The introduction of Rotarix into the Belgian immunization program in June 2006 coincided with an increase of the relative prevalence of G2P[4] strains. However, the genetic composition of these persistent G2P[4] strains has not been investigated. Therefore, we have investigated the NSP4 gene of 89 Belgian G2P[4] strains detected between 1999 and 2013, covering both pre- and post-vaccination periods. The NSP4 genes were divided over seven separate clusters of which six were more closely related to animal than to human strains. The NSP4 genes that clustered more closely to animal DS-1-like strains were isolated after 2004–2005 and were found throughout multiple seasons. Complete genome sequencing of 28 strains identified several other gene segments that clustered more closely to animal than to human DS-1-like strains. These findings suggest that frequent interspecies reassortments may have played a role in the spread of G2P[4] rotaviruses in the post-vaccination period in Belgium. Group A rotaviruses are the most important etiological agent for gastroenteritis in children under 5 years of age346DS-1-like rotaviruses are less prevalent than their Wa-like counterparts and therefore relatively little is known about their long-term evolution81011131415In Belgium, Rotarix was introduced in 2006 and RotaTeq in 2007. Approximately 85% of all administered rotavirus vaccines in Belgium is Rotarix. The introduction of Rotarix coincided with an increased relative prevalence of G2P[4] rotaviruses, which was sustained throughout multiple seasons1920Based on a preliminary phylogenetic analysis of VP7, a selection was made of Belgian G2P[4] rotaviruses collected between 1999 and 2013. In total, 89 strains were selected and the complete NSP4 gene was sequenced. NSP4 sequences were obtained from every rotavirus season between 1999 and 2013 except for the 2002–2003 rotavirus season. Despite repeated attempts no reliable sequences could be obtained for the few available strains from 2002–2003, possibly due to low viral load.The 89 Belgian NSP4 sequences were compared with DS-1-like strains deposited in GenBank  and wereThe third largest cluster  contained five closely related Belgian G2P[4] strains detected between 2006 and 2010 that were probably the result of human-to-human transmission events and were intermingling with other human G2 and G12 strains isolated from the USA, Thailand, India, Pakistan and Australia. Close to this cluster at 1.0–1.6% nucleotide distance a group of bovine rotaviruses isolated in India was found as well as two bovine-like human P[14] strains, RVA/Human-wt/TWN/04-97s379/2008/G8P[14] and RVA/Human-wt/IND/N-1/2009/G6P[14] .RVA/Human-wt/BEL/BE21/2005/G2P[4] and RVA/Human-wt/BEL/BE23/2005/G2P[4] were relatively distantly related to other rotaviruses , but most closely to bovine-like human strains isolated from Denmark, Australia and Japan and animal strains from Ireland (between 5.3–9.4% nucleotide difference). RVA/Human-wt/BEL/BE38/2007/G2P[4] and RVA/Human-wt/BEL/BE41/2007/G2P[4]  were almost identical to RVA/Human-wt/IRL/CIT-H64/2010/G2P[8], and approximately 4.9–6.5% divergent from a group of bovine-like P[14] strains isolated from humans and a sheep in Italy, Belgium and Spain. Two distinct Belgian G2P[4] strains, RVA/Human-wt/BEL/BE80/2010/G2P[4] and RVA/Human-wt/BEL/BE105/2012/G2P[4] , did not cluster together with any other Belgian G2P[4] strains. RVA/Human-wt/BEL/BE80/2010/G2P[4] was most closely related to a bovine-like human strain isolated from Egypt, but still 7.1% divergent on the nucleotide level, followed by bovine and bovine-like human strains from Denmark and Slovenia showing 7.6% and 9.2% nucleotide difference, respectively. RVA/Human-wt/BEL/BE105/2012/G2P[4] was 1.4% different from RVA/Human-wt/RUS/Nov09-D2/2009/G2P[4], and 2.7% different from two bovine rotaviruses from India. None of the Belgian G2P[4] strains possessed a NSP4 gene segment that clustered closely to the RotaTeq vaccine strain .Based on the phylogenetic analysis, Belgian NSP4 genes were classified according to their most likely origin: human  or animal . The temporal distribution of the Belgian bovine and human NSP4 genes showed that before the introduction of Rotarix in Belgium bovine NSP4 genes were only sporadically detected  . The twoTwenty-eight strains were selected for complete genome sequencing based upon the genetic diversity of NSP4, the year of sample isolation and availability of sufficient stool sample . AnalysiThe phylogenetic tree of VP7 showed that Belgian strains were found in three clusters together with contemporary human G2P4] and G2P[6] strains isolated worldwide . One Bel and G2P[The phylogenetic analysis for VP6 showed that 25 out of 28 Belgian strains were closely related to each other (≤3.7%) and clustered together with other contemporary human G2P[4] strains . Three oThe Belgian G2P[4] rotaviruses were also found in human and bovine(-like) cluster for the VP1 gene segment. Similar to the VP6 phylogenetic tree, three out of 28 Belgian strains clustered more closely to bovine and bovine-like human strains than to typical human DS-1-like rotaviruses . BE38 anThe phylogenetic trees of VP2 and NSP2 displayed a similar clustering pattern  and 8 wiFor VP3, 24 out of 28 Belgian DS-1-like strains were most closely related to other typical human DS-1-like rotaviruses isolated all over the world . The othTaken together, the phylogenetic analysis for each gene segment revealed a wide variation in the genetic composition of Belgian DS-1-like rotaviruses. Twelve Belgian G2P[4] strains possessed a completely human DS-1-like genotype constellation, whereas 10 Belgian rotaviruses were most closely related to human DS-1-like strains for ten out of eleven gene segments. Four Belgian strains contained two animal derived gene segments. For BE38 and BE41 the majority of gene segments were more closely related to bovine than to human strains .A relatively high prevalence of G2P[4] strains has been observed in several countries where Rotarix is the mainly used rotavirus vaccine182419Currently, it is unclear whether the emergence of rotaviruses possessing a bovine-like NSP4 gene is a cause, a consequence or completely unrelated to the increased prevalence of G2P[4] rotaviruses after vaccine introduction. Rotaviruses containing a bovine-like NSP4 gene have been detected in other countries with rotavirus vaccination programs, such as Brazil, Australia, the USA and several countries in Sub-Saharan Africa1012151114Interspecies reassortment between bovine-like and human rotaviruses was frequently detected in Belgian G2P[4] rotaviruses, as we have found evidence for at least 8 independent interspecies reassortments in 7 different gene segments . For at least three clusters (two clusters in NSP4 and one in VP3) evidence was found for sustained human-to-human transmission as similar bovine-like genes segments were detected in multiple rotavirus seasons (up to five consecutive rotavirus seasons). However, our data also indicated that human-to-human transmission is rather rare for most of these human-animal reassortants, suggesting that most interspecies reassortments are probably dead-end infections. Although the three human-to-human transmission clusters are unlikely to have originated in Belgium, the interspecies reassortant dead-end infections may have originated in Belgium as they often do not cluster closely to any other known strains from the global collection . All experiments were performed according to the guidelines and regulations of the WIV. Stool samples were collected from patients hospitalized with acute rotavirus gastroenteritis, and the G-genotype and P-genotype of these samples was determined. To minimize redundant sequencing of identical NSP4 genes a preliminary phylogenetic analysis of G2 genotypes was conducted, which was used to select 89 samples that reflected the genetic diversity of the G2-genotype in every rotavirus season. Based on the phylogenetic diversity of the obtained 89 NSP4 sequences, 28 strains were selected for complete genome sequencing. In this way, our sample set is likely to reflect the overall genetic diversity of Belgian G2P[4] samples between 1999–2013, but caution should be taken with quantitative extrapolation as the samples were not randomly selected.Stool samples were collected at multiple hospitals in Belgium in the framework of the Belgian National Reference Center activities for rotavirus at the University Hospital Leuven, organized and approved by the WIV  in Belgium  according to manufacturer’s recommendations. The NSP4 gene segment was amplified with a one-step RT-PCR kit (Qiagen) using forward primer: 5′-GGC TTT TAA AAG TTC TGT TCC-3′ and reverse primer: 5′-GGW YAC RYT AAG ACC RTT CC-3′, and the following conditions: 50 °C for 30 min, 95 °C for 15 min, followed by 35 cycles of amplification , with a final extension of 10 min at 72 °C. The PCR amplicons were purified with EXO-SAP-it™ (Affymetrix) and subsequently sequenced using the same forward primer as used for the RT-PCR with the ABI PRISM™ BigDye Terminator Cycle Sequencing Reaction kit (Applied Biosystems).The complete genome sequences for eleven strains were determined using the primers as described in How to cite this article: Zeller, M. et al. Emergence of human G2P[4] rotaviruses containing animal derived gene segments in the post-vaccine era. Sci. Rep.6, 36841; doi: 10.1038/srep36841 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


IKBKG, CTLA4, NFKB1, GATA2, CD40LG and TAZ as well as previously reported pathogenic variants in STAT3, PIK3CD, STAT1, NFKB2 and STXBP2. AI/I manifestations were frequently encountered in PIDs, including at presentation. Autoimmunity/inflammation was multisystem in those effected, and regulatory T cell (Treg) percentages were significantly decreased compared with those without AI/I manifestations. Prednisolone was used as the first-line immunosuppressive agent in all cases, however steroid monotherapy failed long-term control of autoimmunity/inflammation in the majority of cases and additional immunosuppression was required. Patients with multisystem autoimmunity/inflammation should be investigated for an underlying PID, and in those with PID early assessment of Tregs may help to assess the risk of autoimmunity/inflammation.Primary immunodeficiencies (PIDs) are rare inborn errors of immunity that have a heterogeneous phenotype that can include severe susceptibility to life-threatening infections from multiple pathogens, unique sensitivity to a single pathogen, autoimmune/inflammatory (AI/I) disease, allergies and/or malignancy. We present a diverse cohort of monogenic PID patients with and without AI/I diseases who underwent clinical, genetic and immunological phenotyping. Novel pathogenic variants were identified in  Many PID conditions impair one or more immunological components required for immune system regulation, and AI/I manifestations are prevalent in PID cohorts across a range of monogenic PIDs.3Advances in the treatment and prophylaxis of infection have improved the quality of life and prognosis for patients with PID. Treatments such as immunoglobulin (Ig) replacement and antimicrobial agents are now highly effective at preventing and treating infections in many PIDs. However, with the improved management of infection, AI/I are becoming an increasing cause of morbidity and mortality.2 We evaluated the prevalence of AI/I manifestations in this cohort, and investigated whether any immunological, genetic or phenotypic features correlated with the development of AI/I. We also describe the treatments and outcomes for the AI/I manifestations across the cohort.To investigate the varied presentation and frequency of AI/I diseases in PID we recruited a cohort of monogenic PID patients as classified within the 2015 International Union of Immunological Societies.IKBKG, CTLA4, NFKB1, GATA2, CD40LG and TAZ. Previously reported pathogenic variants were identified in STAT3, PIK3CD, STAT1, NFKB2 and STXBP2 . Other organ-specific AI/I manifestations effected the gastrointestinal , pulmonary (n=3), hepatic (n=2), cutaneous (n=2) and renal (n=1) organ systems  of cases and AI/I disease in 38% (6/16) of cases. During follow-up, a further 3 participants developed AI/I manifestations, resulting in a total 56% (9/16) of the participants in the cohort experiencing AI/I disease that required medical intervention. Autoimmune cytopenias were the most frequently encountered AI/I complication , memory T cells  and effector T cells  was performed   was also performed  and those with AI/I (PID +AI/I) . Analysierformed .6, 7 Anaerformed . Treg pe + cells  but resu + cells .n=9; −1 per day resulted in an initial clinical response in 7/7 participants. All 7/7 participants subsequently required additional immunomodulation due to refractory/relapsed autoimmune cytopenias during prednisolone weaning. As second-line treatment for autoimmune cytopenias, 6/7 relapsed participants received rituximab and 1/7 was given Ig 2 g kg−1. Of the 6 participants who required rituximab, 4/6 needed a further long-term steroid sparing agent due to recurrence of autoimmune cytopenias post rituximab. Sirolimus (1–2.5 mg per day) was the most effective steroid sparing at maintaining remission for autoimmune cytopenias in 4/4 participants.Treatment inventions for AI/I manifestations were initiated based on clinical disease and symptoms. Prednisolone was used as first-line immunosuppression in all participants with AI/I n=9; . AutoimmGI AI/I manifestations partially responded to prednisolone in 4/4 participants. On weaning prednisolone GI disease returned and sirolimus did not adequately control GI AI/I in all 3/3 participants. Pulmonary disease was not controlled by prednisolone monotherapy in any of the participants, and radiological and lung function continue to decline. Liver AI/I responded to prednisolone in 2/2 participants, but relapsed shortly after withdrawal in 1/2 participants.−1 per day in combination with methotrexate (7.5 mg per week), which resolved the cutaneous AI/I .lineages . Therefo3 Our cohort demonstrates similar characteristics with 4/5 participants with predominantly antibody deficiencies suffering AI/I  a significant proportion (4/10) also suffered AI/I  suffered with Evans syndrome, colitis and granulomatous hepatitis. Autoimmune haemolytic anaemia and immune thrombocytopenia have both been reported in IKBKG deficiency, and colitis is a common inflammatory complication.14 Hepatic granuloma have been only been reported in hypofunctional IKBKG due to disseminated mycobacterial infection.13 A liver biopsy performed on P1 found no evidence of mycobacteria or other pathogens, suggesting that the granuloma are sterile and due to immune dysregulation. Larger studies of IKBKG deficiency patients will help to expand the reported phenotype in this condition.P1 (STAT3 p.G618D) and P3 (STAT3 p.V637M), both with hyper IgE syndrome due to loss-of-function variants in STAT3 and did not demonstrate any AI/I manifestations.15 Non-infectious complications are common in hyper IgE syndrome, as was the case in our participants  showed discordance for autoimmune diseases, with P4 having no AI/I disease and P5 suffering from AIHA and lymphocytic colitis. AI/I disease is frequent in PIK3CD GOF patients with 42% of patients having some form of AI/I in reported cohorts.18P4 and P5 (both CTLA4 p.A54T) and P7 (CTLA4 p.V40M) both suffered with multisystem AI/I.20 The clinical phenotype of CTLA4 insufficiency is heterogeneous with a wide range of organ-specific AI/I being described in the disease. Enteropathy is reported in up to 78% of cases and was present in both P6 and P7.19 Interstitial lung disease was also present in P6 and is reported in 66% of CTLA4 cases.19 Autoimmune haemolytic anaemia and immune thrombocytopenia are also commonly encountered at 28% and 35% of cases, respectively, and psoriasis 21% of cases,19 all of which were also present in P7.P6 (STAT1 p.R274Q GOF) did not develop any AI/I disease during follow-up. A large STAT1 GOF cohort reported AI/I in 37% of patients, with a slight preponderance in female patients.21 Thyroid disease was the most common AI/I reported (22%), but skin disease (10%) and autoimmune cytopenias (4%) were also frequently reported. Further reports have further broadened the phenotype of STAT1 GOF to include ‘IPEX-like’ presentations with multisystem AI/I.22 The janus kinase inhibitor ruxolitinib has shown promise in targeted AI/I in STAT1 GOF patients as a targeted immunosuppressive, as well as having benefits on chronic mucocandidasis.23P8.1 and P8.2 (NFKB1 p.S302Ffs*7) both suffered autoimmunehaemolytic anaemia, which is reported in NFKB1 haploinsufficient patients.25 Differing AI/I is observed in patients with NKFB1 mutations, ranging from antibody deficiency, Behcet-like disease, to an autoinflammatory phenotype.26P9.1 and P9.2 (NFKB2 p.R853*) suffered autoimmune alopecia, which is widely reported in patients with dominant negative NFKB2 variants but the renal disease that was present in P10 has not been reported in NKFB2 variants to date.28 The pituitary adrenal axis is often effected in NFKB2, but was normal in P10, although this is not believed to be an AI/I phenomenon; instead due to hypoplasia of the anterior pituitary.29 Further large-scale studies are needed to catalogue the frequencies and phenotype of AI/I in NFKB1 and NFKB2 patients.P10 , myelodysplastic syndromes, acute myeloid leukaemia and Emberger syndrome. Viral and mycobacterial infections are the most commonly encountered pathogens in GATA2 haploinsufficiency.30GATA2 deficiency usually causes cytopenias due to impaired bone marrow haematopoiesis and myelodysplasia, but the elevated levels of autoreactive peripheral CD38− CD21− B cells described in the periphery of GATA2 patients may increase the risk of antibody-mediated autoimmunity,31 and P11 (GATA2 p.T176P) suffered with recurrent Evans syndrome. Lung involvement with alveolar proteinosis occurs in GATA2 haploinsufficient patients due to impairment of alveolar macrophages, but lung fibrosis has also been reported recently and was observed in P11.33STXBP2 c.1247-1 homozygous) developed autoimmune neutropenia primary sclerosing cholangitis with dysgammaglobulinaemia, after initially presenting with haemophagocytic lymphohistiocytosis  presented with raised IgM, absent IgG and IgA, and necrotic pseudomonal tonsillitis. Stimulated CD4+ T cells showed absent expression of CD40L on the cell surface. CD40LG-deficient patients frequently develop autoimmunity, however P13 has no evidence of AI/I disease to date. At odds with reports of reduced Treg frequency in CD40LG patients, P13 has raised Tregs at 15.4%  has significant T-cell lymphopenia, which is one aetiology believed to predispose to AI/I disease in PID.4 The intrinsic apoptosis pathway is also defective in Barth syndrome due to impairment of mitochondria initiation of apoptosis.37 Despite these potential mechanistic risks for AI/I development,4 AI/I are not widely reported in Barth syndrome patients. Recently TAZ has been described to regulate Th17 and Treg development, and TAZ-deficient lymphocytes show impaired Th17 and increased Treg differentiation.38 This lymphocyte defect may protect Barth syndrome patients from AI/I disease.P14 .Whole-blood EDTA and lithium heparinised samples were collected from controls and patients with PID at a single centre. All participants with PID had monogenic diagnoses of PID listed in the International Union of Immunological Societies classification.+, CD4+ or CD8+, CD27+ and CD45RA+), memory , effector  T cells and Tregs  were analysed using FlowJo .Whole-blood lymphocyte immunophenotyping was performed by flow cytometry on a FACS Canto II . T-, B- and natural killer cell phenotyping was performed using CD45-PerCP-Cy5.5 (clone 2D1), CD3-FITC (clone SK7), CD4-PE-Cy7 (clone SK3), CD8-APC-Cy7 (clone SK1), CD19-APC (clone SJ25C1), CD16-PE (clone B73.1) and CD56-PE (clone NCAM 16.2). T-cell memory phenotyping: CD3-PerCP-Cy5.5 (clone 2D1); CD4-PE-Cy7 (clone SK3); CD8-APC (clone SK1); CD27-PE (clone L128); and CD45RA-FITC (clone L48). B-cell memory phenotyping: CD19-FITC (clone SJ25C1); CD27-APC (clone L128); and IgM-PE . αβ and γδ T cells were assessed using CD3-PerCP-Cy5.5 (clone 2D1), αβ TCR-FITC (clone WT31) and γδ TCR-PE (clone 11F2). Tregs were phenotyped with CD3-PerCP-Cy5.5 (clone 2D1), CD4-APC (clone SK3), CD25-PE (clone 2A3) and CD127-BV450 (clone HIL-7R-M21) . Flow cytometry plots for naive . Pneumococcal and tetanus IgG responses were assessed by commercial enzyme-linked immunosorbent assay according to the manufacturer’s instructions .DNA was extracted from EDTA blood samples using QIAamp DNA blood mini kit  according to the manufacturer’s instructions. DNA quality was checked by Nanodrop spectrometry . Genetic analysis was performed by whole-exome sequencing  using the TruSight One panel kit  and by single-candidate gene analysis (P2 and P3). Data were processed according to GATK best practice guidelines and aligned to GRCh37/hg19 reference genome. Variants identified in this study have been submitted to ClinVar NCBI.49 SIFT50 and Exome Aggregation Consortium,51 supported by Sapientia  and Ensembl.52 Variants pathogenicity was grading according to the American College of Medical Genetics criteria.53Variant interrogation was performed using in silico predictive tools Polyphen2,Participants were grouped into those with PID and AI/I manifestations (PID +AI/I) and those without AI/I (PID −AI/I). Both groups had similar characteristics, including mean age .54 Remission=complete normalisation of laboratory parameters and/or complete resolution of clinical symptoms. Partial response=improvement to near normal laboratory parameters with stabilisation of results and/or improvement in clinical manifestations . Relapse=little or no improvement in laboratory parameters and/or no improvement in clinical symptoms  and/or no improvement/progressive deterioration in imaging .Clinical responses were graded similarly to previous studies.U-test was used for analysis . P<0.05 was used as significance cutoff. Graphs display Mann–Whitney U Ranks (Because of skewed distributions of T-cell subsets , unpaire U Ranks . Data di U Ranks . Figures


Previously persistent papillomas disappeared within 2 months after the final vaccination. Coincident virologic clearance was confirmed by in situ hybridization and a failure of disease to recur after CD3 T cell depletion. Vaccination induced strong mE6 and mE7 CD8+ T cell responses in all mice, although they were significantly weaker in mice that initially presented with persistent warts than in those that spontaneously cleared their infection. A human papillomavirus 16 (HPV16)-targeted version of the DNA vaccine also induced L2 antibodies and protected mice from vaginal challenge with an HPV16 pseudovirus. Thus, MmuPV1 challenge of SKH1 mice is a promising model of spontaneous and immunotherapy-directed clearances of HPV-related disease.Mus musculus papillomavirus 1 (MmuPV1/MusPV1) induces persistent papillomas in immunodeficient mice but not in common laboratory strains. To facilitate the study of immune control, we sought an outbred and immunocompetent laboratory mouse strain in which persistent papillomas could be established. We found that challenge of SKH1 mice (Crl:SKH1-Hrhr) with MmuPV1 by scarification on their tail resulted in three clinical outcomes: (i) persistent (>2-month) papillomas (∼20%); (ii) transient papillomas that spontaneously regress, typically within 2 months (∼15%); and (iii) no visible papillomas and viral clearance (∼65%). SKH1 mice with persistent papillomas were treated by using a candidate preventive/therapeutic naked-DNA vaccine that expresses human calreticulin (hCRT) fused in frame to MmuPV1 E6 (mE6) and mE7 early proteins and residues 11 to 200 of the late protein L2 (hCRTmE6/mE7/mL2). Three intramuscular DNA vaccinations were delivered biweekly via IMPORTANCE High-risk-type human papillomaviruses (hrHPVs) cause 5% of all cancer cases worldwide, notably cervical, anogenital, and oropharyngeal cancers. Since preventative HPV vaccines have not been widely used in many countries and do not impact existing infections, there is considerable interest in the development of therapeutic vaccines to address existing disease and infections. The strict tropism of HPV requires the use of animal papillomavirus models for therapeutic vaccine development. However, MmuPV1 failed to grow in common laboratory strains of mice with an intact immune system. We show that MmuPV1 challenge of the outbred immunocompetent SKH1 strain produces both transient and persistent papillomas and that vaccination of the mice with a DNA expressing an MmuPV1 E6E7L2 fusion with calreticulin can rapidly clear persistent papillomas. Furthermore, an HPV16-targeted version of the DNA can protect against vaginal challenge with HPV16, suggesting the promise of this approach to both prevent and treat papillomavirus-related disease. There are over 200 known human papillomaviruses (HPVs)  that inf2–Mucosal HPVs have been categorized based on their oncogenic potential as low-risk HPV (lrHPV) types (typified by HPV6 and -11), which are associated with benign genital warts, and a dozen high-risk HPV (hrHPV) types , which are associated with malignant disease and have been targeted for vaccine development , 12. The13–+) cervical intraepithelial neoplasia grade 2/3 (CIN2/3) exhibited histological regression (E7 doubly mutated to eliminate oncogenicity (detox)  . Since H (detox) . Vaccina (detox) , 29. Findelivery , 31.Mastomys coucha, for which immunology studies are more challenging than for laboratory mice  allele and commonly used to study chemically induced papillomatosis, wound healing, and skin carcinogenesis. Although inbred HRS/J mice, which also carry Hr, have limited splenic CD4+ T cell function (+ and CD8+) responses similar to those of C57BL/6 mice , (ii) transient papillomas that spontaneously regress , and (iii) never any visible papillomas after challenge (n = 10).Previous studies in hairless S/RV/Cvi-ba/ba mice produced only transient disease after MmuPV1 challenge . No papifunction , SKH-1 mL/6 mice , 42. SKHL/6 mice . The timd spread , row 1. + T cells and potent antitumor immunity in mice  compared to the vaccinated group -fused HPV16 E6 and E7 are capable of generating HPV16-specific CD8 in mice , 43. Thedelivery –46, and immunity , 30, andimmunity , row 3. ed group .+ T cell epitopes induced by vaccination, splenocytes were harvested 3 months after the final vaccination and stimulated by using 20-mer overlapping peptide pools that together encompassed MmuPV1 E6 and E7. A strong MmuPV1 E6 CD8+ T cell response to amino acids 75 to 115 (p4) and a weaker MmuPV1 E7 CD8+ T cell response to amino acids 75 to 110 (p4) were identified in all hCRT-mE6/mE7/mL2-vaccinated mice , or those that never developed clinically apparent warts. Interestingly, mice with persistent warts at the initiation of vaccination mounted a significantly weaker (without multiplicity adjustment) MmuPV1-specific CD8+ T cell response for both E6 (P = 0.03) and E7 (P = 0.01) antigens than did the group of mice that had spontaneously cleared their warts prior to vaccination  technique with a probe against full-length E6/E7 mRNA (not shown)  mouse tail cross sections by using a highly sensitive chromogenic RNA t shown) . High let shown) , whereast shown) . For Mmung warts  and D. Cned cell  and F.FPE mouseE6/E7-targeted RNA in situ hybridization (ISH) technique appears to detect not only mRNA but also viral DNA, and thus, it is a highly sensitive system with which to detect the presence of MmuPV1 infection. Staining is strong in the upper levels of the papilloma as a large focal nuclear signal, which is consistent with the staining of viral genomes, whereas the fine-dotted signal in the lower epithelium is consistent with the E6/E7mRNA signal. Indeed, when treated with RNase A at room temperature for 30 min, the nuclear stain remains in the upper layers, whereas the finely grained staining toward the base of the epithelium is mostly removed (not shown).The MmuPV1 Following challenge, the majority of SKH-1 mice never develop visible warts. However, a subclinical reservoir of MmuPV1 might remain . Thus, 1in vivo electroporation to deliver the HPV16-targeted version of pNGVL4a-hCRTE6E7L2 DNA. Additional groups (n = 5) of mice were vaccinated with Gardasil-9 or left unvaccinated. DNA vaccination induced serum antibodies for all three vaccine antigens HPV16 E6, E7, and L2, as determined by an ELISA (P = 0.12) between these groups  . After tan ELISA . Nineteean ELISA , althouge groups .MmuPV1 challenge of mice of the immunocompetent and outbred SKH-1 strain shows considerable promise as a model of HPV disease in humans. Most genital hrHPV infections are asymptomatic and clear within 6 months to a year, but a subset of cases persist and can progress. Likewise, upon MmuPV1 challenge of SKH-1 mice, ∼20% develop persistent papillomas, and ∼15% show transient disease, but most never develop visible papillomas. Thus far, persistent MmuPV1-related papillomas have remained benign. For the study of therapy, for example, it would be beneficial to have a higher proportion of animals that develop persistent papillomas after challenge. Challenge of FVB/NJ mice with MmuPV1 in concert with exposure to UVB radiation, which is associated with systemic immune suppression, was recently shown to produce persistent papillomas . In addiin vivo electroporation three times biweekly, SKH-1 mice developed a strong MmuPV1 E6/E7-specific CD8+ T cell immune response as well as antibody responses to L2. This regimen was sufficient to drive the complete clearance of these otherwise stable papillomas. We note that in a phase I study of the related vaccine pNGVL4a-hCRTE7(detox) in women with HPV16+ CIN2/3, only 8/27 women demonstrated histological clearance postvaccination  gene have been used safely in 3 clinical studies involving pNGVL4a-Sig/E7(detox)/HSP70 , 64 as w70–+ T cell response may correlate with the clinical outcome after MmuPV1 challenge. Notably, systemic DNA vaccination was able to clear skin lesions, demonstrating that local vaccination is not required and that the T cells can home to the lesion site in the epithelium.While SKH-1 mice with persistent papillomas were able to completely clear their warts after vaccination with hCRT-mE6mE7mL2 DNA, their E6-specific CD8 T cell response was significantly lower after vaccination than the responses to hCRT-mE6mE7mL2 DNA seen in mice that cleared their papillomas or never developed papillomas prior to vaccination. In addition, we previously showed that the adoptive transfer of E6-specific T cells cleared MmuPV1 infection. These findings suggest that the ability to generate a robust E6-specific MmuPV1 CD8The onset of warts and the prevalence of nonmelanoma skin cancer in organ transplant patients suggest that patients carry reservoirs of subclinical or “latent” papillomavirus infection that can be reactivated by iatrogenic immune suppression –79. To t–In summary, we have established a new immunocompetent and outbred mouse model of papillomavirus infection that mimics clinical outcomes seen in patients with genital HPV infection. While we studied skin disease here, MmuPV1 also replicates at oral, anal, and vaginal sites, suggesting how this model might be further improved . Using mGuide for the Care and Use of Laboratory Animals (hr) and athymic nude mice [Crl:NU(NCr)-Foxn1nu] were obtained from Charles River Inc. BALB/cJ mice (strain code 000651) were obtained from The Jackson Laboratory.All animal studies were carried out in accordance with the recommendations in the  Animals  and withTC) were used for data analysis. The TC values were plotted against the copy number to generate a standard curve, which displayed an R2 value of 0.97 and a slope of −2.96. The assay showed linearity from 6 to 6 × 106 copies/reaction.As previously described , athymic8 MmuPV genome copies) supernatant was applied, followed by additional trauma by swabbing with endocervical brushes  and, finally, the application of an additional 20 μl of the wart supernatant. All infection challenges were accompanied by challenge of athymic nude mice as a positive control to ensure the viability of the wart extract and effective challenge.Initial trauma for infection challenge of SKH-1 mice was done by using 2 to 5 passes of a Dremel rotary drill (setting 2) until the epidermis was disturbed. Twenty microliters of the wart (1.5 × 10The DNA vaccines targeting MmuPV1 and HPV16 were generated by direct synthesis (GenScript). The DNA vaccine (15 μg of DNA in 20 μl PBS) was intramuscularly injected into the hind leg thigh muscle, immediately followed by electroporation with a pair of electrode needles inserted into the muscle area surrounding the vaccine injection site. Electrical pulses were administered by using a BTX electroporation generator . Eight pulses of 106 V were delivered with a 20-ms pulse at 200-ms intervals.in situ hybridization probes were developed to detect the full-length E6/E7 mRNA sequence of MmuPV1 by Advanced Cell Diagnostics for use with their RNAscope assay kit. CISH was performed according to the manufacturer's instructions for RNAscope2.0 as previously described . After washing, a horseradish peroxidase (HRP)-based amplification system was then used to detect the target probes, followed by color development with 3,3′-diaminobenzidine (DAB).Custom RNA escribed . Briefly+ T cell epitopes.A panel of 20-mer peptides, each overlapping by 15 amino acids, was generated by GenScript Inc. at a purity of ≥70% for both MmuPV1 E6 (25 peptides) and E7 (20 peptides). These 20-mers were then pooled into libraries of 5 peptides covering amino acids 1 to 40 (p1), 25 to 65 (p2), 50 to 90 (p3), 75 to 115 (p4), and 100 to 140 (p5) for E6 and amino acids 1 to 40 (p1), 25 to 65 (p2), 50 to 90 (p3), and 75 to 110 (p4) for E7. The 9-mer peptides, overlapping by 1 amino acid, for E6 amino acids 89 to 104 and E7 amino acids 76 to 95 were synthesized to further define their CD87 cells/well and incubated with the above-described peptide libraries of MmuPV1 E6 or E7 and a Golgi plug for 12 h. Cells were stained by using fluorescein isothiocyanate (FITC)–anti-gamma interferon (IFN-γ)  and phycoerythrin (PE)–anti-CD8a  antibodies and collected for flow cytometry analysis.Splenocytes were harvested from mice and immediately ground through 70-μm cell strainers  into RPMI growth medium with 10% fetal bovine serum (FBS) (Gibco). Splenocytes were divided into 10SKH-1 mice were administered 100 μg of monoclonal antibody to CD3  through intraperitoneal injection. Depletion was initiated by three daily injections followed by a once-weekly injection, which was maintained for 6 weeks.Escherichia coli. Proteins were then column purified by using Ni-nitrilotriacetic acid (NTA)-agarose  in 8 M urea. MmuPV1 L1 VLPs and HPV16 PsV were developed by using previously described protocols  vector and expressed in rotocols . For ELIBALB/cJ mice 2 weeks after the final DNA vaccination (described above) or Gardasil-9 (1/10 the human dose) vaccination were used for challenge. Mice were injected subcutaneously with 3 mg of medroxyprogesterone (Depo-Provera) and held for 4 days before vaginal challenge to synchronize their estrus cycles. Mice were vaginally challenged by using a mixture of 12 μl of HPV16 PsV and 28 μl of 3% carboxymethyl cellulose (CMC). Twenty microliters of the PsV-CMC mixture was injected into the vaginal tract before and after abrasion by an endocervical brush   while mice were anesthetized. Bioluminescence imaging to measure HPV16 PsV infection was conducted with a Xenogen IVIS 200 instrument  3 days after challenge, and images were acquired for 10 min. Region of interest (ROI) analysis was done by using Living Image 2.0 software.293TT cells were transfected by using TransIT-202 transfection reagent (Mirus), using 2.5 μg pNGVL4a-hCRT-E6E7L2 (lot number 35266A)  DNA/well


The bioactive signalling lipid sphingosine-1-phosphate (S1P) was discovered over 25 years ago as a regulator of cell proliferation. Since that time, S1P has been implicated across multiple cellular signalling systems, including cell survival, migration and differentiation. As a result, S1P has been associated with several health conditions and non-communicable diseases including cancer, inflammatory disorders, diabetes and atherosclerosis, much of which is attributed to its role in immune cell trafficking. S1P exerts its cellular effects through G-protein coupled receptors: S1P receptors 1-5 (S1PR1-5) [Levels of cellular S1P are modulated through the deacylation and phosphorylation of ceramide, the interconversion and subsequent ratio of these molecules is referred to as the S1P/ceramide axis. Many of the cellular signalling systems modulated by S1P are conversely affected by ceramide for example S1P promotes growth and inhibits apoptosis whereas ceramide has the opposite effects on these processes .More recently, an emerging role for the S1P/ceramide axis in the regulation of energy balance has been indicated in several studies. In 2014, Silva et al. demonstrated that S1P receptor 1 (S1PR1) was highly enriched in pro-opiomelanocortin (POMC) neurons in the hypothalamus of rats. The hypothalamus is a key sensor and regulator of nutrient signals  and POMCIn addition, chronic exercise increased both S1PR1 and STAT3 phosphorylation in the hypothalamus of middle aged mice, mirroring previous effects seen upon ICV injection of S1P in rats. ICV injection of S1PR1 activators also induced STAT3 phosphorylation in the hypothalamus and reduced food intake in middle aged rats .Da Silva also found that a high fat diet (HFD) resulting in obesity not only caused hypothalamic leptin resistance but also decreased S1PR1 protein levels in both mice and rats . This efConversely, calorie restriction (CR) has also been shown to alter S1P levels, in our recent study mice undergoing calorie restriction (CR) showed a hepatic increase in S1P and sphingomyelin whilst ceramide was decreased. Furthermore, this response was dose dependent, with the response becoming more pronounced with increasing CR (Figure Taken together, these findings suggest a significant role of the S1P/S1PR1 axis in the hypothalamus, adipose tissue and liver. In particular activation of hunger signalling pathways through S1P produces an anti-obesity effect in mice and rats, through increasing energy expenditure, increasing lipolysis, decreasing lipogenesis and food intake. The S1P/S1PR1/ceramide axis, in addition to downstream signalling pathways, seems to be inducible by both exercise and CR. These studies provide insight into how S1P analogues may be used as a therapeutic agent in the treatment of obesity, and the importance of further work to understand the organ specific effects of S1P.


P < 0.01). Age at diagnosis and estrogen receptor (ER) status were associated with SF3B1 mutations in all breast cancers (P < 0.01) and in luminal B and PR-negative subgroups (P < 0.01). The age at diagnosis and ER status combined had a higher sensitivity and specificity for predicting SF3B1 mutations than each factor alone. SF3B1 mutations are a poor prognostic factor in luminal B and PR-negative breast cancer patients. These mutations are significantly associated with age at diagnosis and ER status. SF3B1 mutations may therefore be a novel therapeutic target for breast cancer patients.The purpose of this study was to explore the relationship between SF3B1 mutations and the prognoses of patients with breast cancer. Clinical and SF3B1 mutation data from The Cancer Genome Atlas were analyzed. SF3B1 mutations were evaluated as prognostic factors in all breast cancer patients and specific subgroups through Cox regression and Kaplan-Meier analyses. We also investigated the relationship between traditional parameters and SF3B1 mutations. Receiver operating characteristics curves were used to analyze common risk factors for their sensitivity and specificity in predicting SF3B1 mutations. SF3B1 mutations were a poor prognostic factor in luminal B and progesterone receptor (PR)-negative breast cancer ( SF3B1 gene encodes subunit 1 of the splicing factor 3b, which is important for anchoring the spliceosome to the precursor mRNA [SF3B1 mutations is particularly high among the unique subtypes of myelodysplastic syndrome that are characterized by increased ring sideroblasts, in which mutation frequencies of 66.7–79% have been reported [SF3B1 knockout mice are embryonic lethal at very early developmental stages, whereas SF3B1 heterozygous knockout mice (SF3B1+/−) exhibit mild skeletal alterations [SF3B1 was found to be the second most frequently mutated gene in chronic lymphocytic leukemia (CLL) at 5–15%; SF3B1 mutations are less common in the early stages of CLL and become more prevalent in advanced disease where they tend to be associated with poor prognosis. The K700E mutation accounts for more than 50% of the variants observed, and additional codons 666, 662, and 625 were found to be hot spots for mutation [SF3B1 mutation are also found in solid tumors such as breast cancers (1.8%), pancreatic carcinoma (4%), uveal melanoma (9.7%), and endometrial cancers (percentages not reported) [SF3B1 mutations are reported to have better prognoses [The precise excision of introns from precursor mRNAs in eukaryotes is performed by the spliceosome , a macrosor mRNA , and is sor mRNA . The frereported , 7, 8. Smutation . In addirognoses , 12.Human breast cancers are heterogeneous, and patients have varying clinical outcomes based on their diagnostic and prognostic parameters. These include morphological assessment, basal-like phenotype, and the expression statuses of estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and progesterone receptor (PR). –15. The SF3B1 in spliceosomal component genes was the most common in breast cancers, and was detected in approximately 1.8% of cases. SF3B1 hotspot mutation K700E was detected in 16% and 6% of papillary and mucinous breast cancers, respectively [SF3B1 K700E mutations could lead to differential splicing. Alternative splicing of genes has also been shown to be associated with SF3B1 mutations in breast cancer, such as TMEM14C, RPL31, CRNDE, DYNLL1, MZB1, ICA1, RPL24, MTERFD3, OBSL1, ABCC5, UQCC, GUSBP11, ANKHD1, ADAM12, F8, and GAS8 [SF3B1 are associated with differential splicing activity in breast cancer; they found that patients presenting with mutations in SF3B1 tended to be older [SF3B1 mutations and prognoses in breast cancer patients.RNA splicing dysfunction may be associated with the pathogenesis of breast cancer, as Maguire et al. revealed that mutations in spliceosomal component genes occur in 5.6% of breast cancers. Mutation of ectively . These Swww.cbioportal.org/) revealed 114 SF3B1 K700E/R hotspot mutations across different types of carcinoma, 54 of which were in invasive breast carcinoma. Four patients with invasive breast carcinoma among 27 with various carcinoma types carried the SF3B1 K666E/M/N/Q/T hotspot mutation . There were 2061 and 1221 patients < 65 years and ≥ 65 years, respectively. The Nottingham prognostic index (N-Index) was only found in the METABRIC data sets. The N-Index was < 4.05 in 1075 patients and ≥ 4.05 in 910 patients. Eighty-one of 3817 patients (2.12%) carried SF3B1 mutation was not associated with OS in breast cancer patients; hence, subgroup analysis was performed to further investigate the clinical value of SF3B1 mutations in these patients . In the PR-negative group, the SF3B1 mutation was also significantly associated with OS . Kaplan-Meier survival analysis showed that the SF3B1 mutation was not an independent predictor for OS in breast cancer patients overall (log-rank test: P = 0.385). In the luminal B and PR-negative groups, however, the SF3B1 mutation was a significantly independent prognostic factor for OS   . Age and ER status were significantly associated with SF3B1 mutation specifically in the luminal B and PR-negative subgroups as well (P < 0.02).Among 11 common prognostic factors investigated, univariate analysis showed that age, ER status, PR status, menopausal state, PAM50 and claudin-low subtype, and breast cancer type were significantly associated with SF3B1 mutation. For age at diagnosis, the ROC curves showed a sensitivity of 65.8% and specificity 64.0%, with an area under the curve (AUC) of 0.639 . For ER status, the ROC curves showed an AUC of 0.602 . When age at diagnosis and ER status were assessed for their combined ability to predict SF3B1 mutation, the ROC curve showed a higher sensitivity and specificity of 66.2% and 69.0%, respectively, with an AUC of 0.690 .Receiver operating characteristic (ROC) curves were generated for all patients with breast cancer to identify the sensitivity and specificity of age at diagnosis and ER status in predicting P = 0.051). In the PR-negative group, the ROC curve for age at diagnosis showed a sensitivity of 73.9% and specificity of 67.1%, with an AUC of 0.685 . As for ER status, the ROC curve showed an AUC of 0.702 (95% CI: 0.612–0.793) (P = 0.001). When age at diagnosis and ER status were used in combination to predict SF3B1 mutation, the ROC curve showed a higher sensitivity and specificity of 82.6% and 70.8%, respectively, with an AUC of 0.756   are linked to cancer , 16, 17.patients , 18–20. patients . A studypatients . The higfeatures , 24. TheMAP3K1 and GATA3 mutations were associated with longer survival, and TP53 mutations with shorter survival, in ER-positive patients but not in ER-negative patients. Conversely, PIK3CA mutations were associated with shorter survival in ER-negative patients, but not in ER-positive patients. Their study also showed associations between mutations in driver genes and clinicopathological parameters; for example, mutations in PIK3CA, GATA3, KMT2C, and CBFB were associated with lower grade tumors in ER-positive patients, while TP53 mutations were associated with higher grade tumors. GATA3, CBFB, CDH1, KMT2C, and SF3B1 mutations were also associated with age at diagnosis [A study by Pereira et al. showed that mutations in driver genes were associated with the prognosis of breast cancer patients. For instance, iagnosis .SF3B1 mutations in breast cancer patents. SF3B1 mutations were not significantly associated with survival outcome in breast cancer patients overall. However, these mutations were associated with worse outcomes in PR-negative and luminal B patients. In the PR-negative patient subgroup, SF3B1 mutations were associated with age at diagnosis, ER status, and histologic grade; in the luminal B subgroup, SF3B1 mutations were associated only with the age at diagnosis. The multivariate logistic regression model revealed that SF3B1 mutations were associated with age at diagnosis and ER status in all patients as well as in the PR-negative subgroup.In our study, we further analyzed the clinic value of SF3B1 mutations were associated with worse outcomes in the PR-negative and luminal B subgroups, we analyzed whether these mutations were significantly associated with the age at diagnosis and ER status with the hypothesis that the age and ER status can predicting the existence of an SF3B1 mutation. We found that, when age at diagnosis and ER status were assessed in combination, the prediction of SF3B1 mutations had a slightly higher sensitivity, specificity, and AUC than the age at diagnosis in all patients. In the PR-negative subgroup, age and ER combined had a higher sensitivity (82.6%), specificity (70.8%), and AUC (0.756) in terms of predicting SF3B1 mutations than age alone.Because SF3B1 K700E mutations are associated with differential gene splicing in breast cancer, including of TMEM14C, RPL31, CRNDE, DYNLL1, ICA1, RPL24, and MTERFD3. Cell lines carrying the SF3B1 mutation were sensitive to the SF3b complex inhibitor spliceostatin A, which suppressed tumor growth. Hence, the spliceosome SF3b complex may be a potential therapeutic target [Maguire et al. showed that c target , 25.SF3B1 mutations are frequently found in breast cancer patients, and that they are poor prognostic indicators in PR-negative and luminal B breast cancer patients. SF3B1 mutations were found to be significantly associated with the age at diagnosis and/or ER status in PR-negative and luminal B breast cancer patients. Moreover, combining the age at diagnosis and ER status could better predict the existence of SF3B1 mutations. As demonstrated by spliceostatin A, the SF3b complex may be a novel therapeutic target for breast cancer patients with SF3B1 mutations.In conclusion, our analysis of TCGA revealed that www.cbioportal.org/). We enrolled 2059, 1105, 103, and 100 invasive breast carcinoma patients from the METABRIC, TCGA , broad, and Sanger datasets, respectively, in our study. Our institutional review board approved this study, which was performed according to the principles of the Declaration of Helsinki. There were 81 patients with SF3B1 mutations among 3817 patients with invasive breast carcinoma. Breast cancers were divided into four types: invasive breast carcinoma (979), breast invasive ductal carcinoma (2316), breast invasive lobular carcinoma (347), and breast mixed ductal and lobular carcinoma (122). Fifty-three patients were not classified because of the rarity of their tumor types, including adenoid cystic breast cancer, phyllodes tumor of the breast, and unclassified breast cancer.We analyzed SF3B1 mutation data and clinic data of breast cancer patients from TCGA database ; these cutoff values were based on OS.The following parameters were investigated for their role in the prognosis of patients with breast cancer: age at diagnosis, N-index, ER status, PR status, HER2 status, menopausal state, PAM50 and claudin-low subtype, histological grade, breast cancer type, tumor stage, and primary tumor laterality. Additionally, SF3B1 mutation as independent prognostic factor was evaluated. The Kaplan-Meier method was used to estimate survival function of each variable that was found to be a significant factor. Univariate analysis was performed to identify significant independent prognostic factors for OS. The Cox proportional hazards model was used to estimate the HRs and CIs of potential prognostic factors in all patients, including that of SF3B1 mutation in the aforementioned subgroups. Patients who were positive for significant prognostic factors were subjected to Kaplan-Meier analysis to clarify the role of the SF3B1 mutation on their survival. A logistic regression model was used to analyze associations between the investigated prognostic factors and SF3B1 mutation. Variables identified via univariate analysis (α = 0.25) were subjected to multivariable logistic regression analysis to determine their association with SF3B1 mutations. ROC curves were constructed to predict the sensitivity and specificity of SF3B1 mutations according to the AUC and 95% CI. We also assessed the combined effects of age at diagnosis and ER status on the occurrence of SF3B1 mutations. All analyses were performed using SPSS for Windows, version 20.0 .All dichotomous variables were analyzed by using a Cox regression models in all patients. Next, we divided all dichotomous prognostic factors into two subgroups in which the


PAX8 is associated with thyroid dysgenesis. We aim to identify and characterize PAX8 mutations in a large cohort of congenital hypothyroidism(CH) from thyroid dysgenesis in Chinese population.Based on mutations in PAX8 mutations by sequencing the whole coding regions of PAX8 on genomic DNA isolated from blood. Cell transfection assays using various vector constructs and induced mutagenesis as well as electrophoretic mobility shift assays were used to investigate the effects of selected mutations on the transcribing and binding activities of PAX8 at the promoters of target genes for thyroglobulin (TG) and thyroperoxidase (TPO).We screened 453 unrelated Chinese patients with CH from thyroid dysgenesis for PAX8 mutations were found, yielding a mutation prevalence of 5/453 (1.1%). We selected two mutations in the critical paired domain of PAX8 and generated mutants D94N and G41V. We demonstrated G41V was unable to bind the specific sequence in the promoters of TG and TPO and activate them. D94N could bind to TG and TPO promoters and normally activate the TG promoter transcription but not the TPO promoter transcription. We also demonstrated a dominant negative role of the PAX8 mutants in impairing the function of the wild-type PAX8.Five PAX8 mutations in CH in Chinese population. The study specifically demonstrated the role of novel mutations D94N and G41V in impairing the function of PAX8, providing further evidence for genetic PAX8 defects as a disease mechanism in CH.We for the first time documented the prevalence and characterized the function of  Congenital hypothyroidism (CH), characterized by elevated thyroid-stimulating hormone (TSH) resulting from reduced thyroid function at birth, is the most common congenital endocrine disease, with an incidence of 1 in 2000–4000 newborns . MutatioPAX8 in thyroid dysgenesis has drawn particularly considerable attention. PAX8 is a paired domain-containing protein in the mammalian PAX protein family of transcription factors and encoded by a single gene on chromosome 2q12–q14, which consists of 12 exons [PAX8 being a thyroid-specific transcription factor that regulates thyroid-specific genes, such as thyroperoxidase (TPO), thyroglobulin (TG), and sodium/iodide symporter (NIS), by binding to specific promoter regions via the highly conserved 128-amino acid paired domain [As a major specific regulator of the thyroid gland, the role of 12 exons . In addi12 exons , 12. The12 exons . PAX8 isd domain .Pax8 knockout resulted in thyroid aplasia in mice [PAX8 mutations were associated with various forms of thyroid dysgenesis in humans [PAX8 defect in thyroid dysgenesis. Most of the inactivating PAX8 mutations in thyroid dysgenesis have been located in the mutational hotspots of exons 3 and 4 of PAX8—corresponding to the DNA-binding paired domain [PAX8 mutations with variable penetrance and expressivity [PAX8 mutations vary considerably [Homozygous  in mice  and heten humans , 17, gend domain –23. Autoiderably .PAX8 in thyroid dysgenesis, its genetic molecular pathogenesis has not been uniformly documented and characterized in different ethnic populations, including the large Chinese population. The aim of the present study was thus to screen for PAX8 mutations in a large cohort of Chinese children with thyroid dysgenesis-caused CH and selectively functionally characterize the novel mutations identified here and in a previous study of ours [Even given this knowledge on the role of  of ours .PAX8 in this cohort of patients. One was a missense mutation in one patient that predicted an aspartate to asparaginate substitution at codon 94 in exon 4 (c.280G>A/p.D94N) of PAX8  [We expanded the analysis from our previous small cohort  to the c8 Figure . Analyse/p.G41V) ; its locThe patient with the D94N mutation was a male subject. He was born at 38 weeks of gestation from unrelated parents who had no family history of thyroid disease. His birth weight was 2,850 g with no abnormal physical findings. Neonatal screening at 6 days of age revealed a high TSH (267.62 μIU/ml). At 26 days of age, his body weight was 3,500 g and height/length 54 cm, with a persistently elevated serum TSH (280 μIU/ml) and low FT4 (2.06 pmol/L) and free T3 (FT3) (1.78 pmol/L). A 99mTc scan confirmed thyroid agenesis, which prompted the diagnosis of CH. Levothyroxine (L-4) therapy was instituted with the dose adjusted appropriately to maintain serum TSH, FT4, and FT3 within normal ranges. He was found to have elevated TSH upon brief withdrawal of L-4 at 3 years of age, confirming permanent hypothyroidism. At the writing of this paper, the patient was 6 years old with normal intellectual development.PAX8 mutation was a female subject, born at 38 weeks of gestation from unrelated parents. Her birth weight was 3,600 g and length 51 cm. She was diagnosed with CH by neonatal screening revealing a high TSH (120.5 μIU/ml). On re-evaluation at 14 days of age, she had a persistently elevated TSH (176.1 μIU/ml) and low FT4 (6.5 pmol/L). A 99mTc thyroid scan showed a eutopic normal-sized bilobed thyroid gland  with weak fixation. She received appropriate L-T4 therapy and her development was normal; at 2 years of age, she was 80-cm tall and weighed 1,750 g, with normal intellectual development.The patient with the G41V As G41V and D94N were in the critical functional region of the paired box domain of PAX8, we explored the effects of these mutations on the function of PAX8 protein. We successfully expressed the wild type (WT) and mutant PAX8 in Hela cells. As shown in Figure TPO and TG. Specifically, HeLa cells were transfected with expression vectors encoding WT or mutant PAX8, together with reporter gene constructs containing TPO or TG promoters placed upstream of a coding sequence for the luciferase reporter gene. Compared with WT PAX8, luciferase activities were significantly lower in cells co-transfected with the TPO promoter and G41V or D94N (P < 0.05) , which could be only partially recovered with additional WT PAX8 cotransfection to a level that is still lower than that achieved with WT PAX8 (P < 0.05). In contract, the ability of D94N to promote the TG promoter-driven reporter gene expression was intact. These results thus demonstrated that the ability of G41V and D94N to activate the TPO promoter was severely impaired, but only the ability of G41V, but not D94N, to activate the TG promoter-driven reporter gene was impaired. These results were also consistent with a dominant negative effect of the two novel PAX8 mutants on WT PAX8.We next examined the effects of G41V and D94N mutants on the transcriptional activities of the target genes ) Figure . LuciferTPO and TG promoters, respectively, were synthesized and labelled with Alexa Fluor 680. The in vitro expressed PAX8 proteins were incubated with the labelled oligonucleotides, separated via PAG, and visualized. We found that mutant D94N, like WT PAX8, could normally bind to the specific DNA sequence in the TPO promoter while mutant D41N lost its binding ability , and PAX8 c.-26G>A in CH. Together with the other two novel PAX8 mutations, G41V and R31H, which we had identified in a portion of this cohort of CH [PAX8 mutations in the Chinese patients with TD/CH is 1.1% (5/453). This is consistent with the reported low prevalence of PAX8 mutations in thyroid dysgenesis in other ethnic populations and another Chinese population [PAX8 mutations was found to be, on average, 1.0%, ranging from 0 to 3.4% [PAX8 pathogenic variants was 2.38% among patients with CH in another study in Chinese population [Consistent with the present study on Chinese patients, a number of mutations have been reported in the coding regions of ulations , 27–29. rt of CH , the ovepulation , 26, 30. to 3.4% . The prePAX8 that encodes the protein homeodomain within the paired domain. Based on the published crystal structure generated using paired domain-DNA complexes [TG and TPO. This provides a structural basis for our observation that the two mutations indeed severely impaired the transcribing abilities of PAX8 at the target genes TPO or TG.The G41V and D94N mutations identified in our study are located in the segment of omplexes , 31, theTPO and TG response elements, yet failed to activate TPO promoter-driven reporter gene transcription, and exhibited a dominant negative effect on WT PAX8. However, D94N could activate the TG promoter normally. Differences between TPO and TG promoter structures may explain this disparity in the effect of the PAX8 mutant on the transcriptional activities of the two genes. Binding of G41V to the TPO and TG promoter binding sites was both impaired, accompanied by expected impaired transactivation of the target gene promoters. Moreover, the ability of WT PAX8 to transactivate the target gene reporter was diminished by mutant G41V co-transfection, exhibiting a dominant negative effect on WT PAX8. It appears that D94N has similar binding and transactivation properties as S48F; the latter is a paired domain mutation of PAX8 with dominant negative activity and does not transactivate a PAX8-responsive promoter [TG and TPO promoters, although it has no dominant negative effect on WT PAX8 [TPO and TG genes [in vitro, could be due to diminished transport of PAX8-D94N and PAX8-G41V into the nucleus. The possibility should be further confirmed by immunofluorescence in the future study. And The transcription of thyroid-specific genes is dependent on multiple thyroid-specific transcription factors, it will be more helpful if the experiments are repeated in thyroid cell lines.Interestingly, D94N showed normal binding to promoter . Another WT PAX8 . Our resTG genes , 24, 30.TG genes , 31, butTG genes , 30, 33.TG genes . Hence, PAX8 mutations in a large cohort of unrelated Chinese CH patients with thyroid dysgenesis and identified several novel PAX8 mutations. We extensively functionally characterized two selected mutations G41V and D94N and demonstrated their significant impairing effects on the binding or activating abilities of PAX8 at the promoters of the target genes TG and TPO, establishing their pathogenic role in the development of CH from thyroid dysgenesis. This is the first report on the prevalence documentation and functional characterization of PAX8 mutations in a large cohort of patients with CH from thyroid dysgenesis in Chinese population; it is also the largest study on PAX8 mutations in CH in general. The results also provide new evidence for a dominant negative role of PAX8 mutants as a disease mechanism in the development of CH from thyroid dysgenesis.In conclusion, we have investigated Atotal of 453 thyroid dysgenesis patients  diagnosed during neonatal screening for CH in Linyi, Qingdao, and Yantai in Shandong Province, China from 2009 to 2012 were enrolled. TSH  and free T4 (FT4)  were determined using standard electrochemiluminescence assay. A diagnosis of CH was established by abnornmally high TSH and low FT4. Thyroid location and size were determined by 99mTc thyroid scan and thyroid ultrasonography, based on which the patients of thyroid dysgenesis were divided into four groups: eutopic and normal-sized thyroid , eutopic hypoplasia , agenesis , and ectopic . Thyroid dysgenesis patients included in this study were all unrelated individuals with no other endocrine abnormalities, growth failure, or neurological deficits. Blood samples were collected with informed written consent from patients’ parents/legal guardians. The research project was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University.PAX8  were amplified by PCR using conditions and primers described previously [Genomic DNA from blood samples was isolated using the phenol-chloroform extraction method. Gene fragments covering the coding sequence, the flanking intronic sequence, the 5’UTR, and the 3’UTR of PAX8 mutations, the full coding sequence of PAX8 was amplified by reverse transcription-PCR from human thyroid mRNA using primers 5′-GGGGTACCATGCCTCACAACTCCATC-3′ and 5′-GCTCTAGACTACAGATGGTCAAAGG-3′. PCR products were cloned into the pCDNA3.1 expression vector  using KpnI and XbaI restriction sites introduced into the primers to obtain the wild-type (WT) PAX8-pCDNA3.1( PAX8WT-pCDNA3.1). Mutations were introduced using the Quick Change Mutagenesis kit , following the manufacturer's protocol, and the following primers: PAX8 D94N-pCDNA3.1 construct, 5′-GTGGAGAAGATTGGGAACTACAAACG-3′ and 5′-TCCCAATCTTCTCCACCACCTTGGGG-3′; and PAX8 G41V-pCDNA3.1 construct, 5′-ACCTGGCCCACCAGGTTGTAAGGCCC-3′ and 5′-ACCTGGTGGGCCAGGTCTACGATGCG-3′.For functional characterization of TPO promoter was PCR-amplified using primers 5′-GGGGTACCGAGCTGCACCCAACCCAATCCT-3′ and 5′-CCCAAGCTTAGTAATTTTCACGGCTGTAACT-3′. To create TG-Luc, a 305-bp fragment containing the human TG promoter was PCR-amplified using primers 5′-GGGGTACCCTTGAGCCTGTTCCCTCCAAAG-3′ and 5′-CCCAAGCTTTTCCTGGCCCTTCCTGGGAGGA-3′. These fragments were cloned via the introduced KpnI and HindIII restriction sites into pGL3-basic  to obtain TPO prom-pGL3basic and TG prom-pGL3basic. All constructs were confirmed by sequencing.To create TPO-Luc, a 418-bp fragment containing the human HeLa cells were maintained in DMEM supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum under humidified 5% CO2 at 37°C. Cells were plated in 48-well plates 24 hours before transfection. Once the cells reached 90% confluency, transfection using WT or mutant PAX8 was performed using lipidosome 2000 (Invitrogen), following the manufacturer's instructions. For transactivation assays, cells were cotransfected with TG-luc or TPO-luc (250 ng), the internal control vector pRL-CMV (Promega), and PAX8 (200 ng) (WT or mutant). Cells were harvested 48 hours later and lysed in 65 μl lysis solution. To determine the effect of mutant PAX8 on WT PAX8, equal amounts of each mutant vector was cotransfected with WT and reporter vectors. Protein extract (10 μl) was analyzed sequentially for firefly and Renilla luciferase activities using a dual-luciferase reporter assay system (Promega). The total amount of added plasmid was kept constant. The ratio between measured firefly and Renilla luciferase activities was expressed relative to the ratio obtained in cells transfected with reporter and empty expression vector (pcDNA3.1) only. All transfections were performed in triplicate and three independent experiments were similarly performed.Total RNA was isolated from transfected HeLa cells using Trizol agent. Reverse transcriptase reactions were performed in a reaction mixture of 50 ng total RNA, 50 μM oligo(dT)18, and RNase-free water to a final volume of 6 μl. Reaction mixtures were incubated at 70°C for 10 min and then 2 min on ice. This was followed by addition of 10 U RNase inhibitor, 100 U RTase M-MLV, 1x M-MLV buffer, and dNTPs (each at 0.5 mM) (Takara). The reaction mixtures were incubated first at 45°C for 1 hour and then at 70°C for 15 min. Real-time PCR for each cDNA was performed in triplicate in a 10-μl reaction mixture containing 2.5 ng cDNA, 0.2 μl each forward and reverse primer (both 10 mM), and 5 μl SYBR Green PCR master mix (Applied Biosystems). Reactions were incubated in 96-well plates on an Applied Biosystems 7900HT Fast Real-Time PCR system at 94°C for 10 min, followed by 40 cycles of 95°C for 15 s and 65°C for 45 s. The 2-(ΔΔCt) method was used to determine relative quantitative levels of individual cDNAs. GADPH was used for normalization and values were expressed as relative difference compared to corresponding controls. The primers used were: forward, 5′-ACTACAAACGCCAGAAACCCTACCA-3′ and reverse, 5′-TGAATGGTTGCTGCACTTTGGTCC-3′.Cell lysates were obtained using protein extraction reagent  and protein concentrations determined using the bicinchoninic acid method  with BSA for creating standard curves. Samples containing 30 μg protein were separated on 12% SDS-PAGE gels. Western blotting was performed using rabbit anti-PAX8 primary antibody  and horseradish peroxidase-labeled mouse anti-rabbit secondary antibody.TPO (TGATGCCCACTCAAGCTTAGACAG) and TG (CACTGCCCAGTCAAGTGTTCTTGA) were synthesized and 5′-end labeled with Alexa Fluor 680. Binding reactions (10 μl) containing 1 μg protein, 150 ng poly (dI-dC), 500 ng salmon sperm DNA, 5 mM DTT, 0.5% Tween 20, 10 mM Tris, and 50 mM NaCl (pH 7.5) were preincubated at room temperature for 10 min. Double stranded oligonucleotides were then added to a final concentration of 100 nM and incubated at room temperature for 20 min. One μl gel loading buffer  was then added and samples were loaded on 4% native polyacrylamide gels (PAG), supplemented with 50 mM Tris pH 7.5, 200 mM glycine, and 2 mM EDTA. TBE buffer  served as the running buffer. PAGs were scanned and visualized using the Odyssey Model 9120 and Odyssey v2.1 software .Electrophoretic mobility shift assay (EMSA) was performed to determine DNA-binding properties of WT and mutant PAX8 using nuclear extracts from transfected HeLa cells. Annealed synthetic oligonucleotide sequences


The gf mechanism of C9ORF72 has been studied in various animal models but not in C. elegans. In the present study, we described mutant C9ORF72 modeling in C. elegans and report the finding of two suppressor genes. We made transgenes containing 9 or 29 repeats of GGGGCC in C9ORF72, driven by either the hsp-16 promoters or the unc-119 promoter. Transgenic worms were made to carry such transgenes. Phenotypic analysis of those animals revealed that Phsp−16::(G4C2)29::GFP transgenic animals (EAB 135) displayed severe paralysis by the second day of adulthood, followed by lethality, which phenotypes were less severe in Phsp−16::(G4C2)9::GFP transgenic animals (EAB242), and absent in control strains expressing empty vectors. Suppressor genes of this locomotor phenotype were pursued by introducing mutations with ethyl methanesulfonate in EAB135, screening mutant strains that moved faster than EAB135 by a food-ring assay, identifying mutations by whole-genome sequencing and testing the underlying mechanism of the suppressor genes either by employing RNA interference studies or C. elegans genetics. Three mutant strains, EAB164, EAB165 and EAB167, were identified. Eight suppressor genes carrying nonsense/canonical splicing site mutations were confirmed, among which a nonsense mutation of F57A10.2/VAMP was found in all three mutant strains, and a nonsense mutation of acp-4/ACP2 was only found in EAB164. Knock down/out of those two genes in EAB135 animals by feeding RNAi/introducing a known acp-4 null allele phenocopied the suppression of the C9ORF72 variant related movement defect in the mutant strains. Translational conformation in a mammalian system is required, but our worm data suggest that altering acp-4/ACP2 encoding lysosomal acid phosphatase may provide a potential therapeutic method of reducing acp-4/ACP2 levels, as opposed or complementary to directly reducing C9ORF72, to relieve C9ORF72-ALS phenotypes. It also suggests that the C9ORF72-ALS/FTLD may share a pathophysiologic mechanism with vesicle-associated membrane protein-associated protein B, a homolog of F57A10.2/VAMP, which is a proven ALS8 gene.An abnormally expanded GGGGCC repeat in C9ORF72 is the most frequent causal mutation associated with amyotrophic lateral sclerosis (ALS)/frontotemporal lobar degeneration (FTLD). Both  M. musculus and zebrafish, have been constructed based on this finding  models, testing Repeat-Associated Non-ATG Translation (RANT) of G4C2, have also been developed in S. cerevisiae, human iPSC-derived neuronal cells and Drosophila, which have identified nucleocytoplasmic transportation pathway anomalies as part of the pathogenetic mechanism of C9ORF72 associated ALS/FTLD (C9-ALS)  repeat in a noncoding region of C9ORF72 is the most commonly identified genetic variant associated with both familial amyotrophic lateral sclerosis (ALS) and familial frontotemporal dementia (FTD)  mechanism of C9ORF72 n::GFP and Phsp-16::(G4C2)n::GFP C. elegans expression constructs, we first amplified human C9ORF72 genomic fragment containing the G4C2 repeat: 5′caaggagggaaacaaccgcagcctgtagcaagctctggaactcaggagtcgcgcgcta(ggggcc)nggggcgtggtcggggcgggcccgggggcgggcccggggcggggctgcggttgcggtgcctgc3′. Primers for this reaction were available on request. Healthy human genomic DNA was used as PCR template. The PCR products were subcloned into the TOPO® TA Cloning vector  to get (G4C2)n TOPO constructs, and then sequenced to determine the repeat number of G4C2. The maximal number we could achieve by this method was 29 due to the high GC% of the repeat sequence. The reference repeat number was determined as 9. (G4C2)n TOPO constructs were digested with NotI and KpnI restriction enzymes to generate the fragment containing (G4C2)n, which was ligated with Fire Lab vectors L3787 and L3788 (Addgene) and processed with EagI and KpnI restriction enzymes. By this method, we generated Phsp-16::(G4C2)n::GFP C. elegans expression constructs (n = 29 or 9). The Punc119::GFP C. elegans expression construct, based on ppd95.75, was a gift from Prof. Craig Hunter at the Harvard Medical School. We inserted (G4C2)n fragments from (G4C2)n TOPO constructs into Punc119::GFP constructs using XbaI and KpnI restriction enzymes to generate Phsp-16::(G4C2)n::GFP C. elegans expression constructs (n = 29 or 9). All plasmid constructions were confirmed by sequencing at the Dana-Farber/Harvard Cancer Center DNA Resource Core.To produce Punc-119::(G4C2)n::GFP and Phsp-16::(G4C2)n::GFPconstructs at 40~50 ng/μl into young adult N2 gonads  at ~50 ng/μl was used as a co-injection marker , grown until they were gravid adults, and then washed off with M9 buffer and harvested in several 50 ml Conical tubes. After rinsing with M9 2–3 times, the animals were left on a NUTATOR mixer for 2 h to eliminate food in the gut, then were washed again with M9 2–3 times. The supernatant was removed after centrifugation at 1500 g for 3 min. A total 500 μl worm pellet was achieved by such method. For DNA extraction, we followed the protocol of Gentra Puregene Kit (Qiagen). The concentration of the final DNA preparation was determined by the Qubit assay (Invitrogen) at the Bauer Core Facility, Harvard University. 1 μg DNA was used for further sequencing library construction and next generation sequencing at BGI Americas (Cambridge MA USA). In brief, the sequencing libraries were made per the TruSeq®DNA PCR-free sample preparation kit (Illumina), and next generation sequencing was performed on an Illumina HiSeq seq platform. The sequencing results were analyzed both at BGI Americas Inc. and at the High-performance Computation Facility of the Partners Healthcare System. In brief, the sequencing reads were first mapped to the reference genome, such as WS220, with Bowtie-0.12.7, the variants were called with Samtools, and they were visualized by the Integrative Genomics Viewer developed by the Broad Institute. Nonsense mutations producing nonsense-mediated mRNA decay (NMD) and canonical splicing site mutations were noticed and confirmed by Sanger sequencing.dpy-6 RNAi as the positive control.We employed gene-specific RNAi cultures from Ahringer's RNAi feeding library (Geneservice Ltd) to carry out RNAi experiments, using the Ahringer's feeding RNAi protocol Kamath, . RNAi exE. coli by rinsing and soaking animals in two Petri dishes filled with M9 buffer. Animals were then transferred to NGM plates (35 mm diameter) with 1 ml of M9 buffer and allowed to accommodate for 1 min. We counted the number of thrashes for 20 s under the dissecting microscope. Only continuous movements were scored, and at least 25 animals were tested for each condition.To obtain the developmental data, we bleached the well-grown adult animals using standard bleach solution to obtain synchronized L1s, 25~50 of which were then placed on seeded NGM plates. Afterwards, the plates were observed and the developmental status was recorded every 24 h for 5–10 days. At least three independent experiments were performed for each study and a typical result was shown. For thrashing assays, we minimized contamination with t-tests.Data were expressed as means ± standard deviations. Statistical comparisons were all performed using unpaired, two-tailed Student's hsp-16 promoter, for conditional global expression, or by the unc-119 promoter, for pan-neuronal expression. C9ORF72 genomic fragments of (G4C2)9/29 in those transgenes were inserted between the transcriptional start site and the translational start site, thus mimicking their role as part of 5′-UTR in human C9ORF72 29::GFP transgenic animals (strain EAB 135) displayed severe paralysis by the second day of adulthood, followed by lethality. These phenotypes were not observed in wild-type animals and animals expressing the empty vector (strain EAB141), and were less severe in animals carrying the Phsp−16::(G4C2)9::GFP transgene (strain EAB242). For example, ~85% of EAB135 animals were dead by day 10 of adulthood when cultured at 25°C, compared to ~18% for N2 or EAB242  pathway  or RAN-2 (Ran GTPase activating protein homolog), two conserved components of the NPC pathway, the movements of N2 animals were more severely affected than those of EAB242 animals after treatment with RNAi for 72 h from the L4 stage, as shown in Figure Phsp−16::(G4C2)9/29::GFP carried both signals with a 100% transmission rate. Thus, the possibility that the transgenes were not integrated into the genomes is quite low. In summary, those transgenic animals with global expression of the disease-associated construct displayed a series of repeat-number dependent phenotypes, among which, movement defects and compromised life span resemble some of the features of illness observed in ALS patients.Transgenes were driven by either the 2 Figure . Integra Figures , 2. When Figures . In the (G4C2)9/29 play a role in producing those phenotypes? Most of the above-mentioned phenotypes were also observed in the pan-neuronal expressional strains of G4C2 , but abnormalities were relatively less severe compared to those in the globally expressed versions. Albeit, the promoters had to be different between those transgene constructs. For example, in the swimming assay, Day 10 adults of EAB136 had 45.7 thrashes/20 s, which was much improved when compared to 4.5 thrashes of EAB135 animals  from CGC. VC40826 animals had very obvious movement defects, a phenotype we used for out-crossing with N2 animals, for five times, to get the EAB157 strain. EAB157 still displayed movement defects and we confirmed gk833833 in EAB157 by PCR-sequencing. We crossed this strain with EAB135 animals in order to introduce gk833833 into the EAB135 background, and generated strain EAB158 . Consistent with the lf hypothesis, EAB158 animals had an improved movement phenotype compared to both EAB135 and EAB157, based on their Day 5 data. On Day 3, a substantial difference did not exist , which indicates either that those variants are not disease-causal or that they may not act through an lf mechanism.Overall we identified 27 candidates using Day 1 adulthood animals. Then we performed 6-9 validations. Only those passing all the validations were studied further. 24 of the 27 suppressor strains failed validations at least twice. The remaining three suppressors passed all 9 validations and were named EAB164, EAB165 and EAB167. EAB164 is a stronger suppressor, in terms of movement, developmental measures Figures , 4A and a Figure . The othPhsp16::G229::GFP)lf of F57A10.2/VAPB underlies the toxic effects of C9ORF72 repeats through a RAB pathway. Caution needs to be noted that the effects of (G4C2) repeats may be irrelevant to the normal function of C9ORF72.The genetic interaction between F57A10.2/VAPB and the disease-related repeat of C9ORF72 suggests that F57A10.2/VAPB is required in C9ORF72-pathology. C9ORF72 has been demonstrated to participate functionally in endosomal trafficking, and it co-localizes with Rab proteins  model in C. elegans . RNA-seq experiments identified a series of differentially expressed genes in that alfa-1(lf) animal and N2s (unpublished data). One of the differentially expressed genes, ZK370.8, is an ortholog of human TTC1 (tetratricopeptide repeat domain 1). Removal of TTC1 produces abnormal vesicular transport in human cells . The work was supported by an NIH Clinical Scientist Development award, K08NS002083 (EB), funds of the Program for Neuropsychiatric Research, at McLean Hospital (BC), National Natural Science Foundation grant 81400940 (XW) and a NARSAD Young Investigator Award to EB. BC is the Robertson-Steele Professor of Psychiatry at Harvard Medical School.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Antiretroviral Therapy(ART) remarkably reduced HIV-1 infection-related mortality in children. The efficacy and safety of different ART regimen in pediatric age groups remained issues of debates and available evidence were scarce especially among children taking the of one the two prototypes (NVP or EFV) Non-Nucleoside Reverse Transcriptase Inhibitor(NNRTI) as backbone of ART regimen.versus zidovudine-lamivudine-efavirenz (AZT+3TC+EFV) ART regimen among HIV-1 infected children.Therefore, the objective of this study was to compare clinical, immunological and virological responses of zidovudine-lamivudine-nevirapine (AZT+3TC+ NVP) versus EFV+AZT+3TC ART regimen among HIV-1 infected children. Data were entered into Epi-info version 7.2.2 for clean up and exported to SPSS version 17 for analysis. Paired and Independent t-tests were used to compare the CD4 cell count, weight and virologic level at six months with corresponding baseline value; and the mean weight, CD4 gain and viral suppression across the two ART regimens at six months of ART respectively.A retrospective cross-sectional study was done by reviewing medical records of the patients to evaluate clinical, immunological and virological outcomes of NVP+AZT+3TC Medical records of 122 patients from NVP-based regimen and 61 patients from EFV group were reviewed. After six months of NVP+AZT+3TC treatment, the mean CD4 cell count difference from baseline was 215. From EFV+AZT+3TC group, the mean CD4 cell count difference from baseline was 205. The mean CD4 count difference between the two regimens was comparable (p 0.145). Similarly, optimal viral suppression was achieved in 82% (100/122) of NVP+AZT+3TC regimen and 83% (44/61) of EFV+AZT+3TC regimen which was still comparable across the two groups.There was no difference in clinical, immunological and virological outcomes among patients taking NVP+AZT+3TC or EFV+AZT+3TC ART regimen. The debversus EFV+AZT+3TC ART regimen among HIV-1 infected children.In this study, wedifferences at six months assessed clinical and immunological and virological outcomes of NVP+AZT+3TC 2A cross-sectional descriptive study was conducted from May-August 2017 by reviewing ART documents of children infected with HIV-1 who were on ART at pediatric ART clinic, Asella Teaching and Referral Hospital (ATRH)The hospital is one of the federal teaching and referral hospitals. Pediatric and child health department is one of the major departments delivering patient care service, teaching and research activities under different subunits categories. Pediatric ART clinic is one of the well-organized clinics of the hospital giving care for pediatric HIV/AIDS care and treatment.2.1versus EFV+3TC+AZT. Before and during the study period the utilization of EFV-based was low as the national guideline by then was not strongly recommending this regimen in pediatric age group. Accordingly for one patient included from EFV- based regimen two patients from NVP based regimen were included (1:2) making the sample size of (n1=61) from (EFV+3TC+AZT) and 122 from (NVP+3TC+AZT) . A simple random sampling technique was used to select patient charts from each regimens using computer-generated random number. At the time of data collection about 545 children with HIV infection were on follow up at pediatric infectious disease clinic. The sampling procedure was determined by patients’ ART regimen and to minimize sampling error and to control the effects of other NRTI patients taking AZT+3TC were included and matched under NVP+3TC+AZT 2.2Data were collected from the patients' ART documents  using standardized format. Data entry was made to EPI info software for clean up and analysis; exported to SPSS version 17 for further analysis. Paired t-test was used to compare the CD4 cell count, weight and virologic level at six months with corresponding baseline value and independent t-test was to compare the mean weight and CD4 gain at six months and viral suppression level at six between the two ART regimens.The factors considered to affect the clinical and immunologic outcomes in both groups were assessed using binary logistic regression: baseline CD4%/count, WHO clinical staging, presence of chronic diarrhea, anemia, and baseline weight, occurrence of TB, and switching of ART regimen. 95% CI with p value of less than 0.05 was considered statistically significant.2.3A total of 183 patients were included in the study. ART documents of 122 patients from NVP+3TC+AZT groups and 61 patients from EFV based regimen were reviewed. Males constitute 51% (62/120) while females were 49% (60/120) from NVP+3TC+AZT regimen. From EFV-based regimen, male comprised of 52% (32/61) and female 48% (29/61).The youngest age at the initiation of ART was 5 months from NVP+3TC+AZT regimen and 17 months from EFV-based groups with the highest being 144 and 168 months (IQR age 74-104 and 95-130 months) respectively.Baseline anthropometry showed that 20.4%(25/122) and 1.5% (2/122) from NVP+3TC+AZT regimen and 22% (14/61) and 2.5% (2/61) from EFV+3TC+AZT regimen were having moderate and severe wasting respectively.Anemia was observed in 18.3% (22/122) of patients from NVP+3TC+AZT arm regimen and 14.8% (9/61) of EFV+3TC+AZT at the initiation of ART with the majority of the cases were having mild anemia.1).About 69% (84/122) and 73% (44/61) patients were WHO clinical stage III or IV at the start of NVP+3TC+AZT or EFV+3TC+AZT regimen, respectively (Table The CD4 cell count at the start of HAART ranged from 15-2000 cell/ml with an Interquartile Range (IQR) of 235-325 cell/ml among NVP+3TC+AZT. And among patients taking EFV+3TC+AZT based ART regimen, baseline CD4 count ranged 25-2075 cell/ml with an IQR range of 260-355 cell/ml.The CD4 cell count at six months of ART was compared with baseline and among the two groups. From NVP+3TC+AZT based ART regimen the CD4 cell count range from 75-2400 c/ml with IQR of 468-632 c/ml, and mean CD4 cell count difference of 215, 95% CI (175.414-245.613). From EFV based group, CD4 count ranged from 65-2100 c/ml with IQR of 435-605 c/ml, and the mean CD4 cell count difference of 205, 95% CI (155.404-235.623). The mean CD4 count differences at six months of ART were comparable between the two groups 2).Patients who have been on NVP+3TC+AZT regimen gained mean weight of 0.78kg and 1.81kg with 95% CI (0.588-0.957 and 1.60-2.2 respectively) at three and six months of HAART. Similarly, the mean weight gain at three and six months of EFV+3TC+AZT were 0.75 and 1.70kg with CI (0.588-0.954) and 1.70kg, 95% CI (1.50- 1.95). Still, there is no significant difference in weight gain among the two groups (Table 2).After six months of ART 82% (100/122) from NVP+3TC+AZT and 83%(44/61) from EFV+3TC+AZT were having adequate viral suppression(<1000coppy/ml) .During the first six months of HAART, a total 27 patients developed tuberculosis with comparable incidence across the two groups  patients who were on NVP+3TC+AZT HAART regimen were switched to other line of ART regimen while 4(6.7%) patients from EFV-based regimen switched to other regimen indicating ART switching was more commonly seen among NVP+3TC+AZT(p<0.001).3). Similarly, normal baseline weight, absence of diarrhea and weight gain >10% at three and six months of ART were found to be a good prognostic indicator of clinical, immunological and virological outcome regardless of the ART regimen (see Table 3).Early clinical stage of the disease and mild immune suppression at the start of ART were predicting favorable clinical, immunological and virological outcomes ART among the two groups of patients  in 82%100/122) and 83%44/61) among NVP+3TC+AZT and EFV+AZT+3TC respectively. This is incongruent with WHO recommendation which highlighted no evidence of EFV virologically superior to NVP 2 and 83%. How eve1 among N3 had a significantly lower chance of achieving CD4 count of 500 cells/mm3 compared with patients with baseline CD4 350 cells/mm3 and above [Presence of chronic diarrhea during ART treatment is associated with poor CD4 cell recovery independent of the nutritional status of the patient which is in agreement with most previous studies. The reason was not clearly identified so far but could be because of associated mal-absorption and poor adherence during diarrheal episode. This research also uncovered higher baseline CD4 cell value strongly predicted good six month CD4 cell recovery and viral suppression response in both groups of patients which was supported with a large cohort study involving 861 adult patients living with HIV in Spain that showed patients with baseline CD4 count of 200 and of 201 to 350 cells/mmnd above .This study also showed optimal weight recovery after initiation of ART strongly associated with better CD4 response and viral suppression which is in line with study report from South Africa which elaborated lower percentiles of weight gain after six months of ART were associated with poor subsequent treatment outcomes and a higher risk of mortality independent of other baseline characteristics. Likewise, normal weight at the start of ART was independent predictors of favorable immunological and virological outcomes in both wings which was coinciding with other observational studies where patients who were underweight had a two-fold increased risk of poor treatment outcomes , 21versus EFV+AZT+3TC regimen. The conflicting results from different studies could be explained by study design, patient selection and possible concomitant HIV-TB treatment which could significantly affect the outcomes.This research finding concluded that there is no difference in clinical, immunological and virological outcomes among patient taking NVP+AZT+3TC


Three additional capsular switch 35B variants were identified, 2 of which also involved 35B/ST558 as cps35B donor. Spread of 35B/ST156 is of concern in view of past global predominance of pathogenic ST156 vaccine serotype strains. Protection against serotype 35B should be considered in next-generation pneumococcal vaccines.We used whole-genome sequencing to characterize 199 nonvaccine serotype 35B pneumococcal strains that caused invasive pneumococcal disease (IPD) in the United States during 2015–2016 and related these findings to previous serotype 35B IPD data obtained by Active Bacterial Core surveillance. Penicillin-nonsusceptible 35B IPD increased during post–pneumococcal 7-valent conjugate vaccine years (2001–2009) and increased further after implementation of pneumococcal 13-valent conjugate vaccine in 2010. This increase was caused primarily by the 35B/sequence type (ST) 558 lineage. 35B/ST558 and vaccine serotype 9V/ST156 lineages were implicated as  Both lineages were relatively rare causes of IPD but were geographically widespread in the United States before and after PCV7 introduction  against invasive pneumococcal disease (IPD) persisted a full decade after its introduction in the United States in 2000, the emergence of 19A and other non-PCV7 serotypes reduced the overall benefit  in 2010, 35B became the most common serotype in ABCs, associated with MICs ,cps35B) in each of the variants (We provide a whole-genome sequence (WGS) pipeline−based resolution and description of current 35B lineages within current ABCs surveillance . Serotype 35B IPD isolates described include 132 recovered during 2015 and 67 recovered during ABCs in 2016 (https://wwwnc.cdc.gov/EID/article/23/6/17-0071-T1.htm). The listing of 35B isolates from 2016 is incomplete because we typically receive all ABCs isolates recovered during a given year by the following summer. Relevant ST156 lineage isolates of other serotypes recovered during this and previous periods are shown in The surveillance population of ABCs is ≈32 million persons in 10 states .A χDuring 1998–2001, penicillin-nonsusceptible 35B accounted for 67.6% (108) of serotype 35B ABCs isolates . During rrgA gene (http://pubmlst.org/spneumoniae/), and of ST11818 (highly related to 15A/ST63), an antimicrobial-resistant nonvaccine serotype lineage that has increased in the post–conjugate vaccine era  were serotype 35B. Of 1,528 IPD isolates recovered from partial year 2016 IPD surveillance, 67 (4.4%) were serotype 35B. Most (168/199) of these isolates belonged to penicillin-nonsusceptible clonal complex (CC) 558 (168 isolates) and CC156 (21 isolates) ; Table 1rgA gene , 2, whicOf 168 35B/CC558 isolates obtained, 147 were ST558, 20 were single-locus variants (SLVs) of ST558 corresponding to 13 STs, and 1 was a double-locus variant ; Table 1–The increased incidence of 35B IPD during the post-PCV7 period (2.1–3.7 cases/million population during 2001–2009 vs. 1.2–1.3 cases/million population during 1998–1999) and the post-PCV13 period (3.3–4.8 cases/million population during 2011–2015), combined with the consistent trend of markedly increased proportions of penicillin-nonsusceptible 35B IPD isolates throughout the conjugate vaccine era , is cons,,pbp1a and pbp2x flank opposite ends of the capsular biosynthetic locus and are sometimes cotransferred during serotype switching events  strain serving as the recipient strain for a 35B/ST558 cps35B donor strain  (cps9V locus and flanking PBP markers (2x-18 and 1a-15) with the cps35B locus and its flanking PBP markers (2x-7 and 1a-4). On the basis of available strain data, the original progeny strain is predicted to have been an ST156 strain with the PBP type 4:12:7; a total of 14 of the 19 35B/ST156 lineage strains still shared these characteristics   and 10,8ference) . Thus, aeristics . Five isWe detected the small (491 bp) segment (bases 11349–11839 of progeny)  of clear,A single penicillin-susceptible 35B/ST162 (SLV of ST156) has the completely sensitive PBP type 0:0:0 ,. This stpbp2b locus and flanking ddl sequence to change the PBP type to 4:11:7 and the MLST type to ST12921 through transfer of ddl with the resistance-conferring selectable pbp2b allele  . Althougb allele .cps35B gene deletion event within the wciG gene  underwent a postswitch intra–ciG gene , which iciG gene . Typing cps35B locus. Two progeny strains showed diversification within the first 2–3.7 kb when compared with the other 17 progeny. The 6-kb region immediately to the left of base 1 in all of the progeny strains had <99.2% sequence identity with the most similar potential ST156 parental recipient strains that we analyzed. However, beyond this segment, progeny had sequence identity with the parental strain for >8 kb.Further indication of chromosome-wide postswitch diversification of this single clade was shown in the left-flanking region of the pbp2x marker and distal pbp2b marker (PBP type 4:7:18). Features of this variant have been described  (ermB and tetM) as the currently common 15A/ST63 clone  were of the long-established penicillin-susceptible 35B/ST452 lineage ; Table 3/ST1092) . The 35B–,<5 years of age) ABCs isolates obtained during 1999, 2001, 2002, 2008, 2009, and 2011–2013  arose through a single ancestral recombination event. This event was facilitated through detailed analyses of crossover points and comparisons of corresponding regions of all progeny isolates with likely parental 35B/ST558 and 9V/ST156 strains. The genetic plasticity of the ST156 lineage is also highlighted in this study by detection of postserotype switch changes affecting β-lactam resistance (PBP1a type) and capsular serotype (wciZ deletion), which is potentially reflective of recent antimicrobial drug pressure and immunologic selection pressure.A distinct antimicrobial-susceptible serotype 35B SLV of ST156 (35B/ST162) is included among 35B ABCs of 35B during 2015–2016 by the β-lactam–susceptible PBP type 0:0:0. Thus, our data indicates that ,,cps35B donor .An additional 35B variant within a vaccine serotype lineage is shown with ST1092 that is typically associated with serogroup 6 strains . Because,,,,Although conjugate vaccines have a history of providing effective and durable protection against IPD (invasive serotype 35B pneumococci including an expanding serotype switch lineage, United States, 2015–2016.Additional information on