Influenza A viruses (IAVs) cause seasonal epidemics and occasional pandemics. Most pandemics occurred upon adaptation of avian IAVs to humans. This adaptation includes a hallmark receptor-binding specificity switch of hemagglutinin (HA) from avian-type α2,3- to human-type α2,6-linked sialic acids. Complementary changes of the receptor-destroying neuraminidase (NA) are considered to restore the precarious, but poorly described, HA-NA-receptor balance required for virus fitness. In comparison to the detailed functional description of adaptive mutations in HA, little is known about the functional consequences of mutations in NA in relation to their effect on the HA-NA balance and host tropism. An understudied feature of NA is the presence of a second sialic acid-binding site (2SBS) in avian IAVs and absence of a 2SBS in human IAVs, which affects NA catalytic activity. Here we demonstrate that mutation of the 2SBS of avian IAV H5N1 disturbs the HA-NA balance. Passaging of a 2SBS-negative H5N1 virus on MDCK cells selected for progeny with a restored HA-NA balance. These viruses obtained mutations in NA that restored a functional 2SBS and/or in HA that reduced binding of avian-type receptors. Importantly, a particular HA mutation also resulted in increased binding of human-type receptors. Phylogenetic analyses of avian IAVs show that also in the field, mutations in the 2SBS precede mutations in HA that reduce binding of avian-type receptors and increase binding of human-type receptors. Thus, 2SBS mutations in NA can drive acquisition of mutations in HA that not only restore the HA-NA balance, but may also confer increased zoonotic potential. Influenza pandemics can have devastating effects as was dramatically demonstrated by the so-called Spanish flu in 1918. Such pandemics are caused by animal influenza A viruses (IAVs) that are sufficiently adapted to humans. This adaptation includes among others a change in the receptor-binding properties of the viral hemagglutinin (HA). Some avian IAVs have already acquired the ability to bind to human-type receptors via their HA. We provide novel insights into the evolution of such viruses and elucidate a crucial role for NA herein. This study is expected to contribute to the timely recognition of animal viruses with increased zoonotic potential. Influenza A viruses (IAVs) cause seasonal epidemics as well as occasional pandemics. The latter occur when animal viruses cross the host species barrier and adapt to humans. IAV particles contain hemagglutinin (HA) and neuraminidase (NA) glycoproteins with receptor-binding and -cleavage activities, respectively. HA binds to glycans terminating in N-acetylneuraminic acid  and is a major host tropism determinant. Human viruses prefer binding to α2,6-linked SIA  expressed on human upper airway epithelia whereas most avian viruses prefer binding to α2,3-linked SIA  present on bird intestinal epithelia . Some avAs yet, little is known about the importance of the NA in host tropism changes. Although the NAs of human IAVs are relatively better at cleaving α2,6 SIA they still, like avian IAVs, display a higher cleavage rate cleavage of α2,3 SIAs –8. It haThe NA protein is a type II transmembrane protein assembling into a mushroom-shaped homo-tetramer. Each globular head domain contains a catalytic site, which is highly conserved between IAVs of different subtypes , 7. A grRecently, kinetic assays based on bio-layer interferometry (BLI) were introduced to study the balance between HA and NA in the context of virus particles. These assays allow monitoring of HA binding and NA cleavage in real time using synthetic glycans or sialylated glycoproteins , 24. IAVIn the present study we show that mutation of the 2SBS in NA disturbs the HA-NA balance of avian H5N1 virus. Importantly, we establish the existence of a functional HA-NA crosstalk by showing that a mutated, non-functional 2SBS drives the selection of mutations in HA that modify its receptor-binding properties, thereby restoring the HA-NA balance. As a corollary, an acquired mutation also resulted in concomitant enhanced binding to human-type receptors. These findings are corroborated by phylogenetic analyses of H9N2 viruses. Also for H9N2 viruses mutations in the 2SBS are selected prior to the acquisition of mutations in the receptor-binding site (RBS) of HA that decrease binding to avian-type receptors and increase binding to human-type receptors. Collectively, these results emphasize the importance of the 2SBS for the HA-NA balance of avian viruses and provide an explanation for the evolution of avian viruses that bind to human-type receptors.432E) [432E viruses carrying the same H5 protein, showed that mutation K432E in NA disturbs the HA-NA balance as it resulted in slower virion self-elution (We previously generated an H5N1 virus with mutation K432E in the 2SBS of NA (H5N1432E) , 24 . After embering) ; Fig 2A.mbering) ; Fig 1B.mbering) ; Fig 1B.Two of the observed HA mutations are located in  or close to  the RBS of H5 . Substit432E) obtained significantly differed titers at the early time point, indicating that a disturbance of the HA-NA balance of H5N1 virus, resulting in slower virion self-elution as determined by BLI, negatively affects virus replication.The selection of the mutations in HA indicates that viruses carrying these mutations are more fit and replicate more efficiently in MDCK cells. To analyze the effect of these HA mutations in more detail, we determined virus growth curves on MDCK cells. All mutant viruses reached higher titers than their respective parental viruses at the early time point . Differe223N, H5125N, and H5156T) and analyzed their receptor binding properties. Each of the three mutations negatively affected the H5 protein binding rate to an avian-type synthetic glycan  as observed using BLI (125H/NN1432E). The initial binding rate observed for these latter virus stocks did not differ compared to H5N1432E  displayed much faster self-elution than parental H5N1432E virus. This self-elution was even faster than H5N1 WT virus, carrying the same NA (156TN1432E) or 125 (H5125H/NN1432E) eluted much faster than virus carrying a wild type H5 combined with the same NA (H5N1432E)  . The fas432E  is paired with HA of H1N1 strain PR8 (432E). The selWT virus , upon pa protein . As experain PR8 . Recombirain PR8 . These HR8 (432E), but agaR8 (432E). We conc125H/NN1432E and H5156TN1432E viruses was observed when compared to the control virus H5N1432E with the same NA  by co-expression of human beta-galactoside alpha-2,6-sialyltransferase 1 . The presence of increased and decreased amounts of α2,6 and α2,3 SIAs on 6’LAMP1 compared to ‘standard’ LAMP1 was confirmed by binding of lectins specific for α2,6 (SNA) or α2,3 (MALI) SIAs . As pred H5N1 WT , in cont H5N1 WT . As a co 6’SLNLN , these vLN (432E .https://nextstrain.org)[Our results indicate that mutation of the 2SBS may drive the selection of mutations in HA that reduce the avidity of HA-receptor binding and may affect HA receptor-binding specificity. The 2SBS in NA is highly conserved in avian IAVs with notable exceptions in H9N2 viruses . Currentrain.org)Fig 5), , https:/rain.org), 18. Subrain.org), 31, accWe here show that mutation of the 2SBS in H5N1 NA, resulting in reduced NA activity, disturbs the HA-NA balance of virions, particularly when NA is accompanied by a strong-binding HA. Passaging such viruses rapidly resulted in the selection of mutations in NA and/or HA that restore the HA-NA balance providing further evidence for the importance of a precisely tuned balance for optimal replication and for an important role of the 2SBS therein. Mutations in HA, which resulted in higher virus titers particularly at early time points, reduced receptor-binding avidity either because they affect the RBS directly (S223N and H125N) , 33, or As H5 prefers binding to avian-type receptors, mutations in HAs, upon disruption of the 2SBS, are primarily selected on their ability to reduce binding to these receptors. Mutation S223N, however, simultaneously resulted in increased binding to human-type receptors. This mutation has previously been observed in H5N1 viruses isolated from humans , 38 and It is well established that all human viruses contain substitutions in the RBS of HA e.g. Q226L) that increase binding to α2,6- linked SIAs and reduce binding to α2,3-linked SIAs, when compared to avian viruses 26L that –41. In a432E. The nucleotide sequence encoding the wild-type multi-basic amino-acid sequence in H5 was modified to encode a low-pathogenic cleavage site as described previously [Recombinant viruses were generated in the background of A/Puerto Rico/8/34 H1N1 (PR8 H1N1) by means of reverse genetics, using PR8 plasmids provided by drs Hoffmann and Webster  as described , 44. RecAfter rescue of the viruses in MDCK-II cells, the virus stocks were immediately used in the passage experiments. Viruses were passaged on MDCK-II cells at a multiplicity of infection (MOI) of 0.001 tissue culture infectious dose 50 (TCID50) per cell. After about 44hpi, cells culture media of infected cells were harvested and titers were determined by endpoint titration in MDCK-II cells. For each passage, viral RNA was extracted from cell culture media of infected cells with NucleoSpin RNA virus kit (Macherey-Nagel) according to the manufacturer’s instructions and converted to cDNA using SuperScript III Reverse Transcriptase (invitrogen) with random Hexamers (invitrogen). HA and NA sequences were amplified using the Q5 High-Fidelity DNA Polymerase (NEB) using specific primers . PCR products were sized-separated using agarose gel electrophoresis and purified by NucleoSpin Gel and PCR Clean-upkit (Macherey-Nagel) following manufacturer’s instructions and then analyzed by Sanger sequencing (Macrogen).Human-codon optimized cDNAs encoding the H1 ectodomain of A/Puerto Rico/8/34/Mount Sinai (H1N1)(GenBank accession no. AF389118.1) or H5 ectodomain of A/duck/Hunan/795/2002(H5N1) (GenBank accession no. CY028963.1) were cloned into pFRT or pCD5 expression plasmids as described previously . Expressobs = dB/dT) per virus particle, which quantifies virus binding affinity, were calculated similarly as previously described [BLI assays were performed using the Octet RED348. All the experiments were performed in Dulbecco's phosphate buffered saline (PBS) with Calcium and Magnesium (Lonza) at 30°C and with plates shaking at 1000 rpm. Streptavidin sensors were loaded to saturation unless indicated otherwise with biotinylated synthetic glycans 3’SLNLN, 6’SLNLN, LNLN, or with LAMP1 or 6’LAMP1 glycoproteins. Synthetic glycans were synthesized at the Department of Chemical Biology and Drug Discovery, Utrecht University, Utrecht, the Netherlands and the Complex Carbohydrate Research Center, University of Georgia, Athens, USA . The virescribed , 24. Forescribed . In shorescribed , 24, In 156T, H5125N, H5223N) were precomplexed at 50 μg/mL with StrepMAb-Classic  and goat anti-mouse IgG H&L-AlexaFluor647  in 50 μL TSM binding buffer. After incubation on ice for 30 min, the mixtures were added to the sub-arrays for 90 min. Washes were performed as described above for the plant lectins. Similarly as for the HAs, virus stocks (H5N1 and H5223NN1) were precomplexed with Fi6v3 antibody (5 μg/mL) and anti-human IgG-Cy3  in the presence of OC (10 μM). All wash steps were performed in the presence of OC (10 μM). Washed arrays were dried by centrifugation and immediately scanned for fluorescence on a GenePix 4000 B microarray scanner (Molecular Devices). The detection gain was adjusted to avoid saturation of the signal. The data were processed with GenePix Pro 7 software and further analyzed using our home written Microsoft Excel macro. After removal of the lowest and highest value of the 6 replicates, the mean fluorescent intensities (corrected for mean background) and standard deviations were calculated (n = 4). Data were fitted using Prism software .The 100 μM synthetic compounds in sodium phosphate buffer  were printed on activated glass slides  by piezoelectric non-contact printing  with a drop volume of ~400 pL and 1 drop per spot at 50% relative humidity . CompounS1 Fig(A) Hemagglutination assays were carried out using virus-like particle (VLP) preparations containing similar amounts of N1 protein similarly as described previously . In shor(TIF)Click here for additional data file.S2 Fig432E passaging series A1 (432E passaging series A1 and B2. Mutated residues and passage numbers are indicated.Examples of sequencing results are shown. (A) Sequencing results obtained for NA of H5N1eries A1 . (B) Seq(TIF)Click here for additional data file.S3 FigSequence of H5 protein of A/duck/Hunan/795/2002(H5N1) used in this study is shown. Residues that were mutated in this study are colored magenta . Amino acids of the three structural elements  of RBD are shown in yellow. The four conserved, structurally-important amino acids of RBS are labelled with asterisks. Mutations A156T and A156S result in a N-glycosylation consensus sequence .(TIF)Click here for additional data file.S4 Fig156T runs at a higher position in the gel than the other H5 proteins. (B) After (mock) treatment of H5 with PNGase F, the recombinant soluble H5 and H5156T proteins were examined by gel electrophoresis and GelCode Blue staining. The difference in electrophoretic mobility of the H5 proteins is lost upon removal of the N-glycans with PNGase F, indicating that H5156T contains an additional N-glycan side chain compared to H5. The position in the gel of relevant molecular weight markers is shown on the right side of the gels.(A) Recombinant soluble H5 proteins expressed in HEK293S GnTI(-) were analyzed by gel electrophoresis followed by GelCode Blue staining. H5(TIF)Click here for additional data file.S5 FigBinding of HA to fetuin was analyzed using a fetuin solid phase binding assay as described previously , 53. Bri(TIF)Click here for additional data file.S6 FigGlycan microarray analysis was used to determine the receptor binding specificities of recombinant soluble H5 (A), H5156T (B), H5125N (C) and H5223N (D). All the HAs were precomplexed with antibodies against the Strep tag and goat anti-mouse IgG H&L (Alexa Fluor 647) similarly as described previously . The mea(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.S3 Table(TIF)Click here for additional data file.


Spinocerebellar ataxia type 1 (SCA1) is an adult-onset neurodegenerative disorder characterized by motor incoordination, mild cognitive decline, respiratory dysfunction, and early lethality. It is caused by the expansion of the polyglutamine (polyQ) tract in Ataxin-1 (ATXN1), which stabilizes the protein, leading to its toxic accumulation in neurons. Previously, we showed that serine 776 (S776) phosphorylation is critical for ATXN1 stability and contributes to its toxicity in cerebellar Purkinje cells. Still, the therapeutic potential of disrupting S776 phosphorylation on noncerebellar SCA1 phenotypes remains unstudied. Here, we report that abolishing S776 phosphorylation specifically on the polyQ-expanded ATXN1 of SCA1-knockin mice reduces ATXN1 throughout the brain and not only rescues the cerebellar motor incoordination but also improves respiratory function and extends survival while not affecting the hippocampal learning and memory deficits. As therapeutic approaches are likely to decrease S776 phosphorylation on polyQ-expanded and WT ATXN1, we further disrupted S776 phosphorylation on both alleles and observed an attenuated rescue, demonstrating a potential protective role of WT allele. This study not only highlights the role of S776 phosphorylation to regulate ATXN1 levels throughout the brain but also suggests distinct brain region–specific disease mechanisms and demonstrates the importance of developing allele-specific therapies for maximal benefits in SCA1. Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease caused by the expansion of CAG repeats encoding the polyglutamine (polyQ) tract in Ataxin-1 (ATXN1) . The disPCP2-hATXN1[82Q]) reduced hATXN1[82Q] transgene protein levels and improved the cerebellar motor coordination phenotypes , which recapitulates all major human SCA1 features  displayed a reduced rescue compared with mice with the S776A mutation on only the expanded allele, suggesting that the WT ATXN1 might exert neuroprotective properties.Here, we show that ATXN1 S776 is phosphorylated throughout the brain. We use CRISPR/Cas9 technology to disrupt endogenous S776 phosphorylation, by mutating the serine to alanine (S776A), on the unexpanded WT features . We findATXN1 allele should be selectively targeted for maximal therapeutic benefit.These findings highlight the potential of disrupting S776 phosphorylation as a means for reducing ATXN1 levels throughout the brain, suggest distinct brain region–specific disease mechanisms, and show that the polyQ-expanded PCP2-hATXN1[82Q] SCA1 mouse model reduces hATXN1[82Q] protein levels in the cerebellum . These data show that endogenous ATXN1 is phosphorylated at S776 in various tissues throughout the mouse, suggesting that S776 phosphorylation may regulate ATXN1 levels in cerebellar Purkinje cells and throughout the brain and peripheral tissues.To address these questions, we first used an ATXN1 S776 phospho-specific antibody on protein extracts of C57BL/6J WT mice . Althougd cortex , as wellAtxn1[2Q] allele of C57BL/6J WT mice to generate 2Q[S776A]/2QAtxn1 animals. Additional synonymous mutations were added to facilitate genotyping. Sanger sequencing confirmed the correct mutations in the F1 heterozygous offspring of 2 lines  on the unexpanded  2 lines .2Q/2QAtxn1 (WT), 2Q[S776A]/2QAtxn1 (WT S776A heterozygous), and 2Q[S776A]/2Q[S776A]Atxn1 (WT S776A homozygous) mice  mice . Specifius) mice . We confus) mice , thus vaAtxn1 mRNA levels were unchanged  knockin mouse model, which, similar to SCA1 patients, expresses 1 Atxn1 allele with a CAG-repeat expansion (Atxn1[154Q]) and 1 unexpanded WT Atxn1 (Atxn1[2Q]) allele  lines  allele . We conf) allele . The segA) lines .After backcrossing, we again measured ATXN1 protein levels in the cerebellum, brainstem, and hippocampus. Disruption of S776 phosphorylation in the polyQ-expanded ATXN1 reduced the protein levels of ATXN1[154Q] in all tested brain regions in both founder lines . In contSCA1 mice recapitulate many pathological and behavioral characteristics of patients affected with SCA1 and develop first symptoms of SCA1 around 5 weeks of age , 19. To Two of the most visually striking features of SCA1 mice, the formation of a hunched posture (kyphosis) and the failure to gain weight, remained unaltered in the SCA1 S776A mice . SCA1 miImpaired motor coordination in SCA1 is caused by the degeneration of cerebellar Purkinje cells . TherefoPCP2-hATXN1[82Q] mouse model was previously shown to abolish ATXN1 inclusion formation  mice  mice . Westernts) mice .Similar to SCA1 and SCA1 S776A mice, S776A double mutants develop kyphosis  and failWe observed a similar effect in regard to the life span of S776A double-mutant mice. Confirming our previous results, SCA1 S776A mice had an extension in life span of approximately 6–7 weeks . This reP value) revealed separate clustering of WT, SCA1, and SCA1 S776A samples  compared with WT, whereas SCA1 S776A mice showed drastically fewer transcriptional changes  mice (Atxn1/–– (Atxn1-KO) mice (A1) mice  and AtxnKO) mice  were bac2Q[S776A]/2QAtxn1 and 154Q[S776A]/2QAtxn1 mice were generated via CRISPR/Cas9-mediated gene editing  using the Phusion High-Fidelity DNA Polymerase (New England Biolabs) and the primers gRNA_S776A_For (5′-TTAATACGACTCACTATAGGGGCGCCGGAGACCCGTAAACGTTTTAGAGCTAGAAATAGC-3′) and gRNA_S776A_Rev (5′-AAAAGCACCGACTCGGTGCC-3′). The reaction was run on a low melting 2% MetaPhor Agarose gel (Lonza) at 80 V at 4°C. The corresponding band was excised and purified using the Wizard SV Gel and PCR Clean-Up System (Promega). The PCR product was then in vitro transcribed using the MEGAshortscript T7 Transcription kit (Invitrogen), and the gRNA was purified using the MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific). editing , 26. TheAtxn1 . The additional synonymous mutations were used to alter the protospacer adjacent motif to increase editing efficiency as well as to allow for simple genotyping by differential primer hybridization.A single-stranded oligonucleotide (ssODN) was purchased from Integrated DNA Technologies for homologous-directed recombination to introduce the S776A and additional synonymous mutations in 154Q/2QAtxn1 males, the females’ ova were dissected. An injection mixture containing 30 ng/μL Cas9 protein (PNA Bio), 20 ng/μL sgRNA, and 40 ng/μL ssODN in 10 mM Tris, pH 7.5, and 0.25 mM EDTA was prepared, and 2 pL was injected into the pronucleus of extracted ova. The ova were then transferred into oviducts of pseudopregnant FVB females by the Baylor College of Medicine Genetically Engineered Mouse Core.Female C57BL/6J WT mice (3–4 weeks of age) were purchased from The Jackson Laboratory and super-ovulated via injection of 5 IU PMSG (Thermo Fisher Scientific) and 48 hours later with 5 IU HCG (Thermo Fisher Scientific) in 0.9% NaCl. Upon overnight breeding with Upon birth, the following primers were used to distinguish unmodified (WT) and modified (S776A) alleles: WT/S776A_CRISPR_For (5′-CCAAACTCTCTGTTGGGGACGTCTGCATCTCGCTCAC-3′), WT_CRISPR_Rev (5′-TTCTCCAGTTTACGGGTCTCCGGCGCCGA-3′) and S776A_CRISPR_Rev (5′-GATTTTTCGAGCTTTCGTGTTTCGGGTGCCGCCCATCTC-3′). Note that the forward primer is the same for both reactions. Positive founders were backcrossed to C57BL/6J WT animals (The Jackson Laboratory), and the correct sequence of the F1 offspring was confirmed using Sanger sequencing (Genewiz). The mice were backcrossed for a minimum of 3 generations before conducting molecular and behavioral assays to prevent off-target effects.n = 6 per genotype). Total RNA was isolated using the miRNeasy Mini Kit from Qiagen following the manufacturer’s instructions. RNA was quantified using NanoDrop 1000 (Thermo Fisher Scientific), and random-primed cDNA was prepared from 1 μg total RNA using M-MLV Reverse Transcriptase (Invitrogen). RT-qPCR was then performed with PowerUp SYBR Green Master Mix (Applied Biosystems), and samples were run on a real-time PCR detection system (Bio-Rad CFX96). All samples were analyzed in triplicate, and Atxn1 expression levels were normalized to the expression of the housekeeping gene Gapdh. Primers for Gapdh and Atxn1 were obtained from MilliporeSigma. Primers used for Gapdh were Gapdh_For (5′- AGGTCGGTGTGAACGGATTTG-3′) and Gapdh_Rev (5′-TGTAGACCATGTAGTTGAGGTCA-3′) and primers used for Atxn1 were Atxn1_For (5′-GAGAATCGAGGAGAGCCAC-3′) and Atxn1_Rev (5′- AGACTTCGACACTGACCTG -3′).RNA from different brain regions was isolated from 6-week-old mice (n = 6 per genotype) were prepared by Dounce homogenization in NETN buffer . Samples were sonicated for a total of 10 times, incubated for 30 minutes at 4°C, and spun at 13,000 rpm at 4°C for 15 minutes. Protein concentrations of the supernatant were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Samples were diluted and prepared in NuPAGE sample reducing agent (Invitrogen) and NuPAGE LDS Sample Buffer (Invitrogen). The samples were then boiled for 10 minutes and then run on NuPAGE 4%–12% Bis-Tris gel 1.5 mm 15-well gels (Invitrogen) in MES running buffer . The proteins were subsequently transferred to Immobilon-FL PVDF membranes . After blocking for 1 hour at room temperature with 1:1 Odyssey Blocking Buffer TBS (LI-COR Biosciences) in TBS, membranes were probed overnight at 4°C with anti-ATXN1  for 24 hours and then placed in 30% sucrose in PBS. Once the brains had sunk, they were embedded in OCT media. Cerebellar sagittal sections were cut to a thickness of 40 μm. Free-floating sections were washed 3 times in PBS and blocked in blocking solution (10% goat serum in PBS with 0.2% Tween-20) for 2 hours at room temperature. Cerebellar sections were immunostained with primary anti-calbindin D-28k antibody  in blocking solution overnight at 4°C. Upon 3 washes with PBS, the sections were incubated with Alexa Fluor 488 Goat anti-mouse IgG (H+L)  for 2 hours at room temperature. The sections were washed 3 times with PBS and then stained with DAPI  in PBS for 5 minutes at room temperature. The sections were washed again 3 times with PBS, mounted using ProLong Gold antifade reagent (Invitrogen), and imaged using the Zeiss LSM 710 confocal microscope. Quantification of molecular layer thickness was performed using ImageJ software (NIH).For cerebellar sections, mouse brains were collected at 44 weeks of age and fixed for 24 hours in 10% formalin. The brains were transferred to 15% sucrose in PBS .For the staining of astrocytes in the hypoglossal nucleus, brain tissue was collected from 30-week-old animals and drop fixed in 10% formalin for 24 hours. Standard techniques were used to embed tissue in paraffin and to take coronal sections 10 μm thick in serial sections every 100 μm. Sections were then prepared for staining by being deparaffinized and rehydrated. Slides were treated with antigen retrieval by boiling for 9 minutes in 10 mM sodium citrate, 0.05% Tween 20, pH 6.0. GFAP antibody  was incubated for approximately 12 hours at 4°C. This primary antibody was then detected with biotinylated horse anti-mouse IgG  and visualized using an ABC reagent kit (Vector Laboratories) according to the manufacturer’s recommendations. Brightfield images were acquired using a Carl Zeiss Axio Imager M2 microscope, equipped with an Axio Cam MRc5 color camera (Carl Zeiss). All immunohistochemistry experiments were performed in triplicate, and figures show representative results. Astrocyte density was determined by counting the number of astrocytes per region of interest  were dissected. Total RNA was isolated using the miRNeasy Mini Kit (Qiagen) following manufacturer’s instructions. RNA was submitted to Genewiz for RNA integrity assessment, library preparation, and sequencing on the Illumina HiSeq platform. For each sample, approximately 100 million pairs of 150 bp pair-end reads were generated. Raw reads were trimmed before mapping by Trimmomatic-0.36 using the adapter reference TruSeq3-PE.fa  and to obtain read counts with default parameters  with both forepaws. The mice were pulled away from the bar with a constant slow force until the forepaws released. The maximum force generated was averaged over 3 pulls for each mouse.The pole test was performed at 7 weeks of age. Each mouse was placed head-upward at the top of a 60.5 cm tall vertical threaded metal pole. Time of descent was measured with a 60-second cutoff time. Falls from the metal pole were counted as a 60-second descent. In case the mouse fell off the pole within the first 10 seconds, the trial was not counted, and the mouse was placed on the pole again to ensure proper grip. The test was performed in triplicate for each animal.The rotarod test was performed at 7 and 24 weeks of age to evaluate coordination and motor skill acquisition . The mice were placed on the rotating rod  for 4 trials every day for a period of 4 days. Each trial lasted a maximum of 10 minutes. The rod accelerated from 4 to 40 rpm in 5 minutes and remained at 40 rpm for the remaining 5 minutes. The time the mice took to fall was recorded. Two subsequent rotations around the rod were also counted as a fall.n+1) − breath length (n)]/breath length (n), where abs is equal to the absolute value and n is equal to the chronological number of a recorded breath. Mice with fewer than 100 reliable recorded breaths were excluded from the analysis.Whole-body plethysmography (Buxco) was performed as described previously . Mice att test, whereas multigroup comparisons used 1- or 2-way ANOVAs. Survival analysis used log-rank test. In each case, P < 0.05 was considered statistically significant.Experimental analysis was performed in a blinded manner when possible. Statistical tests were performed in accordance with the experimental design. Simple comparisons used Student’s 2-tailed Animal care and experimental procedures were approved by the IACUC of Baylor College of Medicine, according to NIH guidelines.LN, HTO, and HYZ conceived the study, designed experiments, and analyzed and interpreted data. LN performed molecular and behavioral assays and wrote the manuscript. LN and HYZ edited the manuscript. SLC, EX, and DBE performed molecular and behavioral experiments. JPO performed histology assays. EA assisted with molecular assays. SLC, YD, YWW, and ZL further performed RNA sequencing analysis. All authors reviewed the manuscript and provided input.


Arabidopsis thaliana Col0 mineral nutrition and lipid metabolism during plant development. We reduced the local GMF (about 40 μT) to Near Null Magnetic Field  to evaluate the effects of GMF on Arabidopsis in a time-course (from rosette to seed-set) experiment by studying the lipid content  and mineral nutrients. The expression of selected genes involved in lipid metabolism was assessed by Real-Time PCR (qPCR). A progressive increase of SA with carbon numbers between 21 and 28 was found in plants exposed to NNMF from bolting to flowering developmental stages, whereas the content of some FA significantly (p < 0.05) increased in rosette, bolting and seed-set developmental stages. Variations in SA composition were correlated to the differential expression of several Arabidopsis 3-ketoacyl-CoAsynthase (KCS) genes, including KCS1, KCS5, KCS6, KCS8, and KCS12, a lipid transfer protein (LTPG1) and a lipase (LIP1). Ionomic analysis showed a significant variation in some micronutrients  and macronutrients  during plant development of plants exposed to NNMF. The results of this work show that A. thaliana responds to variations of the GMF which are perceived as is typical of abiotic stress responses.The Geomagnetic field (GMF) is a typical component of our planet. Plant perception of the GMF implies that any magnetic field (MF) variation would induce possible metabolic changes. In this work was we assessed the role of the GMF on  Several environmental factors affect plant growth and developments. Among them, the Earth magnetic field or geomagnetic field  is an environmental component of our planet and its reduction to Near Null Magnetic Field  has been shown to influence many plant biological processes ,2. One oplied MF . TherefoLipids play crucial roles in plant development being both basic components of cellular membranes and constituents of suberin and cutin waxes that provide structural barriers to the environment ,20. TheyKCS) genes, a lipid transfer protein (LTPG1), a lipase (LIP1), and a fatty acid desaturase (ADS4).In order to investigate the role of the GMF on both lipid metabolism and growth of Arabidopsis plants during development, we exposed plants to NNMF conditions. Here we show that the reduction of the GMF to NNMF affects plant nutrition by altering the content of both micro and micronutrients and the lipid content and composition of Arabidopsis by a differential modulation of several 3-ketoacyl-CoAsynthase (p < 0.05) reduction in the content of almost all identified alkanes (with the sole exception of C31) (p > 0.05) differences in the content of these alkanes (p < 0.05) increases of C22, C24, C25, C26, C27, C28, and C30 were observed in plants exposed to NNMF with respect to GMF control plants (p < 0.05) higher in NNMF plant with respect to GMF control plants  to the late phase of plant growth before senescence (seed-set), when compared to GMF . In gene of C31) A. The ma of C31) A. In the of C31) , the maj alkanes B. Howevel plants B. Duringl plants , a stronl plants C. Finalll plants , both NNl plants D. The onl plants D.p < 0.05) higher content of C18:2 with respect to GMF plants, whereas no significant difference was found for the other FAs (p < 0.05) increase of C18:1 and a decrease of C18:0, with respect to GMF plants (p < 0.05) reduction of C18:2 with respect to GMF plants (p < 0.05) increase with respect to GMF plants  were affected by the reduction of the GMF, including palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1), linoleic (C18:2) and linolenic (C18:3) acids . In the ther FAs A. AlmostF plants B. In theF plants C, whereaF plants D. The inLTPG1, KCS1, and KCS12 was correlated to the NNMF-dependent increased content of C18:2  has been shown to be involved in wax biosynthesis of long-chain alkanes [KCS5 and the production of C29 and C31 alkanes. LIP1 drives the synthesis of an active triacylglycerol (TAG) lipase and is capable of hydrolyzing long chain triacylglycerol [LIP1 correlated with the synthesis of NNMF alkanes, particularly during bolting and flowering, where these wax constituents showed the highest content.Among the several genes that are involved in lipid metabolism we selected some genes involved in fatty acids and alkane metabolism. 18:2 see A, whereaand KCS6  was coheand KCS6 A. The exsiliques . While Kynthesis , KCS1 ha alkanes . In the s [LTPG1 , whereass [LTPG1 B was assand LIP1 . In thisP1  reduced in the flowering and seed set developmental stages. As expected, the shoot length was reduced in NNMF treatment from bolting to seed-set, as previously observed [Considering that several physiological processes linked to plant growth are under control of a complex network of lipid-mediated signaling , we inveobserved ,4,5 and We recently demonstrated that NNMF condition impairs nutrient homeostasis in plants  at the ep < 0.05) increased when compared with GMF conditions  was the only micronutrient showing a significant (nditions . This fip < 0.05) increase was observed only for the micronutrient cobalt (Co). Cobalt was found to increase its availability upon treatment of barley (Hordeum vulgare) with magnetic nanoparticles, with a significant increase with respect to control [During bolting, a significant ( control . p < 0.05) increase in their content with respect to GMF-exposed plants. Moreover, micronutrients such as manganese (Mn), molybdenum (Mo) and nickel (Ni) showed a significantly (p < 0.05) higher accumulation under NNMF when compared to plants grown in GMF conditions. Interestingly, during seed set the content of Co was significantly reduced by exposure to NNMF , potassium (K) and calcium (Ca) showed a significant  were vernalized at 4 °C for 72 h in dark condition in order to synchronize the plants. Seeds were then sown in 8 cm diameter polyethylene pots with soil prepared with a mixture of peat and vermiculite (2 parts of peat and 1 part of vermiculite). For each developmental stage, at least 10 pots for both control and NNMF exposed plants were assayed and the experiment was repeated at least three times  with a photoperiod of 14 h light and 10 h darkness. Control experiments (GMF) were performed in the same lab at a distance of 6 m from the NNMF controlling system and the measured levels of power-line frequency (50 Hz), light and temperature (22 °C) of both controls and treatments were similar. Treated plants were grown inside the NNMF (see below) controlling system. All experiments were performed under normal gravity. Because NNMF delays plant flowering [see also . Sown po22 °C ±1. °C with The GMF B values of control plants were in line with the values of the Northern hemisphere . The geoFor both FA and alkane analyses and each time point, Arabidopsis plants exposed to either GMF or NNMF were harvested and immediately frozen in liquid nitrogen and the plant material was stored at −80 °C. The plant material from pooled pots was then used for extractions.w/v in methanol). Fifty μg heptadecanoic acid (C17:0) were added as internal standard [4. FAME identification and quantification was performed by gas chromatography-mass spectrometry (GC-MS)  and by gas chromatography flame ionization detector (GC-FID) , respectively, as reported earlier [The pooled collected leaves from each developmental stage  were extracted using cyclohexane  and then esterified with boron tri-fluoride  of the identified alkanes were: C21 n-heneicosane (296), C22 n-docosane (310), C23 n-tricosane (324), C24 n-tetracosane (338), C25 n-pentacosane (352), C26-hexacosane (366), C27 n-heptacosane (380), C28 n-octacosane (394), C29 n-nonacosane (408), C30 n-triacontane (422), C31 n- hentriacontane (436).The pooled collected leaves from each developmental stage  were extracted with a mixture of 5 ml pentane:hexane (5:1) for 60 s; 30 μg of n-tritriacontane were added as internal standard. The extract was concentrated by a gentle stream of gaseous Nescribed . An aver3 at 120 °C. The mineralized samples were transferred into polypropylene test tubes and diluted 1:40 in MILLI-Q water. Finally, the concentration of metal elements was measured by inductively coupled plasma–mass Spectrometry ICP-MS (BRUKER Aurora-M90 ICP-MS) as previously described [For ionomic analyses, leaves were collected from each time point of Arabidopsis plants exposed to either GMF or NNMF, and dried in an aerate oven at 70 °C for four days. Samples dry weight were measured and digested by using 65% HNOFor each developmental stage, 100 mg of frozen samples exposed to either GMF or NNMF were ground separately in liquid nitrogen with mortar and pestle. Total RNA was isolated using the Machery-Nagel RNA Isolation mini Kit , and RNase-free DNAse, according to the manufacturer’s protocols. RNA quality and quantity were checked as previously described . cDNA wa® as an internal loading standard. The reaction was performed with 25 µL of mixture consisting of 12.5 µL of 2X MaximaTM SYBR Green/ROX qPCR Master Mix , 0.5 µL 1:5 diluted cDNA and 100 nM primers . Controls included non-RT controls  and non-template controls (water template). All primers were designed using Primer 3 software [At1g06350, Δ9 DESATURASE 4 (ADS4); At1g27950, GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED LIPID PROTEIN TRANSFER 1 (LTPG1); At1g01120, 3-KETOACYL-COA SYNTHASE 1, (KCS1); At1g25450, 3-KETOACYL-COA SYNTHASE 5 (KCS5); At1g68530, 3-KETOACYL-COA SYNTHASE 6 (KCS6); At2g15090, 3-KETOACYL-COA SYNTHASE 8 (KCS8); At2g28630, 3-KETOACYL-COA SYNTHASE 12 (KCS12); At2g15230, LIPASE 1 (LIP1). Four different reference genes At2g37620, ACTIN1 (ACT1); At5g19510, ELONGATION FACTOR 1B ALPHA-SUBUNIT 2 ; At1g13440, CYTOPLASMIC GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPC2); and At1g51710, UBIQUITIN SPECIFIC PROTEASE 6 (UBP6), which were used to normalize the results of the qPCR. The best performing of these four genes was selected using Normfinder software  [eEF1Balpha2.All qPCR experiments were performed on a Stratagene Mx3000P Real-Time System  using SYBR green I with ROXsoftware . PrimersAll amplification plots were analyzed with the Mx3000P software to obtain Ct values. qPCR data are expressed as fold change of NNMF with respect to control plants growing in GMF conditions.t test and Bonferroni adjusted probability were used to assess the difference between treatments and the control. ANOVA was calculated in the comparative analysis of plant development data and a Tukey post hoc test was assessed.The data obtained from lipidomics and qPCR were treated by using Systat 10 . Mean values were calculated along with the standard deviation (SD). Paired Arabidopsis thaliana nutrition and lipid composition and this effect is related to the developmental stage of plants and that its presence is necessary for a normal development and metabolism. By reducing the GMF to NNMF we observed an increased amount of epicuticular alkanes and a modulation of FAs unsaturation. These events were associated to alteration in the nutritional status of NNMF exposed plants. Homeostasis and lipid composition are sensitive to several abiotic stresses [Our results show that the GMF modulates stresses ,55,56 anstresses ,16,57,58stresses . Our worstresses ). By constresses ,17 point


Background: Breast cancer (BC) is the most common malignancy in females and remains a main cause of cancer-associated death worldwide. The solute carrier (SLC) groups of membrane transport proteins, which control the influx of zinc, participate in ranging of physiological processes and may provide novel therapeutic targets of cancers. However, the prognostic values of individual SLC family 39 (SLC39A) genes in patients with BC are not clarified.Materials and Methods: The mRNA expression of SLC family 39 genes in BC was evaluated by using the UALCAN database. The prognostic values of overall survival (OS) of SLC family 39 genes in patients with BC were investigated by Kaplan–Meier plotter. The survival analysis of cells was determined by Project Achilles.Results: The analytic results suggested that SLC39A1, SLC39A3, SLC39A4, SLC39A5, SLC39A6, SLC39A7, SLC39A9, SLC39A10, SLC39A11 and SLC39A13 were significantly up-regulated in BC tissues compared with normal breast tissues. However, SLC39A8 and SLC39A14 were expressed higher in normal tissues than in BC tissues. High expression of SLC39A2, SLC39A3, SLC39A4, SLC39A5, SLC39A7, SLC39A12 and SLC39A13 was significantly associated with worse OS in patients with BC. In contrast, high mRNA levels of SLC39A6 and SLC39A14 indicated favorable OS. Through subgroup analysis, all abnormal expressed SLC family members were correlated with prognoses of patients with specific BC. Moreover, SLC39A7 was associated with proliferation and cloning of BC.Conclusions: Our results suggested that SLC family 39 members were promising prognostic biomarkers of BC. The SLC39A7 played a key role in growth and survival of BC cells. Breast cancer (BC) is the most common malignancy in females and remains a main cause of cancer-associated death worldwide . In receThe solute carrier (SLC) groups of membrane transport proteins, which have 65 families, participate in ranging of physiological processes and may provide novel therapeutic targets for human malignances . By tran. have found that SLC39A4 is a potential prognostic and diagnostic marker in pancreatic cancer , SLC39A3 , SLC39A4 , SLC39A5 , SLC39A7 , SLC39A12  and SLC39A13 . In contrast, high mRNA levels of SLC39A6  and SLC39A14  showed favorable OS. Other genes including SLC39A1 , SLC39A8 , SLC39A9 , SLC39A10  and SLC39A11  were not associated with the OS of BC patients.Then, the potential prognostic values of SLC39A genes in BC were investigated by using Kaplan–Meier plotter. As shown in To further to understand the significant roles of SLC39A genes in prognoses of patients with BC, the OS between SLC39A genes expression and various clinicopathological features based on intrinsic sub-types, lymph node status, differentiation and endocrine therapy status of patients with BC was investigated. According to the expression of ER, PR and HER2, patients with BC were divided into four molecular subtypes: Basal (triple negative), Luminal A , Luminal B  and HER2 .As shown in The results of association between SLC39A genes expression and OS in patients with BC based on lymph node status were shown in According to the cell differentiation status, BC was divided into three subtypes, including well, moderate and poor differentiation. The results are shown in Endocrine therapy is an important method of BC treatment according to the intrinsic sub-types. As shown in P<0.001). To further certificate the results, the cell colony assay was performed. As shown in To determine whether SLC39A genes are essential for cell survival of BC, we investigated the data from Project Achilles. CERES dependence score was obtained from Depmap. As shown in P<0.05). T1-4 represents from primary tumor and closely related to tumor size and cell proliferation according to the criteria from the American Joint Committee on Cancer (AJCC). T1 means the tumor is small with minimally invasive within primary organ site. T2-4 means the tumor is larger than T1 with more seriously invasive within or beyond the primary organ site. SLC39A7 expression level was significantly increased in T2-4 stage compared with T1 stage BC tissues (P=0.0104). “Metastasis” is defined as presence of distant locations or spread via blood way or lymphatics beyond. As shown in P<0.05). “Disease free” is defined as disease free status since initial treatment. The expression of SLC39A7 was observed higher in recurrence group (P<0.05) These results suggest that SLC39A7 was associated with BC progression and might have prognostic significance for patients with BC.To investigate the role of SLC39A7 in BC progression, we analyzed the expression level of SLC39A7 in different intrinsic sub-types, TNM statues, and recurrence of patients with BC from TCGA. Totally, 1082 patients were included in the analysis. As shown in ce group E, P<0.05GSEA was performed to evaluate Hallmark effect gene sets. The patients with BC were divided into two groups according to the expression of SLC39A7. As shown in P=0.097, P=0.013, P=0.16, The mRNA expression can’t perfectly represent the protein level. Hence, the protein expression of SLC39A7 in BC from CPTAC was analyzed by UALCAN database. As shown in Intracellular and extracellular Zn levels and distributions were spatiotemporally regulated by the specific Zn transporters regulate in a tissue-specific manner . AberranIn the present study, we analyzed the mRNA expression profiles of all SLC39A genes in BC tissues and analyzed their correlations with overall survival outcomes in patients with BC by using available databases. Our results suggested that SLC39A1, SLC39A3, SLC39A4, SLC39A5, SLC39A6, SLC39A7, SLC39A9, SLC39A10, SLC39A11 and SLC39A13 were significantly higher in cancer tissue than which in normal tissue, whereas, SLC39A8 and SLC39A14 expressed higher in normal tissue. High expression of SLC39A2, SLC39A3, SLC39A4, SLC39A5, SLC39A7, SLC39A12 and SLC39A13 was significantly associated with worse OS in patients with BC. In contrast, high mRNA levels of SLC39A6 and SLC39A14 showed favorable OS. Through subgroup analysis, expression of all SLC39A members was correlated with prognoses of patients with specific BC. Moreover, we identified that SLC39A7 was a key gene in survival of BC, which is especially associated with LumA BC.A line of evidences has demonstrated the importance of SLC39A members in the progression of BC . Knockdoin vitro experiments [By investigating the data from Project Achilles, we identified the SLC39A7 playing a vital role in cell survival of BC instead of other SLC39A members. Taylar et al. first reported the relationship between Zn and tamoxifen resistance therapeutic strategy and indicated that SLC39A7 is abundantly expressed in a BC cell line - tamoxifen-resistant MCF7 . SLC39A7eriments . In addieriments . ThroughTwo limitations were found in the present study. First, all expression and prognostic analysis were calculated by using the TCGA datasets. Lacking original clinical samples and data would limit the precise of last conclusion. Second, in In conclusion, our results suggested that SLC39A members were promising prognostic biomarkers of BC. The SLC39A7 plays a key role in growth and survival of BC cells. Importantly, our research on the findings of SLC39A members in BC is not insufficient, and suggests more basic and clinical studies focusing on their relationships.Click here for additional data file.


Apicomplexa Plasmodium falciparum and Toxoplasma gondii. However, the mechanisms via which it regulates parasite replication have remained unknown. We performed quantitative phosphoproteomics of T. gondii lacking TgCDPK7 to identify its parasitic targets. Our analysis lead to the identification of several putative TgCDPK7 substrates implicated in critical processes like phospholipid (PL) synthesis and vesicular trafficking. Strikingly, phosphorylation of TgRab11a via TgCDPK7 was critical for parasite intracellular development and protein trafficking. Lipidomic analysis combined with biochemical and cellular studies confirmed that TgCDPK7 regulates phosphatidylethanolamine (PE) levels in T. gondii. These studies provide novel insights into the regulation of these processes that are critical for parasite development by TgCDPK7.Apicomplexan parasites are causative agents of major human diseases. Calcium Dependent Protein Kinases (CDPKs) are crucial components for the intracellular development of apicomplexan parasites and are thus considered attractive drug targets. CDPK7 is an atypical member of this family, which initial characterization suggested to be critical for intracellular development of both  Toxoplasma gondii. It regulates the localization of a small GTPase TgRab11a by phosphorylating it at a specific site, which is critical for trafficking of important parasite proteins and is important for parasite division. TgCDPK7 may regulate key enzymes involved biogenesis of phosphatidylethanolamine, which may contribute to the synthesis of this important phospholipid. These and other studies shed light on a novel signaling pathway in apicomplexan parasite Toxoplasma gondii.In this study, we demonstrate that protein kinase TgCDPK7 regulates cellular processes like vesicular trafficking and the synthesis of phospholipids, which are critical for the development of the parasite  Toxoplasma gondii is an obligate intracellular protozoan, which has to invade host cells to proliferate and thus survive. The lytic cycle of T.gondii causes the acute form of the disease through the rapid division of tachyzoites via the process of endodyogeny, which involves the formation of two daughter cells within the mother cell. Tachyzoites replicate within almost any nucleated cell from a warm-blooded host by generation cycles of 6 to 8h (in vitro), until they reach a number of 64 to 128 parasites per infected cell. They can then trigger active egress, which involves lipid signal transduction  ethanolamine (20–40 nCi/nmol) or  choline (0.1 nCi/nmol) in 1 ml of culture medium in glass tubes  followed by lipid extraction as described previously  (15 μCi/reaction) for 40 minutes at 30°C. The reaction was stopped by boiling assay mix in SDS-loading buffer for 10 minutes. Proteins were separated on SDS-PAGE and phosphorylation of proteins was detected by phosphorimaging using a FUJI FLA5000 or GE STORM scanner.Kinase assays were performed by incubating recombinant proteins 2 μg His-TgRab11a or 5.6 μg GST-TgGPAT (aa 205–413) as substrates with 5 μg recombinant TgCDPK7 kinase domain protein in a kinase assay buffer Click here for additional data file.S1 Data1.1: List of phosphopeptides identified in the phosphoproteomic analysis of TgCDPK7 parasites. 1.2: List of hyposphosphorylated sites identified upon depletion of TgCDPK7. 1.3: List of hypersphosphorylated sites identified upon depletion of TgCDPK7. 1.4: Pathways enriched in hypophosphorylated T. gondii proteins. 1.5: Biological processes enriched in hypophosphorylated T. gondii proteins. 1.6: Protein-protein interaction analysis on the differentially phosphorylated T. gondii proteins. 1.7: TMT labeling details for the quantitative phosphoproteomic analysis of TgCDPK7 parasites.(XLSX)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 FigTgCDPK7 promoter with the 7tet-Op SAG1 (TetO7) inducible promoter was introduced by double homologous recombination in TgCDPK7 locus. After transfection, the drug selected parasites were cloned by limiting dilution. B. Genotyping of TgCDPK7-iKD parasites. Although two independent clones were obtained, clone#1 (TgCDPK7-iKD) was used for most reported studies and some experiments were performed with clone #2 (Panel E). PCR amplification of the unmodified and recombined locus was performed using indicated primers Click here for additional data file.S2 FigA. Plaque assays were carried out by infecting HFF monolayer with ΔKu80 or TgCDPK7-iKD in the presence or absence of ATc with or without 200 μM Eth or 200 μM choline after 10 and 15 days post treatment, respectively and number of plaques were counted after the treatment (B).(PDF)Click here for additional data file.S3 FigA-B. Recombinant TgGPAT (aa. 205–413)  and a N-terminal deletion mutant that only has the PH and the kinase domain of TgCDPK7 (ΔTgCDPK7)  were expressed as GST-fusion proteins. TgRab11a (B) was expressed as 6xHis tagged protein. All proteins were purified by affinity chromatography. A SDS-PAGE gel of the purified recombinant proteins, which were used for kinase assays, is shown here.(PDF)Click here for additional data file.S4 FigRight panel, Densitometry of TgCDPK7-HA band in the Western blot was performed to determine the fold change in its expression upon ATc addition.Subsequently, parasites were harvested followed by Western blotting using anti-HA antibody to detect TgCDPK7-HA. *-a possible breakdown/spliced product. Actin was used as a loading control. (PDF)Click here for additional data file.S5 FigA-C.TgCDPK7-iKD parasites were treated with ATc for 72h following which IFA was performed using antibodies against apicoplast protein Cpn60 (A), microneme protein MIC2 (B), rhoptry protein ROP2 (C) along with GAP45. Microneme (B) and rhoptry (C) proteins were mislocalized upon ATc treatment. D. IFA of TgCDPK7-iKD parasites revealed the presence of IMC1 on newly formed IMCs in the absence of ATc. In ATc-treated parasites, IMC was either missing or disorganized (arrows). E. GRASP-YFP  was tran(PDF)Click here for additional data file.S6 FigA-E. DDmycTgRab11a or S207A parasites were grown in the presence or absence of Shld-1 for 4h. GRASP-YFP was transiently transfected to detect Golgi (A) or antibodies against centrin 1 (B), IMC1 (C) and anti-GRA3 (D), MIC2 (E) were used to detect centrosomes, micronemes and dense granules, respectively. IFA revealed abnormal number/location (arrows) or missing/diffuse (*) Golgi in S207A parasites (A). Centrosomes were stretched (arrow) or diffuse/missing (*) in the S207A mutant parasites (B). IMCs were not observed or was diffused (*) or not in sync (arrow) in S207A mutant (C). Dense granules (D) and microneme (E) were almost unaltered in mutant parasites. F. GPAT-myc was transiently transfected in DDmycTgRab11a or S207A parasites, which were treated with Shld-1. IFA with anti-myc antibody revelaed perinuclear ER-like staining for GPAT in both WT TgRab11a and S207A parasites.(PDF)Click here for additional data file.S7 FigDDmycTgRab11a or S207A parasites were grown in the presence or absence of Shld-1 for 16h. Subsequently, invasion assays were performed and infected host cells were determined. There was no significant difference in invasion by Rab11a or S207A overexpressing parasites .(PDF)Click here for additional data file.S8 FigA. IFA was performed on DDmycTgRab11a-T205A or DDmycTgRab11a-S207D overexpressing parasites in presence of Shld-1 for 4h as described in (PDF)Click here for additional data file.S9 Fig2CO3 pH 11.0 or 0.5% Triton X-100. Western blotting using anti-myc antibody was performed on various fractions. B. TgCDPK7-iKD/DDmycTgRab11b in which TgRab11b was expressed in the presence of Shld-1 as N-terminal DD myc tag fusion protein in TgCDPK7-iKD background was treated with ATc followed by IFA using anti-myc antibody for detecting TgRab11b and anti-GAP45. C. TgCDPK7-HA/DDmycTgRab11a WT or S207A parasites were treated with Shld-1 for 4h. Subsequently, IFA was performed using anti-HA (to detect TgCDPK7-HA) and SAG1 antibodies. There was no significant difference in TgCDPK7 localization whereas SAG1 was mislocalized in TgRab11a S207A parasites as described in the main text.A. TgCDPK7-iKD/DDmycTgRab11a parasites were treated with ATc in the presence of Shld-1. Subsequently, parasite pellets were isolated and used for extracting proteins in PBS, Na(PDF)Click here for additional data file.S10 FigProtein-protein interactions were predicted between differentially phosphorylated proteins in TgCDPK7-iKD proteins upon ATc addition , 1.5 usi(PDF)Click here for additional data file.S11 FigWestern blotting using anti-SAG1/ROM4 antibody showed unaltered protein expression upon TgCDPK7 depletion with ATc for 72 hours.(PDF)Click here for additional data file.S12 FigPLs were extracted from TgCDPK7-iKD tachyzoites grown in the presence or absence of ATc as described in Panel A. Relative abundance of individual molecular species in total FA (A), which shows the significant decrease of C16:0, C18:1 Trans and the significant increase in C20:1, C20:4 and C20:5 upon TgCDPK7 depletion. FA profiles were also quantified for the phospholipid classes PE (B) and PC (C) /ANOVA(A)).(PDF)Click here for additional data file.S13 Fig14C-Eth or 14C-Cho in the presence or absence of Shield-1 (16h treatment). Subsequently, lipids were extracted and radiolabeled lipids were detected by phosphorimaging of TLC. There was no significant difference in PC or PE levels. Right Panel, Densitometry of spots corresponding to PC/PE was performed and fold change in PC/PE in S207A mutant with respect to wild type TgRab11a was determined . B. WT TgRab11a or its S207A mutant parasites were incubated with or without 200 μM ethanolamine in the presence of Shield-1 for 16 hours. The number of parasites per vacuole was determined after 24h. Data represent Mean ± SE, n = 3 and at least 200 vacuoles were counted for each condition.A. Metabolic labeling to monitor the synthesis of PC and PE via the parasite Kennedy pathway in parasites overexpressing either WT TgRab11a or its S207A mutant as described earlier in (PDF)Click here for additional data file.S14 FigA. GPAT was myc-tagged at endogenous locus in its linker region as described in (PDF)Click here for additional data file.S15 FigTgCDPK7-iKD parasites were preincubated for 48h with ATc and were subsequently allowed to invade fresh HFFs in the presence or absence of ATc for 24h. In one case, 200 μM of Eth was added for 120h to cultures prior to addition of ATc. Subsequently, IFA was performed for SAG1 and ROM4. The addition of Eth caused a partial restoration of pellicle staining of SAG1. SAG1 was present at pellicle as well as vesicles but the staining was reduced in comparison to -ATc cells.(PDF)Click here for additional data file.


Therefore, a series of single fragment reassortants in MS285 background and two rescued wild-type viruses were generated by using the reverse genetics method, and the pathogenicity analysis revealed that the PB2 gene contributed to the high virulence of YJ4 virus. Furthermore, there were 11 amino acid differences in PB2 between MS285 and YJ4 identified by sequence alignment, and 11 single amino acid mutant viruses were generated in the MS285 background. We found that the R251K mutation significantly increased the virulence of MS285 in mice, contributed to high polymerase activity and enhanced viral genome transcription and replication. These results indicate that PB2-R251K contributes to the virulence of the EA H1N1 virus and provide new insight into future molecular epidemiological surveillance strategies.The Eurasian avian-like swine (EA) H1N1 virus has affected the Chinese swine industry, and human infection cases have been reported occasionally. However, little is known about the pathogenic mechanism of EA H1N1 virus. In this study, we compared the mouse pathogenicity of A/swine/Guangdong/YJ4/2014 (YJ4) and A/swine/Guangdong/MS285/2017 (MS285) viruses, which had similar genotype to A/Hunan/42443/2015 (HuN-like). None of the mice inoculated with 10 Influenza A virus (IAV) is an important respiratory pathogen that continually impacts both the animal industry and human public health. The natural reservoir for IAV is thought to be wild waterfowl, but viruses frequently jump species barriers and infect humans and other mammals, including pigs, cats, and dogs . Among tThe Eurasian avian-like swine (EA) H1N1 virus was first isolated from pigs in Northern Europe in 1979 , after wThere are many contributing factors of the virulence and host range to influenza A virus, and certain amino acid (aa) substitutions in these factors could alter the host range from avian to mammalian species as well as increase virulence in viral infection. For example, the receptor-binding specificity and virus transmission of pdm H1N1 virus is significantly affected by the amino acids at positions 222 and 226 in HA ,19. The 2.The two viruses A/swine/Guangdong/YJ4/2014 (YJ4 (H1N1)) and A/swine/Guangdong/MS285/2017 (MS285 (H1N1)) have been previously described  [6 50% tissue culture infectious dose (TCID50) of each virus in a total volume of 50 μL or mock inoculated with phosphate-buffered saline (PBS). After infection, the mice were monitored for 14 days for body weight and death rate, and mice with loss of more than 25% of their initial body weight were euthanized humanely. At 1, 3, and 5 days post-infection (dpi), three mice in each group were euthanized to determine virus replication in the nasal turbinate and lungs by TCID50 assay in MDCK cells.Six-week-old female BALB/c mice  were anaesthetized with isoflurane and then inoculated intranasally with 10PB2 gene in YJ4 viruses were introduced into the plasmids of MS285 virus using PCR-based site-directed mutagenesis with primers containing the desired mutations [The eight viral segments from YJ4 and MS285 viruses were cloned into the plasmid vector pHW2000 as previously described . A serieutations . Primer 50/cell. After virus contact to the cells for 1 h, the cells were washed with PBS and further incubated in DMEM with 1 μg/mL TPCK-treated trypsin at 33 °C or 37 °C with 5% CO2. Culture supernatants were harvested at 12, 24, 36, 48, and 60 h post-inoculation (hpi) and stored at −80 °C. Viruses in the culture supernatants were titrated for infectivity by TCID50 assays using MDCK cells.The growth kinetics of recombinant viruses were compared in MDCK and A549 cells at a multiplicity of infection (MOI) of 0.01 TCIDPB2 (wild-type or mutant), PB1, PA, and NP genes (300 ng each) of YJ4 and MS285 viruses, together with 100 ng of pPolI-NP-luc; and 10ng of pPL-TK  using 2.5 µL of Lipofectamine 3000 . Transfected cells were incubated at 33 °C or 37 °C. Cell extracts were harvested and then the luciferase activity was measured using a GloMax® Discover System (Promega) according to the manufacturer’s instructions at 24 h post-transfection. All experiments were run in triplicate.As previously described ,33, 293TThe levels of viral NP segment viral RNA (vRNA), complement RNA (cRNA), and viral messenger RNA (mRNA) were measured by quantitative real-time PCR . After iA549 cells were infected with an MOI of 1 of virus, with uninfected cells (mock) as a control. Cells were harvested 3, 6, 9, and 12 h post infection and lysed on ice in RIPA lysis buffer  containing protease inhibitor cocktails . The protein concentration in the lysates was assessed by using a bicinchoninic acid (BCA) protein assay kit . The samples were separated by SDS-PAGE and transferred to a nitrocellulose membrane . The membrane was blocked in 5% skim milk for 2 h, and then incubated overnight at 4 °C with anti-PB2  or anti-NP  primary antibody. The membrane was washed three times with Tris-buffered saline containing Tween (TBST) for 5 min. Then, the membrane was incubated with goat anti-rabbit secondary antibody for 1 h in the dark at room temperature. The membrane was washed one time with TBST and then analyzed by using an Odyssey Infrared Imaging System .t-test using SPSS software (Windows v21.0). The results are presented as the mean ± SD and a p < 0.05 was considered statistically significant.Experimental results were analyzed by one-way ANOVA or Student’s All experimental animal work was carried out in accordance with animal ethics guidelines and approved protocols and was approved by the Experimental Animal Welfare Ethics Committee of the South China Agricultural University . All experiments with live viruses were conducted in a biosecurity level 2 laboratory.6 TCID50 doses of MS285 or YJ4 viruses showed that all mice (n = 5) exhibited sustained body weight reductions . In A549 cells, introduction of 251K to PB2 of MS285 virus significantly enhanced growth ability at 37 °C , but this positive effect was not observed at 33 °C. Correspondingly, back substitution to 251R in the PB2 of YJ4 virus decreased the replication rate in A549 cells , but the differences are less dramatic in MDCK cells  with a 1.86-fold increase (To understand whether the R251K substitution contributes to the polymerase activity of the EA H1N1 virus, we used mini-genome reporter assays in 293T cells at 33 °C and 37 °C, mimicking temperatures of the mammalian upper and lower respiratory tracts, respectively. Consistent with the result of growth kinetics at different temperatures, introduction of 251K into PB2 of MS285 virus significantly enhanced the polymerase activity at 37 °C (increase . There wincrease B. For coPB2 gene is a major determinant of virulence in mice. Further analysis of the PB2 gene suggested that the PB2-R251K mutation is associated with increased virulence in mice. In addition, virus titer studies in nasal turbinates and lungs showed that PB2-R251K is responsible for the increased viral replication efficiency in vivo.In China, EA H1N1 virus first emerged in 2001 and has become predominant in swine since 2005 , one of Several virulence determinants in EA H1N1 viruses have been identified in previous studies. The HA-G225E mutation in EA H1N1 viruses affected receptor-binding preferences and increased the efficiency of virus assembly and budding, thereby increasing the transmission in guinea pigs . The PB2Virulence of influenza A virus is affected by a great deal of factors. The mouse experiments of r/YJ4 and r/YJ4-251R viruses indicated that the single change at position 251 was insufficient to recover lethality of YJ4 virus up to 100% . MoreoveAlong with the PB1 and PA proteins, the PB2 protein is an important component of the polymerase complex of IAVs. The PB2 protein can bind to the 5′ cap of host pre-mRNAs and then participate in the transcription and replication of the viral genome . AlthougPB2 gene segment, implying that introduction of PB2-251K in pdm H1N1 virus may enhance its replication and pathogenicity in humans. In addition, with continued circulation in swine populations, additional mutations are more likely to emerge, which in combination with the PB2-R251K mutation, may confer more efficient replication of the EA H1N1 virus in humans and lead to another human pandemic.In a recent study reported by Joseph et al. , the autTaken together, the results of the present study demonstrated that the PB2-R251K mutation increased viral replication and pathogenicity in mice. This amino acid change conferred increased polymerase activity and more efficient replication in human cells. These findings not only provide new insights into the role of PB2-R251K on IAV pathogenicity but also suggest that PB2-R251K may have great significance for evaluating the health risks of IAVs.


Background: Recent studies have shown that the transcription factor E2F4 is involved in the progression of various tumors, but its expression and influence on immune cell infiltration and biological functions are largely unknown in hepatocellular carcinoma (HCC).Methods: The Cancer Genome Atlas (TCGA) database, the Tumor Immune Estimation Resource (TIMER) and related online tools as well as a tissue microarray (TMA) were used for analyses in our study.Results: E2F4 expression was elevated in HCC tumor tissue compared with adjacent normal tissue at both the mRNA and protein levels. Overexpression of E2F4 was markedly related to a poor prognosis in HCC patients. In addition, positively and negatively correlated significant genes of E2F4 were identified in HCC. Pathway enrichment analyses revealed that the top 100 positively correlated significant genes of E2F4 were closely related to nuclear splicing and degradation-related pathways. Furthermore, nine hub genes correlated with E2F4 expression were validated based on a protein-protein interaction (PPI) network. It was also demonstrated that E2F4 expression was negatively correlated to immune purity and positively correlated to immune cell infiltration.Conclusion: E2F4 could serve as a novel biomarker for HCC diagnosis and prognosis prediction. Hepatocellular carcinoma (HCC) has become the third-leading cause of cancer-related mortality worldwidein vivoE2F, a group of genes encoding a transcription factor protein family, act as regulators of cell differentiation, response to DNA damage, and cell life cycle A growing body of evidence also shows that E2F4 is remarkably correlated with tumor immune cell infiltration To enhance our understanding of E2F4 crosstalk with immune cells, we determined the relationship between E2F4 and programmed death-ligand 1 (PD-L1) expression, and also validated the relationship between E2F4 gene expression and immune cell infiltration. We analyzed the HCC cohort from the TCGA dataset using multiple online resources. We also investigated the expression level, clinical characteristics, mutations, and differentially expressed genes (DEGs) of E2F4. With these data, we assessed the signaling pathways of DEGs and contrasted the protein-protein interaction (PPI) network to identify hub genes. Finally, the expression of E2F4 in HCC and its association with immune cell infiltration were also explored.https://xenabrowser.net/datapages/) as previously described http://ualcan.path.uab.edu/) The RNA sequencing (RNA-seq) and corresponding clinical data from pan-cancer patients as well as HCC patients were downloaded from The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) data collection (https://kmplot.com/) based on TCGA analysis as described previously The survival curves of HCC cohort patients were analyzed online via The Kaplan Meier plotter  The gene mutations and co-expression of E2F4 were computed and analyzed using CBio-Cancer Genomics Portal  databases (https://cistrome.shinyapps.io/timer) is a database resource for systematic evaluations that contains information regarding the clinical impact of immune infiltration among various types of cancer Tumor Immune Estimation Resource  contains various clinical data for pan-cancer types, and all patients were included as described in a previous study LinkedOmics (http://gepia.cancer-pku.cn/) and Cytoscope software (https://cytoscape.org/) were used to determine hub genes and investigate the correlation between hub genes and E2F4 expression.The PPI network and module analysis were performed for further exploration. The top nine genes identified by degree were considered hub genes and were noted for further study. Additionally, the Gene Expression Profiling Interactive Analysis To decipher the potential molecular mechanisms and explore the functional annotation of E2F4-related DEGs in HCC genomic profiles, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed.Table 1.Tissue microarray (TMA) containing 90 HCC and 90 normal liver tissues was purchased from Outdo Biotech . All of the clinical information including age, gender, grade, TNM stage, as well as survival time, was collected and the details are presented in The IHC staining pattern of E2F4 was evaluated. Briefly, after incubation with the E2F4 primary antibody  Abcam, Cambridge, MA), the tissue microarray was probed with biotinylated goat anti-rabbit secondary antibody rabbit IgG, , and the remainder of the process was carried out according to a previously published study t-test with a p value < 0.05 was applied to identify significance with statistical differences. The OS analysis was performed using R programming of “survival” and “survive” packages online. The OS, RFS, PFS, and DSS were separated and calculated by the median gene expression level of E2F4. Spearman's correlation was applied to identify the relationship between E2F4 and associated genes and pathways. A p value < 0.05 was used to identify significant differences.A Figure ). Additionally, the transcriptional level of E2F4 in the TCGA-LIHC cohort was significantly higher compared with normal tissue . To further investigate the levels of E2F4 mRNA in HCC patients, the clinicopathological characteristics of HCC patients were analyzed. In this study, a slight but statistically significant difference was observed between stages 1 and 3. In regards to the tumor grade, significant differences in E2F4 transcription were found between both grade 1 and grade 2 when compared to grade 3 . However, the E2F4 mRNA showed no significant differences in regard to age, race, or node metastasis status among patients with HCC . These findings suggest that E2F4 expression was upregulated in HCC tissues, and it has potential to be a novel biomarker for HCC diagnosis.UALCAN was used to analyze the expression of E2F4 mRNA from the pan-cancer data. The results showed that the transcription of E2F4 was increased in most cancers . Moreover, patients with high expression of E2F4 had worse DSS . There were no significant differences in PFS  or RFS : 1.03-2.17], log rank p = 0.032; Figure ). As shown in the Figure Figure ), which was consistent with the results from the TCGA cohort . In addition, we found that elevated E2F4 expression was positively correlated with the PD-L1 positive subgroup . There was no significant correlation among E2F4 expression and age, gender, grade, TNM stage, cirrhosis, or alpha-fetoprotein (AFP) levels (Table 1). Moreover, we assessed the correlation of E2F4 expression with survival of patients with HCC. Kaplan-Meier curves revealed that patients with increased E2F4 expression had shorter OS times compared with those who had reduced E2F4 expression . Univariable and multivariable Cox regression analyses were adopted to determine the risk factors for OS prognosis. Univariable analysis demonstrated that advanced grade, late TNM stage, and high E2F4 expression were potential risk factors for decreased OS in HCC. Through further analysis, high E2F4 expression was found to be an independent predictor of OS according to multivariable analysis (Table 2). Taken together, these findings demonstrated that up-regulated E2F4 expression could service as a novel predictor for prognosis in HCC.The association between E2F4 protein expression and clinical characteristics of patients with HCC were assessed. Representative micrographs reveal E2F4 IHC staining patterns with different staining scores in HCC and paired normal liver tissues, and the staining patterns were categorized into three groups, 1+, 2+ and 3+, according to staining intensity . Moreover, the correlation between E2F4 expression with the mutations and co-expressed genes was explored. We obtained eight highly co-expressed genes in HCC based on the TCGA database (Table 3). As shown in Figure , E2F4 expression was significantly positively associated with the following genes: enhancer of mRNA decapping 4 , IST1 factor associated with ESCRT-III , nucleoporin 93 , U6 snRNA biogenesis phosphodiesterase 1 , solute carrier family 7 member 6 , nuclear transport factor 2 , NEDD8 activating enzyme E1 subunit 1 , and adrenocortical dysplasia protein homolog . These results provided the mutation characteristics of E2F4 and the distribution of the genes co-expressed with E2F4 in HCC, implying that E2F4 gene expression has close co-relationship with other genes expression.The mutation characteristics of E2F4 were further investigated, and an analysis using the cBioPortal was performed Figure ). Additionally, the heatmaps of the top 10 negatively related genes identified were CYP4A11, G6PC, ALDH5A1, TMEM56, INSIG1, ACOX1, SEPSECS, ABCA6, SLC10A1, and HMGCS2 . The top 10 positively related genes were EDC4, KIAA0714, NUP93, SLC7A6, CIZ1, PRMT1, NIP7, NUF2, NAE1, and DHX38 .To further validate the biological activities of E2F4 in HCC, the E2F4 DEGs were evaluated in HCC. The volcano map identified E2F4-related DEGs with positively related genes on the right of the plot and negatively related genes on the left of the plot . The expression of E2F4 was significantly associated with the expression of the hub genes , especially for SF3B3 (R = 0.81). Based on these results, the relationship between E2F4 and hub genes was a novel reason for tumor progression. Our results demonstrated that E2F4 expression was correlated with numerous crucial genes involved in many biological regulatory activities such as the cell cycle, protein modification, cellular transcription, and glycolipid metabolism.To determine the relationship of the top 100 positively related genes of E2F4 in HCC, a PPI network was established. As shown inFigure ). More specifically, E2F4-related pathways involved in BPs included mRNA splicing via spliceosome, regulation of mitophagy, and positive regulation of protein targeting the mitochondrion. Negative regulation of mRNA splicing via spliceosome revealed that E2F4 actively participates in nucleic acid, mitochondria, and accounting-related protein regulation. In CC analysis, the nucleolus, mediator complex associated pathways were significantly correlated with E2F4 function. In the MF subgroup, nucleotide binding, promoter-specific chromatin binding, and ATP binding pathways indicated significantly positive affiliation to E2F4 gene regulation . Moreover, according to KEGG analysis, RNA degradation suggested that the E2F4 gene plays a crucial role in cellular biology and the E2F4 gene was positively associated with RNA degradation and negatively associated to RNA transport and the spliceosome . These results collectively demonstrated that E2F4 has a wide range of effects on the production of the global transcriptome and the regulation of genes and pathways involved in mitochondrial metabolism.To investigate the regulation of signaling pathways associated with E2F4, the top 100 significantly related genes were used in KEGG and GO pathway enrichment analyses. GO results revealed six significant biological processes (BP), nine cellular components (CC), and three molecular functions . In HCC, E2F4 was positively correlated with immune cell infiltration by cells including B cells , CD4+ T cells , CD8+ T cells , macrophages , neutrophils , and dendritic cells . However, E2F4 expression was negatively related to immune purity . Considering the high frequent of TP53 mutations in HCC, we investigated the effect of mutated TP53 on E2F4 expression. As shown in Figure , we observed that E2F4 expression was elevated in the TP53-mutant group (n = 105) compared with the TP53 non-mutant group (n = 255). The TP53 mutation status was further evaluated regarding the level of immune cell infiltration. In this study, it was discovered that B cells and macrophages were increased with TP53 mutation compared to wide-type TP53 . Our results demonstrated that TP53 mutation might play an essential regulatory role in tumor immune cell infiltration.Next, we evaluated if E2F4 expression was associated with tumor immune status across 32 solid cancer types. E2F4 expression was found to be significantly associated with immune cell infiltration among several cancers, especially in HCC (HCC is a worldwide threat with significant morbidity and mortality based on the stage of the disease at presentation In this study, we observed that E2F4 expression was dysregulated in pan-cancer, and especially in HCC. Our study has provided a more detailed picture of E2F4 expression in relation to clinical characteristics, prognosis, immune cell infiltration, hub genes, and pathway crosstalk in HCC. Our results demonstrated that E2F4 was highly expressed in HCC, and Kaplan-Meier analyses indicated that E2F4 could be a potential prognostic factor in HCC. Similar to our results, Khaleel and colleagues reported that E2F4 could serve as an outcome prognosis predictor for breast cancer A network analyst algorithm was utilized to further explore the relationship between E2F4 and co-expressed genes, and the crucial enriched pathways involved in this crosstalk. We found that E2F4 could be identified as a regulator of hub genes in liver cancer. In support of our results, studies have demonstrated that E2F4 function as crucial regulator in liver cancer + T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells are co-related immune cells, which serve critical roles in HCC immune infiltration. Altogether, these data powerfully indicate that E2F4 could be a crucial factor mediating immune-associated pathways.The tumor microenvironment has a strong influence on the carcinogenesis of HCC To further investigate the signaling crosstalk involved in controlling abnormal E2F4 gene expression, we examined the E2F4 co-expression network. Our study validates that E2F4 influences cell cycle regulation, DNA repair, and replication. At the same time, E2F4 is also active in metabolic associated process, such as mRNA splicing via the spliceosome and regulation of mitophagy, positive regulation of protein targeting to the mitochondrion, nucleolus, mediator complex ATP binding, nucleotide, binding promoter-specific chromatin binding processes. These molecular activities are consistent with tumor carcinogenesis, and E2F4 could participate in tumor progression via the regulation of these pathways.Taken together, these findings demonstrate that the up-regulation of E2F4 could be a promising molecular target for the diagnosis and treatment of HCC. Reasonably, the potential role of E2F4 in immune environment regulation and clinical conditional diagnostic function still requires further validation.Supplementary figure S1.Click here for additional data file.