Saccharomyces cerevisiae are classified into three types based on differences in the structure of the polar head group, and the compositions and quantities of complex sphingolipids in biomembranes are tightly regulated. In this study, we found that the accumulation of inositol phosphorylceramides (IPCs) due to a defect of mannosylinositol phosphorylceramide biosynthesis (sur1∆ csh1∆), i.e., disruption of the balance of the composition of complex sphingolipids, causes hypersensitivity to low pH conditions (pH 4.0–2.5). Furthermore, screening of suppressor mutations that confer low pH resistance to sur1∆ csh1∆ cells revealed that a change in ergosterol homeostasis at plasma membranes can rescue the hypersensitivity, suggesting the functional relationship between complex sphingolipids and ergosterol under low pH conditions. Under low pH conditions, wild-type yeast cells exhibited decreases in IPC levels, and forced enhancement of the biosynthesis of IPCs causes low pH hypersensitivity. Thus, it was suggested that the accumulation of IPCs is detrimental to yeast under low pH conditions, and downregulation of IPC levels is one of the adaptation mechanisms for low pH conditions.All organisms have stress response systems to protect themselves from various environmental stresses, and regulation of membrane lipids is thought to play an important role in acquirement of stress tolerance. Complex sphingolipids in the yeast  Sphingolipids, one of the major components of biomembranes of eukaryotic cells, are thought to play important roles in acquirement of resistance against some environmental stresses. For example, in the budding yeast Saccharomyces cerevisiae, the biosynthesis of sphingolipids is upregulated during heat stress, which is essential for the acquirement of thermoresistance2. Complex sphingolipids, which each comprise a polar head group and a ceramide (Cer) composed of a fatty acid and a LCB, form lipid microdomains together with sterols in eukaryotic biomembranes4. Several proteins involved in acquirement of stress resistance are associated with lipid microdomains in yeast, and thus disruption of the structure of lipid microdomains, which is induced by inhibition of the biosynthesis of complex sphingolipids and sterols, causes mislocalization and dysregulation of these proteins, and can impair the resistance to several stresses7. In addition, it is suggested that complex sphingolipids also contribute to maintenance of the physical properties of plasma membranes including membrane fluidity and thickness9, which may also influence stress resistance.All organisms are exposed to various stresses caused by changing environmental factors, such as temperature, osmotic pressure, pH, nutritional status and chemicals, and thus have various stress response systems to protect themselvesS. cerevisiae, according to differences in the polar head group structure, complex sphingolipids are classified into three types, inositol phosphorylceramide (IPC), mannosylinositol phosphorylceramide (MIPC), and mannosyldiinositol phosphorylceramide (M(IP)2C)3  or their regulatory subunit (CSG2) results in complete loss or drastic reduction of MIPCs and M(IP)2Cs, and accumulation of IPCs11. A defect of MIPC biosynthesis has deleterious effects under some stressful conditions. For instance, MIPC biosynthesis-deficient mutants exhibit hypersensitivity to exogenous Ca2+ and reduction of the rate of cell survival under nitrogen starvation14. The growth defects caused by these stressful conditions are suppressed by inhibition of biosynthesis of hydroxylated IPCs, suggesting that these phenotypes are not due to loss of MIPCs themselves but to the accumulation of hydroxylated IPCs15. sur1∆ csh1∆ cells also exhibit impairment of cell wall integrity; however, the cell wall-defective phenotype is caused by loss of MIPCs themselves but not by accumulation of IPCs16. The biosynthesis of MIPCs is also related with the function and metabolism of glycerophospholipids; that is, the double defect of biosynthesis of phosphatidylserine and MIPC causes a strong growth defect and impairment of a specific vesicular trafficking pathway18. In addition, MIPCs are involved in maintenance of the asymmetry of glycerophospholipids at plasma membranes through regulation of glycerophospholipid flippases-regulating kinase, Fpk119.In C)3 Fig. A,B. In aS. cerevisiae, C-type complex sphingolipids are the most predominant species, and the levels of IPCs and M(IP)2Cs are much higher than that of MIPCs10. However, several lines of evidence have indicated that the compositions and quantities of complex sphingolipids in yeast biomembranes change under certain stressful conditions. S. cerevisiae and Zygosaccharomyces bailii exhibit increases in the IPC, MIPC and M(IP)2C levels in response to acetic acid stress20. Furthermore, the Target of Rapamycin (TOR) Complex 2 (TORC2)- and Ypk1-mediated signaling pathway, which upregulates sphingolipid biosynthesis, plays an important role in acquisition of resistance to acetic acid stress in S. cerevisiae21. Deletion of vacuolar H+-ATPase (V-ATPase), which causes a defect of vacuolar proton homeostasis, causes dramatic alteration of the complex sphingolipid composition including increases in MIPC levels, and it is suggested that the alteration is an adaptation mechanism against a defect of V-ATPase22. These results suggest that regulation of the compositions and quantities of complex sphingolipids in biomembranes is important for the adaptation of cells to certain stressful conditions.In In this study, we found that accumulation of IPCs due to a defect of MIPC biosynthesis causes a strong growth defect when the pH of the extracellular milieu become acidic. Furthermore, cellular IPC levels rapidly decreased under low pH conditions, and enhancement of the biosynthesis of IPCs caused low pH hypersensitivity, suggesting that decreases in IPC levels are one of the adaptation mechanisms for acquisition of low pH resistance. These findings provide new information as to the importance of regulation of complex sphingolipid levels under stressful conditions.SUR2, SCS7, IPT1, SUR1, and CSH1 2C synthase did not confer the hypersensitivity  , they can effectively be incorporated into cells27. Thus, we firstly examined the effects of the organic acids at pH 4.0 . Since sur1∆ csh1∆ cells exhibited delay of cell growth at pH 4.0 even in the absence of organic acids, it is possible that low pH conditions and the presence of organic acids had a synthetic effect on the growth of sur1∆ csh1∆ cells, so we next examined the effect of the organic acids at pH 5.5. It was reported that intracellular acidification occurs on the addition of acetic acid at pH 5.5; however, the effect of acetic acid at pH 5.5 is weaker than that at the lower pH28. Although at pH 5.5 a five-fold concentration of acetic acid or sorbic acid was required for induction of the growth inhibition observed at pH 4.0, no difference in the growth inhibition pattern was observed between wild-type and sur1∆ csh1∆ cells .The intracellular pH is tightly maintained around neutral, but is slightly affected by the extracellular pH+-ATPase (Pma1)29. Thus, we next investigated the effect of repression of Pma1 expression. Replacement of the promoter region of chromosomal PMA1 with a ADH promoter (ADHp-PMA1) resulted in a decrease in the expression level of Pma1 . As expected, in both sur1∆ csh1∆ and SUR1 CSH1 cells, the repression of Pma1 expression enhanced the growth inhibition caused by the addition of 200 or 30 mM acetic acid at pH 5.5 or 4.0 , probably due to a delay of exclusion of the intracellularly accumulated protons. In contrast, the repression of Pma1 expression did not enhance the slow growth of SUR1- and CSH1-deleted cells at pH 4.0 and 3.7 . Thus, it was suggested that the low pH hypersensitivity of sur1∆ csh1∆ cells is caused by the change of pH in the extracellular milieu but not by intracellular acidification. To monitor intracellular acidification, a fusion protein (pHluorin-mRuby2) that contains super-ecliptic pHluorin (a pH-sensitive GFP variation) and mRuby2 (a pH-stable RFP) was used31. As shown in Fig. -ecliptic pHluorin fluorescence, but not in mRuby2 fluorescence, was observed, implying intracellular acidification due to the treatment with acetic acid. Figure -ecliptic pHluorin to mRuby2 fluorescence in wild-type and sur1∆ csh1∆ cells under various conditions. At pH 4.0, the effect of 30 mM acetic acid on intracellular acidification of sur1∆ csh1∆ cells was more severe than that of wild-type cells . On the other hand, incubation of wild-type and sur1∆ csh1∆ cells at pH 3.7 did not have a notable effect on the intracellular acidification  , a culture condition used for organic acid sensitivity   that rescue the low pH hypersensitivity of sur1∆ csh1∆ cells , which suppress the accumulation of IPC-C due to a defect of MIPC biosynthesis15 38 was used, and doxycycline (Dox) was used for repression of expression by the tetracycline-regulatable promoter. Repression of expression of LIP1 also improved the growth of SUR1- and CSH1-deleted cells at pH 3.5 and 3.0 (Dox-treated sur1∆ csh1∆ versus Dox-treated tet-LIP1 sur1∆ csh1∆ cells)   in wild-type cells incubated at pH 2.5 decreased to approximately 75% of that at pH 5.5 42, Kei1 43, Orm1 and Orm2 (negative regulators of SPT)44, and Lag1 and Lac1 (Cer synthases)45, all of which are involved in regulation of the IPC and Cer levels in cells. To detect the expression of these proteins, the chromosomal genes were tagged with 6xHA at the C-terminus or 3xFLAG at the N-terminus in wild-type cells40 AUR1 was overexpressed under the control of a constitutive TEF promoter (TEFp-AUR1)46. The overexpression of Aur1 by the TEF promoter was confirmed by tagging with 6xHA at the C-terminus of Aur1 in cells cultured at both pH 5.5 and 2.5 , and the overexpression of AUR1 enhanced the increases in the IPC levels in orm1∆ orm2∆ cells (orm1∆ orm2∆ versus TEFp-AUR1 orm1∆ orm2∆ cells at pH 2.5)  Fig. B. In con5.5 Fig. B, suggesur1 Fig. C. Under lls Fig. D. Thus, LAM1 and SIP3, which were found in the transposon mutagenesis screening  and plasma membranes, are involved in retrograde trafficking of sterols between the two membranes, and lam1∆, sip3∆, and ysp2∆ cells exhibit hypersensitivity to the polyene antifungal amphotericin B (AmB), which exerts cytotoxicity by binding to ergosterol at plasma membranes, suggesting that these mutations change the distribution pattern of ergosterol at plasma membranes48    Fig. F. In conls) Fig. F. These 53, and degradation of Orm2 is controlled through the endosome and Golgi-associated degradation pathway (EGAD)54. It should be noted that PMR1 encoding P-type Ca2+/Mn2+-ATPase mainly located in the Golgi apparatus, which was found in our transposon mutagenesis screening 58 were used , deletion of ERG6 or ERG2 confers strong resistance to nystatin that binds to ergosterol within plasma membranes and exerts cytotoxicity63, which may suggest that the nystatin-accessible sterol content at plasma membranes is decreased on the deletion of ERG6 or ERG2. Thus, it is also possible that a change in the distribution pattern of sterols at plasma membranes on the deletion of ERG6 or ERG2 affects the pH sensitivity of SUR1- and CSH1-deleted cells. This is consistent with the fact that the deletion of LAM1, SIP3, and YSP2 that causes hypersensitivity to AmB confers the resistance to low pH conditions in SUR1- and CSH1-deleted cells 2Cs are especially enriched in plasma membranes, whereas IPCs are widely distributed in plasma membranes, vacuoles, and the Golgi65. Thus, it is possible that the deletion of SUR1 and CSH1 affects the plasma membrane distribution of complex sphingolipids, and consequently decreases plasma-membrane permeability under low pH conditions.Several groups indicated that ergosterol plays important roles in maintenance of the rigidity of plasma membranes including maintenance of membrane permeability and fluidityons Fig. B. At preSH1 Fig. E. These sur1∆ csh1∆ sur2∆ and sur1∆ csh1∆ sur2∆ scs7∆ cells, but not in sur1∆ csh1∆, or scs7∆ cells8. This phenomenon seems not to be directly related to our results because the low pH hypersensitivity of sur1∆ csh1∆ cells is suppressed by the deletion of SUR2 or SCS7 . However, further detailed evaluation of various plasma membrane properties of sur1∆ csh1∆ cells cultured under low pH conditions is required in the future.Uemura et al. reported that, under neutral pH conditions, the lateral diffusion speed of enhanced GFP (EGFP)-tagged Hxt1, plasma membrane-localized hexose transporter 1, is decreased in CS7 Fig. A. In addSUR2, LAM1, SIP3, and PMR1, LCB4 and XRN1 were also found in the transposon mutagenesis screening . Thus, some relevance between the LCB catabolic pathway and the low pH sensitivity was suggested. Recently, it was reported that Xrn1, a 5′-3′ exonulease involved in mRNA degradation in the cytosol, regulates translation of a specific group of transcripts encoding membrane proteins67. Thus, dysregulation of the expression of some membrane protein(s) due to the deletion of XRN1 may confer resistance to low pH conditions in sur1∆ csh1∆ cells.Besides In summary, the present study indicated the importance of proper regulation of the IPC levels for acquirement of resistance to low extracellular pH. Further detailed analyses of this molecular mechanism will provide a novel insight into the relationship between regulation of complex sphingolipids and environmental stress responses.S. cerevisiae strains used are listed in Supplementary Table kanMX4 marker from a genome from a yeast knockout library or the pFA6a-kanMX4 vector, the hphNT1 marker from the pFA6a-hphNT1 vector68, the natMX4 marker from the p4339 vector (pCRII-TOPO::natMX4)69, the URA3 marker from pRS406, or the LEU2 marker from the pRS405 vector70. For overexpression of genes by a strong and constitutive TEF promoter, a TEF promoter cassette containing the natNT2 marker from pYM-N19 was introduced immediately upstream of the initiator ATG of the chromosomal gene, as described previously46. To generate cells expressing PMA1 under the control of ADH promoter, an ADH promoter cassette containing the kanMX4 marker from pYM-N18 was introduced immediately upstream of the initiator ATG of chromosomal PMA1, as described previously46. To tag the N-terminus of Lag1 or Lac1 with three copies of the FLAG epitope (3xFLAG), a 3xFLAG tag was introduced immediately downstream of the initiator ATG of chromosomal LAG1 or LAC1 without changing the potential promoter region according to the method described previously71. A DNA fragment of the LAG1 or LAC1 ORF without the initiator ATG was amplified by PCR using LAG1-3xFLAG-HindIII-F and LAG1-3xFLAG-BamHI-R (for LAG1), or LAC1-3xFLAG-HindIII-F and LAC1-3xFLAG-BamHI-R (for LAC1), and yeast genomic DNA as a template . A DNA fragment of 3xFLAG-LAG1 or 3xFLAG-LAC1 was amplified by PCR using 3xFLAG-LAG1-F1 and LAG1Hyg-R (for LAG1) or 3xFLAG-LAC1-F1 and LAC1Hyg-R (for LAC1), and the p3xFLAG-CMV-7.1 vector containing the LAG1 or LAC1 ORF as a template. A DNA fragment containing the hphNTI marker was amplified by PCR using LAG1Hyg-F and LAG1-S2 (for LAG1) or LAC1Hyg-F and LAC1-S2 (for LAC1), and pYM1646 as a template. These two DNA fragments were combined by PCR, and the resultant DNA was used to transform cells. The cells were cultured in YPD medium ), or SC (synthetic complete) medium  and 2% glucose) containing nutritional supplements. Buffered medium was prepared by the addition of 50 mM MES and 50 mM MOPS (for pH 5.5), 100 mM Gly-HCl , and 100 mM phospholic acid-sodium dihydrogen phosphate (for pH 2.5). Medium adjusted to pH 2.5 was also prepared by the addition of HCl.The SUR1-6xHA, and its 5′- and 3′-untranslated regions  was constructed as described below. A DNA fragment containing SUR1-6xHA was amplified by PCR using SUR1-6HA-SacI-S and SUR1-6HA-KpnI-A, and genomic DNA of the BY4741 strain expressing SUR1-6xHA (MTY1315)22 as a template. The fragment obtained was subcloned into pRS416. The DNA sequence was verified with an ABI PRISM 3,100 genetic analyzer . pKL06, a plasmid expressing super ecliptic pHluorin-mRuby2 fusion protein, was obtained from Addgene .A single-copy plasmid (pRS416) containing A600 units/ml. All plates were incubated at 30 °C and photographed after 1 or 2 days.Cells were cultured overnight in YPD or SC-Ura medium at 30 °C, and then spotted onto agar plates containing YPD medium buffered to pH 5.5, 4.0, 3.5, 3.0, or 2.5 in tenfold serial dilutions starting with a density of 0.7 A600 units/ml) in fresh YPD medium, and then incubated for 3 h at 30 °C. The cells were resuspended in fresh YPD medium buffered to pH 5.5, 3.7, or 2.5 to 0.7 A600 units/ml, and then incubated for 1, 2, 3, or 5 h at 30 °C. Then equal numbers of cells, as determined from A600, were plated onto YPD plates, and incubated for 2 days at 30 °C, and then the numbers of colonies on the plates were determined. Relative colony forming units (CFU) were calculated as follows: relative CFU (%) = (colony numbers of each sample/colony numbers of wild-type cells incubated at pH 5.5) × 100.Cells were cultured overnight in YPD medium, diluted  and Dr. Akio Kihara . The library was digested with NotI, and the resultant DNA fragments were transformed into sur1∆ csh1∆ cells. The transformed cells were plated on SC plates lacking leucine buffered to pH 3.7, on which a strong growth defect of sur1∆ csh1∆ cells (but not wild-type cells) was observed, and then incubated for 3 days. The mutants showing resistance to the low pH conditions were isolated, and the sites of transposon insertion were identified as described previously73.Mutagenesis by random insertion of the transposon mTn-S. cerevisiae as described previously74 with minor modifications. Briefly, cells (3 A600 U (for detection of complex sphingolipids or glycerophospholipids), 5 A600 U (for detection of Cer-C), or 1.5 A600 U (for detection of ergosterol)) were suspended in 350 µl of ethanol/water/diethyl ether/pyridine/15 M ammonia , and then incubated at 65 °C for 15 min. The lipid extracts were centrifuged at 10,000g for 1 min and then extracted once more in the same manner. The resulting supernatants were dried. For analysis of complex sphingolipids and Cer-C, the lipid extracts were dissolved in 130 µl monomethylamine (40% methanol solution)/water , incubated for 1 h at 53 °C , and then dried. The lipids were suspended in 60 µl of chloroform/methanol/water , and then separated on Silica Gel 60 TLC plates  with chloroform/methanol/4.2 M ammonia  (for detection of complex sphingolipids), chloroform/methanol/acetic acid  (for detection of Cer), or hexane/diethyl ether/acetic acid  (for detection of ergosterol) as the solvent system. Glycerophospholipids were separated on a LK5 silica gel 150A TLC plate , which had been pre-washed in chloroform/methanol  and treated with 2% boric acid in ethanol. The TLC plates were developed two times with chloroform/ethanol/water/triethylamine 75. The TLC plates were sprayed with 10% copper sulphate in 8% orthophosphoric acid and then heated at 180 °C to visualize lipids. Identification of each complex sphingolipid, Cer-C, glycerophospholipid, and ergosterol was performed as described in previous papers75. The relative amounts of lipids were determined with ImageJ software .Lipids were extracted from 76 with some modifications. Yeast cells (2 A600 U) were collected by centrifugation and then washed with water. After the addition of 1 nmol of sphingosine (d18:1)  as an internal standard, lipids were extracted as described above. For acid hydrolysis, the lipids were dissolved in 500 μl of methanol/water  containing 1 M HCl, and then heated at 80 °C for 18 h. After the addition of 500 μl of 3 M NH4OH, the hydrolyzed LCBs were extracted two times with 500 μl of chloroform. The combined chloroform extracts were washed with 300 μl of 3 M NH4OH three times, dried, and then dissolved in 120 μl of ethanol by heating at 67 °C for 25 min. The lipid solution was mixed with 15 μl of OPA  reagent , followed by incubation at room temperature for 30 min. Samples were centrifuged at 10,000g for 5 min, and the resulting supernatants were incubated overnight at room temperature. Then, the samples were resolved by HPLC on a pre-packed C18 reversed-phase column  using an isocratic eluent composition of acetonitrile/water  and a flow rate of 1 ml/min. The OPA derivatives were monitored at an excitation wavelength of 340 nm and an emission wavelength of 455 nm. The areas of peaks of LCBs  and dihydrosphingosine ) were determined using sphingosine as an internal standard.HPLC analysis of sphingolipids was performed as described previously17 with some modifications. For protein extraction, yeast cells grown in YPD medium were collected by centrifugation, washed with water, and then resuspended in 100 µl of 0.2 N NaOH containing 0.5% 2-mercaptoethanol. The suspension was incubated on ice for 15 min. One ml of ice-cold acetone was added to the suspension, followed by incubation for 30 min at -25 °C, and then the proteins were precipitated by centrifugation for 10 min at 10,000g. The pellet was resuspended in 100 µl of SDS sample buffer . The suspension was mixed well, heated for 3 min at 95 °C, and then centrifuged for 2 min at 10,000g. Then the supernatant was separated by SDS-PAGE according to the method of Laemmli77. For Western blotting, anti-Pma1 , anti-HA , anti-FLAG , and anti-Pgk1  were used as primary antibodies. Horseradish peroxidase-conjugated anti-mouse IgG (Thermo Fisher Scientific) was used as the secondary antibody. The relative intensity of each protein band was determined with ImageJ software.Protein extraction, SDS-PAGE, and Western blotting were performed as described elsewhere39.Phos-tag SDS-PAGE was performed as described elsewhereA600 units/ml) in fresh YPD medium, and then incubated for 3 h at 30 °C. The cells were resuspended in fresh YPD medium with or without 30 or 150 mM acetic acid, which was buffered to pH 4.0, 5.5, or 3.7, to 0.7 A600 units/ml and then incubated for 1 h at 30 °C. The cells were viewed under a fluorescence microscope. The fluorescence intensities of super-ecliptic pHluorin and mRuby2 excited at 475 nm and 555 nm, respectively, were quantified with ImageJ software.Cells harboring pKL06 were cultured overnight in SC-Leu medium (pH 6.0), diluted (1 A600 units/ml) in fresh YPD medium, and then incubated for 3 h at 30 °C. The cells were resuspended in fresh YPD medium buffered to pH 5.5 or 3.7 to 0.7 A600 units/ml, and then incubated for 1.5 h at 30 °C. Then, 10 µM rhodamine 6G was added to the culture medium, followed by incubation for 30 min at 30 °C. The cells were collected by centrifugation, washed two times with water, and then viewed under a fluorescence microscope. The fluorescence intensity was quantified with ImageJ software.Cells were cultured overnight in YPD medium, diluted


The as-deposited films were amorphous and showed low electrical conductivity. The films became electrically well-conducting and crystallized into the an anatase structure upon reductive post-deposition annealing at 600 °C in an H2 atmosphere for 30 min. It was shown that the Ti0.72Nb0.28O2/p+-Si heterojunction fabricated on low resistivity silicon (10−3 Ω cm) had linear current–voltage characteristic with a specific contact resistivity as low as 23 mΩ·cm2. As the resistance dependence on temperature revealed, the current across the Ti0.72Nb0.28O2/p+-Si heterojunction was mainly determined by the band-to-band charge carrier tunneling through the junction.Niobium-doped titanium dioxide (Ti However2 thin films grown by atomic layer deposition (ALD) on crystalline silicon substrates were studied recently [2 and silicon substrate demonstrating nonohmic and asymmetric current–voltage characteristics. Usually, TiO2 is treated as an n-type semiconductor with a wide bandgap reaching 3.4 eV, 3.2 eV, 3.02 eV and 2.96 eV for amorphous, anatase, rutile and brookite phases, respectively [2 has a high resistivity and may serve as an insulator for capacitors [2 films can be changed by variations of oxygen concentration during the TiO2 reduction [2 deficiency creates defects such as oxygen vacancies, titanium vacancies, and Ti3+ and Ti4+ interstitials, which may act either as acceptors or donors of electrons [2 is an amphoteric semiconductor, therefore creation of high conductivity nondoped TiO2 films is hindered.The electrical properties of TiOrecently . It was ectively ,18. Therpacitors ,20. The eduction ,18. The lectrons ,22. TiO22/p-Si contact. Furubayashi et al. showed that Nb-doped anatase TiO2 film is an optically transparent electrically conducting oxide [2 films with a Nb concentration exceeding 6% was less than 2.3 × 10−4 Ω·cm at room temperature. Furthermore, Ti1−xNbxO2 films with x ≥ 0.01 showed a metallic behavior. This paper deals with details of fabrication of a highly conducting TiO2/p-Si heterojunction. Transparent TiO2 films were grown by ALD and doped with Nb.High conductivity semiconductors are needed to produce low resistivity TiOng oxide . The res1−xNbxO2 (mixed titanium niobium oxide) layers were formed on glass, low resistivity +p-type and low conductivity p-type silicon substrates, using a “Fiji F200” atomic layer deposition reactor . A modular ALD system was used for layer formation in a moderate vacuum. First, glass and silicon substrates  were cleaned in ethanol and acetone in an ultrasonic bath for 20 min. Then the silicon surface was thermally oxidized in a quartz tube furnace “SDO-125/3“  at 1150 °C in air for 3 h. The thickness of the silicon oxide layer was measured by a profilometer “Dektak 6M” ; it was 150 nm. Round 100-µm diameter holes were formed in SiO2 by means of a photolithography technique. Thin Ti1−xNbxO2 layers were deposited using tetrakis dimethylamido titanium  and niobium ethoxide (Nb(OEt)5, 99.9%, STREM Chemicals Inc.) as precursors for titanium and niobium oxides. Deionized water was used as an oxygen source for both processes. The reactions for both processes are presented below.Thin Ti2:Partial surface reactions for TiO2:Full reaction for TiO2O5:Partial surface reactions for Nb2O5:Full reaction for Nb−2 mbar before the deposition process. The substrates and the reaction chamber were heated up to 170 °C. A constant flow of 100 sccm of pass-thru and 40 sccm of carrier gas (argon) was used during the deposition process. This kept the reaction chamber at ~0.18 mbar working pressure. To reach the desired vapor pressure, TDMAT and Nb(OEt)5 were heated up to 80 °C and 170 °C, respectively. According to the authors of the paper [1−xNbxO2 films deposited at temperatures around 170 °C have maximum electrical conductivity. Deionized water, which was used as an oxidizer, was kept at room temperature. The mixed oxide was formed by inserting a monolayer of niobium oxide after a few consecutive monolayers of titanium oxide (number of TiO2 monolayers was selected depending on desired Ti:Nb ratio) and the process was repeated until the desired thickness was achieved. Fabrication of every monolayer consisted of four steps: precursor pulse/purge/water pulse/purge. Timings used for titanium oxide and niobium oxide were 0.2 s/10 s/0.06 s/5 s and 0.2 s/5 s/0.06 s/5 s, respectively. Four hundred monolayers were deposited to achieve an approximately 25 nm-thick coating.The reaction chamber was evacuated up to 3 × 10he paper , Ti1−xNb1−xNbxO2 layers were examined by a scanning electron microscope (SEM) “Helios NanoLab 650” equipped with energy dispersive X-ray spectrometer (EDX) “INCAEnergy” . Thin Film ID software (Oxford Instruments) was used to estimate the Ti/Nb ratio with a 3% relative error. A relatively low accelerating voltage (7 kV) was used to achieve higher surface sensitivity. For 5:1 titanium oxide and niobium oxide monolayers deposition the resulting atomic ratio was 72 at.% titanium and 28 at.% niobium. For 10:1 titanium oxide and niobium oxide monolayers deposition the ratio was 84 at.% Ti and 16 at.% Nb.Morphology and composition of Ti1−xNbxO2 layers was studied by X-ray diffraction (XRD) using SmartLab HR-XRD diffractometer  with an X-ray tube equipped with 9 kW Cu rotating anode. Grazing incidence diffraction geometry was used with the incidence angle of Cu Kα beam set to 0.5° which enabled investigation of thin films and reduced influence of the substrate. The resistivity of Ti1−xNbxO2 layers was measured using a four-point probe method.Crystallographic structure of Ti2 atmosphere for 30 min. 500 nm-thick aluminum layer was thermally evaporated using“VAKSIS PVD Vapor-5S_Th”  on the p-type Si immediately after its rear side treatment in HF to remove the unnecessary SiO2. To complete the heterojunction devices, ohmic contacts to the n-type TiO2 were fabricated by thermal evaporation of Ti:Au metal layers with respective thicknesses of 20 nm and: 500 nm onto a photo-resistive mask, and contact patterns were formed using the lift-off technique. Schematic cross-sections of the TiO2/p-Si heterojunction device and microphotograph of the contacts on the top of TiO2 are presented in The Si substrates with as-deposited films were divided into two parts, and one part was annealed in the tube furnace at 600 °C in H2/p-Si heterojunction were performed using a E5270B Precision IV Analyzer . The point-contact electrical resistance dependence on temperature was measured in a liquid nitrogen vapor atmosphere from 78 K up to 350 K. The temperature of the sample was controlled using K-type Nickel-Chromium/Nickel-Aluminum thermocouple .Measurements of direct current (DC) current–voltage characteristics of the point-contact TiO1−xNbxO2 films were measured in the 300–1300 nm wavelength range using AvaSpec ULS2048XL spectrometer and AvaLight-DH-S deuterium-halogen light source . A 50 ms integration time and an averaging of 100 measured spectra were used for the measurements.Optical transmission spectra of the Ti0.72Nb0.28O2 film before (as-deposited) and after annealing in an H2 atmosphere. It was seen that the as-deposited film had an amorphous structure (a broad feature with maxima at 2Θ angle of about 21.6°) along with a crystalline TiO2 of anatase structure. The lattice parameters of anatase tetragonal structure of the film (a = 0.3819 nm and c = 0.9541 nm) were increased in comparison to those presented in ICDD data base card #01-075-2545 (a = 0.3799 nm and c = 0.9509 nm). The increase in lattice parameters should be a result of insertion of Nb ions into crystalline lattice of anatase. After the annealing, the XRD peaks of anatase became sharper as a result of an increase in a crystallite size. The XRD pattern of the annealed film presented one additional peak at 2Θ angle of 27.17°, which could be attributed to niobian rutile Ti0.712Nb0.288O2 (#01-072-7371).2O5 were observed in the Nb-doped TiO2 thin film as indicated previously [During the annealing, the amorphous phase transformed into an anatase crystalline phase, as XRD measurements confirmed. No characteristic peaks of Nbeviously .0.72Nb0.28O2 films in H2 atmosphere also resulted in a substantial decrease of its electrical resistivity from 5.0 × 102 to 1.2 × 10−3 Ω cm. In spite of high conductivity, the annealed Ti0.72Nb0.28O2 film was highly transparent in the measured 400–1300 nm spectral region  characteristics of TiO0.72Nb0.28O2/p+-Si heterojunction device decreased as the sample was cooled down. Dependencies resistance-vs.-temperature for TiO1-xNbxO2/p-Si heterojunction devices with different content of x and formed on different Si substrates are depicted in 0.72Nb0.28O2/p+-Si heterojunction (substrate ρSi = 10−3 Ω cm) linearly increased with temperature. Such linear dependence was an inherent feature of the tunnel diode at small bias voltage.It is worth noting that the I–V characteristics were linear; it was another valuable property for the above-mentioned application. The resistance of such TiOp-n junction can be expressed as [A is a constant, cE and vE represent the conduction and the valence band edges, respectively, and k is the Boltzmann constant. Expression (7) shows that the resistance of a tunnel p-n junction at low bias was a linear function of temperature. The Hall effect measurements indicate that electron density in TiO0.72Nb0.28O2 is 3.5 × 1021 cm−3 and therefore it can be regarded as a degenerate semiconductor [+p-Si (ρ = 10−3 Ω cm) is also a degenerate semiconductor. The hole concentration determined from Hall effect measurements in +p-Si was 1.3 × 1020 cm−3. The energy band diagram of mutually heavily doped +n-TiO2/p+-Si heterojunction in equilibrium condition is shown in 2 conduction band could tunnel through the gap to the empty sites of the p+-Si valence band under a small forward bias. Linear dependence of the resistance on temperature supports the assumption that the interband tunneling current took place in the investigated TiO0.72Nb0.28O2/+p-Si heterojunction. The specific contact resistivity of TiO0.72Nb0.28O2/+p-Si heterojunction was 23 mΩ·cm2 at room temperature which was better than 30 mΩ·cm2 achieved in [In general, when the interband carrier tunneling takes place, the tunnel current across a essed as ,27:It = onductor ,28,29. Lt)I, the excess current (xI) and the diffusion current (dI). The diffusion current is responsible for the current rise under high forward biases [dI should be negligible in comparison with the tunnel current at low bias. The excess current at low bias was mainly determined by the multistep tunneling recombination process via surface states at the TiO2/+p-Si interface [sN ~ 7.0 × 1013 cm−2, at the TiO2/+p-Si interface resulting from significant lattice mismatch between the heterojunction components [2/+p-Si tunnel heterojunction. At reverse bias, every recombination center becomes a source of carrier generation, and high electrical conductivity can be reached by thermally generated carriers [0.72Nb0.28O2/p-Si heterojunction on low conductivity (ρSi = 1 Ω cm) p-type silicon substrate . As mentioned above, the as-deposited TiO0.84Nb0.16O2 film had an amorphous structure and therefore its conductivity was low. As a result, the I–V characteristic of the TiO0.84Nb0.16O2/p+-Si heterojunction device was nonohmic and asymmetric, as observed in other works [er works ,30.1-xNbxO2 thin films were ALD-deposited on p-type Si substrates. Reductive post-deposition annealing was required to crystallize amorphous titanium dioxide into the anatase structure and to increase its electrical conductivity. The current-voltage characteristic of the TiO0.72Nb0.28O2/p+-Si heterojunction device is found to be ohmic, and the junction resistance linearly depends on temperature. In this case the current across the heterojunction is mainly stipulated by the interband charge carrier tunneling. When the highly conductive titanium dioxide is deposited on low conductivity (ρSi = 1 Ω cm) p-Si substrate, the current across the TiO0.72Nb0.28O2/p-Si heterojunction is mainly determined by the multistep tunneling recombination process via the surface states. The contact resistivity of the TiO0.72Nb0.28O2/p-Si heterojunction is higher than that of the TiO0.72Nb0.28O2/p+-Si heterojunction. The formed titanium dioxide films also demonstrate excellent transparency with absorption less than 10% in the visible region. Therefore, the TiO0.72Nb0.28O2/p+-Si heterojunction could be a suitable candidate as transparent interconnection in 2-T perovskite/silicon tandem solar cells.Two different (x = 0.16 and x = 0.28) niobium composition containing heavily doped Ti


For SARS CoV, using an assumed UVC dose near the mid-range of measured values, D90 = 40 J/m2, 90% inactivation times T90 are estimated for exposure to midday 10:00–14:00 direct plus diffuse sunlight and for nearby locations in the shade (diffuse UVB only). For the assumed D90 = 40 J/m2 model applicable to SARS CoV viruses, calculated estimates show that near noon 11:00–13:00 clear-sky direct sunlight gives values of T90 < 90 min for mid-latitude sites between March and September and less than 60 min for many equatorial sites for 12 months of the year. Recent direct measurements of UVB sunlight inactivation of the SARS CoV-2 virus that causes COVID-19 show shorter T90 inactivation times less than 10 min depending on latitude, season, and hour. The equivalent UVC 254 nm D90 dose for SARS CoV-2 is estimated as 3.2 ± 0.7 J/m2 for viruses on a steel mesh surface and 6.5 ± 1.4 J/m2 for viruses in a growth medium. For SARS CoV-2 clear-sky T90 on a surface ranges from 4 min in the equatorial zone to less than 30 min in a geographic area forming a near circle with solar zenith angle < 60O centered on the subsolar point for local solar times from 09:00 to 15:00 h.UVB in sunlight, 290–315 nm, can inactivate SARS CoV and SARS CoV-2 viruses on surfaces and in the air. Laboratory exposure to ultraviolet irradiance in the UVC range inactivates many viruses and bacteria in times less than 30 min. Estimated UVB inactivation doses from sunlight in J/mThe online version of this article (10.1007/s11869-020-00927-2) contains supplementary material, which is available to authorized users. Onset of coronavirus induced diseases , 10 years later Middle East respiratory syndrome MERS , and starting in 2019, a new viral mutation, SARS-CoV-2, causing COVID-19, has promoted increased interest in methods of deactivating the virus on surfaces through chemical biocidal agents  needed to reduce the number of active virus particles by 90%. Measurements by Walker and Ko .Inactivation sensitivity to 254 nm UVC radiation is frequently measured in terms of the dose r and Ko  showed am2 Table  to S4. Tm2 Table , b, whice 2 Fig. . A measu/m2 Fig.  showed ui et al. , b estima et al.  on SARS   to estimate the time for 90% inactivation T90 for a large number of cities worldwide  for globally estimating the inactivation of SARS CoV by sunlight using satellite data, the action spectrum from Lytle and Sagripanti , and a nD90, items 1 to 6 in Table 2, but the probably more precise estimation is 3.7 mJ/cm2.” This corresponds to 106 J/m2 and 37 J/m2, respectively, the latter close to the nominal value of 40 J/m2 assumed here.Heßling et al.  discuss A(λ) for measurements made at 254 nm is not needed for estimating T90 as a function of solar UVB irradiance for their measurement conditions. In order to generalize the RS measurements, an estimate of the equivalent 254 nm D90 amount is obtained by matching their measurement conditions and results using the TUV radiative transfer calculations. The estimated 254 nm inactivation D90 of SARS CoV-2  me-2 Table  gives glλ ≤ 315 nm UVB range that reaches the Earth’s surface at significant intensity, a transfer function from 254 nm to UVB (290–315 nm), or action spectrum A(λ) is needed that is normalized to 1 at 254 nm , estimated cloud transmission CT, and absorbing aerosol transmission CA to derive a useful formulation for high-speed evaluation (Herman D) in J/m2 and inactivation time (T90) in minutes from UV solar radiation to achieve 90% inactivation relative to the D90 exposure at 254 nm.To estimate the effect of sunlight in the 290 ≤  nm Fig.   is valid for coronaviruses. The data in Table T90 for aerosolized virus particle in air or on surfaces is shorter than viruses in liquids. The T90 times for both SARS CoV and SARS CoV-2 on surfaces are given as a function of latitude and season using a uniform calculation method for all cases considered.For SARS CoV, estimates of UVB T90 for a coronavirus are (1) an estimate of the normalized action spectra A(λ) representing the relative efficiency for a wavelength λ compared to the much stronger inactivation rate at the UVC wavelength 254 nm; (2) a calculated estimate of the solar irradiance reaching the Earth’s surface as a function of solar zenith angle (θ = SZA), total column ozone amount (Ω = TCO3) over a specified site, fractional cloud transmission CT of UV irradiance using measured Lambert equivalent reflectivity (LER) of the scene, and fractional absorbing aerosol transmission (CA), all as a function of latitude ζ, longitude ϕ, altitude z, and day of the year (DOY). The same method is applied for SARS CoV-2 after calculating the equivalent 254 nm D90.Key components for estimating O × 1O latitude × longitude grid https://avdc.gsfc.nasa.gov/pub/tmp/OMI_Daily_O3_and_LER/. OMI data are filtered to remove data from bad detector pixels and for the known so-called row anomaly . OMI is a polar orbiting nadir and side viewing satellite instrument (2600-km-wide swath on the surface) providing near global coverage (nadir resolution field of view 13 km × 24 km) once per day from a 90-min polar orbit with an equator crossing time of approximately 13:30 local solar time (LST) .Additional data from the Deep Space Climate Observatory (DSCOVR) Earth Polychromatic Imaging Camera (EPIC) in orbit about the Earth-Sun Lagrange-1 point L2 nm) at the Earth’s surface F, calculated from the TUV model, is obtained for 100 ≤ Ω ≤ 600 Dobson Units (1 DU = 2.687 × 1016 molecules/cm2), for SZA range 0O ≤ θ ≤ 80O, and for an altitude range 0 ≤ z ≤ 8 km. The TUV spectral output is multiplied by a normalized action spectrum A(λ) and integrated over the non-zero range of F(λ)A(λ)  can be used as a table look up or converted to a very accurate functional fit (Online Resource 2: Eqns. S2 to S10), where the latter is much faster for computational purposes. The small error estimates for this type of functional fit are also given (Herman The TUV output n Herman .A(λ) , Ω = 325 DU, and θ = 30O. P = 0.0066 ± 0.0013 W/m2 as shown in Fig. P, ± 0.0013, arises from uncorrelated error estimates for A(λ). Figure FOA(λ) peaking near 305 nm. While the amount of clear-sky 305 nm solar irradiance at the ground is small compared to longer wavelengths, because of attenuation by ozone and Rayleigh scattering, it is significant for viral inactivation  for the Earth’s surface at sea level (z = 0) into a 90% inactivation time T90 as a function of SZA. Laboratory values of the logarithmic decay of viruses of starting number NO exposed to 254 nm UV light for exposure D (J/m2) are measured as a function of time to determine the slope k of the decay curve as a function of the survival fraction N/NO , it is an exposure representing 10% survival, N/NO = 0.1. More complicated two-slope inactivation models have been used when the inactivation vs. D show two log-linear slopes FO(λ) and the measured value of D90, the following formula  weighted irradiance (diffuse + direct) for θ < 40O  is 1.4 to 1.7 times T90. Figures D90 = 40 J/m2. For the UVB inactivation measurements on SARS CoV-2  .For UV irradiance in the vicinity of 305 nm, there is considerable scattered diffuse light caused by strong Rayleigh scattering causing a clear atmosphere in longer wavelengths Fig.  to appeaFor airborne virus particles the actinic flux or fluence is of interest, which is the sum of the upward and downward irradiance, increasing the total UVB exposure by about 5% near the surface of the Earth relative to the total downward irradiance. There is evidence that aerosolized airborne virus particles are more susceptible to UVC than samples measured in liquid Table .T90 to use satellite data on a global basis for daily calculations as a function of latitude and longitude, an efficient representation  and R(θ) are numerical fitting coefficients defined below  is an improved version of the radiation amplification factor.To efficiently expand the calculation of tion Eq.  is needeform Eq.  accurateΩ Herman , and altζ, ϕ) is not explicitly indicated:CTcloud transmission fraction between 0 and 1CAaerosol transmission fraction between 0 and 1H(z)topography height factor relative to sea level between 0 and 8 kmDScorrection factor for the distance of the Earth from the sun relative to 1 AUHere, the dependence on latitude and longitude  of 1.7ΔΩ for low θ and 2.4ΔΩ for θ = 80O  to be determined for any location  as a function of DOY (January 2005 to December 2019). Only the 2019 data are shown. T90 is shown for 12 locations representing major cities in the USA, Asia, Australia, Europe, and South America, some strongly affected by COVID-19. A total of 190 cities and 4 land sites are listed , and the number of months Nm (columns 5 and 7 ) in 2019 where noontime <T90> is 120 min or less. These averages include the effects of daily cloud and aerosol cover.Applying the radiative transfer fitting equations to the nearly complete global coverage afforded by OMI’s field of view enables O and 40O  than for Los Angeles (April to September). The scatter in the points is mainly due to clouds causing time-varying CT.For cities at mid-latitudes between 3040O Fig. , there iD90 = 40 J/m2 calculation is applied to four European cities , the periods of 60–80 min inactivation are reduced to where London, England’s and Berlin, Germany’s shortest T90 inactivation times are close to 70–90 min and quickly increase as the local time differs from noon or the month differs from the June solstice (θ increasing). Two other cities, Rome, Italy and Madrid, Spain, have T90 values similar to New York City and have minimum T90 of 60 to 70 min in June, July, and August.When the ies Fig.   it is 2.6 months per 10O of latitude away from the equatorial zone, although there are fewer points to accurately determine the SH slope.Figure Min <T90> 12:00 are less than 50 min  for April 9, 2020 with the superimposed outer white circle representing the calculated 65-min Min <T90> Hour and the inner white circle the 50-min Min <T90> Hour. Additional EPIC images are obtained approximately every 65 min (NH summer) to 108 min (NH winter) as the Earth rotates.Using the SARS CoV results from Figs. θ = 0O with increasing θ radially in all directions away from the subsolar point. Since April 9 is 18 days after the March equinox, the subsolar point is about 7.6O north of the equator. At 05:32:39 GMT, the clear-sky T90(65 min) circle covers all of China and extends into southern Russia, Japan, and Korea. At 10:56:47 GMT, the outer circle covers southern Europe. Approximately 7 h later at 18:08:59 GMT, as the Earth rotates 15O longitude per hour, EPIC is viewing North and South America where the 65-min Min <T90> Hour circle extends as far north as the border of Canada (about 48O N) and as far south as the northern border of Chile (about 18O S).The images Figs.  and 12 cMin <T90> Hour circle extending to the middle of Africa and well south of Cape Town, South Africa. In contrast, on June 2, 2019 at 11:59:12 GMT, the subsolar point is near 23O N and the 65-min Min <T90> Hour circle extends as far north as England and Germany. As the Earth rotates to later GMT, the 65-min Min <T90> Hour circle would contain all of South America and later, on the next calendar day, Australia and New Zealand.Figure O/hour). For D90 = 40 J/m2, the circles at their widest point are about 45O or 3 h. This means that inactivation times are less than 65 min for Δt = 09:00 to 15:00 h for latitudes of the subsolar point in the equatorial region, ± 23.45O. The longitudinal 65-min inactivation time-interval Δt decreases with increasing latitudinal distance away from the subsolar latitude δ, Δt ≈ 3cos(θ - δ) hours for |θ - δ| ≤ 90Ο, where δ = solar declination angle. For the calculated RS SARS CoV-2 D90 = 3.2 J/m2, longitudinal 20-min T90 inactivation time-interval Δt ≈ 4cos(θ - δ) hours for |θ - δ| ≤ 90Ο.Since the EPIC images are synoptic, the longitudes are equivalent to local solar time of day  corresponds to Eq.T is the exposure time (seconds) and D is the UVB dose (J/m2), where D = PT. In RS, P is not weighted with an action spectrum. The slope k  vs. the UVB dose D (J/m2) for three different exposure rates. When P is small, the determination of k from Ln(N/No) is less certain.A recent study by Ratnesar-Shumate et al.  performeodel Eq.  is appliD is plotted against Ln(N/No) for three different UVB irradiances. Of these, the slope k for P = 0.3 W/m2 has the largest uncertainty k(0.3) = − 0.00803 ± 0.00221 compared to k(1.6) = − 0.00305 ± 0.00031. The result is T90(1.6) = 7.9 min, T90(0.7) = 9.5 min and T90(0.3) = 15.9 min, all short inactivation times. Note that T999 values (99.9%) are four times T90, which are still less than 1 h. These k estimates are slightly different than those in RS , but do not significantly affect the current analysis and conclusions. The differences probably arise from different weighting of data points when RS’s exposure times are almost zero  applies, then the 254 nm UVC D90 equivalent is approximately DE90 = 3.2 J/m2 , since the value of D90 was adjusted to match the simulated UVB amounts in RS. RS measurements made in simulated sunlight give an error estimate of ± 10%  = 16.2 min and T90(0.7) = 19.7 min compared to RS’s values of 14.3 and 17.6. As above, using the action spectrum A(λ) gives a 254 nm UVC D90 equivalent of approximately 6.5 ± 1.4 J/m2 /ln(0.37) = 6.9 J/m2, larger than the estimate for D90 = 3.2 J/m2 given above. The methods for obtaining D90 are entirely different. The method used here relies on finding a value of 254 nm D90 that yields approximately the same inactivation time, 6.8 min, as RS finds for midday during the summer solstice at 40O N latitude using the same TUV radiative transfer code. Sagripanti and Lytle  A(λ)d λ in Eq. −2 min−1).” The noontime F(λ) in Eq The values of nd Lytle  are 3 toT90 are used instead of the values in Figs. D90 = 3.2 J/m2 estimated here. The value D90 = 3.2 J/m2 is approximately 12.5 times smaller than the UVC D90 = 40 J/m2 for the SARS CoV virus used in the previous sections leading to T90 of about 4 min at the equator and about 5 min at 40O N during the summer solstice  during the winter months T90 inactivation times are much longer than 2 h and, on many days, no inactivation is possible. For example, Sodankylä, Finland has only 4 months during which the inactivation time T90 < 2 h  (Lytle and Sagripanti A(λ) is used to estimate the value of 254 nm D90 that gives approximately the same T90 derived by RS for a June solstice at 40o N (254 nm D90 = 3.2 ± 0.7 J/m2 for dried virus droplets on a steel mesh surface and 6.5 ± 1.4 J/m2 for viruses in a growth medium).A study of coronavirus inactivation times by UV solar irradiation is presented for two classes of experimental laboratory data. First, are those measurements made at 254 nm and extrapolated to wavelength longer than 290 nm using an action spectrum T90 for SARS CoV and SARS CoV-2 viruses in a realistic atmosphere when exposed to sunlight based on 90% inactivation doses D90 at 254 nm. The method uses OMI satellite data for cloud transmission, ozone, and aerosol absorption over a wide range of latitudes, longitudes, and day of the year. For SARS CoV, a nominal value D90 = 40 J/m2 is used for 90% inactivation at 254 nm combined with the assumed applicable virus inactivation action spectrum A(λ) provided by Lytle and Sagripanti ESM 2(DOCX 88 kb)ESM 3(DOCX 30 kb)


One of the concepts of theranostics in nuclear medicine is peptide receptor radionuclide therapy (PRRT), whereby labeled somatostatin analogs are used for imaging and treating inoperable or disseminated neuroendocrine tumors (NET).90Y /177Lu-DOTATATE in patients with disseminated NET in a multicenter trial.The aim of the study was to determine the therapeutic efficacy and toxicity of tandem 90Y/177Lu-DOTATATE (1:1) with amino-acid infusion for nephroprotection were included in the study.103 patients with NET G1/G2 treated with Overall survival from the disease diagnosis (OS-D) was 127.4 months and from the time of PRRT (OS-T) was 89.5 months. Progression-free survival (PFS) was 29.9 months. An analysis based on the proliferation index revealed a statistically significant impact on PFS and OS-T . The effect of the primary disease site was also analyzed. For pancreatic vs small bowel vs large bowel, the PFS was 30.8 vs 30.3 vs 40.6 months, the OS-T was 94 vs 61.9 vs 131.2 months and OS-D was 130.4 vs 89.2 vs not reached months, respectively. The 2-year risk of progression was 42%. The probability of 2-year and 5-year overall survival was 89% and 62%, respectively. PRRT was well tolerated by all patients. One patient (1%) developed myelodysplastic syndrome. No other grade 3 and 4 hematological or renal toxicity was observed.90Y/177Lu-DOTATATE is highly effective and safe therapy for patients with disseminated NET.This multicenter trial showed that tandem  The term theranostics in nuclear medicine refers to combined diagnostic imaging and therapy using the same molecule. One of the most successful examples of the theranostics concept in nuclear medicine is peptide receptor radionuclide therapy (PRRT) for imaging and treatment of well differentiated neuroendocrine tumors (NET).Neuroendocrine tumors are a heterogeneous group of neoplasms, arising from cells of the endocrine system, with various clinical behaviors. Although they may show hormonal activity , a significant proportion do not produce enough hormones and/or biogenic amines to cause clinical symptoms  –4. AlthoAmong many options for pharmacological treatment of these tumors, long-acting somatostatin analogs which not only reduce symptoms of the disease but also have anti-proliferative effects , 7 remai111In-diethylenetriaminepentaacetic acid (DTPA)0]-octreotide showed limited patient responses which were, in part, related to the physical characteristics of 111In  2.27 MeV) and a long maximum particle range (10 mm) beta-particle emission by 90Y with has been shown to cause significant nephro toxicity. In contrast, 177Lu emits lower energy ([Emax] 0.497 MeV) and shorter particle range (maximum 2–4 mm) beta particles, and is an alternative to 90Y. The clinical results of octreotate (DOTATATE) therapy showed a very promising therapy response rate, with mild bone marrow suppression and low nephrotoxicity  type 1, 1 von Hippel-Lindau syndrome [VHL] with pancreatic tumor, 2 multiple paragangliomas, 1 retroperitoneal area). 39 patients had low grade tumor NET G1 and 64 had intermediate grade tumor G2 NET.Of 103 patients, 32 patients were diagnosed with a pancreatic neuroendocrine primary tumor, 29 with a tumor originating from small bowel, 20 with a tumor originating from large bowel, 4 with a bronchopulmonary tumor, 12 had tumors of unknown primary origin and 6 had tumors arising from other sites  the injected activity was reduced to 2.96 GBq 90Y/177Lu-DOTATATE or they received only 3 cycles.Patients were usually treated with 4 cycles of 3.7 GBq of 90Y/177Lu-DOTATATE treatment with administered cumulative activity of 12.95–14.8 GBq.Three patients received 2 cycles, one because of progression during therapy, two because of patient choice. Eight patients received 3 doses with cumulative activity of 11.1 GBq. The remaining patients (89%) completed their planned four cycles of All patients with NET-related syndrome received long-acting somatostatin analogs, before and during PRRT with 4–5 weeks after injection of octreotide and 5–7 weeks after injection of lanreotide.Imaging response was based on the radiological evaluation according to the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria and SRI.It was performed at the first available checkpoint 3–6 months after therapy with partial response (PR) in 17(17%) patients, stable disease (SD) in 78 (78%) patients and progressive disease (PD) in 5 (5%) patients. At 3–6 months follow up complete response (CR) was not observed, no patients died.Additionally, the imaging evaluation was performed one year after therapy. At one-year follow up eight patients died, CR was seen in 2 (2%) patients, PR in 20 (22%) patients, SD in 59 (64%) patients and PD in 11 (12%)patients.At 3–6 months vs 12 months follow-up, the disease control rate was 95% vs 88%. The objective response rate (ORR), defined as in the proportion of patients achieving PR or CR, was 17% vs 24%, receptively.The median follow-up duration was 44.1 months (interquartile range IQ: 30.5 to 74.6 months). 41 patients died during follow-up.Overall survival was calculated for all patients and a Kaplan-Meier survival curve was generated for all evaluable patients in this study. PFS was also calculated (including deaths).The median overall survival from diagnosis (OS-D) was 127.4 months, and overall survival from the time of PRRT (OS-T) was 89.5 months. PFS was 29.9 months  was taken into consideration, Kaplan–Meier curve analysis showed a significant impact on PFS and OS-T.When the proliferation index of tumors    Fig..Fig. 3TaThe median overall survival time from diagnosis (OS-D) was 102.6 months for G2 tumors, and not defined for G1 tumors. This difference was not statistically significant on the Kaplan–Meier curve.The observed overall 2-year survival from start of PRRT in G1 vs G2 patients was 91% vs 88%, and 5-year survival was 69% vs 56%, respectively. The 2-year risk of progression was 33% in G1 patients and 51% in G2 patients. Examples of therapeutic effect are presented in Figs. The effect of the primary disease site was also analyzed. Tumors were -grouped into those arising from the pancreas, small bowel, and large bowel primary sites.n = 31) PFS was 30.8 OS-T was 94 months and OS-D was 130.4 months.For pancreatic NET  PFS was 30.3 months, and OS-T was 61.9 months. OS-D was 89.2 months.For small bowel NET , PFS was 40.6 months, and OS-T was 131.2 months. OS-D was not reached.For large bowel NET  and small bowel vs large bowel (p = 0.009); in OS-D small bowel vs large bowel NET (p = 0.047). The difference between other locations and survival parameters was not statistically significant.A statistically significant difference in OS-T was observed between primary tumors located in pancreas vs large bowel  and asthenia. No patient had a carcinoid crisis following the treatment or any acute reaction during radionuclide therapy infusion.During the treatment, a transient decrease in platelet and WBC count was observed in three patients (3%).After completing PRRT, toxicity grade 1 leukocytopenia was found in 17 patients (18%) and grade 2 in two patients (2%); grade 1 thrombocytopenia in 5 patients (5%) and grade 2 in two patients (2%); grade 1 decrease in hemoglobin level was seen in 12 patients (13%) and grade 2 in five patients (5%). One patient (1%) developed myelodysplastic syndrome (MDS). Possible predisposing risk factors for MDS included: previous 2 lines of chemotherapy and high tumor load with hepatic, lymph node and bone metastases. No other grade 3 and 4 hematotoxicity was observed.Patients who experienced a degree of nephrotoxicity had at least 2 risk factors . Only 3 of them had normal kidney function before therapy. Grade 1 nephrotoxicity was seen in 14 patients (15%), and grade 2 nephrotoxicity was seen in 3 patients (3%). No Grade 3 or 4 nephrotoxicity was observed.To date, no hepatic toxicity of any grade or other clinically significant toxicity has been observed.111In, afterwards 90Y and now 177Lu has been used for more than 20 years. Numerous phase I and phase II studies have shown favorable PFS and OS in NET patients compared to historical controls octreotate significantly improved PFS compared to Octreotide LAR  in patients with advanced midgut NET. The estimated PFS in the 177Lu-DOTATATE plus Octreotide LAR group was 40 months vs 8.4 months in the Octreotide LAR arm [The NETTER-1, first phase III multicenter, stratified, open, randomized, controlled, parallel-group study demonstrated that [ LAR arm  which is177Lu-DOTATATE had a survival advantage compared to other forms of treatment including targeted therapy with mTOR or kinase inhibitor [These results clearly showed that patients with midgut NET treated by PRRT with nhibitor , 25.90Y and 177Lu, both as single agents and in combination for PRRT. 90Y and 177Lu have different physical properties, such as the half-life and energy of the β-particles.Research continues into the use of 90Y and 177Lu activity for tandem PRRT. This protocol was described in 2006, and continued later [Based on Marion de Jong’s study, we used a 50:50 combination of 90Y and in 0.62 mm for 177Lu, clearly suggest that 90Y is not an adequate isotope for eradicating small or micrometastases [177Lu delivers significantly higher normalized absorbed doses to 1-cm, 1-mm and 100-μm lesions than 90Y  [90Y increased rapidly when sphere size increased [Recently, the size of individual lesions and optimal radionuclide choice was investigated in dosimetry studies and models. The radius within which 90% of energy is deposited in 5.82 mm for tastases . 177Lu dctively) . Howeverncreased .90Y and 50% 177Lu is probably not the best choice. In one Polish center, proportion 1.85 GBq of 90Y and 5.55 GBq of 177Lu was examined, but it resulted in significant nephro- and hematotoxicity (not published data).The optimal composition of isotopes was investigated. The composition for tandem therapy based on 50% 90Y-DOTATOC and 4 GBq 177Lu-DOTATOC seems to be better for patients with larger and moderate size lesions [Based on dosimetry studies, a combination of 2 GBq  lesions .Consequently, in patients with tumors of various sizes and heterogeneous receptor distribution, the use of radionuclide combinations may lead to higher efficacy compared to the use of a single radioisotope , 27, 28.90Y and 177Lu PRRT combination to outcomes after treatment with either of those isotopes alone.There are currently no randomized trial data comparing the outcomes of the Consequently, though potential efficacy has been reported, the benefit of this combination of radionuclides in humans with NET requires further study , 29–32,90Y/177Lu -DOTATATE treatment compared to the group treated with 90Y-DOTATATE alone. The overall survival for 90Y-DOTATATE was 26.3 months and for 90Y/177Lu -DOTATATE was not reached [90Y/177Lu -DOTATATE therapy showed PFS of 32.2 months and OS of 82 months [90Y/177Lu –DOTATATE in a multicenter study showed PFS 29.9 months and OS 89.5 months.Our early results of tandem therapy are encouraging, showing longer overall survival after the combination  reached . Long-te2 months . The sim177Lu DOTATATE (5.55 GBq) and 90Y DOTATATE (2.6 GBq) which induced better objective responses in 42.3% of patients, but shorter median PFS 24 months by comparison with our study [90Y/177Lu-DOTATOC vs 90Y-DOTATOC  [90Y-DOTATOC and 177Lu-DOTATOC, not using both isotopes simultaneously.The use of the combination of both radionuclides has been corroborated by other researchers. Seregni et al. tested a much higher injected activity and different proportion of ur study . Villardctively) , 30. How90Y and 77Lu alone to the combination of both radionuclides [2 papers have been published in the literature regarding studies with three treatment arms, comparing nuclides , 32. Nei90Y or 177Lu only compared to the combination of 90Y with 177Lu as combined or sequential therapy [A German multicenter study showed reduced PFS and OS in a group of patients treated with  therapy .90Y-DOTATOC plus 177Lu-DOTATOC treatment  was correlated with a longer survival compared to 90Y-DOTATOC  or 177Lu-DOTATOC alone  [Superior response to combination therapy was confirmed in a multivariate survival analysis, in which < 0.001) .90Y and 177Lu either as a combination of both radionuclides in one cycle (TANDEM) or sequentially (DUO), or with 90Y and 77Lu alone, best OS (66 months) was achieved with the combination of 90Y and 177Lu for PRRT. Treatment with 177Lu alone resulted in the next longest survival time (44 months) while the shortest time of survival (24 months) was observed in patients treated only with 90Y. The PFS achieved for these 3 patient groups was 24, 17 and 7 months, respectively [In a recently published paper in 1048 patients treated with ectively . Despite18F-FDG PET/CT imaging is a good prognostic indicator, but this is not reimbursed for NET patients in Poland [The survival gain we have observed in our study may reflect the relatively high number of NET G2 subjects in our patient population. . In our study,the proportion of G1/G2 was 37%/63% in comparison to the NETTER-1 study where the proportion G1/G2 was reverse 66%/34% . In patin Poland , 36.In our group of patients, we observed better PFS and OS-T for NET G1 patients. This is contrary to observations published by Horsch et al.  but consPublished articles had limited data concerning the results of the PRRT in correlation with the origin of primary tumor. Horsch D et al. observed that patients with small bowel NET were significantly less likely to die than patients with other primaries, whereas there was no significant difference in the PFS . The samFrom the clinical point of view, treatment related toxicity is an essential consideration. Published studies have reported several serious side effects of PRRT.90Y labeled peptide therapy...Before the introduction of amino-acid infusion, nephrotoxicity was common and was dose limiting, particularly in association with 177Lu-DOTATATE, no evidence of renal toxic effects were reported during 14 months follow-up. Nephrotoxicity is a well-known side-effect, requires a minimum follow-up of 6 months to be observed, more likely 1 year [90Y/177Lu-DOTATATE, grade 3 and 4 toxicity was not observed, even with longer follow-up (median 44.1 months) [In the NETTER-1 trial with y 1 year , 37–39.  months) .Hematological toxicity is usually transient. In the NETTER-1 trial, grade 3 or 4 neutropenia, thrombocytopenia, and lymphopenia were reported in 1%, 2%, and 9% of patients, respectively . In our The present study has some strengths and limitations. The main limitation of the present study is its retrospective design, relatively small patient cohort and lack of dosimetry.177Lu DOTATATE (5.55 GBq) and 90Y DOTATATE (2.6 GBq) did not result in kidney damage and the cumulative BED values were below the toxicity limit in the majority of patients [90Y and much lower activity of 177Lu in smaller cohort. Nevertheless, we obtained similar survival rates with a low incidence of side effects.Seregni et al.  reportedpatients . The dospatients . In our 90Y and 0.7 Gy/GBq for 177Lu; for bone marrow below 0.2 Gy per cycle) [Limited dosimetry data that we obtained at the beginning of therapy showed absorbed doses for kidney and bone marrow comparable to other publications (for kidney 2.6Gy/GBq for r cycle) , 38. On The low incidence of serious adverse events, high probability of disease control and long PFS and OS constitute real benefits for NET patients. However, a limited population size, lack of control group  and lack of randomization will inevitably affect the strength of our conclusions.90Y/177Lu-DOTATATE or sequential 90Y- and 177Lu-DOTATATE treatment compared to single isotope [The NETTER-1 trial strongly confirmed the effectiveness of PRRT, but the optimal radiolabel and role of combination therapy remains controversial. There is no randomized three-arm trial evidence comparing outcomes for simultaneous  isotope , 32. Thi90Y/177Lu-DOTATATE therapy for patients with metastatic neuroendocrine tumors (G1 and G2) is highly effective and safe, with limited side effects. We have also demonstrated statistically significant favorable outcomes of tandem 90Y/177Lu-DOTATATE therapy in patients with NET G1 compared to NET G2.These results of a multicenter trial indicate that tandem radioisotope


Although the two motifs are conserved, some non-H5/H7 subtypes lack one or both GS. Both sites were glycosylated in this H4N2 virus. Deglycosylation increased trypsin-independent replication in cell culture, cell-to-cell spread and syncytium formation at low-acidic pH, but negatively affected the thermostability and receptor-binding affinity. Alteration of 2NYT4 with or without 18NGT20 enabled systemic spread of the virus to different organs including the brain of chicken embryos. However, all intranasally inoculated chickens did not show clinical signs. Together, although the conserved GS near the HACS are important for HA stability and receptor binding, deglycosylation increased the H4N2 HA-activation, replication and tissue tropism suggesting a potential role for virus adaptation in poultry.To date, only low pathogenic (LP) H5 and H7 avian influenza viruses (AIV) have been observed to naturally shift to a highly pathogenic (HP) phenotype after mutation of the monobasic hemagglutinin (HA) cleavage site (HACS) to polybasic motifs. The LPAIV monobasic HACS is activated by tissue-restricted trypsin-like enzymes, while the HPAIV polybasic HACS is activated by ubiquitous furin-like enzymes. However, glycosylation near the HACS can affect proteolytic activation and reduced virulence of some HPAIV in chickens. In 2012, a unique H4N2 virus with a polybasic HACS was isolated from quails but was LP in chickens. Whether glycosylation sites (GS) near the HACS hinder the evolution of HPAIV H4N2 remains unclear. Here, we analyzed the prevalence of potential GS in the N-terminus of HA1,  Orthomyxoviridae. The genome of AIV consists of eight segments of single-stranded RNA of negative polarity [Avian influenza viruses (AIV) belong to the genus Influenza A Virus in the family polarity ,2 which polarity . AIV arepolarity . Low patpolarity ,5.in vivo and in vitro, while the experimentally modified GS reverted after few virus passages [Although the virulence of H5 and H7 viruses is polygenically determined, mutations in the HA cleavage site (HACS) are the main determinant of virulence in poultry. Mutation of the monobasic HACS in LPAIV to a polybasic HACS in HPAIV is a prerequisite for high virulence and replication. The HA of LPAIV is activated by trypsin or trypsin-like enzymes (e.g. the human airway trypsin-like protease HAT or the transmembrane protease serine 2 TMPRSS2) in the gastrointestinal or respiratory tracts of birds. Thus, these viruses cause localized infections and mild, if any, clinical signs. Conversely, HPAIV are activated by furin-like enzymes ubiquitously expressed in all tissues causing systemic infection and death . Intrigupassages .322PEKRRTR/G329 [2NYT4, 18NGT20, 129NGT131, 162NLT164 and 294NVS296 in the HA1 subunit and 482NGT484 in the HA2 subunit [2NYT4 and 18NGT20 are located in the HA1 N-terminus close to the HACS region. It is unknown whether glycosylation near the HACS can affect the virulence of this unique H4N2 virus in chickens.Some non-H5/H7 viruses exhibited high virulence after acquisition of a polybasic CS with or without reassortment with gene segments from HPAIV H5N1 . HoweverRTR/G329 . Neverthin vitro and in chickens in vivo was studied.In this study, we analyzed pGS at positions 2 and 18 in all HA sequences of the HxNy AIV available in the Global Initiative on Sharing Avian Influenza Data (GISAID) and GenBank to 27 January 2020. Using reverse genetics, the wild-type virus (designated rgH4N2) and three recombinant H4N2 viruses with substitutions in position 2 (designated rgN2S), at position 18 (rgN18D) or at both sites (rgN2S/N18D) were rescued. The impact of the N2S and/or N18D mutations on virus fitness The animal experiment in this study was carried out after approval by the authorized ethics committee of the State Office of Agriculture, Food Safety and Fishery in Mecklenburg – Western Pomerania (LALLF M-V permission number 7221.3–1.1-051-12) and according to the German Regulations for Animal Welfare. The commissioner for animal welfare at the FLI representing the Institutional Animal Care and Use Committee (IACUC) approved the experiment, which was conducted in the BSL3 laboratory and animal facilities of Friedrich-Loeffler-Institut (FLI), Insel Riems, Germany.http://swissmodel.expasy.org/) and then viewed in Geneious Prime  and edited manually. Numbering of aa in this study is based on the mature H4 protein after removal of the signal peptide.Sequences of the HA of AIV were retrieved from GISAID and the Influenza Virus Database of the National Centre for Biotechnology Information (NCBI). After removal of identical and laboratory-viruses, sequences were aligned using Multiple Alignment using Fast Fourier Transform (MAFFT) . pGS wasA/quail/California/D113023808/2012(H4N2) was obtained from California Animal Health and Food Safety Laboratory System, Department of Medicine and Epidemiology, University of California, Davis. A/Victoria/1975(H3N2) was kindly provided by S. Pleschka and A. Mostafa from Justus-Liebig-University, Gießen, Germany. A/swan/Germany/R65/2006(H5N1) was obtained from the repository of the FLI kindly provided by Timm C. Harder. pHWS-plasmids containing all gene segments from this H4N2 virus were successfully cloned in a previous study . pCAGGS 2NYT4 and 18NGT20 to 2SYT4 and 18DGT20 to generate N2S and N18D as found naturally in some AIV, respectively, was successfully done using the following primers: N2S_F: 5´-GGGATACTCTCAGAGCTACACAGGAAATCCTGTG-´3 and N2S_R: 5´-CACAGGATTTCCTGTGTAGCTCTGAGAGTATCCC-´3 for deglycosylation at position 2 and N18S_F: 5´ CGTCATGCCGTATCTGATGGAACAATGGTAAAAACC-´3 and N18S_R: 5´-GGTTTTTACCATTGTTCCATCAGATACGGCATGACG-´3 for deglycosylation at position 18. Three HA plasmids carrying N2S, N18D or both N2S and N18D were successfully generated. Furthermore, the HA gene and mutated variants of H4N2 as indicated below were cloned from pHWS-HA into pCAGGS expression vector by conventional restriction digest and ligation. Four recombinant viruses carrying the HA from wild-type H4N2 (designated rgH4N2), N2S (designated rgN2S), N18D (designated rgN18D) or both mutations (rgN2S/N18D) and the other gene segments from rgH4N2 were rescued by transfection of MDCKII/HEK293T co-culture as previously described [2) was tested for bacterial contamination on Columbia sheep blood agar. Virus stocks were stored at −70°C until use. To exclude unwanted mutations, viral RNA was extracted from virus stocks using the QIAamp Viral RNA Mini Kit and transcribed into cDNA using the Omniscript RT Kit  and segments were amplified by Phusion RT-PCR kit [The pHWS-HA plasmid of A/quail/California/D113023808/2012(H4N2) was used for mutagenesis using QuikChange II Site-Directed Mutagenesis Kit . Changing escribed . Two day-PCR kit . Gel sliGrowth kinetics were performed in CEK in 12-well plates at a multiplicity of infection (MOI) of 0.001 pfu for 8 and 24 h post-infection (hpi). Infection experiments of MDCK, MDCK-HAT and MDCK-TMPRSS2 cells were performed for 24 h only. Briefly, cells were infected for 1 h at 37°C and 5% CO2, incubated for 2 min with citric acid buffer (pH 3.0), then washed twice with 1x phosphate-buffered saline (PBS) and covered with Minimum Essential Medium (MEM) containing 0.2% bovine serum albumin (BSA)  with antibiotics (penicillin-streptomycin) in the presence or absence of 2 µg/µl of N-tosyl-L-phenyalanine chloromethyl ketone (TPCK)-treated trypsin . At the indicated time points, cells and supernatants were collected and stored at −70°C until used for virus titration by plaque assay as described below. All experiments were conducted in three independent replicates. Results are expressed as average and standard deviation for all replicates.2. After an hour, cells were washed twice with 1 x PBS and covered by semi-solid BactoTM Agar  with 50% MEM containing 4% BSA and 1 µg/µl trypsin without antibiotics. The plates were incubated for 3 days at 37°C and 5% CO2, and then fixed for 48 hours using 10% formaldehyde containing 0.1% crystal violet. After removal of the BactoTM Agar, plates were rinsed with distilled water, left to dry and the plaques were counted. Viral titers were expressed as plaque-forming units per ml (pfu/ml).Virus titration was performed by plaque assay. Briefly, confluent MDCKII cells in 12-well plates were incubated with 10-fold dilutions of recombinant viruses at 37°C and 5% COTo study the impact of deglycosylation on cell-to-cell spread with and without trypsin, MDCKII cells were infected with recombinant viruses for 1 h and further processed as described above using plaque assay. After fixation with 10% formaldehyde containing 0.1% crystal violet, the diameter of 50 plaques was measured by microscopy using Eclipse Ti-S with software NIS-Elements, version 4.0; Nikon. Diameter of plaques of the rgH4N2 in the absence of trypsin was adjusted to 100%. The plaque size obtained by different recombinant viruses was calculated relative to the rgH4N2. Results are expressed as relative mean and standard deviation.TM Western ECL Substrate . Images were acquired by a Bio-Rad Versadoc 4000 Molecular Imager  and analyzed by Quantity One software (BioRad).Glycosylation of the HA was confirmed using N-Glycosidase F  in standard Western Blot procedures with few modifications . Briefly5 pfu/ml or 32 HA units. Plates pre-coated with 10 µg/ml fetuin  were incubated with 50 μl viruses overnight at 4°C. Unbound virus was aspirated and the plates were washed with 2x PBS and blocked overnight at 4°C by 0.1% neuraminidase inactivated BSA. Afterward the plates were washed with 2x PBS + Tween® 80 (0.01%) and incubated with a 1:2 serial dilution of HRP-fetuin containing α2,3-SA for 1 h at 4°C. The plates were washed again and incubated for 30 min at room temperature (rt) in the dark. The reaction was stopped with 50 μL of 3% H2SO4 and the optical density (OD) was measured at 450 nm using Tecan ELISA Reader.Affinity to the avian α2,3-linked sialic acid (SA) receptors was determined by a solid-phase binding assay ,32. Brie2. Thereafter, supernatant was removed and the cells were treated with MEM containing BSA and 0.05% trypsin for 10 min at rt for proteolytic activation of HA. Furthermore, the cells were incubated for 15 min with MEM containing 10% FCS. Medium was removed and PBS fusion buffers previously adjusted to pH 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8 and 6.0 were added for 4 min. The cells were then washed with 1x PBS, and MEM with 10% FCS was added. After 4 hours incubation at 37°C, 4% paraformaldehyde (PFA) was added for 10 min and cells were washed with PBS. Syncytia formation was measured by fluorescence microscopy  and the average area of syncytia was calculated. The pH threshold was the highest pH value at which fusion was observed.The effect of glycosylation at positions 2 and 18 on pH-dependent fusion activation of the recombinant viruses was studied as previously described ,34, with5 pfu/ml or 32 HA units and incubated at 50°C for 0, 0.5, 1, 2, 4, and 6 hours in duplicates and then stored at −70°C for titration. HA test and plaque assay were used to determine the HA activity and infectivity, respectively, as previously described [Viruses were adjusted to 10escribed . The HA 4 pfu of each virus via the allantoic sac. Eggs were candled daily for 4 days and subsequently collected in 10% neutral buffered formalin. Tissue samples were paraffin-embedded and 2-3-μm-thick sections were stained with hematoxylin and eosin (HE). The severity of necrotizing inflammation was scored on an ordinal 0 to 3 scale: 0 = no change; 1 = mild; 2 = moderate, and 3 = severe necrosis. The distribution of recombinant viruses in different organs and tissues were studied by immunohistochemistry as described [Four 14-day-old SPF ECE were inoculated with 10escribed  using a 5 pfu of each virus on both sides (~100 µL in each side). Clinical signs were monitored daily and mortality for 10 dpi. Oropharyngeal (OP) and cloacal (CL) swabs were collected at 2, 4, 7 and 10 dpi using MEM. Viral RNA was extracted from swab media using Nucleo Mag® VET Kit  according to the manufacturer's instructions using the KingFisher Flex Purification System . The relative amount of viral load in the swabs was determined by generic real-time-reverse-transcription polymerase chain reaction (RT-qPCR) targeting the AIV Matrix gene [SPF ECE from white leghorn chickens were purchased from VALO BioMedia GmbH. Eggs were incubated at the experimental animal facilities of the FLI until hatch. Six-week-old male and female chickens were randomly allocated into four groups. Five birds were inoculated via the oculonasal (ON) route with 10rix gene . StandarStatistical analyses were done by GraphPad Prism 8 software . Differences in replication kinetics, receptor-binding activity, size of syncytia and heat stability experiments were analyzed using ordinary one-way ANOVA with post hoc Tukey tests. Plaque size and RT-qPCR results were evaluated using ordinary one-way ANOVA with Bonferroni correction. A p-value < 0.05 was considered significant.NYTG), other subtypes have overlapping pGS motifs (e.g. NSNT). At the second position, no pGS motif was observed in subtypes H8, H9, H12, H13 and H16. The highest prevalence for the loss of the second pGS was observed in H10 viruses (0.8%), while it was completely conserved in H14 and H15. Although the pGS at position 2 was not represented in tertiary structure, however, it is expected to localize downstream from the HACS. pGS at position 18 is located upstream from the HACS and glycosylation there may sterically hinder the access of proteases  and 66.9% , respectively. The prevalence of mutations, which lead to the loss of glycosylation at the first pGS, ranged from 0.1 to 16.1% and were highest in subtype H14 (16.1%) followed by H9 (3.2%), while the first pGS was conserved in all representatives of subtypes H1, H2, H8, H11, H12 and H15 (Supplementary Table S1). While H4, H7, H9, H10 and H12-H15 had single pGS   panel A.The impact of deglycosylation on thermostability at 50°C was studied. HA of rgH4N2 was stable for 6 hours as determined by HA assay and plaque assay . ConversThe affinity of different viruses to avian-type receptors was studied using a solid-phase binding assay against fetuin labeled with α2,3-SA. H4N2 showed strong binding affinity to avian-type receptors compared to other viruses. Human H3N2 did not bind effectively to avian-type receptors. Deglycosylation at positions 2 and/or 18 clearly reduced the binding affinity to avian-type receptors; although at higher or comparable levels to HPAIV H5N1  panel D.The impact of deglycosylation on pH activation for HA-cell membrane fusion was analyzed by analysis of syncytia formation in quail fibrosarcoma  cell line at different pH values between 4.0 and 6.0. While high-acidic pH (pH = 4) activation revealed no statistical difference in cell fusion among all glycosylation variants, statistical differences in syncytia formation were observed at low-acidic pH values . HA of rTo determine the histopathological changes and distribution of viruses in different tissues, organs of inoculated chicken embryos were subjected to histopathological and immunohistological examination. Necrosis in the gizzard was observed in all groups . InoculaChickens were inoculated with each virus via the oculonasal (ON) route. All inoculated birds remained healthy without clinical signs or mortality. Virus excretion in OP and CL swabs at 2 and 4 dpi was analyzed by RT-qPCR  panel A.322PEKRRTR/G329) in the HACS was isolated from quails, but the virus exhibited low virulence in chickens [HPAIV H5 and H7 evolve from LP precursors after mutation of the monobasic HACS to a polybasic motif. Non-H5/H7 viruses with a polybasic CS are very rare in nature. In 2012, a unique H4N2, the only non-H5/H7 with 4 basic aa (11NST13 in an HPAIV H5N1 or HPAIV H7N1 impaired activation of HA0 and decreased virus growth in cell culture [In this study, sequence analysis of 26,114 HA proteins obtained from GenBank and GISAID showed that pGS at positions 2 and 18 were conserved across AIV HA subtypes, particularly those adapted to poultry, except H9 viruses which lack glycosylation at position 18 which is partially similar to a previous report . Remarka culture . Glycosy culture .11NST13 indicating an impact on structural stability by glycosylation at the N-terminus [11NST13 of an H5 virus [HA0 is a fusion-inactive protein which must be cleaved by host proteases into the HA1/HA2 two subunit complex to expose the fusion peptide in the HA2 . The fusterminus . InteresH5 virus . HoweverChicken embryos as a model have been previously used to assess tissue distribution and virulence of different influenza viruses ,46–48. IIn conclusion, H4N2 with a unique polybasic cleavage site and mutation of GS at position 2 and 18 exhibited increased trypsin-independent replication in chicken cells, increased cell-to-cell spread, triggered fusion activation at low acidic pH and enabled systemic spread in chicken embryos, which were driven mostly by the N2S mutation. Both mutations reduced receptor binding and thermostability. No impact on virulence in chickens was observed, however. Despite the important role of glycosylation in the vicinity of H4N2 HACS for HA stability, removal of the glycosylation at position 2 may play a role in adaptation of non-H5/H7 which is in contrast to similar studies using HPAIV H5N1 or H7N1.Click here for additional data file.