Antigenic drift of the hemagglutinin (HA) and neuraminidase (NA) influenza virus proteins contributes to reduced vaccine efficacy. To analyze antigenic drift in human seasonal H1N1 viruses derived from the 2009 pandemic H1N1 virus (pH1N1-like viruses) accounts for the limited effectiveness (around 40%) of vaccination against pH1N1-like viruses during the 2015–2016 season, nasal washes/swabs collected from adult subjects in the Rochester, NY area, were used to sequence and isolate the circulating viruses. The HA and NA proteins from viruses circulating during the 2015–2016 season encoded eighteen and fourteen amino acid differences, respectively, when compared to A/California/04/2009, a strain circulating at the origin of the 2009 pandemic. The circulating strains belonged to subclade 6B.1, defined by HA amino acid substitutions S101N, S179N, and I233T. Hemagglutination-inhibiting (HAI) and HA-specific neutralizing serum antibody (Ab) titers from around 50% of pH1N1-like virus-infected subjects and immune ferrets were 2–4 fold lower for the 2015–2016 circulating strains compared to the vaccine strain. In addition, using a luminex-based mPlex HA assay, the binding of human sera from subjects infected with pH1N1-like viruses to the HA proteins from circulating and vaccine strains was not identical, strongly suggesting antigenic differences in the HA protein. Additionally, NA inhibition (NAI) Ab titers in human sera from pH1N1-like virus-infected subjects increased after the infection and there were measurable antigenic differences between the NA protein of circulating strains and the vaccine strain using both ferret and human antisera. Despite having been vaccinated, infected subjects exhibited low HAI Ab titers against the vaccine and circulating strains. This suggests that poor responses to the H1N1 component of the vaccine as well as antigenic differences in the HA and NA proteins of currently circulating pH1N1-like viruses could be contributing to risk of infection even after vaccination. Influenza A (IAV) and B (IBV) viruses are members of the family Orthomyxoviridae, whose entire genome is encoded by 8 single-stranded, negative-sense RNA segments [Circulating influenza viruses are estimated by the World Health Organization (WHO) to infect 5–10% of the population every year, causing a morbidity rate of 3 to 5 million and a mortality rate of 250,000–500,000 deaths annually  acid residues of host cell glycoconjugates via the receptor-binding site (RBS), located atop the HA head [The quaternary structure of the influenza HA protein is a homotrimer, which is “primed” for activation at low pH by proteolytic cleavage of the trimeric HA0 precursor to form two subunits (HA1 and HA2), enabling virus entry into cells and HA-mediated fusion activity . At leas HA head . The RBS HA head . HA2 tog HA head ,7.Since 2009, the HA genes from pH1N1 viruses have evolved. For well over a year, viruses in clade 6, and carrying amino acid substitutions of D114N, S202T (in antigenic site Sb), and S220T (in antigenic site Ca1) in the head domain, and E391K and S468N in the stalk domain compared with A/Cal/07/09, have predominated worldwide, with a number of subclades emerging. Most of the viruses characterized since September 2014 carry HA genes in subclade 6B, which is characterized by additional amino acid substitutions of K180Q (in antigenic site Sa), A273T and K300E in the head domain and E516K in the stalk domain compared with A/Cal/07/09. A number of virus clusters have emerged within clade 6B and two of these have been designated as subclades: viruses in subclade 6B.1 are defined by head amino acid substitutions S101N, S179N (in antigenic site Sa) and I233T, while those in subclade 6B.2 are defined by head amino acid substitutions V169T (in antigenic site Sa) and V190I [The NA protein is a receptor-destroying enzyme that functions as a sialidase, facilitating the release of progeny virions by catalyzing the cleavage of terminal sialic acid residues from cellular and viral proteins . In addiHumans are most commonly infected with IAV subtypes H3N2 or H1N1, or one of two IBV lineages. Accordingly, the seasonal influenza vaccines include 3 or 4 viral strains  . AntibodThe HA and NA proteins incorporate mutations through a process called antigenic drift, decreasing vaccine efficacy ,22, somehttps://www.cdc.gov/flu/about/season/flu-season-2015-2016.htm). Moreover, previous work from our group showed that people infected with 2010–2011 and 2012–2013 seasonal H3N2 viruses uniformly had lower Ab titers against the virus they were infected with compared to the strain used for vaccination that season, suggesting that antigenic differences from the vaccine contributed to increased risk of infection [Despite the apparent antigenic similarity of the vaccine strain and circulating H1N1 strains, a study conducted by the United States Flu Vaccine Effectiveness Network found that 41% of the 781 participants infected with an pH1N1-like IAV during the 2015–16 season had previously been vaccinated . Patients were named with a number and the acronym ACU (from Acute influenza). Sera from patients enrolled in the acute influenza surveillance protocol were obtained at the acute visit (day 0), and around 28 days after the acute visit (day 28). Only samples from patients whom tested positive via RT-PCR for IAV H1N1 were included in this study. Some patients had received the 2014–15 or 2015–16 flu vaccine prior to infection . Each st2, in Dulbecco’s modified minimal essential medium (DMEM)  supplemented with 10% (v/v) fetal bovine serum (FBS)  and 10 μg/mL gentamycin . MDCK/H5 cells were grown in media additionally supplemented with 200 μg/mL hygromycin B every other passage to ensure continued expression of H5, as previously described [Madin-Darby canine kidney (MDCK) cells and human embryonic kidney 293T cells were obtained from the American Type Culture Collection . MDCK cells constitutively expressing the HA protein of A/Indonesia/5/2005 H5N1 (MDCK/H5) were previously described . MDCK anescribed ,28.A virus stock of influenza virus A/California/04/2009 , produced in 9 to 11-day-old SPF embryonated chicken eggs, incubated 2 to 3 days at 33.5 C, was used as the reference strain for HAI and MN assays .2, in DMEM supplemented with 0.3% bovine serum albumin (BSA) , 10 μg/mL gentamycin, and 1 μg/mL tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin .MDCK cells were inoculated with nasal washes/swabs from pH1N1-like virus-infected patients. Upon observation of cytopathic effect, supernatants were collected and used to infect fresh cells. If no cytopathic effect was observed, supernatants were collected 72 hours post-infection (hpi) and used to infect fresh cells. A maximum of three serial passages was performed. All infected cells were grown at 37°C in air enriched with 5% COViruses were titrated by immunofocus assay using an IAV monoclonal Ab specific for the viral NP protein (HB-65), as previously described ,30. IAV 5’-GGGGAAAACAAAAGCAACAAAAATG-3’) and H1N1-HA-3’-NCR-RS (GTGTTTTTCTCATGCTTCTGAAATCCTAATG-3’) or NA-A/Cal-1-VS (5’-ATGAATCCAAACCAAAAGATAATAACCATT-3’) and NA-A/Cal-1410-RS (5’-TTACTTGTCAATGGTAAATGGCAACTC-3’), respectively, with the High Capacity cDNA reverse transcription kit .Viral RNA was purified from nasal swabs/washes using the QIAamp viral RNA extraction kit  or from cell culture extracts using the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. Reverse transcription was performed to obtain cDNA of the HA or NA genes using primers H1N1-HA-5’NCR-VS (GGGAGGCTGGTGTTTATAGCACCC-3’) (for HA region 1) and H1N1-HA-793-VS (CTAGTGGTACCGAGATATGCATTCGC-3’) and H1N1-HA-3’-NCR-RS (for HA region 2) or NA-A/Cal-1-VS and NA-A/Cal-755-RS (5’-GAGGCCTGTCCATTACTTGGTCC-3’) (for NA region 1), and NA-A/Cal-652-VS (5’-GAGTTGGAGAAACAATATATTGAGAACACAAG-3’) and NA-A/Cal-1410-RS (for NA region 2), respectively, was performed with the Platinum Pfx polymerase . The VS and RS primers used in each PCR were used for Sanger sequencing. The complete HA and NA sequences were obtained directly amplifying the viral RNA from nine of the nasal swabs/washes. In addition, viruses from 9 of the 14 clinical samples were isolated, and the complete HA  and NA  sequences were obtained from the viruses isolated in MDCK cells.To obtain DNA segments encoding the HA or NA genes to sequence the HA and NA genes, overlapping PCR using primers H1N1-HA-5’NCR-VS and H1N1-HA-923-RS (5’-GATCGCTCTTCTGGGAGCAAAAGCAGGAGTTTAAAATGAATCCAAACCAAAAG-3’) and NCR-SapI-NACal-3-RS (5’-CATCGCTCTTCTATTAGTAGAAACAAGGAGTTTTTTGAACAAATTACTTGTCAATGGT-3’) and the High Capacity cDNA reverse transcription kit (Applied Biosystems) to produce cDNA. These cDNAs were then amplified by PCR using the same primers NCR-SapI-NACal-5-VS and NCR-SapI-NACal-3-RS and the Platinum Pfx polymerase (Life Technologies) to obtain the DNA transcript of the NA gene with SapI restriction sites at the 5’ and 3’ ends. PCR products were digested with the SapI restriction enzyme and cloned into the pDZ plasmid.Ambisense pDZ plasmids  encodingH5-pseudotyped single cycle infectious IAVs (sciIAVs) in which the viral HA gene was replaced by a gene encoding green fluorescent protein (GFP) and the viral NA gene was replaced either by the NA encoded by A/Cal/04/09 (NA-wt) or by the virus isolated from patient 001 (NA-001) were generated in the backbone of A/Cal/04/09 as previously described ,32. BrieSera from ferrets infected with A/Cal/07/09  and patient sera collected at days 0 and 28 were tested in duplicate against A/Cal/04/09 and the virus isolated from patient 022 for hemagglutination-inhibiting (HAI) and HA-specific neutralizing Ab titers.For HAI assays, twofold serial dilutions of human or ferret sera treated with receptor destroying enzyme (RDE)  were mixed with 8 HA units/50 μL of each virus. The HA protein of the A/Cal/04/09 virus used in the assays was identical to the one deposited in GenBank (accession number JF915184.1). The serum/virus mixture was incubated with 0.5% turkey red blood cells  for approximately 30 minutes at room temperature. The HAI Ab titer was measured as the highest dilution of serum at which hemagglutination was inhibited.2, infected cells were fixed and permeabilized with 0.5% Triton X-100 and 4% formaldehyde in PBS, and an immunofocus assay was performed to determine the amount of not-neutralized virus. The HA-specific neutralizing Ab titer was measured as the highest dilution of serum at which more than 50% of FFU were inhibited.For MN assays, twofold serial dilutions of human or ferret sera was mixed with approximately 200 FFU of each virus and was left at room temperature for 60 minutes. Confluent MDCK cell monolayers were then inoculated with the serum/virus mixtures. After an adsorption period of 60 minutes at room temperature, serum/virus mixtures were removed and replaced with DMEM supplemented with 0.3% BSA, 10 μg/mL gentamycin, and 1 μg/mL TPCK-trypsin. After incubation for 8 hpi at 37°C in air enriched with 5% COAll recombinant (r)HA proteins, encoding a C-terminal trimerization domain and a hexahistidine purification tag  were cloPurified rHA proteins for each influenza strain/subtype were covalently coupled to Bio-plex Pro™ Magnetic COOH Beads  using the Bio-Plex Amine Coupling Kit  as previously described . The couQuantitation of Ab levels in human sera was performed as previously described . BrieflySera from ferrets infected with A/Cal/07/09  and patient sera collected at days 0 and 28 were tested in duplicate against sciIAV-NA-wt and sciIAV-NA-001 for NAI Ab titers using a modified enzyme-linked lectin-based assay (ELLA), as previously described ,35,36. BAt least 8 wells on each plate were dedicated to positive  or negative  controls. The average background signal (negative control) was subtracted from all readings, and percent inhibition compared to the average maximum signal (positive control) at each serum dilution was calculated. The NAI Ab titer was measured as the highest dilution at which at least 50% of the maximum signal was inhibited. Only assays in which the background values were less than 10% of the maximum signal were considered.http://platform.gisaid.org/epi3/frontend). 16,981 HA and 15,796 NA DNA sequences remained after removing sequences lacking associated metadata, or containing missing or erroneous  nucleotides. The HA (9) and NA (9) sequences from the Rochester, NY viral isolates were added to the publically available sequences, resulting in 16,990 HA and 15,805 NA DNA sequences which were used for all sequence-based analyses. Sequences were aligned using MUSCLE [19,926 HA and 17,430 NA DNA sequences of pH1N1-like strains isolated from humans since 2009 were downloaded from the GISAID online database at the time of investigation . Clades were identified as previously described [Phylogenies were reconstructed with the neighbor-joining method and bootstrapped 1000 times with ClustalX version 2.1, using 16,990 HA or 15,805 NA aligned DNA sequences . Each inA single-likelihood ancestor counting (SLAC) approach  was usedstats package in R.For each HA sequence, we calculated its hamming distance to all other HA sequences in the data set, as previously described . The hamLog2 transformation was applied to Ab titers to stabilize data variability and normalization. Repeated ANOVA was used to simultaneously study multiple effects (string and time), and to take account of within-subject correlations. Multiple testing adjustment of Tukey method was applied to prevent type I error inflation. The findings were confirmed by pairwise comparisons through Sign test.Influenza virus HA and NA proteins undergo antigenic drift, leading to decreased vaccine efficacy ,43,44. PInfluenza viruses present in nasal washes/swabs from subjects infected during the 2015/2016 season encoded 18 amino acid changes in the HA protein compared to the strain Cal/04/2009, circulating at the origin of the 2009 pandemic . The infOut of these 18 mutations, 7 were located in the HA stalk domain while the other 11 were located in the HA head domain . As showAs for the NA protein , 14 mutations were found when compared to the vaccine strain . As seenTo analyze if the HA and NA protein sequences of viruses isolated in the Rochester, NY area, were representative of viruses circulating globally, these HA and NA sequences were compared to those of pH1N1-like viruses isolated worldwide during the 2015–2016 flu season. Out of the 6,552 HA and 5,462 NA sequences of 2015–2016 isolates uploaded to the GISAID database at the time of investigation, the majority of isolates from different geographic regions encoded HA and NA proteins with mutations identical to those described here  –56 were Of the 469 amino acids in the NA protein of pH1N1-like strains, 11.3% (53 of 469) of codons were subject to positive selection, while 88.7% of codons (416 of 469) were under negative selective pressure . InteresAs expected, mutations have been incorporated by the pH1N1-like virus population over the past seven seasons, beginning with the emergence of the pandemic-causing triple reassortant in early 2009 ,59,60. NP<0.005) increased in patient serum, as expected [HA is the main viral protein inducing antibodies that protect against infection . To analexpected , with thP<0.005) higher after infection (day 28), as expected. The only exception was the titers specific for the vaccine strain, which did not increase in patients 009 and 010 ” (reviewed in ). To relSerum Ab binding to the chimeric HAs cH5/1 and cH9/1 increased more than 1.5-fold in 8 of 9 subjects, indicating induction of Abs reactive to the stalk region of H1 (a group 1 HA) . In mostThree subjects had relatively high day 0 Ab levels against H1s of older H1N1 viruses that were consistent with OAS-type patterns: subjects 010 (b. 1963) and 018 (b. 1973) had high day 0 titers against H1 USSR77, and subject 012 (b. 1991) had high titers against H1 Tex91 . In mostVaccination and infection induce NA-specific protective antibodies ,20. It hP<0.001) increased at day 28 compared to day 0 (P<0.0001). Furthermore, an 8-fold difference in strain-specific Ab titers was observed using ferret serum , we characterized the HA and NA proteins of pH1N1-like IAVs and investigated the antigenic drift of strains circulating during the 2015–2016 season. Using samples collected in Rochester, NY, we found 18 and 14 mutations in the HA  of the flu vaccine during the 2015–2016 season  were predicted most likely to mutate in future pH1N1-like strains in our previous work forecasting the trajectory of pH1N1 evolution . While tTraditionally, antigenic drift has been measured primarily with HAI assays using antisera from ferrets recovering from infection with vaccine strains . HoweverThe WHO has announced that the H1N1 strain component of the flu vaccine will be updated this coming season (2017–2018) from A/Cal/07/09 to a more recently isolated pH1N1-like strain, A/Michigan/45/2015 . RemarkaThe majority of protective antibodies induced by vaccination or infection with influenza virus are specific to the HA protein ,74. As aIt has been shown that glycosylation on the HA head can modulate HA–receptor binding affinity ,78, enabThe HA stalk domain is considered relatively conserved, however genetic and antigenic differences can be detected ,84. InteIn conclusion, we have shown data suggesting that the mutations present in the HA and NA proteins of 2015–2016 season isolates in Rochester (NY) have accumulated in a non-random, directional manner. The HA of pH1N1-like viruses showed limited but measurable antigenic differences to the vaccine strain. In addition, there were demonstrable antigenic differences between the NA of the pH1N1-like viruses and the vaccine strain. Additionally, infected subjects exhibited low HAI serum Ab titers against the vaccine strain, despite being vaccinated. This suggests that poor responses to the H1N1 component of the vaccine as well as antigenic differences in the HA and NA proteins of the circulating pH1N1-like viruses could be contributing to risk of infection even after vaccination. These results are yet another reminder of why we must continue surveillance efforts to better understanding the different mechanisms employed by influenza viruses in evading the Ab-mediated immune response.S1 Fig(A) Sequence-based analysis of the HA subtypes. (B) Sequence-based analysis of the H1 HA proteins. Sequences are colored by subtype in A.(TIFF)Click here for additional data file.S1 TableAge, gender, weight, and vaccination history corresponding to each H1N1-positive patient (by accession number) enrolled in the acute influenza surveillance program at the University of Rochester Medical Center, NY.(TIFF)Click here for additional data file.S2 TableUnderneath in parentheses, the percentage of total strains from the respective region whose HA encoded the mutation. The total number of HA sequences from each region included in the analysis is indicated in parentheses next to the region name. Blue, yellow and pink boxes indicate amino acid residues included in Sa, Sb and Ca1 antigenic sites, respectively.(TIFF)Click here for additional data file.S3 TableUnderneath in parentheses, the percentage of total strains from the respective region whose NA encoded the mutation. The total number of NA sequences from each region included in the analysis is indicated in parentheses next to the region name. Yellow boxes indicate amino acid residues included in antigenic sites.(TIFF)Click here for additional data file.S4 TablePercentage of pH1N1-like strains isolated during the 2016–17 flu season whose HA (A) and NA (B) encoded the same mutations found in the HA and NA of 2015–16 isolates. Mutations within previously defined antigenic sites are shaded gray.(TIFF)Click here for additional data file.


Background: Low 25(OH) vitamin D levels have been associated with several autoimmune diseases and recently with autoimmune thyroiditis (AT). The aim of the study was to investigate the association of AT with low 25(OH) vitamin D levels in the elderly. Methods: One hundred sixty-eight elderly subjects (mean age: 81.6 ± 9.4 years) were enrolled. Serum levels of 25(OH) vitamin D, anti-thyroid peroxidase (TPO-Ab), anti-thyroglobulin (TG-Ab) antibodies, free triiodothyronine (FT3), free thyroxine (FT4) and thyroid stimulating hormone (TSH) were measured. Results: The prevalence of AT was significantly higher in subjects with vitamin D deficiency (25(OH) vitamin D < 20 ng/mL) when compared with subjects with normal 25(OH) vitamin D (25(OH) vitamin D ≥ 20 ng/mL) levels . Patients with AT and vitamin D deficiency had a comparable hormonal profile compared to patients with AT and vitamin D sufficiency in terms of TSH (p = 0.39), FT3 (p = 0.30), FT4 (p = 0.31), TG-Ab (0.44) and TPO-Ab (0.35). Interestingly, a significant correlation between 25(OH) vitamin D and TPO-Ab  and FT3  has been found in subjects with AT while no correlation was found between 25(OH) vitamin D levels and TG-Ab , TSH  and FT4 . Conclusions: These findings suggest that vitamin D deficiency was significantly associated with AT in the elderly. Therefore, the screening for AT should be suggested in subjects with vitamin D deficiency. After ingestion or synthesis, vitamin D is hydroxylated in the liver by Cytochrome P450 Family 2 Subfamily R Member 1 (CYP2R1) to form 25 hydroxyvitamin D (25(OH)D2 or 25(OH)D3), its major circulating form, which has little biological activity. The 25(OH)D is converted in the kidney by Cytochrome P450 Family 27 Subfamily B Member 1 (CYP27B1), to its bioactive hormonal metabolite 1,25 dihydroxy-vitamin D 2D or calcitriol). The 25(OH)D 1α hydroxylase has been also found in extrarenal sites including placenta, monocytes and macrophages [2D binds the nuclear vitamin D receptor, which heterodimerizes with the retinoid X receptor and binds to vitamin D–responsive elements near target genes [3 receptor and the receptor for the thyroid hormone have been reported to have a similar molecular structure, which was due to two regions that they have in common: the first is a 70-amino-acid, cysteine-rich sequence and the second region is a 62-amino-acid one located towards the carboxyl terminus of the protein [Humans derive vitamin D from cutaneous synthesis  . Thanks  . Thanks protein . Lower v protein . The sam protein  in a stu protein  reportedIn polycystic ovary syndrome, we have demonstrated that the levels of 25(OH) vitamin D were significantly lower in women with Polycystic Ovary Syndrome (PCOS) and AT when compared to women with PCOS and without AT, although no correlation was found between 25(OH) vitamin D and anti-thyroid peroxidase antibodies (TPO-Ab), anti-thyroglobulin (TG-Ab) antibodies, free triiodothyronine (FT3), free thyroxine (FT4) and thyroid stimulating hormone (TSH) levels .The main limit of these studies is that the association of 25(OH) vitamin D with AT has been assessed in young and/or middle-aged subjects. To date, no studies have been performed in the elderly. Based on these considerations, the aim of the current study was to investigate the association of AT with low vitamin D levels in the elderly population, where both conditions are quite common.l-thyroxine, antithyroid drugs, antiepileptic, antipsychotics, amiodarone, glucocorticoid, etc.); treatment with vitamin D or calcium supplement; administration of immunomodulatory medications.A total of 168 patients aged 65 years or older were retrospectively enrolled in the Division of General Internal Medicine and Geriatrics—Italian Auxologic Institute—from April 2010 to June 2010, after obtaining written consent. In order to avoid seasonal influences on 25(OH) vitamin D levels, the study has been carried out in a short time-period. Exclusion criteria included: age < 65 years; treatment with medications affecting thyroid function (2). After fasting overnight for 10–12 h, blood samples were collected for the following measurements: 25(OH) vitamin D, FT3, FT4, TSH, TG-Ab, TPO-Ab, hepatic and renal chemistries. Subjects with elevated TG-Ab and/or TPO-Ab serum levels performed thyroid ultrasound in order to confirm the diagnosis of AT.For all subjects, body mass index (BMI) was calculated as weight (kg)/(height (m)Serum levels of 25(OH) vitamin D were determined through electrochemiluminescence binding assay using the Cobas analyzer (Roche Diagnostics) . Serum TAT was defined on the basis of serum TG-Ab (>115 IU/mL) and/or TPO-Ab (>34 IU/mL) positivity, together with characteristic ultrasonographic features .Vitamin D deficiency was defined as 25(OH)D levels < 20 ng/mL while 25(OH)D levels ≥ 20 ng/mL were considered normal . t-test for independent samples and the non-parametric ManneWhitney U-test for independent samples were applied to test for differences between groups. Pearson correlations were used to determine relationships between variables.Statistical analysis was carried out using SPSS 9.0 . Data are expressed as mean ± SD (Standard Deviation). Pearson correlations were used to determine relationships between variables. Depending on the distribution of the data, the Student’s Clinical and biochemical variables of the entire cohort of patients stratified by 25(OH) vitamin D levels are shown in Selecting 20 ng/mL as the cut-off point, low 25(OH) vitamin D levels were detected in 70% of all patients. The overall prevalence of AT was 23%. p = 0.002).We compared the prevalence of AT between patients with vitamin D deficiency (25(OH) vitamin D < 20 ng/mL) and sufficiency (25(OH) vitamin D ≥ 20 ng/mL), finding that AT was significantly more frequent in patients with vitamin D deficiency , FT3  and FT4 levels . As expected, subjects with vitamin D deficiency were significantly older compared to subjects with normal vitamin D levels . Further, we investigated if patients with AT and vitamin D deficiency (n = 21) had a worse hormonal profile compared to patients with AT and vitamin D sufficiency (n = 18), but we did not find significant differences between the two groups .Although the prevalence of AT was higher in patients with vitamin D deficiency, the subjects with vitamin D deficiency did not differ from subjects with vitamin D sufficiency in terms of TSH  and FT3  , TSH  and FT4 .In addition, we focused on patients with AT in order to investigate if vitamin D levels were associated with the grade of severity of AT in the overall population. In this subgroup Pearson coefficient analyses revealed a significant correlation of 25(OH) vitamin D levels with TPO-Ab (= 0.006) , while nThe main finding of this study was that elderly patients with vitamin D deficiency had a significantly higher prevalence of AT compared to elderly patients with vitamin D sufficiency. These results were in agreement with previous studies in younger populations . FurtherThe prevalence of vitamin D deficiency 70%) was high in our elderly population, although this value is consistent with the results reported in European studies assessing vitamin D deficiency in the elderly ,16,17,180% was hi2D is expressed thanks to the binding of 1,25(OH)2D to its receptor [2D inhibits DC maturation and the production of DC-derived cytokines, such as IL-12 and IL-23. This effect of 1,25(OH)2D prompts the T cells to differentiate towards the Th2 phenotype, rather than to the Th1 and Th17 phenotypes [The Vitamin D receptor (VDR) is an intracellular receptor that is expressed by human immune cells, such as macrophages, dendritic cells, and T and B lymphocytes, although the main target is represented by dendritic cells (DC) ,21. The receptor . In partenotypes ,25. A meenotypes . Polymorenotypes . The degree of vitamin D deficiency has been reported to have a tight relationship with antibody levels and with thyroid hormones, thus suggesting that vitamin D levels may have a direct role in determining the grade of severity of autoimmune disease and of the consequent hypothyroidism ,29. In oIn conclusion, within the limitations of a cross-sectional design, the present study shows that vitamin D deficiency was associated with AT in the elderly. Future longitudinal cohort studies along with prospective interventional trials may contribute to better clarifying if vitamin D supplementation may have a role in the prevention and/or therapy of AT in the elderly.


Yet, clinical applications have been hindered by the restricted sensitivity of the surface radiofrequency-coils normally used. These permit the analysis of spectra only from the interventricular septum, or large volumes of myocardium, which may not be meaningful in focal disease. Löring et al. recently presented a prototype whole-body (52 cm diameter) transmit/receive birdcage coil for 31P at 7T. We now present a new, easily-removable, whole-body 31P transmit radiofrequency-coil built into a patient-bed extension combined with a 16-element receive array for cardiac 31P-MRS.Cardiac phosphorus magnetic resonance spectroscopy  birdcage transmit coil was combined with a 16-element receive array on a Magnetom 7T scanner . Electro-magnetic field simulations and phantom tests of the setup were performed. In vivo maps of B1+ of the whole-body coil across the heart was 46.7% (surface coil 129.0%); and the in vivo measured value was 38.4%. Metabolite images of 2,3-diphosphoglycerate clearly resolved the ventricular blood pools, and muscle tissue was visible in phosphocreatine (PCr) maps. Amplitude-modulated saturation bands achieved 71±4% suppression of phosphocreatine PCr in chest-wall muscles. Subjects reported they were comfortable.The combined  setup increased signal-to-noise ratio 2.6-fold 10 cm below the array (depth of the interventricular septum) compared to using the birdcage coil in transceiver mode. The simulated coefficient of variation for BThis easy-to-assemble, volume-transmit, local receive array coil combination significantly improves the homogeneity and field-of-view for metabolic imaging of the human heart at 7T. The PCr in vivo , and whi in vivo  and obes in vivo .31P-MRS is still applied in clinical medicine, principally because of its comparatively long scan times, signal-to-noise ratios that are too low for single-subject comparisons, and the challenges of recording regionally-resolved spectra across the heart.Yet 31P-MRS have used surface coils for transmission. Surface coils’ radiofrequency (RF) transmit field strength (B1+) inherently drops-off rapidly with increasing distance from the coil to the volume of interest [31P-MRS imaging (31P-MRSI) across the heart challenging. Even with custom-built surface transmit coils, and optimised adiabatic B1+-insensitive excitation pulses, regionally-resolved measurement across the whole heart in clinically feasible times remains elusive [Moving to ultra-high field (i.e. 7T) from clinical MR systems (i.e. ≤3T) leads to a 2.5-fold increase in SNR , 9 and iinterest , 13. Thi elusive .31P birdcage RF-coil into the bore of a Philips 7T MR system . This showed relatively uniform spectral profiles for 31P-MRSI examinations with a rectangular RF pulse in a cylindrical phantom and in a human subject [Recently, Löring et al inserted a whole-body-sized (52 cm in diameter)  subject . However31P transmit RF-coil for use on a Magnetom 7T MR scanner , integrated into an extension of the scanner’s motorized patient bed; we use it in conjunction with a 16-element anterior receive array  for cardiac 31P-MRSI at 7T.In this study, we report initial results of a collaborative project to design, build and test a new, easily-removable, high-pass birdcage, whole-body (55cm-diameter) 31P-MR on a Magnetom 7T MR scanner. The standard Siemens 8-kW RF power amplifier was used at this stage.The underlying design of our whole-body coil was similar to that previously reported . However2) surface transmit coil, previously used in our lab for 31P excitation at 7T [Simulations of electro-magnetic fields were performed out using CST Studio Suite 2016 . A whole-body coil matching the proposed design, i.e. a 40-cm long, 20-rung birdcage coil with a 55 cm diameter, was simulated inside an RF shield matching the diameter and length of the shield of the scanner. Two voxel models, “Gustav” and “Laura”, were simulated in order to identify the 10g local, global head and global body specific absorption rate (SAR) values for 1W input power. In order to get the worst case for the 10g local SAR values, one arm of the voxel model was placed in direct contact with the inner lining of the transmit coil. A rectangular (26×28 cm3), filled with NaCl(aq) (73 mM), and a 2cm cube, filled with KH2PO4(aq) (1.8 M), set at a 10cm depth. A series of fully relaxed (TR = 10 s), non-localized, single average 31P-MRS spectra were acquired increasing the peak voltage of a 10 ms rectangular pulse from 25 V to 400 V in 25 V steps. The SNR and peak B1+ were compared between a quadrature surface 31P RF-coil in transceiver mode , the neve loops ,  positioned over the heart. The 1H RF-coil was then replaced with the 16-channel receive array for the 31P experiments. The first experiment recorded high-quality 3D-resolved spectra using our established cardiac 31P-MRS protocol. Specifically: acquisition-weighted 3D-UTE-CSI [3 field-of-view (FOV) in three volunteers, using a 1 ms long amplitude-modulated excitation pulse [Eight healthy volunteers  were approached between October 2016 and January 2017 and all were consecutively recruited for our three in vivo experiments. A written informed consent was obtained in compliance with ethical and legal requirements. Oxford Central University Research Ethics Committee provided approval for this technical development work. No further demographic characteristics were recorded for the recruited volunteers. The individual whose photograph is shown in 3 FOV, and 60 mm slice thickness. Slice selective 2.5 ms long sinc pulses were used for excitation. Relatively short TR = 300 ms was used allowing for 32 averages within 19 minutes 26 seconds. Two amplitude-modulated saturation bands (10 ms duration) were used to suppress signal from chest muscles in one of the acquisitions.The second experiment recorded metabolite maps and tested the performance of amplitude-modulated RF “saturation bands”. Four volunteers were examined in the second experiment using two acquisition-weighted, transverse 2D-UTE-CSI experiments with a 24×24 matrix over 500×500 mm1+ field maps to quantify the transmit B1+ homogeneity of the new whole-body RF-coil in vivo. Four subjects underwent B1+ field mapping using a Bloch-Siegert sequence [The third  experiment recorded Bsequence  with sim1+ field maps were calculated in all voxels with sufficient SNR, as defined in [Signals from individual receive elements were combined using whitened singular value decomposition , and thefined in .2, respectively. Our simulated worst-case local 10 g SAR in the body was 0.145 W/kg and global body SAR was 0.013 W/kg. The mean simulated B1+ for 1 W delivered power was 0.160 ± 0.075 μT for the heart and 0.122 ± 0.068 μT for liver (mean ± standard deviation for voxels of tissue type “heart” or “liver” in the 3D results). The coefficients of variation (CV), i.e. the standard deviation divided by the mean, for simulated B1+ across the “heart” type voxels were 46.7% for the new birdcage coil (55cm-diameter) and 129.0% for the rectangular surface coil.The results of our simulations are depicted in 1+ comparison between the coils are given in 1+ of the combined whole-body transmit and local receivers at a 10 cm depth below the coil was 3.5 times lower compared to the quadrature surface coil, however, the SNR achieved was comparable.The results of the phantom SNR and peak BRepresentative in vivo spectra acquired with the 3D-UTE-CSI protocol in human heart and liver using the combined setup are depicted in 1+ field was calculated in 52 ± 8 voxels satisfying the SNR criteria per subject. The mean measured CV for B1+ was 38.4%. The resulting mean measured 31P B1+ in vivo was 10.4 ± 2.7 μT. A representative in vivo B1+ map and a histogram of B1+ variability are shown in The B31P-MR at 7T. This setup provides homogeneous excitation through the whole chest without the need for adiabatic excitation pulses, as demonstrated by our CST simulations and 31P B1 maps measured in vivo. In addition, the receive array increases the SNR of our setup compared to the body coil in transmit/receive mode, allowing high-resolution 31P-MRSI experiments in vivo. The increased 31P B1+ homogeneity allows the use of conventional amplitude-modulated pulses to suppress chest wall muscles instead of the SAR demanding BISTRO approach [In our study, we propose a new easily removable whole-body RF transmit coil combined with a 16-element receive array for cardiac approach  that hasapproach .31P measurement in vivo. Our simulated SAR efficiency values were similar to the previously reported ones [2 .Our new coil was designed to easily move in and out of the bore of a Magnetom 7T MR scanner using the system’s motorized patient table. Therefore, the maximal inner diameter of the transmit coil was limited by the scanner bore diameter (58 cm). Our constructed whole-body coil had an inner diameter of 55 cm. Although this means reduction of the available patient space, it is still larger than the diameter of the previously reported whole-body coil that had a 52 cm diameter , and sinted ones , i.e. 3.11 and S12 parameters of whole-body coil ports were observed using a network analyser  when the 16-channel array was inserted (regardless of its position). Adding the receive array led to a 2.6-fold increase in achieved SNR in phantom experiments, in comparison to the use of the whole-body coil in transceiver mode alone. This is comparable to the improvement seen on a 3T TIM Trio scanner (Siemens) between body coil 1H MRI and using half of a 32-channel cardiac receive array . A further increase in SNR might be gained by placing another receive array beneath the volunteer. Active detuning of the body coil during signal acquisition could also potentially lead to an increase in SNR. Every increase in single-element SNR will also lead to an increase in the precision of the complex “weights” used in coil combination algorithms, e.g., WSVD. The final SNR will therefore increase both due to the increased single-element SNR, but even more because of the better complex “weights” estimation in the combination step [To increase receive sensitivity, we combined the whole-body transmit coil with a 16-element receive array. No changes in the Sion step .1 achieved by the whole-body transmit coil in the phantom experiments at a depth of 10 cm was 3.5 times lower than that of our dedicated quadrature surface transmit coil as expected. However, the B1+ homogeneity of the whole-body transmit coil, allowing for short-TR scans with uniform Ernst-angle excitation or multi-echo readout [31P RF-amplifier, such as the 35 kW 123 MHz amplifiers used for 1H-MRI on Siemens 3T scanners. Dedicated low-loss transmit cabling could also be used. We estimate that these changes would give peak B1+ output comparable to our quadrature surface coil at 10 cm depth, while also retaining the coverage and uniformity advantages of the whole-body coil.On the other hand, the peak B readout  compensa31P-MRSI maps. The acquired in vivo 31P B1+ maps showed a high level of uniformity with a coefficient of variation <39% across all voxels with sufficient SNR. To demonstrate the use of this B1+ uniformity of the whole-body coil, we showed the effectiveness (>70%) of conventional amplitude-modulated saturation bands to suppress the signal from chest and abdominal muscles that can otherwise contaminate cardiac 31P-MRS data [The in vivo data demonstrate that while our whole-body RF-coil in transceiver mode could be used to acquire PCr signal from skeletal muscles, its SNR is too low to allow detection of metabolites of lower concentration, e.g., 2,3-DPG. However, in combination with the 16-element receive array, good quality spectra and well-resolved maps of PCr and 2,3-DPG can be acquired in vivo. As expected, the PCr signal was localized to the skeletal muscle wall as well as the heart, while the 2,3-DPG signal was restricted only to the ventricular blood pools. This confirms the high spatial resolution of the acquired MRS data , 22.31P-MR at 7T. This apparatus allowed us to measure in vivo the homogeneity of the 31P transmit field, confirming the results of electromagnetic field simulations. It allows us to record anatomically-consistent 31P metabolite maps, and to use saturation bands with amplitude-modulated pulses (and hence low SAR demands) to suppress signals from skeletal muscle. This combination of hardware is a step towards regionally-resolved, whole-heart cardiac 31P-MRS studies at 7T.In conclusion, we have designed, constructed and tested an easily removable, whole-body transmit RF-coil and its combination with 16-element receive array that is straightforward and comfortable to use for cardiac


Using exome sequencing, Kullar  SSBP1, encoding the single stranded binding protein 1 (SSBP1), segregating with hearing loss in a multi-generational family transmitting m.1555A>G, associated with mtDNA depletion and multiple deletions in skeletal muscle. The SSBP1 mutation reduced steady state SSBP1 levels leading to a perturbation of mtDNA metabolism, likely compounding the intra-mitochondrial translation defect due to m.1555A>G in a tissue-specific manner. This family demonstrates the importance of rare trans-acting genetic nuclear modifiers in the clinical expression of mtDNA disease.The m.1555A>G mtDNA variant causes maternally inherited deafness, but the reasons for the highly variable clinical penetrance are not known. Exome sequencing identified a heterozygous start loss mutation in  MTRNR1, is present at an estimated 1 in 385 (0.26%) of the European population, and is necessary but not sufficient to cause maternally inherited deafness , co-segregated with the m.1555A>G variant and the phenotype. This provides an explanation for the variable clinical penetrance of the disorder.Here, we report a multi-generational family where a heterozygous start loss mutation in the core mitochondrial DNA (mtDNA) replisome protein gene, single stranded binding protein 1 (n = 9) or sensorineural hearing loss . The mean age of hearing loss diagnosis was 3.7 years (range 1.6–5.4 years). There was no history of aminoglycoside usage. Neurological examination of all individuals was otherwise normal. There was no clinical evidence of either proximal or distal myopathy. DNA was available from 25 individuals from Generation III and IV. We subsequently also identified the children of two fathers in Generation III [Subject III-6 (P1), father in Family D, n = 9 children; and Subject III-10 (P4), father in Family E, n = 6 children; mean age of children = 10.3 years (range 1–19 years); Forty-six individuals  carrying the m.1555A>G mtDNA variant from Northern Finland A were prSSBP1 variant. Pyrosequencing (Qiagen) was undertaken for allelic quantification of m.1555A>G. Long-range PCR was used to detect mtDNA deletions using primers (D1R m.19-1 and D2F m.1650-1671) amplifying a product encompassing almost the complete mtDNA. mtDNA copy number was determined using real time PCR TaqMan® assays targeting MT-ND1 or MT-CO3 and the nuclear genes, B2M or RNaseP as described  <1% were performed as previously described , mitochondrially encoded cytochrome c oxidase 2 , succinate dehydrogenase complex flavoprotein subunit A , ATP synthase subunit alpha , translocase of outer membrane 20  and glyceraldehyde-3-phosphate dehydrogenase  (Protein extract from fibroblasts was separated by electrophoresis on a 4–12% polyacrylamide gel (NuPAGE). Immunoblotting was performed as described using antibodies to SSBP1, , mitochondrially encoded cytochrome c oxidase (COX), succinate dehydrogenase (SDH) and sequential COX-SDH staining was performed on sectioned skeletal muscle (10 μm transverse sections) as previously described  that abolishes the primary start codon (http://gnomad.broadinstitute.org). All of the variants were found in Finnish individuals , possibly indicating a founder effect within the Finnish population. The variant is predicted to be pathogenic by MutationTaster = 1, SIFT = 0 and LRT = 0  and Family E  carried the c.3G>A SSBP1 but all carried wild-type m.1555A [SSBP1 sequence.Pyrosequencing of leucocyte DNA confirmed all subjects were homoplasmic for m.1555A>G C. Exome rt codon B. The va LRT = 0 A and D. SSBP1 was found between patient and control fibroblasts  and controls (Subjects C1–3) E. No sigroblasts F, consisHaematoxylin and eosin staining of the muscle biopsy from Subject P5 was unremarkable whereas sequential COX-SDH staining revealed evidence of respiratory chain deficiency with a low number of COX-deficient and COX-intermediate fibres  found in the major non-coding of many mitochondrial genomes, formed by stable incorporation of a third, short DNA strand known as 7S DNA. The exact function of 7S DNA is unknown; however, it has been proposed to play a role in replication as an intermediate of prematurely-terminated heavy (H-) strand synthesis and moreover, perturbations in the steady-state levels of 7S DNA have been observed in a mtDNA maintenance disorder . Previoude novo intra-mitochondrial protein translation by incorporation of 35S radiolabelled methionine revealed markedly reduced global mitochondrial protein synthesis in Subject P1, P2 and P3 cells compared to controls [MT-CYB mRNA expression  reducing the likelihood that our observations are the result of a chance finding , that has previously been suggested to modify the phenotype in a subset of m.1555A>G carriers (SSBP1 variant is unlikely to be pathogenic in isolation given that 10/15 (67%), of the children in Families D and E carry the SSBP1 variant in conjunction with wild-type m.1555A and all have normal hearing. In addition, the heterozygous knockout mouse Ssbp1tm1a(KOMP)Wtsi displays only a mild phenotype, with hearing no different to wild-type littermates . P.F.C. is a Wellcome Trust Senior Fellow in Clinical Science (101876/Z/13/Z), and a UK NIHR Senior Investigator, who receives support from the Medical Research Council Mitochondrial Biology Unit (MC_UP_1501/2), the Wellcome Trust Centre for Mitochondrial Research (096919Z/11/Z), the Medical Research Council (UK) Centre for Translational Muscle Disease research (G0601943), EU FP7 TIRCON, and the National Institute for Health Research (NIHR) Biomedical Research Centre based at Cambridge University Hospitals NHS Foundation Trust and the University of Cambridge. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. C.G. is supported by the European Commission under “Marie Skłodowska-Curie Actions”, Individual Fellowship – Reintegration Panel (Mitobiopath-705560). J.M. receives funding from the Instituto de Salud Carlos III (PI14/00005 and PI14/00070); Departamento de Ciencia, Tecnología y Universidad del Gobierno de Aragón (Grupos Consolidados B33) and FEDER Funding Program from the European Union; and Asociación de Enfermos de Patología Mitocondrial (AEPMI). P.A.G. and M.M. are supported by core funding from Medical Research Council (UK) (MC_U105697135).


P = 0.007 and P < 0.001, respectively). Expression of TOPK was positively correlated with Ki67 (P < 0.001). Knockdown of TOPK significantly inhibited cell growth, colony formation and increased sensitivities to temozolomide (TMZ) in U-87 MG or U-251 cells, while TOPK overexpression promoted cell growth and colony formation in Hs 683 or A-172 cells. Glioma patients expressing high levels of TOPK have poor survival compared with those expressing low levels of TOPK in high-grade or low-grade gliomas . The level of TOPK was low in TMZ-sensitive patients compared with TMZ-resistant patients (P = 0.0056). In TMZ-resistant population, patients expressing high TOPK have two months’ shorter survival time than those expressing low TOPK. Our findings demonstrated that TOPK might represent as a promising prognostic and predictive factor and potential therapeutic target for glioma.TOPK is overexpressed in various types of cancer and associated with poor outcomes in different types of cancer. In this study, we first found that the expression of T-lymphokine-activated killer cell-originated protein kinase (TOPK) was significantly higher in Grade III or Grade IV than that in Grade II in glioma ( Glioma represents the most common primary tumor in the Central Nervous System (CNS). Along with the increased incidence of brain tumors, glioma mortality in China increased by as much as 194% in 2008, compared to data from the 1970s [T-lymphokine-activated killer cell-originated protein kinase (TOPK) is a MAPKK-like serine/threonine protein kinase extensively expressed in various types of cancer, such as colorectal cancer , lymphom7-MeG, N3-MeA and O6-MeG by DNA methylation, leading to DNA double strand breaks, thereby exerting its anti-cancer cytotoxicity [6-MeG, is repaired by O6-methylguanine-DNA methyltransferase (MGMT), which is a DNA repair protein [MGMT gene promoter is unmethylated, which enhances tumor resistance to TMZ [Temozolomide (TMZ), a first line chemotherapeutic drug for glioma patients , can indtoxicity . As a pr protein . The leve to TMZ . In addie to TMZ  and basee to TMZ  are invoIn this study, we found that TOPK expression was significantly increased in high-grade gliomas (HGG) (WHO Grade III & IV) patients. TOPK expression was closely associated with glioma grading, poor survival of glioma patients, cell proliferation and tumorigenesis of glioma, and more importantly, with chemotherapeutic resistance to TMZ.P = 0.007 and P < 0.001, respectively). No significant difference in TOPK expression was found between Grade III and Grade IV (P = 0.6973). Expression levels of TOPK were scored from 0 to 12 according the definition described in the materials and methods section. High score of TOPK was only observed in HGG patients. These data suggested that TOPK was related with histological grade and could act as a promising diagnostic factor for differentiating HGG and LGG. Meanwhile, we examine the expression of Ki67, P53 and EGFR, common molecules associated with histological grade or prognosis in human glioma. Ki-67, reflecting the proliferation and malignancy of cancer cells, was significantly increased with the grade of glioma [P = 0.0003 and P < 0.0001, respectively)     or HGG  patient samples by IHC. As shown in Figure f glioma . Our res) Figure . EGFR ge) Figure . Further) Figure , not betleft panel). Next, the anchorage-independent growth of the U-87 MG/shMock or U-87 MG/shTOPK cell lines was examined, and the results indicated that the number of colonies in U-87 MG/shTOPK cell cultures was much less than that in U-87 MG/shMock cell cultures . Similar results were observed in the U-251/shMock or U-251/shTOPK cell lines . ERK has been identified as one of the substrates of TOPK and TOPK-ERK interaction increases tumorigenesis of colorectal cancer cells [ex vivo.It has been reported that TOPK could promote tumorigenesis of colorectal cancer . As shower cells . Next, wex vivo.In order to further confirm the role of TOPK on glioma cell growth, we transfected pcDNA3 or pcDNA3-HA-TOPK into Hs 683 or A-172 cells to generate TOPK overexpression cell lines. Expression of TOPK and phospho-ERK1/2 was increased Figure . Cell pr+/+ MEF and H2AX−/− MEF cells were treated with TMZ, and we observed that knockout of H2AX promoted cell apoptosis . HGG and LGG patients with low TOPK expression has no significant difference for OS  . These results showed that TOPK overexpression obviously shortens the length of OS in glioma, and TOPK is correlated with poor survival outcome, regardless of WHO Grade. We also compared the correlation between Ki67 expression and survival in the same patient samples, and the results indicated that patients with high Ki67 expression have poor survival outcome as well.  . Therefore, we hypothesize that TOPK can act as a prognostic factor similarly to Ki67 in glioma. To identify the potential association between TOPK expression and the efficacy of TMZ chemotherapy, 35 patients received TMZ chemotherapy were studied. The results indicated that patients with high levels of TOPK were insensitive to TMZ therapy compared to those with low levels of TOPK (P = 0.0056) , which suggested that TOPK expression was significantly related to TMZ resistance. No significant correlation between Ki67 and TMZ treatment was identified (P = 0.075)  and a high TOPK expression group (score: 4–12). Patients with high TOPK expression had a shorter median survival time (MST) and overall survival (OS) than patients with low TOPK expression. Log-rank test showed that glioma patients with high levels of TOPK have poor survival outcome compared with those with low levels of TOPK in HGG or LGG  Figure . Therefoisocitrate dehydrogenase 1/2 (IDH1/2) [p53 mutation [EGFR amplification or mutation [The WHO redefined the classification of CNS tumors in 2016. Unlike the previous editions of WHO classification based on histopathological features, through integrating histopathological and molecular features in glioma diagnosis, more prognostic information is provided. The most frequent genetic alterations associated with glioma initiation and progression include mutation of (IDH1/2) , co-dele(IDH1/2) , p53 mutmutation  and EGFRmutation , 29. Themutation , 31. In mutation , 29, becmutation , 32, sugTOPK contribute to tumorigenesis, tumor development and progression , 33–38, MGMT gene mRNA was barely detectable in both U-87 MG and U-251 cells [Currently, the standard treatment strategies for glioma are surgical resection, radiotherapy and adjuvant chemotherapy. TMZ is a commonly preferred chemotherapy drug for glioma patients. MGMT was identified as an important biomarker capable of predicting the sensitivity of glioma patients to TMZ treatment. 50% of glioblastomas patients with low level of MGMT could not benefit from TMZ therapy, which suggested that the additional factors participated in TMZ resistance . In this51 cells . We also51 cells . TOPK co51 cells . We founin vitro and inhibited tumor growth in vivo [TMZ treatment has improved PFS and OS in glioma patients, but its survival benefit remains unsatisfactory because of primary or acquired resistance. In this study, we demonstrated that TOPK was highly expressed in HGG, and high level of TOPK was significantly associated with TMZ resistance and poor survival in glioma patients. TOPK promotes glioma cellular proliferation and tumorigenesis. Either HI-032 treatment or TOPK knockdown can promote cell apoptosis to TMZ treatment. It has been reported that targeting TOPK decreased growth and survival of glioma initiating cells  in vivo . Therefo in vivo , HI-032  in vivo , OTS964  in vivo  and pant in vivo  have beeIn conclusion, our study demonstrates that TOPK is highly expressed in HGG and contributes to TMZ resistance and poor survival in glioma patients. In addition, our results show that TOPK facilitates glioma cellular growth and tumorigenesis. Therefore, TOPK can serve as a promising new diagnostic, prognostic and predictive factor and a potential therapeutic target for glioma.The study materials consisted of 65 cases of malignant gliomas (WHO Grade II~IV), collected from archival files dating from 2015 to 2016 of Department of Pathology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Tissue blocks from three cases of focal cortical dysplasia and one case of pilocytic astrocytoma (WHO Grade I) were designated as the control group. Clinical parameters including age, grade, MST and TMZ treatment were evaluated . For immunohistochemistry (IHC), the paraffin sections were gradually dewaxed into the water, then immersed in 0.01 M citrate buffer and microwaved for antigen retrieval. After incubation in primary antibodies against TOPK, P53, EGFR and Ki67 overnight, the standard DAKO ChemMateTM EnVision Kit, based on the two-step labeled horseradish perioxidase method, were used according to manufacturer's instructions. 3,3′-diaminobenzidine was used as the chromogen. The deposition of granular dark brown pigments in tumor cells were considered as positive staining.et al. [et al. [The hot-spot method was used for semi-quantification of immunohistochemical expression scores. The expression of each protein was evaluated independently by two pathologists using the semi-quantitative classifications proposed by Yuan J, et al.  and Mont [et al. . The semEcoR I and AgeI sites as described by the manufacturer [The plasmids pcDNA3-HA-TOPK and pcDNA3 were provided by our laboratory. Five shRNA sequences were designed to knock down TOPK. These sequences are: 1. 5′-CCGGGGGAACTAGGCCACCTATTAACTCGAGTTAATAGGTGGCCTAGTTCCCTTTTTG-3′; 2. 5′-CCGGGAAGTGTGGCTTGCGTAAATACTCGAGTATTTACGCAAGCCACACTTCTTTTTG-3′; 3. 5′-CC GGGTAATGATCATTATCGAAGTGCTCGAGCACTT CGATAATGATCATTACTTTTTG-3′; 4. 5′-CCGGGCC TTCATCATCCAAACATTGCTCGAGCAATGTTTGGA TGATGAAGGCTTTTTG-3′;5. 5′CCGGGATTCCACA CATTAATCTTTCCTCGAGGAAAGATTAATGTGTGG AATCTTTTTG-3′. The sense and anti-sense oligonucleo tides were synthesized, annealed and cloned into the pLKO.1-TRC cloning vector at the facturer . A shMocThe human glioma cell lines U-87 MG, U-251, A-172, Hs 683 and the normal cell line HEK293T were purchased from American Type Culture Collection (ATCC), The cell lines were cultured in DMEM supplemented with 10% FBS at 37°C, 5% CO2 incubator following the procedures provided by ATCC and were used within 6 months of resuscitation.6) were seeded onto 10-cm-diameter dishes to 70–80% confluence and harvested in 200 μl RIPA buffer. 50–120 μg of protein was separated by 10%–12.5% SDS-PAGE and transferred to PVDF membranes. Then antibodies were used and antibody-bound proteins were visualized by chemiluminescence in triplicate.Cells (2 × 105) were cultured in 10-cm-diameter dishes and counted at different time points in triplicate, using a hemacytometer to count cell numbers and generate a growth curve.Cells (2 × 103 cells/per well into 6-well plates and cultured in 1ml of 0.33% BME agar containing 10% FBS, with an additional 3ml of 0.5% BME agar containing 10% FBS below. After the cells were cultured in a 37°C, 5% CO2 incubator for 5–10 days, at which time colonies were observed by microscopy.Cells were seeded at 8 × 10t-test was used to evaluate the data. Times for OS were defined from treatment initiation to date of death or last follow-up. The correlation between the level of TOPK or Ki67 and OS were assessed by log-rank test. In all tests, differences were considered significant at P < 0.05.All quantitative experiments were performed in triplicate at minimum. Statistical analysis was performed using Graphpad prism. Student's


TATAA motif in U3 promoter region from the majority of HIV-1 strains and a TAAAA motif in CRF01_AE; but also in LTRs from CRF22_01A1 isolates. Although LTRs from CRF22_01A1 specimens were assigned CRF01_AE, they contained two NF-kB sites instead of the single TFBS described in CRF01_AE. Also, as previously describe in clade C isolates, we found no C/EBP binding site directly upstream of the enhancer region in CRF22_01A1 specimens. In our study, one-third of CRF02_AG LTRs displayed three NF-kB sites which have been mainly described in clade C isolates. Overall, the number, location and binding patterns of potential regulatory elements found along the U3R might be specific to some HIV-1 strains such as clade F2, CRF02_AG, CRF01_AE and CRF22_01A1. These features may be worth consideration as they may be involved in distinctive regulation of HIV-1 transcription and replication by different and diverse infecting strains.Functional mapping of the 5’LTR has shown that the U3 and the R regions (U3R) contain a cluster of regulatory elements involved in the control of HIV-1 transcription and expression. As the HIV-1 genome is characterized by extensive variability, here we aimed to describe mutations in the U3R from various HIV-1 clades and CRFs in order to highlight strain specific differences that may impact the biological properties of diverse HIV-1 strains. To achieve our purpose, the U3R sequence of plasma derived virus belonging to different clades  and recombinants  was obtained using Illumina technology. Overall, the R region was very well conserved among and across different strains, while in the U3 region the average inter-strains nucleotide dissimilarity was up to 25%. The TAR hairpin displayed a strain-distinctive cluster of mutations affecting the bulge and the loop, but mostly the stem. Like in previous studies we found a  CCAAT/enhancer-binding proteins (C/EBP) and specificity proteins 1 (Sp1) were correlated with disease severity and neurologic impairment [The high diversity of HIV-1 has led to its classification into four groups named M, N, O and P. Group M which is the most prevalent worldwide has been further subdivided into at least nine genetically distinct subtypes or clades , several circulating recombinant forms (CRFs) and unique recombinant forms (URFs) , 3. Thuspairment , 17.During the life cycle of HIV-1, transcriptional regulation of the integrated provirus is a key step ahead of viral replication and expression. This early step is regulated by a synergistic interaction of host transcription factors and viral proteins that bind to specific targets in the 5’LTR of the HIV-1 genome. The HIV-1 5’LTR is a complex structure of approximately 640 bp in length with a high concentration of transcription factor binding sites (TFBS). It has been divided into the U3, R and U5 functional regions. The U3 region located upstream of the transcription start site was subdivided into the modulatory (nt -454 to -104), the enhancer (nt -105 to -79) and the promoter (nt -78 to -1) segments , 18. TheSixty five high titer virus derived from de-identified plasma specimens obtained from DUKE EQAPOL viral diversity program , and fivReferences and consensus sequences representing the first 1000 nucleotides of the HIV-1 genome from multiple subtypes and CRFs were downloaded from Los Alamos National Laboratory HIV database and aligned in MEGA version 7 software . RegionsReverse transcription was performed by using Superscript III First-strand synthesis System for RT-PCR (Invitrogen 18080–051). The cDNA was amplified by touchdown (TD) polymerase chain reaction (PCR) procedure with 2 rounds of amplification. Each amplification run was performed in a 50μl reaction volume using Promega PCR master mix  supplemented with 0.4μM of primer Miseq  sequencing of the gel purified PCR product was carried out at the core facility of the Food and Drug Administration . Briefly, concentration of purified amplicons was measured by using Qubit dsDNA BR Assay System  and (2 ng of DNA purified product was processed for NGS library preparation using the Nextera XT DNA Sample Preparation Kit. Specimens were run in the Miseq instrument using a MiSeq v2 kit (500 cycles) to produce paired-end reads of approximately 250pb. After automated cluster generation in MiSeq, the sequencing was processed and fastq files were generated.http://gene-regulation.com/pub/programs.html). A pairwise comparison in CLC Genomics Workbench was used to calculate the percentage of identity.All fastq files were imported into CLC Genomics Workbench version 9.0  using default option. Reads were paired and trimmed to obtain reads with an average Phred score ≥ 20. Contigs were generated using de novo assembly tools with the default options. The reads were mapped back to contigs using previous published parameters . For chaNear full length LTR sequences were not obtained for 11% (8/70) of selected specimens due to failure in amplification and /or sequencing. From the 62 specimens successfully sequenced, more than one contig was obtained for several specimens. Thus, we retrieved a total of 80 contigs that were subtyped as follows: clade B (n = 20), CRF12_BF (n = 5), clade C (n = 8), clade D (n = 10), clade F2 (n = 5), clade G (n = 3), clade A1 (n = 10), CRF02_AG (n = 9) and CRF01_AE (n = 10). For majority of the sequences analyzed, the subtype assigned to the 5’LTR of the HIV-1 isolate was concordant with its previously reported primary genotype –46, 52. Compared to HXB2, the U3 region was the most conserved in clade B with about 88% nucleotide identity versus less than 80% in average for all the other strains. The variability of the U3 region was less than 12% between sequences from the same strains and increased to an average of 23% between sequences from different strains. For most of our studied strains, the highest variability was found in the U3 modulatory segment with less than 75% of nucleotide identity. Nevertheless, the enhancer segment of clade C 5’LTRs share less than 65% of identity with the other strains. Although the number of mutations were higher in the modulatory segment than in the other segments of the U3 region, it also appears to be well conserved within sequences from the same HIV-1 strain (>85% of nucleotides identity). In the enhancer segment the intra-strain diversity varied from 2% (CRF01_AE) to 26% (CRF02_AG), while it was about 6% in the promoter segment. With an average nucleotide identity of 85%, this latter segment of the U3 region displayed few changes when sequences from different strains were compared.CTGATTGGC motif (nt -378 to -370) that was conserved in 56% of the sequences analyzed. Its variant AGATTGGCC was mainly observed in the CRFs as well as in clades G and D specimens, while AGACTGGCC was shown in 1/3 of the CRF02_AG specimens (AGAA(C/T)T was conserved in 55% of the sequences analyzed, while other variants such as ACAACT were mainly found in CRF01_AE and CRF22_01A1 viruses  was highly conserved (80%) with few variants such as the consensus G(G/C)GACCA found in clade C LTRs (AGATT(T/C) mostly found in F2, CRF12_BF, G and CRF02_AG LTRs, and AGATA(T/C) predominant in all the other studied strains. Further downstream (nt -333 to -327), the TGACCTT consensus for the reported AP-1(I), was found in all the strains studied except in CRF01_AE, CRF22_01A 1 and in clade A1 viruses. Indeed, the AP-1 (I) variant ATGTTTT was shared by 80% of CRF01_AE and 40% of CRF22_01A1 specimens. In the remaining sequence from CRF22_01A1 viruses, the consensus sequence TG(T/C)TTTG was found. In clade A1 LTRs, the variant TAACATT generates with its 3’end overlapping nucleotides a CATTTG motif (nt -330 to -325), that matches the E-box consensus CANNTG (Numerous transcription factors have been described in this portion of the U3 modulatory segment . Nucleotpecimens . The adj viruses . TFBS sue C LTRs . On the e C LTRs . AP1 (IIs CANNTG .GGATGG (nt -326 to -319) [GGGTGG in CRF02_AG LTRs and in some sequences from various other strains (TGCTACAAG(C/T) (nt -320 to -310) of the first half of the binding site for the negative regulator of transcription (NRT-1)[CCCAAGGGAAGTAG in clade C, to T(G/C)CAAAGGAGG(C/T)AG in clade D, and to TCCAAGAGAAGTAG in CRF22_01A1 isolates. These variants probably contain specific TFBS, as observed with the clade D included motif AAGGAG that matches a recognition site for Ets-2 [Contiguously, the GATA-like binding site to -319) , was mut strains . The conn (NRT-1), was main (NRT-1). In our n (NRT-1), 61, disor Ets-2 .(G/A)AAGGC (nt -283 to -277), fitting the canonical motif for GABP factors [AACAAAG motif (nt -274 to -268) conformed to the LEF-1/TCF consensus [AATAAA ((A/C)AGGAG (nt -270 to -265) which resembles a binding site for NFAT and Ets-2 factors [CAACTG (nt -260 to -255) perfect fit to the E-box /c-Myb consensus [GCTGCA similar to the recognition site for the AP4 factor [C/T)AACAG, predominant in sequences from CRF02_AG (100%), G (67%) and A1 (60%) specimens, was also found in clades C (38%) and B (20%).In the subsequent sequence, motifs such as  factors , was obsonsensus , was con [AATAAA . The ove factors , 65–67, onsensus , 70, was4 factor . On the (C/A)TTNCNN(C/A)A consensus described as the best fit for the binding of the C/EBP family of transcription factors [CTTGTTACA in HXB2 (nt -254 to -246) was reported as a C/EBPβ target (US3). In our study, the US3 consensus (TNNCACTTN) was found in at least 60% of clades B, D and G assigned LTRs, and the variant TTTNCNNCA mainly found in clade C LTR was also shown to be able to bind C/EBP proteins [CCTAT(G/A) (nt -244 to -239) that was found in non-clade G and non–CRFs isolates, the CCCATG variant was shown in LTRs subtyped as CRF01_AE only, while CCCATC was found in clade G and CRF02_AG only. Subsequently, we observed a CREB/ATF/AP-1-like binding site in several studied isolates, except for clade G and CRF02_AG. In these latter specimens, we found an E-box with the sequence motif CATCTG. Majority of sequences subtyped as F2 (60%), CRF12_BF (80%) as well as some clade B (25%) displayed a TATAA motif within nucleotides spanning this location (TGACCC(A/G) in clade B or TGA(G/T)GA(A/G) in clade A1 which resemble were a CREB/ATF/AP-1 target (nt -221 to -212).Although it displayed one nucleotide mismatch with the  factors –74, the proteins . In mostlocation . FurtherGAGAGAGAAGTG in HXB2 (nt -216 to -203), reported to encompass a second NFAT footprint in this segment of the U3 region [AAGAAGT(G/A)GAGA consensus was mainly found in clade B LTRs (45%) include C/EBP-like motif, while the (C/T)A(G/A)A(G/A)ARGTGGA variant predominant in CRF02_AG isolates includes a GATA-like motif. The highest variability of this NFAT footprint was found in clade A1 isolates in which we noticed that 50% of the variants contain a GAAACAT motif (nt -208 to -202) closely related to the LEF-1/TCF recognition site [TAGAGTGGA (nt -201 to -193) displayed some specific motifs in most of the HIV-1 non-B strains with some the variant containing an AP-1/ATF/CREB-like motif  and AGCTTGCTACA (nt -116 to -106) respectively [(A/G)GC(A/C)TTNC(A/G)NCA found in clades B (95%) and A1 (20%) was the only US2 variant conformed to the canonical binding site for C/EBP proteins. Non-canonical distinct variants were observed among non-B strains and some of them displayed up to three nucleotides mismatches with the consensus ((A/G)GCATTNANNCA was found in about 40% of A1 and 45% of CRF02_AG LTRs, while AC(A/G)CAGACANGC predominant in sequences from clade C (88%) was also found in LTRs from CRF22_01A1 specimens (40%). In our study, the sequence CGCAGACA included in this latter US2 variant was no longer strictly associated to clade C isolates as previously reported [AGCA(C/A)GAAAACA was only displayed by CRF01_AE specimens. In all the clade D and in some CRF02_AG LTRs, the US2 variant GA(A/G)CA(A/G)GCATTT includes the E-box motif CATTTG similar to what we reported earlier in clade A1 viruses (nt- 330 to -325); whereas the C/A)TGAGACAAGC(C/A)( consensus in CRF12_BF (80%) and F2 (60%) LTRs, contains a TGAGACA motif closely related to an AP-1/CREB/ATF target.Several consensus binding sites for transcription factors such as C /EBP, USF-1, Ets, LEF-1 and RBF-2 have been described within the nucleotide sequence from position -176 to the end of the modulatory segment , 77–79. ectively . In our onsensus . The conreported , 81, as CACATGGC in HXB2 (nt -166 to -159) contains the well described E-box consensus motif CAC(G/A)TG recognized by bHLHzip proteins such as USF-1 or TF3 [CANNTG consensus . The sequence downstream of the USF-1 motif (nt -153 to -146), includes a potential GATA motif with a high variability across our dataset (AGAAA(C/T)AT predominant in F2 and CRF12_BF LTRs ATCCGGA (nt -148 to -141) binding site for RBF-1/Ets-1 was found to be very well conserved across all our studied HIV-1 strains except for CRF01_AE isolates mainly in which it was changed to a G/A)TCC(T/A)GAC( variant as previously described [CCG(G/C)AGT and T/A)GAGTCC( (mainly CRF01_AE) in 79% and 14% of the analyzed LTRs respectively (TAA(A/G)(A/G)TA variant which includes a TATAA motif was predominant in G (67%) and CRF01_AE (60%) isolates. Nonetheless, this TATAA motif has been shown to be non-functional in CRF01_AE isolates [ACTGAGA was specifically found in F2 and CRF 12_BF sequence. In this study, we noticed that this insert reported as a partial RBE site motif [Contiguously, the consensus motif CGGA nt -8 to -141escribed , 41. Herectively . These tte motif .ACTGCTGA motif of the RBEIII site (nt -129 to -122) targeted by the RBF-2 complex [TGACACA predominant in sequences from non-B, D, G and non-CRF01_AE isolates, the AP-1-like sequence displayed some strains distinctive motifs (A(A/G)(C/A/T)TTTCTACA found in clades B (70%) and D (80%) LTRs, was the only in our dataset to be conformed to a reported binding site for C/EBP proteins. In all the other analyzed 5’LTR sequences, the following variants were found: GTT(G/A)CTGAC(A/G)A in A1, G, and CRF02_AG, GATTCTAA(G/A)AA in F2 and CRF12_BF, while GTTTCTAACAA which overlaps a TCF/LEF-like motif (CTAACTA) was found in CRF01_AE specimens  was slightly less conserved than NF-κB1 (nt -90 to -81) which showed a nucleotide similarity of about 91% with HXB2. Besides the consensus A(G/A)GACTTCC specifically found in CRF01_AE specimens, we observed within the NF-κB2 site some point mutations such as C9A in 20% of CRF02_AG, T6C in 40% of CRF12_BF LTRs and C10T in 40% of the LTRs from CRF22_01A1 isolates  was overall well conserved. This spacer is embedded within the AP-2 recognition sequence CCGCTGGGGA [CTTCCG of overlapping the NF-κB2 site of CRF01_AE viruses only; and with the low affinity to the variant CTTTCCG present in most of the other studied strains [ACTG scattered among several specimens and predominant in CRF22_01A1, 2) (G/A)CCT found in clade G, and 3) AAAG found in CRF12_BF and F2 LTRs only. This latter variant, by overlapping NF-κB2, led to a CTTTCCAAA motif conformed to the canonical consensus for the binding of C/EBP proteins ((C/A)TTNCNN(C/A)A). Furthermore, we noticed that the mutation of the AP-2 target in CRF02_AG appeared only in LTRs containing an extra NF-κB site.The NF-κB2/ NF-κB1 interspace motif GCTGGGGA , 88, and strains . In our 2 Sp III consensus motif GAGGCGTGGC (nt -77 to -68) was found in 20% of clade B LTR only, while specific variants, such as A(A/C)CGAGGCGT was found in clade D isolates, and AGGCG(T/G)GCCGA which include a GABP–like motif (GAAGGC) was found in F2 (60%) and CRF12_BF (80%) LTRs (TGTGGTGAGG (5T10T) predominant in LTRs from clades A1 and CRF02_AG viruses (≥ 80%) was also found in sequences from CRF22_01A1 (50%) and clade C (22%), and TGTGGCGAGG (5T) seen in all the LTRs from CRF01_AE was shared with clade B LTRs as previously observed [GGCG) and the frequent C5T/A mutation [2 Sp II motif TGGGCGGGAC (nt -66 to -57), mainly conserved in clades B and C sequences was mutated to AGTTGGGCGG in LTRs from clade A1 (90%) and CRF22_01A1 (100%) viruses. In our study, Sp II strain specific variants such as AGGAGTTGGGAGGG(G/A)C, TGGG, and (G/A)GGG(A/C)GGAGT were respectively observed in LTRs from clade G, CRF02_AG and CRF01_AE specimens ((G/T)GGGAGTGG(C/T) found in more than 90% of the sequences analyzed.Three sites named Sp III, Sp II and Sp I for the binding of Sp1 and related factors have been described along with the TATA box in this segment of the U3 region. In our study, the Sp binding sites displayed a high degree of intra-clade conservation and some inter-clade variability. Of the three, the sequence of the Sp III proximal to NF-κB1 displayed the highest variability . Thus, t0%) LTRs . For thiobserved , 90. Ovemutation . The HXBpecimens . These tGCGAGCCCT in HXB2 (nt -47 to -39) was conserved in clade B LTRs only  and CRF12_BF (80%) sequences, and the consensus TAACCCTGC observed in most of the other studied LTRs. We noted that these variants contained a potential E-box/c-Myb element. Further downstream, the TATA box motif (TATAA) flanked at its 3’ and 5’ end by an E-box motif, was extremely well conserved in all our strains studied except for CRF01_AE and CRF22_01A1 where it was changed to TAAAA in 60% and 80% LTR sequence respectively  and in CRF02_AG (33%) viruses. However, we noted that in these two HIV-1 strains, a motif similar to the 5’E-box was observed in the sequence adjacent to the US3 target located in the modulatory segment (nt -242 to -237). The 3’E-box consensus sequence CAGCTG reported as a binding site for bHLH factors such as AP-4 [CGCAGC variant was predominant (CTTTT(T/C)GCCT consensus (nt -15 to -6) was the major variant in non-G, non-A1 and non CRFs isolates (CTCGC(C/T)TCTT consensus was shared by clade A1 (30%), clade G (100%) and CRF02_AG (78%) LTRs, and G/T)CGCTTCTTT and the loop .The TAR element (nt 1 to 59) fold into a hairpin structure composed of a bulge, a loop and a stem. In our study, we found that all the constitutive elements of the TAR structure were affected by singles and clustered sets of mutations across different HIV-1 strains. In general, the stem displayed the highest variability followed by the bulge  reported as a AP-1 recognition site [CCGGGA variant in most of the CR01_AE viruses as previously reported [AGAGCTG variant of the loop was observed in clade D LTRs (40%) and in few sequences subtyped as A1 (10%) and B (5%). This latter variant of the loop converted the well conserved GGAGCTCTCG sequence reported as a NF-κB binding site [AGAGCTCTCG motif.In accordance with previous reports, we found a C24T change in most of the CRF02_AG and majority of clades D, F2, and G isolates , 40, bution site . The conreported , and alsing site , into a G (affecting the T13:A48 pairing) was found in most of clade B LTRs, this change was associated with G11T clade D or with 11T22G in CRF22_01A1 specimens  as well as the AP-1 target (nt 88 to 95) [ACTGCTTAAG sequence (nt 60 to 69) which includes a CREB-like recognition site [CTGCTTAAAG in CRF01_AE and to ACTGCTTGAAG in CRF22_01A1 specimens. In CRF12_BF (80%) and F2 (40%) LTRs, a C61T mutation transformed the CREB-like target ACTGCTTAA into an TTGGCTTAAA motif conformed to the canonical sequence for the binding of C/EBP proteins (TCAAGTAGT (nt 96 to 104) ending the R region. Indeed, this motif conserved in less than one-third of our analyzed sequences, was changed in a strains specific consensus such as CT(A/G)AG(C/T)AGT in clade C LTRs and AA(A)iGTGGTT is LTRs subtyped as CRF01_AE (TAAAGCAGTT found in CRF12_BF and F2 LTRs includes a perfect match to the consensus binding site for CREB factors [In the Poly-A hairpin (nt 61 to 105), the polyadenylation signal motif 8 to 95) , 97, werion site , was chaproteins . The higCRF01_AE . The var factors . This poTo the best of our knowledge, this study is the first to describe the diversity of the 5’LTR U3R region from diverse HIV-1 strains including the newly emerging CRF22_01A1 HIV-1 strain. The CRF01_AE subtype assigned to sequences from CRF22_01A1 may be explained by the close relation of the two strains. Indeed, it has been reported that the gag, nef and segments of the env genes of a typical CRF22_01A1 strain, can be subtyped as CRF01_AE , 52. TheThe U3R region of the HIV-1 5’LTR is involved in the control of both basal and trans-activated transcription of the HIV-1 genome though its interaction with several cellular transcription factors. Therefore, mutations affecting its nucleotide sequence might drive potentials differences in the biological property of diverse HIV-1 strains. Here, we found that the degree of genetic variability of the U3R can vary from very low to fairly high, depending on the segment analyzed. Nevertheless, it has been demonstrated that even minor changes in this region could influence the replication rate and the disease severity of HIV-1 infection , 55.TATAA-binding proteins [In the U3 modulatory segment, besides binding sites for the RBE III and Ets-1 sites that were conserved in almost all the LTRs, majority of the TFBS were distinctive of one or few HIV-1 strains. Several motifs aligned with the TFBS reported in HXB2 displayed a sequence that varied from the reported consensus. As most of the regulatory elements that were studied belonged to clade B LTRs, functions of these TFBS described in non-B strains remains to be determined. Although the affinity for C/EBP proteins of the motifs found in non-B, C and D is yet to be investigated, it has been suggested that the binding ability and functions of C/EBP proteins can be modulated by interaction with other factors such as ATF/CREB/ AP-1 , E-boxesproteins . This miproteins . Indeed,proteins . Thus, tTGACACA in CRF02_AG, CRF22_01A1, CRF12_BF, as well as in clades F2, C and A1 isolates may increase their capacity to establish HIV-1 latent infection compared to other strains. This predisposition may be affected by intra-clade variability due to the duplication of this variant within the MNFLP present in some of these specimens. On the other hand, the presence of an insert motif similar to the AP-1 activator of the p53 promoter might contribute to an increase reactivation of HIV-1 in F2 and CRF12_BF, as it has been shown that p53 could reactivate HIV-1 replication from its latency in U1 cells with upregulation/activation of host transcription factors such as AP-1 .Several TFBS have been shown to be able to activate or repress the transcriptional activity of HIV-1 depending on their affinity with the targeted factor and the spanning nucleotides , 111–113In our study, the important TATA box element as well as key TFBS such as NF-κB was fairly well conserved. In accordance with previous reports , 114–116In our study, the bulge residue T23, the loop as well as paired nucleotides below  and above (G26:C39 and A27:T38) shown to be important of the TAR activity were conserved in majority of the sequence analyzed. However, strain specific mutations within and outside these the features, might explained some differences in the transcriptional activity and replication rate between subtypes and CRFs . ChangesIt is known that the differential activity of the LTR due to the lack or mutation of a TFBS can be compensated by various mechanisms such as the gain of additional factors, the interplay between TFBS in close proximity or the delocalization of the TFBS. On the other hand, association of potential strains distinctive mutations in the U3R may contribute to strain-specific function of the 5’LTRs , 116. FoIn this study, we have reported distinguishing patterns within and outside the reported regulatory element in the U3R region from diverse HIV-1 strains. Due to the important role of this region in the control of the HIV-1 transcription, it is reasonable to suggest that strain specific variability greatly contributed to the effectiveness of replication and gene expression of different HIV-1 subtypes and CRFs. Intra and inter-clade variability of the U3R region should be further explored in future studies related to the replicative capacity and pathogenicity of HIV-1, as this may lead to a better understanding of phenotypic effects of HIV-1genetic diversity.S1 Table(XLSX)Click here for additional data file.