The potential role of human leukocyte antigen (HLA)-G as a target for new cancer immunotherapy drugs has increased the interest in the analysis of mechanisms by which HLA-G expression is regulated, and how the expression can be manipulated. We characterized HLA expression in breast cancer and malignant melanoma cell lines and investigated the induction of HLA-G expression by two distinct mechanisms: stimulation with interferon (IFN)-γ or inhibition of methylation by treatment with 5-aza-2’-deoxycytidine (5-aza-dC). The effect of IFN-γ and 5-aza-dC on HLA expression was dependent on the cancer cell lines studied. However, in general, surface expression of HLA class Ia was induced on all cell lines. Surface expression of HLA-G was inconclusive but induction of HLA-G mRNA was prevalent upon treatment with 5-aza-dC and a combination of IFN-γ and 5-aza-dC. IFN-γ alone failed to induce HLA-G expression in the HLA-G-negative cell lines. The results support that HLA-G expression is regulated partly by DNA methylation. Furthermore, IFN-γ may play a role in the maintenance of HLA-G expression rather than inducing expression. The study demonstrates the feasibility of manipulating HLA expression and contributes to the exploration of mechanisms that can be potential targets for immunotherapy in breast cancer and malignant melanoma. The assembly of immune responses is highly dependent on human leukocyte antigen (HLA) molecules. The HLA molecules are encoded in the human major histocompatibility complex (MHC) and mediate immune activation and immune suppression through HLA class Ia and class Ib molecules, respectively. The extent of immune regulation is inevitable in the context of cancer development and treatment and so is the role of HLA-mediated interaction between tumor and immune cells. Progression of cancer is often associated with altered expression of HLA molecules and multiple studies support that upregulation of HLA class Ib expression, especially HLA-G, is a mechanism, whereby tumors are able to escape immunological surveillance . A concoHLA class Ia genes, HLA-A, -B, and -C, are expressed on almost all nucleated cells. Whereas HLA class Ib mRNA expression is frequent in many tissues, surface expression is restricted in healthy tissue. Expression of HLA-G is mainly restricted to the trophoblast cells in the placenta, where it participates in maintaining tolerance at the fetal-maternal interface [The highly polymorphic nterface ,4. Howevnterface . Of the We have previously shown an association between high HLA-G and HLA-ABC expression with tumor aggressiveness and a worse prognosis in a study including 250 malignant melanoma patients . FurtherHLA-G exist in seven well-recognized isoforms. HLA-G1 to -G4 are membrane-bound molecules and HLA-G5- to -G7 are soluble proteins due to alternative splicing and skipping of different exons ,17,18,19The inhibitory role of HLA-G contributes to the idea of HLA-G being an immune checkpoint molecule with therapeutic potential . CurrentInterestingly, HLA-G expression is rarely found on in vitro established cancer cell lines. Most likely, the tumor microenvironment plays a vital role in the induction of HLA-G and investigation of the specific mechanisms of differential HLA-G expression are highly important. Human interferon (IFN)-γ plays a role in tumor immunogenicity widely known as an anti-tumor cytokine but also with a dual role in immune evasion and tumor growth, for example through upregulation of the checkpoint inhibitor PD-L1 and CTLA-4 ,28,29,30A detailed molecular investigation of HLA-G and its regulation in an in vitro breast cancer or malignant melanoma setting will contribute to the search of mechanisms that can be exploited to therapeutically target HLA-G-mediated immune suppression in cancer. In the present study, we evaluated the extend of HLA class Ia and Ib expression in two breast cancer  and two malignant melanoma  cell lines focusing primarily on HLA-G. We investigated the feasibility to manipulate expression of HLA-G by testing the effect of IFN-γ and 5-aza-dC on HLA-G expression in the cell lines. The results indicate that methylation-mediated repression of HLA-G can be reversed by treatment with 5-aza-dC and further induced by IFN-γ. However, HLA-G expression was not affected by IFN-γ alone. HLA-G expression is dominated by the presence of HLA-G1 followed by HLA-G2/4 and HLA-G3 isoforms. Furthermore, treatment with IFN-γ or 5-aza-dC generally promoted upregulation of most HLA class Ia molecules on the surface of the investigated cell lines, however, with some variability between the treatment conditions. Expression of HLA-E and HLA-DR was more heterogeneous between the cell lines.The role of IFN-γ in regulation of immunological mechanisms has been a great area of research. In the current study, the effect of IFN-γ on surface expression of HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, and HLA-DR on MDA-MB-231, MCF-7, FM-55M2, and FM-56 cell lines was investigated by flow cytometry. Based on previously published studies, all cell lines were treated with 24–30 ng/mL IFN-γ and analyzed after two days of treatment . UntreatBased on the comparison of untreated cell lines and an isotype control, it was evident that all investigated cell lines are HLA-G-negative, except for the HLA-G-positive JEG-3 control cell line . Represep = 0.0024; HLA-B, p = 0.0165; HLA-C, p = 0.0093; HLA-E, p = 0.0200; HLA-DR, p = 0.0190)     A. Howeve 0.0123) B. FM-55M 0.0020) C. Simila 0.0001) D. Additi 0.0001)  and the According to previous studies, epigenetic regulation such as DNA methylation plays a role in regulation of HLA expression. To further explore this, breast cancer and malignant melanoma cell lines were treated with the demethylating agent 5-aza-dC and surface expression of HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, and HLA-DR was investigated by flow cytometry. Based on previously published studies, cells were treated with two different concentrations, 10 µM and 100 µM, of 5-aza-dC and analyzed on day three and six after treatment ,35,36. RIt became evident during the analysis that treatment with 5-aza-dC promoted an increase in granularity of the cells causing an increase in autofluorescence for markers detected with fluorochromes with low emission wavelength. This was the case for HLA-C and HLA-G conjugated to PE and BV510 fluorochromes, respectively. The inclusion of isotype controls did not compensate for the increase in autofluorescence potentially causing a false increase in MFI for the treated samples. Furthermore, whether the potential effect of the change in autofluorescence was equal between isotypes and specific markers and between different samples was not transparent and isotype controls was therefore not included in the analysis. Consequently, the interpretation of HLA-C and HLA-G that are poorly expressed proteins must be done with caution.p = 0.0009; HLA-B, p < 0.0001; HLA-G, p = 0.0177; HLA-DR, p < 0.0001)     (p = 0.0317)    A. When i 0.0006) A. Treati 0.0016) B. Furthe 0.0043) B. For MC 0.0123) E. When i 0.0317) E. After  0.0001) F. Furthe 0.0065) F.p = 0.0476; HLA-B, p = 0.0046; HLA-G, p = 0.0180)     A. After  0.0014) B. For FM 0.0166) C. Howeve 0.0039) D.p = 0.0007, p = 0.0011; HLA-B, p = 0.0025, p = 0.0016; HLA-C, p = 0.0025, p < 0.0001; HLA-E, p = 0.0129, p = 0.0002; HLA-G, p < 0.0001, p < 0.0001; HLA-DR, p = 0.0038, p = 0.0163; day three and six, respectively)  C,D. In cp = 0.0109) PCR. The HLA-G-positive choriocarcinoma cell line JEG-3 was included as a control A. As exp 0.0109) E. Howevep = 0.0360; Day 6, p = 0.0029) (p = 0.0029)  A. Treatm 0.0029) A. Notabl 0.0029) C. Treatm 0.0029) C.p = 0.0234)  A. Furthep = 0.0178; 5-aza-dC vs. 5-aza-dC + IFN-γ, p = 0.0112)   B. On day 0.0182) B.HLA-G gene upon treatment with 5-aza-dC was verified by bisulfite sequencing. A comparison of selected CG sites within the CpG island covering exon 2-4 of the HLA-G gene was made between MDA-MB-231 cells and JEG-3 cells , G2/4 (619 bp), -G3 (345 bp), and -G5 (1017 bp). Representative electropherograms are shown in The signal intensity was generally low for breast cancer and malignant melanoma cell lines compared to JEG-3 cells and thus expression levels of the low-expressed HLA-G isoforms presumably fell below the limit of detection preventing a semi-quantitative analysis of the distribution of HLA-G isoforms in the samples. The quality of the original RNA purified from treated cells was checked by automated electrophoresis and was consistently high for all samples, ruling out that poor RNA quality was the cause of missing signals in the fragment analysis (data not shown).HLA gene expression. A summary of the findings can be found in HLA-G is a well-studied molecule and information regarding its regulatory role in cancer is accumulating. To explore how cancer cell lines regulate HLA expression and whether HLA-G expression can be induced by the cytokine IFN-γ and the demethylating agent 5-aza-dC, we treated selected malignant melanoma and breast cancer cell lines with these two compounds, both individually and combined, and measured the effect on mRNA and cell surface expression levels. Expression of HLA-A, HLA-B, HLA-C, and HLA-E were included in this study in order to broaden the knowledge on how IFN-γ and 5-aza-dC affect both classical and non-classical HLA molecules giving that a concomitant deregulation of HLA class Ia and Ib molecules is associated with immune evasion in cancer . FinallyIn the present study, IFN-γ treatment had no effect on HLA-G mRNA level or protein expression in any of the cell lines. The HLA-G mRNA seemed to decrease in FM-56 cells, but a decrease in HLA-G might be attributed to the uncertainty in the noticeably low mRNA level detected with ddPCR analysis. With some variability between cell lines, IFN-γ mainly promoted an upregulation of the other investigated HLA molecules. The ability of IFN-γ to induce HLA class Ia antigen expression in cancer cells has been observed previously. An early study by Jabrane-Ferrat et al. detected HLA class Ia and HLA-DR surface expression on MDA-MB-231 and MCF-7 breast cancer cell lines that were increased by IFN-γ treatment . In the When it comes to HLA-G surface expression, HLA-G expression is rarely detected on in vitro established cancer cell lines making it challenging to study the molecular function of HLA-G. A comprehensive study by Polakova and Russ on 75 different cancer cell lines including melanoma and breast cancer  imply that under normal culture conditions the cell surface expression of HLA-G is absent in most cancer cell lines across different origin . SimilarWhereas we did not see an upregulation of HLA-G upon IFN-γ stimulation, we did see a clear upregulation of HLA-G upon treatment with a combination of IFN-γ and 5-aza-dC. These results indicate that IFN-γ might be involved in maintaining HLA-G expression rather than inducing expression. This is also observed in one of our previous studies with a focus on reproductive immunology .The analysis of HLA-DR expression upon IFN-γ treatment aids to the emerging interest in the role of HLA-DR during cancer immune surveillance. When stimulating the melanoma cell lines with IFN-γ, expression of HLA-DR significantly increased, which was consistent with observations of other studies ,48. AlthHLA gene. Although many variable sites in the promoter region of HLA genes are conserved, variability in the transcription factor target sites most likely influences the differential regulation. The IFN-stimulated response element (ISRE) located in the proximal promoter region of HLA class I genes displays locus-specific nucleotide variation between the different HLAs leading to differences in IFN-γ-induced expression levels of HLA class I genes [HLA-G gene is the most divergent compared to the ISRE consensus sequence preventing binding of interferon regulatory factors and raising the issue of whether the HLA-G ISRE is fully functional [HLA-G gene also contains other regulatory elements, which are not functional . CombinIb genes . The SXYs [HLA-G ,52. MostHLA class I genes was provided by Serrano et al. in 2001, where treatment with 10 µM 5-aza-dC for 15 days restored HLA-ABC expression on the otherwise HLA-ABC-negative melanoma cell line MSR3-mel [HLA-G transcription is partly regulated by DNA methylation. Previous studies have also revealed that HLA-G gene transcription is inhibited by DNA methylation. A previous study has proven an induction of HLA-G in MCF-7 cells upon treatment with 5-aza-dC and IFN-γ [HLA-G in some of the same cell lines showed that the degree of CpG methylation correlated with expression [HLA-G before treatment compared to only 2% after treatment [HLA-G promoter region seem to differ between normal ovarian surface epithelium and ovarian tumors, though not associated with the expression level [HLA-G promoter region and part of the CpG island in colorectal cancer cell lines and choriocarcinoma cell lines have further showed a discrepancy between methylation level and HLA-G expression [HLA-G gene compared with normal breast tissue; however, there is no significant difference in HLA-G expression. Combined, these findings suggest that DNA methylation is not the sole mechanism regulating HLA-G expression, especially in human tumors, where heterogeneous HLA-G expression can represent the complex interaction between cancer cells and factors in the tumor microenvironment that are not present in vitro.The first evidence that DNA hypermethylation is used by cancer cells to switch off MSR3-mel . Upon deMSR3-mel . In the nd IFN-γ . Howevernd IFN-γ . Bisulfipression . Similarreatment . Furtheron level . Interespression ,60. Simipression . Breast Fragment analysis of cells treated with 5-aza-dC revealed that the predominant HLA-G isoform was HLA-G1, followed by HLA-G2/4 and HLA-G3. The HLA-G5 isoform was also observed for the MDA-MB-231 cells. However, there was a low signal intensity in the analysis possibly hiding the presence of other isoforms. This is emphasized by the high signal and clear presence of HLA-G1-5 in the HLA-G-positive JEG-3 cell line. The low signal intensity was presumably due to low mRNA levels in general, in spite of 5-aza-dC-mediated induction of low levels of HLA-G in otherwise HLA-G-negative cell lines. Furthermore, the discrepancy between the HLA-G mRNA levels detected by ddPCR and the low signal obtained by fragment analysis indicate that the latter technique is limited by assay sensitivity. To fully elucidate on the HLA-G isoform profile in the cancer cell lines upon 5-aza-dC treatment a more sensitive method such as RNA sequencing is required. When comparing the isoform profile and the protein level detected by flow cytometry, there is a clear correlation between the results. The HLA-G antibody used detects HLA-G1 and HLA-G5 isoforms and with all cell lines predominantly expressing HLA-G1, the surface expression level reflects the majority of the total HLA-G expression level. However, the surface expression of HLA-G is presumably higher than detected due to expression of HLA-G2-4 isoforms, which are not detected by the antibody used. The level of surface expression might especially be higher for JEG-3 cells that present with a relatively higher level of HLA-G1-5 isoforms compared to the other cell lines.Altogether, our results support that reversal of methylation-mediated repression of HLA-G is a co-acting mechanism by which tumor cells can take advantage of in order to avoid immune recognition. Demethylation is a common mechanism exploited by cancer cells to induce gene transcription and can explain the difference between HLA-G expression in vivo and in vitro when there is no selection pressure from the tumor microenvironment. A similar model has been proposed before for melanoma cells. Expression of HLA-G in melanocytes can be induced during malignant transformation mediated by stress and epigenetic mechanisms otherwise employed by the cancer cells to facilitate the oncogenic transformation. However, adaptation to in vitro culture conditions might lead to methylation due to the lack of stress signals from the tumor microenvironment . Other pIt could be speculated that the HLA expression profile is reflected by the nature of the original cancer giving rise to the cancer cell lines. Both breast cancer cell lines originate from pleural effusion and display similar expression profiles for most HLA molecules except HLA-DR. Treatment with IFN-γ and 5-aza-dC seems to be more potent inducers of HLA surface expression on MDA-MB-231 cells compared to the MCF-7 cells. Whereas the FM-55M2 cells originate from lymph node metastasis, FM-56 cells originate from a primary tumor. Despite the nature of the cancer, the HLA expression profile are similar for the two cell lines except for HLA-A expression which seems to be low or absent on unstimulated FM-56 cells. Furthermore, the effect of IFN-γ and 5-aza-dC on HLA surface expression is similar for most HLAs on both cell lines. The HLA-G isoform profile is also similar for all four cell lines predominantly expressing HLA-G1. Consequently, there is no indication that the nature of the tumor is related to the expression of HLA molecules in the in vitro setting investigated in the present study.A broader understanding of the mechanism by which HLA-G expression is regulated is essential in the development of immune checkpoint inhibitors targeted at HLA-G. The expression of HLA-G in tumor lesions should also be taken into consideration in the design of therapeutic strategies. Assuming that HLA-G is associated with poor clinical outcome the aim of therapeutic interventions should be to prevent a possible induction of its expression in tumors negative for HLA-G. The present study demonstrate two different approaches to manipulate HLA expression in an in vitro breast cancer and malignant melanoma setting and support a role for DNA methylation in regulation of HLA-G expression. The results presented here can pave the way for further research related to methylation-mediated regulation of HLA class I and class II expression. Furthermore, future studies investigating combinations of different cytokines present in the tumor microenvironment will be essential for the understanding of regulatory factors promoting epigenetic changes that affect HLA expression in primary tumors.®-30-2003TM) and MDA-MB-231, FM-56, and FM-55M2 cells were cultured in Roswell Park Memorial Institute 1640 medium . Culture medium was supplemented with 10% heat-inactivated Fetal Bovine Serum . All cells were cultured at 37 °C and 5% CO2 and sub-cultured when they reached 75–80% confluency. For cell passaging and cell harvest, the cells were gently washed in phosphate-buffered saline  and trypsinized using 0.25% EDTA-Trypsin . Cells were pelleted by centrifugation at 300 g for 10 min at room temperature, resuspended in fresh culture medium and transferred to new culture flasks. Cell lines were cryopreserved in FBS supplemented with 10% dimethyl sulfoxide .The current study was performed on four different cell lines from two different cancers: Human breast cancer cell lines MDA-MB-231  and MCF-7 , and human malignant melanoma cell lines FM-56 (primary melanoma) and FM-55M2 (lymph node metastasis), which was kindly provided by Prof. Mads Hald Andersen, Herlev Hospital, Denmark. Human choriocarcinoma cell line JEG-3  was used as a HLA-G-positive control cell line. JEG-3 and MCF-7 cells were cultured in Eagle’s Minimum Essential Medium . On the day of treatment, medium was replaced with fresh medium containing the specified stimulation agents. Cells were treated with either 30 ng/mL human IFN-γ , except for MCF-7 cells that were treated with 24 ng/mL IFN-γ, diluted in deionized water containing 0.1% bovine serum albumin  or 10 μM and 100 μM 5-aza-dC  diluted in DMSO or both 30 ng/mL human IFN-γ and 10 μM 5-aza-dC. Stimulation with deionized water containing 0.1% BSA or DMSO added in the same concentrations as for the respective assays was used as control. All assays were performed in the same cell passage. Cells treated with IFN-γ were incubated for two days and cells treated with 5-aza-dC were incubated for three and six days until analyzed by flow cytometry and harvested for DNA and RNA purification. Cells treated with 5-aza-dC for 6 days had 2 mL medium removed and replaced with fresh medium without 5-aza-dC on day 3.TM buffer  or PBS supplemented with 1–10% FBS. To discriminate live and dead cells, LIVE/DEAD Fixable Violet Dead Cell Stain Kit  was used according to the manufacturer’s protocol. Afterwards, cells were washed once, pelleted by centrifugation and resuspended in 100 μL buffer. Cells were stained for 15 min with antibodies PE anti-human HLA-G , PerCP-Cy5.5 anti-human HLA-A2 , PE-Cy7 anti-human HLA-E , APC anti-human HLA Class I Bw6 , APC-eFluor 780 anti-human HLA-DR  and BV510 anti-human HLA-C . The cells were washed once in 3 mL buffer, resuspended in 200 μL buffer and immediately analyzed on a FACSCanto II flow cytometer  using FACS Diva Software . For each sample, 50,000 events were recorded. Further analysis was performed using FACS Diva Software and FlowJoTM Software .Untreated and treated cells were harvested and analyzed by flow cytometry. All washing and staining of the cells were performed with BD FACSFlowSimultaneous Purification of Genomic DNA and Total RNA from Animal Cells. Cells were lysed and homogenized by vortexing. Purification of RNA alone was performed using the RNeasy® Micro Kit  following manufacturer’s protocol for Purification of Total RNA from Animal and Human cells. DNA was eluted in 50 μL elution buffer supplied with the kit and RNA was eluted in 30 μL RNase-free water supplied with the kits. DNA and RNA concentrations were measured using DropSense 96 . The DNA was stored at −20 °C until use. The total RNA was stored at −80 °C until use. Quality of the purified RNA was assessed from a selection of the samples by automated electrophoresis using the Agilent 2100 Bioanalyzer system  and the Agilent RNA 6000 Pico Kit  following manufacturer’s instructions. First-strand cDNA synthesis with SuperScript® VILO™ cDNA synthesis kit  was performed using maximum 2.5 μg of the extracted RNA and following the manufacturer’s instructions, and cDNA was stored at −20 °C until further use.For DNA and RNA purification, cell pellets were harvested as described above. The harvested cells were quickly stored as a dry pellet at −80 °C. Genomic DNA and total RNA were extracted simultaneously using the AllPrep DNA/RNA Mini Kit  following the manufacturer’s protocol for TM Supermix for Probes , 10 μM of HLA-G forward primer (5′-AGC AGT CTT CCC TGC CCA CC-3′) and HLA-G reverse primer (5′-AGC TCC CTC CTT TTC AAT CTG AGC TC-3′), 4 μM probe (5′-FAM-CCT TGC AGC TGT AGT CAC TGG AGC TGC GGT CG-BHQ-1-3′) and nuclease-free water in a total volume of 20 μL were used for each reaction. Cycling conditions were: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 62 °C for 1 min, 72 °C for 1 min, finally ending reaction at 98 °C for 10 min and hold at 4 °C. Specific amplicons were confirmed by electrophoresis on a 2% agarose gel. Results were analyzed using the QuantaSoft® software version 1.3.2.0 .Droplet digital (dd)PCR was performed using the QX100 Droplet Digital PCR system  as previously described . In shor® Hot Start Master Mix , 1 μM of HLA-G1-6 forward primer (5′-CAC AGA CTG ACA GAA TGA ACC TGC A-3′) and HLA-G1-6 reverse primer (5′-FAM-GGA AGG AAT GCA GTT CAG CAT GA-3′), and nuclease-free water in a total volume of 25 μL for each reaction.. The PCR was run under following conditions: 95 °C for 2 min, 29 cycles of 95 °C for 30 s, 62 °C for 2 min and 72 °C for 2 min, followed by 72 °C for 10 min and ending with hold at 4 °C. The PCR product was used directly for fragment analysis or stored overnight in the dark at 4 °C. For fragment analysis, 10 μL of nuclease-free water and 0.5 μL of GeneScanTM 1200 LIZTM dye size standard  were added to each well containing 2 μL of the PCR product and subsequently denatured at 95 °C for 3 min. followed by incubation on ice for 2 min. Fragment analysis on the ABI3500 Genetic Analyzer  was performed after the cooling. The PCR fragment lengths were analyzed using the GeneMarker® software . Analysis on 10% of randomly chosen samples were performed in duplicates.PCR was performed with 90–320 ng cDNA, using 13.5 μL of GoTaqHLA-G 14 bp ins/del polymorphism in JEG-3, FM-55M2, FM-56, MDA-MB-231 and MCF-7 cells was performed by TaqMan assay as previously described [Genotyping of the escribed . BrieflyHLA-G sequence was obtained from NCBI Reference Sequence NG_029039.1 and prediction of the 5′ CpG island from the UCSC Genome Browser. Target-specific bisulfite sequencing PCR primers with M13 tags were designed in three pairs overlapping and covering the CpG island. Pair F1R2: forward 5′-TTA GGT GAT AGG TTT TTA GAG AAG TTA ATT-3′, reverse 5′-ATT CCA TAT CTC CTC TTC CCA ATA-3′; pair F2R3: forward 5′-TTT AGG TTT TTA TTT TAT GAG GTA TTT TAG-3′, reverse 5′-ACC AAT CAT CCA CTA AAA AAT ATA AAA AC-3′; pair F3R1: 5′-AGA TTT TTT ATT TGG GAG AAT TTT AAG G-3′, reverse 5′-ATA TTA ATC CCA TTT TCC TCC TCT CC-3′. Specificity of bisulfite-specific primers were confirmed using the online tool BiSearch [TM Terminator v3.1 Cycle Sequencing Kit , 2 μL PCR product, 4 μM sequencing primer and nuclease-free water in a total volume of 10 μL for each reaction. The sequencing reaction was carried out at the following conditions: 96 °C for 1 min, 35 cycles of 96 °C for 30 s, 50 °C for 15 s, 60 °C for 4 min, finally ending reaction by hold at 4 °C. The products were purified using CleanSeq  on Biomek 4000. Bidirectional Sanger sequencing was performed on ABI3500 Genetic Analyzer . The results were analyzed using Mutation Surveyor .Bisulfite conversion of 300–350 ng purified genomic DNA was carried out using the EZ DNA Methylation-Lightning Kit  following the manufacturer’s protocol. The BiSearch ,70. In b® software . p-values < 0.05 were considered statistically significant, and marked in the figures accordingly: * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001. Student’s unpaired t-test with Welch’s correction was used for comparison of IFN-γ and 5-aza-dC treated samples to their respective controls for flow cytometry data and ddPCR results.If not otherwise stated, experiments were performed in triplicates. Figures and statistical analysis were executed using GraphPad Prism


Streptomyces sp. strain IB201691-2A from the Lake Baikal endemic mollusk Benedictia baicalensis. In the course of an activity guided screening three new angucyclines, named baikalomycins A–C, were isolated and characterized, highlighting the potential of poorly investigated ecological niches. Besides that, the strain was found to accumulate large quantities of rabelomycin and 5-hydroxy-rabelomycin, known shunt products in angucyclines biosynthesis. Baikalomycins A–C demonstrated varying degrees of anticancer activity. Rabelomycin and 5-hydroxy-rabelomycin further demonstrated antiproliferative activities. The structure elucidation showed that baikalomycin A is a modified aquayamycin with β-d-amicetose and two additional hydroxyl groups at unusual positions (6a and 12a) of aglycone. Baikalomycins B and C have alternating second sugars attached, α-l-amicetose and α-l-aculose, respectively. The gene cluster for baikalomycins biosynthesis was identified by genome mining, cloned using a transformation-associated recombination technique and successfully expressed in S. albus J1074. It contains a typical set of genes responsible for an angucycline core assembly, all necessary genes for the deoxy sugars biosynthesis, and three genes coding for the glycosyltransferase enzymes. Heterologous expression and deletion experiments allowed to assign the function of glycosyltransferases involved in the decoration of baikalomycins aglycone.Natural products produced by bacteria found in unusual and poorly studied ecosystems, such as Lake Baikal, represent a promising source of new valuable drug leads. Here we report the isolation of a new  Angucyclines are by far the largest group of aromatic polyketides solely produced by actinobacteria ,2. TheseSaquayamycins are a large group of angucyclines that, together with urdamycins, derive from the aquayamycin-type aglycone  10]. Mo. Mo10]. d-olivose as in the case of saquayamycins, vineomycins, moromycins, grincamycins and N05WA963 A–C. At the same time, saquayamycins, vineomycins, moromycins, and grincamycins at C-3 have O-glycosidically attached l-rhodinose. N05WA963 A–C do not have a saccharide chain at C-3 anthracene structure. BaiA5/A6 resembles SchP4/P9 (99%/99%) and UrdF/L (73%/87%). baiA7 encodes a close homologue of SrpB (77%) and UrdE (78%) proteins. UrdE is an oxygenase catalyzing hydroxylation of an angucycline aglycone at the C-12 position − that corresponds to the mass calculated for aglycone of baikalomycins A and B   . This cobaiGT2 abolished production of baikalomycins B and C. However, Streptomyces sp. IB201691-2AΔGT2 demonstrated increased accumulation of baikalomycin A that is barely detectable in the parental strain  and two peaks with m/z of 695.2734 [M-H]− (RT 14.46 and 14.83 min) and characteristic for angucylines absorption spectrum could be found in the extract of S. albus J1074/p8-13bai (Streptomyces sp. IB201691-2A. Based on the detected mass and UV spectra these metabolites are proposed to be baikalomycins trisaccharides closely related to Sch-47554/47555. MS2 data support this idea. All five new metabolites have typical angucycline type fragmentation pattern with the characteristic loss of one sugar, most probably at C-3 position (We have cloned the entire m genes, A was expcins A–C . At the p8-13bai . These cposition . Most plS. coelicolor yielded vineomycin A1 with two aculose moieties instead of cineruloses, present in grincamycins. It is obvious that the baikalomycins biosynthetic enzymes behave differently in the natural producer and the heterologous host. In fact, Streptomyces sp. IB201691-2A did not produce angucycline trisaccharides under any of tested cultivation conditions but accumulated large amounts of shunt product rabelomycin and its derivatives. Vice versa, S. albus carrying the bai gene cluster accumulated the angucycline trisaccharides. In conclusion, six gene clusters encoding the aquayamycin-type angucyclines biosynthesis have a high degree of sequence identity (In the latter case, expression of the grincamycin biosynthesis genes in identity B and, at


Drosophila adult flies. RNA-seq analysis revealed that ZIKV infection alters several and diverse biological processes, including stress, locomotion, lipid metabolism, imaginal disc morphogenesis and regulation of JAK/STAT signaling. To explore the interaction between ZIKV infection and JAK/STAT signaling regulation, we generated genetic constructs overexpressing ZIKV-specific non-structural proteins NS2A, NS2B, NS4A and NS4B. We found that ectopic expression of non-structural proteins in the developing Drosophila eye significantly restricts growth of the larval and adult eye and correlates with considerable repression of the in vivo JAK/STAT reporter, 10XStat92E-GFP. At the cellular level, eye growth defects are associated with reduced rate of proliferation without affecting the overall rate of apoptosis. In addition, ZIKV NS4A genetically interacts with the JAK/STAT signaling components; co-expression of NS4A along with the dominant-negative form of domeless or StatRNAi results in aggravated reduction in eye size, while co-expression of NS4A in HopTuml  mutant background partially rescues the hop-induced eye overgrowth phenotype. The function of ZIKV NS4A in regulating growth is maintained in the wing, where ZIKV NS4A overexpression in the pouch domain results in reduced growth linked with diminished expression of Notch targets, Wingless (Wg) and Cut, and the Notch reporter, NRE-GFP. Thus, our study provides evidence that ZIKV infection in Drosophila results in restricted growth of the developing eye and wing, wherein eye phenotype is induced through regulation of JAK/STAT signaling, whereas restricted wing growth is induced through regulation of Notch signaling. The interaction of ZIKV non-structural proteins with the conserved host signaling pathways further advance our understanding of ZIKV-induced pathogenesis.To gain a comprehensive view of the changes in host gene expression underlying Zika virus (ZIKV) pathogenesis, we performed whole-genome RNA sequencing (RNA-seq) of ZIKV-infected This article has an associated First Person interview with the first author of the paper. Summary: Our study provides evidence that Zika virus infection in Drosophila results in restricted growth of the developing eye and wing through regulation of JAK/STAT and Notch signaling, respectively. Zika virus (ZIKV) is an emerging pathogen of substantial public health concern, and belongs to the flavivirus family that also includes dengue, West Nile and yellow fever viruses. Although most infections are asymptomatic or associated with flu-like symptoms , during ZIKV is an obligate intracellular pathogen; therefore, understanding the interaction dynamics between ZIKV and the host and the resulting host pathology is valuable for developing anti-ZIKV therapeutic strategies . In partInvestigating the function of viral components is an elegant strategy to further our understanding of the molecular basis of viral diseases. Structurally, ZIKV is similar to the other flaviviruses and possesses a 25- to 30-nm nucleocapsid surrounded by a host membrane-derived lipid bilayer. ZIKV contains a positive-sense single-stranded RNA of ∼10 kb. Similar to other flaviviruses, host protease-mediated viral processing results in three structural  and seven non-structural (NS) proteins  . In otheDrosophila melanogaster, with a vast number of genetic and genomic tools available and highly conserved developmental signaling pathways, is widely recognized as an excellent model for studying host-pathogen interactions and human disease. The latter is demonstrated by the fact that 70% of Drosophila genes have human homologs and 75% of human disease-associated genes have homologs in the fly  to gain a comprehensive understanding of the differentially regulated genes during infection. We report that ZIKV triggers a large number of biological processes, ranging from misregulation of developmental pathways to perturbed muscle development. Among the developmental signaling pathways, we have shown that ZIKV infection induces negative regulation of JAK/STAT signaling. Eye-specific expression of ZIKV transgenes NS2A, NS2B, NS4A and NS4B results in striking reduction in the size of the developing eye. We also show that the reduced eye size upon ZIKV NS4A protein expression, in particular, correlates with the reduced level of the JAK/STAT reporter, 10XStat92E-GFP. At the cellular level, the expression of ZIKV transgenes results in reduced rate of proliferation in the JAK/STAT-regulated anterior compartment of eye imaginal epithelia without affecting the level of apoptosis. We further demonstrate that ZIKV transgene expression has no effect on the differentiation of the photoreceptors of the eye, and that ZIKV NS4A interacts with the JAK/STAT signaling components. Co-expression of NS4A and the dominant-negative form of domeless or StatRNAi results in aggravated reduction in eye size, while co-expression of NS4A with activated Hop kinase partially rescues the eye enlargement. Finally, the ZIKV NS4A-mediated regulation of growth is also maintained in the wing, where NS4A overexpression restricts the size of the wing pouch. This effect is linked with reduced activity of Notch signaling, which is instrumental for several cell developmental processes, including proliferation and wing development. Altogether, our findings provide the first evidence linking ZIKV-induced pathogenesis and eye/wing development, and reveal a functional link between ZIKV and regulation of JAK/STAT signaling.Here, we have challenged Drosophila wild-type female adult flies infected with the MR766 strain of ZIKV -based analyses . At 8 dpi, 706 genes were differentially expressed, of which 315 genes were upregulated and 391 genes were downregulated (Tables S3 and S4). Among the DEGs regulating immunity, Diedel and genes of the Turandot family of proteins were significantly enriched at both 4 and 8 dpi (Tables S1 and S3). Unlike for Drosophila C virus (DCV) infection . Also, comparing the individual fold change in all three biological replicates revealed that, out of the three replicates, one replicate deviated from the other two replicates, resulting in the higher P-value and hence non-significant upregulation (Table S5). However, consistent with several pieces of evidence showing the interaction of flaviviruses with the regulators of JAK/STAT signaling (E(bx) and Socs36E, were significantly upregulated in ZIKV-infected Drosophila flies at both 4 dpi  and Socs36E through qRT-PCR analysis prompted us to probe the functional significance of the altered expression of these genes, particularly in the context of ZIKV transgene-induced eye development. In order to examine the effect of ZIKV on Drosophila eye development, we took advantage of genetic overexpression constructs. Gal4/UAS-based studies in Drosophila have led to identification of the functions of several viral genes, facilitating a comprehensive understanding of the outcome of viral infection , where Eyeless is one of the most critical transcription factors regulating eye development in Drosophila . Scoring for the time of pupariation further showed that these flies did not exhibit any developmental delay . However, compared to E1-Gal4, overexpression of genes encoding ZIKV non-structural proteins NS2A, NS2B, NS4A and NS4B driven under E1-Gal4 resulted in significantly reduced size of the eye imaginal disc, a phenotype also resulting from loss of function of hop . In corroboration with the reduced size of eye imaginal epithelia, we noticed that the resulting adult flies harboring E1-Gal4-mediated ZIKV non-structural protein overexpression also displayed a significant reduction in eye size (unpaired (upd) , there was 3000-fold enrichment of NS4A in the eye imaginal epithelia when NS4A was overexpressed  . To ascexpressed E.E1-Gal4-driven NS4A overexpression showed significantly reduced expression of the targets of JAK/STAT signaling – including chinmo, Mo25 and domeless – compared to E1-Gal4 , for detecting the mitotically active cells. We noticed numerous PH3-positive cells in both the anterior (first mitotic wave) and posterior compartment (second mitotic wave) in the eye imaginal epithelia in the case of E1-Gal4 A. Upon oE1-Gal4 A. QuantiE1-Gal4 B and secE1-Gal4 C mitoticl4 alone B,C.Fig.E1-Gal4 alone  and dominant-negative form of Upd receptor, Domeless (DNUAS-dome)  resulted in a drastic reduction in the size of the eye imaginal epithelia (domeless (DNUAS-dome) in the background of E1-Gal4-driven NS4A (E1>NS4A) overexpression resulted in aggravated reduction in eye size  . The ovepithelia   results in enlarged eye phenotype . However, after co-expression of ZIKV NS4A and upd together in the developing eye, we still observed an enlarged eye . We then switched to a less-severe eye phenotype to examine the effect of ZIKV NS4A overexpression. Overexpression of activated Hop kinase, HopTuml , also results in enlargement of the eye  marks the posterior compartment of the wing]. We noticed that there was no difference in the size of the posterior compartment (tagged with RFP), compared to the anterior compartment of the wing imaginal disc (shown by the absence of RFP), when engrailed-Gal4-specific ZIKV non-structural proteins were expressed  phenotype marked by thickening of veins and formation of wing deltas . Similar to the engrailed-Gal4 findings, we found that overexpression of NS2A, NS2B and NS4B had no noticeable defect in the adult wing (Table S6). The classical notched wing phenotype is attributed to downregulated Notch signaling. We next examined the underlying mechanism for NS4A-induced notched phenotype. We found that nub-Gal4-driven NS4A expression resulted in drastic reduction in the size of the pouch region of the wing imaginal epithelia  substantially reduces Wg and Cut expression in the pouch domain of the wing imaginal epithelia, where wg and cut are the morphogens and are considered the targets of Notch signaling  fused with a GFP reporter (NRE-GFP was significantly reduced when ZIKV NS4A was overexpressed in the nubbin region (marks the wing pouch encompassing the DV boundary) of the wing imaginal epithelia , showed that, similar to the expression pattern of Cut, Wg and NRE-GFP, there was reduced expression of Notch protein when ZIKV NS4A was overexpressed compared to nubbin-Gal4 alone  and DCV; however, these mutant flies show a rather weak phenotype to Drosophila X virus (DXV), Sindbis virus (SINV) and vesicular stomatitis virus (VSV)  . Whereasnfection .Drosophila, a recent finding indicates that one of the negative regulators of JAK/STAT signaling, Diedel, is significantly enriched in ZIKV-infected flies  results in the restricted growth of this domain. The subdivision of the wing into anterior/posterior (a/p) compartments occurs in the embryonic stage, while the DV boundary is established during proliferation of the imaginal disc , E1-Gal4 , UAS-NS4A, UAS-NS4B, UAS-NS2A, UAS-NS2B, UAS-StatRNAi (Vienna Drosophila Resource Center no. 43866), 1118; P(10XStat92E-GFP)w (Bloomington no. 26197), 1118; P(NRE-EGFP.S)5Aw (Bloomington no. 30727), nubbin-Gal4 (Bloomington no. 25754). UAS-upd and UAS-HopTuml were generously provided by Heinrich Jasper , and DNUAS-dome was provided by Dan Hultmark . Flies were reared on instant Drosophila diet (Formula 4-24 Drosophila medium) supplemented with yeast , and maintained at 25°C and in a 12:12-h light:dark photoperiodic cycle. Female adult flies aged 4-6 days old were used in infection assays with ZIKV.The following fly lines were used:  E1-Gal4 , engrail-mediated germ line transformation. Primers used for PCR were as follows: ZIKV-NS2A: forward, 5′-CCGGAATTCATGGATTACAAGGATGACGACGATAAGGGGTCAACCGATCATATGGAC-3′; reverse, 5′-CGGGGTACCCTACCGCTTCCCACTCCTTGTGAG-3′. ZIKV-NS2B: forward, 5′-CCGGAATTCATGGATTACAAGGATGACGACGATAAGAGCTGGCCCCCTAGTGAAGTTC-3′; reverse, 5′-CGGGGTACCCTACCTTTTCCCAGTCTTCACATAC-3′. ZIKV-NS4A: forward, prime;-CCGGAATTCATGGATTACAAGGATGACGACGATAAGGGAGCGGCTTTGGGAGTAATG-3′; reverse, 5′-CGGGGTACCCTATCTTTGCTTCTCTGGCTCGGG-3′. ZIKV-NS4B: forward, 5′-CCGGAATTCATGGATTACAAGGATGACGACGATAAGAACGAACTTGGATGGCTGGAAAG-3′; reverse, 5′-CGGGGTACCCTAACGTCTCTTAACCAGGCCAGC-3′. Bold italic sequences are restriction enzyme cutting sites and underlined sequences are Flag tag.Four ZIKV open reading frames (ORFs)  in mammalian expression vectors were gifts from Dr Hongjun Liu's laboratory in Johns Hopkins University . To geneThe ZIKV infection protocol and propagation have been described in detail previously. Briefly, the African strain of ZIKV, MR766, was propagated in Vero cells followed by determination of ZIKV titers using plaque assay on Vero cells as described in Total RNA was extracted from ten adult female flies injected with ZIKV and PBS at 4 and 8 dpi in triplicate using Trizol according to the manufacturer's protocol. Total RNA was re-suspended in 30 μl sterile nuclease-free water. RNA concentration was measured using Nanodrop. RNA integrity and quality were assessed on formaldehyde agarose gel.TruSeq Total RNA with Ribo-Zero Gold Kit  was used to generate strand-specific RNA-seq libraries following the manufacturer's recommendations. The libraries were pooled and sequenced in a single lane on an Illumina HiSeq 2500 sequencing instrument with single-end 50 bp reads. RNA-seq was performed at The Genome Technology Access Center (GTAC) based in Washington University in St Louis, MO, USA.GSE130108.RNA-seq reads were aligned to the Ensembl release 76 top-level assembly with STAR version 2.0.4b . Gene coP-values less than or equal to 0.1.All gene counts were then imported into the R/Bioconductor package EdgeR  and TMM http://david.abcc.ncifcrf.gov/). In all cases, analyses were performed using the list of DEGs. The P-value cutoff to determine enriched pathways was 0.1.GO analysis was performed using DAVID 6.8 bioinformatics resources  was used to synthesize cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR experiments were performed in technical triplicates and with gene-specific primers  using iQeyeless-specific overexpression of non-structural proteins were used. Flies were maintained at a constant temperature of 25°C with a 12-h light/dark cycle, and mortality was recorded daily. Log-rank (Mantel–Cox) test was used to analyze the survival curves.For each fly strain, three groups of 20 female flies carrying Two-hour timed egg collections were carried out and L1 larvae were collected 24 h after egg deposition and reared at 30 animals per vial. The number of larvae that had pupariated at a given time after egg deposition was scored every 8 h.Anti-PH3 , anti-CycA , anti-Dcp-1 , anti-Cut , anti-Wg , anti-Elav  and anti-NICD  were used as the source for primary antibodies. Secondary antibodies included Alexa Fluor 488, 555 and 633 (Invitrogen). For nuclear staining, 4′,6-diamidino-2-phenylindole  was used. Standard procedures were followed for immunostaining. The crosses were properly synchronized for the stages and third-instar larvae were collected for dissections. Briefly, fly tissues were dissected and fixed in PBS containing 4% formaldehyde for 30 min. Following double rinsing in PBS containing 0.1% Triton X-100, the samples were incubated overnight at 4°C with the primary antibody. The samples were then blocked with 1% BSA for 2 h followed by a 2-h incubation with secondary antibody at room temperature. Finally, the samples were mounted with Vectashield medium (Vector Laboratories). Images were acquired with a Zeiss LSM 510 confocal microscope and processed using Adobe Photoshop CS6.Relative amounts of fluorescence were measured with ImageJ software by using Shanbhag thresholding on images and calculating the integrated density, resulting area and mean fluorescence of the background. The following equation was used: corrected total fluorescence=integrated density−(area×mean fluorescence of background).The size of the third-instar wing imaginal discs and adult wings were measured using ImageJ. In the case of the wing pouch, the size in different genotypes was normalized to the total size of the wing disc.The photographs and measurements were acquired using a Keyence VHX 5000 Digital Microscope.t-test was used for statistical analysis of data using Prism (GraphPad Software).qRT-PCR results represent the means±s.d. of relative values from three biological replicates. An unpaired two-tailed Student's


Delafloxacin (DFL) is a novel potent and broad-spectrum fluoroquinolone group of antibiotics effective against both Gram-positive and negative aerobic and anaerobic bacteria. In this study, DFL-loaded stearic acid (lipid) chitosan (polymer) hybrid nanoparticles (L-P-NPs) have been developed by single-emulsion-solvent evaporation technique. The mean particle size and polydispersity index (PDI) of optimized DFL-loaded L-P-NPs (F1-F3) were measured in the range of 299–368 nm and 0.215–0.269, respectively. The drug encapsulation efficiency (EE%) and loading capacity (LC%) of DFL-loaded L-P-NPs (F1-F3) were measured in the range of 64.9–80.4% and 1.7–3.8%, respectively. A sustained release of DFL was observed from optimized DFL-loaded L-P-NPs (F3). Minimum inhibitory concentration (MIC) values of the DFL-loaded L-P-NPs (F3) appeared typically to be four-fold lower than those of delafloxacin in the case of Gram-positive strains and was 2-4-fold more potent than those of delafloxacin against Gram-negative strains. The pharmacokinetic study in rats confirmed that the bioavailability (both rate and extent of absorption) of DFL-loaded L-P-NPs was significantly higher (2.3-fold) than the delafloxacin normal suspension. These results concluded that the newly optimized DFL-loaded L-P-NPs were more potent against both Gram-positive and negative strains of bacteria and highly bioavailable in comparison to delafloxacin normal suspension. Staphylococcus aureus (MRSA) [Acute bacterial skin and skin structure infections (ABSSSIs) and community-acquired pneumonia (CAP) are considered as the most common type of infection which require hospitalization, and are the leading cause of mortality and morbidity worldwide ,2,3. Dels (MRSA) .max), usually achieved within 1–2.5 h in healthy volunteers. It is widely distributed to body fluids with the volume of distribution at a steady state (VSS) in the range of 34–41 L and high plasma protein binding of 83–84% [DFL shows rapid absorption after oral administration and peak plasma concentration  and natural  polymers. The use of organic solvents during the synthesis of polymeric nanoparticles limits their application ,18,19. TDFL was purchased from Beijing Mesochem Technology . Stearic acid, chitosan and pluronic 127 were purchased from Sigma Aldrich . All chemicals and solvents used in this study were analytical/HPLC grade. Ultra-pure water was collected from the Milli-Q water purifier unit  and was used for aqueous solution preparation.w/v acetic acid solution containing pluronic 127 surfactant (50 mg) under probe sonication  for 3 min at 60% voltage efficiency at a temperature of 25 °C  and zeta potential (ZP). The mean size and PDI of L-P-NPs (F1-F3) were measured using Malvern zeta sizer  at 25 ± 2 °C, and dynamic light scattering (DLS) was set 90° . The fretotal = amount of DFL added initially in NPs, Dfree = DFL in supernatant, Dentrapped = DFL entrapped in NPs, and Wnanoparticle = total weight of NPs.An indirect method was followed for the measurement of the percent encapsulation efficiency (EE%) and drug-loading capacity (LC%) of DFL-loaded L-P-NPs (F1-F3). Freshly prepared dispersion of nanoparticles was centrifuged  at 15,000 rpm for 10 min at −4 °C to get supernatant. The supernatant was diluted appropriately, and the drug was quantified by UV spectroscopy at 290 nm. The EE% and DL% were calculated using the following equations :EE% = [ were studied using a DSC thermal analyzer . Accurately weighed samples were crimped in an aluminum pan by applying pressure and heated over a temperature range of 50–300 °C at a constant heating rate of 10 °C/min. The thermal samples were purged with an inert nitrogen gas with a flow rate of 15–20 mL/min .−1, and peaks were interpreted with the help of IR software [FTIR spectra of pure DFL and DFL-loaded L-P-NPs (F1-F3) were recorded using the instrument FTIR spectrometer . Each sample was grinded with potassium bromide (10:100), and a transparent film was used by applying pressure. The spectra were recorded in the wavelength range of 4000 to 400 cmsoftware .A comparative release study of pure DFL and optimized DFL-loaded L-P-NPs (F3) was performed using a dialysis bag  . BrieflyThe surface morphology and approximate size of the optimized DFL-loaded L-P-NPs (F3) were examined under transmission electron microscopy . The DFL-loaded L-P-NPs were vortexed for 3 min after dilution with Milli-Q water. A drop of suspended DFL-loaded L-P-NP was pipped out on parafilm, and then the slide of the TEM grid was placed on the drop and left for 15 min. The grid was then removed and placed on tissue paper with the slide for 45 min for drying, then scanned for imaging.Staphylococcus aureus (ATCC 29737), S. aureus (NCTC 6571), Streptococcus pyogenes (ATCC 12344), S. pneumonia (ATCC 10015), S. agalactiae (ATCC12386), Enterococcus faecalis (ATCC 19433), E. faecalis (ATCC 49532), Salmonella typhimurium (ATCC 14028), Pseudomonas aeruginosa (ATCC 10145), Staphylococcus haemolyticus (ATCC 29970), Proteus mirabilis (NCIMB 13283), Enterobacter Aerogenes (ATCC13048), E. aerogenes (NCIMB 10102), Bacillus subtilis (ATCC 11774), Klebsiella pneumonia (ATCC 9633), Bacillus cereus (ATCC 10876) and Staphylococcus epidermidis (ATCC 12228) were examined. In addition, 10 clinical isolates of S. aureus, S. typhimurium, E. coli, S. pneumonia and K. pneumonia were also used. To verify the accuracy of the susceptibility results, strains of Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 29213) and Enterococcus faecalis (ATCC 29212) were used as control organisms.For this study, seventeen type strains: 2 incubator.Strains were cultured in Mueller Hinton and stored as recommended by the manufacturer. Cultures were incubated for 18 ± 2 h at 36 ± 1 °C while streptococcal strains were incubated in Mueller-Hinton agar with 5% sheep blood under microaerobic conditions at 36 ± 1 °C for 24 h in a CO2SO4) with 0.05 mL of 1.175% barium chloride dihydrate (BaCl2·2H2O) in order to estimate bacterial density [5 colony-forming units (CFU) mL−1.McFarland number 0.5 standard was made by mixing of 9.95 mL  were performed on UPLC C18 columns using a mobile phase composition of acetonitrile with 0.1% formic acid and 10 mM ammonium acetate  at a 0.3 mL min−1 flow rate. The electrospray ionization in positive mode was used for triple quadrupole detection. Quantitation was accomplished with multiple reaction monitoring with parent-to-daughter ion transitions of 441.14 → 379.09 for DFL and 436.89 → 144.87 for the internal standard. The sample extractions were performed by the liquid extraction method using ethyl acetate as an extracting agent. The intra and inter-day precision (% RSD) of the method was ≤10.5, whereas the accuracy was within the range of −13.2% to 11.2% (% RE), respectively.Our recently published UPLC-MS/MS method  was usedn = 6 each) and were administered DFL normal suspension and DFL-loaded L-P-NPs (F3) after overnight fasting (20 mg Kg−1). Blood samples were withdrawn at different time intervals . After samples were withdrawn, all blood sample were centrifuged at 5000× g for 10 min, and plasma were separated and stored frozen at 80 ± 10 °C until further UPLC-MS/MS analysis.The comparative bioavailability of newly developed DFL-loaded L-P-NPs (F3) against normal suspension were evaluated in male Wistar albino rats. Twelve animals weighing between 300–350 g were received from the Animal Care Centre, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. The experimental protocol was approved by the Research Ethics Committee (Reference no. KSU-SE-19-27), and the experiment was carried out according to the guidelines of the Animal Care and Use Committee, King Saud University. Animals were divided into two groups (max and the time to reach maximum concentration (Tmax), AUC from 0 to t (AUC0–24) and 0-inf (AUC0–inf), elimination rate constant (kz), half-life (T½) and mean residence time (MRT).The pharmacokinetic parameters were calculated using WinNonlin software , and all values have been expressed as means ± standard deviation (SD). The noncompartmental pharmacokinetic model was used to calculate CThe mean particle size and PDI of DFL-loaded L-P-NPs (F1-F3) were measured in the range of 299–368 nm and 0.215–0.269, respectively. The results are presented in The encapsulation efficiencies of DFL-loaded L-P-NPs (F1-F3) were assessed in terms of EE% and LC%. The entrapment efficiency gives an idea of the entrapped drug, and the drug loading capacity is about the content of drugs in the nanoparticles. The EE% and LC% of DFL in L-P-NPs (F1-F3) were measured in the ranges of 64.9–80.4% and 1.7–3.8%, respectively . It was DSC spectra of pure DFL and DFL in L-P-NPs (F1-F3) were scanned from 50–300 °C as shown −1), –C–H str (3075.94 cm−1), ketone –C=O str (1715.37 cm−1), carboxylic –C=O str (1623.77 cm−1), C–F str (1114.65 cm−1) and C–Cl str (840.63 cm−1) with confirmed drug purity [−1, which resulted from the overlapping of the CS, SA and DFL.The compatibility studies of the polymer (CS), lipid (SA) and drug (DFL) in nanoparticles (F1-F3) were carried out by the FTIR analysis. The comparative FTIR spectra of DFL and DFL-loaded L-P-NPs (F1-F3) were recorded for the identification and characterization of the drug . The majg purity , and a rIn-vitro release studies were performed to know the pattern and mechanism of DFL from lipid polymer hybrid nanoparticles. A biphasic release pattern was observed from DFL-loaded L-P-NPs (F3), as shown in TEM images of the DFL-loaded L-P-NPs (F3) are shown in E. faecalis ATCC 49,532 and two-fold lower with S. epidermidis ATCC 12228. Noteworthy, DFL-loaded L-P-NPs (F3) showed MIC results with Gram-negative strains with a maximal value of 0.250 µg/mL in the case of P. mirabilis NCIMB 13,283 and a minimal value of 0.0156 µg/mL in the case of H. influenza ATCC 10211. Based on the MIC data, DFL-loaded L-P-NPs (F3) were two-to-four-fold more potent than those of DFL and more potent than levofloxacin by ten-fold, as shown with P. mirabilis NCIMB 13,283, where the MIC value decreased from 2.0 µg/mL to 0.250 µg/mL. Higher potency of DFL-loaded L-P-NPs nanoparticles might be due to the unique structure of DFL with its nano-range size and large surface area.Data presented in −1 of DFL-loaded L-P-NPs (F3) and DFL normal suspension are summarized in max (p < 0.01), AUClast and AUCtot (p < 0.05) higher than the normal suspension of DFL. This results to the bioavailability of DFL-loaded L-P-NPs (F3) increased 2.3-fold in compared to the normal suspension of DFL, which proved the high circulation property of the nano-formulation in the circulatory system. Higher bioavailability of DFL-loaded L-P-NPs may be produced due to their ability to encapsulate the hydrophobic molecules with higher drug payloads and the improved drug stability of poorly water-soluble drugs like DFL. It has also been reported that the maintaining of the structural integrity of the nanoparticles is a critical factor for effective oral absorption of the drug to the site of action. Herein, a preparation of the hybrid form of lipid-polymer nanoparticles provides better structural integrity for nanocarriers [−1 are shown in The comparative pharmacokinetic results obtained after an oral administration of 20 mg kgcarriers . Similarcarriers . HoweverIn this investigation, DFL-loaded L-P-NPs (F3) were formulated and optimized with the intention to enhance their bioavailability and antibacterial activity. An in vitro drug release study showed a biphasic release pattern of the optimized formulation of DFL-loaded L-P-NPs. MIC values of the DFL-loaded L-P-NPs (F3) appeared typically to be four-fold lower than those of DFL in the case of Gram-positive strains and was 2-4-fold more potent than those of DFL against Gram-negative strains. The pharmacokinetic study in rats confirms that the bioavailability of DFL-loaded L-P-NPs was significantly higher (2.3-fold) than the DFL normal suspension. Hence, it is concluded that DFL-loaded L-P-NPs promise a better therapeutic efficacy and could be a choice of replacement for the conventional formulation, with benefits of low dose requirements and better patient compliance.Future research will involve the possibility to carry out in vitro activity for several days to confirm the stability of activity and in vivo antibacterial studies in experimental animals.


The impacts of genetic polymorphisms on drug resistance mutations (DRMs) among various HIV-1 subtypes have long been debated. In this study, we aimed to analyze the natural polymorphisms and acquired DRM profile in HIV-1 CRF01_AE-infected patients in a large first-line antiretroviral therapy (ART) cohort in northeastern China.The natural polymorphisms of CRF01_AE were analyzed in 2034 patients from a long-term ART cohort in northeastern China. The polymorphisms in 105 treatment failure (TF) patients were compared with those in 1148 treatment success (TS) patients. The acquired DRM profile of 42 patients who experienced TF with tenofovir/lamivudine/efavirenz (TDF/3TC/EFV) treatment was analyzed by comparing the mutations at TF time point to those at baseline. The Stanford HIVdb algorithm was used to interpret the DRMs. Binomial distribution, McNemar test, Wilcoxon test and CorMut package were used to analyze the mutation rates and co-variation. Deep sequencing was used to analyze the evolutionary dynamics of co-variation.Before ART, there were significantly more natural polymorphisms of 31 sites on reverse transcriptase (RT) in CRF01_AE than subtype B HIV-1 , including five known drug resistance-associated sites . However, only the polymorphism at site 75 was associated with TF . The mutation rate at 14 sites increased significantly at TF time point compared to baseline, with the most common DRMs comprising G190S/C, K65R, K101E/N/Q, M184 V/I, and V179D/I/A/T/E, ranging from 66.7 to 45.2%. Moreover, two unknown mutations (V75 L and L228R) increased by 19.0 and 11.9% respectively, and they were under positive selection  and were associated with several other DRMs . Deep sequencing of longitudinal plasma samples showed that L228R occurred simultaneously or followed the appearance of Y181C.The high levels of natural polymorphisms in CRF01_AE had little impact on treatment outcomes. The findings regarding potential new CRF01_AE-specific minor DRMs indicate the need for more studies on the drug resistance phenotype of CRF01_AE. At the end of 2017, there were approximately 36.9 million people living with HIV, 59% of whom were receiving antiretroviral therapy (ART) . A studyMost drug resistance genotype data are derived from subtype B HIV-1, which is responsible for about 12% of infections worldwide and is mainly epidemic in North America and Europe . HoweverCRF01_AE is the first reported circulating recombinant form (CRF) of HIV-1 and one of the most influential CRFs in the world , accountThe DRM characteristics of CRF01_AE have been reported in several cross-sectional studies. However, most of these population-based studies assessed the prevalence of transmitted or acquired DRMs among populations –21 or coIn this study, we analyzed the natural polymorphisms of CRF01_AE from a large ART cohort in northeastern China and compared the polymorphisms between patients who experienced TF and those who experienced treatment success (TS). The acquired DRM profile was determined using self-control analyses that involved comparing baseline data to data collected at TF time point. Moreover, the potential role of unknown mutations was explored through co-variation analysis and next-generation sequencing (NGS).pol sequences (HXB2: 2253–3269) obtained by Sanger sequencing based on HIV drug resistance genotyping assays [2Two thousand and thirty-four HIV-1 CRF01_AE-infected patients were selected from a long-term ART cohort (follow-up every 3 to 6 months) at the First Affiliated Hospital, China Medical University in Shenyang between January 2002 and December 2017. Partial HIV-1 g assays  for eachs HXB2: 23–3269 obpol sequences of 2034 CRF01_AE-infected patients at baseline were aligned with reference sequences downloaded from the Los Alamos HIV database (https://www.hiv.lanl.gov/) using the ClustalW tool in Mega v7.0 software, and then were manually edited. The models package in Mega v7.0 was used to determine the best nucleotide substitution model for this dataset. The reference sequences included twelve CRF01_AE strains from Africa and Thailand sampled between 1990 to 2001 and the representative sequences from seven major CRF01_AE lineages in China previously reported [For phylogenetic analysis, the reported . FastTrereported .pol sequences of 42 TF patients at both baseline and TF using Mega v7.0. Bootstrap resampling (1000 datasets) of multiple alignments was performed to test the statistical robustness of the trees with the GTR + G + I nucleotide substitution model. A bootstrap value > 70 was identified as a cluster [A maximum-likelihood tree was reconstructed with the  cluster .https://hivdb.stanford.edu/) and interpreted using the Stanford HIVdb algorithm .DRMs were identified using the Stanford University HIV Drug Resistance Database  analyzed at each site . The mutation rates were also compared between 1148 TS patients and 105 TF patients. The HIV-1 strain HXB2 was used as the reference standard. The sites with a different amino acid (compared to the corresponding site in HXB2), and with a prevalence > 1%, were defined as natural polymorphism sites.The mutation rates of amino acids at sites 1 to 240 of reverse transcriptase (RT) region of the The CorMut package  v1.25.0 pol gene was then amplified by two rounds of nested polymerase chain reaction (PCR) using a KOD-Plus-Neo kit  with the following outer and inner primers, respectively: MAW26/RT-21n (5′-GTATTTCTGCATTAAGTCTTTTGATGG-3′), 3-3F (5′-ACAGTACTAGATGTGGGAGATGC-3′)/3-3R (5′-TATATCATTGACAGTCCAGCT GTC-3′). The reaction conditions are shown in Additional file 3.Longitudinal plasma samples between baseline and TF from four cases with Y181C and L228R mutations were selected. Viral RNA was extracted from the plasma samples using a QIAamp Viral RNA Mini Kit  according to the manufacturer’s protocol and reverse transcribed using a Transcriptor First Strand cDNA Synthesis Kit  with the specific primer Rev2–1 (5′-TCCTGCCATRGRAGATGCCTAA-3′). A 453-bp fragment (HXB2: 2868–3320) in the RT region of the The PCR products were purified with Agencourt AMPure XP beads  and then quantified using a Qubit 3.0 Fluorometer . The fragment length was accurately evaluated using an Agilent 2100 Bioanalyzer . Subsequently, the purified PCR products were adjusted to 2.5 ng/μl and indexed with an adaptor using a TruSeq Nano DNA LT library preparation kit  according to the manufacturer’s protocol. The indexed DNA libraries were analyzed using the Agilent 2100 Bioanalyzer and accurately quantified using a Roche LightCycler® 480 (LC480) Real-Time PCR system  and normalized to 10 nM, then pooled, denatured, and diluted to 15 pM, and finally mixed with 50% PHIX Control Libraries  to create a final volume of 600 μl.http://qiime.org/) in Windows operating system. According to the data quality assessment using FASTQC v0.11.7 software, paired-ended sequences were trimmed by 10–15 bp and truncated to 280–285 bp, and the other parameters were set to the default values. The data were denoised and dereplicated using dada2 plugin v2018.4.0 [Deep sequencing was performed using an Illumina MiSeq System . Oracle VM Virtual Box-5.2.22 software was used to build a virtual environment for running QIIME 2 Core-2018.4  treatment-naïve CRF01_AE-infected patients had transmitted DRMs, with the common DRMs comprising K103 N, G190S, K101E, T215S, K65R, and K219Q. In addition to above DRMs, natural polymorphisms of amino acids with a prevalence > 1% were detected at 53  sites in RT, of which nine sites  were known drug resistance-associated sites. Moreover, 31 sites  in CRF01_AE had higher mutation rates than subtype B HIV-1 strains in the Stanford HIV Drug Resistance Database  . Fifty-one and forty-four natural polymorphism sites in lineages 4 and 5 were detected, respectively, with differences in 35 sites between the two lineages . Both lineages had 26 polymorphism sites with higher mutation rates than in subtype B HIV-1globally , including two known drug resistance-associated sites (sites 179 and 238)  sequences of lineage 4 and 1522 (74.8%) sequences of lineage 5  patients experienced TF. We found 13 sites with differences between TF and TS patients , comprising the polymorphisms at sites 75 and 189, which were only found in TF patients, and the polymorphisms at sites 4, 5, 8, 21, 32, 49, 105, 165, 169, 171, and 204, which were only found in the TS patients. The mutation rate of site 75 in TF patients was significantly higher than in TS patients  . The sequences of the baseline and TF time point from each patient of the 42 TF patients clustered with bootstrap value higher than 85 in the phylogenetic tree . Moreover, a new mutation (L228R) was detected at site 228, a non-DRM site in the Stanford HIVdb algorithm, which increased from 0% at baseline to 11.9% at TF time point . We speculated that both V75 L and L228R might be potential new DRMs in CRF01_AE.Forty-two CRF01_AE-infected patients with TDF/3TC/EFV TF were selected according to the flow chart presented in Additional file Y181C-G190S = 22.86, LOD = infinity). V75 L was correlated with known DRMs G190S , K65R , and M184 V . L228R was correlated with known DRMs G190S  and K65R , and strongly correlated with Y181C   and two potential new DRMs (V75 L and L228R) were demonstrated to be under positive selection pressure . Twenty-eight links were detected among these mutations   were detected, which is even higher than the number of polymorphism sites in CRF01_AE strains mainly from Southeast Asia in a global study on non-B HIV-1 over 10 years ago , 32, andCRF01_AE , suggestCRF01_AE .In this study, the polymorphisms at five known drug resistance-associated sites  were polymorphic accessory mutations or other mutations that did not independently decrease drug sensitivity. None of them were associated with TF. Only V75 L, a low-frequency mutation, was associated with virologic failure, implying that most polymorphisms in CRF01_AE seldom lead to TF. A study on a London cohort in the United Kingdom found that different baseline polymorphisms, including V90I, A98S, and K103R, were associated with virologic failure , but theAt present, two NRTIs plus an integrase strand transfer inhibitor (INSTI) are recommended as a first-line ART regimen for adults in developed countries while, in developing countries like China, two NRTIs plus an NNRTI are still recommended as a first-line ART regimen. In this study, we evaluated the DRM profile of CRF01_AE after TDF/3TC/EFV TF. The most common acquired DRMs among CRF01_AE were K65R, M184 V, G190S/C, Y181C, and K103R, all of which are also common among subtype B and other subtypes. Despite some differences in the mutation type and rate, we cannot confirm that the differences were caused by the various subtypes , 37.More importantly, we detected two mutations with significant increases but without annotation in the Stanford HIVdb algorithm, V75 L and L228R. Site 75 is a drug resistance-associated site but no explanation for V75 L is provided in the Stanford HIVdb algorithm. The V75 L mutation has been reported to provide a selective advantage by allowing escape from the host immune responses  and it iThis study had several limitations. First, due to the high success rate of ART in this cohort, only a small number of TF patients could be included in the acquired DRM analysis. Second, the impact of the L228R mutation with or without Y181C needs further validation using virus growth competition and drug resistance phenotype assays. Nevertheless, this study provided more evidence of polymorphisms and DRMs in the non-B HIV-1 strain CRF01_AE.In summary, the high levels of polymorphisms in CRF01_AE had little impact on treatment outcomes, but some unknown mutations associated with TF might be minor DRMs. The results of this study indicate the need for more studies on drug resistance in non-B HIV-1, especially phenotypic studies to strengthen the drug resistance genotype interpretation, and to improve ART efficacy and minimize the transmission of drug-resistant strains.Additional file 1:Figure S1. Flow chart of selection and analysis.Additional file 2: Demographic and clinical characteristics of participants in this study.Additional file3: Reaction conditions of cDNA synthesis and amplification of the target fragment.Additional file 4: Figure S2. Phylogenetic analysis of the CRF01_AE HIV-1 pol sequences.Additional file 5:Figure S3. Phylogenetic analysis of the 42 TF CRF01_AE-infected patients.


Several xenogenic cell-based therapeutic products are currently under development around the world for the treatment of human diseases. Porcine islet cell products for treating human diabetes are a typical example. Since porcine cells possess endogenous retrovirus (PERV), which can replicate in human cells in vitro, the potential transmission of PERV has raised concerns in the development of these products. Four subgroups of infectious PERV have been identified, namely PERV-A, -B, -C, and recombinant PERV-A/C. Among them, PERV-A/C shows a high titre and there was a paper reported that an incidence of PERV-A/C viremia was increased in diseased pigs; thus, it would be important to monitor the emergence of PERV-A/C after transplantation of porcine products. In this study, we developed a highly sensitive method for the detection of PERV-A/C using next generation sequencing (NGS) technologies. A model PERV-C spiked with various doses of PERV-A/C were amplified by RT-PCR and the amplicons were analysed by NGS. We found that the NGS analysis allowed the detection of PERV-A/C at the abundance ratios of 1% and 0.1% with true positive rates of 100% and 57%, respectively, indicating that it would be useful for the rapid detection of PERV-A/C emergence after transplantation of porcine products. In addition to artificial organs, xenogenic cell-based therapeutic products have been expected to become a breakthrough of this problem4, and several products, such as porcine islet cell products for treating human diabetes, are currently under development around the world7.While the development of superior immunosuppressive drugs and improved medical treatments has made organ transplantation an established medical practice, the chronic shortage of human donor organs remains a problem. The Global Observatory on Donation and Transplantation reported that 139,024 organ transplants were performed all over the world in 2017, but this only covers 10% of the global need9. In the case of pigs, which have been well-studied donors for xenotransplantation, it has been demonstrated that most of known microorganisms can be eliminated by performing a caesarean section and by controlling the breeding environment9, with the sole exception of porcine endogenous retroviruses (PERV)11. PERV infected porcine germ cells in ancient times, and thus all porcine cells possess PERV proviruses in their genome14. Four subgroups of PERV have been identified, PERV-A, -B, -C, and recombinant PERV-A/C. Both PERV-A and PERV-B are capable of infecting human cells, whereas PERV-C is not able to infect human cells16. PERV-A/C is a recombinant PERV-C in which a portion of the env has been recombined with that of PERV-A. Although the genome sequence of PERV-A/C has been shown to be almost identical to that of PERV-C17, the recombinant virus can replicate in human cells19. Since PERV-A/C was originally detected after cocultivation of porcine cells with human cells20 and has not been found in the porcine germ line21, the recombination was considered to have happened in human cells. However, PERV-A/C was found in somatic cells of miniature swine, indicating that PERV-A/C could also arise in porcine cells alone without human cell infection25. Because PERV-A/C recombinants replicate rapidly in human cells in vitro20 and an incidence of PERV-A/C viremia is increased in diseased swine26, it would be important to monitor for the appearance of PERV-A/C after xenotransplantation of porcine cell-based therapeutic products19, even though PERV infection in humans exposed to living porcine cells has not been reported thus far27.One of the major concerns in xenogenic cell-based therapeutic products is the transmission of pathogens carried by donor animals to human recipients28. However, since the recombination sites between PERV-A and PERV-C are not fixed23 and these viral genome sequences are highly identical, there is a concern about false positive and negative. Therefore, examining the sequence of PCR-amplified products is thought to be more reliable in the case of detecting the recombinants. There are 2 classical methods based on Sanger sequencing used in detecting mutations: direct sequencing, in which the PCR-amplified target DNA region is read directly, and sequencing after cloning, in which the amplified DNA is incorporated into a plasmid, cloned in E. coli, and then the inserted DNA of the plasmid is read. Although the direct sequencing method is simple, it has the disadvantage of low sensitivity because only the majority sequence can be read. On the other hand, the sequencing after cloning requires an increased number of clones for increased sensitivity, which is laborious and time-consuming. Amplicon sequencing (Amplicon-Seq) refers to deep sequencing of PCR products using next generation sequencing (NGS) technologies, and is performed for various applications, such as to detect somatic mutations in one or several exons and introns29 or to explore microorganisms in samples by sequencing the 16S rRNA30. It also has been used to detect minor variants of viral populations33. In this study, we performed Amplicon-Seq analysis using the primers that amplify the entire env region of PERV-C and PERV-A/C and characterized the ability of this analysis to detect the presence of PERV-A/C recombinants.The PCR assay is considered to be the most sensitive and rapid method for the detection of known microorganisms and there are reliable primers for detecting PERV-A/C recombinantsenv sequences obtained from the NCBI GenBank, with NGS reads being classified by whether or not the read was in the same cluster as previously reported PERV-A/C env sequences  and PERV-A/C (13653)23 synthesized by in vitro transcription reaction were used as model viruses, and analytic samples were prepared by mixing PERV-C and PERV-A/C at ratios of 1 to 0 (negative control), 1 to 0.1, 1 to 0.01, and 1 to 0.001, which were referred to as "0% PERV-A/C", "10% PERV-A/C", "1% PERV-A/C", and "0.1% PERV-A/C", respectively. The entire env region was amplified by reverse transcription polymerase chain reaction (RT-PCR), and the sequence of the amplified products, which were about 2 kbp, was determined by PacBio single molecule, real-time (SMRT) technology. The primers used in this reaction amplified neither PERV-A nor PERV-B. In our preliminary experiment, RT-PCRs using PERV-A&C-For and PERV-C-Rev as primers also showed no fragment amplification from total mRNA of porcine cell lines PKF (IFO50422), PKR (IFO50423), PKS (IFO50421), and LLC-PK1 (JCRB0060) , indicating not only the absence of PERV-C and A/C in the cell lines but also the specificity of the primers. We used the PacBio RS II as the NGS platform, because it is suitable for long-read sequencing. Raw data were initially processed through the PacBio SMRT portal and filtered for a minimum of 6 passes expected for accuracy of 99.9%34.To determine PERV-A/C recombinants, a phylogenetic tree was created from the multiple alignment of the NGS read sequences and PERV ces Fig. . The RNASix independent analyses offered approximately 23,000 to 44,000 sequence reads from each sample Table . As the p < 0.003, two-way repeated measures analysis of variance and the Student–Newman–Keuls’s post-hoc test), indicating that local alignment was more suitable for this analysis. The mean number of the PERV-A/C reads detected in the 0.1% PERV-A/C samples was 0.33 and 0.83 using global and local alignments, respectively. Assuming that the PERV-A/C reads follow the Poisson distribution, the true positive rate of the 0.1% PERV-A/C samples was calculated to be 28% an 57%, when global and local alignments were applied, respectively . Since PERV-A/C was not detected in the 0% of samples in all experiments, the specificity of this analysis was regarded to be 100%. PERV-A/C at the abundance ratios of 10% and 1% was always detectable, regardless of the alignment method . This peak contained about half of total reads, suggesting that the PERV-A/C reads were lost there. Because global alignment compares the end to end sequences, reads which were not read at the full length could be scored as low identity. On the other hand, local alignment compares the most matched portion of the sequences, thus partial reads might have been unaffected by the analysis.38. Since the global alignment is used for clustering and the homology between PERV-C and PERV-A/C is less than 93%, clustering at 95% similarity or more might reduce the data without affecting the detection sensitivity.In this study, the Amplicon-Seq analysis using PacBio RS II showed that more than 100 recombinant reads were detected in the 10%, 6 to 49 reads in 1%, and 0 to 2 reads in 0.1% of the samples. The number of detected reads was roughly correlated with the concentration of the samples, suggesting that the first NGS data would need to be increased by a factor of 10 or more in order to detect the recombinants at a ratio of 0.01% or less in the samples. This increase in the obtained data would be possible by using other NGS equipment, such as PacBio Sequel I or II. On the other hand, the increase of data would lead to a heavy burden in analysis. Multiple alignment especially takes time to be calculated and thus it is important to reduce the number of reads before that in order to process the analysis as smoothly as possible using general computers. Sequence clustering, which sorts nucleotide or amino-acid sequences into clusters based on sequence similarity is used as one of the methods to reduce the NGS dataAlthough PERV-A/C recombinants were detected in 1% of samples in 6 independent experiments, we were unable to detect them when we analysed low-purified RT-PCR products 13 , which have been infected with both PERV-A and -B but not with PERV-C, using an RNeasy Mini Kit  following the manufacturer’s instructions. RT reaction was conducted by using a ReverTra Ace qPCR RT Kit  and 5′- CTAGAGGTCAGTTTCTCCTTGGCTCAGAAGGCC-3′ (PERV-A-Rev) which binds to 3′ end region of PERV-A (Y12238) env as the gene specific primer. PCR was then performed by using KOD -Plus- Neo (TOYOBO) and primers of 5′- ATGCATCCCACGTTAAGCCGGCGCC-3′ (PERV-A-For), which binds to 5′ end region of PERV-A (Y12238) env, and PERV-A-Rev. The PCR cycling condition consisted of 2 min at 94 °C followed by 35 cycles of 98 °C for 10 s and 68 °C for 1 min (two-step PCR). Amplified products (1983 bp) were then cloned into the pTA2 vector (TOYOBO) and its nucleotide sequence was confirmed (pTA2/PERV-A env). To generate PERV-C env cDNA, total RNA was extracted from pig blood , which was collected in accordance with relevant guidelines and regulations at the licensed Rockland facilities , using a NucleoSpin RNA Blood  following the manufacturer’s instructions. RT reaction was conducted using a ReverTra Ace qPCR RT Kit (TOYOBO) and 5′-CTAGCGGCCAGCTTCCCTGCTAGACGG-3′ (PERV-C-Rev) which binds to 3′ end region of PERV-C (AF038600.1) and -A/C (AF417230.1) env as the gene specific primer. PCR was then performed by using KOD -Plus- Neo (TOYOBO) and primers of 5′-ATGCATCCCACGTTAAACCGGCGCCACCTCCCG-3′ (PERV-A&C-For), which binds to 5′ end region of PERV-A (Y12238), -C (AF038600.1), and -A/C (AF417230.1) env, and PERV-C-Rev. The PCR cycling condition consisted of 2 min at 94 °C followed by 35 cycles of 98 °C for 10 s and 68 °C for 1 min. The pig blood was purchased from a commercial source (Rockland) and no pigs have directly been handled as a part of this study. Amplified products (1917 bp) were then cloned into the pTA2 vector and its nucleotide sequence was confirmed (pTA2/PERV-C env).To generate PERV-A env in which a portion of the env has been recombined with that of PERV-A was generated by site-directed mutagenesis using the PCR-mediated overlap primer extension method41. The recombination site of the PERV-A/C was based on a previous report (PERV-AC env (13653))23. To generate the PERV-A/C env, 3 successive PCR reactions were performed using pTA2/PERV-A env and pTA2/PERV-C env as templates. The first PCR reaction used pTA2/PERV-A env as a template and PERV-A&C-For and 5′- GGTGTTGGTGGGATGGGGGAACCTTCCCTATGCAGGTGCCTTTTCC-3′  env (1111–1131)) as primers, and the second used pTA2/PERV-C env as a template and 5′- GGAAAAGGCACCTGCATAGGGAAGGTTCCCCCATCCCACCAACACC-3′  env (1068–1093)) and PERV-C-Rev as primers. The resultant first (1131 bp) and second (849 bp) fragments were used as templates in the third reaction with PERV-A&C-For and PERV-C-Rev as primers. The resultant products (1980 bp) were then cloned into the pTA2 vector and its nucleotide sequence was confirmed (pTA2/PERV-AC env (13653)). The sequence was nearly identical (98.8%) to that of the previous reported PERV-A/C (AF417230.1) env .PERV-A/C env, in vitro transcription reactions were performed using Not I-linearized pTA2/PERV-C env and pTA2/PERV-AC env (13653) as templates . The synthesized RNAs were purified using PureYield RNA Midiprep System (Promega), and the purified RNAs were then used as model viruses.To synthesize RNAs of PERV-C and PERV-A/C env RNA was mixed with serial diluted 0.1, 0.01, or 0.001 ng of PERV-A/C env RNA, and the mixtures were used as templates in RT-PCR reactions. RT reaction was conducted using a ReverTra Ace qPCR RT Kit (TOYOBO) and PERV-C-Rev which binds to 3′ end region of PERV-C (AF038600.1) and PERV-A/C (AF417230.1) as the gene specific primer. PCR was then performed by using KOD -Plus- Neo (TOYOBO) with primers of PERV-A&C-For which binds to 5′ end region of PERV-C (AF038600.1) and PERV-A/C (AF417230.1) and PERV-C-Rev. The PCR cycling condition consisted of 2 min at 94 °C followed by 35 cycles of 98 °C for 10 s and 68 °C for 1 min. Amplified products were then separated by agarose gel electrophoresis and products of 2kbp were purified using the FastGene Gel/PCR Extraction Kit . The purity of the products was confirmed using the Agilent 2100 Bioanalyzer .Briefly, 1 ng of PERV-C DNA samples were further purified using the AMPure XP , blunt-ended, and ligated to SMRTbell adaptors using the SMRTBell Template Prep Kit 1.0  in order to construct SMRTbell libraries. One SMRT cell was used for each library and analysed using the PacBio RS II platform (Pacific Biosciences).42. Global and local alignments were performed using the vsearch (version 2.14.2)43 and the GENETYX-Mac Ver.20 , respectively. After local alignment, using the SeqKit data containing over 1000 reads were divided into several files of less than 1000 reads each. Processed data were analysed by multiple alignment with PERV env sequences obtained from the NCBI GenBank , and multiple alignment data were used for creating phylogenetic trees using the Neighbor-Joining method44. Multiple alignment and construction of phylogenetic trees were performed using the Geneious Prime .The Circular Consensus Sequence (CCS) reads from each SMRT cell were filtered for a minimum of 6 passes, and reads with under 1500 bp length were eliminated by the SeqKit (version 0.12.0)λ), and the probability of detecting k reads in a single experiment was calculated by the following probability mass function of the Poisson distribution:k! is the factorial of k and e is the Euler’s number (e = 2.71828…).The mean number of PERV-A/C reads detected in a single experiment was calculated (P (0).The true positive rate that is the probability of detecting one PERV-A/C read or more in a single experiment was given by 1 – 45, whereas the term “sensitivity” was used in a broad sense.In this study, use of the term “true positive rate” was identical to “clinical/diagnostic sensitivity” as defined in ISO/TS 17822-1:2014The data that support the findings of this study are available from the corresponding author, YS, upon reasonable request.Supplementary Information.


CFTR gene mutations vary considerably between countries and ethnic groups. Russians are an East Slavic ethnic groups are native to Eastern Europe. Russians, the most numerous people of the Russian Federation (RF), make about 80% of the population. The aim is to reveal the molecular causes of CF in ethnic Russian patients as comprehensively as possible. The analysis of most common CFTR mutations utilized for CF diagnosis in multiethnic RF population accounts for about 83% of all CF-causing mutations in 1384 ethnic Russian patients. Variants c.1521_1523delCTT (F508del), c.54-5940_273+10250del21kb , c.2012delT (2143delT), c.2052_2053insA (2184insA), and c.3691delT (3821delT) are most typical for CF patients of Russian origin. DNA of 154 CF patients, Russian by origin, in whom at least one mutant allele was not previously identified , was analyzed by Sanger sequencing followed by the multiplex ligase-dependent probe amplification (MLPA) method. In addition to the 29 variants identified during the previous test for common mutations, 91 pathogenic CFTR variants were also revealed: 29 missense, 19 nonsense, 14 frame shift in/del, 17 splicing, 1 in frame ins, and 11 copy number variations (CNV). Each of the 61 variants was revealed once, and 17 twice. Each of the variants c.1209G>C (E403D), c.2128A>T (K710X), c.3883delA (4015delA), and c.3884_3885insT (4016insT) were detected for three, c.1766+1G>A (1898+1G>A) and c.2834C>T (S945L) for four, c.1766+1G>C (1898+1G>C) and c.(743+1_744-1)_(1584+1_1585-1)dup (CFTRdup6b-10) for five, c.2353C>T (R785X) and c.4004T>C (L1335P) for six, c.3929G>A (W1310X) for seven, c.580-1G>T  for 11 unrelated patients. A comprehensive analysis of CFTR mutant alleles with sequencing followed by MLPA, allowed not only the identification of 163 of 164 unknown alleles in our patient sample, but also expansion of the mutation spectrum with novel and additional frequent variants for ethnic Russians.The distribution and frequency of the  CFTR) gene. CF is a hereditary disease caused by impaired epithelial chloride channel CFTR function. Variants are classified as disease causing, not disease causing, of variable clinical significance, or of unknown clinical significance. More than 2000 different variants of the CFTR gene sequence have been revealed, the pathogenicity of 20% of which is established (2908+1_2989-1)del]  and c.(273-1_274+1)_(869+1_870-1)del(1209-1_1210+1)_(1392+1_1393+1)del  was detected twice. Complex deletion, CFTRdele12,13;del16, was detected for two patients from unrelated families living in the Moscow region; deletion CFTRdel4-7;del9-10 for two families from the Kaliningrad region and the Republic of Buryatia; deletion CFTRdele2 in families from the Transbaikal region and Irkutsk region.CFTR gene.As a result of analysis of the coding sequence and regions of exon-intron junctions and subsequent search for large rearrangements, 163 out of 164 alleles were identified that were not detected after preliminary testing of frequent variants of the CFTR-dependent Cl− currents and HCO3− transport activities. So we might suggest that the clinical presentation in that patient is due to complex allele E217G-M470  . In the 17G-M470 .CFTR gene could not be identified in one sample. Failure to identify the second pathologic mutation in the CFTR gene after sequencing the coding sequence and searching for large rearrangements may be due to the location of the pathogenic variant either in inner regions of the introns, or in regulatory regions of the CFTR gene, or in regulatory regions outside the CFTR gene. Indeed, such variants have been recently identified, for example, c.1680-883A>G, c.2989-313A>T, c.3469-1304C>G, or c.3874-4522A>G, that lead to the creation of a new donor splice site and the activation of a cryptic acceptor splice site, resulting in the inclusion of an additional pseudo-exon (PE) and the loss of wild type (WT) CFTR transcripts [The second mutant allele of the nscripts .CFTR gene was studied. It was shown that, out of 29 identified variants, frequencies of only 10 exceed 1%, and the mutation detection rate of testing did not exceed 85%. Consistent use of sequencing and MLPA methods has allowed us to identify a significant variety of CFTR gene mutations spectrum , to expand the spectrum of frequent variants (c.1766+1G>C (1898+1G>C), c.2353C>T (R785X), c.(743+1_744-1)_(1584+1_1585-1)dup (CFTRdup6b-10), c.4004T>C (L1335P), c.3929G>A (W1310X), c.580-1G>T (712-1G>T), c.1240_1244delCAAAA (1365del5), detected for five and more unrelated patients, to increase the detection rate of identified mutant alleles for Russian CF patients up to 99.4%, consistently using the strategy of Sanger sequencing and MLPA analysis. This information can be useful for the further optimization of medical genetic counseling in CF high-risk families, for improving the neonatal screening program for CF, and for making decision about the possible CFTR modulators therapy in the future. The identification of previously unknown CF-pathogenic or likely-pathogenic variants is a useful piece of information for diagnostic testing not only in Russia, but worldwide, and can be considered as a contribution to the general knowledge about the CFTR variant heterogeneity.In a representative sample of CF patients (ethnic Russians), the spectrum of 33 routinely analyzed (in Russia) variants of the