DNA viruses have evolved complex strategies to gain control over the cell cycle. Several of them target APC/C, a key cellular machinery that controls the timely progression of the cell cycle, by either blocking or enhancing its activity. Here, we investigated the activity of APC/C during the lytic replication cycle of KSHV and found that, in contrast to that of KSHV's close relatives EBV and HCMV, KSHV lytic replication occurs while the APC/C is active. Perturbing APC/C activity by depleting a core protein or the adaptor proteins of the catalytic domain, and hence interfering with normal cell-cycle progression, did not affect virus replication. This suggests that KSHV has evolved to replicate independently of the activity of APC/C and in various cell cycle conditions. The anaphase-promoting complex, or cyclosome (APC/C), is a large E3 ubiquitin ligase composed of 14 subunits. The activity of APC/C oscillates during the cell cycle to ensure a timely transition through each phase by promoting the degradation of important cell cycle regulators. Of the human herpesviruses, cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) both impair the activity of APC/C during their lytic replication cycle through virus-encoded protein kinases. Here, we addressed whether the oncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV) deregulates the activity of APC/C during the lytic replication cycle. To this end, we used the well-characterized iSLK.219 cell model of KSHV infection and established a new infection model of primary lymphatic endothelial cells (LECs) infected with a lytically replicating KSHV BAC16 mutant. In contrast to those of EBV and HCMV, the KSHV lytic cycle occurs while the APC/C is active. Moreover, interfering with the activity of APC/C did not lead to major changes in the production of infectious virus. We further investigated whether rereplication stress induced by the unscheduled activation of the APC/C-CDH1 complex affects the number and integrity of KSHV viral episomes. Deep sequencing of the viral episomes and host chromosomes in iSLK.219 cells revealed that, while distinct regions in the cellular chromosomes were severely affected by rereplication stress, the integrity of the viral episomes remained unaltered.IMPORTANCE DNA viruses have evolved complex strategies to gain control over the cell cycle. Several of them target APC/C, a key cellular machinery that controls the timely progression of the cell cycle, by either blocking or enhancing its activity. Here, we investigated the activity of APC/C during the lytic replication cycle of KSHV and found that, in contrast to that of KSHV's close relatives EBV and HCMV, KSHV lytic replication occurs while the APC/C is active. Perturbing APC/C activity by depleting a core protein or the adaptor proteins of the catalytic domain, and hence interfering with normal cell-cycle progression, did not affect virus replication. This suggests that KSHV has evolved to replicate independently of the activity of APC/C and in various cell cycle conditions. Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma (KS), a common malignancy in people infected with HIV . KS is a2–10–1, APC/C is associated with CDH1 (FZR1), a coadaptor protein that enables APC/C to target a specific subset of substrates for degradation. This prevents cells from proceeding to S-phase and allows licensing factors to associate at the cellular origins of replication , is crucial for cell cycle progression, as it acts as efficient E3 ubiquitin ligase machinery that targets various cell cycle regulators for degradation in a timely manner. APC/C substrates include cyclins, such as CCNB1 and CCNA2, and other key regulators such as GMNN, CDC6, CDC20, AURKA, and AURKB , 18. In lication , 19.At the onset of S-phase, APC/C is inactivated by EMI1, a potent inhibitor of APC/C-CDH1 that occupies the APC/C catalytic domain and acts as a pseudosubstrate , 21. As 2, prior to mitosis, the CDH1 subunit is phosphorylated to facilitate its dislocation from the APC/C-CDH1 complex to allow APC/C to recruit CDC20, the mitotic coadaptor protein. APC/C-CDC20 specifically degrades mitotic regulators like CCNB1, NEK2A, and securin /cell. Using this infection dose, at 48 h p.i., the morphology of all cells changed from cobblestone to spindle cells , and at Overall, these findings show that LECs provide a highly permissive cellular environment for the KSHV-Lyt replication and are a suitable model to study the effects of KSHV lytic replication in primary cells.To test whether KSHV interferes with APC/C, we infected LECs with KSHV-Lyt using four IU/cell. Latent and lytic viral protein expression was confirmed by immunoblotting for LANA, ORF50, ORF57, ORF45, and K8.1 . Additio39–EMI1 and CCNB1 levels were downregulated at 24 h and later upregulated during the KSHV infection. In contrast, GMNN mRNA levels were either similar to uninfected or increased in the late stages of infection by 2-fold  were analyzed after treatment with phosphonoacetic acid (PAA) to specifically block the KSHV genome replication and thus the accumulation of viral DNA in the nucleus , the APC/C inhibitor (EMI1), and two APC/C substrates (CCNB1 and GMNN), as compared to their levels in uninfected cells. Throughout the 72-h time course, we observed that both CDH1 and CDC20 were expressed in KSHV-Lyt-infected LECs, suggesting that both proteins are available as APC/C adaptors. The levels of CCNB1 did not increase markedly beyond the range that was observed in uninfected cells (<2-fold), while the GMNN levels fluctuated in a similar fashion as EMI1 , indicaty 2-fold . Therefoy 2-fold . Next, wed cells  and wher nucleus . AltogetSubsequently, to validate our findings in another infection model, we repeated experiments using the cancer-derived iSLK.219 cell line stably infected with rKSHV.219, which constitutively expresses GFP under the control of the cellular EF1a promoter and RFP from the PAN promoter, an ORF50-responsive viral lytic promoter . In thesTaken together, these results show that during the lytic cycle in both the KSHV-Lyt-infected LECs and iSLK.219 cells, the APC/C remains under the control of its inhibitor EMI1 and retains the ability to degrade target substrates, such as GMNN.GQ994935.1) using GPS-ARM software  does not affect the KSHV lytic replication cycle in either iSLK.219 cells or KSHV-Lyt LECs.Similarly, depletion of APC3, an essential core protein of APC/C, increased the APC/C substrate levels  in both the KSHV-Lyt-infected LECs and iSLK.219 cells  and B. Y2 at 48 h after EMI1 depletion), while most of the LECs remained in G1 with less than 1% of EMI1-depleted LECs undergoing rereplication  is the induction of rereplication stress , 23. Acclication . As a colication . Furtherlication , and thelication , therebyInterestingly, despite these deep alterations , EMI1-depleted iSLK.219 cells were still able to sustain the full KSHV lytic replication cycle and released infectious virus to the same extent as the control (siNeg) treated cells see . TherefoAltogether, these findings indicate that rereplication stress affects the integrity of the cellular genome but not the viral episomes, thus allowing the efficient production of infectious virus from the EMI1-depleted iSLK.219 cells.1 phase where APC/C-CDH1 is active. This is in contrast to the other herpesviruses, HCMV and EBV, which have been found to block APC/C activity during their lytic cycle  compared to uninfected cells. This could suggest that an intrinsic mechanism of KSHV during primary infection is to manipulate EMI1 levels in order to preserve the ability of the infected cells to proceed to S-phase while the virus establishes latency. Further supporting this hypothesis is the observation that GMNN mRNA is also upregulated during the KSHV lytic cycle  and the AT-rich region close to the K5 gene (OriA) , 54, 55.de novo infected with KSHV-Lyt, the latter providing a novel and useful model to study KSHV lytic replication in a physiologically relevant cell type. Overall, our results show that the KSHV lytic replication program occurs efficiently even when the cell cycle is strongly perturbed and is independent of the activity of APC/C.Our findings were confirmed in two different experimental systems, the iSLK.219 cells and LECs l-glutamine, and 1% penicillin/streptomycin. In the medium of iSLK.219 cells, puromycin (10 μg/ml) (Sigma), hygromycin B (600 μg/ml) (Sigma), and G418 (400 μg/ml) (Roche) were further supplied. To induce the KSHV.219 lytic cycle, iSLK.219 cells were treated with Dox (1 μg/ml) and NaB (1.35 mM). Phosphonoaceticacid (PAA) was obtained from Sigma.Primary LECs (C-12216), BECs (C-12211), and HuVECs (C-12203), all from Promocell, were grown in Lonza EGM-2 (00190860) medium supplemented with EGM 2 MV Microvascular Endothelial SingleQuots (CC-4147). Vascular endothelial growth factor (VEGF) supplement was not added to the medium. In experiments, LECs, BECs, and HuVECs were used from passage 3 to 6. U2OS were obtained from ATCC (HBT-96) and iSLK.219 cells, generated by J. Myoung and D. Ganem , were a The design of the lytic KSHV BAC16 is based on the principle described for KSHV-Lyt BAC36 by M. Budt et al. . In thisKSHV-Lyt virus was reconstituted by transfecting purified KSHV-Lyt BAC DNA in LECs using FugeneHD (Promega) at a DNA (μg) to FugeneHD (μl) ratio of 1:3.5 following the manufacturers recommendations. KSHV-Lyt spread to uninfected LECs was monitored by using EGFP, expressed by KSHV-Lyt, as a marker of infection. The supernatants containing KSHV-Lyt were stored at –80°C.To construct the pLenti6.3-GMNN vector, the following primers were used to amplify the GMNN ORF from the cDNA of iSLK.219 and the pLenti6.3 backbone from the pLenti6.3-EGFP vector (provided by Genome Biology Unit Helsinki):Fw 5′-TAGAGGATCCACTAGTCCAGTGTGGGCCACCATGAATCCCAGTATGAAGCAGAAACAAG-3′Rv 5′-GTTAGGGATAGGCTTACCTTCGAACTCATATACATGGCTTTGCATCCGTAGAGG-3′Fw 5′-GTTCGAAGGTAAGCCTATCCCTAAC-3′Rv 5′-GGTGGCCCACACTGGACTAGTGGATC-3′The purified PCR fragments were fused together using NEB HiFi DNA assembly MasterMix . The sequence of GMNN was verified by Sanger sequencing and the backbone integrity by restriction analysis.pLP1, pLP2, and pVSVG  together with pLenti6.3-GMNN lentiviral expression vector were transfected in 293FT cells using Lipofectamine 2000 (Thermo Scientific). The medium was replaced on the next day and harvested 72 h posttransfection. The virus-containing supernatant was filtered and mixed with 5× PEG-it (System Biosciences) and the virus particles were concentrated according to the manufacturer’s recommendations. Lentivirus titers were determined by infecting U2OS cells and by staining infected cells with GMNN antibodies.4 cells/cm2 and LECs were seeded 2.5 × 104 cells/cm2.Nontargeting siRNA  and siRNA targeting the human EMI1 (L-012434), CDH1 (L-015377), CDC20 (L-003225), and APC3 (L-003229) mRNA transcripts were purchased from Dharmacon. iSLK.219 and LEC cells were reverse transfected with 10 nM and 30 nM siRNA, respectively, using Lipofectamine RNAiMax (Thermo Scientific) according to the manufacturer’s recommendation. iSLK.219 cells were seeded at approximately 4 × 10Cells were lysed in 1× Laemmli sample buffer. Proteins were separated using a 4 to15% Criterion TGX Precast midi gel (Bio-Rad) and subsequently transferred onto a Trans Blot Turbo midi nitrocellulose membrane (Bio-Rad). Protein transfer and loading on each membrane was assessed by Ponceau S staining. The membranes were blocked with 5% nonfat dry milk in TBS-T  and were incubated with the indicated primary antibody diluted in 5% nonfat dry milk or bovine serum albumin (BSA) in TBS-T at 4°C overnight. The antibodies detecting GMNN (sc-13015), CDH1 (sc-166714), CCNB1 (sc-245), ORF57 (sc-135746), ORF45 (sc-53883), K8.1 (sc-65446), actin (sc-8432), APC3 (sc-9972), securin (sc-56207), CCNA2 (sc-271682), PLK1 (sc-17783), and p53 (sc-126) were from Santa Cruz Biotechnology (SCBT). EMI1 (37-6600) antibody was from Thermo Scientific, CDC20 (GTX5395) from GeneTex, AURKB (611082) from BD Biosciences, and ORF50 was a gift from Carolina Arias . AURKA (3092), pp53-S15 (9284S), pCHK2-T68 (2197S), and CHK2 (6334S) antibodies were from Cell Signaling Technology (CST). LANA (ab4103) antibody was from Abcam. The membranes were further incubated with appropriate secondary antibody linked to horseradish peroxidase (HRP) diluted in 5% nonfat dry milk in TBS-T. Anti-rabbit IgG HRP-linked (7074S), anti-mouse IgG HRP-linked (7076S), and anti-rat IgG HRP-linked (7077S) secondary antibodies were from CST. The protein bands were revealed using WesternBright Sirius HRP substrate (Advansta) and images were acquired using a ChemiDoc gel imaging system (Bio-Rad).Where indicated, band intensities were quantified using FIJI  softwareCells were washed with phosphate-buffered saline (PBS) pH 7.4 and fixed in 4% paraformaldehyde in PBS, permeabilized using PBS with 0.2% Triton X-100 and blocked using PBS with 0.2% Triton X-100 containing 1% BSA. Cells were then incubated with antibodies against LANA , K8 , ORF57 , ORF45 , K8.1 , 53BP1 , GMNN , GMNN , pH2AX-S139 , pCHK1-S317 , ORF50 , or GFP  where indicated. The cells were afterward incubated with appropriate secondary antibodies coupled to Alexa fluorophores. Anti-rabbit IgG (H+L) Alexa Fluor 488 (A-11034), anti-rabbit IgG (H+L) Alexa Fluor Plus 555 (A-32732), anti-rabbit IgG (H+L) Alexa Fluor 647 (A-32733), anti-mouse IgG (H+L) Alexa Fluor 647 (A-32728), anti-rat IgG (H+L) Alexa Fluor 647 (A-21247), and anti-mouse IgG (H+L) Alexa Fluor Plus 647 (A-32728) were acquired from Thermo Scientific. Nuclei were counterstained with 1 μg/ml Hoechst 33342 (Sigma) and the images were acquired using an AxioImager microscope equipped with an Apotome (Zeiss), ImageXpress Pico (Molecular Devices), or CellInsight High Content microscope (Thermo Fisher Scientific). The quantification and processing of images were done using the Cell profiler 3.0  and FIJICells were fixed with 70% ethyl alcohol (EtOH) and stained with antibodies against ORF57  and anti-mouse IgG (H+L) Alexa Fluor Plus 647 , where indicated. DNA was then stained with FxCycle PI/RNaseA staining solution (Thermo Scientific) for 1 h. The cells were analyzed using a BD Accuri C6 flow cytometer and data were processed using the CFlow Sampler software (BD).For cell proliferation analysis, cells were maintained for 2 h in medium containing 10 μM 5-ethynyl-2’-deoxyuridine (EdU). Subsequently, the cells were fixed in 4% paraformaldehyde in PBS and the EdU incorporated in the cell DNA was coupled to Alexa Fluor 647 according to the manufacturer’s instructions provided in the Click-iT EdU Alexa Fluor 647 imaging kit (Thermo Fisher Scientific). Images were acquired using a CellInsight High Content microscope (Thermo Fisher Scientific) and analyzed using Cell Profiler 3.0 software to quantify EdU-positive cells.Actin , GMNN , CCNB1  EMI1 . The RT-qPCRs were conducted in a Light Cycler 480 qPCR system (Roche).Cellular RNA was extracted using a NucleoSpin RNA extraction kit (Macherey-Nagel) and a total amount of 1 μg of RNA was reverse transcribed to cDNA using oligo(dT) primers and Multiscribe reverse transcriptase (Thermo Fisher Scientific) according to the manufacturer’s instructions. Equal amounts of cDNA were mixed with 2× SYBR reaction mix (Fermentas) together with appropriate dilution of the primer pairs specific for mRNA transcripts of g, 30 min, room temperature [RT]). The next day the cells were fixed and stained with an EGFP antibody  and the nuclei stained with Hoechst 44432 . The cells were imaged using a CellInsight High Content microscope (Thermo Fisher Scientific) and quantified using the Cell Profiler 3.0 Software  and NaB . Afterwards, the virus dilutions were used to infect U2OS cells by spin infection  for 24 h and 48 h or serum starved for 24 h. Cells from each sample (6 × 105) were subjected to genomic DNA isolation using a NucleoSpin Tissue DNA extraction kit (Macherey-Nagel), including an RNase A treatment after proteinase K digestion. Additionally, prior to DNA isolation, bacteriophage Lambda DNA  was added at 10 lambda genome copies/cell as a DNA spike-in control. The DNA sequencing was performed using NextSeq High Output system (Illumina), 1 × 75 bp single-end reads.Latent iSLK.219 cells were seeded at 4 × 10NC_001416), and to the rKSHV.219 sequence, which is identical to the sequence of BAC16 (GenBank GQ994935), using Bowtie , to the phage lambda reference sequence  as the denominator, likewise to the calculations of rKSHV.219 coverage variation data. Circos plots of the resulting coverage variation tracks were then generated with Circos (2 fold differences of >0.2 (red) and <−0.2 (green).Coverage calculation and 0.1-kb window read counting for rKSHV.219 was performed with IGV-Tools . The resol-FREEC  and calch Circos . Colors t tests were carried out using GraphPad Prism 7 software. To test whether differences between specific means were significant, after one-way ANOVA, a Dunnett’s multiple-comparison test was used.One-way ANOVA and Student’s PRJNA549967).Raw sequencing data are available in the Sequence Read Archive (BioProject


Passive immunization with broadly neutralizing antibodies (bNAbs) of HIV-1 appears a promising strategy for eliciting long-term HIV-1 remission. When administered concomitantly with the cessation of antiretroviral therapy (ART) to patients with established viremic control, bNAb therapy is expected to prolong remission. Surprisingly, in clinical trials on chronic HIV-1 patients, the bNAb VRC01 failed to prolong remission substantially. Identifying the cause of this failure is important for improving VRC01-based therapies and unraveling potential vulnerabilities of other bNAbs. In the trials, viremia resurged rapidly in most patients despite suppressive VRC01 concentrations in circulation, suggesting that VRC01 resistance was the likely cause of failure. ART swiftly halts viral replication, precluding the development of resistance during ART. If resistance were to emerge post ART, virological breakthrough would have taken longer than without VRC01 therapy. We hypothesized therefore that VRC01-resistant strains must have been formed before ART initiation, survived ART in latently infected cells, and been activated during VRC01 therapy, causing treatment failure. Current assays preclude testing this hypothesis experimentally. We developed a mathematical model based on the hypothesis and challenged it with available clinical data. The model integrated within-host HIV-1 evolution, stochastic latency reactivation, and viral dynamics with multiple-dose VRC01 pharmacokinetics. The model predicted that single but not higher VRC01-resistant mutants would pre-exist in the latent reservoir. We constructed a virtual patient population that parsimoniously recapitulated inter-patient variations. Model predictions with this population quantitatively captured data of VRC01 failure from clinical trials, presenting strong evidence supporting the hypothesis. We attributed VRC01 failure to single-mutant VRC01-resistant proviruses in the latent reservoir triggering viral recrudescence, particularly when VRC01 was at trough levels. Pre-existing resistant proviruses in the latent reservoir may similarly compromise other bNAbs. Our study provides a framework for designing bNAb-based therapeutic protocols that would avert such failure and maximize HIV-1 remission. Antiretroviral therapy (ART) can control but not eradicate HIV-1. Stopping ART leads to rapid viral resurgence and progressive disease. ART is therefore administered lifelong. Tremendous efforts are ongoing to devise strategies that will enable stopping ART and yet prevent viral resurgence. One such strategy involves the administration of broadly neutralizing antibodies (bNAbs) of HIV-1 at the time of stopping ART. This strategy is expected to delay if not prevent viral resurgence. Surprisingly, treatment with VRC01, a potent bNAb, resulted in hardly any improvement in viral remission. In this study, we elucidate the cause of this failure. We hypothesized that VRC01-resistant strains may pre-exist in latently infected cells, which are unaffected by ART. They can thus outlast ART and get reactivated, triggering VRC01 failure. We built a detailed mathematical model based on this hypothesis and showed that it quantitatively captured observations of VRC01 failure in clinical trials on chronic HIV-1 patients. Our study thus identifies a potential vulnerability of bNAbs, namely, bNAb-resistant strains pre-existing in latently infected cells. Our model offers a framework for predicting bNAb-based treatment protocols that would preclude failure due to pre-existing resistance and maximally prolong remission. Antiretroviral therapy (ART) for HIV-1 infection rapidly suppresses viremia to undetectable levels and curtails disease progression but is unable to eradicate the virus . The rebOne strategy that holds promise is to administer broadly neutralizing antibodies (bNAbs) of HIV-1 for a short period post-ART, i.e., during an analytical treatment interruption (ATI) ,14. bNAbin vivo /ln[W(t)/W(0)]+δt], where W(t)/W(0) is the fold expansion of wild type virus at time t; H(t)/H(0) is the fold change of the mutant-to-wild type ratio at time t; and δ is the death rate of infected cells. Rewriting this equation as Y = sX, where Y = ln[H(t)/H(0)] and X = ln[W(t)/W(0)]+δt, we estimate s by fits (lines) to corresponding data (symbols) [Y vs. X for three mutants: (A) R456W, (B) G458D, (C) N279K-N280D-R456W-G458D. The best-fit estimates (95%CI) of s are -0.568  for R456W; -0.5091  for G458D; and -0.5672  for N279K-N280D-R456W-G458D. The relative replicative fitness of the respective strains are obtained as ζ = (1+s).We applied a previous formalism  to analysymbols)  of Y vs.(TIF)Click here for additional data file.S2 FigPredictions of stochastic simulations (Eqs. S1-S11), showing (A) time-evolution of the latent cell pool, (B) distribution of the waiting time for the reactivation from latency, (C) time-evolution of activated or productively infected cells, and (D) distribution of the waiting time for the first productively infected cell carrying a VRC01-resistant strain (bar graph). (In (A) and (C), the different lines represent different stochastic realizations.) Variation of the latter distribution is shown with (E) initial latent cell pool size (cells), (F) VRC01 efficacy, and (G) viral production rate (virions/cell/day). In (D)-(G), the corresponding probability density function calculated using the deterministic formalism (Eqs. S12-S17) is shown as solid lines. (H) Expected waiting time, τm, for the formation of a productively infected cell carrying a VRC01-resistant provirus, calculated using the deterministic formalism as a function of the initial latent pool size for the VRC01 efficacy indicated, which is the efficacy against the wild-type averaged over the dosing interval.(TIF)Click here for additional data file.S3 Fig(A)(B)L0 (cells) from the different distributions indicated. The red line in (B) is data from the A5340 trial.Model predictions similar to those in (TIF)Click here for additional data file.S4 FigIC50 (μg/mL) indicated. The red line in (B) is data from the A5340 trial.Model predictions similar to those in (A) (TIF)Click here for additional data file.S5 FigIC50 (instead of p) in each virtual patient drawn from a log-normal distribution ) (Inset). The red line in (B) is data from the A5340 trial.Model predictions similar to those in (A) (TIF)Click here for additional data file.S6 Figη1, has been varied accordingly. The initial total antibody concentration (A1+A2) has been varied while keeping the ratioA1/A2 fixed, with the maximum (A1+A2) (in μg/mL) corresponding to the 40 mg/kg dosage of VRC01 , which (TIF)Click here for additional data file.


Although MG II may be the most abundant planktonic archaeal group in surface water, their lipid composition remains poorly characterized because of the lack of cultured representatives. Circumstantial evidence from previous studies of marine suspended particulate matter suggests that MG II may produce both GDGTs and archaeol-based lipids. In this study, integration of the 16S rRNA gene quantification and sequencing and lipid analysis demonstrated that MG II contributed significantly to the pool of archaeal tetraether lipids in samples collected from MG II-dominated surface waters of the Northwestern Pacific Ocean (NWPO). The archaeal lipid composition in MG II-dominated NWPO waters differed significantly from that of known MG I cultures, containing relatively more 2G-OH-, 2G- and 1G- GDGTs, especially in their acyclic form. Lipid composition in NWPO waters was also markedly different from MG I-dominated surface water samples collected in the East China Sea. GDGTs from MG II-dominated samples seemed to respond to temperature similarly to GDGTs from the MG I-dominated samples, which calls for further study using pure cultures to determine the exact impact of MG II on GDGT-based proxies.Planktonic archaea include predominantly Marine Group I Thaumarchaeota (MG I) and Marine Group II Euryarchaeota (MG II), which play important roles in the oceanic carbon cycle. MG I produce specific lipids called isoprenoid glycerol dibiphytanyl glycerol tetraethers (GDGTs), which are being used in the sea surface temperature proxy named TEX Planktonic archaea are dominated by Marine Group I Thaumarchaeota MG I;  and MariMembers of MG II have not been cultured; however, information on their lifestyle has been obtained through metagenomic studies. It is suggested that MG II live heterotrophically and occur mostly in the photic zone . Howeversn-1 hydroxyl position (sn-1 position of the glycerol moiety (MeO-AR) and macrocyclic archaeols (MARs) and unsaturated archaeols  allows the identification of a wide range of archaeal membrane lipids e.g., . Archaeaposition . The corposition , one or position , or doubposition . AR contuns-ARs;  are also86 index; Nitrosopumilus maritimus, the first representative of MG I isolated from marine environments, reported lipids including GDGTs with zero to four cyclopentyl moieties and crenarchaeol, a GDGT containing one cyclohexyl and four cyclopentyl moieties   of epipelagic waters from the eastern tropical North Pacific, equatorial Pacific and off the coast of Cape Blanc  with four double bonds were recently suggested as potential biomarkers for MG II based on analyses of uns-ARpe Blanc . Using ape Blanc . This colization . TEX86 w by MG I . Thus, tIn this study, we characterized and quantified archaeal membrane lipids in surface water samples from the Northwestern Pacific Ocean (NWPO) and East China Sea and supported these measurements with DNA sequencing and determination of cell density in order to determine the sources of the archaeal lipids. Both sample sets differed markedly in their archaeal community members with MG II being dominant in NWPO samples and MG I in East China Sea samples. Accordingly, the combined sample set was ideally suited to constrain the contribution of MG II to the marine archaeal lipid pool, to evaluate its effect on archaeal lipid based proxies, and to test previous hypotheses regarding candidate lipids of MG II . In addiDongfanghong II during the East China Sea 2014 cruise  and the NWPO 2015 spring cruise . In situ temperature and salinity were measured by a conductivity-temperature-density (CTD) unit (Sea-Bird 911 CTD). Samples for inorganic nutrients  were filtered through 0.45 μm cellulose acetate filters and stored at −20°C until analysis with an AutoAnalyzer 3 HR .Samples were collected on board the R/V During each cruise, samples of SPM were collected from surface water  of a freeze-dried GF/F filter. The filter was cut into slices and extracted using a modified Bligh and Dyer method . In brie18 column  maintained at 45°C (Lipid separation was achieved on an ultra-high performance liquid chromatography (UHPLC) system (Dionex Ultimate 3000RS) in reversed phase conditions with an ACE3 C at 45°C . Target  at 45°C  on a tri46-GTGT; 46-GTGT standard. Similarly, the abundance of C-GDGTs was corrected by the response factor of purified GDGT-0 standard versus the C46-GTGT standard. The abundances of C-AR, C-uns-ARs and MeO-AR were corrected by the response factor of a core archaeol standard  versus the C46-GTGT standard. In this study, C-uns-ARs are presented as C-uns-AR (u), where u = the number of double bond equivalents (DBE), representing either double bonds or rings and thus including both unsaturated and macrocyclic archaeols.Quantification of lipids was achieved by adding an internal glycerol trialkyl glycerol tetraether standard (CDNA was extracted from 12% (1/8) of a GF/F filter  using the FastDNA SPIN Kit for Soil  with a final elution in 100 μL deionized water.The archaeal 16S rRNA gene was quantified in all samples by quantitative polymerase chain reaction . Abundance (cells per liter) was normalized according to the dilution folds of DNA template and the volume of filtered seawater. The qPCR primers were Arch_787F . Sets of sequences with at least 97% identified were defined as an OTU , and chimeric sequences were identified and removed using UCHIME  and Arch_958R (5′ YCCG GCGTTGAVTCCAATT 3′), which showed higher coverage for archaeal 16S rRNA gene as described recently . Sequencg UCHIME . The taxd of 70% .Cell densities of MG I, MG II, and MG III were inferred based on total archaeal community composition derived from sequencing  and totaThe cellular lipid content (fg/cell) was calculated using the equations below for the MG I community only (Eq. 2) and for the whole archaeal community (Eq. 3), where n = cell density (cells/L) and a = lipid abundance (ng/L). Intact polar GDGTs (IP-GDGTs) were defined as the sum of HPH-, 2G-OH-, 2G-, 1G-OH-, and 1G-GDGTs. Total GDGTs are the sum of IP- and C- GDGTs.Ring Index (RI) was calculated using Equation 4 according to p-values were obtained by analysis using R software. The neighbor-joining trees were constructed using MEGA software.Cluster analysis in this study was performed by PAST software using the unweighted pair-group average algorithm. Correlation coefficients and In situ temperature, salinity and nutrient content were determined for every sample. Salinity varied little between the two sample sets ; the average nitrate content of the E-samples was 1.1 μmol/L with a range of 0.53 to 2.05 μmol/L (n = 10). The average nitrite content of the N-samples was 0.16 μmol/L with a range of 0.02 to 0.27 μmol/L, while that of the E-samples was 0.04 μmol/L with a range of 0.01 to 0.14 μmol/L.The fixed nitrogen contents in the N-samples were two to four times higher than those in the E-samples. The average nitrate content of the N-samples was 4.19 μmol/L with a range of 0.77 to 8.84 μmol/L (n = 5) in the N-samples and 0.05 μmol/L  in the E-samples. Silicate content averaged 2.45 μmol/L (ranging from 0.11 to 4.68 μmol/L) in the N-samples and 0.95 μmol/L (ranging from 0.24 to 1.57 μmol/L) in the E-samples . OTU 310, OTU 89 and OTU 233 had higher relative abundance in E-samples . E-samples showed a slightly higher average archaeal cell density of 3.3 × 107 cells/L  than in E-samples . As a result, MG I ranged between 1.7 × 10The total archaeal lipid content varied greatly within each sample set. Total archaeal lipids ranged from 2.9 to 12 ng/L (average was 6.6 ± 3.5 ng/L) in N-samples and from 1.4 to 22.3 ng/L (average was 8.4 ± 6.1 ng/L) in E-samples .Except for sample N-B3 (characterized by a predominance of C-GDGTs), all N-samples were dominated by 1G-GDGTs followed by 2G-GDGTs and C-GDGTs . These tARs accounted for 1.4 to 37.3% of total archaeal lipids  with corA cluster analysis performed on the comprehensive lipid distribution showed distinct lipid distributions between the N- and E- samples. The N-B3 sample had a significantly different lipid distribution from other samples and was thus subsequently considered as an outlier. Lipid distributions of N- and E- samples mainly differed upon the relative abundance of C-GDGTs, 2G-GDGTs, 1G-GDGTs, 2G-OH-GDGTs, and C-uns-ARs . In MG IPrevious studies showed that MG II usually inhabit the surface photic zone while MG I are found in deeper layers of the water column e.g., , which iThe unambiguous difference in archaeal community structure between the two sample sets offers the opportunity to analyze in detail the lipid contribution by the uncultivated MG II archaea in the samples they dominate. For this purpose, IPLs were analyzed from the same filters used for archaeal community analysis. IPLs, particularly those with phosphate head groups, are assumed to be rapidly degraded upon cell death  and hencMG I are considered as the dominant source of IPL-GDGTs in the ocean . In thisIn the MG II-enriched N-samples, assuming all GDGTs derive from MG I cells, the calculated cellular archaeal lipid quota would be 5.16 to 165 fg/cell (51 fg/cell in average) for total GDGTs and 1.99 to 132 fg/cell for IP-GDGTs  and low values in the N-samples , which behaves similarly to TEX86  in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject number CM and CZ developed the idea and designed the study. CM extracted lipids and analyzed DNA. CM, SC, and JL analyzed lipids. CM, SC, JL, K-UH, and CZ wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The sensitivity of qualitative [18F]FES and [18F]FDG PET/CT interpretations was assessed, comparing them with histological diagnoses.Our database of consecutive patients enrolled in a previous prospective cohort study to assess [18F]FES PET/CT was 71.1% , while that of [18F]FDG PET/CT was 80.0%  with a threshold of positive interpretation, and 93.3%  when a threshold of equivocal was used. There was no significant difference in sensitivity between [18F]FES and [18F]FDG PET/CT (P = 0.48) with a threshold of positive [18F]FDG uptake, but the sensitivity of [18F]FDG was significantly higher than [18F]FES (P = 0.013) with a threshold of equivocal [18F]FDG uptake. One patient with a benign lesion showed negative [18F]FES but positive [18F]FDG uptake.Of the 46 enrolled patients, 45 were confirmed as having recurrent breast cancer, while one was diagnosed with chronic granulomatous inflammation. Forty (89%) patients were ER-positive, four (9%) were ER-negative, and one (2%) patient did not undergo an ER assay. The sensitivity of [18F]FES PET/CT as an initial test when standard imaging studies are equivocal or suspicious.The restaging of patients who had ER-positive primary breast cancer and present with recurrent disease may include [ Breast cancer is one of the most frequently diagnosed cancers and is the leading cause of cancer death in women . About 718F]fluorodeoxyglucose ([18F]FDG) would only be used in situations where standard staging studies are equivocal or suspicious [18F]FDG PET/CT over standard imaging studies [18F]FDG PET/CT results [18F]FDG PET/CT [18F]FDG PET/CT may differ between studies, depending on how to determine the foci of increased [18F]FDG uptake are related to benign conditions. Equivocal decisions are therefore likely to be inevitable FDG PET/CT [18F]FDG uptake correlates with histologic grade and tumor proliferation index [18F]FDG uptake is higher in ER-negative tumors [18F]FDG uptake in ER-positive breast cancer may affect the diagnostic accuracy. To date, the accuracy of [18F]FDG PET/CT for the diagnosis of recurrent breast cancer has not been separately reported in patients with ER-positive primary breast cancer.The histological and biological characteristics of breast cancer have an important impact on tumor visualization with [G PET/CT . [18F]FDon index , 21, 22,e tumors . Accordi18F]fluoroestradiol ([18F]FES) provides a unique method to noninvasively assess molecular information on ER expression [18F]FES PET/CT for the assessment of ER status in recurrent or metastatic lesions is good overall [18F]FES PET/CT [18F]FES studies. Therefore, [18F]FES PET/CT can be used as an alternative to tissue biopsy and ER assays [18F]FES PET/CT in the diagnosis of recurrent breast cancer in comparison with [18F]FDG PET/CT has not so far been studied.PET/CT with FES PET/CT for ER status in recurrent lesions in patients with breast cancer [18F]FDG PET/CT for clinical reasons. The direct comparison of [18F]FDG and [18F]FES PET/CT in the same population is robust, as differences in potential modifiers associated with the population, study methods, and technologies can be eliminated. In the current study, we aimed to compare the diagnostic sensitivity of [18F]FDG and [18F]FES PET/CT for breast cancer recurrence in patients with ER-positive primary breast cancer who were enrolled in a previous cohort study.Recently, we conducted a prospective cohort study to assess the diagnostic accuracy of [t cancer . The inc18F]FES PET/CT for the assessment of ER status because of recurrent or metastatic breast cancer lesions [18F]FDG PET/CT studies performed to identify unsuspected regional nodal disease and/or distant metastases were retrospectively reviewed. Our institutional review board approved this study protocol and waived the requirement for informed consent. This study was conducted in accordance with the Declaration of Helsinki and our institutional guidelines.The study patients were identified from our database of a prospective patient cohort who underwent [ lesions . The pat18F]FES PET/CT, and underwent [18F]FDG PET/CT within 60 days before needle biopsy or surgery. Patients were excluded if they had ER-negative or ER-unknown primary breast cancer. The complete selection criteria, setting, location, and dates of the prospective cohort study were previously reported FES was described previously [18F]FES [The production of [eviously . PET/CT [18F]FES . PET/CT 18F]FDG PET/CT images were obtained from the skull base-to-upper thigh using one of several different PET/CT scanners , 50–70 min after intravenous injection of 5.2–7.4 MBq/kg (0.14–0.2 mCi/kg) of [18F]FDG as described in a previous report [[s report .18F]FES PET/CT reviewed core-needle biopsy and surgical samples in accordance with routine standard procedures. The recurrent or metastatic specimens taken from the breast, liver, ovary, uterus, or bone were excluded from the analysis. Clinical information and other laboratory test results were available to the assessors of the histological examinations. Immunostaining for ER was performed using the 6F11 mouse monoclonal antibody , as previously described [Experienced breast pathologists who were blinded to the results of the [escribed . Semi-quescribed . ER stat18F]FES PET/CT images in a random sequence. The physicians qualitatively assessed the [18F]FES uptake of reference lesions, as well as up to five additional non-reference lesions in the order of highest intensity relative to background activity [18F]FES assessments were positive.The recurrent lesion from which a sample was taken for histological examination was selected as the reference lesion for the sensitivity analysis, as reported previously . Three bactivity , 37. Les18F]FDG PET/CT images in consensus using a syngo.via image display system , with the images displayed in a random sequence. These two physicians were also blinded to all patient information and the [18F]FES PET/CT images; they only had knowledge of the location of the biopsy sites for [18F]FDG PET/CT image evaluation. The lesions were classified as positive, equivocal, or negative lesions by qualitative analysis. An [18F]FDG focus was interpreted as negative when it was related to a non-malignant process or the physiological biodistribution of [18F]FDG. Focally abnormal [18F]FDG uptake higher than that of surrounding tissues and that could not be related to non-malignant or physiological uptake was interpreted as positive. [18F]FDG uptake that could not be characterized was defined as equivocal [Two board-certified nuclear medicine physicians (H.J.S. and D.Y.L.) interpreted the [quivocal .18F]FDG SUV results were harmonized across different PET/CT scanners by matching the recovery coefficient profiles without partial volume correction, as described previously . The FES PET/CT [18F]FES and [18F]FDG PET/CT were included in this study. Thirty-five of 46 patients (76%) underwent [18F]FDG PET/CT before needle biopsy or surgery, 22 of whom were initially diagnosed as having recurrent tumors by ultrasonography or computed tomography, and the 13 of whom were detected by [18F]FDG PET/CT. The remaining 11 (24%) had [18F]FDG PET/CT on the day of or after tissue biopsy. The clinical and histological characteristics are listed in Table Between November 2013 and November 2016, 90 patients completed [S PET/CT . Figure 18F]FES and 11 days (IQR 5–21) for [18F]FDG. The median time interval between [18F]FDG and [18F]FES PET/CT was 10 days (IQR 7–19). All but three patients underwent the [18F]FDG PET/CT at our institution. The injected dose of [18F]FDG ranged from 218 to 440 MBq (5.9–11.9 mCi). The uptake time before [18F]FDG PET/CT ranged from 50 to 67 min. The median blood glucose level was 99 mg/dl (IQR 88–108).The median time between PET/CT and biopsy or surgery was 5 days (IQR 1–11) for [18F]FES and [18F]FDG uptake in the 45 patients with recurrent breast cancer. Of these 45 patients, [18F]FES PET/CT was positive in 32, and the sensitivity for diagnosing recurrent breast cancer was 71.1% . [18F]FDG was positive in 36 when a positive interpretation was defined as the threshold, and 42 when a positive or equivocal interpretation was used, with the sensitivities being 80.0%  and 93.3% , respectively. When a threshold of positive [18F]FDG uptake was used, there was no significant difference in sensitivity between [18F]FES and [18F]FDG PET/CT (P = 0.48). However, when a threshold of equivocal uptake was used, the sensitivity of [18F]FDG PET/CT was significantly higher than that of [18F]FES PET/CT (P = 0.013).Table 18F]FES PET/CT was positive in 31 and [18F]FDG PET/CT with thresholds of positive or equivocal uptake was positive in 31 and 37 patients, resulting in sensitivities of 77.5% , 77.5% , and 92.5% , respectively, with there being no significant difference between them (P > 0.10). Five of six patients with equivocal [18F]FDG and two of three with negative [18F]FDG uptake showed a positive [18F]FES PET/CT result  and negative  qualitative interpretations (P < 0.0001). There was also a significant difference in the maximum SUVs of [18F]FDG between positive , equivocal , and negative  interpretations (P = 0.0012).The maximum SUV of [18F]FES-positive non-reference lesions , regional lymph nodes (n = 10), distant lymph nodes (n = 11), lung (n = 11), pleura (n = 4), and breast (n = 1). Interestingly, three of nine patients who had ER-positive/[18F]FES-negative reference lesions had [18F]FES-positive non-reference lesions. One non-reference lesion of the three patients was confirmed as ER-positive metastatic breast cancer by histologic analysis .Of the 45 patients with recurrent breast cancer, 25 patients (56%) had a total of 47 [sis Fig. . If thes18F]FES PET/CT had comparable diagnostic sensitivity to [18F]FDG PET/CT for the diagnosis of recurrent breast cancer when positive [18F]FDG uptake was used as a threshold. However, [18F]FES PET/CT had significantly lower sensitivity than [18F]FDG PET/CT if equivocal [18F]FDG uptake was included as a positive threshold. When ER-positive recurrent breast cancer was analyzed, there was no significant difference in sensitivity between [18F]FDG and [18F]FES PET/CT. One patient with a benign lesion showed true-negative [18F]FES but false-positive [18F]FDG uptake. To the best of our knowledge, this is the first study evaluating the diagnostic performance of [18F]FES and [18F]FDG PET/CT relative to histological results in patients with ER-positive primary breast cancer.In this study, we showed that [18F]FES PET/CT would be likely to show high sensitivity. The sensitivity of [18F]FES PET/CT was comparable to or lower than that of [18F]FDG, depending on the threshold used. All four patients with ER-negative recurrence showed negative [18F]FES PET/CT. However, the false-negative [18F]FES PET/CT results were of relevance, because discordant ER expression between primary and recurrent breast cancer was accurately determined. We also had nine false-negative patients who displayed ER-positive immunohistochemistry but negative [18F]FES. As seven of the nine [18F]FES-negative patients showed a positive [18F]FDG uptake, the false-negative results cannot be explained by the limited spatial resolution of PET/CT, but rather reflect the low ER expression levels in tumor, or inherent differences between ER assays and [18F]FES uptake [18F]FES-negative tumors may represent a functionally endocrine therapy-resistant breast cancer [18F]FES-negative.In this study, we included only ER-positive primary cancer patients in whom FES PET/CT strategy for diagnosing recurrent breast cancer would have false-negative results, which would generally lead to delayed treatment [18F]FDG PET/CT may be offered for patients who are [18F]FES-negative. Despite the theoretically lower sensitivity of [18F]FES PET/CT for breast cancer recurrence, positive [18F]FES uptake could replace histological diagnosis of recurrent breast cancer and positive ER expression [18F]FDG PET/CT [18F]FES PET/CT would be of additional value in whole-body imaging for tumors expressing ER, to identify the subset of patients with mixed [18F]FES uptake, as shown in the three patients with heterogeneous uptake [18F]FDG PET/CT-first strategy may have lower false-negative results. Nevertheless, positive [18F]FDG results still require histological confirmation and assessment of ER status, and even negative [18F]FDG cannot completely exclude the possibility of recurrent breast cancer; therefore, biopsies of possible breast cancer recurrence may still be performed. If collecting a biopsy sample is not feasible or associated with risk, positive [18F]FES PET/CT results can adequately diagnose ER-positive recurrent breast cancer and represent an alternative to histological assessment. Our results suggest that although [18F]FES PET/CT may have a slightly lower sensitivity than [18F]FDG, staging evaluation of women who had ER-positive primary breast cancer and then present with recurrent disease could include [18F]FES PET/CT. An [18F]FES PET/CT-first strategy for recurrent cancer may lead to changes in patient management and have an impact on outcomes.An [reatment . Therefopression  and coulG PET/CT . [18F]FEs uptake . However18F]FES PET/CT for the assessment of ER status in recurrent lesions, and included consecutive patients who were typical of those presenting for first breast cancer recurrence. However, our patient inclusion and exclusion criteria may have resulted in potential risks and limited applicability. We enrolled only those patients who had histological confirmation and who underwent [18F]FES PET/CT for restaging to guide optimal therapy. The diagnostic sensitivity may be higher if lesions are large enough for a tissue biopsy to be attained. In addition, PET/CT studies might have not been performed in situations where standard staging studies are equivocal or suspicious, as recommended by the guidelines. Nevertheless, we believe that the primary purpose of this study, which was to compare sensitivity between [18F]FDG and [18F]FES PET/CT, is unlikely to be influenced by our patient selection. We excluded only one patient who had no [18F]FDG PET/CT. In most patients, the [18F]FDG studies were performed for clinical reasons. There are no other potential risks of bias or applicability regarding the quality of the diagnostic sensitivity data [18F]FES positivity threshold could be precisely determined, as suggested by a very low P level for the differences in SUV results between the dichotomous qualitative [18F]FES categories.Diagnostic accuracy studies are at risk of bias due to shortcomings in their design and conduct, and the results may not be applicable to other patient populations . We retrity data . Further18F]FES PET/CT compared with [18F]FDG PET/CT; however, the main concern was how the sensitivity of [18F]FES differs from that of [18F]FDG in patients with ER-positive primary breast cancer. A comparison of the specificity of [18F]FES and [18F]FDG PET/CT can be inferred from previous studies. Second, we did not include asymptomatic patients with elevated tumor markers. Our results may not be applicable to the specific setting of rising tumor markers. Third, this study may have verification bias in which the positive results of [18F]FDG PET/CT in 13 patients could affect the biopsy decision leading to a high sensitivity estimate. Nonetheless, the sensitivity of [18F]FES PET/CT was comparable to that of [18F]FDG PET/CT when positive interpretation was defined as positive for the presence of recurrent breast cancer. Finally, we did not assess the diagnostic value of [18F]FDG and [18F]FES PET/CT in addition to or instead of standard imaging tests. [18F]FES PET/CT can be performed as an initial test in the setting of recurrent disease when standard imaging studies are equivocal or suspicious.There are several limitations to this study. First, we enrolled only one patient with a benign inflammatory lesion. It was not possible to directly demonstrate a high specificity of [18F]FES PET/CT in patients who had ER-positive breast cancer and presented with recurrent disease was comparable to or lower than that of 18F-FDG, depending on the threshold used. Although we were not able to directly demonstrate a high specificity in this study, the well-established high specificity indicates that positive [18F]FES PET/CT results can adequately diagnose ER-positive recurrent breast cancer. [18F]FES PET/CT would be of additional value in whole-body imaging of tumor ER expression, to identify additional patients with recurrence with mixed [18F]FES uptake. We conclude that the restaging of women who have ER-positive primary breast cancer and present with recurrent disease may include [18F]FES PET/CT. Large-scale prospective intervention studies are needed to investigate the potential utility of [18F]FES PET/CT as a replacement for, rather than an addition to, standard imaging studies, and to determine the impact of [18F]FES PET/CT on patient outcomes.The sensitivity of [