids –6. Hencety acids . Phosphorophages . PEPCK psistence . Except I FBPase –14. Euka regulon . The compathways ; howeverdentity) . The gendentity)  suggestsglpX (Rv1099c) with flanking DNA, 893bp upstream and 970bp downstream of the gene, was amplified by PCR and cloned into p2NIL [glpX gene by inverse PCR, using glpX_Rev1 and glpX_Rev2 primers (lacZ and sacB genes from pGOAL19 into the vector’s PacI site (producing FM152); the DNA was introduced into M. tuberculosis H37Rv by electroporation. Mutagenesis was carried out as previously described [Mutagenesis was essentially carried out as previously described . The codto p2NIL , produci primers ., The PCescribed . To confglpX gene together with 445 bp of upstream sequence was produced by PCR of M. tuberculosis genomic DNA using primers tbglpX_up and tbglpX_end , and 0.05% Tween 80. For growth with defined carbon sources, 7H9 medium was used with 0.05% Tyloxapol and a carbon substrate at 0.1% or 0.2% (wt/vol).glpX gene knockout on in vivo growth. For each strain, 36 mice were infected via aerosol delivery. At indicated time points 6 mice from each group (infected with the respective strain) were sacrificed via carbon dioxide asphyxiation and the lungs from individual mice were aseptically removed, homogenized and CFU were determined.BALB/c mice (approximately eight weeks old) were used for evaluating the effect of Bacteria were grown to mid-log phase, disrupted in a bead beater, the cell-extracts were clarified and FBPase assays were conducted as described previously .Mtb cultures were grown in 7H9 medium + OADC (100ml) until late log or early stationary phase (Klett units ~ 150) and then treated with 1ml of 500mM hydrogen peroxide solution (effective peroxide concentration = 5mM). 1 ml samples were taken as controls before hydrogen peroxide treatment (referred to as 0 hr /input sample). Additional samples (1 ml) were taken at 2 hrs, 6 hrs and 12 hrs after hydrogen peroxide treatment.Mtb cultures were treated with 6ml of sterile 2 mM acetic acid solution (effective pH ≈ 4.5). 1 ml sample was taken as control before addition of acetic acid (referred to as 0 hr /input sample). Additional 1ml samples were taken at 2 days, 4 days and 6 days after pH change to ≈ 4.5 units.Late log or early stationary phase of 100 mL Mtb cultures were treated with 10ml of sterile 2.2 mM DETANO (Diethylenetriamine/nitric oxide) solution (effective DETANO concentration = 0.2 mM). 1ml sample was taken as control before DETANO treatment (referred to as 0 Hr /input sample). Additional 1ml samples were taken at 1 day, 2 days and 3 days after DETANO treatment.Late log or early stationary phase of 100 mL MIC values for standard drugs were obtained using the standard Microplate-based Alamar Blue Assay (MABA) assay .glpX (Rv1099c) was constructed in M. tuberculosis H37Rv producing strain HG1. PCR and Southern blotting analysis were used to confirm the presence of the mutation  through de-esterification [glpX does not significantly affect the growth profile of Mtb, in enriched media  as indicated in the growth profile . It is clear that the ΔglpX strain failed to efficiently replicate during the acute phase (i.e. first 30 days post-infection) of infection as compared to WT Mtb. The CFU for the ΔglpX strain were consistently 2–3 log10 lower than those for WT Mtb. The CFU for ΔglpX in lungs started to decline rapidly after day 57 (glpX expressed from its native promoter (glpX complement strain) restored survival and replication through both acute and chronic phases, although the bacterial load in the lungs did not reach the WT level (data not shown). Similar to the in vitro growth profile of the glpX complemented strain on fatty acids or gluconeogenic growth substrates, growth in vivo was not fully restored to WT levels , and while the glpX complement strain restored the FBPase activity levels (FBPase activity = 21.03 ± 2.31 nmol/min/mg), it does not restore it to the WT levels (FBPase activity = 33.78 ± 2.67 nmol/min/mg). 2 mM of F1,6BP was used as substrate in this assay.The total cellular FBPase activity in WT, ΔglpX and complemented strains indicated that the deletion of The survival of the knockout under various in vitro stress conditions  was measured and no significant changes were observed in comparison with Wild type (data not shown).The inhibitory concentrations for both strains were not strikingly different except for PA-824 .Mtb strain grew fastest and achieved the highest growth rate on glycerol, followed by dextrose and acetate, similar to the earlier studies [ΔglpX to grow on glycerol or acetate as a sole carbon source was severely compromised as compared to WT Mtb, ΔglpX grew twice as fast as WT Mtb on dextrose as a sole carbon source. The slower growth profile of ΔglpX on glycerol or acetate was expected since glpX encodes a functional fructose 1,6-bisphosphatase (FBPase) required for growth on gluconeogenic substrates (glycerol/fatty acids/acetate) [Mtb grows fastest in vitro on glycerol, less quickly on dextrose, and least quickly on acetate.As expected, in aerated batch cultures, WT  studies . While tacetate) , 20. Preacetate) , 26–30 iΔglpX on dextrose is most likely a result of the disruption of a possible regulatory mechanism of FBPase with the phosphofructokinase (encoded by pfk1 and pfk2 in Mtb) enzyme . However the presence of such a regulatory mechanism for FBPase is not experimentally verified in Mtb. Taken together the observations indicate the ΔglpX does not display an eugonic growth phenotype on gluconeogenic substrates , rather it is dysgonic. This observation is concordant with the expected disruption of FBPase activity in Mtb by deletion of the glpX gene. The growth of WT Mtb on an equimolar mixture of glycerol and acetate exceeded that achieved with either constituent alone. Similar effects were observed using a mixture of dextrose and glycerol and a mixture of acetate and dextrose. The compromised growth of ΔglpX on glycerol or acetate was not completely rescued by their combination either. However, with the presence of dextrose in the medium (dextrose + glycerol or dextrose + acetate), the growth profile was almost similar to WT Mtb. A reason for such a phenotype is that both glycerol and acetate are gluconeogenic in nature and therefore cannot be effectively utilized by the ΔglpX. However, dextrose being a preferred glycolytic nutrient for ΔglpX easily rescues the glpX mutant strain and the combinations involving dextrose grew in a manner similar to WT Mtb. Central carbon metabolism in Mtb is a peculiar case involving compartmentalization of metabolic pathways and thereby the metabolites. As understood from our results and the model of compartmentalized central carbon metabolism [Mtb preferentially utilizes glycerol over dextrose and acetate. The predominant pattern of distribution for glycerol and acetate combination as carbon source requires a functional glpX/FBPase. However, since ΔglpX does not possess a fully functional FBPase, its growth is severely compromised on gluconeogenic substrates like glycerol and acetate. The reduced growth/growth-compromised phenotype of ΔglpX is rescued to some extent by dextrose in combination growth media since with dextrose as a carbon source, FBPase activity becomes dispensable (predominant pattern of distribution is not gluconeogenic but rather glycolytic).The fast growth of tabolism , Mtb preglpX gene deletion adversely affected the FBPase activity levels in ΔglpX. However, FBPase activity was not completely abolished in the ΔglpX indicating a possible compensation by other genes/alternate proteins having FBPase activity. The residual FBPase activity in ΔglpX can be attributed to several other gene products/ other classes of enzymes such as inositol monophosphatases (IMPase). The M. tuberculosis genome encodes four IMPase like genes, ImpA, SuhB (Rv2701c), ImpC (Rv3137) and CysQ (Rv2131c) [Mtb has been extensively characterized both structurally and biochemically [(Rv2131c) gene product, in addition to having IMPase and FBPase activity, showed substrate specificity that was broader than those of several bacterial and eukaryotic IMPase. The dimeric enzyme exhibited dual activities of IMPase and FBPase, with Km of 0.22 ± 0.03 mM for IMP and Km of 0.45 ± 0.05 mM for FBP. All four IMPases in Mtb share very limited sequence homology with each other ). Both ImpA and ImpC share similar sequence homology (20–25% sequence identity) with TM1415 IMPase/FBPase–Thermotoga maritime (PDB id: 2P3N) [Methanococcus jannaschii (PDB id: 1G0H) [ΔglpX is attributable to CysQ or ImpA and ImpC.As anticipated, Rv2131c) , 31, 32.emically . SuhB apd: 2P3N)  and MJ01d: 1G0H) . It is aglpX is required to achieve an initial instillation dose and also maintain high bacterial loads at later time points (acute and chronic phases). The initial instillation dose for ΔglpX is significantly lower than the WT Mtb and complement respectively. There are several reports wherein the initial bacterial loads of certain gene deletion strains of Mtb are significantly different than the control WT Mtb strain [et al., in describing the phenotype of ΔdlaTΔpdhC, the deletion strain was not recovered post-day one despite equal input into the aerosolizer. However, by day 7, the ΔdlaTΔpdhC and ΔlpdC mutants were both recovered at 100 CFU, suggesting that the ΔdlaTΔpdhC mutant was viable but non-culturable at day one. We expect a similar case for ΔglpX, where the deletion of glpX has limited the viability of the bacteria. The lower bacterial counts at later time points could be due to an initially low instillation dose (<100 CFU at 1 day post infection), however the rate of growth differs during the initial 30 days post-infection. The significance or essentiality of genes can be efficiently studied at varying stages of disease progression (acute vs. chronic infection) by generating conditional mutants to understand the function of such essential genes. ΔglpX strain like WT Mtb, is sensitive to stress factors likely to be encountered inside the host, such as low pH, hydrogen peroxide, and nitric oxide indicating that the growth suppressed phenotype in vivo is primarily due to a drastic reduction of FBPase activity and not to any other underlying effects.The in vivo survival profile suggests that b strain , 36, 37.glpX strain showed similar MICs for all the drugs tested except for PA-824. The glpX gene deletion reduces the inhibitory activity of PA-824 but not OPC-67683, a structurally similar nitroimidazole. Rv3547, encoding a 151 amino acid protein with no similarity to any protein with a known function, was characterized as a F420-dependent nitroreductase [Rv3547 catalyzed reduction of this compound. Mutations in the mycobacterial Rv3547 gene are found in OPC-67683-resistant Mtb strains as well, suggesting that the Rv3547-encoded enzyme is required for activation of both PA-824 and OPC-67683 [ΔglpX strain is comparable to that of WT Mtb strain. The reduced FBPase activity in ΔglpX strains could result in lower intracellular F6P/G6P levels and hence reduced G6P dehydrogenase activity and the corresponding reduced hydride donor activity of F420. Thus the modest resistance of ΔglpX to PA-824 may be due to reduced intracellular G6P/F6P levels.The Δeductase , 39. F42PC-67683 . HoweverglpX gene encodes a functional FBPase and is essential for both in vitro and in vivo growth and survival of Mtb. The loss of this gene results in drastic reduction of FBPase activity but not complete abolition. Our findings verify the glpX encoded FBPase II in Mtb can be a potential target for drug discovery.In conclusion the


CD147 is a widely expressed integral plasma membrane glycoprotein and has been involved in a variety of physiological and pathological activities in combination with different partners, including cyclophilins, caveolin-1, monocarboxylate transporters, and integrins. Recent data demonstrate that both CyPA and CD147 significantly contribute to renal inflammation, acute kidney injury, renal fibrosis, and renal cell carcinoma. Here we review the current understanding of cyclophilin A and CD147 expression and functions in kidney diseases and potential implications for treatment of kidney diseases. FurtherMore recently, Zhu et al. suppose through investigating CD147 in hepatocellular carcinoma that more CD147 expression might arouse the response of inflammatory cytokines and T cells to improve the immune environment to delay the progression of hepatocellular carcinoma , similarLupus nephritis (LN) usually results in higher mortality and poor quality of life. The diagnostic accuracy for many biomarkers, such as neutrophil gelatinase-associated lipocalin (NGAL) and monocyte chemoattractant protein-1 (MCP-1), remains unsatisfactory . CD147 wβ, MMPs, and others.Renal fibrosis is the important determinant and prognostic predictor of chronic progressive kidney failure, which accounts for approximately 8–10% of individuals in the developed countries –90. The β [A large body of research has shown that macrophage infiltrating into injured interstitium could increase the product of cytokines related to fibroblast proliferation and activation. As previously mentioned, CD147 is expressed on macrophages and acts as a ligand of E-selectin. Mice with a triple knockout of E-, P-, and L-selectin indicate a significant reduction in macrophage recruitment after unilateral ureteral obstruction surgery . The datβ .β1 to induce fibroblast-to-myofibroblast differentiation and to elevate cell contractility in tissue remodeling [−/− mice in the interstitium using a unilateral ureteral obstruction model had much less fibrosis than Bsg+/+ ones at 14 days after operation [−/− mice. Furthermore, TGF-β response was lower in primary Bsg−/− tubular epithelial cells. The above-described results demonstrated that CD147 promotes the formation of renal fibrosis and suggested that CD147 would have multiple effects on promoting renal fibrosis such as the hyaluronan production, the regulation of MMPs, and macrophage infiltration and might be a novel candidate target gene for the prevention of organ fibrosis and therapeutics targeting. In the future, blockade of CD147 by siRNA or anti-CD147 antibody could be performed in vivo and in vitro to further verify the effects of CD147 on renal fibrosis.It is well known that CD147 induces the production of MMPs and CD147 expression in fibroblasts is increased by TGF-modeling . Therefomodeling . And theperation , and MMP−/− mice with coxsackievirus B3-induced myocarditis demonstrated a significantly reduced T cell and macrophage recruitment at 8 days compared with wild-type mice [β [CyPA and CD147 were found to be upregulated in patients with inflammatory cardiomyopathy. CyPAype mice . Treatme mice [β . TherefoBased on the above various studies reviewed, it is clear in kidney diseases that both CD147 and CyPA have multifunctional properties, both independently and as an interacting complex. Increasing evidence demonstrates a crucial role of CD147 in kidney diseases including renal carcinoma and AKI probably through regulating different cell signal pathways. Therefore, targeting CyPA and CD147 appears to represent a promising strategy for treating kidney diseases. siRNA specific targeting CD147 is a useful tool to silence CD147 gene and inhibit its activity . However


Rapid pacing during hypercalcemia + hypothermia (5.5 mM and 34°C) produced significantly earlier excited beats  with wider QRS durations  than suppressed beats. Digoxin + hypothermia (0.75 μM) produced the most excited beats with significantly earlier RCL  and wider QRS  ratios relative to suppressed beats. Increasing pacing further shortened RCL . In a prospective study, TTX (100 nM) increased RCL ratio  without changing the QRS duration of excited beats. The algorithm was compared to a cross-correlation analysis with 93% sensitivity and 94% specificity. This ECG based algorithm distinguishes between triggered and automatic activity.Rapid ventricular pacing rates induces two types of beats following pacing cessation: recovery cycle length (RCL) prolongation (overdrive suppression) and RCL shortening (overdrive excitation). The goals of this study were to compare common experimental protocols for studying triggered activity in whole-heart preparations and differentiate between recovery beats using a new methodology. Post-pacing recovery beat cycle length (RCL) and QRS were normalized to pre-paced R-R and QRS intervals and analyzed using a K-means clustering algorithm. Control hearts only produced suppressed beats: RCL ratio increased with rapid pacing (25 ± 4.0%,  However, at times electrical activity can originate prematurely at ectopic sites, leading to arrhythmias. Early work in canine purkinje fibers using cardiac glycosides found premature low amplitude membrane depolarizations following rapid pacing  Differentiate between OS and OE beats with a new methodology, (2) Develop a robust protocol for creating OE beats that can be studied, and (3) Demonstrate that OE beats can be pharmacologically manipulated.Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health  and has been approved by Institutional Animal Care and Use Committee (IACUC) at Virginia Polytechnic Institute and State University.The investigation conforms to the n = 37) were anesthetized with sodium pentobarbital (325 mg/kg) and injected intraperitoneally with heparin to prevent blood clotting. Atria were excised to remove competitive stimulation from atria, and ventricles were perfused in a Langendorff system with oxygenated Tyrode solution  at 37°C and 50 mmHg. Motion was reduced using 7.5 mM 2,3-diacetylmonoxime.Retired breeder male guinea pigs (2), or the sodium channel inhibitor tetrodotoxin . Hearts were maintained at normal (37°C) and hypothermic (34°C) temperatures. ECG measurements were made 10 min after the start of perfusion. RCL and QRS ratios were then normalized to the pre-paced beat's cycle length (BCL) and the QRS, called RCL and QRS ratios, respectively. The recovery window was 1500 ms. A k-means two cluster analysis, which is an automated clustering algorithm that calculates the distance from each data point to a continuously calculated centroid of each cluster, was used to separate the control, hypercalcemic, digoxin, and hypothermic data into two clusters (Cluster 1 and Cluster 2).A volume-conducted bath electrocardiogram (ECG) similar to lead I was continuously recorded to determine recovery cycle length (RCL) and QRS duration of the QRS complex immediately after the cessation of pacing. Hearts were paced with plunge bipolar electrodes fixed in the basal interventricular septum at pacing rates from 200 to 375 bpm for 15 s. Hearts were also perfused with the glycoside digoxin (0.75 μM), high extracellular calcium  were calculated and recovery beats for digoxin + tetrodotoxin were stratified into two clusters depending on the centroid they were closest to.To determine sensitivity and specificity of our new algorithm, cluster classification was compared to a previously validated cross-correlation analysis as the gold-standard  and QRS morphology of native beats, and therefore the recovery beat. For example, any intervention which increased native rhythm rates would likely decrease all recovery beat RCL. This analysis could classify the recovery beat as an OE beat without accounting for the fact that BCL changed. A similar argument can be made for QRS.To account for BCL and QRS variability, we chose to normalize recovery beat RCL and QRS. In Figure in vivo and ex vivo guinea pig measurements with atria intact  and 2  in whole hearts have included analyzing recovery beats: (1) of ventricular origin, (2) that have a shorter cycle length than the longest post-paced R-R interval, (3) with preceding delayed afterdepolarizations (DADs), or (4) with preceding spontaneous calcium releases  using a QRS cross-correlation analysis as the gold standard . This is consistent with the finding that non-specific sodium channel blockers can delay the time to spontaneous calcium release  was used in this study. This compound alters intracellular calcium concentrations and handling, and therefore may impact OE beat formation (Backx et al., The authors have submitted a University Invention Disclosure to Virginia Tech in regards to the quantitative methodology described in this study.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.


Human cytomegalovirus (HCMV) latency in the myeloid lineage is maintained by repressive histone modifications around the major immediate early promoter (MIEP), which results in inhibition of the lytic viral life cycle. We now show that pharmacological inhibition of histone deacetylases (HDACs) relieves this repression of the MIEP and induces transient expression of the viral lytic immediate early (IE) antigens but, importantly, not full virus reactivation. In turn, these latently infected cells now become targets for IE-specific cytotoxic T cells (CTLs) which are present at high frequency in all normal healthy HCMV positive carriers but would normally be unable to target latent (lytic antigen-negative) cells. This approach of transiently inducing viral lytic gene expression by HDAC inhibition, in otherwise latently infected cells, offers a window of opportunity to target and purge the latent myeloid cell reservoir by making these normally immunologically undetectable cells visible to pre-existing host immune responses to viral lytic antigens. Human cytomegalovirus (HCMV) causes significant morbidity and mortality in the immunocompromised and the immunonaïve1in vivoLike all herpesviruses HCMV persists for the life-time of the host after primary infection which is, at least in part, due to the consummate ability of HCMV to avoid immunosurveillance. However, this persistence is underpinned by the ability of the virus to establish a latent infection in certain cell types in vivo are cells of the myeloid lineage, including CD14+ monocytes and their CD34+ progenitors786710111314One established site of HCMV latency In the context of an inadequate immune response , these reactivation events can result in virus dissemination to multiple target organs and subsequent clinical diseaseImportantly, we show here that the treatment of latently infected cells with the Class II -specific HDAC inhibitor (HDACi) MC1568 leads to the transient expression of viral lytic gene products which then makes these cells novel targets for pre-existing HCMV-specific CTLs, routinely present at high frequency in all immunocompetent virus carriers. These observations are proof of principle that the use of HDAC inhibitors can result in removal of latently infected cells which may be a useful novel therapeutic strategy in certain clinical settings.We have previously shown that latent infection of myeloid cells results in major changes in cellular gene expressionin vivo and in vitroWe reasoned, therefore, that induction of histone deacetylases by latent virus may be important for the latency-associated repression of viral lytic gene expression and that deacetylase inhibition might impact on this regulation of viral gene expression during latency. Consequently, we tested whether inhibition of HDAC4 during latent infection had any effect on IE gene expression using the Class II-specific HDAC inhibitor MC1568, which is known to inhibit HDAC4 activity Interestingly, this induction of viral IE gene expression by MC1568 did not result in reactivation of the full lytic cycle, even after 7 days continuous treatment, as no expression of viral UL99 (pp28), a true late viral gene only expressed after viral DNA replication, was detected . ConsistTherefore, importantly, these cells - expressing IE antigens but stalled in full reactivation -would be prime targets for the well established immunodominant IE-specific T cell responses known to be at high frequency in normal HCMV carriers.Between 0.5–10% of total circulating CD8+ CTLs in healthy HCMV seropositive donors are specific for the viral major IE lytic geneAlthough the data, so far, suggest that the IE-specific CTLs were recognising and killing the latently infected cells, which had been induced to express IE antigens by HDAC inhibitor treatment, it is possible that this removal of monocytes expressing IE antigen resulted from CTL-mediated repression of IE gene expression and that these monocytes still carried reactivatable virus. In order to confirm that the disappearance of monocytes transiently expressing IE was due to T cell killing, rather than perhaps re-repression of IE gene expression, we confirmed the expression of CD107a (LAMP-1 an indication of cytotoxic T cell degranulation and part of the CTL killing mechanism) in these IE-specific T cells after surveillance of MC1568-treated latent monocytes . Figure However, a key question is whether drug treatment permits naturally latently infected cells in the peripheral blood of healthy donors to become targetable by blood resident HCMV-specific CTLs. To address this we carried out analyses on total peripheral blood mononuclear cells (PBMC) from two seropositive individuals to allow assessment of the effectiveness of MC1568 in clearing naturally latent HCMV from the peripheral blood. Taken together, these data argue that pre-existing autologous IE-specific T cells in the peripheral blood compartment of healthy HCMV carriers are able to target and purge naturally latently infected monocytes after these monocytes have been treated to induce IE expression transiently with HDAC inhibitors.Our view is that such novel immunotherapeutic strategies could lend themselves to, for example, the treatment of stem cell donations from HCMV seropositive donors to reduce latent load prior to engraftment but, as a number of HDAC inhibitors are already in clinical use, could also be used in donors prior to donation.8 CD14+ monocytes were isolated as describedCell isolations and establishment of experimental latency using HCMV TB40E or IE2-EYFP-tagged TB40E have been described previously5 cells, the number of IE positive cells following reactivation routinely ranged from 5 to 500 IE expressing cells per well between experiments. Consequently, data were normalised to numbers of IE-expressing cells after differentiation.All assays quantifying IE reactivation in response to treatments were normalised to the number of IE positive cells following reactivation by differentiation of CD14+ or progenitor cells into dendritic cells as described previously6 cells routinely resulted in 2–10 IE positive foci of infection and these numbers are directly graphically represented. Unless otherwise stated, each experiment was carried out a minimum of two times with at least duplicate samples.For natural latency, differentiation of approximately 10Error bars represent standard deviation and P values were determined using the Student’s t-test. Values are represented by the standard asterisk system where P < 0.05* is statistically significant; P < 0.01** is of medium significance and P < 0.001*** is statistically highly significant.Western blotting was carried out as described previouslyCells were fixed and stained as described previously5 per well of a 96 well plate and then co-cultured in X-Vivo 15 supplemented with 2 mM L-Glutamine with donor-matched (HLA-A2) EBV or HCMV-specific CD8+ T cells at a ratio of 5:1. The number of HCMV IE-expressing monocytes were enumerated over several days as indicated.CD14+ monocytes were plated at 1 × 10ex vivo were isolated from total PBMC by magnetic activated cell sorting (MACS), were co-cultured with donor-matched IE-specific CD8+ T cells in 100 μl RPMI-10 per well ). Plates were incubated for 48 hours at 37 °C in 5% CO2, and developed according to manufacturer’s instructions. Plates were read using an ELISPOT plate scanner  and spots enumerated using ImageJ .ELISPOT plates were prepared, coated and blocked according to manufacturer’s instruction (eBioscience). CD14+ monocytes, isolated directly 2 atmosphere. CD8+ T cells were harvested and washed, then stained with a combination of surface antibodies  at 37 °C in a humidified COtibodies  and LIVEtibodies  at 4 °C MC1568 (Sigma) was tested for cytotoxicity  and usedGCAATGC with the probe [FAM]CCAATGGCTGCAGTCAGGCCATG[TAM] and GAPDH GGAAGCTTGTCATCAATG and CCCCACTTGATTTTGGAG with the probe [JOE]ATCACCATCTTCCAGGAGCGAG[BHQ1]. Viral transcript UL138 was detected with primers CGCTGTTTCTCTGGTTAG and CAGACGATACCGTTTCTC with probe [Cy5]CCGACGACGAAGACGATGAAC[BHQ2] and viral transcript UL99 (pp28) with the primers CGAACTCTGCAAACGAATA and GAGGGATGTTGTCGTAGG with the probe [Cy3]CGTAGAGACACCTGGCGACC[BHQ2]. Samples were analysed with an ABI 7500 Fast Real Time machine using MicroAmp Fast Optical 96 well reaction plates using the RT parameters: 50 °C for 20 min followed by heat inactivation 95 °C for 5 min then PCR steps: 50 cycles, 95 oC for 15 s and 60 oC for 45 s. Data were accrued from reactions carried out in the presence and absence of RT and the minus RT values subtracted from the plus RT samples to obtain the RNA levels. All primers were tested against a standard curve to allow comparison between samples.RNAs were isolated using the miRNeasy mini-kit and amplified using the Quantitect virus + ROX virus kit (Qiagen). The viral IE transcript was amplified alongside the housekeeping transcript GAPDH using the following primers and probes: IE: CAAGAACTCAGCCTTCCCTAAGAC and TGAGGCAAGTTCToC for 15 s and 60 oC for 45 s.Chromatin immunopreciptition assays were carried out as described previouslyTo analyse the levels of cell death, cells were stained directly with trypan blue in PBS (a live dead discriminator). Alternatively, the viability of cells was determined using the LIVE/DEAD Fixable dead cell stain kit  and analysed by FACS.All human samples were obtained under ethical approval and after approval of protocols from the Cambridgeshire 2 Research Ethics Committee (REC reference 97/092) and that these protocols were conducted in accordance with the Declaration of Helsinki. Informed written consent was obtained from all of the volunteers included in this study prior to providing blood samples and all experiments were carried out in accordance with the approved guidelines.How to cite this article: Krishna, B. A. et al. Transient activation of human cytomegalovirus lytic gene expression during latency allows cytotoxic T cell killing of latently infected cells. Sci. Rep. 6, 24674; doi: 10.1038/srep24674 (2016).


The present study aimed to standardize pregnancy detection in big cats like the tiger (Panthera tigris), jaguar (Panthera onca) and lion (Panthera leo) using fecal samples. High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LCMS) were performed to identify PGFM in feces. An EIA developed against 9α,11α-dihydroxy-15-oxo-prost-5-en-1-oic acid-BSA was used to assay PGFM in fecal samples of the Bengal tiger, Asiatic lion and jaguar. The PGFM levels increased after 9 weeks of pregnancy and remained elevated until parturition. All animals showed elevated levels of PGFM in the last trimester of pregnancy, thus making PGFM a reliable tool for pregnancy diagnosis during this period that can be useful in captive breeding programs in these species.Non-invasive monitoring of hormones using feces has become a vital tool for reproductive management and reliable pregnancy diagnosis in big cats. Previous studies described the PGF However, many felids reproduce poorly due to inappropriate captive management practices  intensity was measured at 450 nm in an ELISA reader  and one jaguar . Separation of analytes was performed according to our standard protocol . AliquotMass spectrometry was carried out on an API3200 QTrap LC/MS/MS system with a Turbolon Spray electrospray ionization (ESI) source  using multiple reactions monitoring and equipped with a Z-spray ESI interface as described earlier . ChromatTo confirm PGFM and to identify other metabolites in fecal extracts detected by the PGFM antibody, 20 μL of the immunoreactive HPLC fractions were subjected to LCMS analysis. The presence of three transitions with a signal-to-noise ratio above three associated to the expected retention time allows identification of the analyte.For all animals the values  are represented as means ± standard error of the mean (SEM). Because the frequency of sample collection varied, weekly means were calculated for all the individuals and this was plotted together with the SEM in the graph. Non-pregnant samples were unavailable, hence, samples from before mating and after parturition were considered, to calculate the baseline PGFM levels in non-pregnant animals. The Mann–Whitney U test (M–W test) was used for testing differences in PGFM levels between weeks and terms. To examine differences in PGFM concentrations, the entire pregnancy period was divided into three equal periods as early (1–5 weeks), mid (6–10 weeks) and late term (11–15 weeks) for Asiatic lion, Bengal tiger and jaguar. We used the Friedman nonparametric repeated measures ANOVA to check for differences of fecal PGFM concentrations between the terms. All statistical analyses were carried out using SPSS 17.0.The HPLC immunoreactive chromatogram  shows twA comparison of the immunoreactivities in pregnant samples between days 89 and 100 in tiger and between 70 and 77 in jaguar revealed PGFM as a minor portion whereas the major portion consists of the unknown metabolite in fraction 25 varying distinctly between different days of pregnancy . No tend2 = 18.00, P = 0.006). The mean basal PGFM levels during the first 7 weeks following conception ranged between 830 and 1100 ng/g. Further, PGFM concentrations started to increase from week 8 onwards (1512 ± 236 ng/g) until parturition (4987 ± 615 ng/g). Compared to basal concentrations (830–1100 ng/g), this increase in PGFM concentration was significant from week 9 onwards (P<0.05). Altogether, PGFM concentrations varied significantly among early, mid and last term of pregnancy . Thereafter, PGFM concentrations dropped to baseline levels (1500 ng/g) the week following parturition. PGFM levels showed significant differences between the terms  and significant increases from the first to last term  and its metabolites (5α-pregnan-3α-ol-20-one) can be used for pregnancy detection in several species of felids , 13. HowIn this study, the fecal PGFM concentrations remained distinctly elevated during the last trimester (last 30 days) of pregnancy, peaking towards parturition in all animals studied, the pregnant lion, tiger and jaguar. Compared to previous results  in felidThe HPLC immmunograms of extracted fecal samples in the three felid species showed a similar pattern, consisting of only minor portions of authentic PGFM  whereas the major portion consists of an unknown immunoreactive component. This fits with our earlier data from other felid species in which PGFM was detected in more or less significant proportions but turned out not to be the major proportion of immunoreactivities .The comparison of HPLC immunograms generated from different days of pregnancy revealed a high intra-individual variation in immunoreactive prostaglandin metabolites, but there was no pattern of metabolite composition that could be related to the stage of gestation. Thus, changes in metabolite composition seemed to occur at random, which confirms our earlier results. We assume that these differences may reflect changes in intestinal eicosanoid metabolism . HoweverUnfortunately, we were so far unable to identify the unknown metabolite in fraction 25. Previous studies on the metabolism of radiolabelled PGF2α in rabbits and sheep revealed that PGF2α and PGFM were undetectable but found other metabolites , 41. A rBased on the facts that similar prostaglandin metabolites might be excreted with urine and feces , whereaspanthera species analyzed here, an elevation of fecal PGFM metabolites over a threshold level of 1.5 μg/g dry feces can serve as pregnancy sign, allowing diagnosis with very few samples if not just a single one. This is a major advantage of PGFM pregnancy diagnosis in a captive animal setting, when regular fecal sample collection is unattainable. In such a setting, the fecal PGFM test is superior to the use of fecal gestagen assays. Another management tool in high demand is the prediction of parturition. Our data suggest that elevations in PGFM over 5 μg/g feces might be indicative for delivery within a week. However, drying fecal samples, shipping samples overnight to a laboratory, and performing the EIA generates a time lapse until the availability of results making this type of parturition prediction for now less feasible. Although this conclusion or application recommendation remains speculative until it has been validated on more individual profiles in different zoos.Irrespective of the chemical character of the unknown major PGF2α metabolite in feces, our results confirm that the PGFM test can be used in big cats for pregnancy diagnosis. In all three


Vaccinia virus is a large, enveloped virus of the poxvirus family. It has broad tropism and typically virus replication culminates in accumulation and lytic release of intracellular mature virus (IMV), the most abundant form of infectious virus, as well as release by budding of extracellular enveloped virus (EEV). Vaccinia viruses have been modified to replicate selectively in cancer cells and clinically tested as oncolytic agents. During preclinical screening of relevant cancer targets for a recombinant Western Reserve strain deleted for both copies of the thymidine kinase and vaccinia growth factor genes, we noticed that confluent monolayers of SCCF1 cat squamous carcinoma cells were not destroyed even after prolonged infection. Interestingly, although SCCF1 cells were not killed, they continuously secreted virus into the cell culture supernatant. To investigate this finding further, we performed detailed studies by electron microscopy. Both intracellular and secreted virions showed morphological abnormalities on ultrastructural inspection, suggesting compromised maturation and morphogenesis of vaccinia virus in SCCF1 cells. Our data suggest that SCCF1 cells produce a morphologically abnormal virus which is nevertheless infective, providing new information on the virus-host cell interactions and intracellular biology of vaccinia virus. Vaccinia virus (VV) is a large, enveloped virus belonging to the poxvirus family. Currently, the virus can only be found as a laboratory strain used for the study of poxviruses or cancer treatment. It has a linear, double-stranded DNA genome approximately 190 kbp in length encoding for approximately 250 genes . Since tReplication-competent oncolytic vaccinia viruses have shown great promise as a potential cancer treatment . Over thImportantly, because of its broad tropism, vaccinia virus is also being developed for virotherapy in domestic animals and is currently being tested in clinical trials at least in pet dogs . ConversFeline squamous cell carcinoma cell line (SCCF1) was kindly provided by Dr. T. Rosol . SCCF1 has been developed from a laryngeal SCC of a cat. Keratinocytes were maintained in culture for greater than 50 passages. SCCF1 had strong cytokeratin immunohistochemical staining, weak vimentin staining and no p53 staining . Also aflacZ gene in vgf site coding inactive but immunogenic beta-galactosidase enzyme. Wild-type control was not included in the studies due to the biosafety regulations. In addition, vvdd-luc has insertion of luciferase gene and vvdd-tdTomato encodes fluorescent protein tdTomato. The viruses were engineered as previously described [The viruses used in the study were all cancer specific double deleted vaccinia viruses (vvdd) of Western Reserve strain and they have total deletion of vaccinia growth factor (vgf) and a partial deletion of thymidine kinase (tk) gene. All viruses have also insertion of escribed , 12. Vvdescribed .ueous One Solution Proliferation Assay, Promega, Madison, WI). Inactivated vaccinia virus was used as a non-replicating control, inactivation of the virus was done as described previously [Cell viability after viral infection was measured using a colorimetric cell lysis test (MTS). Ten thousand cells per well were plated on 96-well plates in growth medium (GM) supplemented with 5% fetal calf serum (FCS). Confluent cell layers were infected in triplicates with different concentrations of virus for 1 hour in 2% FCS GM. After that, 5% FCS GM was added into wells and cells were incubated until cell viability was measured by MTS according to manufacturer’s instructions . Transduction of tdTomato was also confirmed by infecting cells with 0.2, 1 or 5 pfu/cell and visualizing the tdTomato expression 24 hours later with fluorescent microscope.Presence of viable virus inside the cells was also confirmed by plating 200,000 cells per 24-well in 5% FCS GM. Next day, the cells were infected in triplicates at 0.01 pfu per cell in 2% FCS GM for 1 hour. After infection, 5% FCS GM was added and cells were collected after 24, 48 and 72 h. After 3 freezing-thawing cycles a standard plaque forming assay was performed.Ten thousand and 50,000 cells were plated on 6-well plates and infected 48 h after with 10 pfu per cell for 1 h in 2% FCS GM. Cells were scraped from the wells and collected with supernatant at 24 and 72 h. Different amounts of cells were plated in triplicates on 6 well plates. One or 3 days after plating, colonies were stained with crystal violet and counted.Infected A549 cells and SCCF1 cells were collected and virus was extracted and purified to determine the amount of virus produced by the cells. Also supernatant of the infected SCCF1 cells was collected on different time points and plaque assay was performed to determine the production rate of secreted virions. To study the efficacy of secondary infection, collected supernatant was also used to infect either SCCF1 or A549 cells and transduction assay was performed as described previously.Supernatant of infected SCCFI cells was collected and incubated with or without NR-417 monoclonal anti-vaccinia antibody  for 1 hour in +37°C. Standard plaque assay was performed to determine the amount of neutralized virus.Two EM specimen preparation methods, negative staining of whole mount viral particle and thin sectioning of resin embedded cells with viruses, were used in this study.Negative staining: this method is used to study surface structures of purified, whole-mount vaccinia viral particles in the supernatant of infected SCCF1 and A549 cells. The method detects viral particle as a whole in 3 dimensions. For asymmetrical particle like vaccinia virus, the orientation is not random, with the largest, flat surface attached and facing up predominantly. To get purified viral particle, supernatant of infected cells was collected and the virus was purified through sucrose cushion as described previously . Viral p3. Fresh glutaraldehyde fixative was added on top of the pellet and fixed further for half an hour. The pellets were then post-fixed by 2% osmium tetroxide for 1 hour, dehydrated in series of ethanol and embedded in LX-112 resin. Ultra-thin sections were cut at thickness of 60–80 nm, mounted on 200 mesh EM specimen supporting grids, stained with uranyl acetate and lead citrate in Leica EMstain automatic stainer  according to the manufacturer´s recommendations and then observed by EM.Resin embedded thin section electron microscopy: this method is used to study internal structure of viral particle and infected host cells. It can reveal structures of vaccinia viral particles at any orientation without bias as the embedding and cutting direction is random relative to the particles. For doing this, cultured SCCF1 and A549 cells were infected with vaccinia and 48 hours after the infection the cells were fixed on-site in culture dish immediately out of incubator with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 minutes and the cells were scraped off the dish, collected by centrifuge as a small pellet of 2–3 mmBoth negative stained specimen and thin sections were observed by using JEM 1400 transmission electron microscope , at 80 KV acceleration voltage. Digital electron micrographs were taken by a side-mounted Morada TEM digital camera . Images were processed (brightness and contrast operation only) by iTem software from the camera manufacturer. Adobe Photoshop software was used for annotation.P<0.05 was considered statistically significant.All values are indicated as mean standard error of the mean (SEM). Differences between groups were calculated using two-tailed Student’s t-test (GraphPrism software). Fig. 1A). With extremely high amount of virus, cytotoxicity could eventually be achieved on day 10 (data not shown). However, vvdd proved significantly more effective (p = 0.0073) cytotoxicity in A549 cells compared to SCCF1 cells when both cell lines were infected with 1 pfu/cell and viability was measured on day 3 . When SCCF1 was growing as an intact monolayer, infected cells were not completely lysed even 10 days after infection . Successful virus entry and early gene expression in the SCCF1 cells was confirmed by virus-mediated expression of firefly luciferase . Viable virus was also shown to be located inside the SCCF1 cells by plaque assay .Vvdd was unable to effectively lyse SCCF1 cells when pfu/cell values 0.01, 0.1 or 1 were used . Secreted particles were also used for re-infecting SCCF1 and A549 cells and in both cell lines these particles were able to cause secondary infection indicating that these produced virions were infectious . To further characterize the nature of the particles neutralization assay was performed showing that basically all the particles can be recognized and neutralized by anti-vaccinia antibody .In order to assess whether vaccinia-mediated reporter gene expression was associated with productive virus replication, we titered chronically infected confluent SCCF1 cell culture supernatant on different time points. We observed low but constant secretion of virus at least up to 12 days post infection . All of the viral particles were brick-shaped and are surrounded by an outer envelope (OE). In contrast, all virions produced by SCCF1 cell line were misshaped immature virions. Further, no outer envelopes are detected and particles were loosely packed with irregular surface and irregular staining intensity . Normal ultrastructural organelle morphology of SCCF1 cells is shown in Electron microscopy on purified, secreted virus particles revealed clear differences between particles produced by SCCF1 versus A549 cells. A549 cells were able to produced mature, enveloped virions with features typical of mature vaccinia viruses . Nomenclature of the different particle structures is inconsistent in the literature for historic reason. Moss proposed a simplified naming system for normal, mature poxvirus  using onFig. 4A) showing numerous viral particles, most of them are mature virus (MV), majority of the MV are wrapped or enveloped intracellular enveloped virus (IEV) by membrane from trans-Golgi cistern, and the extracellular viral particles are wrapped by double membrane (EEV) with outer membrane from cell plasma membrane. Some EEV are still associated with cells (CEV). Inserts with high magnification show them with clear membrane layers. Findings from electron microscopy sections of infected cells are in agreement with the findings from negative stained whole mount viral particles, and provided more details about the maturation status of the viral particles. Infected A549 cells produce the whole range of viral particles at different maturation stages. A low magnification outlook of two adjacent cells  showing Fig. 5A), and a few of them have DNA injected to form nucleoids (IVN) . In some areas, the replication factory contains abnormal granular virosomes (VS), with a few IVs scattered around . Even at the peripheral region, where viruses are going to be release outside of the cells, there were only enlarged IVs inside cytoplasm and no membranous envelope obtained. Also, no EEVs were found outside the cells .Inside the infected SCCF1 cells, the picture was different: the replication factory is inconspicuous, there were just some immature viruses (IV) of different size  stay inside cells until lysis; constituting robust, stable virions well suited for transmission of infection between hosts. Intracellular enveloped virus (IEV) is formed by wrapping of IMV with intracellular membranes, and is an intermediate between IMV and cell-associated enveloped viruses (CEV) enabling efficient virus dissemination to the cell surface, utilizing microtubules . CEV indSeveral virus-encoded proteins have been identified to have a role in virus morphogenesis. For example, the VV encoded I7 protein participates in proteolytic processing of the vaccinia virus membrane and core components, and is critical for transition from IVN to MV . Lack ofAs mentioned above, the viral protein defects affect replication, maturation and morphogenesis of vaccinia virus. However, our data suggests that the similar effects might also be brought out by the host cells. It seems that the vaccinia viral particles within SCCF1 cells were not able to form the core unit from viral DNA and thus no MV was detected. One possible explanation is that although the crescent can close to form IV, the viral genome DNA is not packed or condensed to form the core unit. The loose nucleosome threads need some core nucleoproteins to get further condensation and encapsidation. The IVN to MV transition appeared to be completely arrested in SCCF1 cells, as we did not see any MV by electron microscopy. However, it is important to keep in mind that thin section is not completely conclusive as a very small fraction of the cell volume is being visualized in each case. As all the core proteins and membrane proteins needed for virion maturation are encoded by the viral genome, it seems that some cellular co-factor target of a viral protein affecting the replication of the virus is missing or defective in the SCCF1 cell line. Alternatively, the cells might produce some anti-viral cytokines or interferons which can arrest the life cycle of the virus. Identifying the mechanism would yield interesting insight into both viral and cellular biology.Notably, not much has been reported about vaccinia and feline cell lines. For domestic cats more than 400 cases of Cowpox virus (CPXV) infections have been described , but it To our knowledge, this is the first time this kind of replication pattern of vaccinia virus has been reported. These findings could provide valuable insights into molecular mechanisms underlying host cell infection, viral replication as well as virulence and life cycle of vaccinia virus.S1 FigInfectious virus was recovered from SCC-F1 cells by plaque forming test after infection of cells with 0.01 pfu/cell.(TIF)Click here for additional data file.S2 FigA) The SCC cell line was derived from squamous cell carcinoma and maintains most of the morphological features of squamous epithelial cells; nucleus with loosely packed chromatin (Nu), cytoplasm is rich in different organelles like mitochondria (Mit), ribosomes and endoplasmic reticulum, patches of glycogen (gl), and most prominently, bundles of tonofibrils (Tf) consisting of keratin intermediate filaments. B) Cells also display characteristics of cancer cells such as large nucleus filled with low electron density euchromatin (Nu) and prominent nucleolus (No). D) Mitochondria within the cells were in a high dynamic process of fusion and fission resulting in numerous long, irregular, branching mitochondria, phenomena common when cells are under stress.(TIF)Click here for additional data file.S1 TableSCCF1 cells were infected with vvdd-tdTomato 10 pfu/cell in duplicates and 10,000, 1,001, 100 or 10 infected cells were grown 1 or 3 days to evaluate if cell were able to form colonies.(DOCX)Click here for additional data file.


The zebrafish has various advantages as a model organism to analyze the structure and function of neural circuits but efficient viruses or other tools for fast gene transfer are lacking. We show that transgenes can be introduced directly into the adult zebrafish brain by herpes simplex type I viruses (HSV-1) or electroporation. We developed a new procedure to target electroporation to defined brain areas and identified promoters that produced strong long-term expression. The fast workflow of electroporation was exploited to express multiple channelrhodopsin-2 variants and genetically encoded calcium indicators in telencephalic neurons for measurements of neuronal activity and synaptic connectivity. The results demonstrate that HSV-1 and targeted electroporation are efficient tools for gene delivery into the zebrafish brain, similar to adeno-associated viruses and lentiviruses in other species. These methods fill an important gap in the spectrum of molecular tools for zebrafish and are likely to have a wide range of applications. In rodents and other vertebrates, gene transfer in the brain is often accomplished by the injection of viral vectors, particularly adeno-associated viruses (AAVs) or lentiviruses  , the teleost homolog of olfactory cortex.Experiments were performed in wild-type zebrafish (Danio rerio) of both sexes that were raised at 25–28°C on a 14/10 h on/off light cycle. Adult fish were > 3 months old. All experimental protocols were approved by the Veterinary Department of the Canton Basel-Stadt (Switzerland).For surgical procedures, fish were anesthetized with 0.01% tricaine methanesulfonate . Larvae were embedded in low-melting agarose using standard procedures. Adult fish were held dorsal side up by a fish holder made from wet sponges inside a flexible plastic tube. The body of the fish was held by the sponges while the head was free. The tube was integrated in a custom-made stereotactic chamber with lateral stabilizers that were used when high spatial precision and stability was required. The chamber was placed on a tilted stage under a stereomicroscope  and returned to their home tanks afterwards.HSV-1vectors were obtained from three different sources: (1) BioVex , (2) SinoGenomax (China), (3) the Massachusetts Institute of Technology (MIT) viral core (USA). Note that sources (1) and (2) have recently discontinued the custom production of HSV-1. All HSV-1 viruses used in this study and their inserts, sources, titers, and production methods  or, in a few cases, in 0.9% NaCl. Plasmid concentrations were between 0.2 μg/μl and 4 μg/μl. In most experiments, a concentration of approximately 1 μg/μl was used. Co-electroportation of two plasmids was performed using equal concentrations of each plasmid.Plasmids used for electroporation are summarized in Table Virus suspensions were injected into the dorsal telencephalon , the olfactory bulb, or Dp. All procedures were performed under a stereomicroscope. Experiments in the dorsal telencephalon did not require high spatial precision. In these cases, the fish was held by the sponge holder without lateral stabilizers. A craniotomy was made over the dorsal telencephalon near the midline using a dentist's drill. Micropipettes were inserted vertically through the craniotomy into the dorsal telencephalon using a manual 3-axis manipulator  using an electrode puller . The tip was broken to obtain a diameter of 10–20 μm. At each injection point, the capillary was pressurized using a syringe connected with flexible tubing and the ejected volume was measured by monitoring the movement of the meniscus inside the capillary.in vivo and to test the efficiency of different promoters.Stereotactic procedures for electroporation were equivalent to those used for viral injections. For electroporation in the dorsal telencephalon using external electrodes, 100–300 nl of plasmid suspension was injected at each of three injection points approximately 250, 350, and 450 μm below the level of the bone Figure , left. TTargeted electroporation in Dp using internal electrodes was performed using lateral stabilizers in the stereotactic chamber. DNA solution was loaded into a micropipette that was held vertically by a manual 3-axis manipulator as described above Figure . A pair The two electroporators used in this study included a basic instrument  and a more advanced instrument . Targeted local electroporation was performed exclusively using the NEPA21 electroporator because this instrument allowed for fine tuning of the pulse protocol based on tissue impedance. Pulse trains consisted of a pair of high-amplitude poring pulses with opposite polarity followed by a train of lower-amplitude transfer pulses. The polarity of the transfer pulses was reversed after 50% of pulses were applied Figure , right. ex-vivo preparation of the adult zebrafish brain as described , a Ti:Sapphire laser , a custom-built unit containing galvanometric scanners  and custom-built external detection optics with photomultipliers . GFP/YFP were excited at 860 or 980 nm; red-fluorescent proteins were excited at 980 nm. Fluorescence emission was detected in two channels using green (535/50 nm) and red (640/75 nm) emission filters. A third channel was used to acquire the signal of a position-sensitive detector for transmitted infrared light. This channel produced a contrast-enhanced transmitted light image that was used to direct the recording pipette for patch clamp recordings. The microscope and related equipment were controlled by ScanImage and Ephus software , a Multiclamp 700 B amplifier (Molecular Devices) and Ephus software  was added to the ACSF.Electrical stimulation in the olfactory bulb was performed by placing a glass pipette  filled with 1 M NaCl at the posterior end of the olfactory bulb. A train of 10 pulses  was delivered 10 times at an inter-trial interval of 12 s. In order to induce slow, epileptiform population activity, the GABA2). At least 15 trials were acquired for each cell. In some electrophysiological recordings, optical stimulation caused a small stimulus artifact that was removed from the recorded traces by replacing voltage values with a constant value.Optical stimulation of ChR2 with blue light was performed with a strong LED  that was mounted in the epifluorescence lamphouse attached to the Olympus BX-51 microscope body. Optical stimuli consisted of trains of light pulses  as described . The synaptic latency was estimated as the time between the offset of the 10 ms light pulse and the first inflection of the current trace. The inflection point was usually sharply defined within a time window of < 2 ms and determined manually by inspection of each trace. The amplitudes of averaged EPSCs and IPSCs were measured as the peak currents within a 20 ms time window after the offset of the light pulse relative to pre-stimulus baseline.Calcium signals (ΔF/F) were calculated as changes in fluorescence intensity (ΔF) relative to a baseline period of 2–4 s before response or stimulus onset (F). To quantify calcium signals of individual neurons, regions of interest were outlined manually on time-averaged ΔF/F maps. In order to quantify fluorescence changes of GECIs during epileptiform activity, 9 neurons from 3 fish were analyzed for each GECI. In each neuron, ΔF/F of calcium transients were measured at the soma and at a dendritic location that showed large ΔF/F values in time-averaged maps. Amplitudes of multiple large calcium transients were then averaged for each neuron, and mean calcium transients were then averaged over neurons for each GECI.In order to assess the fluorescence intensity in the dorsal telencephalon through the intact skull, individual fish were anesthetized and viewed through a fluorescence stereomicroscope. Fluorescence intensity was scored manually relative to the mean fluorescence intensity observed 10 days after electroporation of plasmid #10. This plasmid contained the hSyn promoter and was chosen as a reference because it produced an intermediate fluorescence intensity.in vivo by imaging fluorescence through the intact skull at different time points after injection. In addition, high resolution images of neurons expressing fluorescent markers were obtained by multiphoton microscopy in an ex-vivo preparation of the brain  within a volume of approximately 200 × 200 × 100 μm3 around the injection site.Most HSV-1 viruses produced fluorescence that could be observed through the intact skull. Fluorescence was first observed 2 days post-injection (dpi), reached a maximum around 9 dpi and declined slowly thereafter Figure , often ln = 3 fish). Few labeled neurons were found in telencephalic areas between the olfactory bulb and Dp and no labeled neurons were found in deep layers of the olfactory bulb  also produced robust fluorescence for > 2 weeks around the injection site . We refer to this approach as “external-electrode-electroporation” (EEP).We first developed a simple procedure to electroporate plasmids into neurons of the dorsal telencephalon without high spatial precision. Plasmid DNA , strong and widespread fluorescence was observed through the intact skull in 11/12 fish. Fluorescence was first detected 3 days post-electroporation (dpe), peaked around 8–12 dpe, and thereafter remained high for weeks Figures . Multiphn = 3 fish). Moreover, co-electroporation of a plasmid containing a Tet driver construct (plasmid #12) and a second plasmid containing a Tet responder construct (plasmid #13) resulted in transgene expression in a substantial number of cells  or plasmid #2 , reporter gene expression in Dp was observed in 85% of fish . The morphology of GFP-expressing neurons was normal without obvious signs of damage. In most cells, expression appeared strong compared to the expression of the same or similar transgenes in stable transgenic lines (not shown). Strong fluorescence was observed even for transgenes that are usually difficult to express at high levels such as fusion proteins containing ChR2 . Fluorescence intensity was scored relative to the signal observed 10 days after electroporation of construct #10 (hSyn::ChR2wt-GFP-mbd), which produced intermediate expression levels. The intensity and time course of expression varied between constructs but only one promoter  failed to produce detectable expression. The fastest onset of expression was produced by construct #14 (CMV::mRuby), reaching peak levels at 5 dpe. Thereafter, expression gradually declined until it became undetectable at 45 dpe. Expression driven by other constructs usually peaked at 10 dpe and declined more slowly. Three constructs  still generated substantial expression at 45 dpe. These constructs contained the human EF1α promoter (hEF1α), the EF1α promoter from Xenopus (xEF1α), and a heat-shock promoter from zebrafish (zHsp70l).EEP was performed in the dorsal telencephalon and fluorescence was examined through the intact skull at different time points , also produced the highest peak fluorescence signals. Somewhat weaker but still substantial fluorescence was observed after electroporation of plasmids #7, #10, and #11, which harbored the human synapsin-1 promoter (hSyn), the zebrafish Elavl3 promoter (HuC) and the chimeric CAG promoter . Strong and widespread expression using the zHsp70l promoter was observed without application of a heat shock. Expression may therefore be driven by basal activity of the promoter or activated by a cellular response to the electroporation event. These results indicate that the zHsp70l promoter is particularly effective in driving expression of transgenes in a wide range of neurons when introduced into the adult zebrafish brain, consistent with previous reports  was particularly strong and outlasted the fluorescence signals of other plasmids containing the same reporter Figure . To furtn = 4 for each plasmid). Moreover, we used targeted IEP to express plasmid #2 in Dp neurons. All constructs produced high-level expression. Labeled neurons had normal morphologies except for those electroporated with plasmid #3 (ChR2tc-mEos2), which sometimes showed unusual dendritic shapes and hot spots of fluorescence that may reflect protein aggregation. Targeted whole cell patch clamp recordings were performed from neurons expressing ChR2tc-GFP (n = 11 neurons in Dp) or ChR2wt-GFP-mbd  in an explant preparation of the whole brain  and different durations  were delivered at 1 Hz. In all neurons, action potentials were triggered reliably (probability > 90 %) when the duration of light pulses was 10 ms or longer . Excitatory post-synaptic currents (EPSCs) and inhibitory post-synaptic currents (IPSCs) were measured by holding the recorded neurons close to the reversal potentials for chloride currents (−70 mV) and cation currents (0 mV), respectively. EPSCs time-locked to the optical stimulus were observed in only one neuron, and stimulus-locked IPSCs were observed in two neurons  in Dp neurons by targeted IEP and prepared brain explant preparations at 7–10 dpe. Neurons were optically stimulated with trains of wide-field light pulses . Whole cell voltage clamp recordings were performed from ChR2tc negative neurons in Dp that were usually intermingled with ChR2tc positive neurons . At 7–10 dpe, fluorescence was examined by multiphoton microscopy in the ex-vivo preparation. In order to produce large changes in intracellular calcium concentration we applied the GABAA receptor blocker Gabazine (1 μM) through the bath. This treatment is known to induce epileptiform bursting of many neurons in the forebrain at low inter-burst frequency . Particularly large fluorescence changes were observed with GCaMP6s . Calcium transients observed with GCaMP6s  by IEP we also measured fluorescence changes evoked by odor stimulation. Previous electrophysiological studies showed that many Dp neurons receive depolarizing synaptic input during odor stimulation but only a small subset fires action potentials (Yaksi et al., We report methods to directly express transgenes in neurons of the adult zebrafish brain by HSV-1 or electroporation. Both methods are simple, efficient and can produce strong and long-lasting gene expression without obvious toxicity. In other species, fast gene transfer can be achieved using AAVs or lentiviruses but comparable methods have been lacking in zebrafish. This gap in the molecular toolbox for zebrafish may therefore be filled by HSV-1 and targeted electroporation.HSV-1 is used for gene transfer in other species because it exhibits a high potential to infect neurons and low levels of toxicity. Our observations in zebrafish are fully consistent with these properties of HSV-1. However, we observed substantial variation in reporter gene expression between different HSV-1 viruses, presumably depending on the method of virus production and other factors. HSV-1 can produce dense expression of transgenes, which is important to target large populations of neurons. The ability of HSV-1 to retrogradely infect neurons via their axons can be exploited to target defined projection neurons. Conceivably, additional cell type selectivity may be generated by the choice of the promoter, which can be exchanged using established procedures (Simonato et al., We established reliable protocols for gene transfer by electroporation using external or internal electrodes. Compared to gene transfer by HSV-1, transgene expression was sparser but strong. One advantage of electroporation is that plasmids can be introduced directly into neurons without the need to package genetic material into viruses or other vectors (Barnabé-Heider et al., Electroporation has been used previously to introduce DNA into individual or small groups of neurons (Haas et al., Efficient gene expression in the adult brain was achieved with a wide range of promoters that drive broad gene expression at early developmental stages. This result was not necessarily expected because gene expression in stable transgenic lines often becomes restricted in the adult brain (Stamatoyannopoulos et al., We took advantage of electroporation to express a variety of ChR2 variants and GECIs in the adult telencephalon. Using a simple procedure to assess basic functional properties of GECIs in the intact brain we observed functional differences between GECIs that corresponded well to previous observations in other assays and species (Akerboom et al., Dp is a telencephalic brain area that is homologous to mammalian olfactory cortex (Mueller and Wullimann, 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.


Hephaestin is a vertebrate multicopper ferroxidase important for the transfer of dietary iron from intestinal cells to the blood. Hephaestin is mutated in the sex-linked anemia mouse, resulting in iron deficiency. However, sex-linked anemia mice still retain some hephaestin ferroxidase activity. They survive, breed, and their anemia improves with age. To gain a better understanding of the role of hephaestin in iron homeostasis, we used the Cre-lox system to generate knockout mouse models with whole body or intestine-specific (Villin promoter) ablation of hephaestin. Both types of mice were viable, indicating that hephaestin is not essential and that other mechanisms, multicopper ferroxidase-dependent or not, must compensate for hephaestin deficiency. The knockout strains, however, both developed a microcytic, hypochromic anemia, suggesting severe iron deficiency and confirming that hephaestin plays an important role in body iron acquisition. Consistent with this, the knockout mice accumulated iron in duodenal enterocytes and had reduced intestinal iron absorption. In addition, the similarities of the phenotypes of the whole body and intestine-specific hephaestin knockout mice clarify the important role of hephaestin specifically in intestinal enterocytes in maintaining whole body iron homeostasis. These mouse models will serve as valuable tools to study the role of hephaestin and associated proteins in iron transport in the small intestine and other tissues. Every day, billions of iron atoms from the diet must be transferred from intestinal cells to the blood in order to maintain iron balance. The transfer of iron across biological membranes is usually associated with the oxidation or reduction of the iron, and current evidence supports an important role for the vertebrate multicopper ferroxidase (MCF) hephaestin (HEPH) in the export of iron from intestinal enterocytes sla) mouse sla mouse model arose in the 1950s in an irradiated mouse colony and has a deletion in the gene encoding HEPH (Heph), corresponding to a large internal portion of the protein Sla mice have a defect in iron absorption sla mice still remain iron-deficient throughout life relative to wild-type controls HEPH was identified in 1999 by Vulpe et al. who mapped the mutated gene responsible for the phenotype of the sex-linked anemia . The Hephint/int model was designed to enable the study of the specific role of HEPH in the intestine, since HEPH is expressed at low levels in other tissues as well. We also directly compared the phenotypes of Heph-/- and sla mice in order to determine potential differences in these models which could provide insights into HEPH function.The phenotype of In vivo experiments are reported here in accordance with the ARRIVE guidelines All work performed was in accordance with the National Institutes of Health (NIH) guidelines, as described in the Guide for the Care and Use of Laboratory Animals of the NIH, and with approval from the Office of Laboratory Animal Care at the University of California, Berkeley, and the QIMR Berghofer Medical Research Institute Animal Ethics Committee. -/- and Hephint/int mouse models Heph is located on the X chromosome, and thus males only carry one allele of Heph. The generation of the Heph floxed mouse strain  has been recently described Heph gene. We crossed Hephfl/fl mice (kindly provided by Professor Joshua Dunaief at the University of Pennsylvania and previously backcrossed onto the C57BL/6J background) with C57BL/6J transgenic mice bearing the Cre recombinase transgene driven by either the EIIa promoter, which is ubiquitously active C5379Lmgd, The Jackson Laboratory), or the Villin promoter, which is only activated in the intestine 997Gum, The Jackson Laboratory), in order to generate the Heph-/- and Hephint/int knockout strains, respectively. Expression of Cre recombinase leads to excision of the region in the DNA between the LoxP sites (exon 4 of Heph). Exon 4 is the second protein coding exon in Heph and contains residues required for the trinuclear copper cluster site that is needed for ferroxidase activity -/- strain, the EIIa-Cre recombinase transgene was bred out of the line.We used the Cre-lox system to create the Heph-/y and wild-type (WT) littermates for experiments, heterozygous Heph+/- females were bred with Heph-/y males. Male Hephint/y and Hephfl/y littermate controls were generated by crossing Hephfl/fl females with Hephint/y males. In this latter cross, all mice and progeny were homozygous or hemizygous for floxed Heph alleles, and the Hephint/y breeders and male Hephint/y and female Hephint/int progeny carried only one copy of the Villin-Cre transgene.To generate male Hephsla mice were from stocks maintained at QIMR Berghofer but were originally from The Jackson Laboratory . Tamoxifen-inducible FPN1 intestinal knockout mice (Fpn1int/int) were generated as previously described by crossing Fpn1fl/fl mice on a mixed background (kindly provided by Professor Nancy Andrews at Duke University) with mice bearing the VilERT2-Cre recombinase transgene (Tg(Vil-Cre/ERT2)23Syr) Fpn1 knockout in Fpn1 floxed mice bearing the VilERT2-Cre transgene, pups (7 days old) were injected once daily for three days with tamoxifen. The tamoxifen solution was prepared fresh on the first day of injection by dissolving tamoxifen powder  in 100% ethanol to 50 mg/mL. The dissolved tamoxifen was then diluted to 8.3 mg/mL in sunflower seed oil (Sigma) and then injected subcutaneously at 75 µg tamoxifen/g body weight. The remaining tamoxifen solution was stored at 4°C, and then warmed by hand and vortexed to ensure solubilization prior to use on subsequent days. Mice were closely monitored for any adverse reactions. Mice for all studies were on the C57BL/6J background unless otherwise noted.The EIIa- and Villin-Cre mouse strains were obtained from The Jackson Laboratory . The 59Fe were provided with iron-free deionized water and housed in grid-bottom cages designed to minimize coprophagia. Unless otherwise noted, all mice were allowed unlimited access to food and water.Mice were maintained on a standard rodent chow diet containing approximately 160 ppm iron  or, for iron absorption studies as indicated, an iron-deficient diet made in-house and described previously 2 gas inhalation or anesthetized by a single intraperitoneal (IP) injection of 44 mg/kg ketamine and 8 mg/kg xylazine in saline. Blood was then collected from the posterior vena cava and heart. Approximately 250 µL of blood was collected into potassium EDTA tubes  for whole blood analysis. Tissues were harvested, trimmed to remove connective tissue, and either immediately snap frozen in liquid nitrogen for future RNA/protein analyses or incubated in 10% phosphate buffered formalin overnight at 25°C for future processing for Perls' Prussian blue staining for iron. Intestinal enterocytes were collected as previously described g for 5 minutes at 4°C, and the supernatant was discarded. Blood and other non-enterocyte cells, if present, form a small visible layer on top of the enterocytes after centrifugation. This layer was removed by aspiration if present. Enterocytes were then resuspended in cold PBS by inversion and centrifuged again. The wash and centrifugation steps were repeated twice more, and the cells were then snap frozen. Previous analyses have indicated that >95% of the cells collected by this method are enterocytes Mice were euthanized by CO3) of frozen tissue was first homogenized in 500 µL ice-cold lysis buffer containing 25 mM tris(hydroxymethyl)aminomethane (Tris), pH 7.4, 125 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), and 0.2% polysorbate 20 (Tween-20) by vigorous shaking with 1.4 mm ceramic beads  in a Precellys24 tissue homogenizer  at 4°C. DNA was prepared from tail tip and ear punch pieces or 5 µL of other tissue homogenates using the QuickExtract DNA Extraction Solution  as per the manufacturer's instructions, but with only 50 µL extract solution per sample. PCR was performed using MangoTaq DNA Polymerase master mix  as per the manufacturer's instructions. Primers, annealing temperatures, and expected product sizes are described in Mice were routinely genotyped by PCR using DNA obtained from a 1–2 mm piece of the distal tail tip or from a small ear punch sample. For genotyping of other tissue samples, a small piece (∼2–3 mmHprt). All primers  and RNA was extracted as per the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using oligo(dT) primers and Moloney Murine Leukemia Virus reverse transcriptase (Invitrogen) as per the manufacturer's instructions. Real-time quantitative PCR (RT-qPCR) was performed using LightCycler (LC) 480 SYBR Green I Master Mix in an LC480 machine  as per the manufacturer's instructions, and the data were analyzed by calculating the concentration of each sample from its Ct value using a standard curve made from pooled sample cDNA as described previously  primers  were valg for 30 minutes at 4°C to pellet debris, and the supernatant was then collected and assayed for protein concentration using the Pierce BCA Protein Assay Kit . Total protein (60–80 µg) was mixed with SDS loading buffer with fresh dithiothreitol (DTT) and heated at 75°C for ten minutes . The samples were then separated on 10% (for HEPH and FPN1) or 15% (for FTN) Tris-glycine SDS polyacrylamide denaturing gels, and then transferred to Immobilon-FL polyvinyl difluoride (PVDF) membranes . Membranes were blocked for one hour at room temperature with shaking in blocking buffer ). Membranes were then incubated with the relevant primary antibody diluted in blocking buffer for one hour at room temperature  with protease inhibitors as per the manufacturer's instructions . The homogenized sample was centrifuged at 16,000×Tissue was paraffin processed, sectioned, and the sections stained for ferric iron using Perls' Prussian blue stain by the Histotechnology Facility at QIMR Berghofer Blood samples were analyzed on a Coulter Ac•T diff Analyzer using the Veterinary Application Software  and reticulocytes were counted at the Pathology Department of the Royal Brisbane and Women's Hospital  using a Sysmex XE-5000 automated hematology analyzer .59Fe followed by whole body counting. Mice initially maintained on a chow diet were switched to an iron-deficient diet and subsequently housed in cages with wire grid bottoms. After a specified period of time on the diet, mice were dosed by gavage with 100 µL of a freshly prepared solution containing 200 µM ferrous sulfate in 10 mM HCl, spiked with 3 µCi 59Fe radiolabeled iron  per dose. In the iron absorption study where mice were put on the iron deficient diet for one week prior to dosing, mice were fasted but provided with water ad libitum for approximately four hours both before and after dosing. The radiation in each mouse was measured using a RAM DA gamma counter with a PM-11 tube  at a fixed distance just after dosing, and then again five days later. The second reading was taken five days after dosing to give time for complete turnover of the intestinal epithelial cells, so that only 59Fe that had been transferred from enterocytes to the body would be detected Whole animal absorption measurements were carried out by giving mice an oral dose of All values are expressed as mean ± standard deviation (SD). Tests for significance were performed in GraphPad Prism version 6.0c for Mac OS X. For comparisons between two groups, Student's t-test and the F test for equal sample variance were performed. For comparisons among multiple groups, one- way analysis of variance (ANOVA) with Tukey's post test and the Brown-Forsythe test for equal sample variance were performed. When the sample variances were determined to be unequal, Welch's correction was applied to t-tests and the Kruskal-Wallis test followed by the Dunn Multiple Comparison post test was performed on data analyzed by one-way ANOVA. Pearson's chi-squared test was used to compare actual versus expected genotype ratios obtained in our breedings. A P value of 0.05 percent or less was considered significant.-/y and WT control littermates  bred with male Heph knockout hemizygotes, revealed slightly lower numbers of Heph-/y versus WT and Heph-/- versus heterozygous littermates , but results were not statistically different from the expected 50∶50 ratios  as determined by Pearson's chi-squared test. Heph-/y and WT male littermates exhibited no significant differences in body weight when weighed weekly from 3–7 weeks of age  from 34 litters born to 8 female heterozygotes . Heph-/y and sla mouse blood also contained significantly less hemoglobin overall, and the average concentration of hemoglobin in the red cells of these mice was low. Both genotypes also had nearly twice the number of reticulocytes as WT controls. Older (10–12 weeks of age) male Heph-/y and sla mice were no longer anemic. Heph-/y and sla mice had significantly more red cells than WT controls, but the red cells were still, on average, microcytic. The average concentration of hemoglobin per red cell no longer differed from that of WT mice and neither the hematocrit nor the total amount of hemoglobin in the blood was lower in 10–12 week old Heph-/y and sla mice than in WT controls.Adult Hephals aged . At 6–7 -/y and sla mice relative to controls at both 6–7 and 10–12 weeks of age  protein, a marker of iron stores, was examined by immunoblotting and levels were approximately four times higher in Heph-/y enterocytes than WT controls  of any mice, and there were no differences between the levels of radioactive iron in the GI tract of WT and Heph-/y mice  at 6–7 weeks of age by one-way ANOVA followed by Tukey's post test, or the Kruskal-Wallis test followed by the Dunn Multiple Comparison post test where appropriate, Heph-/y and sla mice had significantly smaller red cells, lower hemoglobin and hematocrit levels, and more reticulocytes than Hephint/y mice, indicating a more severe anemia , suggesting that systemic upregulation of CP is not involved. Future studies in mice with double knockout of CP and HEPH will allow us to better address the role of CP in intestinal iron absorption. More recently, a third MCF, zyklopen, was identified which is expressed in some tissues but not intestinal enterocytes, so it is unlikely to compensate for loss of HEPH Additional ferroxidases may compensate for the lack of HEPH in the intestine. A cytosolic ferroxidase activity has been detected in extracts from isolated enterocytes from Heph-/y and Hephint/y mice indicate that HEPH function in intestinal enterocytes alone is particularly important for maintaining systemic iron homeostasis. This observation is consistent with the critical role of these cells in body iron acquisition, as well as the high level of HEPH expression in this tissue. However, the milder presentation of Hephint/y mice, as revealed in their hematology and liver Hamp1 expression, indicates that HEPH plays additional extra-intestinal roles in iron metabolism.The similar phenotypes of the Heph-/y versus control mice at 10–12, 23–26 or 76–79 weeks of age (data not shown). The brain and eyes, however, were not studied. Because HEPH is also ablated in the intestine of Heph-/y mice, less iron is available to load other cells in the body, and thus any iron-loading phenotypes may not be readily apparent without administration of parenteral iron to bypass intestinal iron absorption. As has been shown in the eye, some cell types express other ferroxidases that could at least partially compensate for the lack of HEPH, so double knockout models may provide a more sensitive means to determine the importance of HEPH in other cell types If HEPH is involved in efficient iron efflux from other cell types, it might be expected that some other cell types would show iron accumulation in the whole body knockout model. Currently HEPH is only known to play an important role in the intestine, eye, and brain, with cells in these tissues accumulating iron when HEPH expression is perturbed Figure S1Verification of intestine-specific knockout of Heph at the DNA level. DNA extracted from a panel of homogenized tissues from Hephfl/y and Hephint/y littermates was amplified by PCR. Top panel, results for the “Heph knockout” reaction, which yields a strong band at 230 bp if a Heph knockout allele is present. Bottom panel, results from the “Heph wild-type” reaction, which yields a strong band at 240 bp if a wild-type Heph DNA is present and a band at 300 bp if Heph floxed DNA is present. Tissues from two mice of each genotype were tested.(TIF)Click here for additional data file.Figure S2int/y mice.Verification of HEPH knockout in the enterocytes of Heph HEPH protein expression in enterocytes from Hephfl/y and Hephint/y mice (N = 6 per genotype) was examined by SDS-PAGE followed by immunoblotting with the HEPH D4 antibody. Actin expression in the same samples is shown below.(TIF)Click here for additional data file.Figure S3Dmt1-/y,  mRNA levels in the enterocytes of Hephslaint/y mice, and Hephfl/y and WT controls., and Heph The expression of Dmt1 mRNA as measured by RT-qPCR using cDNA prepared from RNA from isolated proximal small intestinal enterocytes from 6–7 week old male mice, N = 11–19 mice per genotype. Dmt1 expression was normalized to the expression of the Hprt housekeeping gene. Results are presented as mean ± SD. Statistical analysis was carried out across all five groups by the Kruskal-Wallis test followed by the Dunn Multiple Comparison post test. Groups that share at least one letter are not significantly different.(TIF)Click here for additional data file.


Mycobacterium avium subspecies paratuberculosis (MAP), a slow-growing, acid-fast bacterium. It is a part of the Mycobacterium avium complex (MAC), which also comprises of opportunistic pathogens of humans, as well as innocuous, environmental bacteria [Mycobacterium tuberculosis glycolipids by TLR2 and thereby restraining the expression of MHC-II molecules and processing of antigen and presentation of MHC restricted antigens by macrophages in a TLR2-dependent manner [M. paratuberculosis evades and resists this active CD4+ T-cell response and survives and infects other macrophages, a hallmark of mycobacterial infections. In this context, the identification of antigenic proteins is useful in understanding the immune evasion mechanism of MAP within host macrophages.Johne’s disease is a chronic mycobacterial infection of the small intestine affecting ruminants worldwide. It is estimated that over 50% of the European Union (EU) dairy holdings is infected . The caubacteria . MAP genbacteria ,4. It hat manner . HoweverIn this study, a comparative proteomic analysis of an orthologous putative gene MAP1138c or LprG and H37Rv1411c (LprG) was done using online bioinformatics tools namely ProtPram and SignalP 4.1, Phosphor 2.1, ProtScan and Hydropathy plot. The theoretical 3D structure of MAP1138c was generated using SWISS MODEL server using H37Rv1411c (LprG) as template. The secondary and super secondary structures of MAP1138c protein were identified and analyzed using PROMOTIF. The theoretical 3D structure generated was further assessed for its reliability using QMEAN, ANOLEA and GROMOS structure assessment tools of SWISS MODEL server.Gapped BLAST of MAP1138c identified a homolog having sequence identity match of 70 % and positive match of 83%, respectively with crystal structure of H37Rv1411c (LprG). Proteomics analysis reveals that MAP1138c is a prolipoprotein and is translocated to the cell membrane using Tat pathway and probably the mature protein is secreted out after cleavage by SPase I peptidases. The ProtPram analysis shows similarity in physiochemical parameters namely Grand average of hydropathicity, aliphatic index and instability index for both H37Rv1411c (LprG) and MAP1138c proteins  in the cavity and initiates a TLR2 mediated immune evasion. The analysis of QMEAN , ANOLEA and GROMOS structure assessment tools reveal that the global and local properties of MAP1138c protein model generated by automated SWISS-MODEL server using H37Rv1411c (LprG) as template is reliable and can be used for further analysis (Figure  agonistsin silico analysis of MAP1138c, we identified several key aspects fundamentally important to propose a function for this putative protein. These studies reflect upon the possible role of MAP1138c in inhibiting MHC-II Ag processing leading to reduced recognition of infected macrophages by CD4+ T cells. This may be an important mechanism for immune evasion during persistent M. paratuberculosis infection in ruminants and humans.Through our comprehensive