Recent studies suggest an important role of immunoglobulin E (IgE) as an alternative pathogenic pathway in the development of bullous pemphigoid (BP), as the most frequent subepidermal blistering disease of the skin Use of IgE targeted therapies, such as omalizumab, has been shown promising in recent studies. The aim of this study was to assess the effect of omalizumab on FcεRI and IgE expression on circulating basophils and on lesional intradermal cells in BP to generate insight into the immunological effects of omalizumab in BP. We report two cases of BP patients treated with omalizumab. Efficacy of treatment was assessed clinically 4 months after initiation of the therapy. Lesional and non-lesional skin biopsies where taken before and 4 weeks after initiation of omalizumab therapy. In addition, FcεRI expression on circulating cells and IgE levels in serum and in the skin samples, as well as anti-BP180 and anti-BP230 in serum and eosinophils and basophils counts in blood were assessed before and during treatment. Both patients showed a marked improvement after 4 months, with no adverse effects. Down-regulation of FcεRI, IgE in lesional skin and on circulating basophils were observed in parallel with clinical improvement. The current case study supports the role of omalizumab in the treatment of a subset of BP patients. Our observations suggest that omalizumab represents a valuable therapeutic option in the management of BP patients. Its efficacy might be related to reduction in FcεRI+ and IgE+ basophils and intradermal cells. Bullous pemphigoid (BP) is a blistering antibody-mediated autoimmune skin disease. BP is associated with tissue-bound and circulating autoantibodies directed against two hemidesmosomal proteins, BP180  and/or BP230  . As a reTwo patients with severe recalcitrant BP treated with omalizumab at Department of Dermatology, Bern University Hospital were included after informed consent. Both patients have suffered from intense pruritus since long time. The previous standard local and systemic therapies could not improve their complaints.Efficacy of treatment was assessed clinically 4 months after initiation of the therapy. Lesional and non-lesional skin biopsies where taken before and 4 weeks after initiation of omalizumab therapy. In addition, FcεRI expression on circulating cells and IgE levels in serum and in the skin samples, as well as anti-BP180 and -BP230 in serum, eosinophils and basophils counts in blood were assessed before and during treatment, as discussed before , 21.After 4 months of therapy (300 mg subcutaneously every 4 weeks), the patients were clinically disease-free and the pruritus subsided completely. The patients did not report any adverse events .The eosinophils, basophils and IgE level in serum were variably affected and differed between the two patients. The BP-180/230 level decreased also slightly in our patients under Therapy. However, levels of FcεRI on circulating basophils decreased dramatically. Furthermore, a significant reduction of IgE+ and FcεRI+ cells in dermis were observed in the immunohistochemistry stainings after 4 weeks of omalizumab treatment , 3. To iOur study in two patients with therapy-resistant BP in which omalizumab was used as rescue therapy has assessed the impact of omalizumab on levels of eosinophils, basophils, and IgE in the circulation, on FcεRI expression on circulating basophils, and on lesional expression of IgE and FcεRI in skin biopsy samples. Our observations suggest that changes in lesional IgE and FcεRI expression might be useful to distinguish the subset of BP patients who benefit from omalizumab therapy.Frequent detection of high levels of IgE autoantibodies against BP180 and BP230, increased serum levels of IgE and the reported beneficial effect of anti-IgE therapy in small case series support the idea that IgE autoantibodies are involved in BP pathogenesis –24. Howe+ cells and IgE+ cells in lesional and non-lesional skin (dermis) of patients   .BP180 and BP230 ELISAs are highly sensitive methods for the diagnosis of BP, in particular BP180 ELISA, is a sensitive tool for monitoring disease activity –33. HoweOur findings support the idea that IgE autoantibodies contribute to tissue damage. Prospective, randomized controlled trials are necessary to confirm that omalizumab represents a new treatment option for BP. However, the current literature suggests that only a subset of patients responds to omalizumab therapy. Therefore, it is paramount to identify biomarkers that predict the best treatment for the individual BP patient. Biomarker development typically relies on a solid understanding of the pathomechanisms at play. In this context, the exact mechanisms by which IgE contributes to pathogenesis in BP is not completely clear. Our data now suggests that optimal clinical responses to anti-IgE treatment in BP might be associated with dissociation of the IgE-FcεRI complex, consecutive down-regulation of FcεRI expression, and finally a decrease in lesional IgE-bearing immune cells. These observations provide novel insight into the immunomodulatory effects at the tissue level of anti-IgE treatment in BP and lay an important basis for future studies.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.Patients gave written informed consent for research use of their data.SS, KG, LF, LB, NY, and CS designed the study and performed acquisition, analysis, interpretation of data, and critical revision of the manuscript for important intellectual content. SS, KG, and CS wrote the manuscript.NY has received honoraria for consulting and advisory board attendance from Novartis. The remaining 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.


Variances in polymers processed by single-screw extrusion are investigated. While vortical flows are well known in the fluids community and fountain flows are well known to be caused by the frozen layers in injection molding, our empirical evidence and process modeling suggests the presence of vortical fountain flows in the melt channels of plasticating screws adjacent to a slower-moving solids bed. The empirical evidence includes screw freezing experiments with cross-sections of processed high-impact polystyrene (HIPS) blended with varying colorants. Non-isothermal, non-Newtonian process simulations indicate that the underlying causality is increased flow conductance in the melt pool caused by higher temperatures and shear rates in the recirculating melt pool. The results indicate the development of persistent, coiled sheet morphologies in both general purpose and barrier screw designs. The behavior differs significantly from prior melting and plastication models with the net effect of broader residence time distributions. The process models guide potential strategies for the remediation of the processing variances as well as potential opportunities to achieve improved dispersion as well as complex micro and nanostructures in polymer processing. The global plastics industry exceeds one billion kg in production output per day, with practically all of this material processed by one or more plasticating screws. While extrusion is a steady-state process, it is highly transient in the reference frame of the polymer. In this article, we seek to investigate and model the plastication behavior in the widely used general purpose and barrier screw designs. Our research is guided by modeling and simulation, which support the understanding of the interaction between the machine design, material properties, and processing condition. We are specifically interested in understanding the polymers’ states as a function of position and time within the extrusion screws during plastication.Maddock  performeThe five-zone melting model  shown inL:D) ratios. The reason for this trend is that longer screw lengths provide greater residence times and associated heat transfer by viscous dissipation to ensure plastication at higher material flow rates. Screw design guidelines have been established [The historical trend has been to increase processing throughput using longer screws and higher length:diameter  single-screw extruder with a bore diameter of 38 mm (1.5 in) was instrumented with an auxiliary process controller  that incorporates real-time computing capabilities with 23 wired analog inputs and 11 wired digital inputs. The instrumentation includes a melt pressure transducer , die pressure transducer , infrared thermocouple , intrusive melt thermocouple , laser micrometer , puller velocity , system power meter , absolute rotary encoder , water bath thermocouple, and others.An extrusion grade of high-impact polystyrene  was processed with the melt properties described in The black, blue and violet blends’ master batches were all polystyrene-based  to ensure compatibility with the HIPS. To investigate the viscosity behavior of the neat HIPS and colored blends, a Dynisco LCR7000 capillary rheometer  was used in compliance with ASTM D3835–08 . The viscosity at different shear rates and three different temperatures  was measured, and is plotted in The extruder was operated at 40 RPM during the addition of the three charges each having 5% of the black, blue, and violet blends. Each charge was added when the prior material being processed fully cleared the feed channel in the first two turns of the screw. Screw freezing experiments (similar to Maddock’s ) were thu as a function of time t and space in three dimensions x by solving the momentum equation [T is predicted according to the energy equation including heat conduction, heat convection, and internal viscous heating given the predicted shear stresses and shear rates PC and thermal conductivity k are modeled as a function of position x and temperature T according to the linear interpolation of the data in A process simulation was developed to investigate the melting and flow behavior of the polymer during plastication with the general purpose screw. The simulation predicts the flow velocity field equation :(1)ρDuDtLF model ,16 with dH/dL at the end of the feed section. Inspecting rows six through eight of 1. Feed Section Packing: Comparing the external images of the general purpose screw of The results suggest that the feed section in the general purpose screw could be shortened by at least two turns, or the rate of taper in the transition section increased so as to more efficiently utilize the full length of the screw.2. Axial Velocity Variation in General Purpose Screw: The varying downstream velocities of the different regions of the channel section are clearly evident from the outer surfaces in 3. Axial Velocity Variations in the Barrier Screw: Similar velocity variation is also observed in the barrier screw, for example with violet material flowing ahead of the blue material at turn 12, as well as blue material flowing ahead of the black material at turn 20. Indeed, there is even more rapid propagation of the melt stream due to the barrier flight, with the blue blended polymer racing far ahead of the black polymer. The sections of dH/dL. However, overall, the melting rate in the solids bed of the barrier screw is very impressive compared with that of the general purpose screw.4. Tadmor and Shapiro Melting Models Inappropriate for Barrier Screw: Viewing the cross-sections of vortical morphology [5. Flow Conductance in Recirculation Zone: The recirculation zones are clearly evident within the general purpose screw, as shown by concentric ovals within the melt pool at the right of the channel (adjacent the active flank) for the cross-sections of turns 14 to 26 in rphology ,19. Simirphology .Regarding the arc length development of the vortex, the tangential velocity of the recirculation zone is much less than the circumferential velocity of the screw relative to the barrel. The cross-section at turn 16 of fountain flow effect [Considering the flow in these recirculation zones, it is recognized that these are areas of relatively high shear, and thus, further viscous heating. The flow conductance is thus higher in the melt channels than the adjacent solids bed, even though they are adjacent in the channel, which explains the previously described axial velocity variation. This axial velocity variation provides a w effect ,22 where6. Significant Variances in the Processed Polymer: The varying polymer structures observed in the sections of the general purpose screw may be problematic for at least two reasons. First, there is a very broad residence time distribution since the polymers in the different regions of the channel are flowing at different rates. The black material stagnating early in the screw channel  will tend to increase the vortical fountain flow due to less time for heat conduction concurrent with greater shear heating. We now believe that this vortical fountain flow is a predominant cause of the “surging” phenomenon in extrusion and loss of control at higher screw speeds.Incorporate additional mixing sections within screw designs. Since the vortical fountain flow is caused by persistently growing melt recirculations in the melt pool adjacent the active flank, intermittently breaking up the melt channel will cause the vortical fountain flows to disperse. All the results, both experimental and theoretical, suggest that the use of early, coarse mixing channels would be highly effective.The evidence suggests that vortical fountain flows have significant effects in polymer plastication. We believe that the developed coiled sheet morphology could be predicted and exploited to achieve improved dispersion or create micro or nanostructured morphologies within the screw channel. However, higher fidelity material constitutive and processing models are needed to guide researchers and practicing engineers to improve processing capabilities and fully exploit the potential of the processed polymeric systems and composites. For now, variances in temperature and flow rate and pressure will adversely impact most polymer processing applications. To minimize the vortical fountain flow and its adverse effects, practitioners should:


Small reactive oxygen and nitrogen species (ROS/RNS) driven signaling plays a significant role in wound healing processes by controlling cell functionality and wound phase transitions. The application of cold atmospheric pressure plasma (CAP), a partially ionized gas expelling a variety of ROS and RNS, was shown to be effective in chronic wound management and contrastingly also in malignant diseases. The underlying molecular mechanisms are not well understood but redox signaling events are involved. As a central player, the cellular tumor antigen p53 governs regulatory networks controlling proliferation, death, or metabolism, all of which are grossly modulated by anti- and prooxidant signals. Using a human skin cell model, a transient phosphorylation and nuclear translocation of p53, preceded by the phosphorylation of upstream serine- (ATM) and serine/threonine-protein kinase (ATR), was detected after CAP treatment. Results indicate that ATM acts as a direct redox sensor without relevant contribution of phosphorylation of the histone A2X, a marker of DNA damage. Downstream events are the activation of checkpoint kinases Chk1/2 and several mitogen-activated (MAP) kinases. Subsequently, the expression of MAP kinase signaling effectors , epithelium derived growth factors, and cytokines (Interleukins 6 + 8) was increased. A number of p53 downstream effectors pointed at a decrease of cell growth due to DNA repair processes. In summary, CAP treatment led to an activation of cell repair and defense mechanisms including a modulation of paracrine inflammatory signals emphasizing the role of prooxidant species in CAP-related cell signaling. Their occurrence can be controlled by plasma source design and discharge parameter engineering, especially the composition of the working gas. In vivo, stimulatory effects in chronic or acute wound healing  or in humans  were reported [Cold physical plasma (CAP) is an emerging biomedical technique and found to interfere with processes controlled by redox signaling d cancer –6. Diffed cancer –10. In preported , 11–14. reported –20. Incrreported , 22. Thereported . Furtherreported , 25. A wreported –28.A complex, not fully understood relationship evolved around p53 and redox signal transduction checking free reactive oxygen or nitrogen species (ROS/RNS) levels and controlling cell fate . The actPublished data on CAP effects in cells or tissues suggest a role for p53, its downstream targets, and connected pathways such as the mitogen-activated protein (MAP) kinases in governing the cellular response towards CAP-derived ROS/RNS . PotentiA strong activation of the MAPK – p53 axis was found after CAP, emphasizing that plasma-derived ROS/RNS have a significant impact on cell fate and performance. The involvement of p53 phosphorylation indicates a substantial impact on cellular processes necessary to adapt to CAP treatment. An activation of cell protective processes accompanied by an increased expression of growth factors and cytokines relevant in wound underlines the use of CAP in wound management and other redox-signaling related conditions.2 [6 cells were seeded in 60 mm dishes . As cold physical plasma source, the kINPen 09  was utilized. This plasma jet consists of a central pin-type electrode that ignited a plasma by applying a voltage of 2–6 kV at a frequency of around 1 MHz. Argon  was used as feed gas (3 standard liters per minute). For all experiments, an indirect treatment regimen was chosen to assure homogeneity of the treatment and it was achieved by exposing 5 ml of RPMI w/ all supplements to the plasma effluent at a distance of 9 mm using an automated xyz-table. The treated liquid was transferred immediately to the prepared cells.HaCaT keratinocytes were cultivated in RPMI 1640 cell culture medium containing 8% fetal bovine serum , 2 mM glutamine, 0.1 mg/ml streptomycin, and 100 U/ml penicillin  at 37°C, 95% relative humidity, and 5% CO2DCF-DA . Cells were stained with 1 μM of dye for 20 min, prior to plasma-treated medium was added, and evaluated 5 min thereafter by fluorescence microscopy. Cell viability was assessed using the CellTox™ Green Cytotoxicity Assay . Briefly, 15,000 cells were seeded in 96 well plates 24 h prior to experiment. 24 h after indirect treatment, the dye was added. After 15 min, the fluorescence intensity was measured at λex 490 nm and λem 525 nm using a microplate reader . To determine late apoptosis, a Gallios flow cytometer and Kaluza software  were applied 18 hours after indirect treatment using Green Caspase-3 Kit  according to the manufacturer's protocol.Changes of intracellular redox levels were determined using CM-Hw/v) paraformaldehyde for 20 min, permeabilized with PBS/0.1% Triton X-100 for 20 min, and treated with 3% bovine serum albumin (BSA) in PBS. Slides were washed twice with 1% BSA in PBS and incubated at 4°C overnight with p53 antibody . Afterwards, cells were washed twice with PBS and incubated with secondary antibody Alexa Fluor 488-conjugated goat anti-rabbit  for 1 h. Coverslips were washed again with PBS and mounted using VECTASHIELD® with DAPI . Samples were observed using an Axio Observer Z1 .HaCaT cells were grown on glass coverslips for 24 h, and plasma treated as indicated. After appropriate time points, cells were fixated with 4% , and total mRNA was reversely transcribed using Transcriptor First Strand Synthesis Kit . Primer specificity was confirmed by separating PCR amplification products in an agarose gel. Quantitative real-time PCR was performed using the Fast Sybr Kit  and a LightCycler 480 . Gene specific primers for BAX, BBC3, GADD45, and CDKN1A were used –47 at a β-mercaptoethanol, 0.001% bromophenol blue). Proteins were separated on a 10% SDS-PAGE Gel  and blotted onto a Roti®PVDF membrane . After blocking in TBS-T , blots were incubated with Erk1/2 (#9102), Mek1/2 (#9126), Sapk/Jnk (#9258), p38 (#9212), p53 (#2527) as well as phospho-specific antibodies for p-ATM , p-ATR , p-Chk1 , p-Chk2 , p-Erk1/2 , p-p38 , p-Mek1/2 , p-Sapk/Jnk , p-HSP27 , p-p53 , and p-p53 , all 1 : 1000 in TBS-T at 4°C overnight . Then, procedure was preceded by 1 h incubation with secondary antibody  1 : 10,000 in TBS-T and followed by incubation with ECL reagent. Chemiluminescence was detected by ImageQuant LAS 4000 and analyzed by ImageQuantTL . Phosphorylated protein levels of p53-dependent kinases were normalized to β-actin (housekeeping). Analyses of secreted proteins were performed using the enzyme-linked immunosorbent assay (ELISA). Human IL-6, IL-8, and GM-CSF were detected using ELISA Max™ kits  and human VEGF-A using ELISA . Procedures were performed according to the manufacturers protocols.Cells were plasma-treated, rinsed with ice-cold PBS, and then lysed in ice-cold RIPA lysis buffer containing protease and phosphatase inhibitors  and 2 mM phenylmethanesulfonylfluoride . The protein concentrations were equalized and samples were heated to 95°C for 5 min in Laemmli buffer . Statistical comparison between experimental groups was done using one-way analysis of variances followed by 2DCF  showed an intensely increased Ser37 phosphorylation, whereas Ser15 phosphorylation level was not affected (data not shown).The nuclear localization of p53 is caused by activation of p53 through phosphorylation of serine 15 (Ser15) and serine 37 (Ser37). The phosphorylation levels one hour after plasma treatment showed a clear dependence on treatment intensity. Phosphorylation on Ser15 was increased after 60 s and more clearly after 180 s . In compμM H2O2) led to a very slight rise in p-ATR, whereas the p-ATM levels raised profoundly (data not shown). No impact on phosphorylation status and total protein expression of γ-H2AX by indirect plasma treatment was detected .Phosphorylation level of Chk1, the kinase downstream of ATM, was tightly coupled to plasma treatment and incubation time. A more than twofold increase of Chk1 phosphorylation after 60 s and a sixfold upregulation after 180 s of treatment was detected . After t2O2 did not lead to increased Erk phosphorylation but induced phosphorylation of p38 and Jnk only (data not shown). All three MAP kinase phosphorylation levels returned to their baseline 24 h after plasma treatment.The phosphorylation level of the mitogen-activated protein kinases Erk1/2 (MAPK1/2) increased steadily with treatment intensity and peaked at nearly fivefold increase after 180 s . A similTo clarify the signaling cascade, we checked the downstream activation of p53 target genes by quantitative real-time PCR (qPCR). The expression levels of BAX and BBC3 (proapoptotic pathway), GADD45 (DNA repair), and CDKN1A/p21  were not significantly altered by 60 s of plasma treatment after 3–12 h . The six2O2 control, phosphorylation of Hsp27 was only mildly stimulated (data not shown).The MAP kinase and ROS/RNS-associated heat shock protein 27 (Hsp27), a downstream effector of the p38 signaling cascade, showed a clear correlation of phosphorylation level with treatment time. Similar to the p38, the maximal phosphorylation level was reached after 180 s of treatment . In H2O2e.g., growth factors, and cytokines are strongly regulated by the MAP kinase activity. The plasma treatment had a significant impact on mRNA level resulting in a three- to fivefold upregulation of growth factors such as the vascular endothelial VEGFA and heparin-binding growth factor HBEGF as well as the colony-stimulating factor CSF2. For IL-6 as a proinflammatory cytokine, the mRNA levels showed a significant upregulation of DNA transcription, in contrast to IL-8.Gene expression and protein secretion of several cell-signaling molecules, The secretion of the two cytokines (IL-6/8) and growth factors (GM-CSF and VEGFA) was further measured using ELISA. Cytokine profiling indicated that IL-6 and IL-8 are most significantly increased in 180 s treated cells after 12 h . The secCold atmospheric plasma generates a mixture of reactive oxygen or nitrogen species (ROS/RNS) in the gas phase that in contact with liquids lead to the deposition of secondary/tertiary species. Among those, singlet oxygen, atomic oxygen, hydroxyl radicals, and hydrogen peroxide are major contributors –50. Sinc2-DCF (after ester cleavage) since no reporter dyes that are accepted of being able to distinguish between different intracellular ROS are commercially available. Intracellular compartmentalization increases complexity that is not addressed by the simple dye. A promising but demanding approach in this regard is thiol switch dyes (HyPER) [2O2 without the presence of DNA strand breaks [γ-H2AX was only nonsignificantly increased by CAP seems to contribute to the above conclusion as only a strong γ-H2AX activation points at DNA damage as initial effect. Additionally, DNA damage was not reported a major route in vitro and in vivo for the plasma device investigated here [γ-H2AX phosphorylation in human colon cells, combined with an increase of Chk2 activation [γ-H2AX seen by the authors. Paralleling the low increase of γ-H2AX, only small changes in ATR phosphorylation were seen for all plasma treatment times. This indicates that only a limited and transient oxidative damage to cellular DNA occurred. The activation of ATR is also reflected by the Ser37 phosphorylation of p53 that the kinase can be responsible for; yet, also ATM is able to phosphorylate p53 at Ser37 to a minor extent [After CAP treatment, an increase of intracellular ROS was observed in HaCaT cells using an unspecific redox-sensitive dye which is in line with previous findings , 63. How (HyPER) . Subsequ (HyPER) . Reports (HyPER) . Besides (HyPER) –69. It wd breaks . Observad breaks . In an Ad breaks , 73. Pred breaks . It remated here –76. Howetivation . In contr extent . Indicatr extent , 75, 79.2−, 1O2) at the cell membrane via oxidation of the epidermal growth factor receptor [2O2 which is able to cross the cell membrane via aquaporins [A connected pathway is the mitogen-activated protein (MAP) kinase signaling. MAP kinases have a direct role in translating physical stimuli into biochemical signals. The phosphorylation of p53 at serine 15 by p38 kinase was detected after UVB irradiation . In addireceptor  or lipidreceptor , 87. Theuaporins . Downstruaporins –91. Hsp2uaporins . Furtheruaporins . Having uaporins . Plasma uaporins . An overThere is mounting evidence that p53 modulates the wound healing processes , and theCold plasma treatment leads to complex, interwoven redox signaling events in mammalian cells via the impact of short- and long-lived ROS/RNS. Signaling related to the p53 axis was found to be a major hub of cold plasma-cell interaction, along with the upstream redox sensors ATM and ATR. Further, MAP kinase signaling accompanied and modulated the p53 signals. Both, negative (p53 activation) as well as positive (Erk activation) effects on cellular longevity were detected for human keratinocytes. A treatment intensity dependence of the observed proapoptotic, proinflammatory, and prosurvival effects as well as a general activation of redox-related proteins showed a timeline dependency of all changes. Functional consequences with regard to the clinical use of plasmas in wound treatment are a reduction in cell proliferation, a transient increase in cell migration, and secretion of immunomodulatory signal proteins.


Eighty-one percent of the screened flocks (n = 22) were positive for IBV infection. Thirty of these isolates were successfully characterized using molecular tools targeting the most variable spike (S) 1 gene. IBV isolates from this study clustered into three genotypes based on partial S1 gene variability. The majority of the IBV isolates (70%) were Massachusetts (Mass) type, and the rest were either Connecticut (Conn) type or an uncharacterized genotype with genetic characteristics of Mass and Conn types. Since the majority of the IBV isolates included within the Mass type, we used a Mass type IBV isolate to reproduce SES in specific pathogen free (SPF) white leghorn chickens in lay. Further studies are warranted to investigate whether other IBV isolates can cause SES, to clarify the pathogenesis of SES and to develop a vaccine in order to prevent SES as observed in Western Canadian layer flocks.A disease with a sudden drop in egg production and shell-less eggs called, shell-less egg syndrome (SES) has been observed in Western Canada egg layer flocks since 2010. The etiology of this disease is not known. We hypothesize that SES is caused by an infectious bronchitis virus (IBV) strain since it is known that IBV replicates in the shell gland causing various eggshell abnormalities. In this study, we screened egg layer flocks, in the provinces of Alberta (AB) and Saskatchewan (SK), with and without a history of SES for the presence of IBV infection. During 2015–2016, a total of 27 egg layer flocks were screened in AB ( Since 2010 an increasing number of observations of transient shell-less egg production in layer operations located in Saskatchewan (SK), Canada was reported, and recently, this was also reported in Manitoba and Alberta (AB). These are sporadic outbreaks in layers of varying ages, during lay and affected flocks generally show a drop in egg production in addition to the production of shell-less eggs. This condition is known as “shell-less egg syndrome” (SES) and is thought to be caused by a strain of IBV since some strains of IBV are known to target the shell gland of the reproductive tract where the shell of the egg is formed .Egg production in laying chicken can be affected by a number of avian viruses including hepatitis E virus, Newcastle disease virus (NDV), low pathogenic avian influenza virus (LPAIV), avian encephalomyelitis virus (AEV), infectious bronchitis virus (IBV), and egg drop syndrome virus (EDSV). All these viruses except AEV can affect the internal and external quality of eggs ,7,8,9,10Coronaviridae, however, thus far there are no reports that indicated IBV could cause shell-less eggs. Although no specific reproductive-tract-tropic IBV strains are known, some IBV strains can cause lesions in the reproductive tract and the respiratory tissues [IBV is a host-specific respiratory pathogen, which belongs to the family  tissues . IBV rep tissues . Disrupt tissues . Also, i tissues .spike protein (S) 1 gene is the most variable and the main inducer of virus-neutralizing antibodies, while S2 is relatively conserved [S1 gene is the foremost technique that is used to differentiate IBV strains into various genotypes [S1 gene obtained during IBV infection outbreaks in Ontario, Quebec, British Columbia, Nova Scotia, and Newfoundland and Labrador. The characterized IBV isolates of these Canadian provinces belonged to nine genotypes [S1 gene amplification, detected a new phylogenetic cluster [S1 gene was used for characterization of five field isolates from Ontario, Canada, and this study reported the amino acid similarity of 55 to 71% to the M41 reference and MILDVAC-Ma5 vaccine strains [The IBV genome undergoes mutations and recombination which can lead to changes in virulence and disease pathogenesis ,18,19. Tonserved ,21,22,23enotypes ,25. IBV enotypes ,27 throuEthical approval to carry out this study was obtained from the Health Science Animal Care Committee (HSACC) of the University of Calgary, protocol number AC15-0140, permission date (30 October 2015).Fertile specific-pathogen-free (SPF) eggs and chickens were purchased from the Canadian Food Inspection Agency (CFIA).n = 20) and AB (n = 7) from the beginning of 2015 to the end of 2016. In SK, 8 out of 12 flocks had a history of SES. In AB, 4 out of 7 flocks had a history of SES. The production-related data along with IBV serology results of the observed layer flocks are given in the Trachea, lung, kidney, uterus, and cecal tonsil (CT) samples were obtained from layer farms with and without a history of SES in the Western Canadian provinces of SK (nucleoprotein (N) gene was used. The real-time PCR positive samples were subjected to conventional PCR assay targeting partial S1 gene. Then, the samples that were negative in partial S1 conventional PCR assay were propagated in 9–11 days old SPF embryonated eggs. After each round of propagation, collected allantoic fluid was subjected to a conventional PCR assay to amplify the partial S1 gene for sequencing.To identify IBV positive samples, a real-time polymerase chain reaction (PCR) targeting the conserved g at 4 °C for 10 min and the supernatant was stored at −80 °C until further processing.A piece of tissue (40 mg) was homogenized in 1 mL of cold PBS using a Pro200 Power homogenizer  on ice. The tissue homogenates were centrifuged at 1000× ® reagent  following the manufacturer’s protocol. The RNA concentration was determined using a Nanodrop 1000 spectrophotometer at 260 nm wavelength  and the RNA quality was determined using the Nanodrop based on the 260:280 UV absorbance ratio. About 2000 ng of extracted RNA was converted to complementary DNA (cDNA) using random primers (hexamers of d(N)6) . Also, a ‘no reverse transcriptase’ (NRT) control was included in the cDNA conversion step to detect cDNA contamination.Total RNA was extracted from 250 µL of tissue homogenate using the Trizol LSN gene using primers: Fw-5′GACGGAGGACCTGATGGTAA3′ and Re-5′CCCTTCTTCTGCTGATCCTG3′, as described previously [Previously published real-time PCR assay was used to amplify a 200 nucleotide fragment of the IBV-eviously . Melt cueviously .N-gene real-time PCR positive samples that were negative in partial S1 conventional PCR were passaged in nine days of age embryonated SPF chicken eggs and PBS was used as a negative control. Embryos were inoculated with homogenates originating from tissue samples in the allantoic cavity. Allantoic fluid was harvested at three days post-infection (dpi) and stored at −80 °C in aliquots of 0.5 mL.Briefly, tissue homogenates of S1 gene  using the primers Fw-5′GTKTACTACTAYCAAAGTGCCTT-3′ and Re-5′GCATGCWARCAARCCTCTAGG-3′ [® gel purification kit  following the manufacturer’s instructions.A conventional two-step reverse transcriptase (RT)-PCR assay was carried out to amplify a fragment of 620 nucleotides of the IBV CTAGG-3′ . Both po® 10.1.3, Biomatters Ltd., Auckland, New Zealand) with the highest sensitivity recommended for Sanger sequencing products. The aligned sequences were trimmed at both ends to the size of the shortest sequence read obtained. The resulting partial IBV S1 nucleotide sequences (508 nucleotides) from the current study were deposited in the GenBank repository with the accession numbers ranging from MH509448 to MH509477 and were aligned with the published sequences of IBV reference strains and serotypes including Canadian strains [® 10.1.3); mBed cluster size was set to 100. Forty IBV sequences that were retrieved from the NCBI GenBank database using the nucleotide BLAST® search tool representing common serotypes, vaccine strains and Canadian field IBV strains are listed in Sanger sequencing was performed at the Center for Applied Genomics  for both forward and reverse directions. The resulting sequences were visually checked. Sequences were manually edited and proof-read with reference to IBV M41 (GenBank accession: AY851295). Both forward and reverse contigs were de-novo assembled into one contig using Geneious assembler . Possible clustering of new IBV isolates was determined using the generated phylogeny along with similarities and dissimilarities to the reference serotypes used. Translate function of the Geneious software was used along with open reading frame (ORF) 1 following the standard genetic code. Translated amino acid sequences were realigned using the Global alignment with Blosum62 cost matrix. The RAxML phylogenetic tree was generated using RAxML plugin version 8.2.7 under the protein model GAMMA BLOSUM62 with Rapid bootstrapping and search for best-scoring ML tree with 1000 bootstrap replicates, with parsimony random seed is set to 1 [Aligned nucleotide sequences were used to construct an unrooted phylogenetic tree by Randomized Accelerated Maximum Likelihood (RAxML) method  using thset to 1 . Furtherset to 1 .Since 70% of IBV isolates originated in our survey were Mass type, we hypothesized that SES is likely to be caused by a Mass type IBV isolate originating from Western Canada layer flocks. The Mass type IBV isolates 15AB-01 (originating from AB) and 15SK-02 (originated from SK) were selected for experimental reproduction of SES. Before being used, the inoculums of 15AB-01 and 15SK-02 were screened for the presence of LPAIV , EDSV 3, and NDVn = 4) and allowed to acclimatize for 2 weeks in high containment poultry isolators. The feed was a commercial layer ration, which contained 17% of calcium and birds were provided 12-h light/dark cycle a day throughout the experimental period. At 26 weeks of age, 1 and 2 chickens were infected with the IBV isolate 15AB-01 and 15SK-02, respectively, with a dose of 1 × 106 EID50 per bird under isoflurane anesthesia. A total volume of 400 µL per bird was used with 250 µL intratracheally, 100 µL intranasally, and 50 µL via the intra-ocular route. One chicken was treated as an uninfected control . Daily egg production was noted and checked for any shell abnormalities for 12 dpi.Twenty-four-week old white leghorn SPF chickens in lay were obtained  and stored at −20 °C. The tissues were also collected into 10% neutral buffered formalin for histological observations. Chickens were also bled at 3, 7, 15, 18, and 21 dpi into plain tubes containing no anticoagulants. Blood tubes were left on the bench at room temperature for 30 min and centrifuged at 2000× g for 10 min at 4 °C, the serum was separated, and aliquots were stored at −80 °C until further analysis.Six 18-week old SPF laying chickens were obtained from the CFIA, Ottawa, ON and transferred to high containment poultry isolators. Then the chickens were allowed for a 7-week period of acclimatization and stabilization of egg production with access to ad libitum feed and water. The feeding and lighting were done as indicated in the pilot experiment. At 25 weeks of age, 3 chickens were infected with the Mass type IBV field isolate (15AB-01) with a dose of 1 × 10Formalin-fixed terminal tissue samples (21 dpi) were processed at the Diagnostic Services Unit of the University of Calgary’s Faculty of Veterinary Medicine including staining of sections with stained hematoxylin and eosin (H & E). Sections were examined under the light microscope and photomicrographs were taken under 40× magnification.Serum anti-IBV antibody titers were determined using the IDEXX IBV antibody test kit  following the manufacturer’s instructions. The plates were read at 650 nm absorbance using SpectraMax M2 microplate reader .IBV RNA copies per 200 ng of cDNA were estimated based on the standard curve generated using a serial dilution of IBV plasmid.t-test. The Chi-square test with Yates correction was used to compare the total marketable egg production between the infected and uninfected control groups during the observation period. The Fisher’s exact test was used to compare the difference in marketable egg production between the infected and uninfected control groups at each day. IBV genome loads of the tracheal and cloacal swab samples at different time points were analyzed using Two-way ANOVA followed by a Bonferroni post-hoc test. IBV genome loads between tissues collected at 21 dpi were compared with One-way ANOVA followed by Tukey’s multiple comparisons. All statistical tests were performed using the built-in statistical analysis feature of the GraphPad™ Prism 7 . Differences were considered significant at p < 0.05.Geometric mean IBV ELISA titers of the surveyed flocks with and without a history of SES were compared using student’s p = 0.7230) that had a history of SES (4147.86 ± 1067.39) and the flocks without a history of SES (3596.00 ± 918.13).The relevant background data including IBV vaccination history and IBV specific antibody titers of the surveyed layer flocks with and without a history of SES are given in the N gene real-time PCR assay. IBV positive samples originated from 22 farms (81% of the examined flocks), and 4/7 and 18/20 farms were positive for IBV genomes in AB and SK, respectively  belonging to 27 farms with and without a history of SES and of which 79 samples (13% of the examined samples) were positive in the IBV ectively . Overallectively .S1 conventional PCR without being propagated in eggs. Nine real-time PCR positive samples that were negative for partial S1 conventional PCR were egg passaged. About 6–7 cycles of repeated egg passages were required for these 9 samples to obtain adequate IBV titers to amplify partial S1 gene by conventional PCR. Based on the phylogram drawn using the IBV S1 short fragment nucleotide sequence, 21 (70%) IBV isolates clustered with the Mass type IBV. Three (10%) IBV isolates formed a sub-cluster within the Conn type while 6 (20%) isolates formed a separate uncharacterized cluster . However, the difference in marketable egg production between the two groups at each day was not statistically significant (p > 0.05).All the chickens infected with 15AB-01 IBV isolate did not produce regular eggs for 10 dpi. The IBV infected group of chickens produced 2 and 1 shell-less eggs on 8 and 9 dpi, respectively. Thereafter for 3 days, the infected chickens produced 1 regular egg per day. On 16 dpi, the infected chickens produced 2 regular eggs and 1 shell-less egg. From 18 dpi until the end of the study (21 dpi), the infected chickens produced 2 regular eggs per day . In compp < 0.0001).The uninfected controls showed no detectable levels of the IBV genome in any tissue or tracheal and cloacal swabs throughout the observation period. p < 0.05).IBV genome loads in tissues collected at 21 dpi can be found in p < 0.01) onwards.The antibody response in serum collected from IBV infected and control chickens is illustrated in The mucosal folds of the shell gland of 2 out of 3 infected chickens showed edema a–c when The described studies led to several major findings. First, IBV infections in Western Canada layer flocks are caused mainly by strains related to Mass and Conn. Second, we did not observe a difference in IBV infection between flocks with and without a history of SES in Western Canada. Third, the majority (70%) of IBV strains detected in layer flocks were Mass-type IBV. Fourth, the field IBV isolates that formed a separate cluster II did not align with any of the known reference sequences. Fifth, SES recently observed in Western Canada layer flocks is linked to a Mass type IBV isolate.The IBV strains detected in AB and SK are unknown until now and the current investigation records IBV strains isolated from layer flocks in these two provinces of Canada. Unlike in other Canadian provinces, the IBV strains observed in Western Canada are mainly of Mass and Conn genotypes. However, further studies are required in order to classify these IBV isolates serologically. Some data on the incidence and distribution of IBV isolates have been published in Canada, but there was limited information available about strains in the Western regions, notably AB and SK. In Quebec, between 1976 and 1980, 24 isolates of IBV have been characterized based on serum neutralization assay  which inIBV infection in very young chickens can cause permanent damage to the oviduct, resulting in the cystic oviduct and fully grown false layer chickens . IBV infIn conclusion, we found that the majority of the IBV strains isolated from layer flocks affected with SES in SK and AB were Mass type. Similar to EDSV, the Mass type IBV isolate, 15AB-01 was capable of experimentally inducing shell-less eggs in SPF chickens, consistent with the observed association of this isolate with SES in the field outbreaks. Further investigations are warranted to elucidate the pathogenesis of SES and identifying an appropriate vaccine for the control of SES in layer flocks in Western Canada.


Aromatic 1H CPMG relaxation dispersion experiments in phenylalanine, tyrosine and the six-ring moiety of tryptophan, however, are affected by 3J 1H–1H couplings which are causing anomalous relaxation dispersion profiles. Here we show that this problem can be addressed by site-selective 1H/2H labeling of the aromatic side chains and that artifact-free relaxation dispersion profiles can be acquired. The method has been further validated by measuring folding–unfolding kinetics of the small protein GB1. The determined rate constants and populations agree well with previous results from 13C CPMG relaxation dispersion experiments. Furthermore, the CPMG-derived chemical shift differences between the folded and unfolded states are in excellent agreement with those obtained directly from the spectra. In summary, site-selective 1H/2H labeling enables artifact-free aromatic 1H CPMG relaxation dispersion experiments in phenylalanine and the six-ring moiety of tryptophan, thereby extending the available methods for studying millisecond dynamics in aromatic protein side chains.Aromatic side chains are often key residues in enzyme active sites and protein binding sites, making them attractive probes of protein dynamics on the millisecond timescale. Such dynamic processes can be studied by aromatic  This has been demonstrated on the folding/unfolding of protein GB1 at high temperatures.Here we demonstrate that in site-selective 1H and site-selective 13C labeled, using 2 g/L 2-13C1 glucose  or 440 µM (site-selective 1H labeled) protein in 20 mM HEPES and 10% (v/v) D2O at pH 7.0. Small amounts of NaN3 were added.Uniformly 1H CPMG relaxation dispersion experiments were performed using a relaxation compensated approach as published before . No 2H decoupling was used for the experiments. Experiments were typically performed with 72 number of scans resulting in a experimental time of 2 days. This was done to assure the highest data quality possible for the establishment of the method. Further the dispersion step is quite moderate and there are only three positions for the global fit. Spectra were processed with NMRPipe , 2 Phe (F30 and F52) and 3 Tyr (Y3 and Y33 and Y45).13C labeling in Phe ε* (F30 and F52), Tyr ε* (Y3 and Y33 and Y45) and Trp ζ3 (W43). In the uniformly 1H labeled sample (based on 2-13C1 glucose) Trp δ1 and ζ2 . If normalized by the protein concentration the site-selective 1H labeled sample will display 5.5 times the signal strength for Phe ε* (F30 and F52) and three times the signal strength for Trp ζ3 (W43). In case of Phe ε*, with about 20% 13C incorporation from 2-13C1 glucose . In case of Tyr the site-selective labeling did not work well (around 4% 13C incorporation). The reason for this is not clear. As a direct consequence of this only a limited number of experiments could be performed on Tyr. On the other hand this highlights the selective incorporation of the Phe precursor in Phe. Results of 13C incorporation are summarized in Table Both labeling methods result in site-selective 3J) to the 13C bound proton we performed 2D 1H13C-HSQC-1H1H-TOCSY experiments. For the uniformly 1H labeled samples we observe strong cross signals to vicinal (and for Trp ζ3 4J) protons  or a high amount of  deuteration of vicinal protons, that are believed to cause artifacts in aromatic 1H CPMG relaxation dispersion experiments  or pretty good (Trp ζ3) in terms of producing high amounts of isolated 1H–13C spin pairs in aromatic side chains. Labeling for Tyr ε* are shown to be on the same level in previous attempts  and 40 °C (exchange conditions) , 0.29 s−1 (F52) and 0.39 s−1 (W43). Furthermore, R2 values at high refocusing frequencies are around 15 s−1 higher in the uniformly 1H labeled sample. 8 s−1 can be directly attributed to the 3J 1H–1H coupling , the artifact is reduced but still there. Under exchange conditions at 40 °C one can record pronounced relaxation dispersion profiles for the site-selective labeled sample , and established that this problem can be eliminated by the use of site-selective 1H labeled precursors.Next, we recorded 1H CPMG relaxation dispersion experiments using site-selective 1H labeled samples on a known exchanging system, GB1 at 40 °C (Weininger kex) of (94 ± 5) s−1 and a population of the unfolded state (pu) of (2.8 ± 0.1)%. The derived chemical shift differences from the relaxation dispersion profiles are in excellent agreement with shift differences directly derived from spectra at 40 °C, that show the characteristics of an unfolded protein  3J 1H–1H coupling constants. Correct parameters of unfolding of GB1 could be derived by 1H CPMG relaxation dispersion experiments on a site-selective 1H and 13C labeled sample. By site-selective 1H/2H labeling one can therefore extend the positions suitable for aromatic 1H CPMG relaxation dispersion experiments to Fε and Wζ3 specifically and Yε in principal (this work) and Fδ, Fζ, Yδ, Wε3, Wη2 and Wζ2 (using suitable precursors), thereby extending the arsenal of aromatic probes for the study of ms dynamics.We have demonstrated that artifact-free aromatic


Plant phenology will likely shift with climate change, but how temperature and/or moisture regimes will control phenological responses is not well understood. This is particularly true in Mediterranean climate ecosystems where the warmest temperatures and greatest moisture availability are seasonally asynchronous. We examined plant phenological responses at both the population and community levels to four climate treatments  embedded within three prairies across a 520 km latitudinal Mediterranean climate gradient within the Pacific Northwest, USA. At the population level, we monitored flowering and abundances in spring 2017 of eight range‐restricted focal species planted both within and north of their current ranges. At the community level, we used normalized difference vegetation index (NDVI) measured from fall 2016 to summer 2018 to estimate peak live biomass, senescence, seasonal patterns, and growing season length. We found that warming exerted a stronger control than our moisture manipulations on phenology at both the population and community levels. Warming advanced flowering regardless of whether a species was within or beyond its current range. Importantly, many of our focal species had low abundances, particularly in the south, suggesting that establishment, in addition to phenological shifts, may be a strong constraint on their future viability. At the community level, warming advanced the date of peak biomass regardless of site or year. The date of senescence advanced regardless of year for the southern and central sites but only in 2018 for the northern site. Growing season length contracted due to warming at the southern and central sites (~3 weeks) but was unaffected at the northern site. Our results emphasize that future temperature changes may exert strong influence on the timing of a variety of plant phenological events, especially those events that occur when temperature is most limiting, even in seasonally water‐limited Mediterranean ecosystems. At the community scale, we focused on the seasonality of growth and senescence of canopy biomass. We asked: (a) How will the phenology of individual species, as well as prairie plant communities, respond to climate change across a latitudinal gradient? (b) Will range‐restricted species’ phenological responses and flowering abundances differ in direction and/or magnitude when planted within versus beyond their current northern range limits? And, (c) will changes to soil temperature or moisture be more predictive of phenological responses?22.1The study was conducted at three sites from southwestern Oregon to central‐western Washington in the Pacific Northwest (PNW)  were randomly assigned to one of four climate treatments with five replicates each: control (ambient temperature and precipitation), warming (canopy temperature raised by 2.5°C), warming with additional precipitation , and drought . The southern and central sites were part of a previous experiment from 2010–2012 with a different set of treatments consisting of control, warming by 2.5°C, increased precipitation intensity by 20%, and warming by 2.5°C + increased precipitation intensity by 20%  had unique sources for each site; the remaining four species  had single sources for all sites.Between October 2014 and January 2015, all plots at the southern and central sites were mowed and raked while the new northern plots were treated with Glyphosate 2%  to remove standing biomass. By February 2015, all plots were seeded with a common mix of 29 native grass and forb species found in PNW prairies , we tallied the total number of reproductive stalks containing spikelets (hereafter considered flowers). For all species, we recorded the total number of flowering individuals. From these observations, we identified the first flowering dates (FFD) and peak flowering dates (PFD) for each species in each plot. Additionally, we calculated temperature sensitivities (change in days per °C) for each species at each site as: /ΔT, where ΔT is the difference in temperature between the warmed and ambient plots, or 2.5°C.From April to mid‐June 2017, we collected flowering and abundance data on our eight focal species on a weekly  or biweekly (southern site) basis. For each forb species, we tallied the total number of open flowers (defined by the presence of exposed stamens or stigmas) within each plot. For the lone grass species , which calculates the normalized difference vegetation index (NDVI) from measurements taken above each plot canopy. NDVI is an index of “greenness” on a scale of −1 to 1, with increasing values indicating a greater quantity of live biomass  and the warming and warming + ppt treatments only marginally differed for PFD for one species , we collapsed the climate treatments into two temperature categories: ambient (control and drought) and warming (warming and warming + ppt) and reran analyses. Due to site × warming interactions, we tested for site effects using ambient plots only. Within sites, we tested for an effect of warming using two‐tailed t tests. PFD data for C. grandiflora at the northern site were excluded due to an overwhelmingly large sample size (>500 plants per plot) which made it logistically impossible to count flowers during its peak growing period.All analyses used R version 3.3.2 . We selected the model with the lowest AIC value and tested for goodness‐of‐fit with a chi‐square test. Finally, we identified significant effects using likelihood‐ratio chi‐square tests. When a significant site x climate treatment interaction was present, we repeated this process within each site to test for climate treatment effects.p > 0.10), so we collapsed to the two ambient and warming categories and reran all NDVI analyses.We analyzed multi‐year NDVI variables  with repeated measures ANOVAs with site and climate treatment as between‐subject effects and year as a within‐subject effect. Following significant year interactions, we tested these variables (and 2018 growing season length) within years against site, climate treatment, and their interaction with ANOVAs. Additionally, to test for differences in NDVI on sampling dates across the duration of measurement, we conducted repeated measures ANOVAs with sampling date as a within‐subject effect. We used logit‐transformations to improve normality and Greenhouse–Geisser corrections when sphericity was violated. Following site × date interactions, we conducted repeated measures ANOVAs within each site. Lastly, following date × climate treatment interactions, we performed one‐way ANOVAs on each date. Again, we found no differences between the control and drought treatments and between the warming and warming + ppt treatments for any of these analyses , mean winter , and mean spring  soil temperatures. For soil moisture variables, we looked at the annual number of days below wilting point ; the mean annual matric potential ; and the date of first wilting point (DFWP). Strong correlations among the three temperature variables as well as the three soil moisture variables made it inappropriate to include all variables in multiple regression. Instead, for each response variable, we created 15 total models: each combination of one temperature variable with one moisture variable (nine models), and each temperature and each moisture variable alone (six models). Then for each response variable, we used the MuMIn package  over all models that include the explanatory variable  nor the ambient plots (n = 10), so we were unable to perform statistical tests on this species at this site. PFD advanced under warming relative to ambient temperature for three of five species at the southern site, four of six species at the central site, and all seven species with PFD data at the northern site  and peak (PFD) flowering dates at all sites. FFD advanced under warming for four of five species at the southern site, three of five species at the central site, and all eight species at the northern site Figure . A fourts Figure a; howevee Figure .C. grandiflora flowered earliest in the southern site, but A. mollis, P. nothofulvus, and P. congesta all flowered earliest at the central site. There was no effect of site on FFD for the two perennial species, F. roemeri and S. malviflora , P. nothofulvus at the southern site compared to the central (p = 0.091), P. congesta at both the southern and northern sites compared to the central (p ≤ 0.036), and S. malviflora at the northern site compared to both the southern and central   of temperature and moisture explanatory variables from 2016–2017 for FFD and PFD of each species .Across all plots, peak biomass was reached 20.6 ± 4.3 days earlier under warming than under ambient temperatures, regardless of site or year . Warming did not affect the 2017 date of senescence at the northern site  and 17.1 ± 5.5 days earlier than the northern site (p = 0.012). In 2018, senescence occurred 16.2 ± 4.3 days earlier under warming compared to ambient, regardless of site . The southern site senesced 20.3 ± 5.3 days earlier than the central site, which senesced 17.9 ± 5.3 days earlier than the northern site .Contrary to the results for peak biomass, the effect of warming on the date of senescence varied by site and year  compared to ambient (−0.011 ± 0.001 Δ NDVI day−1) at the central site in 2017 (p = 0.029), but no treatment differences for either the southern or northern sites in 2017 or any site in 2018 (p ≥ 0.134). In ambient plots in 2017, the rate of senescence was greater in the south (−0.017 ± 0.001 Δ NDVI day−1) than both the central (−0.011 ± 0.001 Δ NDVI day−1) and northern sites . Similarly, in 2018 across all plots, senescence rate was again greater in the south (−0.018 ± 0.001 Δ NDVI day−1) than both the central (−0.011 ± 0.001 Δ NDVI day−1) and northern sites .The effect of warming on the rate  of senescence was also dependent on site and year (site × warming and site × year interactions: p < 0.001). At the central site, warming increased biomass from November 2016 through April 2017. By the end of June 2017, there was a shift to warming‐induced biomass suppression, which continued until mid‐October 2017, and again from mid‐May 2018 to the end of sampling in August . At the northern site, warming increased biomass from late February to mid‐April 2017, then in mid‐June, and again from late‐October 2017 to May 2018. Warming suppressed biomass relative to ambient for only one sampling date in 2017 (9 May) and only two dates in 2018 (13 June and 19 July) .The effect of warming on NDVI value depended on site and date, with warming effects shifting from overall negative (suppressing biomass relative to ambient) to positive (increasing biomass relative to ambient) from south to north Figure . At the p = 0.076). Warming resulted in a net reduction in the southern site growing season length by 20.5 ± 6.1 days (p = 0.004) and in the central site growing season length by 21.6 ± 9.4 days (p = 0.037). There was no effect of warming on the northern site growing season length (p = 0.965). Under ambient conditions, the southern site had the shortest growing season  while the central (232.7 ± 4.2 days) and northern sites (238.4 ± 4.2 days) did not significantly differ from one another (p = 0.615).The effect of warming on the 2018 growing season length was also dependent on site  included mean annual temperature, while mean annual matric potential appeared in one candidate model for each variable (four models)  modeled against an equal number of temperature and moisture predictor variables. Furthermore, none of the population‐ or community‐level variables had different responses to control (ambient temperature and precipitation) versus drought (ambient temperature and −40% precipitation), nor did warming (+2.5°C and reduced soil moisture) ever differ from warming + ppt (+2.5°C and ambient soil moisture).Our 2.5°C increase in temperature in the warming treatments reflects expected future temperatures for the region, with models projecting ~3°C increase by the end of the 21st century , rather than simply the magnitude of total precipitation, for moisture regime changes to meaningfully impact the timing of many phenological events in this system.It is important to place our results that changes in temperature are likely to be more impactful than changes in soil moisture under future climate in the context of a Mediterranean climate system. Many prairie plants that are adapted to Mediterranean climates limit the timing of their reproductive events to the spring, prior to the extremely water‐limited summer months. Plant growth and canopy development follow a similar trend. Thus, we propose that plants adapted to a Mediterranean climate are predisposed to temperature regulation for many aspects of their phenology. Our soil moisture data provide evidence that PNW Mediterranean ecosystems are buffered against large relative changes in precipitation during much of the year. From late fall to early spring, rain was frequent enough that the soils remained near saturation point (0 kPa) regardless of climate treatment. Over the summer, however, drought severity was so extreme that the soils remained well below permanent wilting point , also regardless of treatment. These observations held true for both 2017 and 2018, which were relatively wet and dry years, respectively, for the southern and central sites, and relatively wet and average years for the northern site. From August 2016 to August 2017, precipitation for our southern, central, and northern sites was 163%, 119%, and 132%, respectively, of the 30‐year averages from 1981–2010, while from August 2017 to August 2018, precipitation was 78%, 74%, and 106% of average, respectively . DespiteIt is also important to note that different phenological events are likely to have different mechanistic triggers, especially in a Mediterranean climate system in which high temperatures are asynchronous with the wet season. For example, Peñuelas et al.  found prM. laciniata at the northern site) to 12.4 days per °C , with a total mean advancement of 5.3 days per °C across species and sites. PFD advancements ranged from 1.4 days per °C (M. laciniata at the northern site) to 8.8 days per °C (P. nothofulvus at the northern site), with a total mean advancement of 4.7 days per °C. These values fall very much in line with evidence and predictions from other studies suggesting flowering times advance on average at a rate of ~2–7.5 days per °C . Thus, the flowering phenology results presented here have largely been consistent during other years of this experiment.At the population level in 2017, all eight of our focal species experienced some degree of advancement in first flowering dates (FFD) and peak flowering dates (PFD) with warming. Warming treatments advanced FFD from 2.1 days per °C  exhibited greater sensitivity at the northern site, but two other cases (FFD for P. nothofulvus and P. congesta) exhibited greater sensitivity at the southern site.Some studies have found that phenological temperature sensitivity is greater at higher latitude , and both C. grandiflora and P. congesta are known host plants of the Taylor's checkerspot butterfly (Euphydryas editha taylori), two federally listed endangered species  to be affected by warming in the following ways: consistent suppression at both the southern and central sites during the late spring to summer of both relatively wet (2017) and dry (2018) years, and suppression at the northern site during the summer of an average rainfall year (2018). However, we also found positive effects of warming at the central site during the winter and spring of the wet year (2017) and at the northern site during the winter, spring, and fall of both wet and average years. Thus, for the central and northern sites, there appear to be interactions between the effect of warming and annual rainfall on live biomass across parts of the year, a phenomenon that has been previously documented  are reported to be increasing with global warming (Ibáñez et al., 5°N are r2 sequestration (Cleland et al., Shifts in the phenology of canopy biomass may have implications for community dynamics and ecosystem processes. Changes to growing season lengths are known to affect water cycling, rates and amount of carbon sequestration, and nutrient uptake from the soil (Ibáñez et al., Overall, our study offers substantial evidence that future changes in temperature may have great influence on the timing of many key plant phenological events in a Mediterranean climate system and that effects due to changes in soil moisture may be buffered from even large changes in the amount of precipitation so long as the timing and duration of the rainy season are unchanged. We observed a strong influence of temperature on flowering phenology in eight native plant species both within and beyond their current geographic ranges, as well as for canopy biomass phenology at the community scale. Additionally, we found that the majority of our eight focal species are experiencing considerable reductions in their abundances near or south of their northern range limits, suggesting that the clock is ticking on their ability to persist within their current ranges.None declared.PR analyzed the data and wrote the manuscript. SB, LPM, BR, BJ, and PR designed the study. All authors contributed to data collection and revisions, with substantial edits made by SB, LPM, BR, BJ, and SH. Click here for additional data file.


This study suggests that the phosphorylation status of Y78 regulates IAV replication by inhibiting the nuclear export and ubiquitination of NP. Overall, these findings shed new light on the biological roles of NP phosphorylation, especially its negative role in NP ubiquitination.Phosphorylation and dephosphorylation of nucleoprotein (NP) play significant roles in the life cycle of influenza A virus (IAV), and the biological functions of each phosphorylation site on NP are not exactly the same in controlling viral replication. Here, we identified tyrosine 78 residue (Y78) of NP as a novel phosphorylation site by mass spectrometry. Y78 is highly conserved, and the constant NP phosphorylation mimicked by Y78E delayed NP nuclear export through reducing the binding of NP to the cellular export receptor CRM1, and impaired virus growth. Furthermore, the tyrosine kinase inhibitors Dasatinib and AG490 reduced Y78 phosphorylation and accelerated NP nuclear export, suggesting that the Janus and Src kinases-catalyzed Y78 phosphorylation regulated NP nuclear export during viral replication. More importantly, we found that the NP phosphorylation could suppress NP ubiquitination  Influenza A virus (IAV) is an enveloped virus belonging to the Orthomyxoviridae family . Its genPhosphorylation is an important posttranslational modification of IAV proteins. It has been reported that multiple sites on NP of IAV can be phosphorylated, and these phosphorylated NPs are involved in a number of activities in IAV life cycle. The phosphorylation sites on NP are predominantly serine (S), threonine (T), and tyrosine (Y). Hutchinson et al. identified 12 phosphorylated sites on NP  . Among tApart from phosphorylation, ubiquitination is another key posttranslational modification of NP to regulate IAV replication. K184 ubiquitination of NP was reported to increase virus RNA replication and cellular deubiquitinating enzyme USP11 decreased polymerase activity and viral replication . UbiquitIn our study, Y78 of NP was identified as a phosphorylated site. We demonstrated that the phosphorylation and dephosphorylation of Y78 regulated the virus genome replication, transcription, and NP nuclear export. Interestingly, we also found that there is a cross-talk between the phosphorylation and ubiquitination of NP.2. The influenza virus A/WSN/1933 (H1N1) strain was rescued using a 12-plasmid reverse genetic system  and propagated in 9-day-old embryonated chicken eggs .The human embryonic kidney (293 T) cells, human lung alveolar epithelial (A549) cells, and Madin-Darby canine kidney (MDCK) cells  were maintained in Dulbecco’s modified Eagle’s medium  with 10% fetal bovine serum  at 37°C and 5% COPhos-tag acrylamide was purchased from Wako. Dasatinib was purchased from MCE. Imatinib and AG490 were purchased from CST. An RNase inhibitor was purchased from Promega. Protease inhibitor cocktail and phosphatase inhibitor phosSTOP were purchased from Roche. Anti-FLAG (M2) Affinity Gel was purchased from Sigma-Aldrich. Pierce™ Protein G Agarose (20397) was purchased from Thermo Scientific. Rabbit polyclonal antibody against NP and mouse monoclonal antibody against M1 were generated as previously described . Anti-HSThe full-length NP sequence from the A/WSN/33 virus was cloned into the pcDNA4.0/TO, pCMV-MYC, and pCDNA3.0-FLAG vectors. FLAG or MYC-tagged NP mutants, including Y78F and Y78E were generated using a site-directed mutagenesis kit . The following primers were used: NP-Y78F-up, 5′AGGAGGAATAAATTTCTAGAAGAACATCCCAGTGCGGGGA-3′; NP-Y78F-dn, 5′-ATGTTCTTCTAGAAATTTATTCCTCCTCTCGTCAAAAGCA-3′; NP-Y78E-up, 5′-AGGAGGAATAAAGAGCTAGAAGAACATCCCAGTGCGGGGA-3′; and NP-Y78E-dn, 5′-ATGTTCTTCTAGCTCTTTATTCCTCCTCTCGTCAAAAGCA-3′. The full-length TRIM22 and CRM1 were synthesized according to NCBI Reference Sequence NM_006074.5 and NM_003400.4, respectively, and then cloned into the pcDNA3.0-Flag and/or pCMV-MYC vectors. The single ubiquitin molecule was synthesized according to NCBI Reference Sequence NM_021009.6, and cloned into the pCMV-HA vector. The expression plasmids for the PA, PB1, and PB2 genes from A/WSN/33 virus were generated by cloning into the pcDNA3-FLAG vector as described previously . pHH21-c2+-Phos-tag SDS-PAGE as described previously (2 (Sigma) added to the separating gel before polymerization. The gel was silver stained, and the separated bands were subjected to nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) identification .IAV-infected 293 T cells were lysed in lysis buffer  supplemented with complete protease inhibitor cocktail and a phosphatase inhibitor phosSTOP. The NP was purified with protein G agarose beads pre-bound to a rabbit anti-NP polyclonal antibody for 3 h at 4°C. Proteins were separated by 12% Mneviously . Briefly2, and the supernatant containing the recombinant viruses was harvested and then centrifuged at 2,000 g for 10 min to remove the cell debris.The wild-type (WT) A/WSN/1933 (H1N1) virus and its NP mutants were generated by using a 12-plasmid-based reverse genetic system as described previously . Briefly2. The virus inoculums were removed by washing with PBS. Cell monolayers were incubated in DMEM with 1% FBS and 1 μg/ml TPCK-treated trypsin at 37°C. After infection, 500 μl of supernatant was collected at 24, 36, 48, 60, and 72 h and stored at −80°C. At the same time, 500 μl of fresh medium was added back to each dish. Finally, virus titers were determined by plaque assays as previously described (MDCK cells grown to monolayers in 10-cm cell culture dishes were washed with PBS and infected with virus (MOI = 0.001) for 1 h at 37°C in 5% COescribed .The plasmids for expression of the PA, PB1, PB2, NP (WT or mutant) proteins, and luciferase reporter plasmids  were co-transfected into 293 T cells as previously described . Cells tTotal RNA was extracted from 293 T cells by use of TRIzol (Invitrogen) according to the manufacturer’s instructions. Samples were digested with DNase I and subjected to reverse transcription-PCR (RT-PCR). RNA was reverse transcribed using the following PCR primers: mRNA primer, oligo(dT); cRNA primer, 5′-AGTAGAAACAAGG-3′; and vRNA primer, 5′-AGCGAAAGCAGG-3′. A mock reaction was performed with no reverse transcriptase added to the reaction mixture. The analysis of relative M1 gene expression was performed using the following PCR primers: M1-up (5′-TCTGATCCTCTCGTCATTGCAGCAA-3′) and M1-dn (5′-AATGACCATCGTCAACATCCACAGC-3′). GAPDH served as an internal control, using the PCR primers GAPDH-up (5′-GGTGGTCTCCTCTGACTTCAACA-3′) and GAPDH-dn (5′-GTTGCTGTAGCCAAATTCGTTGT-3′). The cycling conditions comprised an initial denaturation step of 30 s at 95°C, followed by 40 two-step cycles (95°C for 5 s and 60°C for 31 s). Dissociation curve analysis was performed after each assay to ensure specific target detection.g for 15 min. Lysates were incubated with anti-FLAG M2 affinity gel for 2 h. Following four washes in wash buffer , the precipitated proteins were separated by SDS-PAGE and then transferred to immobilon polyvinylidene difluoride (PVDF) membranes . The membranes were blocked for 2 h at room temperature in blocking solution , and proteins were detected using appropriate antibodies, followed by the addition of anti-rabbit or anti-mouse secondary antibody coupled to horseradish peroxidase. Proteins were visualized by use of chemiluminescence detection reagents.293 T cells were lysed in lysis buffer , supplemented with complete protease inhibitor cocktail and phosphatase inhibitor phosSTOP. After an incubation period of 30 min at 4°C, insoluble components were removed by centrifugation at 12,000 Indirect immunofluorescence assays (IFAs) were performed with a Leica SP8 confocal laser scanning microscope. Coverslips carrying the cells were washed with PBST (PBS plus 1% Triton X-100) and fixed with 4% paraformaldehyde. Cells were then blocked with 4% bovine serum albumin (BSA) dissolved in PBST and stained with anti-NP and anti-FLAG antibodies. Secondary antibodies were tetramethyl rhodamine isocyanate (TRITC)-conjugated anti-rabbit IgG and fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG. Finally, the location of cell nucleus was labeled with 4′,6-diamidino-2-phenylindole (DAPI). The wavelengths of TRITC, FITC, and DAPI are 561 nm, 488 nm, and 405 nm.2PO4, 10 mM HEPES, 5 mM NaHCO3, 1 mM CaCl2, 0.5 mM MgCl2, 5 mM EDTA, and Complete protease inhibitor cocktail). The cells were allowed to swell for 5 min on ice and then dounce-homogenized 50 times. After centrifugation at 7,500 rpm for 2 min at 4°C, the pellets represented nuclei plus debris, and the supernatants were cytosol plus plasma membrane. The nucleus-debris pellet was then resuspended in 1 ml of TSE buffer  and dounce-homogenized 30 times, followed by centrifugation at 5,000 rpm for 2 min. The pellet was resuspended and washed twice to obtain the final nuclear pellet. The final pellet was resuspended in 30 μl of TSE buffer.293 T cells in 6-cm dishes were harvested into 300 μl of CLB buffer  plasmids or MYC-TRIM22 plasmid. The cells were harvested at 30 h after transfection. Western blotting was probed with anti-NP, anti-c-MYC, and anti-β-actin antibodies.293 T cells were transfected with expression constructs for FLAG-tagged NPs and HA-tagged ubiquitin or MYC-TRIM22. Thirty-six hours post-transfection, cell extracts were then immunoprecipitated with FLAG-M2 beads. The eluted proteins were analyzed by western blotting using an anti-HA, anti-FLAG, and c-MYC antibodies.Sequence alignments were performed using MegAlign software . Statistical analyses were performed using Prism6 software .pY). We found that the phosphorylation level of NP Y78F mutant was greatly decreased compared with that of WT NP  (WSN) and NP was purified by protein G agarose beads pre-bound to rabbit anti-NP polyclonal antibody. Phos-tag SDS-PAGE was used to purify the phosphorylated NP . After sof WT NP , demonstThe schematic diagram of NP functional domains demonstrated that Y78 belongs to the basic loop region (residues 72-92) . At the To investigate the role of NP Y78 phosphorylation in influenza virus replication and packaging, we attempted to rescue the WT and mutant recombinant WSN viruses. We introduced glutamate (E) substitution at Y78 to mimic the constitutively phosphorylated residue and F at Y78 to mimic the constitutively dephosphorylated residue. The 12-plasmid reverse genetic system containing WT or mutant NP plasmid was transfected into 293 T cells to rescue the WT or NP mutant virus, and the supernatants were harvested at 72 h post-transfection (h.p.t.) for plaque assays. In three independent viral packaging experiments, both the WT and Y78F WSN viruses were successfully rescued, and the titer of Y78F mutant virus was greatly reduced compared with that of WT virus . HoweverNP is required for viral RNA synthesis , and binIn addition, NP has long been recognized as the second most abundant protein of influenza virus and an important component of the vRNP complexes. It plays a major role in the overall structural organization and stabilization of the complexes, then affects the polymerase activity of vRNP . TherefoThe dynamic cellular localization of NP is critical for efficient viral replication and transcription , and NP Previous studies have suggested that a classical cytoplasmic receptor protein CRM1 can interact with NP and transport NP into the cytoplasm . To deteThe nuclear export of vRNP complex is mediated by the CRM1-NEP-M1-vRNP complex, during which the nuclear transport capacity of the NEP and M1 plays an important role . MoreoveWe further investigated which kinds of tyrosine kinases play the dominant role in Y78 phosphorylation and their effect on NP sub-cellular localization. The tyrosine kinase inhibitors, including Imatinib (Bcr-Abl inhibitor), Dasatinib (Src inhibitor), and AG490 (JAK inhibitor), were used to treat WSN or Y78F mutant virus-infected cells. We detected the effect of these three tyrosine kinase inhibitors on the tyrosine phosphorylation level of WT and Y78F NP in A549 cells. We found that all of these three tyrosine kinase inhibitors decreased the tyrosine phosphorylation of WT NP , left. MSeveral lines of evidence indicate that the phosphorylation-mediated subcellular localization of substrates regulates the ubiquitination . Thus, wIt has been established that the E3 ligase TRIM22 mediates ubiquitin-proteasome degradation of NP , and CNOE3 ligases usually interact with substrates, so we wondered whether Y78 phosphorylation affected NP ubiquitination by regulating the binding of NP to TRIM22. The interaction between NP  and TRIM22 was determined by CO-IP assays. 293 T cells were transfected with MYC-NPs and FLAG-TRIM22. We found that the Y78E mutant reduced the binding of NP to TRIM22, while the Y78F mutant enhanced NP-TRIM22 interaction . The aboAs two types of the posttranslational modifications in eukaryotic biology, phosphorylation and ubiquitination can take a cross-talk . PhosphoPhosphorylation and dephosphorylation is a reversible dynamic equilibrium process. IAV NP underwent phosphorylation and dephosphorylation at various stages during viral multiplication, and the exposed NP phosphorylation sites might also be different during various infection periods . In the Phosphorylation levels of proteins are regulated by kinase-catalyzed phosphorylation and phosphatase-catalyzed dephosphorylation. There are quite a few studies related to the regulation of influenza virus replication by kinases. Activated human protein kinase C (PKC) family member PKCδ interacts with the polymerase subunit PB2 and regulates NP oligomerization and RNP assembly in the course of IAV infection . Serine/To be the major structural component of vRNP, NP performs a vital function in the replication of influenza virus by nuclear-cytoplasmic shuttling . NP on vvia a CRM1-dependent pathway.Two NLSs on NP control the nuclear import of vRNP , and thrIn conclusion, we identified the tyrosine phosphorylation site Y78 on NP and discovered that Y78 phosphorylation regulates the nuclear export and ubiquitination of NP, which is vital for the replication of IAV. Our data further expand the biological functions of NP phosphorylation and provide a potential target for the development of antiviral agents.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.All embryonated chicken eggs experiments were approved by the Research Ethics Committee of Chinese Academy of Sciences and complied with the Beijing Laboratory Animal Welfare and Ethical Guidelines of the Beijing Administration Committee of Laboratory Animals.WL conceived the study. LS and LC designed the experiments, analyzed the data, and wrote the manuscript. LC performed the experiments. WZ provided experimental technical guidance and results analysis. ML, XB, and WY helped with some experiments. JL, WF, and GG helped analyze the data and revise the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chlamydomonas sp. UWO241, a psychrophile residing in the deep photic zone. Recently, we found that UWO241 has lost the genes responsible for light-independent chlorophyll biosynthesis, which is surprising given that this green alga comes from a light-limited environment and experiences extended periods of darkness during the Antarctic winter. Why discard such a process? We argued that it might be linked to the very high dissolved oxygen concentration of Lake Bonney at the depth at which UWO241 is found. Oxygen is the Achilles’ heel of the key enzyme involved in light-independent chlorophyll biosynthesis: DPOR. If this hypothesis is true, then other algae in Lake Bonney should also be susceptible to losing DPOR, such as Chlamydomonas sp. ICE-MDV, which predominantly resides in the chemocline, a depth with an even higher oxygen concentration than that where UWO241 exists. Here, we report that, contrary to our earlier prediction, ICE-MDV has maintained the genes encoding DPOR. We briefly discuss the implications of this finding in relation to the loss of light-independent chlorophyll synthesis in UWO241.The cold, permanently ice-covered waters of Lake Bonney, Antarctica, may seem like an uninviting place for an alga, but they are home to a diversity of photosynthetic life, including  Chlamydomonas sp. UWO241 [Chlamydomonas sp. ICE-MDV [No two lakes are the same. But Lake Bonney in the McMurdo Dry Valleys of Victoria Land, Antarctica, is perhaps one of the most unique and uninviting lakes on Earth. Its frigid waters (around 40 meters deep) are permanently enclosed by one storey of ice, resulting in a chemically stratified environment, largely cut off from the outside world . Neverth. UWO241 ,7 and Ch ICE-MDV ,8. Both There are better places than an ice-covered Antarctic lake to perform photosynthesis. Within Lake Bonney, UWO241 and ICE-MDV must endure sustained cold (~5°C year-round), high salinity (up to 0.7 M NaCl in the deep layers), and perpetual low irradiance, not to mention 24-hours of darkness during peak austral winter . Here, wa biosynthesis: light-dependent and light-independent protochlorophyllide oxidoreductase  [por, is ubiquitous among photosynthetic eukaryotes [chlB, chlL, and chlN, can facilitate chlorophyll synthesis in the dark [Most eukaryotic algae contain two distinct nonhomologous enzymes for the penultimate step of chlorophyll ctively) ,11. LPORkaryotes  and is okaryotes . Evidenckaryotes , which pthe dark . DPOR isthe dark , which ipor but lacks chlB, chlL, and chlN, indicating it has discarded DPOR and is now entirely dependent on LPOR for making chlorophyll [Chloroplast and nuclear genome sequencing demonstrated that UWO241 contains orophyll . Why wouorophyll  and remaorophyll ,18, wherorophyll , is not orophyll . If the So, what of ICE-MDV? It can be found in the shallow zone of Lake Bonney (5 m) and is particularly dominant in the chemocline (15 m) where the dissolved oxygen concentration is even higher than in the deeper photic zone where UWO241 exists . Hence, chlB, chlL, and chlN were easily located in the draft genome assembly of ICE-MDV, which comprised 331,087 contigs, averaging 812 nt. Note: the sequences were identified by blasting (BLASTN) the UWO241 chlB, chlL, and chlN genes against the ICE-MDV contigs. Adding to our disappointment, these genes were clearly chloroplast located and harbored no signs of deleterious or knockout mutations (please see GenBank accessions MN046391-MN046393). A nuclear-located gene for por was also found in the assembly. Thus, despite existing in an aquatic environment with a remarkably high dissolved oxygen concentration, ICE-MDV clearly retains functional genes for light-dependent and light-independent chlorophyll synthesis.Alas, complete sequences of chlB, chlL, and chlN genes share 100%, 99.8%, and 100% nucleotide sequence identity, respectively, with those from another Antarctic green alga: Chlamydomonas sp. ICE-L [What does this mean for our earlier conjectures on the forces driving the loss of DPOR in UWO241? Surely, it weakens them. But it is worth highlighting an additional feature of the ICE-MDV data we collected. Specifically, the ICE-MDV p. ICE-L . (Nucleop. ICE-L . UWO241,p. ICE-L . Lastly,p. ICE-L .chlB, chlL, and chlN genes. Moreover, we currently do not know if DPOR is active in the high-oxygen natural habitat of ICE-MDV and whether this alga has retained the ability to synthesize chlorophyll in the absence of light. Whatever the reasons for the presence of DPOR in ICE-MDV and its absence in UWO241, these data further emphasize just how unique UWO241 is relative to other green algae [The close phylogenetic affiliation of ICE-MDV and ICE-L could be an indication that the former is part of population that extends beyond the bounds of Lake Bonney – potentially into an environment where oxygen concentrations are low enough to not inhibit DPOR. If such a scenario is true, then it might be preventing the loss of the genes encoding for light-independent protochlorophyllide oxidoreductase within ICE-MDV. Alternatively, the close phylogenetic relationship between ICE-MDV and ICE-L could suggest that ICE-MDV arrived in Lake Bonney more recently than UWO241 and has not yet had time to lose the en algae , both in