Infectious bronchitis virus (IBV) causes a major disease problem for the poultry industry worldwide. The currently used live-attenuated vaccines have the tendency to mutate and/or recombine with circulating field strains resulting in the emergence of vaccine-derived variant viruses. In order to circumvent these issues, and to develop a vaccine that is more relevant to Egypt and its neighboring countries, a recombinant avirulent Newcastle disease virus (rNDV) strain LaSota was constructed to express the codon-optimized S glycoprotein of the Egyptian IBV variant strain IBV/Ck/EG/CU/4/2014 belonging to GI-23 lineage, that is prevalent in Egypt and in the Middle East. A wild type and two modified versions of the IBV S protein were expressed individually by rNDV. A high level of S protein expression was detected in vitro by Western blot and immunofluorescence analyses. All rNDV-vectored IBV vaccine candidates were genetically stable, slightly attenuated and showed growth patterns comparable to that of parental rLaSota virus. Single-dose vaccination of 1-day-old SPF White Leghorn chicks with the rNDVs expressing IBV S protein provided significant protection against clinical disease after IBV challenge but did not show reduction in tracheal viral shedding. Single-dose vaccination also provided complete protection against virulent NDV challenge. However, prime-boost vaccination using rNDV expressing the wild type IBV S protein provided better protection, after IBV challenge, against clinical signs and significantly reduced tracheal viral shedding. These results indicate that the NDV-vectored IBV vaccines are promising bivalent vaccine candidates to control both infectious bronchitis and Newcastle disease in Egypt.The online version of this article (10.1186/s13567-019-0631-5) contains supplementary material, which is available to authorized users. Gammacoronavirus in the family Coronaviridae. The viral genome is a single-stranded, positive-sense RNA of about 27.6 Kb in length , denatured by heating in boiling water bath for 5 min, resolved by SDS–PAGE and analyzed by Western blot using chicken polyclonal anti-IBV antibody.To determine the incorporation of the S protein of IBV into the NDV particles, recombinant viruses were partially purified by ultracentrifugation. Briefly, 10 mL of infective allantoic fluid of each recombinant virus was passed through 30% sucrose cushion for 2 h at 28 000 rpm at 4 °C and the virus pellet was resuspended in 500 µL PBS. Partially purified LaSota virus was used as a negative control. The resuspended pellet was then mixed with SDS-PAGE sample buffer, heat denatured, resolved by SDS-PAGE, and analyzed by Western blot using chicken polyclonal anti-IBV antibody.−6 to 10−9 were inoculated into four 10-day-old embryonated eggs via allantoic sac route. Eggs were incubated at 37 °C and examined three times daily for 7 days to record times of embryo mortality. The MDT was determined as mean time (h) for the minimum lethal dose of virus to kill all the inoculated embryos.The pathogenicity of each recombinant NDV-vectored IBV vaccine candidate virus was compared to that of parental LaSota virus by the mean death time (MDT) assay in 10-day-old SPF ECE . Briefly6 PFU/100 µL of rLaSota, rLaSota/wt.S, rLaSota/S(Y1145A) + Fct12 and rLaSota/SΔct + Fct12, respectively, via oculonasal route. A fifth group, containing 10 birds, served as unvaccinated control.To evaluate the protective efficacy against IBV, sixty 1-day-old SPF White Leghorn chickens were divided into 5 groups and housed in negative pressure isolators with feed and water provided ad libitum. Four groups containing 15, 10, 15 and 10 birds were vaccinated with 104.2 EID50 of IBV/Ck/EG/CU/4/2014 via oculonasal route. Birds were observed and scored for clinical signs until 10 days post-challenge (dpc) before they were euthanized using CO2 euthanasia procedure approved by IACUC, University of Maryland. Scores include, 0 for normal, 1 for mild ocular discharge and mild nasal discharge and/or sneezing, 2 for heavy ocular discharge and heavy nasal discharge and/or coughing, and 3 for tracheal rales and/or mouth breathing. At 5 dpc, tracheal and oropharyngeal swabs were collected from all birds to detect the level of viral shedding based on IBV-specific iTaq™ one-step universal probe RT-qPCR kit [Three weeks post-immunization, blood samples were collected from all birds to determine the level of hemagglutination inhibition (HI) antibody titers in the serum against NDV  and the qPCR kit .12 in addition to the unvaccinated control group were challenged with 100 chicken lethal dose 50% (CLD50) of virulent NDV strain Texas GB, via oculonasal route, in the ABSL-3 + facility. The NDV strain Texas GB is used as a NDV challenge strain in the US. Birds were observed for clinical signs and mortality until 10 dpc. In IBV and NDV challenge studies, death/mortality was not the endpoint, birds were scored for physical appearance, physical activity, neurological and/or respiratory signs. When reached a cumulative score of 6, birds were euthanized using CO2 euthanasia procedure approved by IACUC, University of Maryland. Euthanized birds were scored as dead on the same day.To evaluate the protective efficacy against NDV, 5 birds from groups vaccinated with rLaSota and rLaSota/S(Y1145A) + Fct6 PFU of rLaSota, rLaSota/wt.S, rLaSota/S(Y1145A) + Fct12 and rLaSota/SΔct + Fct12, respectively, at day 1 of age and boosted at day 14 of age via oculonasal route. Birds in group number 5 served as unvaccinated control. On day 35 of age (3 weeks post-boost), birds in all groups were challenged with 105.2 of IBV/Ck/EG/CU/4/2014 via ocular route. Clinical signs were recorded daily for 10 dpc. At 5 dpc, tracheal and oropharyngeal swabs were collected from each bird to determine the level of viral shedding using IBV-specific iTaq one-step universal probe RT-qPCR kit [Fifty 1-day-old SPF White Leghorn chicks were divided into 5 groups of 10 birds each and housed in negative pressure isolators with feed and water provided ad libitum. Birds in groups 1, 2, 3 and 4 were immunized (primed) with 10qPCR kit . Blood sIBV-specific immune response was determined using ELISA. Briefly, ELISA plates (Nunc Maxisorp) were coated with heat-inactivated purified IBV/Ck/EG/CU/4/2014 in carbonate-bicarbonate buffer, pH 9.8, at 4 °C overnight. The IBV-coated plates were then blocked by 5% skim milk powder in PBS overnight at 4 °C. Plates were then loaded with serum samples diluted 1:100 in 5% skim milk for 2 h at room temperature. Plates were washed, and IBV-specific antibodies were detected using the reagents in the commercial IBV ELISA kit  according to the manufacturer instructions. Absorbance  was measured at 405 nm using BIOTEK ELx808 ELISA reader.P value < 0.05.Statistical significance between different groups were determined using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test using Prism 7  with 12 and rLaSota/SΔct + Fct12, were successfully rescued. The RT-PCR and DNA sequencing confirmed the presence of the three different forms of the IBV S gene in the rLaSota genomes. After the fifth passage in ECE, the presence of IBV S genes were detected by RT-PCR in the three recombinant viruses and confirmed by DNA sequencing, indicating that all recombinant viruses were genetically stable. The expression of S protein by rNDVs was detected by Western blot. Western blot analysis of the infected DF-1 cell lysates revealed three bands indicating S, S1 and S2 proteins of IBV. However, no bands were detected in mock and in rLaSota-infected DF-1 cells  + Fct12 and rLaSota/SΔct + Fct12 but not in rLaSota/wt.S or in rLaSota. This indicates that fusion with the last 12 amino acids of NDV fusion protein resulted in the incorporation of IBV S protein into the NDV virion  + Fct12 and rLaSota/SΔct + Fct12 were evaluated in DF-1 cells in the presence of exogenous protease. The growth of all three recombinants were comparable with that of the parental rLaSota. The growth of rLaSota/wt.S, rLaSota/S(Y1145A) + Fct12 and rLaSota/SΔct + Fct12 were slightly lower than parental rLaSota till 48 hpi but by 60 hpi they reached titers similar to parental rLaSota. rLaSota, rLaSota/wt.S and rLaSota/S(Y1145A) + Fct12 reached maximum titers of 106.6, 106.9 and 106.3 TCID50/mL, respectively, at 60 hpi, and rLaSota/SΔct + Fct12 reached a maximum titer of 106.6 TCID50/mL at 48 hpi  + Fct12 and rLaSota/SΔct + Fct12 showed MDT values of 134 h, 137 h and 139 h, respectively, which were higher than that of the parental rLaSota (98 h) indicating that they are slightly attenuated than the parental rLaSota.The pathogenicity of each rNDV was evaluated in ECE by MDT assay. rLaSota/wt.S, rLaSota/S(Y1145A) + Fct12 and rLaSota/SΔct + Fct12 showed significantly milder signs than birds in the control and rLaSota-vaccinated groups, especially at 4 and 5 dpc  + Fct12 and rLaSota/SΔct + Fct12 showed significant increase in ELISA antibody titers compared to the control non-vaccinated group . However, the chickens vaccinated with rLaSota and rLaSota/wt.S did not show IBV specific ELISA antibody titers  + FctA + Fct1212 did not show any clinical signs or mortality, whereas all birds in the control group showed severe nervous signs and were euthanized at 4 dpc. These results suggest that the rNDV vectored IBV vaccines provided protection also against NDV.The protective efficacy of rNDVs expressing IBV S protein was evaluated against virulent strain of NDV. Upon challenge with velogenic NDV strain Texas GB, all birds in groups vaccinated with rLaSota and rLaSota/S(Y1145A) + Fct12 and rLaSota/SΔct + Fct12 reached log2 values of 3.2 ± 0.29, 3.0 ± 0.19 and 3.4 + 0.22 which were significantly lower than that in the rLaSota-vaccinated group (5.1 ± 0.23) , the means of serum HI antibody titers against NDV in groups vaccinated with rLaSota/wt.S, rLaSota/SY1145A) + FctA + Fct1212 and rLaSota/SΔct + Fct12 showed significantly lower clinical signs scores than birds in control and rLaSota-vaccinated groups at 6 and 7 dpc (P < 0.01)  compared to the control group. Whereas, birds vaccinated with rLaSota/S(Y1145A) + Fct12 and rLaSota/SΔct + Fct12 showed very little, but not significant reduction of IBV shedding compared to the control group  + Fct12 and rLaSota/SΔct + Fct12 showed significantly higher ELISA antibody titers compared to the control non-vaccinated group (P < 0.01). However, the chickens vaccinated with rLaSota and rLaSota/wt.S did not show IBV specific ELISA antibody titers  + Fcts Figure A.Figure 2 ± 0.31–0.42 which were significantly lower than that induced by the parental rLaSota (4.9 log2 ± 0.43). Three weeks after boosting , the mean serum HI titers in these groups increased to 5.7–6 log2 ± 0.26–0.32 which were comparable to that of rLaSota (6.2 log2 ± 0.35) , the mean serum HI titers against NDV induced by the three recombinant NDVs expressing the different forms of IBV S gene reached 2.4–3 logIB is a major disease problem in the poultry industry in Egypt . Current12 and rLaSota/SΔct + Fct12. The IBV S protein in rLaSota/S(Y1145A) + Fct12 and rLaSota/SΔct + Fct12 were modified to abolish the intracellular retention signal and to facilitate the transport of the IBV S protein to the cell surface [In this study, three NDV-vectored vaccine candidates were generated using three different forms of the S protein of Egyptian IBV strain EG/CU/4/2014: rLaSota/wt.S, rLaSota/SY1145A) + Fct45A + Fct surface .The whole S protein was used as the vaccine antigen instead of S1 or S2 protein because the S protein is highly conformation dependent. Some of the critical neutralizing epitopes on the S protein are formed by amino acids from both S1 and S2 proteins and these epitopes will be lost if they are separated. This was recently confirmed by Shirvani et al. . In the 8 HAU) which makes them suitable for mass vaccine production. All the vaccine candidates were slightly attenuated compared to the parental rLaSota virus by MDT assay.In this study, NDV was chosen as the vaccine vector for IBV considering that the vaccine can protect against both IB and ND , which aOne-day-old chickens immunized by all three rNDVs expressing the IBV S protein showed clinical protection against challenge with the Egyptian IBV strain EG/CU/4/2014. Although the three IBV vaccine candidates significantly reduced the severity of the clinical signs, they did not show reduction in tracheal viral shedding, which is another important criterion for determining the efficacy of an IBV vaccine. This result indicates that the immune responses elicited by single immunization may not be high enough to stop the virus shedding from the respiratory tract. It should be noted that IBV is a highly infectious and a fast replicating virus . Therefo12 and rLaSota/SΔct + Fct12 provided only protection against the clinical disease but did not significantly decrease the tracheal viral shedding. This could be because the IBV S protein in case of rLaSota/wt.S elicited more effective cell mediated immune response, which is crucial for clearance of the IBV from the respiratory tract [12 and in LaSota/SΔct + Fct12 could have changed the conformation of the IBV S protein and/or rendered loss of critical T cell epitopes necessary for effective blocking of IBV infection. Our results suggest that wild type S protein of IBV is the best protective antigen. Any modification to the S protein can affect the protective efficacy of the protein.In the prime-boost immunization study, rLaSota/wt.S provided significant protection in terms of alleviation of clinical signs and reduction of tracheal viral shedding after IBV challenge. However, rLaSota/SY1145A) + Fct145A + Fcry tract –54 and ary tract , 56. Ano5.2 EID50 per bird. This indicates that the prime-boost vaccination strategy can provide a protective immune response against a high dose IBV challenge. There is also a need to investigate further if single-dose vaccination of chickens at older age (2-week and older) may be enough to provide a protective immune response against IBV challenge due to the well-developed immune system at this age [Notably, the rLaSota/wt.S provided significant protection against IBV challenge in the prime-boost vaccination experiment even though the challenge dose was increased to 1012 and rLaSota/SΔct + Fct12 showed a significant increase in the IBV-specific antibody titers. A possible explanation is that the surface expression of the IBV S protein and its incorporation into the NDV virion, in case of rLaSota/S(Y1145A) + Fct12 and rLaSota/SΔct + Fct12 elicited much stronger humoral immune response compared to the rLaSota/wt.S which showed only intracellular expression of the S protein. Moreover, coating the ELISA plates with an inactivated whole IBV could have also increased the detection threshold of the IBV-specific humoral antibodies which were raised against S protein alone. Similarly, Eldemery and coworkers reported a higher level of antibodies against IBV S protein when S-ectodomain-coated-ELISA plates were used compared to whole-virus-coated-plates [The ELISA results showed that rLaSota/wt.S did not produce significant increase in the IBV-specific serum antibody titers. On the other hand, the rLaSota/S(Y1145A) + Fctd-plates .In summary, recombinant NDV strain LaSota expressing three different forms of the chicken-codon-optimized S protein of a prevalent Egyptian IBV variant strain were generated and evaluated for use as bivalent vaccines in Egypt. This study confirmed and extended the work of Shirvani et al.  that theAdditional file 1.Cloning of three different forms of IBV S protein into pUC57. (A) Chicken-codon-optimized wild type IBV S gene cloned into pUC57 (pUC57-CO.S). IBV S protein domains: ectodomain, transmembrane (TM) and cytoplasmic tail are represented in blue boxes, preceded by NDV gene-end , intergenic , gene-start  and Kozak (in white) sequences, and flanked by PmeI sites. Dotted numbers indicate amino acid positions in the cytoplasmic tail of IBV S protein. Arrows with circled numbers indicate the primers used to induce the S protein modifications using pUC57-CO.S as template (refer Table 1 for the primers). (B) Primers 1, 2, 3 and 4 used to induce the “Y1145A” mutation and add the last 12 aa of the cytoplasmic tail of NDV F protein  to create pUC57-CO.S(Y1145A)+Fct12 (C) Primers 1 and 5 used to replace the cytoplasmic tail of IBV S protein by the last 12 aa of the cytoplasmic tail of NDV F protein to create pUC57-CO.SΔct+Fct12.


Phosphorylation was confirmed by mass spectrometry and [32P]PO4 incorporation, and in silico and in vitro assays using purified ORP4L identified putative proline-directed kinases that phosphorylate the site. The functional significance of the phospho-site was assessed by mutating serine 762, S763, S766 and S768 to aspartate or alanine to produce phosphomimetic (S4D) and phosphorylation-deficient (S4A) mutants, respectively. Solution binding of 25-hydroxycholesterol and cholesterol by recombinant ORP4L-S4D and -S4A was similar to wild-type but ORP4L-S4D more effectively extracted cholesterol from liposomes. ORP4L homo-dimerization was unaffected by phosphorylation but gel filtration of ORP4L-S4D indicated that the native conformation was affected. Confocal microscopy revealed that ORP4L-S4D also strongly associated with bundled vimentin filaments, a feature shared with ORP4S which lacks the PH and dimerization domains. We conclude that phosphorylation of a unique serine/proline motif in the ORD induces a conformation change in ORP4L that enhances interaction with vimentin and cholesterol extraction from membranes.The family of oxysterol binding protein (OSBP) and OSBP-related proteins (ORPs) mediate sterol and phospholipid transfer and signaling at membrane contact sites (MCS). The activity of OSBP at MCS is regulated by phosphorylation, but whether this applies to ORPs is unknown. Here we report the functional characterization of a unique proline/serine-rich phosphorylation motif (S Phospholipids were purchased from Avanti Polar Lipids . Antibodies against giantin and polypeptide N-acetylgalactosaminyltransferase (GALNT) , vimentin  and V5  were used for immunofluorescence microscopy and immunoblotting. Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies  and IRDye 680LT- and IRDye 800CW-conjugated secondary antibodies  were used for immunofluorescence and immunoblotting, respectively.Cholesterol and 25-hydroxycholesterol were purchased from Steraloids . 25OH was performed in the presence or absence of 40-fold excess unlabelled sterol to determine specific binding as previously described PO4 (0.5 mCi/ml) for 16–20 h in phosphate-free DMEM supplemented with 2% FCS as previously described [32P]PI(4)P (60:20:10:10:2000 DPM/assay), and were prepared by extrusion through 400 nM pore membranes using the AVESTIN LiposoFast system . Liposomes for cholesterol extraction assays were composed of [14C]PC/PE/PS/lactosyl-PE/[3H]cholesterol (59:20:10:10:1) and were prepared as described above.For PI(4)P extraction assays, radiolabelled PI(4)P was prepared by incubating HeLa cells with PO4 (200 μCi) for 6 hours. Cells were harvested in lysis buffer  on ice for 15 min. Supernatants were prepared by centrifugation at 10,000xg for 10 min and incubated with a V5 monoclonal antibody for 16 h at 4°C. Immune complexes were isolated on protein A-Sepharose and resolved by SDS-8%PAGE. Gels were dried and exposed to film at -80°C for 6–12 h. The [32P]-labelled supernatants were also immunoblotted for ORP4L and ORP4S expression using a V5 monoclonal antibody. [32P]PO4 incorporation into wild-type and ORP4 phospho-mutants was determined by densitometry and expressed relative to total ORP4 protein expression (quantified by infrared imaging of immunoblots as described above).To quantify phosphorylation of ORP4, HeLa cells transiently expressing wild-type or phospho-mutants of ORP4L or ORP4S were incubated in phosphate-free DMEM containing 2% FBS, 100 nM okadaic acid and [32P ]ATP (0.8 μCi) and incubated at room temperature for 30 minutes. The assay was terminated by spotting 10 μL of the reaction mixture onto a multiscreen phosphocellulose P81 plate, which was washed 3 times for 15 minutes each in 1% phosphoric acid. Radioactivity on the P81 plate was measured in scintillation fluid using a Trilux scintillation counter.Protein kinase assays were performed using purified ORP4L and ORP4L-S4A (20 μg) as substrates in a final volume of 25 μL . Samples were mixed with the kinases (10–50 nM each) and the reaction was initiated by the addition of 5 μl of PO4 and the proteins were subsequently immunoprecipitated, resolved by SDS-PAGE and identified by autoradiography  most which (16) are in the N-terminal region excluding the ORD. Since the serine/proline-rich motif in ORP4L could be minimally phosphorylated or obscured by phosphorylation in the N-terminal region, we examined the phosphorylation status of the ORP4S phospho-mutants. Wild-type ORP4S has fewer phosphorylation sites and incorporated approximately 20% less [32P]PO4 relative to ORP4L. Importantly, phosphorylation of the ORP4S-S4A and -S4D was reduced to 25–50% of wild-type levels , seven kinases were predicted to have the highest probability of phosphorylating serine 762, 763, 766 and/or 768; the proline-directed kinases glycogen synthase kinase (GSK) 3a and 3b, cJun N-terminal kinase 1 (JNK1), extracellular signal-regulated kinase 1 (ERK1) and cyclin-dependent kinase 1 (CDK1), as well as the non-proline-directed casein kinase 1 alpha 1 (CK1a1). These kinases were tested for their ability to phosphorylate ORP4L and ORP4L-S4A . The amount of [32P]PO4 incorporation into ORP4L was expressed relative to ORP4L-S4A to identify those kinases with the highest activity and specificity for the phosphorylation site (The human ORP4 phosphorylation site is punctuated by two proline residues and was therefore predicted to be a substrate for proline-directed kinase(s). Using an ion site . CK1a1, Kd 45±12 nM), ORP4L-S4A (Kd 62±28 nM) and ORP4L-S4D (Kd 60±18 nM) for [3H]25OH were not significantly different (Kd160±40 nM), ORP4L-S4A (Kd 255±90 nM) and ORP4L-S4D (Kd 144±43 nM) also had similar affinity for [3H]cholesterol P from liposomes by ORP4L-S4D was increased slightly but did not reach significance  unmodified peptide 760–772 differs from the mass of the (B) phosphorylated peptide 760–772 by one phosphate molecule (80 Da). Sequence-related information from a second fragmentation step confirmed individual phosphorylation of serine 762, 763, 766 and 768 (data not shown).ORP4L was subjected to trypsin digestion and LC-MS/MS analysis as described in the Materials and Methods, and peptide corresponding to amino acids 760–772 was identified using the Sequest HT DB search engine. A first fragmentation shows that the mass of the (TIF)Click here for additional data file.S2 FigWhole cell lysates were immunoblotted with a vimentin monoclonal and OSBP polyclonal antibodies, followed by IRDye 680LT- and 800CW-conjugated secondary antibodies.(TIF)Click here for additional data file.


P < 0.01) compared to cells treated with L-alanine in both models of muscle cell atrophy. The mTORC1 inhibitor rapamycin prevented the glycine-stimulated protection of myotube diameter, and glycine-stimulated S6 phosphorylation, suggesting that mTORC1 signaling may be necessary for glycine's protective effects in vitro. Increasing glycine availability may be beneficial for muscle wasting conditions associated with inadequate nutrient intake.Glycine supplementation can protect skeletal muscles of mice from cancer-induced wasting, but the mechanisms underlying this protection are not well-understood. The aim of this study was to determine whether exogenous glycine directly protects skeletal muscle cells from wasting. C2C12 muscle cells were exposed to non-inflammatory catabolic stimuli via two models: serum withdrawal (SF) for 48 h; or incubation in HEPES buffered saline (HBS) for up to 5 h. Cells were supplemented with glycine or equimolar concentrations of L-alanine. SF- and HBS-treated myotubes  were ~20% and ~30% smaller than control myotubes. Glycine-treated myotubes were up to 20% larger ( SF was d in HBS , 10 withd in HBS , 9, 10.Rapamycin  was used to inhibit mTORC1 activation .DMEM media was formulated without glycine (Life Technologies). Basal levels (0.4 mM) or additional amounts (2.5 mM) of glycine (Sigma-Aldrich) were added when appropriate to serum free or differentiation media, as specified.Cells were washed 2 × 5 min in phosphate buffered saline (PBS) and then fixed with 4% paraformaldehyde/PBS for 15 min. Cells were then washed in PBS (3 × 5 min), permeated with 0.1% TritonX-100/PBS, washed in PBS (3 × 5 min) and then incubated in 3% bovine serum albumin (BSA)/PBS for 2 h. Cells were incubated with primary antibody overnight at 4°C. MF20  in 3% BSA/PBS was used to stain myosin heavy chain (MHC). Cells were then washed with PBS (3 × 5 min) and incubated in goat-anti-mouse IgG2b Alexa555 secondary antibody  and DAPI  for 2 h in 3% BSA/PBS. Cells were washed in PBS (3 × 5 min) and then imaged on a Zeiss Axiovert 40 CFL inverted microscope using a 10X objective. Four images were taken in each well from pre-defined locations within each quadrant. Myotube diameter was measured using AxioVision software . A total of ~50–80 myotubes were measured per well and the average diameter of each well was used for statistical analysis as described previously , 9, 10.Myotubes were grown and treated as described previously. To determine the rate of protein synthesis we utilized SUnSET methodology, as described , 11. BriFigure 3) or by BLOT FastStain™ as per manufacturer's instructions .Cell lysates were prepared using RIPA lysis buffer  including protease and phosphatase inhibitor cocktails (Sigma-Aldrich). Lysates were rotated end over end at 4°C for 30 min, then centrifuged at 13,000 RPM at 4°C for 10 min to collect the supernatant. Protein content was determined via Bio-Rad DC protein assay as per manufacturer's instructions . Briefly, samples were separated by SDS-PAGE using Criterion™ TGX Stain-Free™ Precast Gels (Bio-Rad) and proteins were transferred to 0.45 mm PVDF via Trans-Blot® Turbo™ transfer system (Bio-Rad). Gels were visualized using a ChemiDoc™ imaging system (Bio-Rad) and images were captured prior to and following transfer. Membranes were blocked for 2 h at room temperature (RT) in 5% (w/v) bovine serum albumin  or 5% Skim Milk (for puromycin) in Tris-buffered saline-Tween 20 (TBST). Membranes were incubated overnight at 4°C with primary antibodies. The following day membranes were incubated for 1 h at RT in horseradish peroxidise-conjugated secondary antibodies. Membranes were treated with enhanced chemiluminescence  and proteins were visualized using ChemiDoc™ imaging system (Bio-Rad). Blots were quantified using ImageLab® software (Bio-Rad), and normalized to total protein as quantified from the Stain Free Gel imaging (pAkt (S473), Akt, pmTOR (S2448), mTOR, pp70S6K (Thr389), p70S6K, pS6 (S235/236), S6, p4EBP1 (T37/46), 4EBP1, pSTAT3 (Tyr705), and STAT3 were purchased from Cell Signaling Technologies  and diluted 1:1,000 in 5% BSA/TBST. Secondary antibodies  were diluted in 5% BSA/TBST.To assess whether protein expression of key structural protein were affected by glycine in the wasting models, MF20  was diluted 1:50 in 5% BSA/TBST. α-actin (Sigma-Aldrich) was diluted 1/3,000 in 5% BSA/TBST. Secondary antibodies  were diluted in 5% BSA/TBST.Anti-puromycin  was diluted 1:5,000 in 1% BSA/TBST. Secondary antibodies  were diluted 1:50,000 in 5% milk/TBST.post hoc test was used to determine significant differences between individual groups and P values reported in figures. For transparency, we report both significant differences (P < 0.05) and trends (P < 0.1) are reported where appropriate.All values were expressed as mean ± SD. Data were normalized to the appropriate control group for ease of visualization, unless otherwise stated. Two-way ANOVAs (time and treatment) were used to compare between groups for dose-response, time-course, and rapamycin experiment, while one-way ANOVAs were used for all other comparisons. Tukey's P < 0.05; P < 0.01) compared with SF containing L-alanine-treated myotubes (SF ALA+). We have not further investigated why the beneficial effects of glycine were lost at a concentration of 5 mM as we used the dose-response studies to select the most appropriate and effective dose. All further experiments in SFM were performed using 2.5 mM glycine or L-alanine.Growth factor deprivation (48 h) significantly reduced myotube diameter  did not augment the effects of SF alone  from SF  induced myotube wasting similar to that caused by nutrient deprivation (SF). Differentiation media without basal glycine reduced myotube diameter by 17% compared to DM  than myotubes treated with L-alanine (HBS ALA+). The beneficial effects of glycine were lost at a concentration of 5 mM. All further experiments in HBS were performed using 2.5 mM glycine or L-alanine.Nutrient starvation (5 h) reduced myotube diameter . There were no significant effects of rapamycin on myotube diameter in L-alanine treated cells  and starvation models . No changes in AKT, mTOR, p70S6K or 4EBP1 phosphorylation were observed under any treatment condition (Myotubes were incubated with the mTORC1 inhibitor rapamycin to determine the involvement of mTORC1 in the protective effect of glycine in the nutrient starvation and growth factor deprivation models. We measured the effects of rapamycin on myotube diameter and the phosphorylation status of downstream targets of mTORC1. In a model of 4 h nutrient starvation (HBS), rapamycin attenuated the protective effects of glycine by 13%  has been shown previously to dramatically reduce protein synthesis and mTORC1 signaling and reduce myotube diameter . SimilarIn C2C12 myotubes incubated in HBS, glycine supplementation attenuated myotube wasting, maintained protein synthesis and promoted S6 phosphorylation. In both wasting models, the glycine-induced increases in S6 phosphorylation were completely prevented by rapamycin. Likewise, rapamycin inhibited the glycine-stimulated protection of myotube diameter, indicating that mTORC1 signaling is necessary for glycine's protective effects on myotube size in both growth factor deprivation and nutrient starvation models. These results are consistent with the effect of glycine on C2C12 myoblasts where acute exposure (30 min−6 h) promoted proliferation, cell viability and protein synthesis in the absence of serum, in an mTORC1-dependent manner . HoweverUpon activation, mTORC1 phosphorylates and activates two parallel signaling pathways. S6 kinase 1 phosphorylation leads to activation of the ribosomal protein S6, while phosphorylation of the eukaryotic initiation factor 4E (eIF4E)-binding protein (4EBP1) ultimately allows for the synthesis of new proteins. Despite inhibition of S6 phosphorylation by rapamycin in glycine treated cells, there was no alteration in phosphorylation of mTOR, P70S6K and 4EBP1 in either of the nutritional wasting models after 4 h of treatment suggesting that upstream signaling occurs earlier. Divergence in signaling between S6 and 4EBP1 has been described previously in glycine treated C2C12 myoblasts , and myo− channels composed of three different subunits: (1) the ligand binding α subunits ; (2) a structural β subunit (GLRB); and (3) a cytoplasmic anchoring protein known as gephyrin (GPHN). GlyR mediated cell-cell communication is enabled by gephyrin, an anchoring protein that provides the scaffolding needed for cytoplasmic GlyRβ binding (The way in which many amino acids signal to mTORC1 is still relatively unknown. It is possible glycine may signal to mTORC1 via a non-canonical mechanism using Vps34 –19. Howe binding . Interes binding , 21. Alt binding –24. Furtin vitro. Rapamycin inhibited the glycine-stimulated protection of myotube diameter, and the glycine-stimulated S6 phosphorylation, indicating that mTORC1 signaling is necessary for glycine's protective effects in vitro. The removal of glycine from differentiation media for 48 h highlights that glycine is required to maintain cellular homeostasis under normal differentiation conditions. Our results suggest that increasing glycine availability may be beneficial for muscle wasting conditions associated with inadequate nutrient intake.In summary our results in C2C12 myotubes demonstrate a role for glycine in the protection of skeletal muscle wasting The datasets generated for this study are available on request to the corresponding author.MC, DH, GL, and RK interpreted results, conception and design of research, edited and revised manuscript, and approved final version of manuscript. MC, DH, JC, and JT performed experiments. MC and DH analyzed data. MC drafted 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.


Water turbidity was measured continuously throughout the year with moored sensors, supplemented with turbidity profiles and bottle samples, from sites on the lake. During summer there was a gradual exponential (e-folding time of 25 days) decrease in epilimnetic turbidity due to relatively steady settling of solids from the epilimnion through the thermocline to the hypolimnion. Rapid oscillations (periods of approximately 1 day) in turbidity during summer were due to wind driven internal waves. Convective cooling and wind-shear driven stirring during fall storm events increased the turbidity to an annual high just before ice-on. During these storm events, similarity scaling indicated wind shear imparted greater energy than convective cooling to the turbulence at the base of the water column. Ice suppressed wind forcing and resulted in a rapid decrease in turbidity. The rate of decrease in turbidity following ice-on was initially greater than the rate of decrease in the epilimnion during summer, and then slowed until the under-ice turbidity was a relatively constant value which was sustained during the latter half of the ice-on period. The minimum turbidity during winter was greater than the minimum during summer. Following ice-melt in spring, wind driven stirring increased turbidity until summer stratification began.We investigated temporal variations in turbidity in Base Mine Lake, an oil sands pit lake, located in northeast Alberta, Canada. The lake has a surface area of 7.8 km Turbidity is an important characteristic of aquatic ecosystems. It controls the penetration of sunlight into the water column, thereby affecting the absorption of solar energy and the resulting distribution of heat , 8, the Dissolved organic matter (e.g. humic acids), suspended organic particles (e.g. phytoplankton), and suspended inorganic particles  a unique man-made water body in the Athabasca oil sands region in Alberta, Canada Fig. . The lak2) is located in a mined out oil sands pit at the Syncrude Canada Ltd. base mine site in northeast Alberta, Canada  piped into the lake from a nearby reservoir. The net inflow from surface runoff plus precipitation onto the lake surface minus evaporation from it was negligible. The volume of the water increased from approximately 35 × 10A meteorological station was mounted on a floating platform anchored in the center of the lake  and then at both the northeast platform  and southwest platform  using self-contained temperature loggers  on moorings. Each of these temperature chains included two pressure loggers to verify the vertical stability of the mooring. In May of 2015 two self-contained turbidity loggers (RBRDuo with Seapoint turbidity sensor) were added to the temperature chain at the southwest platform, P3, at depths of 2.5 m and 7.5 m. The temperature and pressure loggers sampled every 10 s, and the turbidity loggers sampled every 30 s. During May, June and July of 2015 an RBRConcerto was used at P3 in place of RBRSoloTs. This instrument had an accuracy of approximately 0.25 °C.In the first year of the study, monthly from May to October 2013 and in February 2014, turbidity and total suspended solids (TSS) data were obtained from bottle samples collected throughout the water depth at various locations. The samples were analyzed in the laboratory with a calibrated bench top turbidity meter. Coincident water temperature data were collected in the field with a YSI Castaway (accuracy 0.01 °C). We will focus on the turbidity measurements in the present paper, because of its relative simplicity  compared to the sampling and laboratory work associated with determining TSS, and because it is a more direct measure of water clarity (and light penetration). Preliminary analyses (not shown) indicate the turbidity in NTU is approximately twice the TSS in mg/L.Monthly from May to October 2014, 2015 and 2016, and in March 2015, turbidity and temperature profiles were obtained with a Seabird 19plus (accuracy 0.0005 °C) equipped with a Seapoint turbidity sensor at multiple locations.Base Mine Lake exhibits dimictic behaviour, mixing throughout the water column in May (immediately following ice off), and in September and October, following the breakdown of summer thermal stratification and before ice-on Fig. a. Over tThe timing of turnover and stratificationThe timing of ice cover and lake turnover varies from year to year  show both a relatively uniform turbidity of 200 NTU  spherical particles fall through the water column at the Stokes settling velocity:The epilimnion is subject to daily vertical mixing so that the particle concentration, like turbidity, is considered uniform. The total mass of particles in the epilimnion is n. Smith  shows thParticles are falling from the epilimnion into the hypolimnion with velocity, The average depth of the epilimnion, 6 m3/month Here we apply the approach of Smith  and ReynEquating the rate of change of particle mass in the epilimnion with the flux of particles settling into the hypolimnion gives:The e-folding time scale equals the time it would take for a particle to settle through the epilimnion. Typically the epilimnion in Base Mine Lake has a depth of about 5 m so that in both 2015 and 2016, Every summer, wind events drive tilting, upwelling and oscillations of the thermocline in lakes, including Base Mine Lake. Here we examine one of these events from the summer of 2015 and the connection between motion of the thermocline and variations in turbidity.We focus on the variations that were excited by a strong wind (> 6 m/s and sustained for more than 6 h) from the north on 17 July 2015 (day 198), see Fig. The turbidity measured at a depth of 7.5 m at the southwest platform shows a large drop during the wind event Fig. e. To undThis pattern repeats itself during every wind event throughout summer stratification see Fig. . Note, tIn August, the epilimnion cools and deepens to the bottom of the water column Fig. a. There At the onset of fall turnover (about day 246 in 2015) internal wave activity ceases and the turbidity at both 2.5 m and 7.5 m increase almost linearly until ice-on , was 5, 10, 36, and 16 NTU in 2013, 2014, 2015 and 2016, respectively.In terms of timing of events, 2016 showed the latest onset of summer stratification (June 23) compared to previous years of late May/early June. Fall turnover dates ranged from Aug. 27 to as late as Sept. 7. The timing of ice-on and ice-off was fairly consistent from year to year , there are several notable differences between the years in the turbidity levels and the timing of various events Fig. .First, aOnce ice formed on the lake in November 2015 turbidity initially decreased far more rapidly than during summer, see Fig. One hypothesis for elevated winter turbidity may be gas ebullition from the FFT, and with bubbles rising through the water column In addition to the decline in turbidity after ice-on, the turbidity exhibits variations at shorter time scales < 10 days) throughout the winter. In Fig.  days thrSimilarly, the largest variations in turbidity under the ice are not explained by downwelling of cooler, clearer water from above or by upwelling of warmer, more turbid water from depth. Note that downwelling appears as falling isotherms whether temperature is decreasing with depth  in turbidity during summer are due to wind-driven internal seiches. Convective cooling and wind during fall storm events increase the turbidity to an annual maximum just before ice-on. Ice-on suppresses wind forcing, resulting in a rapid decrease in turbidity. By mid-January the turbidity falls to a relatively constant value that is higher than the summer minimum. The reason(s) for this relatively high minimum turbidity in winter is, as yet, unexplained; however, we note that it may be sustained convection driven by salt exclusion as ice thickens, heating from warm underlying FFT, or by ebullition from the FFT. Following ice-melt, wind increases turbidity until summer stratification begins and turbidity declines.


In the high-frequency range near 2 f0 or larger, the P-wave amplification is obvious, which maybe explain the discrepancy between the HVSR and the transfer function.The horizontal-to-vertical spectral ratio (HVSR) and empirical transfer function analyses were performed on the S-wave recordings from two vertical borehole strong motion arrays: the Garner Valley Downhole Array in southern California, and the KiK-net Ichinoseki-Nishi Vertical Array in West Ichinoseki, Japan. The results show that the discrepancy between the HVSR and the transfer function is mainly caused by the significant site response of the vertical component, thus, vertical incident P-waves are proposed to play an important role in the vertical amplification. The P-wave amplification is frequency-dependent. In the low-frequency range within  The evaluation of site-effects due to local geology or topography has become a standard requirement in microzonation studies or site evaluation for important facilities . Many emThe HVSR method was initially used to estimate the fundamental period and was subsequently extended to determine shear-wave velocity of near-surface soils using ambient-noise/microtremor measurements –7. HowevIn this article, strong motions recorded by two vertical borehole arrays,the Garner Valley Downhole Array (GVDA) and the KiK-net Ichinoseki-Nishi Vertical Array (IWTH25), are analyzed. First, the discrepancy between HVSR and transfer functions are revealed and discussed. Second, the 1-D seismic response method which can deal with site response under the excitation of P or SV waves in frequency domain and the wave number integral method used for construction of synthetic seismograms are applied to interpret site specific wave propagation characteristics. Finally, the applicability of HVSR on site response is explored.Hs, and the vertical Fourier amplitude of ground motion, Vs, at the free surface:The HVSR method was originally used to estimate the characteristics of the ground motion from the employment of the microtremor observation .Then it Lermo and Chavez-Garcia  found thSi(f) is the source term of the ith event, Pij(f) is the path term between the jth station and the ith event, Gj(f) is the site term for the jth station, and Ij(f) is the instrument response term for the jth station. The spectral ratio is obtained by dividing the Fourier spectrum of the acceleration at the jth station by the spectrum at the kth reference station as follows:Since introduction of the Empirical Transfer Function , many stj and k is much less than their hypocentral distances from the source, the source and path term would be eliminated. The instrument response can be removed from the data under the assumption that both instruments are the same. The exact site response can thus be obtained from formula  in this study. The Hs and HB are the horizontal Fourier amplitude motions recorded on an alluvium site and on the basement respectively.If the distance between stations  formula . The phyin situ evidence for the research of the seismic response theory, method and applicability of this geotechnical model. In recent years, many downhole arrays have been built all over the world, and seismic records of two vertical arrays, GVDA and the IWTH25, were incorporated in our dataset.The increase in the number of borehole instruments provided a significant step forward in directly measuring the effects of surface geology and critical constraints on our methods for interpreting surface observations . Boreholhttp://www.nees.org/). The main Garner Valley station is instrumented with several downhole accelerometers (from 6 m sub-surface down to 150 m depth) . The near-surface soil types present are silty sand, sand, clayey sand, and silty gravel. The alluvium gradually transitions into decomposed granite at depths between 18 m and 25 m. Decomposed granite consisting of gravely sand exists between 25 and 88 m. At a depth of 88 m the contact with granodiorite of the Southern California Peninsular Ranges batholith is reached. Bonilla [Fig 1C.The GVDA is a ground motion research site in a seismically active region in Southern California, US, maintained by NEES  Site is one of the KiK-net stations, which is equipped with three-component accelerometers, installed at the surface and the bottom of a 260m borehole. Its velocity profile is presented in Fig 3 presents an example of observations at different depths for the three components of ground acceleration from IWTH25 site. When the seismic waves propagate from the bedrock through the soil column, the surface to bedrock amplification is significant. The HVSR curves are determined using spectral ratio of S-wave part of horizontal and vertical components. The TF curves are determined using spectral ratio of S-wave part of components at surface over incident upgoing S-wave at borehole site. A more than 10s window beginning 0.5–1 sec before the onset of the S-wave is taken from each records. A 5% Hanning taper was applied to all time windows. All the S-wave Fourier spectra are smoothed by using the logarithmic smoothing function proposed for this correction [HVSRs can be obtained through strong motions recorded by the sensors located at different depths for every seismic event. Fig 4 respectively shows HVSRs and TFs at different depths for two vertical arrays. In this Figure the shaded areas constitute the average of results of all the corresponding considered events with ±1 standard deviation, while the TFs are evaluated using traditional spectral ratios of the S-wave, as explained above.Considering the difference of HVSR derived from different components, we calculated HVSRs through different components separately. For the GVDA and IWTH25 site, these are EW/UD and NS/UD. Fig 4, firstly, the HVSRs and TFs on the surface of sites yield quite similar results in the whole considered frequency band. They have the same spectrum shapes. The predominant frequencies obtained from HVSRs are in good agreement with TFs. Since it has been recognized that the TF is a reasonable indicator of site-effects, whose peak frequency usually is the predominant frequency of a site, the agreement of spectrum shapes between HVSRs and TFs further verified the applicability of HVSR in pinpointing the resonant frequency, as previous studies proved before [As we can see from d before –14. In od before  researchSH/BH)/(SH/SV) = SV/BH. Under the assumption that the HVSR in the firm substrate could be regarded as a constant, the shape of the ratio SV/BH is consistent with the vertical TF, which is denoted by the ratio SV/BV. The TFs of the GVDA and IWTH25 are also shown in In order to interpret the amplitude discrepancy between the HVSR and the TF for different arrays, the ratio of the average TF to the average HVSR is introduced, as shown in Fig 5, the transfer matrix method [Fig 1C and Table 1 were adopted. Considering the downgoing wave effect, we induced the borehole response and outcrop response, as proposed by Bonilla et al. [Kawase et al.  proved tx method  was appla et al. . The borFig 6A, the spectral shapes of vertical seismic responses at surface are shown for the GVDA. Also shown in Fig 6A are the theoretical and synthetic borehole response, and the theoretical outcrop response. The peak frequencies of the theoretical borehole response of GVDA are about 4.5 Hz, while corresponding peak frequencies of outcrop response are about 4 Hz, closer than the observed peak value of 3 Hz. It has to be mentioned that the theoretical borehole and outcrop response are obtained under the condition of vertical incident P-waves in a linear regime, ignoring any effect of material damping and incident angle. To consider possible effects of these two factors, the wave number integral method [L 5.4 earthquake of 7 July 2010 and the ML 5.4 earthquake of 12 June 2005), which are offered by Harvard University  are presented in Table 3. The synthetic borehole responses in Fig 6A are derived from a P-wave window of vertical component in Fig 6B. The amplitudes of synthetic accelerograms of these two events are much bigger than the observation, so we adjust the amplitudes of synthetic accelerograms and put them together with the observations. Anyway, the comparison of the shape of synthetics and observed time histories shows that the P waves explain the largest part of the vertical amplification. The synthetic borehole responses are in good agreement with observed vertical transfer function, especially for event A, which further verify that the vertical amplification can be explained by the borehole response of incident P-waves.In l method  is introl method . The focas a suitable reference site, as defined by Steidl et al. [Vs 1810m/s) of the IWTH25 site indicates that at this depth, the site may undergo amplification that cannot be ignored. It also appears that one of the principle assumptions that H/V is equal to unity on the bedrock does not necessarily apply in all circumstances.According to analysis in previous section, based on observation, it is clear that we cannot always assume a borehole sensor located below the soil column in competent granite rock l et al. , with a -wave wavelength is about two times larger (depending on the Poisson ratio) than the S-wave wavelength. The fundamental frequency of the site can be evaluated using the equation f0= Vs/4h, the fundamental frequency under the excitation of P-wave should be about 2 f0. Generally, in the low-frequency range, the P-wave wavelength is much larger than the depth of soil strata, so the size of the soil column can be ignored compared to the P-wave wavelength. As the frequency increases, the P-wave wavelength reduces and becomes comparable to soil profiles, so that the effect of the soil strata becomes more obvious. In our study, it is obvious that in the low-frequency range within f0, the level of the vertical P-wave amplification is slight, while in high-frequency range near 2 f0 or larger, the P-wave amplification is obvious. Based on these considerations, we can deduce that HVSRs and TFs should be almost exactly the same for those frequency ranges in which the P-wave amplification can be completely ignored. We casually selected ABSH01 from many KiK-net borehole arrays and analysed 14 events with PGA 1.1–9.5cm/s2, recorded between 26 September 2003 and 15 May 2015. Fig 7 presents the obtained TF, HVSR and vertical response. In the range of 0.4-2Hz, there is no P-wave amplification, as shown in Fig 7B. So we can deduce that in this frequency range, the observed HVSR should be consistent with the TF, which indeed verified by Fig 7A.We compared HVSR and TF for the two sites (GVDA and IWTH25), and proved that their differences are based on vertical site responses, through theoretical solution of wave propagation in elastic layered site and synthetic accelerograms, which revealed that the vertical amplification caused by the vertical incident P-wave is probably the main reason for the vertical site responses. This result demonstrates that the other principle assumption of the HVSR method, namely that “the vertical motion is not significantly amplified by the surface layers”, is not generally applicable.However, there remains no doubt that HVSR resembles TF for the two vertical arrays, as the shapes of HVSRs agree quite well with TFs, which further confirmed its validity in determining the predominant frequency of a site. The discrepancy between HVSR and TF is closely related to the level of the vertical P-wave amplification, and the amplification is frequency dependent. Commonly, the P2, whereas only two events (recorded by GVDA) have PGAs > 50cm/s2, so the performance of these sites is closer to a linear regime. However, a similar conclusion can be obtained even considering the site nonlinearity under severe earthquakes. We take two stations in western China as examples. Rong et al. [Fig 8 presents the theoretical spectral ratios (defined as TFs in [2) or strong motions (with PGA 560 cm/s2 for 51SFB and 960 cm/s2 for 51WCW), the HVSRs agree quite well with the theoretical spectral ratios, which illustrates that the HVSRs and the theoretical spectral ratios can still reveal the amplification of the site consistently even when velocity profiles change during severe earthquakes. Under the strong motion excitation, the nonlinear deformation process of soil will manifest itself in two ways in seismological observation: wave velocity decreases and resonance frequency will be therefore shifted downward. Simultaneously, increased dissipation will reduce soil amplification in the strong motion compared to the weak motion as shown in Fig 8. The theoretical borehole responses are linear results which indicate the site response. In the relatively low frequency range within f0 (about 7Hz for 51SFB and 10Hz for 51WCW), the theoretical borehole response can be ignored, the HVSR and transfer function agree quite well. In the relatively higher frequency range (>10Hz) where the theoretical borehole response can’t be ignored, the discrepancy between the HVSR and the transfer function is obvious.In our dataset, most of observed PGAs of the considered events for two vertical arrays are less than 50cm/sg et al.  conductes TFs in , evaluatf0, the level of the vertical P-wave amplification is slight, while in high-frequency range near 2 f0 or larger, the P-wave amplification is obvious. Otherwise, the HVSRs and TFs are compared considering the site nonlinearity under severe earthquakes, the results show that in the frequency range in which the P-wave amplification can be ignored, the HVSRs and TFs are in good agreement in amplitude and spectrum.The observations of ground motion in two vertical arrays presented and analyzed here have provided fundamental data for the comparison of HVSR and TF. The discrepancy between HVSRs and TFs has been explored and interpreted by comparing vertical site response to theoretical and synthetic borehole or outcrop response. In addition, the applicability and conditions of using HVSR as TF have been studied. From what we have discussed above, we can come to the conclusion that the HVSRs from observed earthquakes resemble the TFs for horizontal components, the HVSR method can be used to determine predominant frequency. But discrepancy still exists in absolute amplitudes of HVSRs and TFs. The consistency of them depends on the vertical seismic response of the site, which is also frequency-dependent. The vertical site response can be explained by the P-wave amplification.Commonly, in the low-frequency range within


Dear Editor,Proline-rich antimicrobial peptides (PrAMPs) are a class of antimicrobial peptides containing a high content of proline residues. PrAMPs selectively target Gram-negative bacteria through special transporters such as SmbA to enter cytoplasm . Oncocin has high antibacterial activity as its minimal inhibitory concentration (MIC) is around 1 μg/mL  were used (Table S1). They all encode a common VF motif, which was placed at the 0, 2, 4, 6, 8, or 10 codon positions after the AUG start codon. Each active ribosome translating through the common VF motif produced one smFRET event as shown in Fig. We examined how deletions in Onc112 affect its ability to inhibit elongation . OncΔNr, a C-terminally truncated derivative, affected elongation in a similar manner as Onc112. However, OncΔVD, an N-terminally truncated derivative, caused minor inhibition. Our results agreed with previous findings that the N-terminus of Onc112 plays a key role in binding and deactivating the ribosome and its C-terminus mainly functions in cellular uptake  appeared when Cy3-F was delivered into the A-site  when Cy3-F was translocated to the P-site  and the appearance of steady Cy3 and FRET intensities −1 to 3.5 ± 0.3 s−1(μmol/L)−1 in the presence of Onc112 −1  to a steady high FRET state (E = 0.5–0.6) until fluorescence signals disappeared due to photo-bleaching  to another transient high FRET state (E = 0.6) Below is the link to the electronic supplementary material.


C-ρ1R relates to its structure. To address this, we tested the ability of the structurally related compounds homotaurine, hypotaurine, and isethionic acid to modulate GABAC-ρ1R.The amino acid taurine (2-Aminoethanesulfonic acid) modulates inhibitory neurotransmitter receptors. This study aimed to determine if the dual action of taurine on GABAXenopus laevis oocytes, hypotaurine and homotaurine partially activate heterologously expressed GABAC-ρ1R, showing an increment in its deactivation time with no changes in channel permeability, whereas isethionic acid showed no effect. Competitive assays suggest that hypotaurine and homotaurine compete for the GABA-binding site. In addition, their effects were blocked by the ion-channel blockers picrotixin and Methyl phosphinic acid. In contrast to taurine, co-application of GABA with hypotaurine or homotaurine revealed that the dual effect is present separately for each compound: hypotaurine modulates positively the GABA current, while homotaurine shows a negative modulation, both in a dose-dependent manner. Interestingly, homotaurine diminished hypotaurine-induced currents. Thus, these results strongly suggest a competitive interaction between GABA and homotaurine or hypotaurine for the same binding site. “In silico” modeling confirms these observations, but it also shows a second binding site for homotaurine, which could explain the negative effect of this compound on the current generated by GABA or hypotaurine, during co-application protocols.In C-ρ1R that occupy the agonist binding site. The dual effect is unique to taurine, whereas in the case of hypotaurine and homotaurine it presents separately; hypotaurine increases and homotaurine decreases the GABA current.The sulfur-containing compounds structurally related to taurine are partial agonists of GABAThe online version of this article (10.1186/s12868-018-0448-6) contains supplementary material, which is available to authorized users. Incorporation of sulfur into amino acids, proteins, enzymes, vitamins, and other biomolecules makes sulfur essential for biological systems . In mamm2+ channels and neurotransmission  code: 4COF). The structures were drawn using the PyMOL program.


As of March 2018, the database includes data collected at 35 sites in 15 Köppen climate zones from the years 2004 to 2017. The key variables in each dataset are hourly values of δ18O and δD in atmospheric water vapour. To support interpretation of the isotopologue data, synchronized time series of standard meteorological variables from in situ observations and ERA5 reanalyses are also provided. This database is intended to serve as a centralized platform allowing researchers to share their vapour isotope datasets, thus facilitating investigations that transcend disciplinary and geographic boundaries.The isotopic composition of water vapour provides integrated perspectives on the hydrological histories of air masses and has been widely used for tracing physical processes in hydrological and climatic studies. Over the last two decades, the infrared laser spectroscopy technique has been used to measure the isotopic composition of water vapour near the Earth’s surface. Here, we have assembled a global database of high temporal resolution stable water vapour isotope ratios (δ Water vapour mixing ratios are expected to rise in the future warmer climate, leading to more extreme weather events4. Quantification and elucidation of processes underlying the variations in atmospheric water vapour remain one of the grand challenges in water cycle science5.Water vapour in the atmospheric boundary layer, the lowest layer of the Earth’s atmosphere and about 1 km in thickness, is a key component of the hydrological cycle and the climate system. It is estimated that this reservoir contains the majority of the atmospheric vapour, contributing to about 2/3 of the Earth’s natural greenhouse effect216O (about 99.731%), HD16O (about 0.003789%), H217O (0.037%) and H218O (about 0.2%)6. Isotopic compositions indicate the relative abundance of a heavier isotope (HD16O or H218O) to the lighter H216O isotope. Kinetic and equilibrium fractionation processes occur during transitions between the vapour phase and the condensed phases (liquid or solid), such that the vapour phase is generally more depleted in the heavier isotopes than the condensed phases. The degree of fractionation is strongly influenced by the atmospheric conditions  during the phase transitions. The isotopic compositions of water vapour can therefore provide an integrated perspective on the hydrological history of air airmasses and has been widely used for tracing physical processes in hydrological and climatic studies7. Water vapour isotopic measurements provide additional constraints on state variables of the atmosphere simulated by general circulation models8–10. By tagging atmospheric moisture with the isotopic tracers, it becomes possible to trace the origin of precipitation water with isotope-enabled Lagrangian trajectory models12. By incorporating information on vapour isotopes, an atmospheric data assimilation system yields more accurate simulations for meteorological fields than if such information is left out13.There are four primary isotopologues of water: H14. The Global Network of Isotopes in Rivers (GNIR) is another public data depository developed recently for archiving isotopic compositions of river water15. Both databases are for isotopes in the condensed phases of water. To our knowledge, our database is the first to archive vapour isotope measurements for public use without restriction.Currently, two databases on stable water isotopes are publicly available. The Global Network of Isotopes in Precipitation (GNIP), administrated by the International Atomic Agency, has been archiving precipitation isotope data for several decades16. The SWVID is intended to serve as a platform similar to GNIP and GNIR, allowing researchers to share their vapour isotope datasets at a centralized depository and thus facilitating investigations that transcend disciplinary and geographic boundaries. We expect that data in this archive will be useful for 1) validation of isotope-enabled atmospheric models, 2) calibration of vapour isotope observations from satellites, 3) investigation of isotopic fractionations under varied surface conditions, and 4) improvement of atmospheric data assimilation.In this paper, we describe a Stable Water Vapour Isotope Database (SWVID) that archives high-frequency observations of water vapour isotopes collected using isotopologue-resolved infrared laser spectroscopy. Before this technology became available, measurements of vapour isotopic compositions required collection of water vapour using cryogenic trapping and subsequent analysis by mass spectrometry, a labor-intensive operation that does not permit high-frequency measurements. Thanks to the development of research-grade and commercial water vapour isotope spectroscopic analysers, it is now possible to measure isotopic compositions of water vapour continuously, in-situ, and at high temporal frequencies (seconds to hours). Such measurements have improved our understanding of the mechanisms underlying hydrological processes, such as diagnosing moisture paths, partitioning evapotranspiration, and interpreting convective mixinghttp://vapor-isotope.yale.edu/). The key variables in each dataset are 18O and D isotopic compositions of atmospheric water vapour. To support interpretation of the isotopic data, synchronized time series of standard meteorological variables  are also provided. The measurement period for each site is shown in As of March 2018, the datasets in this database (Data Citation 1) have been collected in 15 Köppen climate zones (17. All isotope compositions are expressed in delta notation in reference to the VSMOW (Vienna Standard Mean Ocean Water) standard.We have compiled in-situ water vapour isotope measurements and the corresponding climatic data from 35 sites . Measure18 located in Connecticut, United States. The forest is dominated by red maple, eastern white pine, beech and hemlock. Mean tree height varies between 16 and 22 m. The average annual temperature is 7.0° C, with the average total annual precipitation of 133 cm. The water vapour isotope experiment was conducted from May 2005 to September 2005, using a tunable diode laser (TDL) trace gas analyzer . The analyzer measured the isotopic composition at 10 Hz and the data were averaged to hourly intervals for archiving and analysis. The measurement height was 30.7 m above the ground. Standard meteorological variables were measured simultaneously at the forest.Great Mountain  is a forest site19 located on the coast of the Atlantic Ocean in Connecticut, USA. Water vapour isotope experiments were conducted from December 2003 to December 2004 and from March 2007 to May 2008. The isotopic composition of atmospheric vapour was measured with the same TDL analyzer as Great Mountain. The air was drawn from 21.7 m above the ground. The average annual temperature is 10.5° C. The average annual precipitation is 120 cm. Standard meteorological variables were measured at the Tweed Airport 10 km away from the isotopic measurement site.New Haven  is an urban site20–22, located at the Rosemount Research and Outreach Centre, the University of Minnesota, near the Minneapolis – St Paul, Minnesota, USA. The crop species are corn and soybean, which are typical of the US Corn Belt. The annual mean air temperature is 7.6 °C and precipitation amount is 92 cm. The site includes two isotopic measurement datasets. One dataset was obtained from April to October 2007 at the G21 site, which is part of the AmeriFlux network, and the other was obtained at tall tower site from January 2010 to April 2014. The instrument at G21 was a TDL analyzer  and that at the tall tower site was also a TDL analyzer . The sample inlets were located at a height of 185 m and 3 m above the ground surface at the tall tower and the G21 site, respectively. The instrumental temporal resolution is 2 Hz. The averaging interval is 1 h.Rosemount  is a farmland site23 in Ontario, Canada. The forest is composed of red maple, eastern white pine, large-tooth aspen and white ash. The mean tree height is 21 m. Annual mean air temperature is 6.4 °C and annual mean precipitation is 86 cm. The measurement was made with a TDL analyzer (model TGA100A) with the intake height of 37 m above the ground. The sampling frequency is 2 Hz and the averaging interval is 1 h. The experiment was carried out from May to August 2009.Borden  is an Environment Canada research station located in a forest site24 is a forest site located in Colorado, Unite States and operated as part of the AmeriFlux network. This is a subalpine mixed coniferous forest  with a mean tree height of 11 m. A cavity-enhanced near infrared laser (CIL) analyzer  was used to measure water vapour isotopes at 1 Hz. The averaging interval is 1 h. The height of the sampling inlet is 20 m above the ground.Niwot Ridge 25 is located in Colorado, USA. The site is dominated by a mature open-canopy ponderosa pine forest with an average height of 18.5 m. The experiment was conducted from May to September 2011 using a CIL isotopic analyzer  with the inlet positioned at a height of 27.1 m above the ground. The sampling frequency is 0.6 Hz, and the averaging interval is 1 h.Manitou Experimental Forest Currently six datasets in Europe are available in our database, including Camargue in France, Rietholzbach in Switzerland, Kourovka in Russia, Karlsruhe in Germany, and Teide and Izana in Spain.26, continuous measurement of the isotopic compositions of atmospheric water vapour was performed between July and August 2011. The site is 170 m away from a lagoon and 12 km from the Mediterranean Sea. This area is controlled by the Mediterranean climate. The annual mean temperature is 14.0° C. The annual precipitation is 70 cm. The Camargue site is situated in a vast wetland area consisting of variable saltpans, lakes, lagoons, saline, freshwater marshes and temporary wetlands. The site is flooded during the wet season (September – March) while is dry in other months. The isotopic analyzer type is CIL . The sampling frequency is 0.2 Hz, the averaging time is 1 h and the air intake height is 1.75 m above the ground.At the Camargue site 27 is located in the Kourovka Astronomical Observatory near the western boundary of western Siberia, Russia. This site is dominated by pristine peatland with dense pine forests. The mean tree height is about 15 m. Local climate is continental. The annual mean temperature is −1.0 °C and the annual mean precipitation is 46 cm. The water vapour isotopic measurement was performed from September 2012 to August 2013 with a CIL analyzer . The sampling frequency is 2 Hz, the averaging interval is 1 h, and the inlet height is 8 m above the ground.The Kourovka site 28. The Rietholzbach site has a temperate humid climate, with an annual mean precipitation is 146 cm and the mean temperature is 7.1°C. The water vapour isotope measurement was performed from August to December 2011 with a CIL analyzer . The sampling frequency is 0.2 Hz, the averaging interval is 1 h, and the intake height is at 1.5 m above the ground.The Rietholzbach site is situated in the northeastern Swiss Prealps . The catchment is sparsely populated and primarily used as pastureland, while about 25% of the area is forested29. The measurement was conducted in an urban area near the KIT campus. The site has an oceanic climate, with an annual mean precipitation is 79 cm and the mean temperature is 11 °C. The analyzer type is CIL . The sampling frequency is 0.6 Hz, the averaging interval is 1 h, and the inlet height is 28 m above the ground level. The measurement took place between September 2012 to August 2013.The Karlsruhe site is located 12 km north of Karlsruhe in southwestern Germany 30. The area where these two sites are situated has a Mediterranean climate. The vegetation in the surrounding area consists of sparse brooms. The Izana Observatory is a mountain-top station located at 2367 m above the mean sea level , and the Teide Observatory is located at a volcanic cone at 3550 m above mean sea level . During the measurement period, air temperature varied between −6° C and 15° C at Izana and between 6° and 11 °C at Teide. The annual mean precipitation, measured at a weather station (52 m above mean sea level) on Canary Islands, is 23 cm31. The analyzer type at both stations is CIL . The sampling frequency is 0.6 Hz and the averaging interval is 1 h. The measurement started at Izana in March 2012 and that at Teide in July 2013 and they are still ongoing. The most recent data are available upon request to the current station PI, O. E. Garcia. At Izana, the sampling inlet is at a height of 30 m above the ground. At Teide, the inlet height is 6 m above the ground.Izana and Teide are two mountain sites on Canary Islands, SpainCurrently, we have eight sites in Asia, including eight sites in China , two sites in Japan (Mase and Nagoya) and one site in Taipei.33. The annual mean air temperature is 12 °C. The annual mean precipitation is 58 cm. A TDL trace gas analyzer  was used in this site. The sampling frequency is 10 Hz, the averaging interval is 1 h, and the inlet height is at 10 m above the ground.At the Beijing site , the measurement was made from December 2006 to December 2007 in an urban neighourhood34 and the ChinaFLUX eddy flux network, in southeast China. The dominant vegetation types are slash pine and cypress, with a mean height of 14.3 m. The annual mean temperature is 17.1 °C. The annual mean precipitation is 138 cm. The isotopic measurements were conducted from December 2006 to December 2007, using a CIL analyzer . The sampling frequency is 2 Hz, the averaging interval is 1 h, and the air intake height is 27 m above the ground.Qianyanzhou  is a forest site operated as part of the Chinese Ecosystem Research Network (CERN)35–37 is a winter wheat and summer maize double-cropping system in north China. This site is also part of the CERN. Wheat was planted in November 2007 and harvested in mid-June 2008, with a maximum height of 0.75 m. Corn was planted at the beginning of June 2008 and was harvested in mid–September 2008. The annual mean temperature is 12.2 °C. The annual mean annual precipitation is 48 cm. The isotopic experiment was conducted between April and September 2008, using a TDL analyzer . The sampling frequency is 10 Hz, the averaging interval is 1 h, and the inlet height is 1.6 m to 4.2 m above the ground depending crop height.The Luancheng site 36, Institute of Botany, Chinese Academy of Sciences. The dominant vegetation species are Stipa krylovii, Agropyron cristatum, and Artemisia frigida. The mean air temperature is 2.1 °C. The annual mean precipitation is 38 cm. The isotopic measurement was conducted from June to September 2009 using a TDL analyzer . The sampling frequency is 10 Hz, the averaging interval is 1 h, and the inlet height is 1.8 m above the ground.The Duolun experiment was conducted in a steppe ecosystem in northwest China at the Duolun Restoration Ecology Research Station 38. The experiment was conducted in an arid oasis spring maize, with an area of 5.5 km × 5.5 km. The annual mean temperature is 7.4° C and the mean annual precipitation is 13 cm. The isotopic experiment was conducted from May to September in 2012, using a CIL analyzer . The sampling frequency is 0.6 Hz, and the averaging interval is 1 h. The intake height is 1.5 m to 3.6 m above the ground, depending on crop height.The Heihe site, located in in the middle reaches of the Heihe River watershed  in northwest China, is part of the Heihe Watershed Allied Telemetry Experimental Research (HiWATER) program2) and shallow (mean depth 1.9 m) freshwater lake in the Yangtze River Delta, China39. The annual mean air temperature is 16.2°C and annual mean precipitation is 112 cm. The measurement took place over the water, at a location about 250 m from the north shore . The analyzer type is DLT . The air inlet height is 3.5 m above the water surface. The sampling frequency is 2 Hz, and the averaging interval is 1 h. The measurement started in August 2014 and ended in December 2016.The Taihu site  is part of the Taihu Eddy Flux Network. Lake Taihu is a large , the isotopic measurement was performed in July 2015. The experimental site is located in the Zhanjiang Mangrove National Nature Reserve, close to Yingluo Bay, near Beihai city. This area is influenced by tropical monsoon climate and marine climate, with distinct wet and dry seasons. The total annual rainfall is about 160 cm. The annual mean temperature is 23.0 °C. The Gaoqiao site is a vast wetland consisting of six mangrove species with a mean canopy height of 1.5 m. The analyzer type is CIL (Picarro L2130-i). The sampling frequency is 0.2 Hz, and the averaging interval is 1 h. The analyzer air intake is located at a height of 4 m above the plant canopy.41. The field is sparsely covered by weeds in the spring (March-April), and by rice plants from May until harvesting at the end of August. The maximum canopy height is 1.1 m. The mean annual air temperature of 13.8 °C and the mean annual precipitation is 120 cm. The isotopic measurement was made with a CIL analyzer  with an inlet height of 2 m above the ground. The sampling frequency is 0.6 Hz, and the sampling interval is 1 h.The Mase site , which is part of the AsiaFlux eddy flux network, is located in a rice paddy field in Tsukuba, Japan43, with a sampling frequency of 2 Hz.Nagoya is an urban site located in an urban neighborhood of the City of Nagoya  on the Pacific side of central Japan. The mean annual temperature is 16.0 °C. The mean annual precipitation is 150 cm. The isotopic measurement was conducted from September to November 2011. The inlet height is 15 m above the ground. The analyzer type is DLT (model DLT-100)44. The distance between the two sites is about 200 m. These sites are located in the City of Taipei  on the western Pacific side of northern Taiwan. The annual mean temperature is 23.0 °C. The mean annual precipitation is 240.5 cm. The analyzer type is CIL . The sampling frequency is 0.6 Hz. Meteorological data are obtained from a nearby meteorology station operated by the Center for Weather Bureau, Taiwan (site number: 466920).Water vapour measurement at the Taipei site was carried out with the analyzer’s inlet above a cabin top (inlet height about 3 m above the ground) in the grassland , managed by the Department of Atmospheric Science, National University of Taiwan (NUT), from 11th January to 14th August 2012) and with the inlet at the 7th floor (inlet height 30 m above the ground) of the Department of Geography building on the NUT campus, from 15th August 2012 to 31st March 201346. The isotopic measurement started in February 2010 at an eddy covariance tower in the Center . This site is located in a semiarid mixed savanna ecosystem, which receives an annual mean rainfall of about 50 cm. The annual mean temperature is 20.3° C. The vegetation surrounding the tower has an average height of approximately 4 m and is a sparse mixture of mainly Acacia woody species and C4 grasses. The measurement was made with a CIL analyzer  at a sampling frequency of 1 Hz. The analyzer’s intake height is 22.5 m above the ground.The Mpala Research Center located in Kenya is operated by Princeton University47. The intake height is 3 m above the ground. The mean annual maximum (minimum) temperature is 29.0 (20.8) °C. The mean annual rainfall is about 199 cm. The measurement was conducted over an urban area. The experiment experienced the passage of Cyclone Ita. The averaging interval is 25 seconds.The isotope measurement was made at Trinity Beach, Australia  from April 10 to 13, 2014, using a CIL analyzer  at a sampling frequency of about 1 Hz48 in northwest Greenland  during the summer field campaigns of 2010 (24 May to 3 August), 2011 (5 July to 3 August), and 2012 (21 May to 3 August). The annual mean precipitation is about 20 cm. The annual mean air temperature is about −29 °C, with summer mean temperature of –8.3° C. Water vapour isotope observations were conducted at a 2 Hz with an LGR DLT100 analyzer and a 0.6 Hz Picarro L2120i analyzer (2010 season) and a Picarro L2120i analyzer (2011 and 2012 seasons). The intake height is 1 m and 13 m (2010 season) and 3 m (2011 and 2012 season) above the snow surface. The averaging interval is 24 h.Atmospheric isotopic monitoring was carried out as part of the international deep drilling program at the NEEM siteIvittuut, southern Greenland  is a coastal site 100 m away from the Arsuk Fjord, at approximately 5 km east of the open ocean and 10 km west of the Greenland Ice Sheet. Arctic vegetation, mainly bushes and grasslands, is present in at the site. The annual mean temperature is 2 °C. The annual mean precipitation is 113 cm. The measurement started in September 2011 and ended in September 2012. The instrument type is CIL . Its inlet height is 5 m above the ground. The sampling frequency is 0.6 Hz, and the averaging interval is 24 h.49. While the local mean temperature is –54.3 °C, it was –32.4 °C during the campaign of 2014/2015, reaching a maximum value of –24.5 °C. Ice core data suggest an average annual snow accumulation of 2.7 ± 0.7 g cm–2 yr–1. The isotopic measurement was conducted from December 2014 to January 2015 with a CIL analyzer  at a sampling frequency of 2 Hz. The averaging period is 1 h. The inlet height is 2 m above the snow surface.The Concordia station is located near the top of Dome C , about 950 km from the coast−1 and typical Katabatic winds form around 3 h and vanish around 15 h local time. The mean temperature for the 2014 campaign was similar to the average from 1998 to 2013 (−25° C). Water vapour isotope measurements were performed using a Los Gatos Research (LGR) Inc. analyzer (type DTL-100) from 17th December 2013 to 21th January 2014. The sampling inlet height is 3 m, the sampling frequency is 2 Hz and the average period is 11 min.The Kohnen station  is a summer station located on the Antarctic Plateau, 2892 m above sea level and 550 km from the coast. The surface slope on this site is 1.3 ± 0.3 m km50. On each cruise, the ship left Fremantle in Western Australia in late November, sailed to the Syowa station  on East Ongul Island, Antarctica in late December, and stayed in the Lutzow-Holm Bay near Syowa to early February. The ship then sailed back to Australia, arriving at Sydney in Eastern Australia or Fremantle by the middle of March. The measurement was made on the top deck of the ship with a LGR TDL100 analyzer. The inlet height is 30 m above the sea surface. The sampling frequency is 2 Hz, and the averaging interval is 1 h. Hourly meteorological variables were measured at the height of 30 m above the sea surface.The vapour isotopic measurement was conducted on the icebreaker Shirase along the Japanese Antarctic Research Expedition (JARE) cruise track during two Australian summers in 2013/2014 (JARE55) and 2014/2015 (JARE56)et al.51 and the data  are also available at Data Citation 3.Isotopic measurements were made in the Atlantic marine boundary layer on five research cruises, including STRASSE, PIRATAFR24, RARA, ACTIV and BERMUDA, between 2012 and 2015. These measurements were made with laser spectroscopy . The analyzer’s inlet height varies between 10 and 20 m above the sea level. These cruises took place in the Atlantic Ocean between 4° S to 63° N. The average period is 15 min. More detailed information about these five cruises is given by Benetti 52. Measurements of water vapor mixing ratio and its deuterium isotope ratio δD were made with a custom-designed tunable diode laser spectrometer ISOWAT II at altitudes between 150 m and about 7000 m above sea level. The sampling frequency is 1 Hz. A custom designed working reference generator was used to perform in-flight measurements of a certified water working reference. Extensive pre- and post-flight calibrations were conducted on the ground with a custom designed calibration unit. The in-situ data measured during MUSICA are freely available at: http://share.lsdf.kit.edu/imk/asf/musica/INSITU/.Vertical profiles of water vapor isotopes were measured during the MUSICA project over the Atlantic Ocean near the Canary Islands, Spain. Seven research flights were conducted from the Las Palmas Airport  on 21, 22, 24, 25, 30, 31 July and 01, August 201353, on board of a research aircraft in September to November 2012 (Data Citation 4). The flight altitude varies from 0 to 4500 m above the ground surface. The instrument is Picarro L2130i analyzer customized with a triple-laser setup to allow fast measurement. The data acquisition rate is 1 Hz.Vertical profile measurements were made during the HyMeX SOP1 campaign in the western Mediterraneanhttp://vapor-isotope.yale.edu/.The isotopic and meteorological data are archived at Data Citation 1 and an interactive map of these data are available at The isotopic and meteorological data are recorded in comma separated ascii- format in csv files, and the meta information is stored as PDF files. The meta file contains information about time stamps, data format, time zone convention, missing data flags, variable definitions, reference papers and site contact.18O, standard deviation of δ18O, δD, standard deviation of δD, air temperature, relative humidity, air pressure, precipitation, net radiation, wind speed, wind direction, and air temperature, relative humidity, air pressure, precipitation, net radiation, wind speed, and wind direction extracted from ERA5 , Latitude, Longitude, water vapor mixing ratio, δAn interactive map enables the user to view individual site information and download the dataset. By clicking on the site of interest on the map, the user is directed to the digital data and the metafile for chosen site.The vapour isotope analyzers used by the site investigators fall into two broad categories: tunable diode laser absorption spectroscopy (TDLAS) and cavity-enhanced near infrared laser absorption spectroscopy (CEILAS). Both techniques measure isotopologue concentrations on the principle of the Beer–Lambert Law of light absorption. Detailed information on the analyzer types is given in 18O at the dewpoint temperature of 15 °C32, and 0.2‰ for δ18O at the dewpoint temperature of −10.8 °C19.TDLAS analyzers were used in New Haven, Borden Beijing, Luancheng, Duolun, Great Mountain, Rosemount  and Las Palmas . Laboratory tests of the TGA analyzers show a 1-h precision (one standard deviation) of 1.1‰ for δD and 0.07‰ for δ54. ISOWAT II was used during the MUSICA aircraft campaign . Instead of relying on laboratory bench-test uncertainty estimations which can significantly underestimate in-flight uncertainties, this instrument featured a custom-designed in-flight working-reference source to check the instrument performance several times throughout each flight and at different altitudes of the vertical profile. In addition, extensive pre- and post-flight calibrations were conducted on the ground using a custom-designed calibration gas source. Uncertainties of the in-flight data were dependent on absolute humidity, which changed by 2 orders of magnitude between 25000 ppm near surface and as low as 100 ppm in the upper troposphere. The humidity-dependent uncertainties are provided in the data archive for each 1-second data point. For the majority of in-flight measurements, we found the total uncertainty (combined relative and absolute uncertainties) to be around 10‰ for δD.The ISOWAT II tunable diode laser spectrometer was an advanced second-generation instrument based on the ISOWAT prototype, which was operated onboard the IAGOS-CARIBIC passenger aircraft48. LGR analyzers were used in Qianyanzhou, Taihu, Nagoya, Mapla and Ship-JARE site, while Picarro analyzers were used in Manitou Forests, Niwot Ridge, Kourovka, Rietholzbach, Camargue, Karlsruhe, Teide, Izana, Heihe, Gaoqiao, Mase, Taipei, Trinity Beach, Ivittuut, NEEM, Dome C, Ship-Bermuda, Ship-PIRATA, Ship-RARA, Ship-STRASSE, Ship-ACTIV and Flight-HyMeX SOP1. The key difference between these two types is that CRDS requires smaller cavity volumes due to its on-axis beam geometry while ICOS provides much faster time response and wider dynamical ranges. The precision for 10- min averages is better than 0.04‰ and 0.1‰ for δ18O and δD, respectively, for Picarro analyzers, while for LGR analyzers, the precisions on δ18O and δD measurements are about 0.02‰ and 0.20‰, respectively48, although this hugely depends on the humidity level and there needs to be frequent calibration to reach this point.The Picarro analyzers use cavity-ring down spectroscopy (CRDS) and the LGR analyzers use a later CEILAS technique called off-axis integrated-cavity-output spectroscopy (OA-ICOS)18O, and the difference between the laser measurement and that predicted from a Rayleigh distillation device is 1.1‰ for D/H and −0.06‰ for 18O/16O32. (2) In New Haven, a comparison of the laser measurement of δ18O with cold trap/mass-spectrometry and a Rayleigh distillation device reveals a difference of −0.36 and −0.11‰, respectively55. (3) At Dome C, Allan-Werle variance measurements of the Picarro L2130i analyzer and a high-finesse water isotope spectrometer  shows almost no loss of precision with the analyzer down to a humidity of 450 ppmv (0.2‰ for δ18O and 1.1‰ for δD) to 200 ppmv. (4) At the NEEM site, inter-comparison between Picarro L1102-I and LGR 100 A analyzers  reveals acceptable reproducibility, stability, and precision with standard deviations of 0.23‰ for δ18O and 1.4‰ for δD.Four of the stationary sites have documented cross-validation against other measurement techniques. (1) In Beijing, the difference between the laser measurement and cold trap/mass-spectrometer analysis is 1.2 ± 6.0‰ for δD and 1.1 ± 1.3‰ for δWhile measurement errors can be related to fundamental optical effects such as the coherent nature of laser radiation, the main errors in the isotopic field measurements arise from two sources: (1) absolute isotope correction or standardization to the VSMOW scale, and (2) humidity dependence. Calibration procedures for removing these errors vary between instruments as well as over time. Humidity-dependent calibrations were not always applied, but all measurements were calibrated and reported to the VSMOW scale. The calibration cycle is typically 3 to 6 h, by introducing one or more reference waters into a vapourizer.In CEILAS-based analysis, multiple isotope standards are used for calibration. During the calibration cycle, the analyzer is switched from the ambient vapour sample to the calibration reference stream. Correction to the absolute VSMOW scale and removal of instrument drift are made with a curve fit between the measured values and the known values of the water standards. At a number of sites , the calibration was accomplished with an automated Picarro Standards Delivery Module (SDM). At Nagoya, Mapla, Trinity Beach, NEEM, Ship-JARE and Flight-HyMeX SOP1, an LGR water vapour isotope standard source  with a single or multiple water standards was used for calibration. Some sites, including Borden, New Haven, Great Mountain, Rosemount, Beijing, Luancheng, Ship-Bermuda, Ship-RARA, Ship-ACTIV and Flight-LasPalmas, used a self-made calibration module. Due to difficulties in field operation, the analyzer at Dome C was calibrated in the laboratory after field deployment. The delta values of the calibration standard waters used in each site are summarized in 51. The level of humidity dependence is instrument-specific. Generally, humidity dependence is less significant at humidity levels greater than 5000 ppmv. For drier situations where humidity varies between 2000–5000 ppmv, errors on the order of 1.8‰ for D and 0.6‰ for 18O can be expected56. At very low humidity levels (below 2000 ppmv), standard calibration devices, such as the SDM from Picarro, are not able to generate stable humidity, and confidence is low for composite isotopic variables such as deuterium excess and custom-made calibration devices has therefore been developed.Humidity dependence is characterized by measuring a reference water vapour stream of the same isotopic compositions across a range of humidity conditionset al.19. This system was used in Beijing, Duolun, Heihe, Luancheng, Great Mountain, New Haven, Qianyanzhou and Rosemount. For Teide, Izana, Mase, Gaoqiao, TrinityBeach and Taipei sites, no humidity correction was applied. The humidity dependence correction information also available in Meta file for each site.In Manitou Forests, Niwot Ridge, Kourovka, Rietholzbach, Camargue, Karlsruhe, Nagoya, Mapla, NEEM, Ivittuut, Dome C, Kohnen, ship cruises  and flight cruises , corrections were made for humidity dependence, assuming that the measurement drift is linear or non-linear function of water vapour concentration. Another procedure for removing the humidity dependence is to dynamically adjust the humidity of the calibration stream to match the humidity of the ambient air. This dynamic calibration system is referred to as the ‘dripper’ by Lee The datasets archived on this site are contributed by individual scientists and institutes and have been quality-controlled by the site investigators. In most cases, the data have been published in peer-reviewed journals. We encourage interested parties to contact the site investigators to explore possible collaboration opportunities based on these data.How to cite this article: Wei, Z. et al. A global database of water vapor isotopes measured with high temporal resolution infrared laser spectroscopy. Sci. Data. 6:180302 https://doi.org/10.1038/sdata.2018.302 (2019).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.