This study aims to investigate the time-dependent prognostic utility of two fibrosis markers representing organ fibrogenesis  and type IV collagen 7S (P4NP 7S)) in patients with acute heart failure (HF).390 patients with acute HF were dichotomised based on the median value of fibrosis markers at discharge. The primary outcome measure was a composite of cardiac death and HF hospitalisation.P4NP 7S significantly declined during hospitalisation, whereas PIIINP did not. The cumulative 90-day and 365-day incidence of the primary outcome measure was 16.6% vs 16.0% (p=0.42) and 33.3% vs 28.4% (p=0.34) in the patients with high versus low PIIINP; 19.9% vs 13.0% (p=0.04) and 32.3% vs 29.0% (p=0.34) in the patients with high and low P4NP 7S, respectively. After adjusting for confounders, high P4NP 7S correlated with significant excess risk relative to low P4NP 7S for both 90-day and 365-day primary outcome measure , which was driven by significant association of high P4NP 7S with higher incidence of HF hospitalisation. Furthermore, P4NP 7S exhibited an additive value to conventional prognostic factors for predicting 90-day outcome . High PIIINP did not correlate with significant excess risk for both 90-day and 365-day outcome.This study suggests a possible role of P4NP 7S in the risk stratification of patients with acute HF. Systemic venous congestion in heart failure (HF) causes end-organ injury, which adversely affects prognosis. It has been recently reported that the serum 7S domain of the collagen type IV N-terminal propeptide (P4NP 7S), an established marker of liver fibrosis correlated with liver function tests, pulmonary capillary wedge pressure and right ventricular and atrial pressure in patients with HF. In addition, in a single centre exploratory analysis, P4NP 7S exhibited a potential prognostic utility.In patients hospitalised for acutely decompensated heart failure (HF), P4NP 7S at discharge correlated with significant excess risk for HF hospitalisation. P4NP 7S was superior to conventional liver function tests and another liver fibrogenic marker, N-terminal propeptide of procollagen III (PIIINP) for the prognostic utility.The evaluation of P4NP 7S at discharge might facilitate the identification of those patients at high risk for persistent end-organ injury and subsequent heart failure-related events.Systemic venous congestion is a hallmark of heart failure (HF).4Fibrogenetic cascade is a common pathological response in numerous tissues. Even in HF, congestion-induced organ injury could evoke a fibrogenetic response.7Hence, we conducted a prospective cohort study to evaluate in-hospital changes of two collagen markers  and P4NP 7S) and to confirm the utility of these markers for predicting early and late clinical outcome.The present study is a prospective cohort study enrolling consecutive patients admitted for ADHF at three tertiary referral hospitals in Japan . We excl10.1136/openhrt-2019-001041.supp1Supplementary dataBlood samples, collected at admission and immediately before discharge, were centrifuged, and the serum was transferred to a central laboratory for PIIINP and P4NP 7S measurement. Owing to the limited serum volume, PIIINP was measured in 330 patients on admission and 384 patients at discharge. Other clinical biomarkers, including brain natriuretic peptide (BNP), renal function and LFTs (liver function tests), were simultaneously assessed as a routine clinical practice.The prespecified primary outcome measure in this study was a composite of 365-day cardiac death and HF hospitalisation. The secondary outcome measures were individual components of the primary outcome measure and all-cause death. In addition, our prior exploratory analysis and the previous study by others suggested that the prognostic effect of LFTs and collagen markers might be time-dependent and that the impact of these markers on short-term and long-term outcomes might be different.2 tests. We tested the correlation between clinical parameters using the Spearman’s correlation coefficient. We dichotomised patients based on the median value of each collagen marker at discharge and estimated cumulative incidences of clinical events across PIIINP and P4NP 7S using the Kaplan-Meier method. The differences in cumulative incidences of clinical events were assessed by the log-rank test. We estimated the risks related to high PIIINP and P4NP 7S for the primary and secondary endpoints relative to the low PIIINP and P4NP 7S using the Cox proportional hazard model, which are presented as HR and 95% CI. Clinically relevant factors, including age, sex, ejection fraction (EF) <40% and estimated glomerular filtration rate (eGFR), were incorporated as risk-adjusting variables. We investigated the time-dependent prognostic utility of each collagen marker by the landmark analysis at 90 days after discharge. Surviving patients with HF hospitalisation within 90 days were included for the analysis beyond 90 days. We also evaluated the HR related to the abnormal value of individual LFTs .During hospitalisation, P4NP 7S significantly decreased from 6.1 ng/mL on admission to 5.1 ng/mL at discharge . Patientr=0.31; p<0.0001). Of note, %ΔPIIINP and %ΔP4NP 7S only weakly correlated with each other  for cardiac death and HF hospitalisation at 90 days postdischarge was 0.654 . When adThe main findings in the present study were (1) P4NP 7S declined in parallel to a decline in BNP, whereas PIIINP did not markedly change during hospitalisation; (2) patients discharged with high P4NP 7S were at high risk of HF readmission, especially within 90 days of discharge; and (3) P4NP 7S, when added on the established risk factors better improved the prediction of early cardiac death and HF hospitalisation than conventional LFTs and PIIINP.Congestion is the major clinical manifestation in most patients with ADHF.2 12 13Organ congestion could cause fibrosis.In this study, we observed that P4NP 7S significantly decreased during hospitalisation with a smaller decline in patients with high P4NP 7S at discharge than in patients with low P4NP 7S. Moreover, high P4NP 7S at discharge was associated with high incidence of HF readmission, with additive prognostic value to conventional prognostic factors. Reportedly, most patients with ADHF experience a marked improvement in clinical congestion during hospitalisation.This study has several limitations. First, peripheral biomarkers of collagen turnover might be affected by conditions other than HF. Although we carefully excluded patients with severe comorbidities, other undetected factors might have affected the values of collagen markers. Second, data regarding the severity of congestion, such as jugular venous pressure, oedema, weight changes during hospitalisation and inferior vena cava diameter, are lacking. Although P4NP 7S exhibited significant correlation with severity of haemodynamic congestion and right-sided pressure in the previous studies,


Ageratum leaf curl Sichuan virus (ALCScV) is a novel monopartite begomovirus, which was identified from Ageratum conyzoides plants in Sichuan Province, China. In this study, we showed that ALCScV can induce typical dwarf and downward leaf-curling symptoms in Ageratum conyzoides, Helianthus annuus, and Nicotiana benthamiana plants and that the noncognate betasatellite can enhance disease symptoms and increase viral accumulation. Expression of the ALCScV-encoded V2, C1, and C4 proteins through a Potato virus X (PVX) vector caused severe symptoms in N. benthamiana. Further study revealed no symptoms in N. benthamiana plants inoculated with infectious ALCScV clones lacking the C4 protein and that the relative viral DNA accumulation levels significantly decreased when compared with ALCScV-inoculated plants. Thus, our mutational analyses demonstrated that C4 is a pathogenicity determinant that plays key roles in symptom formation and virus accumulation. Furthermore, we also demonstrated that the second glycine of C4 was critical for ALCScV pathogenicity. Begomovirus, Mastrevirus, Grablovirus, Capulavirus, Eragrovirus, Curtovirus, Becurtovirus, Topocuvirus, and Turncurtovirus)  . Among torldwide . Furtherorldwide . Bipartiorldwide , whereasorldwide . Previouorldwide . No sequIn general, for monopartite begomoviruses, the DNA A component contains six open reading frames (ORFs). ORFs V2 and V1 are contained in the sense virion strand. ORF V2 encodes the movement protein (MP), which mediates viral systemic infection of the host plant , and ORFMalvastrum yellow vein virus (MaYVV) C4 protein suppresses RNA silencing and can enhance disease symptoms (Mungbean yellow mosaic virus (MYMV) is essential for viral pathogenicity, and a C4 mutant showed significantly decreased viral DNA accumulation compared with wild-type MYMV  C4 protein was indispensable for virus movement (Tomato leaf curl Yunnan virus (TLCYnV) C4 protein promoted host cell division by interacting with SKη kinase  is a newly discovered monopartite begomovirus that is not associated with alphasatellites or betasatellites  were analyzed via expression with the Potato virus X (PVX) vector, which revealed V2 as a virulence factor that could induce systemic necrosis in N. benthamiana. The C1 induced severe dwarfing symptoms compared with control treatment. The C4 induced severe leaf hyperplasia and petiole elongation. Furthermore, mutational analyses showed that the C4 plays critical roles in symptom formation and virus accumulation. In addition, the second glycine (Gly) residue of C4 was indispensable for ALCScV pathogenicity.tellites . In 2018N. benthamiana, Ageratum conyzoides, and Helianthus annuus were grown in an insect-free greenhouse, held at a constant temperature of 26°C, with a 16-h light/8-h dark cycle.Wild-type via Seamless Cloning (To test the pathogenicity of ALCScV (GenBank No. MG917698), an infectious ALCScV clone (isolate SC782) was constructed  Cloning . The SC7 Cloning Table S1mC4 (1089 bp) (we used the Fast Mutagenesis System to mutate the start codon (ATG) in the C4 gene to ACG, without changing the amino acid sequence of the C1 protein), C2 (408 bp), C3 (405 bp), and C4 (303 bp) were amplified using specific primers , V2 (351 bp), C1 (1089 bp), C1 primers Table S1Fast Mutagenesis System  and the SC782-mC4-F1/SC782-mC4-R1 primer set . Then, we used the imer set Table S1G2A, C4C8A, C4C10A, and C4C8/10A, the full-length C4 gene sequence was amplified using the C4-F/C4-R primer set and cloned into the pGEM-T Easy vector . Then, we used the Fast Mutagenesis System and a mutation primer .To investigate the subcellular localization of C4 and C4er pairs Table S1To further investigate the function of the second amino acid position of C4, we used the same method described above to construct an infectious clone (pBinPLUS-1.9A C4m2) that only had a mutation in the second amino acid position of C4.Agrobacterium tumefaciens strain GV3101 (pMP90 pSoup). The A. tumefaciens bacteria were grown on LB solid medium containing rifampicin (20 μg/ml) and kanamycin (50 μg/ml) in a 28°C incubator for 48 h. Then, single clones were expanded into LB liquid medium containing rifampicin (20 μg/ml) and kanamycin (50 μg/ml) and incubated at 28°C for 12 h. The A. tumefaciens bacteria were collected by centrifugation and resuspended with inoculation buffer  to and OD600 of 1.0. After standing at room temperature for 2 h, an aseptic syringe was used to inoculate N. benthamiana plants (leaf infiltration) at the 4–6 leaf stage.The recombinant PVX overexpression vector and pBinPLUS-based expression vectors were transformed into 2O. The endogenous Nb25S ribosomes gene was detected as an internal control , 1-μl forward primer (10 μM) and 1-μl reverse primer (10 μM), 1-μl total DNA (100 ng/μl), and 7-μl RNase-free ddH2O, and the PCR cycle number is 25. Then, the PCR products were separated by a 1% agarose gel electrophoresis.For semi-qPCR analysis, the quantity and purity of total DNA were measured by spectrophotometer. The total DNA concentration were normalized (100 ng/μl) prior to semi-qPCR. The quantitative primers 782-qPCR-F/782-qPCR-R were also used to semi-qPCR, and the endogenous Ageratum conyzoides, Helianthus annuus and N. benthamiana plants inoculated with ALCScV , leaf curling and shrinking started to appear on N. benthamiana plants. To investigate the effect of noncognate DNA β on ALCScV pathogenicity, we selected the Tomato yellow leaf curl virus (TYLCV) China betasatellite (Y10β) (GenBank No. AJ781300), MaYVV (Y47β) (GenBank No. AJ421482), Tobacco curly shoot virus betasatellite (Y35β) (GenBank No. AJ421484), and MaYVV Yunnan betasatellite (Y250β) (GenBank No. AJ786712) for further studies. N. benthamiana plants were inoculated according to the following combinations: (i) ALCScV, (ii) ALCScV + Y10β, (iii) ALCScV + Y35β, (iv) ALCScV + Y47β, and (v) ALCScV + Y250β. At 21 dpi, the results showed that co-inoculation with ALCScV and noncognate DNA β induced more severe symptoms in N. benthamiana plants than inoculation with ALCScV alone , in contrast to plants inoculated with the empty PVX vector   to ACG, without changing the amino acid sequence of the C1 protein. Then, the N. benthamiana were inoculated separately with ALCScV-mC4 or ALCScV. At 28 dpi, the results showed that typical dwarf enation and downward leaf-curling symptoms were observed with N. benthamiana inoculated with ALCScV, whereas no symptoms were observed with N. benthamiana inoculated with ALCScV-mC4 . The results revealed that the C4 protein has one potential N-myristoylation site (the second Gly residue of the C4 protein) and two potential S-palmitoylation sites [the eighth and tenth cysteine (Cys) residues of the C4 protein]  was expressed in N. benthamiana from the PVX vector. At 7 dpi, the plants inoculated with PVX-C4C8A, PVX-C4C10A, and PVX-C4C8/10A showed similar phenotypes, compared with plants inoculated with PVX-C4. However, the PVX-C4G2A-inoculated plants only showed mild mosaic symptoms, and no other typical symptoms were observed , Zhan et al. observed necrotic lesions in PVX-V2 inoculated plants . Previous research also showed that the C1 protein of Mulberry mosaic dwarf-associated virus can cause similar symptoms in host plants   . However protein . Additio4 mutant . Carlucc4 mutant .G2A variant (affecting the putative N-myristoylation site) only showed mild mosaic symptoms, and no leaf hyperplasia and petiole elongation symptoms were observed. However, further research showed that the putative N-myristoylation site of C4 protein was critical for ALCScV pathogenicity. Many previous results were consistent with our current findings. For example, the N-myristoylation site of C4 protein encoded by the African cassava mosaic Cameroon virus (EACMCV) was essential for membrane localization of the C4 protein, which served as the basis for EACMCV pathogenesis  that the noncognate betasatellite can enhance disease symptoms and viral accumulation, (iii) that the ALCScV V2 protein can induce systemic necrosis, and (iv) that the C1 protein can induce severe dwarfing symptoms in N. benthamiana plants. The ALCScV C4 protein functioned as a pathogenicity determinant and the second Gly residue of C4 was indispensable for ALCScV pathogenicity.In summary, our results suggest (i) that ALCScV can successfully establish systemic infection and induce typical viral symptoms in Ageratum leaf curl Sichuan virus is MG917698.The datasets generated for this study can be found in the: The GenBank accession number of LQ conceived and designed the experiments. PL, CJ, HR, ZJ, and HG conducted experiments. GW, ML, and PL analyzed experimental data. All authors contributed to the article and approved the submitted version.This research work was supported by the Fundamental Research Funds for the Central Universities (Grant No. XDJK2017A006).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.


Enhanced amino acid supply and dysregulated integrin signaling constitute two hallmarks of cancer and are pivotal for metastatic transformation of cells. In line with its function at the crossroads of both processes, overexpression of CD98hc is clinically observed in various cancer malignancies, thus rendering it a promising tumor target.Methods: We describe the development of Anticalin proteins based on the lipocalin 2 (Lcn2) scaffold against the human CD98hc ectodomain (hCD98hcED) using directed evolution and protein design. X-ray structural analysis was performed to identify the epitope recognized by the lead Anticalin candidate. The Anticalin - with a tuned plasma half-life using PASylation® technology - was labeled with 89Zr and investigated by positron emission tomography (PET) of CD98-positive tumor xenograft mice.Results: The Anticalin P3D11 binds CD98hc with picomolar affinity and recognizes a protruding loop structure surrounded by several glycosylation sites within the solvent exposed membrane-distal part of the hCD98hcED. In vitro studies revealed specific binding activity of the Anticalin towards various CD98hc-expressing human tumor cell lines, suggesting broader applicability in cancer research. PET/CT imaging of mice bearing human prostate carcinoma xenografts using the optimized and 89Zr-labeled Anticalin demonstrated strong and specific tracer accumulation (8.6 ± 1.1 %ID/g) as well as a favorable tumor-to-blood ratio of 11.8.Conclusion: Our findings provide a first proof of concept to exploit CD98hc for non-invasive biomedical imaging. The novel Anticalin-based αhCD98hc radiopharmaceutical constitutes a promising tool for preclinical and, potentially, clinical applications in oncology. Elevated amino acid transport, altered cell adhesion and migration as well as adhesive signaling constitute crucial factors for cancer development and growth and play a critical role for metastatic transformation of cells. The cluster of differentiation 98 heavy chain  is involved in both pathophysiological processes and therefore represents an interesting therapeutic target Figure ). First, it acts as a molecular chaperone for membrane trafficking, stabilization and proper function of the CD98lc Structurally, CD98hc is a type-II-transmembrane glycoprotein which is disulfide-linked to one of six known multi-pass CD98 light chains (CD98lc), all of which are permease-type amino acid transporters with different specificities In line with its important role in cellular metabolism and adhesive signaling, overexpression of CD98hc has been detected in solid and hematological human malignancies, including colorectal cancer Likewise, elevated expression of the covalently associated CD98lc components Lat-1 18F]-fluoroethyl-L-tyrosine (FET) and trans-1-amino-3-[18F]-fluorocyclobutanecarboxylic acid (FACBC). While FET is widely used in Europe for PET imaging of brain tumors 89Zr-labeled αLat-1 mAb has shown tumor uptake in a colorectal cancer xenograft model Therefore, the specific recognition and/or targeting of CD98hc by appropriate protein reagents offers potential for cancer theranostics. To date, the most advanced protein drug candidate is the humanized αhCD98hc monoclonal antibody (mAb) IGN523, which has shown robust preclinical anti-tumor activity in patient-derived lymphoma as well as NSCLC xenograft tumor models and a favorable safety profile in a phase I clinical study Here, we describe the development and preclinical investigation of a hCD98hc-specific Anticalin. Anticalins constitute an emerging class of artificial binding proteins obtained by combinatorial design based on the compact and robust human lipocalin scaffold 10 variants based on Lcn2 randomized at 20 positions within the structurally variable loops 12) and, following extensive washing, bound phagemids were eluted under denaturing conditions using 4 M urea. After five consecutive cycles, pooled phasmid DNA from the enriched library was prepared and subcloned on pNGAL98 in order to perform high-throughput ELISA screening from microcultures expressing the soluble lipocalin variants with a C-terminal Strep-tag II, as previously described StrepMAB-Immo . After 1 h incubation and washing steps, bound lipocalin variants were incubated with the biotinylated hCD98hcED, followed by detection of lipocalin•target complexes using ExtrAvidin/alkaline phosphatase (AP) conjugate (Sigma-Aldrich). Signals were developed with 0.5 mg/mL p-nitrophenyl phosphate in AP buffer  and absorbance was measured at 405 nm with a Synergy 2 photometer . For clones showing significant binding towards hCD98hcED the expression cassette on the phasmid was sequenced, and the soluble lipocalin variant was subsequently produced at preparative scale in E. coli JM83 with a C-terminal His6-tag using the plasmid pNGAL118 2PO4, 160 mM Na2HPO4, 115 mM NaCl pH 7.4) on a 24 mL Superdex 75 10/300 GL column . Plasmid construction and production of PASylated lipocalin variants D11vs-PAS200, D11vs-PAS200-Cys and wild type (wt) Lcn2-PAS200-Cys was performed according to published procedures Selection of hCD98hc-specific lipocalin variants was performed via filamentous phagemid display from a combinatorial library comprising 1×10E. coli or in MEXi-293E cells  was calculated as koff/kon. To test competitive binding of the lipocalin variants P3D11, P3A12 and P1E4 to immobilized hCD98hcEDg (cf. ).Real time surface plasmon resonance (SPR) spectroscopy was performed on a BIAcore 2000 system  at 25 °C using HBS-T  Tween 20) as running buffer. The purified biotinylated m/hCD98hcED produced in cEDg cf. , 100 nM BstXI, the randomized DNA fragment was ligated with the backbone of pNGAL146 and used for electrotransformation of E. coli JK321 9 transformants, which were plated on LB agar medium supplemented with 100 µg/mL ampicillin (LB/Amp). The bacterial lawn was scraped from the plate(s), resuspended in 50 mL liquid LB/Amp medium to reach an initial OD550 = 0.15 and incubated at 37 °C. Gene expression was induced at OD550 = 0.5 by addition of 10 ng/mL anhydrotetracycline (aTc) for 2.5 h. Approximately 2×108 cells were sedimented by centrifugation and resuspended in PBS in the presence of biotinylated hCD98hcED at different concentrations  followed by incubation for 1 h at 4 °C. After centrifugation and one washing step with PBS, the bacteria were incubated with 25 µg/mL streptavidin/phycoerythrin (PE) conjugate  and 3 µM Fab A3C5-DY634 conjugate Stability and affinity engineering of the lipocalin variant P3D11 was accomplished by directed evolution using bacterial surface display (BSD) as previously described 3 and stable L-glutamine; Biochrom, Berlin, Germany), supplemented with 10% (v/v) fetal bovine serum  at 37 °C under humidified 5% CO2 atmosphere. The HEK293T cell line and a corresponding CD98hc knock-out clone 2+ and Mg2+; Biochrom) and detached using PBS (w/o Ca2+ and Mg2+) supplemented with 0.5 mM EDTA for 10 min at 37 °C. Cells growing in suspension  were directly washed with PBS (w/o Ca2+ and Mg2+). For each measurement, 250,000 viable cells were resuspended in 250 µL FACS buffer (PBS + 10% (v/v) FBS) supplemented with 1 µM D11vs-PAS200-Cy5.5  FBS, until 60-70% confluence was reached. After three times washing with PBS, the cells were incubated with 1 µM D11vs-PAS200-Cy5.5 for 1 h at 37 °C, followed by another three washing steps. Ramos cells, on the other hand, were grown in suspension and stained, after centrifugation and resuspension, in a similar manner, followed by transfer to a PDL coated Lab-Tek II chamber slide. For control experiments, the D11vs-PAS200-Cy5.5 conjugate was either premixed with a 10-fold molar concentration of unlabeled D11vs-PAS200 or wtLcn2-PAS200-Cy5.5 was applied. Cells were fixed and counterstained by 5 min incubation with ice-cold methanol containing 1 µg/mL 4',6-diamidino-2-phenylindole . Digital fluorescence images were recorded on an Axiovert 40 CFL microscope equipped with an AxioCam MRm camera  using 365/12 nm (DAPI) and 716/40 nm (Cy5.5) band-pass filters.PC-3 and Caco-2 cells were cultivated at 37 °C on poly-D-lysine (PDL) coated Lab-Tek II chamber slides (Thermo Fisher Scientific) in RPMI 1640 medium containing 2.0 g/L NaHCOi pH 7.5, 50 mM K2SO4 and applied at a concentration of 1 µM. In order to identify the wavelength with maximum change in CD signal for thermal denaturation studies, spectra were first recorded with a 1 mm path length quartz cuvette  from 190 to 250 nM both at 20 °C and 90 °C. Thermal unfolding of the lipocalin variants was then measured at a wavelength of 214 nm by heating from 20 °C to 90 °C at a rate of 60 K/h. Data were fitted to an equation for a one-step unfolding transition, and the melting temperature (Tm), enthalpy of unfolding (∆Hm) and Gibb's free energy of unfolding (∆G0) at ambient temperature were calculated as previously described CD spectra and thermal unfolding of purified lipocalin variants were measured using a J-810 spectropolarimeter  equipped with a PT-423S Peltier element and controlled by Spectra manager software (ver. 1.53.05). Proteins were dialyzed against 20 mM KP6 PC-3 or Ramos cells. On day 12 post injection (p.i.) the tumor had reached a diameter of ~0.5 cm and the mice were used for in vivo studies.Animal experiments were conducted with permission from the District Government of Upper Bavaria (application no.: 55.2-1-54-2532-216-15). CB17-SCID mice at an age of 6 weeks (♂ and ♀) were purchased from Charles River Laboratories  and held under specific pathogen free (SPF) conditions. Mice were injected subcutaneously above the right shoulder with 100 µL RPMI 1640 medium (without supplements) containing 6×1089Zr-labeling of Dfo-conjugated 89Zr(IV), obtained in 1 M oxalic acid  and neutralized with 3 M Na2CO3. After 1 h incubation at 37 °C, radiolabeling was confirmed by radio thin layer chromatography (radio-TLC) on chromatography paper strips  using Na-citrate pH 5.0 as mobile phase. The radio-labeled protein tracer was separated from reagents by gel filtration using a PD-10 column  equilibrated with 0.9% NaCl solution . Radiochemical purity of the protein tracer was confirmed by radio-TLC prior to its use for injections.For in vivo PET and a threshold of 10 kBq/mL for ex vivo PET images.For the biodistribution study, mice were injected with the protein tracer via the tail vein at a dose of 3.96 ± 0.12 MBq. For blocking experiments, a 100-fold or 250-fold molar equivalent of unlabeled D11vs-PAS200 was injected 2 h prior to the tracer injection. Static PET/CT imaging was performed at indicated time points under isoflurane anesthesia in an Inveon PET/CT small-animal scanner  with 20 min PET and 5 min CT signal acquisition. Data were analyzed with the Inveon Research Workplace software  and reconstructed using the 0.8 mm high resolution OSEM-3D algorithm. Quantification of regions of interest (ROI) was performed by threshold-based image segmentation with a lower threshold at 50% of the hottest voxel for 2 automatic gamma counter (Perkin Elmer). 1% of the injected protein tracer dose was applied as reference. Uptake values were calculated as %ID/g and corrected for radioactive decay from the time point of injection.Tumor, blood and organs were dissected from the sacrificed animals at the end of the experiment in order to determine weight and quantify radioactivity using a 2480 WizardAfter radioactivity measurement, the tumor was transversally bisected and used for immunohistochemistry and autoradiography analysis. For autoradiography, the tumor was embedded in Tissue-Tek O.C.T.  in a mold and frozen. Subsequently, 10 µm sections were cut using a CM1950 cryostat  and the sections were mounted on a superfrost microscopic slide (Thermo Fisher Scientific). The slide was placed, together with a 1:2 dilution row of the radiotracer, in silicon isolators  on a storage phosphor screen BAS-IP super resolution film  for one week, followed by quantification using a CR 25 BIO autoradiography scanner  and data analysis with AIDA software ver. 4.24.036 . Pictures chosen show the sections with the highest exposure level in order to allow optimal comparison of blocked and unblocked tumor sections.Tumor tissue was fixed in 10% (w/v) neutral-buffered formalin solution  for 48 h and stored at 4 °C until the radioactivity had decayed. Tissue samples were dehydrated using an automated system  and subsequently embedded in paraffin. Serial 2 µm sections were prepared with a rotary microtome  and subjected to histological and immunohistochemical analysis. Hematoxylin and eosin (H&E) staining was performed on deparaffinized sections with Eosin and Mayer's Haemalaun . Immunohistochemistry was performed using a Bond RXm system (Leica Biosystems) with primary antibodies against CD98hc  and CD31  using 1:750 and 1:100 dilutions, respectively. Briefly, slides were deparaffinized using deparaffinization solution (Leica Biosystems), pretreated with Epitope retrieval solution 1  for 30 min (CD98hc) or pretreated with Epitope retrieval solution 2  for 30 min (CD31). Bound antibody was detected with a polymer refine detection kit without post primary reagent and with an intermediate rabbit anti-rat secondary antibody  and signals were developed with 3,3'-diaminobenzidine (DAB). Representative images were collected on an Aperio AT2 digital pathology slide scanner using ImageScope (ver.12.3) software (both from Leica Biosystems).t test for unpaired data was used. p values ≤ 0.05 were considered statistically significant . Experimental data were analyzed using Prism 6 , and standard deviations (S.D.) are indicated.For mean comparison between two groups of animals, the Student's E. coli (hCD98hcED) and fully glycosylated in human embryonic kidney (HEK) 293E cells (hCD98hcEDg) . The four experimentally verified N-glycosylation sites Table S1). This extensive glycosylation of hCD98hcED may hamper in vitro phage display selection of cognate Anticalins due to limited sterical accessibility of potential surface epitopes, as previously seen in the selection of Anticalins against the prostate-specific membrane antigen (PSMA) E. coli as the initial molecular target for phage display selection. N-terminal fusion of the biotin acceptor peptide (BAP) in vivo situation for the type II membrane protein, rendering its epitopes accessible for binding of lipocalin variants during phage display selection, ELISA screening and in vitro binding studies.The soluble monomeric extracellular domain of human CD98hc  was produced both as unglycosylated protein in 10, which had served for the development of other hapten- and protein-specific Anticalins in the past E. coli at the shake flask scale and purified to homogeneity, resulting in monomeric proteins with apparent molecular sizes similar to recombinant wild type (wt) Lcn2 . To determine the rate constants of association and dissociation of the selected Anticalin candidates, SPR real-time interaction analyses were performed using both the glycosylated and unglycosylated immobilized CD98hcED. The three lipocalin variants showed equilibrium dissociation constants (KD) in the single-digit to sub-nanomolar range . The lipocalin variant P3D11 revealed a particularly low KD value of 150-170 pM for hCD98hcED and hCD98hcEDg, as well as a long complex half-life of 6 h, hence suggesting suitability for tumor targeting in vivo . Interestingly, competitive SPR experiments indicated that all three Anticalins recognize overlapping epitopes on hCD98hcED ( shown for P3D11 and P3A12), despite considerable sequence deviation especially between the variants P1E4 and P3D11 (cf. ).Anticalin candidates were selected from a Lcn2 random library with a combinatorial complexity of 1×10Strep-tag II) after isolation of the 1:1 complex via SEC . Crystals belonged to the space group C2, with two complexes per asymmetric unit, allowing collection of a synchrotron X-ray data set and refinement of the structure to a resolution of 1.8 Å (Table S4).To identify the epitope region and to understand the structural mechanisms of the tight molecular interaction between P3D11 and the CD98hc ectodomain, we crystallized this lipocalin variant in complex with hCD98hcED . The β-barrel axis of P3D11 is tilted by ~45° with regard to the TIM barrel axis of hCD98hcED. The contact interface between both proteins involves a buried surface area (BSA) of 1473 Å2  as well as 15 hydrogen bonds and 3 salt bridges (Table S5), resulting in the largest interface observed for Anticalin•protein complexes thus far I values of 8.8 and 5.2 for P3D11 and hCD98hcED, respectively .hCD98hcED revealed a glycoside hydrolase fold, comprising a central TIM barrel core domain (residues 114-438) and a C-terminal β-sandwich domain (residues 439-529) as previously described Table S5). Further to the direct protein•protein contacts, 16 water molecules mediate hydrogen bonds at the interface. Based on the distribution of hydrogen bond donors, acceptors as well as water molecules, the P3D11 binding site towards hCD98hcED can be dissected into two areas, a rather polar region around the lipocalin loops #1 and #2 as well as a predominantly apolar region around loops #3 and #4 .The epitope of hCD98hcED essentially comprises two loops, L1 (residues 128-137) and L2 (374-404), which follow on the first and the eighth β-strand of the TIM barrel, respectively. Almost 75% of the contact interface is contributed by L2, which penetrates deeply into the binding pocket of the engineered lipocalin, while L1 contributes approximately 20% to the total BSA . Likewise, P3D11 shows conformational differences among its loops #1-#4 compared with wtLcn2 (PDB code 1L6M); yet, these may also be caused by the many sequence changes , as observed for other Anticalin structures before.Interestingly, binding of P3D11 causes an induced fit on the side of hCD98hcED. Superposition of the hCD98hcED Cα positions in the complex with P3D11 with those for hCD98hcED crystallized alone (PDB entry 2DH2) Tables ). Modeling of the glycan moieties on the hCD98hcED shows that the Anticalin binds the protruding L1/L2 epitope in close proximity to two of the N-linked oligosaccharides without steric hindrance . Interestingly, P3D11 does not show any crossreactivity with the murine antigen (mCD98hcED) as investigated via SPR measurements (data not shown), which can be explained by significant differences both in the amino acid sequence and in the glycosylation pattern between the two CD98hc orthologs. In particular, loop L2 of mCD98hcED is shorter by one residue, carries an additional N-glycosylation site and shows considerable sequence changes Although the selection of P3D11 was performed with the unglycosylated hCD98hcED, its affinity for the glycosylated hCD98hcEDg was indistinguishable (Table 1). To tackle this issue, stability engineering was performed via directed evolution using bacterial surface display (BSD) starting from an error-prone library with moderate amino acid mutation rate . To select variants of P3D11 with enhanced thermal stability, expression of the membrane-anchored protein in E. coli was induced at 37 °C  and only bacteria showing both a strong hCD98hcED binding signal and high cell surface display level were gated in the FACS experiment .Despite its tight binding activity towards hCD98hcED, the lipocalin variant P3D11 suffered from low thermal stability, a caveat for further preclinical as well as clinical development , whereas just very modest binding was detected for P3D11 under these conditions, and no signal for wtLcn2, as expected. Considering that P3D11 exhibits already quite a low KD value of 150 pM for hCD98hcED, the increased binding signal is likely caused by the more efficient bacterial surface display due to improved protein folding and/or stability of its variants. Subsequent sequence analysis revealed that replacement of Gly81 and Phe71 in the P3D11 sequence by Val and Ser, respectively, were crucial in this regard . Consequently, the variant D11vs, which carries both mutations, was produced as a soluble protein at preparative scale and characterized with respect to its thermal stability and target affinity.After six enrichment cycles, the lipocalin library exhibited a pronounced average binding signal after incubation with 1 nM hCD98hcED, whereas almost no signal was detectable for the initial clone P3D11 if tested under the same conditions. Single clone analysis with 1 nM hCD98hcED verified strong binding of several new P3D11 variants  for D11vs (Tm = 63.5 °C) compared to P3D11 . Importantly, also the cooperativity of the unfolding transition was much steeper, corresponding to a higher enthalpy of denaturation (∆Hm) as well as extrapolated free energy of denaturation (∆GU) under standard conditions (25 °C). This is in line with our observation of a lower tendency of D11vs to aggregate during incubation at 37 °C compared to P3D11 . Additionally, D11vs showed even higher affinity towards hCD98hcED, with a KD value of 60 pM, due to both faster association and slower dissociation rate constants (1).CD thermal unfolding studies at physiological pH demonstrated a significant rise by 8.3 °C in the melting temperature (T). However, due to the lack of a β-carbon, glycine causes a higher backbone conformational flexibility ), of which also Tyr68 was introduced during the initial selection. In addition, Val81 increases the contact area with hCD98hcED, which likely contributes to the enhanced affinity . On the other hand, replacement of Phe71 by Ser enables the formation of stabilizing hydrogen bonds to Asn174 and Lys50 within the Anticalin , and the smaller side chain may enable its loop #2 to bend slightly away from the β-barrel axis, thus creating a better sterical situation for target binding.The enhanced stability and affinity of D11vs can be explained on the basis of the crystal structure solved for the P3D11•hCD98hcED complex: Gly81 was introduced during the initial selection campaign, where it replaced Arg81 of wtLcn2, thus creating space for tight interaction with the target , thus increasing its hydrodynamic molecular volume, which leads to a retarded kidney filtration in vivo imaging applications, previous investigations have shown that fine-tuning of the pharmacokinetics in this manner can boost protein tracer accumulation and tumor contrast in vitro and in vivo imaging as well as drug delivery (Table S2).For Figure ). Competition experiments with excess unlabeled D11vs-PAS200 as well as soluble hCD98hcEDg resulted in signal suppression. The specificity of the Anticalin towards native hCD98hc was further confirmed by using a HEK293T knock-out cell line with an inactivated SLC3A2 gene (encoding the CD98hc membrane protein) Figure S9). Furthermore, immunofluorescence microscopy experiments with D11vs-PAS200-Cy5.5 revealed pronounced membrane and also cytoplasmic staining of Ramos, PC-3 and Caco-2 cancer cells . Cell staining was effectively blocked when adding a 10-fold molar concentration of the unlabeled lipocalin variant, again demonstrating target specificity. As expected, no fluorescence staining for any of the tested cell lines was detected when using the Sulfo-Cy5.5-labeled PASylated wtLcn2.The ability of D11vs-PAS200 to bind hCD98hc in a native cellular environment was investigated by cytofluorometry and immunofluorescence microscopy using human cancer cell lines of different origins that exhibit high expression of this tumor marker. Flow cytometric analysis of the B-cell lymphoma cell lines SU-DHL-4, Raji and Ramos, the prostate carcinoma cells DU-145 and PC-3 and the colorectal adenocarcinoma cell line Caco-2 using Sulfo-Cy5.5-labeled D11vs-PAS200 revealed strong binding to the membrane-associated target  and five with a PC-3  xenograft, were injected with ~3 MBq of the radiolabeled protein, followed by PET/CT imaging at time points t = 24, 48 and 72 h p.i. To assess target specificity of the injected Anticalin in PC-3 xenografts, two of the five mice per group received a 100-fold molar amount of the unlabeled PASylated Anticalin 2 h before 89Zr-tracer injection. After 24 h, the best imaging contrast and high accumulation of 3.0 ± 0.3 %ID/g was observed for the PC-3 tumors, with decreasing tumor to background contrast after 48 and 72 h.The applicability of the PASylated Anticalin D11vs for the Figure ) showed a significant (p = 0.024) difference in radiotracer accumulation between tracer-injected mice (N=3) and those mice which were additionally injected with unlabeled Anticalin (N=2). Apart from the strong uptake by the tumors, elimination-related accumulation of radioactivity in liver, kidney and bladder was observed. Moreover, signals were seen in the joints, which can be explained by accumulation of liberated Zr(IV) due to the limited complex stability of the non-cyclic chelator Dfo Figure ). Furthermore, the 89Zr-labeled D11vs-PAS200-Dfo tracer allowed good visualization of the Ramos xenografts 24 h p.i., yet with lower contrast as compared to the PC-3 tumors. Of note, the Ramos xenografts grew faster and more infiltrating than the PC3 xenografts, which made it difficult to accurately delineate these tumors on the PET images. Consequently, the PC-3 xenografts were chosen for further experiments.Threshold-based segmentation of the PET images , with signals strongly reduced in tumors collected from the blocked mice. Concomitant immunohistochemical analysis of vessel distribution (via staining of CD31) as well as hCD98hc abundance, using cognate antibodies, revealed a well-vascularized tumor with high expression of hCD98hc for both PC-3 and Ramos xenografts .After ex vivo biodistribution experiments at the optimal time point 24 h p.i., a second study using male CB17-SCID mice bearing PC-3 tumors was conducted . Two cohorts, each with five mice, were injected with either 3.96 ± 0.12 MBq 89Zr-labeled D11vs-PAS or with the protein tracer in combination with a 250-fold molar amount of the unlabeled PASylated Anticalin (at t = -2 h). After 24 h, PET/CT scans were performed, tumors were explanted, subjected to an ex vivo PET scan and, finally, a biodistribution analysis was conducted. PET/CT images of the mice without prior blocking of hCD98hc showed pronounced accumulation of radioactivity in the tumor, whereas the tumors were barely visible in the blocked mice, in line with the ex vivo PET/CT ROI segmentation analysis (p = 0.027). Finally, the biodistribution study revealed high accumulation of radioactivity (8.6 ± 1.1% ID/g) in the tumor , with good tumor-to-blood and tumor-to-muscle ratios of 3.5 and 11.8, respectively. Radioactivity in the excretion-related organs matched the observations above, with additionally pronounced accumulation in the spleen. This can be explained by the formation of insoluble Zr-phosphate In order to confirm these imaging results and to obtain quantitative data from in vivo imaging, and only few therapeutic drug candidates addressing this target, are available to date.Due to its pathophysiological significance, CD98hc constitutes a highly attractive biomedical target for diagnostic as well as therapeutic applications in oncology. However, no CD98hc-specific reagents for D values already in the pico- to single-digit nanomolar range as well as stable monomeric behavior, which once again validates the utility of this protein scaffold and the library design.We have developed an Anticalin with exquisite specificity and high (picomolar) affinity towards the extracellular domain of hCD98hc. The initial Anticalin candidates were selected from a random library based on human Lcn2, an abundant siderophore-binding plasma protein. This previously designed library 2. Competition analysis for binding of the glycosylated hCD98hcg showed that all selected Anticalins in this study, including P3D11, recognize the same epitope region. P3D11 and P3A12 share a set of amino acid exchanges relative to wtLcn2, with 8 identical residues among the 20 randomized positions. Both Anticalins exhibit highly similar sequence stretches in loop #1, loop #2, as well as their adjoining β-strand segments A/B and C/D , which contribute to the interface in the P3D11•hCD98hcED complex (Table S5).Based on its favorable association and dissociation rate constants, with a remarkable complex dissociation half-life of ~6 h, P3D11 was chosen as the lead candidate for further characterization and optimization, eventually resulting in the Anticalin D11vs. X-ray structural analysis of the P3D11•hCD98hcED complex revealed deep penetration of the epitope loop L2, located in the membrane-distal part of the hCD98hc ectodomain, into the cup-shaped ligand pocket of the Anticalin, with a buried paratope surface area of 1424 ÅFigure ). A similar kind of structural paratope plasticity was previously observed for a set of Anticalins directed against the extra-domain B of oncofetal fibronectin While N-glycosylation of CD98hc has no influence on the target affinity of both P3D11 (or its improved version D11vs) and P3A12, binding of the third Anticalin candidate, P1E4, is significantly diminished (~100-fold). This behavior can be explained by minor variation in the mutual orientations between hCD98hcED and the different Anticalins, which may influence the sensitivity towards sterically demanding carbohydrate side chains in the neighborhood despite recognition of overlapping epitopes , thus nicely illustrating the power of directed evolution for stability engineering of Anticalin proteins .In spite of its remarkable antigen affinity, suitable for 89Zr-labeled D11vs radiotracer, whose plasma half-life was optimized using PASylation technology, showed high accumulation in PC-3 xenograft tumors with good imaging contrast and pronounced specificity, as blocking of CD98hc binding by co-application of unlabeled D11vs-PAS200 reduced tracer accumulation by ~40%. These findings were in line with in vitro cytofluorometry and immunofluorescence microscopy, where highly specific binding of D11vs on several CD98hc-positive human cancer cell lines, but not under conditions of a blocked CD98hc epitope, was evident. Tracer uptake in kidney, liver, spleen and joints of the xenograft mice was as expected due to the known in vivo release of 89Zr(IV) from the radiochelator 89Zr-chelating reagents with higher in vivo stability, as for example fusarinine C, a cyclic siderophore from Aspergillus fumigatusSmall animal PET/CT imaging and biodistribution analysis with the in vivo imaging study of CD98hc overexpression on tumor cells described in the literature. Considering the clinical importance of elevated CD98hc expression in several human cancers, this Anticalin, charged with appropriate radionuclides or toxic payloads, constitutes a promising new tool for preclinical and, potentially, clinical applications in oncology.In conclusion, we have selected and engineered a human CD98hcED-specific Anticalin with high affinity and successfully applied it for CD98hc-targeted PET imaging. This novel radiopharmaceutical demonstrated high tumor uptake in prostate carcinoma as well as B-cell lymphoma xenograft mouse models with excellent tumor visualization. To our knowledge, this is the first Supplementary methods, figures, and tables.Click here for additional data file.


Our findings revealed that gestational dietary methionine could affect maternal and placental H2S concentrations. With the increase of dietary methionine, maternal plasma and placental H2S concentrations changed quadratically, which was consistent with placental vascular density and reproductive performance. The decrease in H2S production caused by an increase in dietary methionine was likely to be the cause for the increase in the rate of low birth weight piglets and needs further study.Hydrogen sulfide  were randomly allocated into five groups, with feed diets comprisingstandardized ileal digestible methionine/lysine (Met/Lys) ratios of 0.27 (nutrient requirements of swine (NRC); 2012 level), 0.32, 0.37, 0.42, and 0.47, respectively. The litter size and weight at birth were measured and recorded. Maternal blood samples were obtained at embryonic day (E) E40 d, E90 d, and E114 d of gestation. The placental samples were collected at parturition. The results showed that maternal plasma H2S concentration was not affected at E40 d. However, the maternal plasma H2S concentration changed quadratically with the dietary Met/Lys ratio at E90 d (p < 0.01) and E114 d (p = 0.03). The maximum maternal plasma H2S concentration was at the dietary Met/Lys ratio of 0.37. Meanwhile, maternal plasma H2S concentration was positively correlated with piglets born alive (p < 0.01) and litter weight (p < 0.01). Consistent with the maternal plasma, the placental H2S concentration also changed quadratically with the dietary Met/Lys ratio (p = 0.03); the Met/Lys ratio of 0.37 showed the maximum H2S concentration. In conclusion, our findings revealed that the gestational dietary Met/Lys ratio could affect maternal and placental H2S concentrations, which may be an important molecular mechanism affecting placental angiogenesis and piglet development.The placenta is a unique bond between the mother and the fetus during pregnancy, and a proper placental angiogenesis is vital for fetal development. H Methionine (Met) is an essential amino acid in the diets of pigs ,2. In ad2S) is a ubiquitous signaling molecule with important functions in many mammalian cells and tissues and is now considered to be the third gas-phase signaling molecule, in addition to carbon monoxide (CO) and nitric oxide (NO) [2S is mainly produced by Met and the cysteine transsulfuration pathway (TSP), via the direct enzymatic action of cystathionine-γ-lyase (CGL) and cystathionine-β-synthase (CBS) [2S production, placental angiogenesis, and blood flow velocity [2S donors significantly attenuated placental vascular dysplasia and IUGR, caused by CGL inhibition [2S produced by trophoblast cells can promote angiogenesis in placental artery endothelial cells [2S production in sow plasma and placenta. The goal of the current study was to investigate the role of the gestational methionine/lysine ratio on maternal and placental H2S production, both in vivo and in vitro. Hydrogen sulfide (Hide (NO) ,8,9. Endse (CBS) ,11. Studvelocity ,13. Howehibition . In vitral cells . Howevern = 65) were randomly allocated into five groups and fed diets with standardized ileal digestible methionine/lysine (Met/Lys) ratios of 0.27 (nutrient requirements of swine (NRC); 2012 level), 0.32, 0.37, 0.42, and 0.47 respectively. The diet was maintained at constant levels of Lys (average SID Lys 0.71%); the ingredients and nutrient compositions of the experimentaldiets are listed in The experimental design and the dietary formula were described previously . In thisThe sample collection of blood and placenta was described previously . Twelve 2PO4 buffer (pH 7.4) and was centrifuged for 5 min at 13,000 rpm. The supernatant wasused for H2S content and production assay. The protein concentration was determined by the bicinchoninic acid (BCA) protein assay kit .The placenta was selected based on the average pig birth weight ± one standard deviation of each group. A total of 100 mg of placental tissue was separated and ground to a fine powder using a porcelain mortar and pestle, chilled with liquid nitrogen. Next, 10% of the tissue homogenate was made with 100 mM of ice cold KH2 incubator. Before the Met treatment, the PIEC cells were seeded in a 12-well plate (2 × 105 cells/well); 80% confluent PIEC cells were placed in serum-free basic medium for 18 h, followed by Met-free RPMI 1640 medium for another 6 h, then treated with different concentrations of Met  for 24 h (n = 4). The supernatant culture medium was collected for H2S content assay.Porcine iliac artery endothelial cells (PIEC) were obtained from the cell bank of the Chinese Academy of Sciences , cultured in Roswell Park memorial institute (RPMI) 1640 medium , and supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin solution  at 37 °C in a 5% CO2S levels were measured through the formation of methylene blue [2PO4 buffer (pH 7.4) and 250 μL of 1% (w/v) zinc acetate; for the culture medium and placental tissue homogenate, 500 μL samples were mixed with 250 μL of 1% (w/v) zinc acetate. The reaction mixture was incubated at 37 °C for 2.5 h. The protein content in the samples was removed by adding 10% trichloroacetic acid (250 μL) to the reaction mixture, which was pelleted by centrifugation. The supernatant was then mixed with 100 μL of N-dimethyl-p-phenylenediamine sulfate (20 mM in 7.2 M HCl) and 100 μL of FeCl3 (30 mM in 1.2 M HCl) in a test tube. The mixture was incubated at room temperature for 15 min. The absorbance of the resulting solution was measured at 670 nm in a 96-well plate with a microplate reader. The H2S concentration was calculated using a standard (NaHS) calibration curve  .In this study, the Hene blue ,17. For 2S concentrations, as shown in p < 0.05 was considered to indicate statistical significance. Regression analyses were performed to evaluate the quadratic effects of dietary Met/Lys ratio and H2S concentration, as shown in 2S concentration are shown in Statistical analysis was performed using the SAS statistical package . A one-way analysis of variance (ANOVA) followed by a least-significant-difference (LSD) post-hoc test was conducted to explore the impact of the gestational dietary Met/lysine ratio on maternal plasma and placenta H2S concentration, we collected maternal plasma at different stages of pregnancy. We found that by E40 d, increasing the Met/Lys ratio in the diet had no effect on the maternal plasma H2S concentration, as shown in 2S concentration changed quadratically, with an increase in the dietary Met/Lys ratio, as shown in 2 + 1577x −288.7, R2 = 0.19, p < 0.01) and at E114 d , the maximum maternal plasma H2S concentration was at the dietary Met/Lys ratio of 0.37. These results indicated that maternal plasma H2S concentration could be affected by the dietary Met/Lys ratio.To study the effect of the gestation dietary Met/Lys ratio on maternal plasma H2S in the reproductive performance of the sows, we analyzed the correlation between the maternal plasma H2S concentration and the number of piglets born alive, the litter weight, and average pig birth weight at E114 d. We found that the maternal plasma H2S concentration had a positive correlation with the number of piglets born alive and the litter weight, as shown in 2S may have played an important role in the sows’ reproductive performance.To determine the involvement of H2S concentration. The placenta was selected based on the average pig birth weight ± one standard deviation of each group. Similar to the maternal plasma, the placental H2S concentration also changed quadratically with the dietary Met/Lys ratio, as shown in 2 + 105.6x −9.17, R2 = 0.12, p = 0.03), the maximum placental H2S concentration was at the dietary Met/Lys ratio of 0.37.We further examined the effect of the gestation dietary Met/Lys ratio on the placental H2S production in vitro, PIEC cells were treated with variable mediums which were (1) Met-free (0 μM), (2) had a physiological concentration of Met (50 μM), and (3) had a high concentration of Met , respectively. We found that the H2S concentration was highest in the Met-free medium condition and lowest in a physiological concentration of Met (50 μM), as shown in 2S production increased initially (from 50 to 500 μM) and then maintained relative stability (from 500 to 1000 μM).To further confirm the effect of Met on H2S is an endogenous stimulator of angiogenesis that promotes vascular endothelial cell proliferation, migration, and angiogenesis by activating the NO/cGMP pathway, opening KATP channels, and promoting the phosphorylation of PI3K/AKT and MAPK pathways [2S concentration was positively correlated with the sows’ reproductive performance. With an increase in Met/Lys ratio in the sows’ diet, the maternal plasma and the placental H2S concentrations changed quadratically. These results indicated that there was a dose-effect of Met concentration on H2S production.Hpathways ,18,19. I2S production, recent studies have found that dietary restriction (50% restriction) or Met restriction is the most effective way to increase H2S production. Studies have found that limiting the feed intake can increase liver CGL expression, increase endogenous H2S production, and alleviate liver ischemia-reperfusion injury [2S production can increase in the skeletal muscles by up-regulating the GCN2/ATF4 pathway and activating NAD+/SIRT1 signaling, thus triggering skeletal muscle angiogenesis and delaying muscle aging [2S concentrations were highest under Met-free conditions (0 μM), whereas relative to physiological concentrations, high concentrations of Met did not significantly inhibit H2S production in PIEC cells, which suggested that there were some differences between in vitro and in vivo experiments.Although Met is a precursor for Hn injury . Addition injury  and mainn injury . Recentlle aging ,23. We f2S concentrations were consistent with the sows’ reproductive performance. The decrease in H2S production caused by an increase in dietary Met was likely to be the cause for the increase in the rate of low birth weight, along with the failure to further improve the reproductive performance of the sows. Studies of rodents have found that a high concentration of Met can significantly inhibit CGL activity in peritoneal macrophages, reducing serum H2S content and increasing inflammatory reactions [2S could promote mammary epithelial cell proliferation by activating the PI3K/Akt-mTOR pathway in porcine [2S producing enzymes, or increasing the H2S content by using H2S donors, may be an effective way to alleviate the adverse effects of high Met production. Our current results found that H2S donors diallyl trisufide(DATs) could significantly promote PIEC angiogenesis (unpublished data), which suggests that H2S is a potential target for improving the reproductive performance of sows.In modern animal production, gestational restriction feeding is an effective and widely used practice to improve the reproductive performance of sows. Our previous study found that under the restriction feeding conditions, increasing the Met/Lys ratio from 0.27 to 0.37 can significantly increase the average pig birth weight and the litter weight in the pigs that are born alive . Howevereactions . A recen porcine . Therefo2S concentration, which may be an important molecular mechanism affecting placental angiogenesis and piglet development.In conclusion, our findings revealed that gestational dietary Met could affect maternal and placental H


There was a history of close contact with pigs 3 days before symptom onset. The child recovered within 3 days. No other transmissions were observed. Serological investigations of the virus isolate revealed cross-reactions with ferret antisera against influenza A(H1N1)pdm09 virus, indicating a closer antigenic relationship with A(H1N1)pdm09 than with the former seasonal H1N1 viruses.A zoonotic A/sw/H1 During routine surveillance at the National Influenza Centre in Germany in June 2020, a nasal swab was conspicuous because qPCR for the influenza A virus matrix protein (MP) and N1 neuraminidase (NA) genes were positive, whereas the haemagglutinin (HA) qPCR gave no results. The sample underwent whole genome sequencing and results pointed to a zoonotic influenza virus originating from swine. Here we describe the clinical features of the infection as well as the results of antigenic and genetic characterisation of this zoonotic influenza virus.The diagnostic sample originated from a 2.5-year-old child who lived on a farm, had regular contact with pigs, most recently 3 days before symptom onset, and was not vaccinated against influenza. The child had influenza-like illness over 3 days, displaying fever up to 40 °C, a sore throat, rhinorrhoea, headaches, myalgias, some fussiness and one episode of emesis, and slept a lot. Afterwards, they recovered quickly and fully. The child was not treated with antiviral drugs. No other family member, including the child’s 5-month-old sibling, showed any symptoms, although some of them had been in close contact with the pigs. Four weeks later, 15 pigs of all age groups held at the farm and six family members were swabbed. All nasal swabs were negative, indicating absence of further virus circulation at this location. Four family members tested positive for rhinoviruses, but not the child who had had influenza. Because these swabs were qPCR-negative, virus isolation was not attempted from the pigs’ swabs.The pig herd of the farm has 600 fattening pigs. Every 4 weeks, 120 new pigs  are introduced from another farm in Germany. The pig farm is situated outside of the village and no one except the farmer, his family and the veterinarian have access to it. The pig feed is generated by the farm from its own harvest. The pigs are not vaccinated against influenza. Two weeks before the child was infected, a new batch of pigs arrived at the farm. At that time, some pigs were displaying a cough, for which they were treated with antibiotics. Thus, the infection was most probably introduced to the herd via the new batch of pigs. avN1 virus . Antigenic characterisation showed that cross-reactivity was highest with swine hyperimmune serum directed against influenza A/sw/H1N1 virus  [1]. FurBlood samples from 14 of 15 pigs were found to be seropositive against the infecting virus (HES/2020). In haemagglutination inhibition (HI) tests against HES/2020, titres ranged from 1:10 to 1:160. All pig sera were negative against influenza A(H1N1)pdm09 virus (A/Brisbane/2/2018).avN1 and A(H1N1)pdm09 viruses , G , I , 1C.2.2 (HA), F , 1F (NA), F (MP), 1E  ,6. It isSupplementary Figures S1–S10). To detect substitutions with potential functional relevance in the HES/2020 genome, the FluSurver online tool was employed (https://flusurver.bii.a-star.edu.sg/), identifying substitutions in the HA receptor binding domain (D222E) [Supplementary Table S1). The substitutions NP-Q357K, PA-X-R57K, PA-R57K, PA-T639A are present in both zoonotic viruses and in both analyses . They displayed nucleotide identities of 95% each, whereas all other coding sequences displayed nucleotide sequence identity > 95% relative to the reference sequence. Reference sequences were swDUEL/2012 for HA and NA, swLUED/2013 for MP, NP, NS and PB1, swHEIST/2012 for PA and swOOST/2012 for PB2). Amino acid (AA) sequence variation was highest over the regulator proteins of the host innate immune response, NS1, PA-X and PB1-F2  [ (D222E) , R351K;VWhile HES/2020 does not exhibit NA or PA mutations conferring resistance against neuraminidase inhibitors or baloxavir marboxil, its M2 sequence contains the AA substitutions L26I, V27A and S31N, all of which are associated with adamantane resistance (amantadine and rimantadine). Phenotypic susceptibility testing against oseltamivir, peramivir and zanamivir confirmed that HES/2020 was sensitive to all neuraminidase inhibitors authorised in Europe.avN1 and A/sw/H3N2 in Lower Saxony, in 2010: A/sw/H1avN1 in Lower Saxony, in 2011: A/sw/H1huN2 and A/sw/H1avN1 in Lower Saxony) [avN1 infections were the most common [This is the sixth zoonotic swine influenza virus infection in humans investigated at the German National Influenza Centre (in 2007: A/sw/H1 Saxony) . Of the t common . All preavN1 are the predominant swine influenza viruses in Germany [avN1 1C.2.2 and H1avN1 1C.2.1. Other swine influenza viruses include H1huN2 and H3N2 viruses as well as H1pdmN1 and H1pdmN2 viruses [The genetic diversity of influenza A viruses in the European pig population is increasing -17. A/sw Germany . Among t viruses -18. An iavN1 viruses which confers adamantane resistance via the M2-S31N mutation in MP gene 2 [Swine influenza viruses acquired adamantane resistance in the late 1980s [P gene 2 . This muP gene 2 . In addiP gene 2 . Swine influenza viruses have acquired some resistance genes against human myxovirus resistance protein MxA during their evolution in pigs, facilitating their transmission to humans . Pig-to-avN1 viruses in ferrets. This is in line with previous findings that influenza A(H1N1)pdm09 infection induces broadly neutralising (not strain-specific) antibodies [avN1 viruses in the human population are rare [avN1 virus at varying microneutralisation titres and none was negative [avN1 influenza virus. Our serology investigations indicate some level of cross-reactivity between influenza A(H1N1)pdm09 virus and A/sw/H1tibodies . Antibodare rare ,28. On tnegative . AlthougavN1 viruses in the future. In particular, swine reassortant viruses may quickly acquire antigenic changes, and this is where substantial zoonotic potential may arise.However, the rising genetic diversity among swine influenza viruses, involving antigenic drift and shift, may increase divergence from influenza A/sw/H1


Two antiviral classes, the neuraminidase inhibitors (NAIs) and polymerase inhibitors  can be used to prevent and treat influenza infections during seasonal epidemics and pandemics. However, prolonged treatment may lead to the emergence of drug resistance. Therapeutic combinations constitute an alternative to prevent resistance and reduce antiviral doses. Therefore, we evaluated in vitro combinations of baloxavir acid (BXA) and other approved drugs against influenza A(H1N1)pdm09 and A(H3N2) subtypes. The determination of an effective concentration inhibiting virus cytopathic effects by 50% (EC50) for each drug and combination indexes (CIs) were based on cell viability. CompuSyn software was used to determine synergism, additivity or antagonism between drugs. Combinations of BXA and NAIs or favipiravir had synergistic effects on cell viability against the two influenza A subtypes. Those effects were confirmed using a physiological and predictive ex vivo reconstructed human airway epithelium model. On the other hand, the combination of BXA and ribavirin showed mixed results. Overall, BXA stands as a good candidate for combination with several existing drugs, notably oseltamivir and favipiravir, to improve in vitro antiviral activity. These results should be considered for further animal and clinical evaluations. Since 1977, two subtypes of influenza A viruses, H1N1 and H3N2, along with influenza B viruses, have co-circulated in the human population. These viruses are responsible for seasonal epidemics and may cause global pandemics ,5 but thMadin–Darby canine kidney cells over expressing the α2,6 sialic acid receptor   were maMucilAirTM reconstituted HAE, issued from primary cells obtained from pools of nasal biopsies, were provided by Epithelix SARL  and maintained in air–liquid interphase with specific culture medium in Costar Transwell inserts , according to the manufacturer’s instructions. The A/California/7/2009 (H1N1)pdm09, A/Switzerland/9715293/2013 (H3N2) and A/Texas/50/2012 (H3N2) influenza viruses were obtained from NIBSC  and from the Centre National de Référence des Virus des Infections Respiratoires, Lyon, France Sud.Baloxavir acid was synthesized at Shionogi & Co., Ltd.  and diluted in dimethyl sulfoxide (DMSO) to a stock concentration of 10 mM. Favipiravir (T-705) was purchased from Adooq Bioscience  and diluted in DMSO to a stock concentration of 100 mM. Oseltamivir carboxylate (the active form of oseltamivir) was synthesized by Hoffmann-La Roche  and diluted in sterile water to a stock concentration of 10 mM. Zanamivir and ribavirin were purchased from Sigma  and diluted in sterile water and phosphate-buffered saline (PBS), respectively, to stock concentrations of 10 mM and 100 mM, respectively. Peramivir was synthesized by Biocryst  and diluted in sterile water to a stock concentration of 10 mM.2 atmosphere, followed by the addition of BXA, each NAI, favipiravir and ribavirin in two-fold serial dilutions. For A(H1N1)pdm09 infections, concentrations of 0.0175–10 nmol/L were used for BXA; 2.34–1000 nmol/L for oseltamivir; 2.34–1000 nmol/L for zanamivir; 0.468–400 nmol/L for peramivir; 78.125–80,000 nmol/L for favipiravir; 78.125–40,000 nmol/L for ribavirin, diluted in MEM and TPCK (L-1-Tosylamide-2-phenylethyl chloromethyl ketone) trypsin (1 mg/mL). For A(H3N2) infections, concentrations of 0.39–200 nmol/L were used for BXA; 7.8–4000 nmol/L for oseltamivir; 35.15–16,000 nmol/L for zanamivir; 1.56–800 nmol/L for peramivir; 312.5–160,000 nmol/L for favipiravir; 78.125–40,000 nmol/L for ribavirin, diluted in MEM and TPCK trypsin (1 mg/mL). These dilutions were based on Fukao et al. [ST6-GalI-MDCK cells (5000 cells/well) were seeded in 96-well plates and inoculated with 200 Median Tissue Culture Infectious Dose per well (TCID50/well) of each of the influenza strains pdm09 and A/Switzerland/9715293/2013 (H3N2)). Infected cells were incubated for 60 min at 37 °C in a 5% COo et al.  and on oo et al. ,29. Theno et al. .Briefly, the weighted average CI (CIwt) was calculated for each combination as (CI50 + 2 × CI75 + 3 × CI90 + 4 × CI95)/10 to estimate drug combination effects at high levels of virus inhibition and to increase therapeutic relevance . Drug combination effects were defined as CIwt < 0.7, synergism; CIwt > 0.7 and <0.9, moderate synergism; CIwt > 0.9 and <1.2, additivity; CIwt > 1.2 and <1.45, moderate antagonism; CIwt > 1.45, antagonism.® culture medium  were applied through basolateral poles. At 24, 48  and 72 hpi, apical washes with 150 μL OptiMEM were performed and stored in two aliquots at −80 °C for TCID50 viral titration. At 24, 48 and 72 hpi, transepithelial electrical resistance (TEER) between the apical and basal poles of the HAE, considered as a surrogate of epithelium integrity, was measured using a dedicated volt-ohm meter (EVOM2) and compared to baselines values measured before infection (t = 0).The apical poles of HAE were gently washed twice with warm OptiMEM medium ) and then infected with a 150 μL dilution of A/California/7/2009 (H1N1)pdm09 or A/Texas/50/2012 (H3N2) in OptiMEM, at a multiplicity of infection (MOI) of 0.1 or 0.01, respectively. For mock infection, the same procedure was followed using OptiMEM as inoculum. After 1 h incubation at 37 °C, the viral inoculum was removed. At 5, 24 and 48 hpi, treatments with specific dilutions of antiviral molecules alone or in combination in MucilAirAll experimental assays were performed in triplicate at a minimum, and representative results are shown. HAE viral titers in the drug combination groups were compared against those of both the untreated (*) and most performant single-treated (#) groups using mixed model two-way analysis of variance (ANOVA) with Bonferroni post hoc test. The testing level (α) was 0.05. Statistical analyses were performed on all available data, using GraphPad, Prism 7.As shown in Combination drug experiments against the A(H1N1) strain A showed In the A(H3N2) background B, BXA alBased on the results obtained in ST6-GalI-MDCK cells, we further evaluated the efficacy of the selected two-drug combination ratios determined in In the case of A(H3N2), apical viral titers peaked at 72 hpi, reaching mean values of 7.5 (±6.4) × 109 TCID50/mL, in the untreated group . InteresAntivirals play a crucial role in the treatment and control of influenza epidemics and pandemics . The M2 In this study, we aimed at investigating the potential benefits of combining BXA to most approved antivirals against seasonal influenza A(H1N1) and A(H3N2) strains using both a classic cell line and a physiologically and predictively relevant HAE model.Firstly, we observed that BXA had the lowest EC50  against Secondly, our results show that combinations of BXA with NAIs  have higher potency compared to individual drugs, against A(H1N1)pdm09 and A(H3N2) viruses. Such synergistic effects were confirmed in the HAE model, for which the physiological relevance and predictive value have been previously described by our group and others . These fCombinations of other polymerase inhibitors with NAIs, as well as combinations of two polymerase inhibitors, have also been studied. Ormond and colleagues demonstrated that the combination of oseltamivir with favipiravir negatively impacted viral replication after several passages . These rIn our study, we also assessed two polymerase inhibitors: favipiravir and ribavirin (a transcriptase inhibitor of Polymerase Basic 1—PB1) ,48 in coAdditional in vitro studies using resistant strains (I38T or H275Y variants) should be done to better understand drug activities and to evaluate the impact of combination therapy on mutant viral replication. Additionally, further animal studies should be conducted to evaluate the effectiveness of our synergistic combinations in a complete system.In summary, our results suggest that BXM stands as a potential good candidate for combination therapy with several existing drugs to improve antiviral activity and eventually delay drug resistance of influenza type A viruses. Combination therapies with BXM should be further considered for animal studies and clinical investigations.


Individuals with serum 25(OH)D ≥75 nmol/L had significantly lower waist circumference (p < 0.0001), C-reactive protein (p = 0.003), glycated hemoglobin (p < 0.0002), fasting triglycerides (p < 0.0001), total homocysteine (p < 0.0001), and insulin resistance (p = 0.0001) and had significantly higher HDL-cholesterol (p < 0.0001) and maximal oxygen uptake (marker for CRF) (p< 0.0009) compared to those with 25(OH)D <30 nmol/L. In conclusion, serum 25(OH)D ≥75 nmol/L is associated with positive indicators related to cardiometabolic diseases in US adults.A serum vitamin D  concentration of ≥75 nmol/L is recommended for optimal health. We investigated the relationship between serum 25(OH)D and metabolic syndrome (MetS), diabetes, cardiometabolic biomarkers, and cardiorespiratory fitness (CRF) in US adults using clinical cut points recommended by health organizations. Data from USA’s National Health and Nutrition Examination Surveys were used. Prevalences and likelihood of having MetS and diabetes according to clinical cut points for serum 25(OH)D  were determined with multivariate logistic regression. Relations between serum 25(OH)D and various cardiometabolic biomarkers, CRF, MetS, and diabetes were tested using multivariable adjusted regression. Prevalence of MetS and diabetes were significantly lower in individuals with serum 25(OH)D ≥75 nmol/L  compared to those with 25(OH)D <30 nmol/L  ( Serum vitamin D  concentration is used as a biomarker of vitamin D status. 25(OH)D in the circulation represents endogenously synthesized vitamin D in the dermis and from diet and supplements. Recently there has been an increased interest in vitamin D due to widespread prevalence of suboptimal serum 25(OH)D concentrations in the US and across the globe. The prevalence of hypovitaminosis D [<50 nmol/L of 25(OH)D] was ≈32% in the US population . Based oThe role of vitamin D in bone metabolism is well documented . Recent 2 max), a marker of cardio-respiratory fitness (CRF) is associated with increased risk for CVD , a hormone form, into the 3rd ventricle of the brain improved glucose tolerance and hepatic insulin sensitivity through VDR action [2D into the brain dramatically decreased the food intake and body weight in obese rodents. Although a clear cause and effect for vitamin D in glucose and insulin metabolism and body weight has been established, it is surprising that studies on vitamin D supplementation and CVD have yielded inconsistent results [We found individuals with <30 nmol/L of serum 25(OH)D have ≈183% higher diabetes prevalence compared to those with ≥75 nmol/L 25(OH)D. This translates into 70% increased risk of having diabetes in those with <30 nmol/L serum 25(OH)D compared to those with ≥75 nmol/L serum 25(OH)D. In a recent meta-analysis based on 28,258 older subjects, Lucato et al.  found thR action . They al results ,45,46,472 max (a marker of CRF) compared to those with <30 nmol/L. Growing evidence links low CRF to increased risk for CVD and cancer [2 max in professional soccer players. Further Lammle et al. [2 max relates to the protective effect of vitamin D on lung function [2 max, the difference in VO2 max we observed between the highest and the lowest serum 25(OH)D concentrations cut points was ≈1.9 mL/kg/min. Whether this small difference in VO2 max has any clinical relevance is not known. Controlled studies are needed to establish a cause and effect relation between vitamin D and CRF.We report, for the first time in a large US sample that individuals with ≥75 nmol/L 25(OH)D had ≈5.9% higher VOd cancer . Koundoud cancer  reportede et al.  found a function . Adolescfunction . Althougp < 0.003). A recent meta-analysis demonstrated that vitamin D supplementation lowered C-reactive protein [High circulating acute phase inflammatory markers, i.e., C-reactive protein and fibrinogen have been linked to chronic inflammatory diseases such as MetS, diabetes, obesity, and CVD ,51,52. U protein  suggesti protein . The exap < 0.008). Individuals with ≥75 nmol/L 25(OH)D had ≈3.7% lower diastolic blood pressure than those with <30 nmol/L 25(OH)D. Support for an inverse association of 25(OH)D with blood pressure from epidemiological and prospective studies is very strong [2D, and 1-α hydroxylase are found in vascular endothelial cells, a function for vitamin D and VDRs in blood pressure has been proposed [2D administration reduced the expression of renin [3 (33000 IU) administration for every 2-weeks for six months reduced systolic blood pressure significantly [Although individuals with serum ≥75 nmol/L 25(OH)D had significantly lower risk of MetS, only the association between 25(OH)D and diastolic blood pressure was significant D concentration in the analysis. We were unable to separate the data by diabetes type 1 or type 2 status because NHANES survey did not contain that specific data. The MetS sample contains all diabetes cases who had met the MetS diagnostic criteria (please see 2.3 above). Therefore, there was an overlap of subjects between MetS and diabetes logistic regression analysis. Whether this overlap of diabetes cases in the MetS analysis lead to errors is not known. Overall, US adults with serum 25(OH)D <30 nmol/L have significantly higher prevalence of MetS and diabetes and higher inflammatory markers and reduced CRF compared to those in the serum 25(OH)D ≥75 nmol/L group. Recent evidence from controlled trials and meta-analysis is promising with regard to reporting beneficial effect of increased vitamin D in reducing the risk of cardiometabolic diseases. Our findings support the evidence that serum 25(OH)D concentrations ≥75 nmol/L are associated with improved metabolic pathologies related to inflammation, insulin resistance, MetS, and diabetes ,73,74. G


Recently, we found that sustained stimulation of the calcium-dependent PKCβII isoform leads to decrease in KCNQ1 subunit membrane localization and KCNQ1/KCNE1 channel activity, although the role of KCNE1 in this regulation was not explored. Here, we show that the auxiliary KCNE1 subunit expression is necessary for channel internalization. A mutation in a KCNE1 phosphorylation site (KCNE1(S102A)) abolished channel internalization in both heterologous expression systems and cardiomyocytes. Altogether, our results suggest that KCNE1(S102) phosphorylation by PKCβII leads to KCNQ1/KCNE1 channel internalization in response to sustained PKC stimulus, while leaving KCNQ1 homomeric channels in the membrane. This preferential internalization is expected to have strong impact on cardiac repolarization. Our results suggest that KCNE1(S102) is an important anti-arrhythmic drug target to prevent IKs pathological remodeling leading to cardiac arrhythmias.The slow cardiac delayed rectifier current (IKs) is formed by KCNQ1 and KCNE1 subunits and is one of the major repolarizing currents in the heart. Decrease of IKs currents either due to inherited mutations or pathological remodeling is associated with increased risk for cardiac arrhythmias and sudden death. Ca Axopatch 200B (Axon Instruments) was used for data acquisition and current data were analyzed using Clampfit software. Peak tail currents were fit with a Boltzmann equation, G(v) = Gmax/[1 + expS2 Fig). Because of the very high membrane localization of the KCNQ1 channels in cells expressing KCNQ1 alone, in order to further investigate effects of cPKC on channel localization, we also studied changes of cytoplasmic channel expression, which may be more sensitive to cPKC treatment. Consistent with our previous results, the cytoplasmic KCNQ1 expression was significantly increased after cPKC stimulation in the presence of KCNE1 subunit . In the Although our previous work had shown internalization of the KCNQ1 subunit in response to sustained cPKC activation when KCNE1 subunits were expressed, we did not explore whether the KCNE1 subunit was internalized together with KCNQ1. To investigate whether KCNE1 subunits membrane expression was affected by sustained cPKC stimulation, we used FLAG-tagged KCNE1 subunits expressed together with KCNQ1 and labeled the cells with anti-FLAG antibodies in cells treated with a cPKC activator peptide . Our data showed that membrane localization of KCNE1 subunits decreased in response to sustained cPKC activation . To asceBecause multiple stoichiometries between KCNQ1 and KCNE1 have been suggested , we inveKCNE1(S102) has been shown to be important for channel acute regulation by cPKC , 43–46. To investigate the role of KCNE1(S102) to cPKC regulation of channel function, we first measured the effect of cPKC activation in cells expressing untagged KCNQ1 subunits co-expressed with either KCNE1(WT) or KCNE1(S102A) subunits. For cells expressing KCNE1(WT), currents were inhibited by the cPKC activator treatment, while KCNE1(S102A) expressing cells show no response to cPKC . The unsTo investigate the role of KCNE1(S102) on cPKC regulation of IKs channel membrane localization in the native system, KCNQ1-GFP was expressed with either wild-type KCNE1(WT) or KCNE1(S102A) in cardiomyocytes using adenoviral infection. We have previously shown that prolonged α1-AR activation inhibits channel membrane localization via cPKC activation, and inhibits KCNQ1/KCNE1 currents in cardiomyocytes , 21, altTo confirm KCNQ1/KCNE1 channel regulation is independent from subunit stochiometry in cardiomyocytes, we also expressed human IKs channel subunits at an increased KCNQ1 ratio . Cardiac cells were stimulated with Phe . As for cells with the 1:1 infection ratio , KCNQ1-GHere we found that homomeric KCNQ1 complexes are not regulated by sustained stimulation of calcium-dependent PKC isoforms. In addition we show that KCNE1(S102) phosphorylation site is critical in regulation of the channel membrane localization by calcium-dependent PKC isoforms. These findings provide a novel mechanism by which cardiomyocytes can preferentially internalize IKs complexes (KCNQ1/KCNE1), leaving homomeric KCNQ1 complexes at the plasma membrane. As KCNE1 is critical for KCNQ1 characteristic channel function , modulatXenopus oocytes [The C-terminus of KCNQ1 has been shown to interact with the C-terminus of KCNE1, and form a macromolecular complex with other molecules, including PIP2, calmodulin, Nedd4.2 and AKAP-Yotiao, responsible for proper assembly, function and regulation of the channel –51. KCNE oocytes , suggest oocytes . In addiKCNQ1 subunits expressed alone have been shown to undergo dynamin-dependent internalization in response to AMP-activated protein kinase (AMPK) activation in kidney cells , 54. PKCKCNE1(S102) is a conserved putative PKC phosphorylation site in KCNE1. The equivalent residue to KCNE1(S102A) has been suggested to underlie the inhibition of mice and rat IKs in response to PKC . KCNE1S and 4. IS1 FigLeft, representative confocal images of HEK293T cells expressing KCNQ1 and α1A-AR, treated with Phe (30 μM for 90 min). Right, summary data of experiments conducted. Control = vehicle. Scale bars, 5 μm. n = number of cells tested.(DOCX)Click here for additional data file.S2 Fig(A) Representative traces of current elicited from a +20mV depolarizing step from a holding potential of -80mV in HEK293T cells transiently expressing KCNQ1 alone (left), and KCNQ1 and KCNE1 in either the absence (middle) or presence (right) of cPKC activation . Control cells were treated with control peptide (C1). The early current was measured 0.25 s after the depolarizing step (Iearly), and the late current was the current activated between 0.25 s and 2.75 s after the depolarizing step (Ilate), as indicated. Dashed lines marked the 0 current. (B), Summary data of the ratio of Ilate:Iearly for depolarizing steps from 0 to +40 mV. *p<0.05.(DOCX)Click here for additional data file.S3 Fig(A), Representative confocal images of HEK cells expressing KCNQ1-GFP in the presence and absence of KCNE1 subunit, in the presence and absence of cPKC activation . Cytoplasmic fluorescence was measured as indicated and normalized to background fluorescence (intracellular fluorescence-background fluorescence)/ (background fluorescence). (B), Summary data of KCNQ1 cytoplasmic fluorescence in experiments conducted as in the top panels. CTRL = vehicle. Scale bars, 5 μm. *p<0.05. (n = cell number).(DOCX)Click here for additional data file.S4 FigLeft, Representative immunofluorescent images of fixed HEK cells expressing KCNQ1 and FLAG-KCNE1 after sustained cPKC stimulation . Nuclei were labeled with DAPI (in blue). Right, Summary data of normalized KCNE1 membrane localization. (B) Left, Representative Western Blots of whole-cell extracts obtained after sustained cPKC activation . UNT. was untransfected sample. Data were normalized to GAPDH loading control expression. Right, Average of KCNE1/GAPDH expression ratio in 3 experiments. Control = control peptide C1. Scale bars, 5 μm. *p<0.05, (n = number of cells).(A) (DOCX)Click here for additional data file.S5 FigWestern Blots of whole-cell extracts obtained after sustained cPKC activation . UNT. marks untransfected samples.(DOCX)Click here for additional data file.S6 Fig1/2 (right) of KCNQ1/KCNE1(S102A) channels at control condition  and sustained cPKC activation (1 μM cPKC activator peptide KAC1-1) for 90 min (in red). The solid lines in the left panels are Boltzman fits of experimental data. *p<0.05, n = the number of cells measured.Average I-V plots (left) and V(DOCX)Click here for additional data file.S7 FigLeft, Representative images of cardiomyocytes expressing adenovirus of KCNQ1-GFP and KCNE1 at a ratio of 3:1, in the presence and absence of sustained α1-AR stimulation , and cPKC inhibition . Right, Summary data of membrane localization of KCNQ1 from experiments conducted as in the left panel. *p<0.05, n = number of cells measured.(DOCX)Click here for additional data file.S8 Fig(A) Left: representative confocal images of HEK cells expressing KCNQ1-GFP and the KCNE1 subunit, in the presence and absence of dorsomorphine (90 min). Cytoplasmic fluorescence was measured at the indicated region. Right: Summary data of KCNQ1 normalized membrane localization in experiments conducted as in the left panels. (B) Top: representative confocal images of HEK cells expressing KCNQ1-GFP and KCNE1 treated with Phe  in the presence and absence of dorsomorphine (90 min). Bottom: Summary data of KCNQ1-GFP cytoplasmic fluorescence in experiments conducted as in the top panels for cells treated with either Phe  or cPKCact  as indicated. For all conditions, control is significantly different from treated cells. Scale bars, 5 μm. *p<0.05. (n = cell number).(DOCX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


H9N2 avian influenza virus is one of the most widely circulating viruses in poultry and poses a huge potential threat to human health due to its frequent gene reassortment with other influenza viruses. In this study, we generated a series of H9N2-H7N9 reassortant viruses and examined their pathogenicity in a mouse model. We found that HA or combined HA and NA replacement on the H9N2 background led to no substantial change in the virus-induced pathogenicity, whereas H9N2 virus containing H7N9 internal genes had significantly higher virulence in comparison to the parental H9N2 virus. This increased pathogenicity is associated with enhanced viral replication both in mice and in MDCK cells. We further demonstrated that the viral ribonucleoprotein complex from H7N9 virus possessed higher activity than that from its H9N2 counterpart. Collectively, our data demonstrated that genetic compatibility between H9N2 and H7N9 viruses facilitated the reassortment between H7N9 and H9N2 viruses co-circulated in poultry and that internal gene replacement would convert H9N2 virus into a novel threat to human health. Moreover, viral genome analysis revealed that HA genes of the human H9N2 isolates belonged to the G1-W, G1-E, or BJ94 lineages and were related closely to the viruses isolated from local poultry (H9N2 avian influenza virus (AIV) is the most widely circulating AIV in poultry populations globally. H9N2 viruses were first isolated from turkeys in American in 1966 . In the  poultry .H9N2 viruses are often found to be co-circulated among poultry with other AIV subtypes, increasing the rates of reassortment between these viruses and thus posing a huge threat to public health. H9N2 AIV could serve as gene donor to supply internal genes to other viruses, including the H7N9, H10N8, and H5N6 influenza viruses that recently caused human infection . ReciproH9N2 infections, distinct from H7N9 infections, normally only cause mild symptoms in human, and therefore, the potential risk of enhanced virulence and/or transmissibility resulting from reassortment with other viruses is easy ignore. In this study, we aimed to address the risk that H7N9 human influenza virus will provide internal genes to H9N2 AIV.AY253750.1-AY253756.1, AY743216.1) isolated from chicken in China was conserved at our laboratory. A/Shanghai/4664T/2013(SH/4664) H7N9 virus (Genbank accession number: KC853225.1-KC853232.1) was conserved at a Biosafety Level 3 (BSL3) lab at Shanghai Public Clinical Center. For the generation of recombinant virus by reverse genetics, the cDNA of genes from CK/F98 or SH/4664 was PCR-amplified and inserted into pHW2000 vector through BsmBI cutting sites. Viruses were rescued by using an eight-plasmid reverse genetics system A/Chicken/Shanghai/F/98 (CK/F98) H9N2 virus (Genbank accession number: 6EID50 per 50 μl virus (eight mice per group). Additionally, five mice were inoculated with 50 μl PBS buffer, serving as negative controls. Among the experimental group, five mice were monitored daily for 14 days post-inoculation (dpi) for body weight and mortality. Mice with 30% body weight loss or more were considered dead and were euthanized. The other three mice were sacrificed at 4 dpi to determine virus titers in the lung, nasal turbinate, liver, spleen, kidney, and brain. For virus titration, tissues were weighed and homogenized using the TissueLyser II (Qiagen) to produce a concentration of 0.1 g/ml (wt/vol) homogenate in 0.01 M PBS. After centrifugation at 12,000 rpm for 10 min, 100-μl aliquots of the supernatants were collected, and a series of dilutions were made for inoculating 9-day-old embryonated eggs. Virus titers were subsequently determined by the Reed–Muench method.Six-week-old female BALB/c mice  were maintained under specific pathogen-free conditions at the animal facilities of Shanghai Public Health Clinical Center. All animal studies were performed in strict accordance with the Home Office regulations and were approved by the Shanghai Public Health Center Local Ethical Committee. The experiments involving live viruses were carried out in a BSL3 facility at Shanghai Public Clinical Center conforming to the institutional biosafety manual. For virus challenge, mice anesthetized with 5% pentobarbital were intranasally inoculated with a dose of 1 × 10A minigenome assay was used to measure the activity of reconstituted viral ribonucleoprotein (vRNP), as reported previously . BrieflyL-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK). Infected cells were further incubated at 37°C, 5% CO2 in a humidified incubator. Aliquots of cell-culture supernatants were collected at indicated time points post-infection and used for virus titration in MDCK cells.Confluent MDCK cells in a 25-cm flask were infected with the indicated viruses at a multiplicity of infection (MOI) of 0.001. The viral inoculums were removed after 1 h of adsorption at 37°C and washed three times with PBS, followed by the addition of serum-free Dulbecco’s Modified Eagle Medium supplemented with 1 μg/ml trypsin treated with t-test. When more than two groups were compared, one-way ANOVA was applied in combination with the post hoc Bonferroni test.All of the statistical analyses were performed using Prism 7 software . Data were expressed as mean ± SD, except for analysis of mouse body weight change, where mean ± SEM was used. For comparison between two groups, statistical significance was assessed using a two-tailed Student’s 6 EID50. All of the infected mice survived up to 14 days after virus challenge. Both viruses caused a slight weight decrease in the infected mice in the early period that lasted for 5 and 8 days for CK/F98-4664HA and CK/F98 virus, respectively. After that, the weight of the mice in both groups start to increase. CK/F98-4664HA infection had weaker weight effects than the parental virus CK/F98, including a higher nadir and faster rebound. Accordingly, compared to the weight before infection, the former group attained an average weight of approximately 5% higher at 14 dpi, whereas the CK/F98 group showed essentially no gain  virus on the background of H9N2 virus. To evaluate the pathogenecity of that virus, mice were intranasally infected, as described above. All of the CK/F98-4664HA/NA-infected mice survived at 14 dpi. As compared to the CF/F98 group, the CK/F98-4664HA/NA group displayed more rapid weight loss in the early stage, reaching the nadir at 4 dpi . In addi6 EID50/g, which was significantly higher than that of CK/F98-infected mice  by reverse genetics. This reassortant virus, designated as CK/F98-4664Int, was successfully generated and subsequently examined in a mouse infection model. Unlike the surface gene replacement viruses and parental virus CK/F98, CK/F98-4664Int caused drastic weight loss, reaching a nadir of 28% weight loss within 9 dpi . There wP < 0.001; 10 TCID50 in the supernatant at 24 hours post inoculation (h.p.i.), whereas there was no virus detectable in the supernatant of cells infected with the latter. The virus titer of CK/F98-4664Int-infected cells was 100–1000 times higher than that of CK/F98 infected cells at 48 and 72 h.p.i. (P < 0.001; We further analyzed the kinetics of replication of CK/F98-4664int versus parental CK/F98 on MDCK cells. MDCK cells were infected with each of the two viruses at an MOI of 0.001, and the virus titers in the supernatant were tested on MDCK cells at different numbers of hours post-infection. CK/F98-4664Int proliferated significantly faster than CK/F98, and the titer reached 3.5 logP < 0.001; To test the activities of vRNPs, minigenomes derived from H9N2 and H7N9 viruses were analyzed on 293T cells. The relative activity of vRNPs from H7N9 virus was about 7-fold higher than that of vRNPs from H9N2 virus (As one of most widely circulating influenza viruses in chickens , H9N2 isPrevious studies have suggested that H9N2 AIVs can act as either donors or recipients of genes when co-circulated with other virus strains . Here, win vivo pathogenicity was highly correlated with virus titers. Thus, the CK/F98-4664Int virus with internal genes entirely from the H7N9 strain combined with a couple of H9N2 surface genes yielded significantly higher viral titer in lung and nasal turbinate as compared to H9N2 CK/F98, in line with substantially increased mortality. On the contrary, either the CK/F98-4664HA or CK/F98-4664HA/NA virus, with HA or both HA and NA from H7N9 virus and retaining the CK/F98 internal gene constellation, showed a similar low level of viral replication to CK/F98 in infected animal and consequently remained low in pathogenicity. That CK/F98-4664Int virus grew better than CK/F98 virus in cultured MDCK cells further indicated that the replication difference could be attributed at least in part to direct functioning of the internal gene constellation. This notion was further evidenced by the higher activity of H7N9-derived vRNP when compared to that of H9N2-derived vRNP in minigenome assay. Thus, the subunits of viral polymerase, PA, PB1, and PB2, along with NP, are strong candidates for virulence determinants distinguishing H7N9 from H9N2. However, we cannot exclude the candidacy of the other two internal gene-encoding segments, the NS and M segments, which respectively produced two viral proteins, namely NS1 and NS2 and M1 and M2, by alternative splicing. Indeed, previous studies have suggested the involvement of NS and M genes in the emergence of reassortants between H9N2 and H5N1 viruses. NS1 protein, which is only expressed in infected cells, is particularly of interest, given its critical role in evading host innate immunity. Thus, it will be important for future studies to pinpoint which component or combination of internal genes from H7N9 is responsible for the conversion of H9N2 to a mammalian-adapted virus on the advent of H7N9-H9N2 reassortment.Our animal studies revealed that the In summary, our study demonstrated the genetic compatibility between human-origin H7N9 and avian-origin H9N2 virus and revealed gain of the internal gene cassette from human H7N9 virus by reassortment as a potential path by which for avian H9N2 to evolve to become a causative agent in mammals. This is in agreement with the concept that the zoonotic potential of an influenza virus is determined by two critical factors: the entry and spread capability associated with HA and cellular permissiveness dictated by internal gene products. A future comparison between CK/F98-4664Int and parental 4664 virus will be needed to validate that the internal gene cassette is fully responsible for the difference between the replication capabilities of H9N2 CK/F98 and H7N9 SH/4664 strains in mammalian cells. It will also be of great interest to determine the detailed mechanism by which gene acquirement shapes the virulence of H9N2 virus. We propose that it is important to closely monitor for the emergence of H9N2 reassortant virus containing a human-derived H7N9 internal gene cassette, which might pose a serious threat to human health.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Shanghai Public Health Clinical Center.MZ, JX, XZ, and ZL designed the experiments, analyzed the data, and wrote the manuscript. MZ, HC, QT, CZ, SY, LJ, XL, and DF performed the experiments. All authors reviewed and revised the first and final drafts of this 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.