In this work, we evaluate the impact of low [O2] on the physiology of SCAPs isolated and processed in parallel at 21% or 3% O2 without any hyperoxic shock in ambient air during the experiment performed at 3% O2. We demonstrate that SCAPs display a higher proliferation capacity at 3% O2 than in ambient air with elevated expression levels of two cell surface antigens: the alpha-6 integrin subunit (CD49f) and the embryonic stem cell marker (SSEA4). We show that the mesodermal differentiation potential of SCAPs is conserved at early passage in both [O2], but is partly lost at late passage and low [O2], conditions in which SCAPs proliferate efficiently without any sign of apoptosis. Unexpectedly, we show that autophagic flux is active in SCAPs irrespective of [O2] and that this process remains high in cells even after prolonged exposure to 3% O2.Stem cells isolated from the apical papilla of wisdom teeth (SCAPs) are an attractive model for tissue repair due to their availability, high proliferation rate and potential to differentiate in vitro towards mesodermal and neurogenic lineages. Adult stem cells, such as SCAPs, develop in stem cell niches in which the oxygen concentration [O Different stem cell models are under investigation for cell therapy purposes, all having their advantages and concerns. Among the different models, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) or adult stem cells, mesenchymal stem cells (MSCs), show great promise in the field of regenerative medicine . Indeed,A unique population of MSCs referred as SCAPs , resides in the apical papilla of human immature wisdom teeth. SCAPs appear to be the source of odontoblasts that are responsible for the formation of root dentin ,6. Compa2 secrete FGF2 (Fibroblast Growth Factor 2) and display improved survival rates when injected into experimental animals ) is autophagy. Indeed, autophagy plays a key role in stem cell homeostasis as it acts as a robust quality control mechanism [One well-known process regulated by low oxygen concentration  at low [O2] on these properties. This study established the advantage and limitations of SCAP isolation and expansion at low [O2].Human immature wisdom teeth are commonly extracted in teenagers for orthodontic reasons. In this study, we compared the properties of SCAPs (isolated from three teenagers) that were isolated and expanded in parallel under continuous low or high  as that used for their isolation. SCAPs were isolated from three different individuals using these conditions and named thereafter: UBx-SCAP-N1, N2 and N3 (21% O2) or UBx-SCAP-H1, H2 and H3 (3% O2). These settings are summarized in 2] cell chamber was an Invivo2 300 RUSKINN in which all experiments at low O2 concentration were performed. When stipulated, early passages were up to P7 and late passages were from P11 up to P22. The detailed experimental procedure for the preparation of SCAPs for EXP III is provided below.Experiment III (EXP III): SCAPs from two teeth from the same patient were isolated and expanded at either 3% or 21% O2, the medium (40 mL) was conditioned for 1 h under 3% O2. Teeth were then processed in a sterile hood at 21% or 3% O2 as previously described , (EXP II), grew faster: doubling population times were 50 h at 21% O2 and 31 h at 3% O2 and cumulative population doubling were higher at 3% versus 21% O2  or only the expansion process (at 21% or 3% O2) which was important to improve proliferative efficacy, we undertook EXP III with SCAPs isolated from the same individuals, isolated and grown in parallel under 3% and 21% O2 , was plotted for each patient, at early and late passages , which were used as positive controls  and also that of other markers described as “stemness/proliferative-linked” markers like CD49f, stage-specific human embryonic antigen-4 (SSEA4), CD24 and PDGFRβ ,45,46,47nditions A. Howevereported , while to 21% O2 A. Expres of MSCs  data not of MSCs ,24. Undecontrols B [49]. 2 behave as classical MSCs, we undertook in vitro differentiation experiments towards mesodermal lineages. Cells from the three individuals at early or late passages were induced to differentiate as described in the Materials and Methods, then stained with an OsteoImage™ kit , with red oil  or Alcian blue (for staining of glycosaminoglycans produced by chondrocytes). As shown in 2] was detected, indicating that cells kept their stemness properties in both conditions in vitro. At later passages, we observed a loss in osteogenic and adipogenic differentiation potential at 3% O2, while chondrogenesis differentiation remained, indicating that low [O2] might preserve this unipotent mesodermal feature of SCAPs.To determine whether SCAPs grown under 21% or 3% O2 and 3% O2 without chloroquine but highly increased after the addition of chloroquine. These results demonstrated that the autophagic flux was well activated in both O2 conditions, at early  at low [O2]. This study, performed with SCAPs from three individuals allowed a large spectrum of analyses regarding their properties such as proliferation, clonogenicity, cell cycle, multipotency and autophagic flux both at early and late passages  in which we observed high proliferation and no apoptosis of cells  after at least one month at 3% O2.Previous analyses performed with stem cells and in particular with BM-MSCs show many advantages for cells grown under low [O2] ,52,53,542] 2 ,55. Howe. Howe2 [2] led to an increased proliferation capability of SCAPs and to a preserved expression of the classical MSC markers with a slight decrease of CD105, as already reported [2 and expanded at either 21% or 3% O2 (as in our EXP II) also demonstrated an advantage of cell growth under low [O2] compared with ambient air [2] in comparison with ambient air. In contrast, PDGFRβ, which is enriched in human placental MSCs with elevated colony forming unit efficiency [2]. In addition, CD24 expression, which may refer to the number of committed progenitors cells in the papilla tissue, was not differentially expressed in our cell growth conditions [2]-dependent markers. CD49f/6 integrin, whose deletion in mouse models leads to a disabling skin disease (epidermolysis bullosa), is part of the laminin receptor in epithelial cells which plays a critical structural role in the hemidesmosome [2], we performed the test at 21% O2 to determine whether cells maintained their clonogenicity advantage. We observed that clonogenicity of SCAPs was variable among the three tested individuals and that the switch from 3% to 21% O2 was deleterious at late passage for SCAP clonogenicity. A direct comparison of clonogenicity efficiency of SCAPs grown at 3% or 21% O2 should help to assess whether low [O2] maintains better the self-renewal capacity of SCAPs. SSEA4, which is highly expressed in human ESCs and iPSCs, is a stemness marker enriched in cells maintaining their stemness properties  at early passages (up to P7) but was restricted to chondrogenesis at late passages under 3% O2, in agreement with previous reports showing that osteogenesis was reduced under low [O2]  ,62. The AP cells . In addiAP cells . In addi2]. As expected, we found that the activation of autophagy was associated with high LC3 puncta and with the induction of BNIP3 (a key mitophagy player) in SCAPs switched from 21% to 3% O2 . This observation questions the potential involvement of BNIP3 in mitophagy since LC3 and BNIP3 usually colocalize when an active mitophagy process is engaged. Further investigation is needed to determine the significance of the heterogeneous expression of autophagy/mitophagy-involved markers in SCAP physiology. Nevertheless, our work suggests that autophagy is not an acute stress response in SCAPs and its impact in stemness and proliferation of cells should be further analyzed.Autophagy is known as a stress-induced mechanism that allows cells to recycle macromolecules and organelles to cope with stress conditions and thereby promote cell survival . Hypoxiato 3% O2 . Interesd injury ,67. In Sd injury ,69. Ther2 did not reach their exhaustion phase, up to 24 passages, in contrast with SCAPs isolated and grown at 21% O2 that we could not keep growing after 20 passages. However, at low [O2] and late passages, SCAPs lost some of their stemness properties in classical differentiation medium, that was established for experiments performed at 21% O2. Future work with a larger sampling of cells will aim at deciphering the role of autophagy in increased proliferation of SCAPs grown at 3% O2. A key future challenge will be also to determine how pre-conditioned SCAPs at 3% O2 survive after transplantation in experimental animal models and to understand better the mechanism of their adaptation to [O2] in vivo.In this study, we have produced SCAP banks from EXP I, II and III and characterized many features of the cells which are now available for further fundamental and applied research. SCAPs directly isolated at 3% O


The H9N2 influenza viruses that have become established in Bangladeshi live poultry markets possess five gene segments of the highly pathogenic H7N3 avian influenza virus. We assessed the replication, transmission, and disease potential of three H9N2 viruses in chickens and New World quail. Each virus replicated to high titers and transmitted by the airborne route to contacts in both species. Infected chickens showed no disease signs, and the viruses differed in their disease potential in New World quail. New World quail were more susceptible than chickens to H9N2 viruses and shed virus after airborne transmission for 10 days. Consequently, New World quail are a potential threat in the maintenance and spread of influenza virus in live poultry markets. Previous studies have shown that bobwhite quail support influenza virus replication with receptors in their respiratory tract.Gallus domesticus) aged 6 weeks were also used. The number of birds and the sampling times differed between species due to the space limitations in our BSL3 facilities and not handling two different species on the same day. For each virus, five donor quail and five donor chickens were infected by the natural route with 106 EID50 in 0.5 mL total volume phosphate‐buffered saline  as others have done.50) was calculated by the method of Reed and Muench.The three specific viruses used in this study were A/environment/Bangladesh/10306/2011 H9N2) , A/chicken/Bangladesh/10450/2011 (H9N2) (GenBank KC757840), and A/quail/Bangladesh/19462/2013 (H9N2) (GenBank KJ643700). The viruses were propagated and titrated in the allantoic cavities of 10‐day‐old embryonated chicken eggs at 35°C for 48 hours as described previously.N2 (isola50 5.4‐6.4 log10/mL) in the upper respiratory tract of both chickens and quail (Table 10/mL). In donor chickens, peak shedding of virus occurred at 3 dpi and the titers fell to background levels by Day 7. Similar virus titers were measured for each of the three viruses at 3, 5, and 7 dpi (data not shown). Shedding in quail donors peaked at 4 dpi, and shedding was sustained beyond 8 dpi in three of five donor birds for each virus. At least one donor quail in each group shed virus orally for 10 days.Each of the H9N2 influenza viruses from chickens, quail, and the environment replicated to a high titer  virus showed no overt disease signs, whereas birds infected with the two isolates from quail  showed varying levels of disease signs (Table 2This study shows that the New World quail (Bobwhite species) is more susceptible to infection with Bangladeshi H9N2 influenza virus than chicken. Each of the H9N2 viruses replicated to a high titer in the respiratory tract of both chickens and quail, with low‐level shedding in the feces. The viruses all transmitted by direct contact and airborne transmission in chickens and by airborne transmission in quail, but the efficiency of airborne transmission to chickens varied. The differences in morbidity and mortality caused by the three H9N2 viruses in quail may be associated with sequence differences.In this study, quail were more severely affected by the H9N2 viruses containing five gene segments from HPAI H7N3 than were chickens. H9N2 viruses from Pakistan containing four gene segments  from HPAI H7N3‐affected quail more severely than chickens and caused sporadic deaths .Although fewer airborne‐contact chickens (3 per group) were used than in New World quail (5 per group), differences in airborne transmissibility are apparent and emphasize the risk factors of New World quail present in live bird markets: Variable transmission occurred in chickens among viruses tested, but high transmission occurred in New World quail. Quail (Old World) have been recognized as efficient spreaders of H9N2 influenza viruses and have been banned from live poultry markets in Hong Kong since 2002.


BRCA1 and BRCA2 spliceogenic variants are often associated with an elevated risk of breast and ovarian cancers. Analyses of BRCA1 and BRCA2 splicing patterns have traditionally used technologies that sample a population of cells but do not account for the variation that may be present between individual cells. This novel proof of concept study utilises RNA in situ hybridisation to measure the absolute expression of BRCA1 and BRCA2 mRNA splicing events in single lymphoblastoid cells containing known spliceogenic variants (BRCA1c.671-2 A>G or BRCA2c.7988 A>T). We observed a large proportion of cells (>42%) in each sample that did not express mRNA for the targeted gene. Increased levels (average mRNA molecules per cell) of BRCA2 ∆17_18 were observed in the cells containing the known spliceogenic variant BRCA2c.7988 A>T, but cells containing BRCA1c.671-2 A>G were not found to express significantly increased levels of BRCA1 ∆11, as had been shown previously. Instead, we show for each variant carrier sample that a higher proportion of cells expressed the targeted splicing event compared to control cells. These results indicate that BRCA1/2 mRNA is expressed stochastically, suggesting that previously reported results using RT-PCR may have been influenced by the number of cells with BRCA1/2 mRNA expression and may not represent an elevation of constitutive mRNA expression. Detection of mRNA expression in single cells allows for a more comprehensive understanding of how spliceogenic variants influence the expression of mRNA isoforms. However, further research is required to assess the utility of this technology to measure the expression of predicted spliceogenic BRCA1 and BRCA2 variants in a diagnostic setting. BRCA1 and BRCA2 variants can confer an increased risk of breast and ovarian cancers, and many of these variants are known to disrupt mRNA splicing, as seen in BRCA1 and BRCA2 mRNA splicing patterns have been explored in depth using a number of advanced sequencing technologies to highlight significant diversity, both in the number and level of the alternative events expressed naturally by these genes [Germline se genes . This dise genes ,5.BRCA1 and BRCA2 mRNA expression levels may be influenced by such fluctuations. Expression based assays (such as reverse transcriptase-PCR and RNA-seq) measure average mRNA expression in a population of cells. However, the individual cells of the population are likely to be producing transcripts at very different levels, potentially providing an additional measure of mRNA expression patterns associated with spliceogenic variants. Advanced in situ hybridisation (ISH) technologies provide the means to observe actual levels of mRNA transcripts in single cells, in addition to detail on spatial expression within a cell. Measuring gene splicing at the single cell level may provide a more comprehensive method of evaluating the biological and clinical significance of alternative isoforms. In this study, we utilised RNAscope [BRCA1 and BRCA2 spliceogenic variants on mRNA transcript expression in single cells.In particular, individual cells are known to express genes in a stochastic manner, occurring in bursts, irrespective of the transcription factors present ,7, suggeRNAscope , a uniquBRCA1 and BRCA2 mRNA splicing events in single cells, from individuals with or without germline spliceogenic variants. Results showed that the proportion of cells expressing BRCA1 transcripts ranged from 4% (2/50) in the control to 44% (22/50) in the BRCA1c.671-2 A>G carrier, as seen in BRCA2 transcripts ranged from 24% (12/50) in the control to 58% (29/50) in the BRCA2c.7988 A>T carrier. Results from the negative and positive control probes from the RNAscope Multiplex Fluorescent Detection Kit are shown in the BRCA1 mRNA transcript expression ranged from 0 to 17 (mean = 2.1) BRCA1 molecules per cell in the BRCA1c.671-2 A>G carrier, and 0 to 10 (mean = 0.3) in the control. The BRCA2 mRNA transcript expression ranged from 0 to 29 (mean = 3.2) molecules per cell in the BRCA2c.7988 A>T carrier, and 0 to 5 (mean = 0.6) in the control as shown in BRCA1 and BRCA2 mRNA molecules per cell compared to non-treated LCLs , as seen in In this study, RNAscope allowed the detection of specific  to 58% 2/50 in thBRCA1 mRNA expression in LCLs carrying the spliceogenic variants BRCA1c.671-2 A>G and BRCA2c.7988 A>T have reported increased expression of the ∆11 and ∆17_18 splicing events, respectively. However, these studies were unable to elucidate BRCA1 and BRCA2 expression patterns at the single cell level. Using RNAscope, we show that the BRCA1c.671-2 A>G variant carrier LCL showed no significant difference in the total number of detected BRCA1 ∆11 mRNAs compared to the control LCL across the cells assayed in each sample, demonstrated by p = 0.48 in BRCA1 ∆11 splicing event was shown to be significantly greater in the variant carrier compared to the control  and to identify cell-specific overexpression in different tissue types. mRNA isoforms detected in samples at very low levels might actually be highly abundant in a small number of cells, but this observation can only be detected at the single-cell level, as seen in BRCA1 and BRCA2 transcripts, and an increase in the number of cells containing transcripts, in the treated samples compared to the untreated samples. Higher expression in the treated samples facilitated the detection of the targeted alternative splicing event, suggesting that NMD inhibitor treatment is advantageous when analysing BRCA1 or BRCA2 transcript expression with RNAscope.Nonsense-mediated decay inhibitors are often used in splicing analysis to prevent the degradation of mRNA , thus imn = 50), which might have led to an under-representation of some isoforms, such as those that were lowly expressed. Increasing the total number of cells counted in each sample may provide a more accurate survey of isoform expression patterns. However, counting a greater number of cells would be relatively laborious using manual assessment. This challenge could be alleviated by using image analysis software, such as the Aperio RNA ISH Algorithm (Leica Biosystems), or Cell Profiler (http://cellprofiler.org/). It is important to note that there is paucity in published data relating to the efficacy of these image analysis software tools. It is unclear why some cells contained a greater number of C1 (targeting the deletion of interest) probe signals compared to the reference C2  probe signals as seen in BRCA1 and BRCA2 is 4.5 and 4.8 h [BRCA1 (∆3) and BRCA2 (∆12) [Our study is proof of concept, demonstrating the utility of RNA in situ hybridisation to characterise the effects of spliceogenic variants at the single cell level. Our study had several potential limitations, including the use of a single control and the number of cells analysed , specific mRNA transcripts are now able to be detected using shorter probes [BRCA1 ∆11 using BaseScopeTM probes spanning the exon 10_12 junction will not identify the BRCA1 ∆9_11 splice event, even though both isoforms contain a deletion of the large exon 11 sequence. Thus, assays targeting exon 11 may be utilised to examine potentially ‘leaky’ variants, which give rise to multiple alternative isoforms.The unique probe design RNAscope uses to obtain high specificity also limits the number of isoforms that can be detected with this technique. Three probe pairs are required to return a detectable signal, with each pair taking up 36–50 nucleotides of the target sequence, while 10–20 pairs are recommended to gain optimal detection . This lir probes . HoweverBRCA1 and BRCA2 mRNA expression patterns at the single cell level in LCLs. This technology could potentially play an important role in assessing variable expression levels of BRCA1 and BRCA2 isoforms by establishing whether mRNA isoforms detected in samples at very low levels may actually be highly abundant, but in a small number of cells. Such findings may be associated with an increased risk of disease, yet might go undetected using cell population-based methods because the number of cells expressing the aberrant change is likely to be insufficient to indicate an association with disease. Spliceogenic variants previously dismissed as low-risk might need to be re-evaluated at the single cell level to determine if aberrant mRNA isoforms are being expressed at high levels but in a low number of cells. Furthermore, single cell expression analysis provides an important means for investigating the poorly explored link between many human diseases and gene expression variability (variance) [To our knowledge, this is the first study to reveal ariance) ,16. SuchBRCA1c.671-2 A>G and BRCA2c.7988 A>T) and two from healthy controls, were obtained from the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer . The variant carriers were selected for analysis as they contained variants known to disrupt normal splicing patterns [2 atmosphere. mRNA was extracted from LCLs cultured with and without the nonsense mediated decay (NMD) inhibitor cycloheximide (100 µg/mL) for 4 h.Four lymphoblastoid cell lines (LCLs), two derived from rare variant carriers (patterns . Cell liBRCA1 ∆11 upregulated in BRCA1c.671-2 A>G and BRCA2 ∆17_18 upregulated in BRCA2c.7988 A>T variant carriers [green and C2red) were designed for BRCA1 and BRCA2. C1green probes were designed to target the deleted mRNA region (BRCA1 exon 11 or BRCA2 exons 17_18). C2red probes were designed to target a large transcript region, with the pretext that at least part of that region is represented in all expressed transcripts as seen in green and C2red probe signals are detected, whereas the expression of each of the targeted alternative splicing events of interest was calculated by subtracting the number C1 signals from the number of C2 signals in each cell, as seen in UBC (positive control) tissue and bacterial gene dapB (negative control) were included for each assay.Labelled probes were designed to detect splicing alterations known to be upregulated in the variant carriers, namely, carriers ,9. Probe® Fluorescent Multiplex kit  according to the manufacturer’s instructions specified in RNAscope® Fluorescent Assay for PBMC and Non-Adherent Cells (Part 1), and the RNAscope® Fluorescent Multiplex Kit User Manual Part 2 [RNA in situ hybridisation was performed once for each sample using the RNAscopel Part 2  . The EC plan-Neofluar 40×/1.3 oil Dic M27 objective was used with DAPI, TRITC and FITC filters. Fluorescent signals were counted independently for each wavelength in 50 non-overlapping cells. Accurate counting was conducted by scanning through different focal planes in the molecule .BRCA1 C1green (exon 11) and C2red (exons 1–10) fluorescent signals in a cell, we determine that all detected transcripts contain exon 11 as seen in red to C1green signals indicates an exon-skipping event (∆11), as shown in The number of transcripts containing the targeted deletion event in each cell was determined through differences in the number of fluorescently labelled probes detected for each colour channel. For example, where there is an equal number of


Cancer is associated with various genetic and environmental risk factors. Beside the mutations or aberrant expression of protein-coding genes, the genetic deregulation of non-coding RNAs has also an important role during tumor progression and metastasis. Long non-coding RNAs (lncRNAs) are a class of ncRNAs larger than 200 nucleotides that may function as tumor-suppressor or oncogene.There is a raising trend of cancer incidence among Iranian population during the last decades. Therefore, it is required to prepare a general population specific panel of genetic markers for the early detection of cancer in this population. The tissue-specific expression characteristics and high stability in body fluids highlight the lncRNAs as efficient diagnostic and prognostic noninvasive biomarkers in cancer. In present review we summarized all of the lncRNAs which have been reported until now in different tumors among Iranian patients.This review paves the way of introducing a population based noninvasive diagnostic panel of lncRNAs for the early detection of tumor cells among Iranian population. Cancer is one of the main global health challenges and is the leading and second causes of deaths among developed and developing nations, respectively . Cancer In present review we have summarized all of the lncRNAs with significant roles during tumor progression which have been reported until now among Iranian cancer patients to pave the way of introducing a non-invasive population based diagnostic panel of lncRNAs Table . We cateSOX2 overlapping transcript (SOX2OT) is an lncRNA located on 3q26.33. The SOX2 is located within the third intron of SOX2OT gene which is transcribed in the same orientation. There are 13 splice variants for SOX2OT . SOX2OT LincRNA-Regulator of Reprogramming (lincRNA-RoR) regulates the reprogramming process in pluripotent stem cells. Moreover, it is associated with iPSC derivation and ESC pluripotency . AberranLnc-POU3F3 is located on the reverse strand of chromosome 2q12.1 and POU3F3 upstream which is a member of the class III POU family of transcription factors . POU3F3 Prostate cancer associated transcript 1 (PCAT1) acts as an oncogene through inhibition of BRCA2 and stimulation of MYC. It promotes tumor cell invasion through targeting RBM5 and KLF6 in pancreatic and ovarian cancer , 55. It Prcat17.3 and Prcat38 are located upstream of TMPRSS2 gene, which are used to distinguish malignant and nonmalignant prostate tissues. Prcat17.3 and Prcat38 are positively correlated with TMPRSS2 in prostate cancer. Cat2184.4 is located upstream of PMEPA1 which acts as a tumor suppressor in prostate tumorigenesis. It has been shown that there were significant up regulations of Prcat17.3 and Prcat38 and significant down regulation of Cat2184.4 in prostate cancer (PCa) tissues compared with benign prostate hyperplasia (BPH) among a group of Iranian subjects. Moreover, the results showed a significant up regulation of Prcat17.3 level in urine samples of PCa patients compared with BPH patients. According to the ROC curve analysis, it has been demonstrated that the Prcat17.3 urine assay has a better sensitivity in distinguishing PCa from BPH compared with urine PCA3 tests. Therefore, Prcat17.3 and Prcat38 can be suggested alone or in combination with PCA3 as diagnostic markers of PCa among Iranians .HOTAIR is an oncogenic factor transcribed from the antisense strand of HOXC locus Which is involved in epigenetic regulation through interaction with Polycomb Repressive Complex 2 (PRC2) . HOTAIR ANRIL is an lnc-RNA which binds with CBX7 and SUZ12 within the PRC1 and PRC2 to regulate the transcriptional repression , 63. It Prostate cancer associated non-coding RNA 1 (PRNCR1) is located in 8q24.21 and highly expressed in aggressive PCa. It has a critical role in PCa progression through regulation of androgen receptor (AR) . BesidesLong intergenic non-protein coding RNA 355 (LINC00355) is associated with various cellular processes such as apoptosis, proliferation, and migration , 73. It SOX2OT is an lncRNA that is de-regulated in tumor tissues and is down regulated during the cell differentiation , 37. MorThe findings demonstrated that the relative expression of HOTAIR long non-coding RNA was significantly up regulated in GC tissues compared with normal margins in a sample of Iranian subjects. There was also association between HOTAIR expression level with TNM staging and lymph node metastasis. Moreover, they showed a significant direct association between the levels of HOTAIR and SUZ12 expressions . AnotherTUG1 plays a critical role in epigenetic regulation through interaction with PRC2 or PRC1 . It induGrowth arrest specific 5 (GAS5) is an ncRNA that inhibits the glucocorticoid receptor via binding with its DNA binding domain. It regulates gastric cell proliferation, apoptosis, and migration through CDK6, YBX1, and P53 –102. It ANRASSF1 is an antisense lncRNA of RASSF1 involved in epigenetic regulation through binding with PRC2 that is required for recruiting PRC2 to the RASSF1A promoter region. This process results in accumulation of the H3K27me3 and decreases the RASSF1A protein. It has been observed that there were significant increased levels of ANRIL and ANRASSF1 in GC tumors compared with the normal margins among a subpopulation of Iranian patients . ANRIL wProtein tyrosine phosphatase receptor type G antisense (PTPRG-AS1) is an antisense lncRNA of PTPRG which has important roles in cell growth, differentiation, and neoplastic transformation. PTPRG-AS1 is correlated with ER+ and ER −  subtypes, tumor grade, and clinical results . It funcIt has been reported that there were a significant association between HOTAIR polymorphisms and risk of breast cancer in a sample of southeast Iranian population in which rs920778 polymorphism significantly increased breast cancer risk while the rs12826786 and rs1899663 polymorphisms significantly decreased breast cancer risk. Moreover, rs920778 and rs12826786 were significantly correlated with ER status . SimilarH19 is a paternally imprinted gene associated with the human Beckwith-Wiedemann syndrome that is located on chromosome 11p15.5. H19 acts as an oncogene in bladder cancer, breast cancer, and hepatocellular carcinoma. It is associated with E2F1 transcription factor to induce breast cancer cell cycle progression . H19 is Steroid receptor RNA activator (SRA) regulates gene expression by steroid hormones. SRA is involved in regulation of physiological processes such as NR signaling, steroidogenesis, and mesenchymal fate. SRA functions as a molecular scaffold to regulate the transcription by various co-regulators and chromatin-modifying factors in both activating and repressive complexes . GAS5 isPRNCR1 is a critical AR regulator. It has been reported that there were significant increased levels of PRNCR1 expressions in breast tumor tissues compared with normal margins among a sub population of Iranian subjects. Moreover, there were significant association between PRNCR1 overexpression and clinical-pathological features such as tumor size and lymph node involvement . SimilarThe NAV2-AS2 is a lncRNA located in 11p15.1 and the minus strand of NAV2 sequence . NAV2 isLong non-coding upstream of MYCN (lncUSMycN) is transcribed from the 14-kbp upstream of the MYCN transcription start site. It has been reported that the lncUSMycN up regulated N-Myc mRNA expression through non O protein. It has also important roles in cell proliferation and tumorigenesis in neuroblastoma. There was significant up regulation of lncUSMycN among a sample of Iranian breast cancer patients compared with healthy controls. Moreover, levels of lncUSMycN expression was significantly associated with the early stages of breast cancer .LncRNA ZEB1 antisense 1 (ZEB1-AS1) is transcribed from the ZEB1 promoter sequence and it regulates the ZEB1expression levels. ZEB1-AS1 binds with MLL1 to induce H3K4me3 in ZEB1 promoter sequence. It promotes colorectal tumor cell proliferation through p15 inhibition . It has LOC100287225 is an lncRNAs located in the long arm of the chromosome 18, upstream region of deleted in colorectal carcinoma (DCC). DCC is a trans-membrane cell adhesion protein belonging to the immunoglobulin family which is associated with axon attraction and apoptosis induction. It has been demonstrated that there were significant LOC100287225 down regulations in tumor tissues compared with adjacent tumor-free tissue among a sample of Iranian CRC patients , 143.VIM-AS1 RNA is located in 10p13 that is transcribed from a shared bidirectional promoter with Vimentin mRNA . It has Lnc-LAMC2-1:1 is located in 1q25.3 and overlaps with LAMC1. It has been reported the genetic variation in lnc-LAMC2-1:1 was associated with CRC by affecting miRNA binding . The lncOIP5-AS1 functions as a sponge for miR-448 to regulates BCL-2 in lung adenocarcinoma cells . OIP5-ASThere are various diagnostic methods for the early detection of cancer. However, majority of routine methods are invasive. The deregulated pattern of lncRNAs in tumor tissues is mirrored in body fluids which make these factors as a non-invasive candidate with better tolerance for the cancer patients compared with biopsy. Therefore, this review summarized all of the lncRNAs with significant roles during tumor progression which have been reported until now among Iranian cancer patients. This review paves the way of introducing a population based non-invasive panel of lncRNAs for the early detection of cancers among Iranian population.


The World Health Organization (WHO) prequalification of Two hundred and fifty (250) patients were recruited from the HIV outpatient clinic at ITM. Accuracy and precision of CD4 T cell counting on fresh EDTA anticoagulated venous blood samples were assessed in the laboratory on a Muse Auto CD4/CD4% system. Extensive precision analyses were performed both on fresh blood and on normal and low stabilized whole blood controls. Accuracy ((bias) was assessed by comparing results from Muse CD4/CD4% to the reference . Clinical misclassification was measured at 500, 350, 200 and 100 cells/μL thresholds.Intra-assay precision was < 5%, and inter-assay was < 9%. CD4 T cell counts measured on Muse Auto CD4/CD4% System and on the reference instrument resulted in regression slopes of 0.97 for absolute counts and 1.03 for CD4 T cell percentages and a correlation coefficient of 0.99 for both. The average absolute bias as compared to the reference was negligible (4 cells/μL or 0.5%). The absolute average bias on CD4 T cell percentages was < 1%. Clinical misclassification at different CD4 T cell thresholds was small resulting in sensitivities and specificities equal or >90% at all thresholds except at 100 cells/μL (sensitivity = 87%). All samples could be analyzed as there was no repetitive rejection errors recorded.The Muse Auto CD4/CD4% System performed very well on fresh venous blood samples and met all WHO acceptance criteria for analytical performance of CD4 technologies. In 2017, about 36.7 million people were living with HIV of which 20.7 56.4%) were receiving antiretroviral treatment (ART) % were re.in vitro diagnostics assessment includes an independent analytical performance evaluation of CD4 counting instruments, in particular of those meant for use in resource-limited settings [in vitro diagnostic (IVD) flow cytometer installed in a well-equipped accredited clinical laboratory with excellent performance in external quality assessment (EQA) schemes and operated by highly-skilled laboratory personnel [Ideally, laboratory evaluations of new CD4 technologies are conducted independently from the manufacturer. The WHO prequalification of settings . During ersonnel .Most currently used IVD flow cytometers are sophisticated large, heavy and very expensive instruments which are operated by highly skilled and dedicated personnel. They are usually used in large hospitals, are semi- or fully automated and have a high sample throughput. Fortunately; several less complicated and more affordable instruments, dedicated to CD4 T cell counting, have been developed the past two decades . As a reThe gap between the large IVD instruments and the small POC devices is currently filled by a number of simplified, small flow cytometers which still require a laboratory and trained personnel but which are much easier to set up in small laboratories near the point-of-care. An example of such “intermediate” flow cytometer dedicated to CD4 counting, prequalified by WHO, which has been around for more than 20 years is the FACSCount ,15. In 23-EDTA vacutainer tubes for routine CD4 T cell counting. The same blood sample was used for CD4 T cell counting on Muse Auto CD4/CD4% System. The WHO study protocol requires collection of at least 50 blood samples from patients with <200 CD4 T cells/μL, 100 blood samples from patients with 200–500 CD4 T cells and 50 blood samples from patients with >500 CD4 T cells [Adult patients presenting for routine CD4 T cell enumeration at the HIV outpatient clinic of the Institute of Tropical Medicine (ITM) Antwerp, Belgium (in April-June 2017) provided a venous blood sample in 3 ml K T cells . The stuThe laboratory evaluation was conducted with two Muse Auto CD4/CD4% Systems. The Muse CD4/CD4% System is a small capillary flow cytometer with dedicated software for CD4 T cell counting of both percentages and absolute CD4 T cell counts. The instruments needs to be installed on a steady table and requires stable external power supply. It has a large color touch screen user interface to enter patient information and reagent batch numbers. The Muse Auto CD4/CD4% reagents are a cocktail of fluorochrome-conjugated antibodies to identify all lymphocytes and CD4+ T lymphocytes. The test uses a lysing solution to lyse red blood cells prior to CD4 T cell counting. The test system comes with a Muse system check which are bead controls for system check and proficiency testing of operator (correct volume pipetting). The test requires mixing 10μL of fresh blood (<48h) with 10μL of monoclonal antibodies followed by an incubation of 15 minutes. Subsequently, 380 μL of lysing solution are added  and after another incubation of 15 minutes the tube can be analyzed on the Muse instrument. The instrument reports both CD4 T cell percentages and absolute CD4 T cell counts (cells/μL).μL. The CD4 category was determined by the first CD4 result of a venous blood sample measured on FACSCalibur using Trucount tubes.The coefficient of variation (CV), also known as relative standard deviation (RSD), was used as a standardized measure of dispersion (precision) and was calculated as standard deviation (SD) divided by the mean of 10 repeat observation. The assessment of intra-laboratory variation of test results of Muse Auto CD4/CD4% system included instrument precision, intra-assay variation, inter-instrument variation, and inter-assay variation, using venous blood specimens. Blood samples were stratified in categories around clinically relevant CD4 thresholds: 200 (150–250), 350 (300–400) and 500 (450–550) CD4 cells/Instrument precision (run-to-run) consisted of re-reading a single stained venous blood sample ten times on the same Muse Auto CD4/CD4% System. For intra-assay precision (tube-to-tube variability), staining and reading was repeated 10 times on the same blood sample and analyzed on the same Muse Auto CD4/CD4% System. For inter-instrument precision (instrument-to-instrument variability), each blood sample stained ten times and each tube was analyzed on each of the two Muse Auto CD4/CD4% Systems. The average of the obtained results was calculated for each instrument. The inter-instrument variability (CV inter-instrument) was calculated using the average result and SD of the 2 different instruments for each patient sample.The inter-assay precision (day-to-day variability) was measured on venous blood samples analyzed within 6 hours after blood collection and reanalyzed within 48 hours after blood collection.The CD4 reference method was performed in an ISO15189:2012 certified laboratory which participates in external quality assessment programs (QASI and UK-NEQAS). The service maintenance of the CD4 reference instrument  is assured twice a year by a service engineer from BD Biosciences. The CD4 reference instrument is calibrated daily, and checked by running Multicheck Normal and Low process controls (BD Biosciences) . Four different batches of Normal controls and Low controls were used during the three-month duration of the study in Antwerp.The Muse Auto CD4/CD4% System is supplied with control beads which have to be run daily as system and proficiency check prior to analyzing patients samples. Multicheck Normal and Low controls were run daily on the Muse Auto CD4/CD4% Systems to assess reproducibility of test results during the entire study period. For each control blood sample, the reported results were compared to the sample validation range provided by the manufacturer.Venous blood samples were alternatively analyzed on one of two different Muse Auto CD4/CD4% Systems each day. Absolute CD4 T cell counts and CD4 T cell percentages measured on the Muse Auto CD4/CD4% System were compared to the corresponding CD4 counting results from the FACSCalibur . CD4 counts on FACSCalibur were measured according to the manufacturer's recommendations using Multiset reagents  and BD Trucount tubes . Acquisition on FACSCalibur was automated but the individual sample analyses were done manually using CellQuest software in accordance with the accredited procedure used for routine CD4 counting in the laboratory.The agreement between Muse Auto CD4/CD4% System results and those obtained on the reference method was assessed by Passing-Bablok regression analysis , SpearmaThe bias of Muse Auto CD4/CD4% system test results at different clinically relevant CD4 thresholds  and at 1000 cells/μL was calculated using Passing-Bablok regression analysis with bootstrapping of the confidence intervals of the bias at different CD4 thresholds . EstimatClinical misclassification was calculated for CD4 counts at clinically relevant CD4 thresholds of 200, 350, 500 cells/μL, using the reference method results as the “true” value for sensitivity and specificity. Inter-rater agreement was calculated with the Kappa coefficient .Statistical analyses were done with MedCalc version 18.2.1 .Invalid results or sample rejection was defined as the inability of Muse Auto CD4/CD4% System to provide a CD4 results from a patient blood sample. If the analysis was aborted/rejected, a second sample was prepared and read. Upon a second rejection, the test result was recorded as sample rejection.In total, two hundred and fifty patients were included in this study. Clinical data was available for 236 patients, of whom 234 were HIV infected.The median age (minimum–maximum) of the participants was 46 years (15–85) and 78% were males. Most patients (86%) were receiving antiretroviral treatment. The median (inter quartile range) CD4 T-cell count was 410 (252–582) cells/μL; 52 had CD4 < 200 cells/μL (median 129 (65–180) cells/μL), 113 had between 200–500 CD4 T cells/μL (median 386 (312–437) cells/μL), 85 had > 500 CD4 T cells/μL (median 753 (581–948) cells/μL). None of the patients presented with malaria; 4 patients had clinical features suggestive of pulmonary tuberculosis. From 14 patients clinical data was not available.The mean instrument precision for absolute CD4 T cell counts and CD4 T cell percentages in venous blood varied between 2.5 and 5.5% depending on the CD4 category. The intra-assay CV varied from 2.8 to 4.6%. The inter-assay CV varied from 3.5 to 8.1%. The inter-instrument CV varied from 2.5 to 4.4%. The individual CV for the different CD4 categories and for each type of precision assay are presented in Four different Multicheck batches (lots) of Normal and Low controls were used over the entire laboratory evaluation including 36 consecutive measurements of a Normal and Low CD4 count control over the entire study duration of 3 months. The Low controls were stabilized blood samples with CD4 counts between 100–200 CD4 T cell counts/μL. The Normal controls were stabilized blood samples with CD4 T cell counts between 600–1000 CD4 T cells/μL. The average CV for absolute CD4 T cell counts over the entire study duration was <5% for Normal CD4 count controls and <8% for Low CD4 T cell count controls. The overall CV for CD4 percentages was <2% for Normal controls and <6% for Low controls.Agreement between Muse Auto CD4/CD4% System and the reference method is shown in Fitted line equation, the number of analysed samples (n) and Spearman rank correlation are depicted in the legends.Absolute bias on absolute CD4 T cells counts (top left), absolute bias on CD4 T cells percentages (top right). Relative bias on absolute CD4 T cells counts (bottom left), relative bias on CD4 T cells percentages (bottom right).The absolute and relative bias was calculated for absolute CD4 T cell counts and for CD4 T cell percentages .As the relative bias can be large at low CD4 T cell counts, without being meaningfull, the relative bias was recacluated for patients with > 100 CD4 T cell counts resulting in a smaller, and thus better limits of agreement . As the Misclassification analysis is shown in During the study, the rejection rate was 0/250 (0%). All blood samples could be analyzed on Muse Auto CD4/CD4% System and all results were included in the study.Technicians found the Muse Auto CD4/CD4% instrument easy to use after a short 4-hour training.The keypad on the screen used to enter details on the sample, user, batch numbers etc was considered a bit small and technicians with larger fingers  had trouble pressing the correct keys. The small blood volume (10μL) and reagent volume (10μL) required to perform the test was a concern at the start of the study but, as supported by the results, that did not have any impact on the precision and accuracy of the measurements. The system check at start-up which includes an operator proficiency check is considered an asset as it will assure accurate pipetting. The instrument has no sample loader and samples are analyzed on the instrument manually. The manufacturer recommends an extra instrument cleaning step after 20 measurements to prevent needle clogging. Since preparation of 20 samples takes about 45min to 1 hour and the analysis of 20 samples on the instrument take another hour, 80 samples can be analyzed on an 8h working day per technician and per instrument. In theory, a first result can be generated after 1 hour.The Muse CD4/CD4% instruments are much smaller than conventional large IVD flow cytometers, easier to move around (mobility) and simple to operate (much less training required).The system reported warnings but the frequency was low (<5%). Most of the warnings were related to a concern about correct automatic gating and required visual inspection and adjustment of the gate. Setting the instrument takes about 15 min (complete system clean + system check) before blood samples can be analyzed. Technicians consistently implemented the wash procedure which can optionally be skipped although it is not recommended to do so. At the end of a day, the flow cell needs to be cleaned by a thorough cleaning procedure (designated “Extreme Clean Procedure”) which takes about 15 min. The technicians did not experience any clogging of the needle during the study. The capillary needle of the flow cell in the instrument is very delicate and replacement should be done very carefully. Technicians never had to replace the flow cell during the study.The laboratory evaluation of the Muse Auto CD4/CD4% system was conducted in accordance with the WHO prequalification evaluation protocol to verify whether this system met the WHO acceptance criteria for CD4 technologies . Those aAccuracy (bias) was assessed by comparing results of the Muse Auto CD4/CD4% to the results obtained by an established reference method/instrument. This is the easiest way to assess accuracy of a new CD4 technology as there are no international reference blood samples to assess the bias Alternatively, the bias can be assessed by comparing the results a CD4 technology to the consensus value of an external quality assessment program (EQA). The consensus value is defined as the average CD4 result of all laboratories participating laboratories in the EQA program and the difference between the individual result and the consensus value is a good estimated of the bias. During the present study, the average difference (bias) between Muse Auto CD4/CD4% and the FACSCalibur results were negligible (0.5% or 4 cells), and well within the maximum allowable relative bias of 10%. This small bias is confirmed by the regression analysis with a slope close to 1 (0.97), and a correlation of 0.99. There was a very slight tendency towards larger underestimation at higher CD4 T cell counts as the relative bias increased from -0.7% to -2.1% between 200 and 1000 CD4 T cell counts .In case the Muse Auto CD4/CD4% would be used for taking decisions with regard to treatment initiation, the present study found that sensitivity at 100 CD4 T cells/μL was 86% and ≥90% at the higher thresholds with a specificity of ≥95% indicating that the number of misclassified patients would be relatively low .The instrument uses small volumes of blood and reagent (10μL each). This could be considered as a concern as pipetting of such small volumes may introduce larger errors if pipets are not properly calibrated and technicians not properly trained. However, this was not found to be an issue during this study as the precision studies produced very good results. The obligatory pipetting proficiency test (pass/fail) prior to running blood samples is certainly helping to assure good pipetting practice.Until today, two field studies of the Muse Auto CD4/CD4% have been published, both by the same group ,19. One The observation that the Muse Auto CD4/CD4% is small but precise and accurate instrument is encouraging and instruments like the Muse could be used to monitor HIV disease and to screen patients for the risk of opportunistic infections. Even when CD4 T cell counting will lose importance in monitoring ART, instruments like Muse Auto CD4/CD4% are still considered to be as an essential disease-specific IVD tool for health care facilities with clinical laboratories [The limitation of the present study is that the analytical performance was conducted in a Western accredited laboratory with well-trained technicians. This study did not assess information on robustness and temperature and humidity fluctuations common to rural areas. Although our study validated the analytical performance characteristics of the instrument, more field studies are required to assess the performance of the Muse Auto CD4/CD4% in real-life conditions typical of rural settings.In conclusion, the Muse Auto CD4/CD4% system showed a very good analytical performance on fresh venous blood samples. The instrument was considered easy to use, with minimal training and infrastructure required, making it suitable for decentralization of CD4 T cell counting in rural resource-limited settings with a small laboratory, trained technicians and stable power supply.S1 Database(XLSX)Click here for additional data file.


Following publication of this work , the folFig 1A (ii) Stat3 panel appears to similar to the Input Src panel when horizontally flipped, and to the IP: Stat3/Src blot panel in Fig 1C (i) when flipped verticallyFig 2A (i) Input: EGFR panel appears similar to Fig 1A (i) Input: EGFR panelFig 3A the Stat3 blots for input samples appear similar in (i) and (ii)Fig 3C the EGFR and Src blots shown for input samples appear to be similar in (i) and (ii)Fig 3B EGFR lanes 1–3 appear similar to lanes 7–9Fig 6B (i) Src panel appears similar to Fig 3A (i) Src panelFig 6B (ii), in the β-actin panel there appears to be a vertical discontinuity between lanes 3 and 4, and lane 4 appears to have a different background as compared to other lanes in the panelThe authors have noted that all figure panels represent independent experiments. However, they have explained that in Fig 3A, the same Stat3 input panel was presented in panels (i) and (ii) because the same input control data was relevant to both Stat3 immunoprecipitation experiments. The immunoprecipitation experiments shown in panels (i) and (ii) used the same amount of the same samples. In Fig 6B (ii), authors commented that differences in background could be attributed to lower levels of protein in lane 4 compared to lane 3.While the authors stand by the validity of the experimental data and the conclusions reached in the study, owing to the time passed since the experiments were completed they were unable to provide any of the original images or raw underlying data in the article. Hence, we have been unable to resolve the above concerns.PLOS ONE Editors retract this article.In light of the image concerns and the unavailability of primary data, the


Human embryos are usually cultured to blastocyst stage by Day 5 or 6 after insemination. However, some embryos grow slowly and reach blastocyst stage at Day 7. Acceptable live birth rates have been reported after transfer of Day 7 blastocysts resulted from fresh oocyte in vitro fertilization (IVF). It is unknown whether an extended embryo culture to Day 7 is necessary for cryopreserved oocyte IVF to obtain more transferrable blastocysts.In this study, 455 oocytes from 57 cycles were warmed, inseminated, and the resulting embryos were cultured by Day 7 to examine blastocyst development after extended culture. Fifty one blastocysts from 16 cycles were biopsied to examine embryo aneuploidies.It was found that 35.1% of the cycles had Day 7 blastocysts, and 3.5% of the cycles had only Day 7 blastocysts. Day 7 blastocysts accounted for 15.6% of total blastocysts. The proportion of top quality of blastocysts was lower at Day 7 than at Day 5 or 6. However, no differences were observed on aneuploid blastocyst rates among Days 5, 6 and 7. Similar clinical pregnancy, ongoing pregnancy and embryo implantation rates were obtained after Day 7 blastocyst transfer as compared with Day 5 or 6 blastocyst transfer.These results indicate that embryos from oocyte warming cycles should be cultured to Day 7 if they do not reach to blastocyst stage by Day 6 so that number of usable blastocysts can be increased. Oocyte cryopreservation has become one of the most important human assisted reproductive technologies, which provides approaches of fertility preservation for women who want to delay childbearing , and forThe procedures for in vitro fertilization (IVF) and embryo culture did not have any difference between cryopreserved/warmed oocytes and fresh oocytes. However, recently, it has been found by time-lapsing analysis and morphokinetic that cryopreserved/warmed oocytes had about 1 h of delayed fertilization and embryo development . DelayedFor cryopreserved oocytes from oocyte bank, oocyte number is very limited, usually 6–8 per cycle, thus the number of embryos developed to blastocyst stage is much fewer than that from most fresh oocyte IVF cycles. Therefore, it may be necessary to extend embryo culture to Day 7 so that slow growing embryos can reach to blastocyst stage. In our clinic, we started to Day 7 culture for all oocyte warming cycles from 2019, and our preliminary data showed that implementation of Day 7 culture for oocyte warming IVF cycles increased overall blastocyst number.In this study, 455 cryopreserved donor oocytes were warmed for 57 recipient cycles. Warmed oocytes were inseminated by intracytoplasmic sperm injection and all embryos were extended to Day 7 culture if they did not reach blastocysts by Day 6. As shown in Table Day 7 blastocysts had poorer quality as compared with Day 5 or Day 6 blastocysts , 11, 12.Although previous studies found that more Day 7 blastocysts were aneuploid as compared with Day 5 or Day 6 blastocysts, suggesting that slow growing embryos may be resulted from oocyte aneuploidy , 11, 12.In the present study, there were 35 fresh blastocyst transfers and 16 frozen embryo transfer (FET). There were no statistical differences between fresh blastocyst transfer and FET in terms of clinical pregnancy rates (60.0 vs 56.3%), ongoing pregnancy rates (54.3 vs 50.0%) and embryo implantation rates (53.9 vs 58.8%).Acceptable live birth rates have been obtained after Day 7 blastocyst transfers –10. HoweFor fresh oocyte donation, recipients usually receive many oocytes and high quality of oocytes usually produces enough blastocysts at Days 5 and 6, thus very few laboratory may culture embryos from donor oocytes to Day 7. This may be the reason that no data is published on the transfer of Day 7 blastocysts resulted from donor oocytes. However, most IVF clinics now switch fresh oocyte to frozen oocyte IVF when donated oocytes are used , and 6 oIn conclusion, our preliminary data with limited case numbers indicate that Day 7 culture is necessary for cryopreserved/warmed oocyte IVF cycles, especially for those cycles with limited number of oocytes being warmed. The proportions of cycles with Day 7 blastocysts and proportions of blastocysts obtained at Day 7 appear to be higher than those observed with fresh oocyte IVF. In addition, our limited data indicate that aneuploid rate with frozen donor oocytes did not differ between Day 7 blastocysts and Days 5/6 blastocysts although quality of Day 7 blastocysts is not as good as quality of Day 5/6 blastocysts. Transfer of Day 7 blastocysts resulted in successful embryo implantation and clinical pregnancy, and both embryo implantation rate and clinical pregnant rate were not different from those with Day 5 or Day 6 blastocyst transfer. Putting together, these data suggest that the implementation of Day 7 culture for those embryos that have not developed to blastocysts by Day 6 is necessary in oocyte warming cycles.


Dear Editor,De novo lipogenesis and gluconeogenesis in the liver contribute, at least in part, to the dynamic homeostasis of lipid and glucose levels  and CREB regulated transcription coactivators (CRTCs)  was also elevated in Crtc2−/− mice as judged by real-time quantitative PCR (qPCR)  . Together, these results indicate that CRTC2 inhibits SREBP1c activity during fasting.To investigate the effect of CRTC2 on SREBP1c processing during fasting, we fasted mice for 12 h and evaluated SREBP1c processing. We found that higher levels of mature SREBP1c accumulate in ons Fig. . TherefoInsig2a expression and thus attenuated SREBP1c processing in wild-type mice during fasting  enhanced Insig2a is a predominant isoform of Insig2 in the liver  and constitutively active (CRTC2/AA)  treatment, while Crtc2 deficiency by knockdown or knockout attenuated Insig2a-luc activity  studies to determine whether Insig2a is a direct target of CRTC2/CREB. Supporting this notion, glucagon stimulation dramatically increased both CRTC2 and CREB occupancy on the Insig2a promoter . This highlights the important role of the CRTC2/CREB complex in SREBP1c maturation during fasting. Meanwhile, our results fill the gap in our understanding of how glucagon or fasting signaling modulates SREBP1c processing.It is well known that fasting or glucagon attenuates SREBP1c processing .All institutional and national guidelines for the care and use of laboratory animals were followed. Yuanyuan Zhang, Yi Liu, Liqun Chen, Yiguo Wang and Jinbo Han declare that they have no conflict of interest.Yuanyuan Zhang and Jinbo Han designed the study and analyzed the data. Yuanyuan Zhang, Yi Liu and Liqun Chen performed the experiments. Jinbo Han wrote the paper. All authors reviewed and commented on the manuscript.Supplementary material 1 (PDF 807 kb)Below is the link to the electronic supplementary material.


On page 4 of the original publication , the cor570 was measured using microplate reader at 200 μl per well.Quantitative measurement was performed by dissolving CV stained pellicle in 4 ml of 85% ethanol and the ODTable


Jumping translocations of 1q refer to the break-off of chromosome 1q as a donor fusing to two or more recipient chromosomes. We detected jumping translocations of 1q in three patients with initial diagnosis of myelodysplastic syndrome (MDS) and later progression to acute myeloid leukemia (AML). Review of literature found jumping translocations of 1q in 30 reported cases of MDS and AML. The cytogenetic findings from these 33 cases showed that seven cases had a stemline clone and 26 cases had de novo jumping translocations of 1q in which 5% of cell lineages had additional structural rearrangements. In 75% of cases, the 1q donor jumped to the short arm of recipient acrocentric chromosomes. Approximately 82% of the fusions occurred in the telomeric regions of short and long arms and 18% occurred in the pericentric or interstitial regions of recipient chromosomes. Hypomethylation of the donor 1q pericentromeric region and shortened telomeres in recipient chromosomes were associated with the formation of jumping translocations. Jumping translocations of 1q as an indication of chromosomal instability pose high risk for progression of MDS to AML and a poor prognosis. Further understanding of underlying genomic defects and their clinical significance will improve overall treatment and patient care. Jumping translocations are a rare type of cytogenetic abnormality initially detected in a case of acute monocytic leukemia and later found in various types of leukemias –3. JumpiA retrospective review of cases with MDS and AML from the CytoAccess database in Yale Cytogenetics Laboratory found three cases with jumping translocations of 1q . ChromosThis male patient was first evaluated at age 59 with an indication of leukopenia and mild anemia; cytogenetic analysis found a normal result. Two years later, this patient was diagnosed with MDS and chromosomal analysis on a bone marrow aspirate showed an abnormal clone with jumping translocations of 1q. The karyotype was as follows: 46,XY,der(15)t[7]/46,XY,der(21)t[3]/46,XY,dup(1)(q21q42)[2]/46,XY,der(14)t[2]/46,XY[6]. FISH analysis using dual color probes for the PBX1 gene at 1q23.3 and TCF3 gene at 19p13.3 detected three copies of 1q in 32% of bone marrow cells. This patient started chemotherapy and then received a bone marrow transplant in two months. Six months later, the patient progressed to AML and died at age 62. Final chromosomal analysis revealed a karyotype of 46,XY,der,del(20)(q11.2q13.3)[15]. FISH tests confirmed three copies of 1q and a deletion at 20q in 83.5%-96.5% of bone marrow cells.This patient, a 63-year old male, was referred with MDS by an indication of MDS with excess blasts. Chromosomal analysis showed a karyotype of 46,XY,der(15)t[4]/46,XY,der(22)t[2]/46,XY[7]. FISH analysis using dual color probes for the CDKN2C gene at 1p32.3 and CKS1B gene at 1q21.3 confirmed three copies of 1q in 5.5% of bone marrow cells. Over the next six months, the percentage of abnormal cells increased despite two rounds of chemotherapy and the patient progressed to AML. A transit clone with the der(15)t and a deletion at 11q was noted. The last chromosome analysis revealed a karyotype of 46,XY,der(15)t[13]/46,XY,der(22)t[2]. FISH detected three copies of 1q in 94% of bone marrow cells. This patient died eight months after the initial finding of the jumping translocations of 1q.This patient was a male at age 73 when diagnosed with MDS. Chromosomal analysis showed a karyotype of 46,XY,der(15)t[13]/46,XY[7]. FISH confirmed three copies of 1q in 82.5% of bone marrow cells. Two years later, the patient progressed to AML and died soon after the last chromosome analysis which revealed a karyotype of 46,XY,der(15)t[9]/46,XY,der(22)t[4]/46,X,der(Y)t,t[3]. FISH detected three copies of 1q in 94% of bone marrow cells.The cytogenetic findings of successive studies from these three patients all showed persistent jumping translocations of 1q with an increased percentage in bone marrow cells. The chromosome patterns of jumping translocations of 1q are shown in The results from these 33 cases revealed the chromosomal fusion patterns between the 1q donor chromosome and different recipient chromosomes. However, all studies done by routine chromosome analysis lacked the molecular characterization of breakpoints involving the chromosome fusion, underlying genomic imbalances, and tumor gene mutations. A recent study using genomic array testing and targeted gene panel next-generation sequencing detected the 100–107 Mb duplication of 1q with breakpoints in 1q21.1-q21.3 region and recurrent mutations in the TET2 and SF3B1 genes , 13.Approximately one-third of MDS/AML cases with jumping translocations of 1q either emerged from a stemline abnormal clone or evolved additional clonal abnormalities and two-thirds were detected as de novo chromosomal abnormalities. Shortened telomeres and many variant telomeric repeats were detected in the fusion points from jumping translocations of 1q in a case of MDS progressed to AML; this result suggested that the extended proliferation of cancer cells might be related to the loss of telomeric function . Copy nuThe progression of MDS to AML and poor prognosis were noted in the present three cases with jumping translocations of 1q. This result was consistent with previous findings in which jumping translocations of 1q were associated with a high risk of progression to AML and poor prognosis and thus warranted aggressive therapy , 10. CurIn summary, jumping translocations of 1q have been shown as an indication of chromosomal instability likely induced by epigenetic alterations of hypomethylation and shortened telomeres. The jumping translocations of 1q showed unique rearrangement patterns with multiple cell lineages which all have a gain of donor chromosome segment and lack other complex structural or numerical rearrangements. It is not clear whether these epigenetic alterations occur independently as a two-step event or concurrently by driving genetic defects. Further molecular analyses to understand the genetic defects causing jumping translocations of 1q and related disease-causing mechanism can facilitate targeted therapy for this type of MDS and AML.


ALGA0110477 (SSC12), explained 9.3% of the genetic and 6.2% of the phenotypic variance for viral load. Dissection of the SSC12 QTL based on gene annotation, genomic and RNA-sequencing, suggested that a missense mutation in the SYNGR2 (SYNGR2 p.Arg63Cys) gene is potentially responsible for the variation in viremia. This polymorphism, located within a protein domain conserved across mammals, results in an amino acid variant SYNGR2 p.63Cys only observed in swine. PCV2 titer in PK15 cells decreased when the expression of SYNGR2 was silenced by specific-siRNA, indicating a role of SYNGR2 in viral replication. Additionally, a PK15 edited clone generated by CRISPR-Cas9, carrying a partial deletion of the second exon that harbors a key domain and the SYNGR2 p.Arg63Cys, was associated with a lower viral titer compared to wildtype PK15 cells (>24 hpi) and supernatant (>48hpi)(P < 0.05). Identification of a non-conservative substitution in this key domain of SYNGR2 suggests that the SYNGR2 p.Arg63Cys variant may underlie the observed genetic effect on viral load.Porcine circovirus 2 (PCV2) is a circular single-stranded DNA virus responsible for a group of diseases collectively known as PCV2 Associated Diseases (PCVAD). Variation in the incidence and severity of PCVAD exists between pigs suggesting a host genetic component involved in pathogenesis. A large-scale genome-wide association study of experimentally infected pigs (n = 974), provided evidence of a host genetic role in PCV2 viremia, immune response and growth during challenge. Host genotype explained 64% of the phenotypic variation for overall viral load, with two major Quantitative Trait Loci (QTL) identified on chromosome 7 (SSC7) near the swine leukocyte antigen complex class II locus and on the proximal end of chromosome 12 (SSC12). The SNP having the strongest association,  SYNGR2 gene that influences the ability of the PCV2 virus to replicate, which can affect pig growth and immune system following infection. This research will aid in the development of genetic tests with the ability to predict PCV2 susceptibility. Early identification of pigs that are susceptible to PCV2 infection will result in an improvement in the general health and welfare of pigs.The cost of managing Porcine Circovirus 2 (PCV2) associated diseases in the US alone costs the swine industry more than $250 million a year. This virus is found in all swine populations in the US, but only a few pigs get sick and show signs of disease. Previous anecdotal field data showed differences between pig breeds in both incidence and severity of PCV2-associated diseases, supporting the role of host genetics in disease susceptibility. This research, including over 1,000 experimentally infected pigs with PCV2, is the largest study ever conducted to understand the role of host genetics in PCV2 related illnesses. We found that a pig’s own genetics can impact the ability of PCV2 to multiply and cause disease. Specifically, we found a mutation in the  Circoviridae family and the smallest virus known to infect mammalian cells. Despite its small size, this single-stranded circular DNA virus has been identified as the causative source of a set of systemic disorders known as Porcine Circovirus Associated Diseases (PCVAD), which includes Post-Weaning Multi-systemic Wasting Syndrome (PMWS). PMWS is characterized by severe weight loss, respiratory and enteritic conditions that can lead to mortality [Porcine Circovirus 2 (PCV2) is a member of the ortality . Other sortality . Currentortality .in vitro siRNA and gene editing validation models to elucidate the role of host genetics in pathogenesis by identifying genes and genetic variants that could influence PCV2 susceptibility.Anecdotal field data and initial experimental evidence , 5 descrSubstantial variation in the timing and magnitude of immune response, and in the efficiency of PCV2 replication, was reported in our previous studies of experimental infection with a PCV2b strain , 8. The SLAII) at 24–25 Mb while the other was located near the proximal end of SSC12, at 3–4 Mb. The SNPs associated with the largest genetic variance, ALGA0039682 and ALGA0110477, in each of these two windows explained 65.1% and 99.7%, respectively of the genetic variance explained by their respective windows.The initial GWAS was performed using a BayesB-based approach where individual SNPs and successive 1 Mb windows of the genome were evaluated for association with phenotypic variation . BayesiaALGA0110477) associated with the largest effect on viral load explained 9.3% of the genetic variance and 6.2% of the phenotypic variance for PCV2 viral load . Interestingly, SNPs in the genomic region encompassing these markers did not show strong evidence of association with viral load despite LD with ALGA0110477. A more nuanced analysis fitting haplotypes across the region rather than individual SNPs using BayesIM [ALGA0110477, providing support for the initial discovery based only on ALGA0110477. The unplaced scaffold containing the SSC12 marker, ALGA0110477, did not contain any annotated candidate genes that might underlie the observed effects, and the available sequence surrounding the marker only extended for 84 bp. Using inverse PCR (iPCR), the proximal sequence was extended to 1,252 bp. This extended sequence was used to interrogate contigs from an early version of a long read-based genome assembly of a pig (accession NPJO00000000), [ALGA0110477 used for identification of candidates genes described below. The recent release of a long read-based improved reference assembly, Sscrofa 11.1 (GenBank accession GCA_000003025), supported more accurate ordering and placement of markers, including ALGA0110477 .The SNP  are located at the proximal end of SLAII with DRA being the closest gene (24.8 Mb) from the SLAII complex. Combined, these two SNPs explained 3.8% of the genetic variance for viral load. While the role of SLAII in antigen recognition and immune response in a variety of infectious diseases is well established, highly polymorphic genes and extended LD are the main factors limiting the discovery of functional variants. The SNP ALGA0039710, was still associated with the largest effect in viral load in an analysis of a subset of samples (n = 268) with extreme phenotypes that included novel SNPs located in genes in the QTL such as DRA, C2, CFB, NELFE, SKIV2L [One of the genomic regions associated with viral load was located on SSC7, in the vicinity of , SKIV2L .Sscrofa 11.1, we used the un-annotated long-read scaffold to identify the genes surrounding the SSC12 marker ALGA0110477. Ab initio gene prediction [-64 and a pBLAST score > 200 were identified. Five of these genes were found to be expressed in RNA-seq data of peripheral blood from pigs subjected to PCV2. These genes are involved in immune response and cytokine signaling (SOCS3), inhibition of apoptosis and promotion of cell proliferation (BIRC5), membrane trafficking and transport (SYNGR2) and transmembrane ion channels (TMC6 and TMC8). The number of isoforms observed across these genes varied from one (SOCS3) to more than 10 (TMC6).Previous to the recent release of the ediction  and pBLAALGA0110477 homozygotes exhibiting extreme viral load following PCV2 challenge uncovered missense (n = 4), synonymous (n = 11), and UTR (n = 10) SNPs and an UTR indel across the 5 candidate genes located in the QTL region. In addition, 1–2 kb sequencing upstream of the Transcription Start Site (TSS) for BIRC5, SOCS3 and SYNGR2 uncovered 32 SNPs and 4 short indels. These novel polymorphisms and 580 SNPs from the Porcine SNP60 BeadArray were mapped to the 19 Mb scaffold using BLAT. The highest LD between ALGA0110477 and the polymorphisms mapped on the scaffold was with a SNP from the Porcine SNP60 BeadArray  located 24.5 kb away followed by a group of 3 SNPs from SYNGR2 (r2 = 0.42–0.48) including the missense polymorphism SYNGR2 p.Arg63Cys located 123.7 kb away. Using an additive linear mixed model and a subset of pigs with extreme high and low viral loads (n = 268) genotyped for all polymorphisms mapped to the scaffold (n = 629), we found that the SYNGR2 p.Arg63Cys SNP and a 1bp indel located 343 bp upstream of BIRC5 TSS were associated with the largest effects on PCV2 viral load (P < 0.0001). The phenotypic variance explained by each of these novel polymorphisms was substantially larger (21–23% +/- 6.1–6.4%) compared to the original QTL SNP ALGA0110477 (12.6 +/- 4.8%). As expected, these polymorphisms were associated with large effects on all weekly viremia measures (P < 0.0001), and on PCV2-specific antibodies, starting from 14 dpi for IgM and 21 dpi for IgG (p < 0.0001). The effects on growth during the challenge were most evident after 14 dpi as well as during the entire challenge period .RNA-seq analysis of alternate SYNGR2 p.Arg63Cys SNP  is located in the first loop of synaptogyrin-2 (SYNGR2) in a region conserved across mammals [Arg residue is prevalent in other species  sometimes being replaced by His , Lys  or Gln  while the Cys residue appears to be specific to swine  being lower compared to the heterozygote  and alternate homozygote . The favorable homozygous genotype was also associated with lower weekly viremia  was detected between SYNGR2 p.Arg63Cys and the SNPs associated with the largest effects from the QTL detected on SSC7 (ALGA0039682 and ALGA0039710). The effect of this SNP was confirmed in an independent validation data set consisting of 71 pigs infected with the same PCV2b strain and representing all three SYNGR2 p.Arg63Cys genotypes. This SNP had an effect on viremia starting from 14 dpi to 42 dpi (P < 0.05). The viremia of the homozygous genotype for SYNGR2 p.63Cys allele was lower than the alternate homozygote  and the heterozygote .The SYNGR2 p.63Cys allele (Q = 1.2%) compared to Engle et al. (2014) dataset (Q = 18.3%), which is less favorable for detecting associations in additive statistical models, and 2) lower ability of the ALGA0110477 to capture the low viremic effects of SYNGR2 p.63Cys. While in the Engle et al. (2014) dataset the presence of the ALGA0110477 C variant had a probability of 65% to be located on the same haplotype with SYNGR2 p.63Cys, in McKnite et al. (2014) this variant is found in similar proportions in haplotypes that carry different SYNGR2 alleles .Our inability to uncover the QTL located on SSC12 in our previous report  was baseBIRC5 (BIRC5 g.-343delA) was found to be in high LD (r2 = 0.83) with SYNGR2 p.63Cys allele and as expected was associated with low viral load (P < 0.0001). The deletion was predicted to affect a potential motif for NR5A2, a DNA-binding zinc finger transcription factor. However, no significant difference in expression was observed between BIRC5 genotypes across time points following in vivo PCV2 challenge (P < 0.17). At 14 dpi the homozygotes for the insertion exhibited an elevated nominal expression compared to the other genotypes, but the difference was not significant (P = 0.061).The 1bp deletion located 343 bp upstream of the TSS of SYNGR2 p.Arg63Cys and BIRC5 g.-343delA (r2 = 0.83) hampered the ability to distinguish their individual effects in the in vivo challenge dataset. In contrast, the LD between SYNGR2 p.Arg63Cys and other SYNGR2 SNPs was limited (r2 < 0.26), as well as the LD between BIRC5 g.-343delA and other BIRC5 polymorphisms (r2 < 0.16). A very defined LD block exists from ALGA0110477 to SYNGR2 that includes 16 DNA polymorphisms. Within this block, there were 9 haplotypes with individual frequencies greater than 1% that accounted for 85% of the haplotypes present. A single haplotype (Hap 1) carried the SYNGR2 p.63Cys allele. The frequency of this haplotype in our resource population was 0.28. The remaining eight haplotypes carried the SYNGR2 p.63Arg allele. A haplotype substitution effect demonstrates that Hap 1 was associated with the lowest viral load  and indigenous Chinese breeds , as well as wild pigs such as common Warthog (Phacochoerus africanus), Java (Sus verrucosus) and Visayan (Sus cebifrons) warty pigs. Phylogenetic analysis based on the polymorphisms from this haplotype block separated the breeds into paternal  and indigenous Asian breeds while the maternal breeds (Large White and Landrace) were located in between these two groups  was the closest to Pietrain and Duroc. The other haplotypes present in our resource population were similar to those identified in Berkshire (Hap 4), Hampshire (Hap 5), Meishan (Hap 2), or Jinhua (Hap 9). This finding suggests that SYNGR2 p.63Cys is the predominant allele in Duroc and Pietrain, breeds in which more emphasis is placed on growth related traits compared to breeds such as Large White and Yorkshire. Our analysis of SYNGR2 p.Arg63Cys in pure breeds showed that SYNGR2 p.63Cys has a frequency of 0.25 in Yorkshire, 0.53 in Landrace and 0.78 in Duroc.The haplotype that carried the SYNGR2 p.Arg63Cys substitution is in a conserved domain involved in vesicle formation [SYNGR2 affecting replication of a tick-borne human RNA virus [SYNGR2 may play a role in the internalization and early release of PCV2 from endosomes influencing its replication. The Porcine kidney 15 cell line (PK15) has an epithelial origin and is a well-established model system for PCV2 innate immunity and cellular pathogenesis [SYNGR2 p.63Arg and the insertion of BIRC5 g.-343delA variants associated with high-viremia. Expression of SYNGR2 did not differ across time points following PCV2 infection of PK15 cells, corroborating in vivo findings. In order to validate a role of SYNGR2 in PCV2 replication, we transfected PK15 with siRNA targeting the mRNA of SYNGR2. We evaluated two siRNA (siRNA-01 and siRNA-03) at two different concentrations (10 nM and 20 nM) and found that siRNA-01 was the most efficient to knock-down mRNA level of SYNGR2 compared to the cells subjected to a scramble siRNA control. A substantial reduction (>75%) in SYNGR2 mRNA level was observed starting 24 hours after transfection  when compared to scramble siRNA and non-transfected control cells, indicating a role of SYNGR2 in viral replication . This deletion is predicted to cause a shift in the reading frame and an altered protein (195 residues) beginning at amino acid residue 42 compared to the wildtype SYNGR2 sequence (224 residues). The deleted fragment included the conserved motif located in the first loop while the shift in the reading frame affected the C-terminus of SYNGR2. A significant reduction in viral titer starting at 24 hpi in cells (PK15 edited clones were generated by CRISPR-Cas9 Ribonucleoprotein (RNP) complex approach with a pair of guide RNAs (31_AC/40_AC) targeting the second exon of morphism . Sequencin cells  and 48 hin cells  was obseSYNGR2 was observed in E1 cells compared to wildtype PK15 cells with a significant difference observed at 24 hpi , a novel tick-borne bunyavirus in humans [SYNGR2 mRNA had been upregulated more than 200-fold at 36 hpi with SFTSV. In vitro silencing of SYNGR2 resulted in a decrease in viral replication and a reduction in the number and size of the inclusion bodies, further substantiating the role of SYNGR2 in facilitating SFTSV infection [Substantial variation in efficiency of viral replication and specific immune response was reported in our previous studies of experimental infections with PCV2 , 8. Hostocytosis , 18. Then humans . SYNGR2 nfection .SYNGR2 in PK15 cells was associated with a significant reduction in PCV2 titer, indicating a role of SYNGR2 in promoting viral replication. SYNGR2 p.Arg63Cys, the only missense polymorphism identified in SYNGR2, is characterized by a predicted change in charge and hydrophobicity of the first loop that connects two essential transmembrane domains, and is located in a region conserved across mammals. In rats, the first intraluminal loop and the C-terminus of SYNGR2 were found to be crucial for successful incorporation of the protein into vesicular membranes and vesicle formation [SYNGR2 p.63Arg allele  in Duroc than in Large White or Landrace . This selection pressure and the presence of mild PCV2 strains could have resulted in a rapid increase in the frequency of the SYNGR2 p.63Cys allele in both Pietrain and Duroc.Predominance of the SYNGR2 mRNA levels following in vitro or in vivo infection with PCV2. This may reflect important distinctions between SFTSV and PCV2. For instance, SFTSV is an RNA virus with the capacity to replicate within intracytoplasmic inclusion bodies, or viral factories. As Sun et al. (2016) demonstrated, SYNGR2 is a component of these vesicles and necessary for their formation [SYNGR2 p.Arg63Cys on overall protein folding or conformation of the first loop. A shift in the position of the second transmembrane helix as a result of the substitution in the loop region was predicted by HMMTOP software [In our research we did not observe an increase in ormation . As SFTSormation   partial deletion of a key domain by gene editing, provided evidence of the involvement of SYNGR2 in PCV2 infection. Since the SYNGR2 p.Arg63Cys polymorphism is the only missense mutation within the entire gene and located in this key domain, it is a plausible QTN (Quantitative Trait Nucleotide) candidate for PCV2 susceptibility. However, future studies of the mechanistic role of SYNGR2 and specifically of SYNGR2 p.Arg63Cys substitution will be required to provide additional experimental evidence of their role in PCV2 replication and pathogenesis.In this study, decreased viral titer by 1) exogenous reduction of The experimental design and procedures used during this research project were approved by the Institutional Animal Care and Use Committee of the University of Nebraska -Lincoln.Erysipelothrix rhusiopathiae, Clostridium perfringens, Leptospirosis and Colibacillosis and tested negative for Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). Prior to experimental infection, the pigs tested negative for presence of PCV2 in peripheral blood by real time quantitative PCR (qPCR) and had a sample/positive ratio (S/P) lower than 0.4 for IgM and 0.3 for passive IgG, the PCV2-specific antibodies [Experimental PCV2b challenge was conducted in nine batches that varied in size from 81 to 141 pigs, with a total of 974 pigs. The genetic makeup of this resource population consisted of crossbred pigs produced by 14 genetic lines generated by seven genetic programs. The dams of the experimental pigs had been vaccinated for PCV2 at 3 weeks of age with a single dose of Ingelvac CircoFLEX vaccine (Boehringer Ingelheim). The suppliers of the pigs also had vaccination programs for Porcine parvovirus, SYNGR2 p.Arg63Cys genotypes infected with the same PCV2b strain at 5 weeks of age was generated using the same experimental conditions. A group of 40 pigs (SYNGR2 p.63Arg/63Cys and 63Arg/63Arg) vaccinated for PCV2 at 3 weeks of age were used as controls. The vaccinated pigs were housed in the same room with the experimentally infected pigs, but in different pens.A validation dataset consisting of a group of 71 pigs representing all three 4.0 50% tissue culture infection dose (TCID50) intranasally and intramuscularly.The PCV2b strain (UNL2014001) used for the experimental infection was obtained from a pig with symptoms characteristic to Post-weaning Multisystemic Wasting Syndrome (PMWS), which is the most common PCVAD syndrome. The strain was sequenced (accession KP016747.1) using dye terminators and the sequence was compared to PCV2 strains available in GenBank . The strPCV2 specific antibodies, IgM and IgG, were profiled weekly from serum using ELISA (Ingenasa) as described in McKnite et al. (2014). Estimates of the number of PCV2b copies, or viremia, was performed using viral genomic DNA isolated by QIAamp DNA Minikit (Qiagen) and quantified by qPCR using TaqMan Master Mix and ABI 7900 Real Time PCR System (Thermo Scientific). The viral load for each pig during the entire challenge was represented as area under the curve (AUC) based on an algorithm that takes into account viral levels observed at each time point following infection  fitting a smooth curve over the 28 days and summing the areas in 0.01 time increments [Sscrofa 11.1 porcine reference genome assembly and used in GWAS via GenSel software package [The DNA was isolated from ear and tail tissue clips using DNeasy or Puregene blood and tissue kits (Qiagen). The experimental animals were genotyped using either the first or second generation of the Porcine SNP60 BeadArray (Illumina) that contain 62,183 and 61,565 SNPs, respectively. Only the common SNPs present in both BeadArray versions  were mapped on  package . DNA sam package .SOCS3, BIRC5 and SYNGR2 and their 2–4 kb region upstream of the transcription start sites (TSS) was performed using dye terminators and ABI PRISM 3100 Genetic Analyzer (Thermo Scientific) on high and low viremic samples. Discovery and validation of the polymorphisms detected by RNA-seq was based on alignment of DNA sequences using Sequencher software (Gene Codes). Potential impact of the polymorphisms located in the proximal promoter on important regulatory motifs was evaluated using FIMO (version 4.11.3) [Targeted DNA sequencing of candidate genes in the SSC12 QTL region including  4.11.3)  and the SOCS3, BIRC5, SYNGR2, THA1, TMC6 and TMC8 was performed by multiplex assays using Sequenom MassARRAY platform and Sequenom iPLEX chemistry based on the manufacturer protocols .Genotyping of polymorphisms located in the transcribed regions and proximal promoters of π equal to 0.99 that assumed a prior probability of 1% of the SNPs having a non-zero effect. The Markov chain included 40,000 samples with the first 1,000 being removed as burn-in. Markov chain was set to use every 40th sample to estimate posterior distribution for the genetic variance explained by each 1 Mb window of the reference genome. This distribution was used to estimate the probabilities of each 1Mb window having a variance greater than 0 or greater than the average variance explained by each 1Mb window as described in McKnite et al. (2014).The proportion of phenotypic variance explained by host genetics for PCV2-viremia, PCV2-specific antibodies (IgM and IgG) and average daily gain (ADGi) during experimental infection was estimated based on Porcine SNP60 BeadArray genotypes using a BayesB model  and GenSBayes Interval Mapping (BayesIM) was implemented to derive haplotype effects across the genome on PCVAD-related traits as described in Kachman (2015) and Wilson-Wells and Kachman (2016) . BrieflyALGA0110477 to SYNGR2 that includes 16 DNA polymorphisms, was estimated as haplotype substitution effects. Contrasts between haplotypes were estimated using a linear mixed model as described above including one variable for each haplotype with values 0, 1, and 2 corresponding to the animal having 0, 1, or 2 copies of the haplotype in question. The haplotype substitution effects were presented as deviations from the mean of the haplotypes.Associations between the single marker genotypes and phenotypic variation were tested using a linear mixed model fitted by JMP 10.0 (SAS Inst. Inc.) that included marker genotype and batch as fixed effects, litter and pen as random effects while age at infection and IgG were used as covariates. Additive and dominance effects were estimated for each of the targeted DNA polymorphisms. A similar model was used to estimate the interaction between SNPs. The potential effect on viral load of the haplotypes in the defined LD block from ALGA0110477 sequence, a SNP previously unmapped on the Sscrofa 10.2 reference genome. A genomic scaffold (19 Mb) of the proximal end of SSC12 was constructed based on Pacific Biosciences sequencing reads [ALGA0110477 sequence and all SSC12 mapped and unmapped SNPs were determined on the genomic scaffold using BLAT. Annotation of the QTL region on the SSC12 scaffold was based on RNA-seq alignments and BLAST but also using ab initio approaches such as GenScan [Inverse PCR (iPCR), using four  and six cutter  restriction enzymes (New England Biolabs), T4 DNA ligase (New England Biolabs) and nested PCR using AmpliTaq Gold 360 DNA polymerase (Thermo Scientific), was employed to expand the genomic DNA sequence surrounding the short  GenScan , 27 in cTT = 6) and low (NCC = 5) viremic genotypes for ALGA0110477 at 0, 7 and 14 dpi were subjected to RNA sequencing. RNA was extracted from peripheral blood collected in Tempus tubes using the Tempus Spin RNA Isolation Reagent Kit (Thermo Scientific). RNA samples were sequenced using Ion Proton technology as described in the manufacturer protocol (Thermo Fisher Scientific Inc.). The adaptor-free sequencing reads were trimmed and filtered using Trim galore (version 0.4) [Sscrofa 11.1. The number of reads mapped to each gene in the annotated QTL region was obtained using HTSeq (version 0.6.1p1) [In order to profile transcriptome changes and sequence variation related to PCV2 infection, peripheral blood samples collected from the validation group of pigs that exhibited high (Nion 0.4)  with lowion 0.4) . The rea0.6.1p1) .SOCS3, BIRC5 and SYNGR2 across time points following PCV2 infection was quantified using TaqMan Master Mix and CFX384 Real Time PCR (BioRad). The qPCR assays were designed using IDT Realtime PCR Tool software (www.idt.com) and sequences generated based on RNA-seq alignments. RNA was extracted from peripheral blood samples collected in Tempus tubes from a subset of pigs representing all genotypes from the validation data set that displayed extreme viral load (high vs low) (n = 40) from 0 to 21 dpi using the Tempus Spin RNA Isolation Reagent Kit (Thermo Scientific). Complementary DNA (cDNA) was obtained using a mix of random hexamers and poly dT primers using First strand cDNA Synthesis Kit . Expression of ribosomal protein L32 (Rpl32) gene was used for normalization. Mean normalized expression (MNE) values were calculated based on cycle crossing thresholds (CT) obtained for the technical triplicates taking qPCR efficiencies into account [ALGA0110477, SYNGR2 p.Arg63Cys or BIRC5 g.-343delA genotypes and time point following infection were log10 transformed and compared by t-test.Expression of the candidate genes  account . MNE val2) with 5.0x105 cells per well and infected with UNL2014001 PCV2b strain (TCID50 = 104) when 80–100% confluent at MOI = 0.00025. One hour following infection, cells were washed and fresh media was added (DMEM high glucose and 2% FBS). The cells were incubated at 37 °C with 5% CO2 for up to 5 days. Control cells were maintained the same way and mock-inoculated with plain DMEM high glucose media. Supernatants and cells were collected at specific time points and frozen at −80 °C. Viral DNA was extracted from supernatants using QIAamp DNA Mini kit (Qiagen). RNA, viral and host DNA was extracted from PK15 cells using AllPrep DNA/RNA Mini kit (Qiagen). TaqMan Master Mix and CFX384 Real Time PCR Detection System were used for quantification of PCV2 and expression profiling of BIRC5 and SYNGR2 from PK15 cells. Dideoxy sequencing of the cDNA and genomic DNA was used to profile the sequences and to genotype BIRC5 and SYNGR2 variants in PK15 cells.The porcine kidney cell line (PK15) was grown in DMEM high glucose media supplemented with 10% FBS and 1% Penicillin-Streptomycin . Cells were cultured in 12-well plates (4 cm2) with 2.5x105 cells per well when ~80% confluent with two siRNA oligos  targeting SYNGR2 mRNA and the AllStars Negative Control siRNA  at 10nM and 20nM concentrations. Transfection was performed using Lipofectamine RNAiMAX transfection reagent (Invitrogen) following the manufacturer’s protocol. Cell samples were collected 24, 48, 72, and 96 hours post transfection in PBS and centrifuged at 16,000xg for 1 minute to pellet the cells. RNA was extracted using RNAeasy Mini kit (Qiagen). Real Time PCR was used to profile SYNGR2 expression. siRNA oligo 01 and the AllStars Negative Control siRNA (20nM) were used for subsequent transfections prior to infection. The siRNA transfected cells were inoculated 24 hr after transfection following the same infection protocol described above. Statistical differences in viral titer between cell lines across time points were tested using a linear model fitted by JMP 10.0 (SAS Inst. Inc.) that included batch and cell line as fixed effects. Pairwise comparisons between least-squares means of the viral titers were based on the Tukey test.PK15 cells were transfected 24 hours after plating in 12-well plates (4 cmSYNGR2 were designed and ordered (IDT), three located upstream (5’) and three located downstream (3’) of the SYNGR2 p.Arg63Cys polymorphism. Each guide RNA was hybridized with fluorescently labeled Alt-R CRISPR-Cas9 tracrRNA ATTO 550 (IDT) and Alt-R S.p. Hifi Cas9 Nuclease V3 (IDT) following the manufacturer’s protocol to form Ribonucleoprotein (RNP) complexes. These RNP complexes were reverse transfected into PK15 cells using Lipofectamine RNAiMAX transfection reagent (Invitrogren) at a final concentration of 10nM. After 48 hours post transfection, genomic DNA was extracted using QIAamp Blood DNA Mini Kit (Qiagen) and amplified via PCR using LongAmp Taq DNA polymerase (NEB) with primers located in the introns flanking the second exon of SYNGR2 . The amplicons were subjected to T7 endonuclease I (NEB) digestion following the manufacturer’s protocol and visualized by agarose gel electrophoresis to assess cutting efficiency of each individual guide RNA. The ability of guide RNA pairs (upstream/downstream) to generate partial deletions of the second exon was assessed following the same RNP transfection protocol with a final RNP concentration of 20nM (10nM/guide RNA). After 48 hours post transfection, genomic DNA was extracted, amplified, and visualized via agarose gel electrophoresis.Six potential guide RNAs targeting the second exon of SYNGR2 mRNA sequence  to amplify full-length transcripts, which were sequenced using Dideoxy sequencing. A single clone (E1) homozygous for a 106bp deletion was plated and infected with PCV2b inoculate as previously described. Wildtype PK15 cells were concurrently infected and served as a control.A single guide RNA pair (sg31_AC/sg40_AC) was selected to generate PK15 edited clones. After 24 hours post transfection, the cells were collected and sorted using Fluorescence Activated Cell Sorting (FACS) into 96 well plates to generate single cell clones. Genomic DNA from each single cell clone was extracted using QuickExtract DNA extraction solution (Lucigen) following the manufacturer’s protocol and genotyped by PCR amplification and gel electrophoresis. RNA was extracted from selected clones using All Prep DNA/RNA Mini kit (Qiagen) and PCR was performed with primers located in the 5’ and 3’ UTRs of the SYNGR2 led to nonsense-mediated mRNA decay, expression of SYNGR2 was profiled by qPCR at 0, 24 and 48 hrs in edited and wildtype PK15 cells.To determine if induced changes in the mRNA sequence of S1 FigSscrofa 11.1 assembly. The y-axis represents the contribution of each SNP to the genetic variance. Alternate colors represent autosomes, from SSCs 1 to 18, chromosome X and Y followed by a set of SNPs without a genomic location.Each dot represents the proportion of genetic variance explained by an individual SNP. The x-axis represents the position of each SNP in the swine genome using (TIF)Click here for additional data file.S2 FigSscrofa 10.2 assembly including ALGA0110477 were excluded from the analysis. Each dot represents the model frequency associated with 50kb QTL. The X-axis represents the position of the 50 kb loci across the swine genome using Sscrofa 10.2 assembly. The Y-axis represents the model frequency of the association between a QTL and PCV2b viral load.Alternate colors represent autosomes, from SSC1 to 18. Unmapped SNPs in the previous (TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 FigSscrofa 11.1 assembly. The Y-axis represents the model frequency of the association between a QTL and PCV2 viremia. Alternate colors represent autosomes, from SSC1 to 18.Each dot represents the model frequency associated with each 50kb QTL. The X-axis represents the position of the 50 kb loci across the swine genome using (TIF)Click here for additional data file.S5 FigSscrofa 11.1 assembly. The Y-axis represents the model frequency of the association between a QTL and IgM following PCV2 infection. Alternate colors represent autosomes, from SSC1 to 18.Each dot represents the model frequency associated with each 50kb QTL. The X-axis represents the position of the 50 kb loci across the swine genome using (TIF)Click here for additional data file.S6 FigSscrofa 11.1 assembly. The Y-axis represents the model frequency of the association between a QTL and IgG following PCV2 infection. Alternate colors represent autosomes, from SSC1 to 18.Each dot represents the model frequency associated with each 50kb QTL. The X-axis represents the position of the 50 kb loci across the swine genome using (TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S9 Fig(TIFF)Click here for additional data file.S10 Fig(TIFF)Click here for additional data file.S11 Fig(TIFF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.