Poly (PVA) has emerged as the most potent mimic of antifreeze (glyco)proteins ice recrystallization inhibition (IRI) activity, despite its lack of structural similarities and flexible, rather than rigid, backbone. The precise spacing of hydroxyl groups is hypothesized to enable PVA to recognize the prism planes of ice but not the basal plane, due to hydroxyl pattern matching of the ice surface giving rise to the macroscopic activity. Here, well-defined PVA derived from reversible addition–fragmentation chain-transfer (RAFT) polymerization is immobilized onto gold nanoparticles to enable the impact of nanoscale assembly and confinement on the observed IRI activity. Unlike previous reports using star-branched or bottle-brush PVAs, the nanoparticle–PVA retains all IRI activity compared to polymers in solution. Evidence is presented to show that this is due to the low grafting densities on the particle surface meaning the chains are free to explore the ice faces, rather than being constrained as in star-branched polymers. These results demonstrate a route to develop more functional IRI’s and inclusion of metallic particle cores for imaging and associated applications in cryobiology. Nature has evolved a diverse range of biological macro-molecules which can modulate the formation and growth of ice to enable extremophiles to survive at subzero temperatures.28Despite this diverse range of materials shown to have IRI activity, there is an outstanding question of how the different natural and synthetic antifreeze macromolecules interact with ice. We do not know the roles of bound and unbound components,37An advantage of synthetic over natural polymers is the ability to tune their architectureConsidering the above, in this work we designed and synthesized metal-core nanoparticles bearing multiple copies of poly on their surface, using RAFT polymerization to enable control over molecular weight and to install a thiol-anchoring group. We identify polymers which lead to colloidally stable gold particle dispersions to enable the ice recrystallization activity and ice shaping activity of the hybrids to be quantified and compared to free polymers in solution.ω-chain-ends via xanthate chain-transfer agent,S-benzyl O-ethyl carbondithioate to give a panel of poly(vinyl acetates)’s (PVAc’s) , and IR (infrared) spectroscopy. Mn values of the resulting polymers were in reasonable agreement with the theoretical values; dispersity values were rather broad as expected for polymers derived from lesser activated monomers1H NMR as well as complete removal of carbonyl stretches (1729 cm−1) and appearance of hydroxyl (3300 cm−1) in the infrared  was exploited to install a protected thiol at c’s) see , which winfrared .4; 4 nm diameter) were synthesized by reducing a solution of gold(III) chloride and trisodium citrate with sodium borohydride (see 4) were fully characterized by TEM  bands of the gold coupling due to aggregation of the nanoparticles.max. TEM analysis confirmed that the particle size and distribution did not change after polymer functionalization. ζ potential measurements showed an increase (from −19.4 to −5 mV) upon polymer coating, due to displacement of the citrate stabilizing ligands, supporting successful surface coating. To enable chemical characterization of the surface, XPS was employed on dried-down nanoparticles . IRI activity was evaluated by monitoring the growth of polynucleated ice crystals (using a “splat” assayThese PVA-coated nanoparticles were designed to enable the effect of macromolecular architecture on IRI activity to be probed. Previous results have shown that adding a branch (3-arm versus 2-arm) to polymers of PVA did not lead to any increase in activity.−1, reinforcing that this is the most potent nonprotein inhibitor known.98@Au4 was not stable under the assay conditions, with some aggregation occurring. This gave rise to potentially false-negative results with it apparently losing activity, and hence these data are excluded  to that of free PVA chains. On a molar basis (assuming one particle is a very large molecule) this would represent a dramatic increase in activity, but such a comparison is not valid for large multivalent systems. The limiting concentration for inhibiting all growth was ~0.1 mg mL−1. To explain the retention of activity, how PVA could bind ice must be considered. Molinaro et al. used molecular simulations to hypothesize that PVA can bind to both the primary and secondary prism planes of ice via its hydroxyl groups. The distance between hydroxyls in ice on the primary and secondary prismatic faces is 2.76 and 2.74 Å, respectively, and between consecutive hydroxyls in PVA is 2.92 Å enabling a good fit for pattern matching . This low density means the polymers are free to interact with the ice faces, and to target the desirable prism faces unlike in highly grafted systems  was used to obtain ice crystals, whose growth could be monitored. For aqueous solutions, rounded disc shaped ice crystals were observed . HoweverConsidering the above data, it is clear that the lightly grafted PVA/AuNP hybrids retain the full activity of the IRI active polymers despite a chain-end being immobilized onto the particle. This confirms that, to assemble larger nanoscopic IRI active materials suitable for application, the overall grafting density and flexibility must be considered to ensure the material can access all ice faces and appears to confirm the simulation/theory evidence for how this apparently simple polymer interacts with the complex ice/water interface.Here we have demonstrated the first synthesis and characterization of poly gold nanoparticle hybrids by using RAFT polymerization and grafting-to methodology. PVA chain lengths were identified which gave colloidally stable particles, due to the relatively low grafting densities achieved by this strategy, and the surface-bound polymers were observed to retain their full ice recrystallization inhibition activity. We hypothesize that the low grafting density on the particle surface is beneficial as it enables the polymers to explore the ice surface so each can engage with prism faces, which have the correct hydroxyl spacings to enable PVA binding. This is in contrast to confined  polymers which are sterically constrained so only a small proportion can engage in favorable prism-face interactions. This hypothesis was supported by ice shaping assays with addition faceting/dendritic ice observed for the nanoparticles. These challenge the previous observations, showing that complex architecture can be used to modulate and maintain IRI activity by controlling the PVA density. These results will not only guide the development of more active IRIs, but also enable their application in more fields where their nanoscopic dimensions, or optical properties, are useful, such as in high-resolution imaging.S-benzyl O-ethyl carbondithioate was synthesized as previously described.20Phosphate-buffered saline (PBS) solutions were prepared using preformulated tablets (Sigma-Aldrich) in 200 mL of Milli-Q water (>18.2 Ω mean resistivity) to give [NaCl] = 0.138 M, [KCl] = 0.0027 M, and pH 7.4. Vinyl acetate (>99%), 4,4′azobis (>98%), and benzyl bromide (98%) were purchased from Sigma-Aldrich. Vinyl acetate was filtered through a plug of basic alumina to remove inhibitors prior to use. The 4,4′azobis was recrystallized from methanol and stored at −18 °C in the dark. Potassium ethyl xanthate (98%) was purchased from Alfa Aesar. 1H and 13C NMR spectra were recorded on Bruker Avance III HD 300 MHz, HD 400 MHz, or HD 500 MHz spectrometers using deuterated solvents obtained from Sigma-Aldrich. Chemical shifts are reported relative to residual nondeuterated solvent. All size exclusion chromatography (SEC) data were recorded in THF on Agilent 390-LC MDS instruments equipped with differential refractive index (DRI) detectors. Systems were equipped with 2× PLgel Mixed D columns (300 × 7.5 mm) and a PLgel 5 μm guard column. The eluents are THF with 2% TEA (triethylamine) and 0.01% BHT (butylated hydroxytoluene). All samples were run at 1 mL min−1 at 50°C. Poly(methyl methacrylate) standards  were used for calibration. Analyte samples were filtered through a nylon membrane with 0.22 μm pore size before injection. Respectively, experimental molar mass  and dispersity (Đ) values of synthesized polymers were determined by conventional calibration using Agilent GPC/SEC software.e DSC/TGA instrument under nitrogen from 30 to 600 °C using standard alumina 70 μL crucibles and a blank in each run.Nanoparticle size and dispersity was measured by transmission electron microscopy (TEM), UV–vis spectroscopy, and size measurements by dynamic light scattering (DLS). DLS was performed on a Malvern Instruments Zetasizer Nano-ZS instrument with 4 mW HeNe laser 632.8 nm. UV–vis spectroscopy was performed on a BioTek Synergy HT microplate reader. TEM was performed on a JEOL 2100 LaB6 high-resolution microscope. X-ray photoelectron spectroscopy (XPS) was carried out on the Kratos Axis Ultra instrument with a delay-line detector. TGA was performed on a Metter Toledo STARx/0.45/∞/0-2/FN22 lens  and a Canon EOS 500D SLR digital camera was used to obtain all images. Image processing was conducted using ImageJ software, which is freely available from http://imagej.nih.gov/ij/.Sucrose assays were performed on, and ice wafers were annealed on, a Linkam biological cryostage BCS196 instrument with T95-Linkpad system controller equipped with a LNP95-liquid nitrogen cooling pump, using liquid nitrogen as the coolant . An Olympus CX41 microscope equipped with a UIS-2 20A 10 mL droplet of polymer in PBS solution is dropped from 1.4 m onto a glass microscope coverslip, which is on top of an aluminum plate cooled to −78 °C using dry ice. The droplet freezes instantly upon impact with the plate, spreading out and forming a thin wafer of ice. This wafer is then placed on a liquid nitrogen cooled cryostage held at −8 °C. The wafer is then left to anneal for 30 min at −8 °C. Three photographs are then taken of the wafer in different locations at 20× zoom under cross polarizers. The number of crystals in the image is counted, again using ImageJ software, and the area of the field of view divided by this number of crystals to give the average crystal size per wafer, reported as a percent of area compared to PBS control.−1 until few ice crystals remained and then cooled at 0.05 °C min−1, and the shape of ice crystals was observed. Micrographs were obtained every 0.1 °C.Samples dissolved in PBS buffer containing 45% sucrose were sandwiched between two glass coverslips and sealed with immersion oil. Samples were cooled to −50 °C. The temperature was then increased to −8 °C and held for 1 h to anneal. The samples were then heated at 0.05 °C min−1 (0.08 mg mL−1), aqueous solution of HAuCl4 was prepared at room temperature in glassware washed with aqua regia [CAUTION. HANDLE WITH CARE]. A 13.8 mg (0.05 mmol) portion of trisodium citrate was added, followed by 5 mL of an ice-cold 0.1 M  solution of NaBH4. The solution was stirred at room temperature overnight. Small AuNPs were isolated by taking the supernatant after centrifugation at 13.2k RPM for 10 min at 25 °C. A 1 mg portion of polymer was added to 1 mL of Au4, and the solution was agitated for 60 min at room temperature. These solutions were washed three times and concentrated in Amicon Ultra 0.5 centrifugal filter units with an Ultracel-30 membrane before being redispersed in the same volume of PBS.The 4 nm citrate-stabilized AuNPs were synthesized as described by Ieong et al.ACS Publications website at DOI: 10.1021/acs.langmuir.8b01952.The Supporting Information is available free of charge on the 1H NMR spectra, FTIR spectra, UV–vis spectra, photographs, TGA analysis, XPS survey scans, and IRI activity assessment (PDF)Additional synthesis details and figures including Supp Info


Saccostrea glomerata), blue mussel (Mytilus edulis), saucer scallop , and southern calamari , examining cross‐reactivity between species and with blue swimmer crab tropomyosin, Por p 1.Shellfish allergy is an increasing global health priority, frequently affecting adults. Molluscs are an important shellfish group causing food allergy but knowledge of their allergens and cross‐reactivity is limited. Optimal diagnosis of mollusc allergy enabling accurate advice on food avoidance is difficult. Allergens of four frequently ingested Asia‐Pacific molluscs are characterized: Sydney rock oyster . IgE‐reactive Sydney rock oyster proteins were identified by mass spectrometry, and the novel major oyster tropomyosin allergen was cloned, sequenced, and designated Sac g 1 by the IUIS. Oyster extracts showed highest IgE cross‐reactivity with other molluscs, while mussel cross‐reactivity was weakest. Inhibition immunoblotting demonstrated high cross‐reactivity between tropomyosins of mollusc and crustacean species.These findings inform novel approaches for reliable diagnosis and improved management of mollusc allergy. Additional mollusc allergens have been reported, but not yet fully characterized.Shellfish allergy is an increasingly important global health priority, often presenting in childhood and typically persisting into adulthood.As for other shellfish groups, tropomyosin has been identified as a major allergen for several mollusc species.Food processing, including heating and digestion, can affect IgE reactivity of allergens by chemically altering protein structures via different processes including aggregation, polymerization, and degradation.Saccostrea glomerata , Mytilus edulis (blue mussel), Amusium balloti , and Sepioteuthis australis . SRO allergens were identified by mass spectrometry, the novel oyster tropomyosin Sac g 1 was sequenced, and cross‐reactivity with other mollusc and crustacean tropomyosins assessed. The effect of heat treatment of mollusc extracts on IgE reactivity was also examined. The findings of this study will inform novel and reliable diagnostic approaches to enhance management of mollusc allergy.We report here the characterization of allergens in four commonly consumed Asia‐Pacific mollusc species: 22.1–1; Phadia Pty Ltd, Uppsala, Sweden; TableSera were obtained from 13 patients recruited from the Alfred Hospital Allergy Clinic with a clinical history of seafood allergy and positive mollusc‐specific IgE by ImmunoCAP (>0.35 kUA L2.2S. glomerata) with  or without visceral mass (ROvm), blue mussel , saucer scallop , and southern calamari  was removed from its shell, sliced into fine pieces and incubated in PBS pH 7.2 overnight at 4 °C with constant mixing. After centrifugation at 13 000 × g at 4 °C for 20 min, the supernatant was collected, filter‐sterilized, and stored at −80 °C. For cooked SRO with (CO) or without visceral mass (COvm), blue mussel (CM), saucer scallop (CS), and southern calamari (CC) extracts, the whole molluscs were cooked in boiling PBS for 20 min prior to extract preparation as above. Extract protein concentrations were determined using the Bradford assay kit .Flesh from fresh Sydney rock oyster  and screened with patient sera diluted 1:10 in 1% skim milk powder/0.05% Tween/PBS. IgE binding was detected using rabbit anti‐human IgE  and goat anti‐rabbit IgG‐HRP .IgE ELISA was performed as described previously.450 nm ≥ 1.5 at 1:10 dilution) by direct ELISA for the relevant coating extract: for CC—A8, A10, A13; CM—A8, A11; CS—A8, A10, A13; CO—A7, A8, A10, A11, A13, and COvm—A2, A8, A10, A13. Sera were first titrated for IgE reactivity with the mollusc extract to determine the serum concentration at which the O.D.450 nm was ≈1 and within the linear phase of the titration curve. Using this dilution, patient sera were preincubated with increasing concentrations of mollusc extracts or ovalbumin  for 1 h at room temperature before testing by ELISA as above. The percentage inhibition was calculated as 100 − [(O.D.450 nm of serum with inhibitor/O.D.450 nm of serum without inhibitor) × 100]. To allow comparison between patients, the concentration required for 50% inhibition of IgE binding was determined. To assess nonspecific inhibition by mollusc extracts, serum from a non‐seafood allergic, atopic (ryegrass pollen‐sensitized) subject was incubated with the above inhibitors, and then tested for IgE reactivity with ryegrass pollen extract in comparison with untreated serum.Inhibition IgE ELISA was performed as described2.4–1) for 20 min at 37 °C. Basophil activation was assessed by flow cytometry by determining the percentage of viable, high IgE‐expressing, CD63+ cells (gating strategy shown in ref. −1) and stimulation buffer alone as negative controls.In vitro basophil activation by the mollusc extracts was assessed as described previously.2.5Mollusc extract proteins were separated by SDS‐PAGE and stained with Coomassie Brilliant Blue as described previously.–1, 2 μg mL–1, and 10 μg mL–1), OVA , or the same mollusc extract (20 μg mL–1) as used for the binding assay (positive control). The preincubated sera were then tested for IgE reactivity with the mollusc extracts by immunoblotting as above. Nonspecific inhibition by mollusc extracts was assessed as described for the inhibition IgE ELISA above.For IgE inhibition immunoblotting, sera  were preincubated for 1 h at room temperature with rPor p 1 . Briefly, the protein bands were destained, reduced, and alkylated. The gel pieces were washed and dehydrated, followed by trypsin digestion. The gel pieces were then sonicated and analyzed by LC‐MS/MS on a HCT ULTRA ion trap mass spectrometer  coupled online with an Ultimate 3000 nano HPLC . Data were exported in Mascot generic file format and searched against the Swiss‐Prot databases using the MASCOT search engine  with all taxonomy selected.2.7Crassostrea gigas) tropomyosin (GenBank accession number AB444943.1). PCR products were cloned into a sequencing vector, pCR 2.1, using the TOPO TA cloning kit  and transformed into TOP10 chemical competent Escherichia coli. Positive colonies were confirmed by blue–white screening and colony PCR using gene‐specific oligonucleotide primers for the presence of inserts. Plasmids were purified from overnight culture using an AxyPrep Plasmid Miniprep Kit  and sequenced .To determine the amino acid sequence of SRO tropomyosin, cDNA was amplified and sequenced as we described for blue swimmer crab.2.8p < 0.05. The Spearman's rank correlation test was used to assess correlation between IgE ELISA and ImmunoCAP values. Analyses were performed using GraphPad Prism for Windows .The Friedman test in conjunction with Dunn's multiple comparison test was used to compare serum IgE reactivity between mollusc extracts. Differences were defined as statistically significant at 33.1FigureAnalysis of mollusc extracts by SDS‐PAGE revealed a range of proteins from 5 to >188 kDa , particularly for the raw extract likely due to removal of endogenous proteinases. However, there was no overall difference in protein profile except for the more prominent band at 20 kDa seen only in the ROvm extract Figure .3.2FigureIgE ELISA showed that all cooked mollusc extracts had higher overall IgE reactivity than their corresponding raw extract, although this difference was not statistically significant for scallop or oyster without visceral mass Figure. For indr2 = 0.7, p  =  0.005) and cooked  oyster extracts. Removal of the visceral mass did not impact this correlation. There was a strong correlation between mussel ImmunoCAP and cooked blue mussel (CM) IgE ELISA values , but interestingly, no correlation for the raw mussel extract. IgE ELISA reactivity for raw or cooked scallop and calamari extracts showed no correlation with respective ImmunoCAP values.Comparison between patient oyster ImmunoCAP and IgE ELISA values revealed a positive correlation for both raw  with the mollusc extracts and flow cytometry Figure. Activat3.4FigureFigureIgE‐reactive proteins within each of the mollusc extracts were examined by immunoblotting Figure. The resFor the cooked calamari and mussel extracts, the bands seen at 31 kDa and 55 kDa, respectively, for all subjects were shown in control experiments to be due to nonspecific binding of the secondary antibody to the proteins in the absence of patient serum (data not shown).3.5FigureInhibition IgE ELISA was used to assess IgE cross‐reactivity between whole mollusc extracts and the effect of cooking on cross‐reactivity Figure. Sera we3.6FigureInhibition IgE immunoblotting was used to examine the effect of cooking on mollusc tropomyosin cross‐reactivity with the well‐characterized blue swimmer crab tropomyosin, rPor p 1Two relevant subjects for each cooked mollusc extract, that is, with IgE reactivity to a band in the tropomyosin region, were selected. The crustacean tropomyosin rPor p 1 inhibited IgE reactivity to the “tropomyosin” band for all four mollusc species. In most cases, the inhibition was dose dependent, but for one subject's serum IgE reactivity to mussel extract Figure C there w3.7TableTo further characterize SRO allergens, we excised IgE‐reactive bands for mass spectrometric identification Table. Bands a3.8Figurehttp://www.allergen.org/index.php) based on subsequent further testing of IgE reactivity of the purified protein (data not shown). Alignment of known tropomyosin sequences for different shellfish species shows that SRO tropomyosin has very high sequence identity with tropomyosin from another oyster species (Pacific oyster), and lower identity with two bivalves  and the cephalopod Japanese flying squid  was cloned and reverse transcribed into cDNA to obtain the complete sequence (4S. glomerata) tropomyosin, Sac g 1, was sequenced and cross‐reactivity with other mollusc and crustacean tropomyosins assessed.Diagnosis of mollusc allergy is often difficult due to unreliable clinical history as patients may be unaware of the specific trigger of their food‐allergic reaction. Currently, food challenge is the most reliable way to identify food allergy. However, this procedure may be associated with risk of serious adverse reactions in the case of shellfish challenge due to high allergen potency and adult patient comorbidities. For practical reasons, this diagnostic procedure is seldom conducted in adults. A reliable laboratory‐based assay is needed. Hence, we sought to characterize allergens in four commonly consumed Asia‐Pacific mollusc species, assessing frequency of recognition by patient IgE and cross‐reactivity. The effect of heating on IgE reactivity of mollusc extracts was also examined. SRO  was used. In this assay, native allergen extract was used, presenting conformational as well as linear IgE epitopes. Basophil activation following in vitro allergen challenge as assessed by CD63 expression is a well‐recognized correlate of histamine release and clinical reactivity ,IgE reactivity and allergen sensitization can be affected by food processing.While many molluscs are cooked before ingestion, thereby potentially presenting heat‐modified allergens to the patient's immune system, this is not usual for oysters. An alternative explanation for the increased IgE reactivity of cooked extracts is that cooking in the presence of endogenous or exogenous sugars results in generation of advanced glycation end products due to the Maillard reaction.Clinical management of mollusc allergy includes avoidance of the offending species. It is therefore critical to understand the cross‐reactivity between different mollusc species to avoid accidental allergen exposure and potentially severe allergic reactions. Allergen cross‐reactivity between mollusc species and other animals has been suggested both experimentally and clinically.Patients with putative mollusc tropomyosin reactivity also demonstrated IgE cross‐reactivity with the major crustacean tropomyosin Por p 1 previously identified by us.Taken together, our novel findings on the characterization of allergenic proteins of mollusc species should inform development of reliable component‐resolved diagnostic assays for mollusc allergy and enable accurate dietary advice.The authors declare no conflict of interest.


Mid-Holocene climate was characterized by strong summer solar heating that decreased Arctic sea ice cover. Motivated by recent studies identifying Arctic sea ice loss as a key driver of future climate change, we separate the influences of Arctic sea ice loss on mid-Holocene climate. By performing idealized climate model perturbation experiments, we show that Arctic sea ice loss causes zonally asymmetric surface temperature responses especially in winter: sea ice loss warms North America and the North Pacific, which would otherwise be much colder due to weaker winter insolation. In contrast, over East Asia, sea ice loss slightly decreases the temperature in early winter. These temperature responses are associated with the weakening of mid-high latitude westerlies and polar stratospheric warming. Sea ice loss also weakens the Atlantic meridional overturning circulation, although this weakening signal diminishes after 150–200 years of model integration. These results suggest that mid-Holocene climate changes should be interpreted in terms of both Arctic sea ice cover and insolation forcing. Mid-Holocene climate was characterized by strong summer solar heating that decreased Arctic sea ice cover. Here the authors show that this sea ice loss had profound effects on the climate system, distinct from direct effects of solar heating, over North America, northern Asia, and the North Atlantic. The thermal maximum was particularly pronounced at high latitudes including Greenland, the Canadian Arctic, and Alaska6. Proxy data also indicate that Arctic sea ice cover was smaller than the present during the mid-Holocene (around 6 ka BP) over Fram Strait, Baffin Bay, and the Labrador Sea7. This is because summer insolation can warm the Arctic rapidly throughout the season, as sea ice decline can amplify the warming via various feedbacks, referred to as polar amplification10. These paleo-proxy records are supported by climate models that simulate substantial reduction of the summer and autumn sea ice cover during the mid-Holocene relative to the pre-industrial climate12.Northern Hemisphere summer solar insolation was anomalously strong during the early-mid Holocene, causing the Holocene thermal maximum from around 9000 years to 5000 years before present (BP)13, we anticipate that Arctic sea ice decline may have contributed substantially to mid-Holocene climate change. Indeed, modeling studies associate the recent and future declines of Arctic sea ice cover with significant impacts on Northern Hemisphere weather patterns21. In particular, polar stratospheric warming, a negative Arctic Oscillation (AO)-like weakening of mid-high latitude westerlies, and a slight cooling over Central–East Asia appear robustly in the climate model simulations. In contrast, there has been little research on how Arctic sea ice cover contributed to mid-Holocene climate change.Because Arctic sea ice influences surface albedo and ocean–atmosphere heat exchange, as well as ocean’s freshwater content22. The root-mean-square errors of sea ice extent and volume between CESM1–CAM5 and observations are one of the lowest23 among 49 climate models that have participated in phase 5 of the Coupled Model Intercomparison Project (CMIP5).In this study, we show that the Arctic sea ice decline had profound effects on mid-Holocene climate throughout the year. Specifically, we present modeling evidence that the impact of sea ice decline on mid-latitude temperature might be as large as that of the direct effect of insolation. We perform three simulations to separate the effect of sea ice loss from that of direct insolation: one with pre-Industrial insolation and sea ice, one with mid-Holocene insolation and sea ice, and one with mid-Holocene insolation and pre-Industrial sea ice (see Methods). We use a state-of-the-art climate model, the Community Earth System Model, version 1 (CESM1) with the Community Atmospheric Model, version 5 (CESM1–CAM5)12. Figure 4, has previously been reported as a response to Arctic sea ice loss25. The impact of Arctic sea ice loss is not limited to high latitudes: sea ice loss warms the North Pacific Ocean and North America, but slightly cools the North Atlantic Ocean. The longitude–time Hovmöller plot of SAT averaged between 45°N and 62°N  in summer-autumn decreases by 30–35% over wide areas of the Arctic relative to the pre-industrial climate Fig. , which i2°N Fig.  shows th2°N Fig.  are comp2°N Fig. . The inslot Fig.  verifieslot Fig. , the annlot Fig. .Fig. 1Si20. This Eurasian continent cooling occurs mostly in early winter (December–January), during which SAT decreases by 0.8 K over wide areas of Central and East Asia  8.5. The development of high pressure in sub-Arctic Russia often causes cold eastern Eurasian winters29. Over Central and East Asia, both sea ice loss and insolation forcing act to decrease temperature  multi-model average  zonal-mean westerlies from the lower troposphere to the stratosphere  changes. Figure 36, Fig. 37 showing that the AMOC weakening is a transient response to the Arctic sea ice loss. Therefore, it is unlikely that the AMOC weakening and the associated North Atlantic cooling persisted throughout the mid-Holocene. While the AMOC weakening signal substantially diminishes after year 200, the North Atlantic cooling still persists , whereas Arctic sea ice loss opposes this cooling by increasing SAT over North America by 1–2 K. Our model results suggest that Arctic sea ice cover and direct insolation forcing played distinct roles in mid-Holocene climate change. This separation offers a conceptual framework for interpreting Holocene proxy records of the mid and high latitudes.In summary, our model simulations demonstrate that Arctic sea ice loss driven by anomalously strong summer insolation in mid-Holocene has a profound effect on weather and climate. During mid-Holocene winter, Arctic sea ice loss weakens the mid-high latitudes westerlies, which has been shown to be a robust response to the projected future Arctic sea ice loss across climate models22. The atmospheric model is the Community Atmospheric Model version 5 (CAM5) with 30 vertical levels, and the ocean component is the Parallel Ocean Program version 2 (POP2) with 60 vertical levels. The land and sea ice components are the Community Land Model version 4 (CLM4) and the Los Alamos sea ice model version 4 (CICE4), respectively.We perform simulations NCAR CESM1.2.1, a fully coupled model with approximately 1° horizontal resolution (f09g16)21 also used this method  to isolate the effect of Arctic sea loss on atmospheric circulations.To distinguish the climatic responses to sea ice loss and anomalous insolation forcing in the mid-Holocene, we perform three different model simulations. First, we perform a 335-year-long Pre-industrial simulation that has constant year 1850 radiative forcing and greenhouse gas concentrations. Second, the mid-Holocene simulation with 6 ka BP insolation and greenhouse gas concentrations is branched off from year 31 of the Pre-industrial simulation and runs for 315 years. Third, another simulation with mid-Holocene forcing is branched off at year 31 of the Pre-industrial run, except the albedo of sea ice is increased globally, and throughout the year, from 0.73 to 0.91 to reflect more sunlight, while the snow albedo over sea ice is not changed. A recent studyThe three different model simulations can be summarized as:0 k: Pre-industrial control simulation (335-year duration: from year 1 to 335)6 ka: Mid-Holocene climate simulation (315-year duration: from year 31 to 345)6 ka with 0 k sea ice: Mid-Holocene climate with sea ice albedo is increased to 0.91 (316-year duration: from year 31 to 346)The contributions of Arctic sea ice loss and direct insolation anomalies to mid-Holocene climate, relative to the preindustrial, can be separated as follows:The contribution of Arctic sea ice loss: (6 ka)–(6 ka with 0 k sea ice)The contribution of insolation forcing: (6 ka with 0 k sea ice)–(0 k)40. Discarding the first 50 years of model integration, therefore, captures the equilibrium response of atmosphere, whereas the deep ocean circulation takes several hundred years to reach an equilibrium41 , especially net surface shortwave flux (FSW↑−FSW↓): Fnet↑=FSW↑−FSW↓+FLW↑−FLW↓+SHF↑+LHF↑, where FSW↑ and FSW↓ are upward and downward shortwave radiative fluxes, respectively. FLW↑ and FLW↓ are upward and downward longwave radiative fluxes, respectively, and SHF↑ and LHF↑ denote sensible and latent heat fluxes, respectively. The net surface shortwave flux simulated by the increased mid-Holocene ice albedo experiment is almost identical to that of the pre-industrial control simulation .The increased ice albedo in the 6 ka with 0 k sea ice experiment prevents Arctic sea ice loss in summer–early autumn in response to the anomalously strong mid-Holocene insolation. However, this increase in ice albedo has little influence on Southern Hemisphere sea ice  had equal means or not. These statistically significant values are presented as ‘hatches’ in the figures.To assess whether these model simulation produced different climate conditions, a statistical significance test was performed for seasonally averaged variables. Specifically, a two-sample To assess the realism of mid-Holocene climate simulated by CESM1, we also examine ten coupled GCMs from the PMIP3 and CMIP5. We evaluated the SAT and SST fields by examining the differences between PMIP3 mid-Holocene and CMIP5 Pre-Industrial simulations. A list of the PMIP3–CMIP5 models and their Atmosphere and Ocean resolutions are provided in Table Code from this study (mostly MATLAB scripts) is available from the corresponding author upon request.Supplementary InformationPeer Review File


DAPK2  is a member of the DAPK family of proteins: death associated Ser/Thr kinases that belong to the calmodulin (CaM)-regulated kinase superfamily , but speIn our recent study, we identified AMPK as a positive upstream regulator of DAPK2 . AMPK isNotably, Ser289 phosphorylation by AMPK is specific to DAPK2 as this mode of regulation is not shared with DAPK1. Despite the fact that this residue is located in a region that is homologous between the two kinases and is conserved in both, we found that it is not phosphorylated in DAPK1 upon AMPK activationin cells. However, this site was previously found to be phosphorylated in DAPK1 by RSK, a member of the MAPK family . This diL and thus promoting autophagy induction P at the autophagosome assembly site, which is the first step in autophagosome assembly. A previous screen for protein-protein interactions within the cell death network identified Atg14 as a binding protein of DAPK2 , implyin


Plasmodium enolase is a target for the growth neutralizing antibodies. Interestingly, the three invasive stages i.e. sporozoites, merozoites, and ookinetes express this protein on their cell surface. Polyclonal anti-Plasmodium falciparum enolase (Pfeno) antibodies disrupt traversal of ookinete through mosquito mid-gut wall as well as have inhibitory effect on parasite growth at erythrocytic stage. In a recent study, it was observed that immunization with a unique epitope of parasite enolase (EWGWS) could confer partial protection against mouse malaria. Further validation is needed for the protective potential of this unique epitope in otherwise highly conserved enolase.P falciparum enolase, a monoclonal antibody specific to EWGWS is generated. In vitro parasite growth inhibition assays and passive immunization of Plasmodium yoelii (or Plasmodium berghei) infected mice were used to assess the parasite growth neutralizing activity of the antibody.In order to investigate the efficacy of growth inhibitory potential of the epitope of P. yoelii or P. berghei infected mice showed significant reduction in parasitemia as compared to controls (p < 0.001). Surface Plasmon Resonance measurements indicated high affinity binding of H12E1 to P. falciparum enolase (KD ~ 7.6 × 10−9M).Screening a panel of monoclonal antibodies raised against recombinant Pfeno that were specific to EWGWS resulted in isolation of H12E1. This antibody recognized only EWGWS epitope containing enolases. H12E1 strongly inhibited parasite growth in culture. This inhibition was strain transcending. Passive infusion of this antibody in A monoclonal antibody directed against EWGWS epitope of Pfeno was shown to inhibit the growth of blood stage malarial parasites. This inhibition was species/strain transcending and is likely to arise due to blockade of enolase on the surface of merozoites, functionally implicating Pfeno in invasion related events. Presence of enolase on the cell surface of merozoites and ookinetes could potentially result in inhibition of host cell invasions at erythrocytic and transmission stages in the parasite life cycle. It is suggested that antibodies against EWGWS epitope have the potential to confer dual stage, species and strain transcending protection against malaria.The online version of this article (10.1186/s12936-018-2455-6) contains supplementary material, which is available to authorized users. Plasmodium, being an intracellular parasite, needs to invade host cells to establish infection. There are three invasive stages  in the life cycle of Plasmodium, two of which (sporozoites and merozoites) get briefly exposed to the humoral branch of the human immune system, rendering the molecular machinery of merozoites and sporozoites involved in invasion as attractive targets for anti-malarial vaccine.Despite recent progress in malaria prevention and control, the disease continues to take a heavy toll . It is bPlasmodium and its multistage complex life cycle are to combine multiple antigens that are valid targets at various stages in the parasite life cycle as well as their orthologues from different species/strains to obtain an effective multistage, species and strain transcending malaria vaccine  = 0 µg/ml) was also measured microscopically at all the three time points . Parasite growth with time at various concentrations of the antibody is shown in Fig. 50 values obtained from these titrations were 28.5 ± 2.7 (from 48 h data) and 16.6 ± 1.3 µg/ml (96 h data), respectively  for two strains of P. falciparum ranged between 13.0 and 28.5 µg/ml (Table The nature of antibody induced growth inhibitory effect on parasite (reversible or irreversible) was investigated in in vitro cultures. Culture samples containing synchronized d RBCs ~ % pRBCs wd RBCs ~ % pRBCs wP. yoelii 17XL or P. berghei strain ANKA and injected with purified IgG on the same day. The protective effect of H12E1 was assessed by measuring the effect of injected antibody on the parasitemia and comparing that with the pre-immune IgGs injected controls. Results obtained for two different species of mouse malarial parasites are shown in Fig. P. yoelii challenged group, passive infusion of H12E1 clearly attenuated parasitemia in experimental animals compared to the controls  yielded a dissociation constant KD = 7.58 × 10−9 M. Considering the KD value obtained here, it can be argued that at a mAb concentration of ~ 1–2 µg/ml, ~ 50% of the antigen would be in bound state. The value of the association rate constant obtained here falls well within the range suggested earlier (1 × 105/M/S) /60*kd) of rPfeno-H12E1 complex was also computed (t1/2 = 10.5 min). Since merozoites reinvade fresh erythrocytes in a couple of minutes of their egress, it requires high concentrations of high specificity antibodies that can bind rapidly (high ka) and stably (long t1/2) so as to have a substantial fraction of merozoites saturated with antibody to generate the inhibitory effect [Binding affinity and half-life of an antibody-antigen complex are important determinants of an antibody’s potency to neutralize a pathogen , 43. Dir105/M/S) . A simily effect , 45. ThePlasmodium has a single gene for enolase, inhibition of this activity could result in parasite growth inhibition. To determine whether the binding of H12E1 antibody had any effect on the enzyme activity, rPfeno (80 nmoles) was incubated with different amounts of mAb  and the activity of rPfeno-mAb complex was measured. The activity measurements showed that the binding of enolase to H12E1 had no effect on its catalytic activity  (PDB: 3OTR). Structural mapping of the four functional sites: (i) the plasminogen binding site 277DKSLVK282 (blue) [Enolase is a glycolytic enzyme that catalyzes the inter conversion of 2-phosphoglycerate and phosphoenolpyruvate. As ity Fig. a indicat2 (blue) ; (ii) th2 (blue) ; (iii) t2 (blue)  (green);Plasmodium, enolase localizes in multiple organelles  where it is likely to participate in different physiological functions [277DKSLVK282 domain of Pfeno, while merozoite inhibitory antibody (H12E1) functions through the 104EWGWS108 epitope. The underlying molecular mechanisms in the two cases are likely to be quite different. In ookinetes, Pfeno functions as a receptor for plasminogen as well as a ligand for mosquito mid-gut epithelial cell receptor [Enolase is a glycolytic enzyme with a highly conserved structure and multiple moonlighting functions , 48. Sevunctions . Presencunctions , 23, 50.unctions , 50 whilunctions . Data prreceptor , whereasP. falciparum reticulocyte binding protein homolog 5 (PfRH5) had shown similar inhibition [Plasmodium  and different strains of P. falciparum (3D7 and INDO). Given the highly conserved nature of the target sequence and its location in the polypeptide  have been tested for their ability to protect against malaria and block its transmission , 9, 62. For the development of a multivalent vaccine that is effective against multiple species and strains of the parasite, with an ability to neutralize growth at multiple stages, one will need to combine several antigens , 17, 62.P. falciparum (3D7 and INDO) and three different species of Plasmodium  indicates the species and strain transcending nature of this epitope. Plasmodium spp. enolase has thus emerged as a unique antigen that has surface expression at all three invasive stages; is an invasion target in at least two stages (merozoites and ookinetes); the target epitope of which is highly conserved, is monomorphic and has the ability to induce protective antibodies on immunization. Poor immunogenicity of this epitope is a concern that needs to be addressed. Thus, antigenic epitope EWGWS from Pfeno could be a good candidate for the development of a two-stage malaria vaccine.In this paper, we have described the generation of a monoclonal antibody H12E1 that has a potent merozoite neutralizing activity and targets the EWGWS epitope in Pfeno. The mAb induced an almost complete inhibition of parasite growth in in vitro cultures of the asexual stages (> 90% inhibition at < 100 µg/ml). Active immunization of mice with an antigenic peptide containing EWGWS epitope is known to confer partial protection . PassiveAdditional file 1. Purification, specificity and isotyping of mAbs. Three mAbs that had high reactivity with WT-ePfeno were purified. (A) Coomassie stained 10% SDS-PAGE for the Protein-A Sepharose purified IgGs from hybridoma supernatants of the three mAbs. Two bands corresponding to heavy and light chain of antibody are observed. (B) Western blot of purified rPfeno with each mAb and (C) isotyping of mAbs. All three mAbs were of IgG2b class of immunoglobulins.Additional file 2. Quantitation of un-ruptured schizonts in anti-rPfeno antibody treated cultures at 48 h post antibody addition. Control samples had no schizonts. Data are expressed as percent of total Schizonts present at 24 h post antibody addition (see Fig. see Fig. .


Hylobius abietis) damage to newly planted conifer seedlings. Site preparation is one of the most important measures for reducing these risks and as several studies have shown the damage is highly dependent on the amount of pure mineral soil around the seedlings. We investigated effects of three site preparation techniques: (1) disc trenching with a conventional Bracke T26, (2) MidiFlex unit and (3) soil inversion with a Karl Oskar unit on characteristics of the planting spots, growth and pine weevil damage and survival rates of untreated and insecticide treated planted Norway spruce (Picea abies) seedlings. All three site preparation techniques reduced pine weevil damage in comparison with no site preparation, and the proportion of spots with pure mineral soil they created was inversely related to the rate of mortality caused by pine weevil. The results indicate that the quality of the planting spots depends on the technique used. In areas where pine weevil is the major threat to seedling survival, the amount of mineral soil in the planting spots is the most important factor in order to protect the seedling from damage. Without site preparation most planting spots consisted of undisturbed humus. Generally, the Karl-Oskar created the most spots with pure mineral soil, but on very stony soils the Bracke T26 created more mineral soil spots than other methods. Site preparation is a valuable tool in order to improve survival in regeneration areas and it is of great importance to make the right choice of technique depending on the particular circumstances on the actual site.In northern Europe, there are high risks of severe pine weevil ( The management of vegetation competition in forests is an integral part of silvicultural practices in many parts of the world , the most serious pests of planted conifer seedlings on forest land in northern Europe  seedlings.The aim of this study was to investigate effects of three site preparation techniques  on characteristics of the planting spots, and growth, pine weevil damage and survival rates of untreated and insecticide treated Norway spruce (A control treatment (no site preparation) was also included. Various studies have compared seedling growth and survival rates following site preparation by different techniques, but few have also compared the nature of the planting spots.For example, we wanted to test if a site preparation technique that can adjust the planting spots according to differences in soil moisture conditions and stoniness can generate a higher proportion of high quality spots.In this study area the quality of the planting spot was correlated to the achieved amount of mineral soil in order to reduce the risk of damage by pine weevil.Classification of planting spots, the effect of site preparation technique on sites with various stoniness and soil type class, as well as the effects on seedling height growth and survival rates could be of high interest for any other countries that uses site preparation as a regeneration tool.3 ha−1 year−1, with original stands dominated by Norway spruce (Picea abies L. Karst.) Sandy silt was the most common soil texture, and only two of the sites consisted of sandy till. The mean annual precipitation (data from 1960 to 1990) was 629 mm for the sites in Scania (1–5) and 1032 and 812 mm respectively for Västergötland (site 6) and Småland (site 7). The mean temperature (data from 1962 to 1990) was 6.6, 5.8 and 6.1 °C respectively. .Seven experimental forest sites were used in this study. They were clear-cut during the winter of 2010–2011 or 2011–2012 and planted during the following spring .The Norway spruce seedlings were obtained from a commercial nursery, Södra Odlarna, Falkenberg (Sweden) and originated from Bredinge seed orchard located in southern Sweden . They were grown for 2 years in 90 cmPlanting spots (except the controls) were prepared at each study site using a Bracke T26 disc trencher , a Midiflex disc trencher , both mounted on forwarders and a Karl Oskar  unit mounted on an excavator. For practical reasons different persons were hired to perform the site preparations at the different occasions, i.e. in total three operators for Bracke T26, one operator for Midiflex and two operators for Karl Oskar.The Bracke T26 is a conventional two-row, rear end-mounted disc trencher, designed to create tracks or furrows and berms of inverted soil (from the tracks) covering the soil beside them, so the surface of the berms consists of mineral soil. The Midiflex is also a two-row disc trencher but with the unit and discs mounted in the middle of the forwarder in front of the rear boogie. It creates rows of track and berms, like a conventional disc trencher, but the rear bogie runs over and thus compresses the berms. The Karl Oskar is an excavator-based preparation unit that is specially designed for creating various kinds of planting spots. The operator can use it for either inverting, mounding or patch scarification. Thus, the method applied to create each planting spot can be adapted in accordance with the characteristics of the spot and seedling to be planted.At each site, rows of planting spots were created by each of the three machines, in blocks of six prepared tracks, two per machine, and two tracks with no site preparation Fig. .Fig. 2ScThe order in which the machines made the tracks was randomly selected. Each block covered approximately 20 × 20 m, and their positions were intended to ensure that site conditions were as homogeneous as possible within them and the soil moisture in each block was classified as one of three classes: dry, mesic or moist  were planted in spots created by each treatment. Thus, 80 seedlings were planted in each block (at ca. 2 m spacing), and there were 37 blocks in total, so 2960 seedlings were used in the experiment.No site preparation avoid planting in low spots.Bracke T26 On dry or mesic soil, the operator should try to create shallow tracks with mineral soil surfaces and the seedlings should be planted in the middle of them. If a spot in the middle of a track is too low so there is a risk of standing water and drowning of the seedling as a consequence, the seedling should be planted at a higher level. On moist soil, the driver should still aim to create tracks of mineral soil, but the seedlings should be planted in the area between the track and the berm  rather than in the middle of the track.Midiflex The operator should try to get plenty of mineral soil on the compressed berms and the seedlings should be planted on them. If there is insufficient mineral soil on the berm, they should be planted in the track, but avoiding low spots. If no planting spots containing mineral soil are created, the seedlings should be planted in the berms.Karl Oskar The operator was instructed to select an appropriate type of preparation depending on the stoniness and other site conditions: mounds should be made in moist spots, inverted spots in all other areas where possible, and patch spots otherwise.The following instructions were given to the operator of each machine and the staff planting the seedlings.Stoniness was assessed by pushing a metal pin into the ground to a depth of 20 cm ; track or patch ; mound or berm ; or inverted . The soil moisture within this area was also recorded, as dry, mesic or moist.Within a radius of 10 cm around each seedling the dominating soil type, i.e. soil type class was determined in four classes: undisturbed humus, disturbed humus, mixture of humus and mineral soil, and pure mineral soil. The height of each planting spot was measured to the closest centimetre, relative to the undisturbed ground bordering the site preparation spot, and could be negative if the spot was lower than the undisturbed ground. The shortest distance (cm) from the seedling to disturbed humus  was measured, to the closest centimetre, at the same time.2. The severity of damage was recorded subjectively using a six-level scale: 0 = undamaged, 1 = slightly damaged, 2 = uncertain or insignificant damage, 3 = severely damaged, 4 = life-threatening damage, and 5 = dead. Damage due to other causes  was also determined.Directly after planting, the height of the aboveground part of each seedling was measured, in late autumn (October) during each of the first 3 years, we registered its height, length of its leading shoot and damage by pine weevil the length measures were done to the closest centimetre and the debarked area of the stem to the closest 0.1 cm0 is the intercept and β1–β7 the regression coefficients, X1 is the stoniness and the last X variables represent the indicator variables for the four site preparation treatments . Chi square tests were used to assess statistical differences between site preparation methods. Effects of stoniness of the ground on site preparation results, in terms of soil type class and distance from undisturbed humus in planting spots were also analyzed. A first-order regression model, with soil type class or distance from undisturbed humus as the response variable, and stoniness as the explanatory variable, was applied using PROC GLM in SAS 9.4. In the model, site preparation treatment was included as a qualitative predictor variable and the effect of interaction between stoniness and site preparation was tested. For example, the model used to investigate the effects of the four site preparation treatments was:2) in each year was analyzed using data for all living seedlings, and seedlings that were attacked and killed during that year.When analysing seedling height (cm), and length of leader (cm) data recorded for all seedlings classified as living seedlings were used. The mean area debarked (cmi is the random effect of site (i = 1–7), bjk is the random effect of block (j = 1–21) within planting year (k = 1–2), γl is the fixed effect of site preparation treatment (l = 1–4) and δm is the fixed effect of insecticide treatment (m = 1–2). The interaction between site preparation and insecticide treatment was included in the model as a fixed effect. To test effects of soil type on seedling height, length of leader and debarked area 3 years after planting, the same model was used, but instead of site preparation the fixed effect of the four soil type classes was tested. A similar model was used to test for differences in height of planting spot and distance to disturbed humus between site preparation methods and planting spot class. Satterthwaite approximation was used to determine appropriate degrees of freedom in all analyses. When significant differences between treatment means were detected, they were separated by least square means, with Tukey–Kramer adjustment of p-values. An α-level of 0.05 was used in all analyses.The accumulated debarked area during the experimental period was based on all planted seedlings. Both height and debarked area adequately followed a normal distribution with equal variances, and were analysed with a mixed model implemented in PROC MIXED in SAS 9.4:When analysing effects of the explanatory variables on the proportion (η) of attacked or killed seedlings after the first three growing seasons, the input data adequately followed a binomial distribution to use generalized linear mixed models implemented in PROC GLIMMIX in SAS 9.4. Total seedling mortality was defined as the proportion of planted seedlings that had been killed at the end of the third year. The proportion of seedlings that had been attacked by pine weevil was analysed both per year and as an accumulated value over the experimental period. For these analyses, a logit link function was used:mentclass2pt{minimWithout site preparation, almost all planting spots were classified as “no scarification (undisturbed humus)”. After preparation using the Bracke T26, most planting spots were classified as tracks or patches, regardless of soil moisture class. Of spots created by the MidiFlex, most were mounds/berms and about a third were tracks/patches. As expected, many spots created by the Karl Oskar were classified as inverted, but the results also reflected the instructions and the unit’s capacity to create diverse kinds of planting spots in accordance with site conditions. In dry blocks, the planting spots were almost evenly distributed between tracks/patches, mounds/berms and inverted. In mesic blocks most spots were inverted, while in moist blocks they were mostly mounds/berms Table .p = 0.0001 for all soil type classes). Without site preparation, almost all planting spots (94%) consisted of undisturbed humus  and shortest distance from the seedling to disturbed humus were also affected by the site preparation treatment and planting spot class Table . Plantin2) of 0.48, the model did not fit the data as well as the model describing the effect of stoniness on soil type class (R2 = 0.91). However, it revealed the same patterns, and both models detected highly significant effects on site preparation (p < 0.001). The Karl Oskar created spots with the longest distances between planted seedlings and disturbed humus, but as stoniness increased, these distances decreased. In areas with a stoniness index ≥ 70, the Bracke T26 created planting spots with longer distances to disturbed humus than the other units.The effect of stoniness on distance to disturbed humus was also tested Fig. . With a p < 0.0001). After 3 years, the overall rates of mortality caused by pine weevils, including both untreated and treated seedlings, were 9, 14, and 18% following site preparation with the Karl Oskar, Bracke T26 and MidiFlex units, compared with 58% following no site preparation. On average, after 3 years 11 and 39% of the seedlings treated and not treated with insecticides had been killed, respectively, and the difference was statistically significant (p < 0.0001). A significant interaction between the site preparation and insecticide treatments was detected (p = 0.0039), showing that the Karl Oskar reduced the mortality of seedlings that had not been treated with insecticide more than the other units  after 3 years.Most of the seedling mortality caused by pine weevils occurred during the first growing season, but a substantial amount of seedlings were also killed during the second and third year in some of the treatments Fig. . SeedlinMost of the seedlings (80.3%) were attacked, but not necessarily killed, by pine weevils during the experimental period. During the first growing season, both the insecticide treatment and site preparation strongly reduced attack rates Table . The KarDuring the second growing season the insecticide treatment had the opposite effect, as more seedlings treated with insecticide at the time of planting were attacked and debarked than the others. However, this increase in attack frequency did not lead to an increase in seedling mortality in the second year Fig. . During At the time of planting all seedlings had similar heights Table . During p < 0.0001). It declined with increases in the amount of mineral soil in the planting spot, from undisturbed humus through disturbed humus and a mixture of mineral soil and humus to pure mineral soil (Table p < 0.0001 for both), but the only significant differences were that seedlings in planting spots with undisturbed soil had lower heights and shorter leading shoots than those in other soil type classes  in inverted spots created by the Karl Oskar In mounds created by the Bracke T26 and Karl Oskar the distance to humus reached around 7 cm, and in other types of planting spot the distances were shorter. The area debarked by pine weevils reportedly decreases with increasing distance to humus, but only weakly at distances > 10 cm (Örlander and Nordlander The height of the planting spots (topography), well described by Sutton  differs Several studies shows that experience and motivation of the operator influences the efficiency and quality of the work. Karl-Oskar is probably the technique where you have the most possibilities to affect the result. Different operators work differently and might, for example, compress the soil in the planting spot to varying degrees with the aggregate. This might be one reason why the inverted spots created in this study were somewhat elevated. The Bracke T26 created the highest mounds, probably because mounds created by the Midiflex and Karl-Oskar (but not the Bracke T26) are compressed by the unit per se or by the rear bogie.Structural characteristics of the ground, such as stoniness, will affect the quality of the planting and site preparation (Arnkil and Hämäläinen Without site preparation, the seedling mortality rate was very high, almost 80% after three growing seasons, and the main cause was damage by pine weevil. These findings are consistent with observations of seedlings planted on fresh clear-cuts in southern Sweden in several previous studies (Nordenhem During the experimental period, most seedlings were attacked by pine weevils, but not necessarily killed or severely damaged. During the first growing season, both the site preparation and insecticide treatments significantly reduced frequencies of attacked seedlings and debarked areas. In contrast, during the second year, seedlings treated with the insecticide before planting tended to be attacked more frequently, and debarked more severely, than untreated seedlings. Wallertz and Petersson  recordedSeveral studies have also shown that the tolerance of seedlings to pine weevil damage is influenced by their stem base diameter (Örlander and Nilsson In general, the results from the present study highlights the importance of choosing the right method of site preparation for a given site. Disc trenching; either with Bracke T 26 or Midiflex, creates relatively homogenous characteristics of planting spots over the prepared area. In contrast, Karl Oskar due to its flexibility can select the best site preparation method for each individual potential planting spot by using either inverting, mounding or patching. The advantage of this is reflected in the results since Karl Oskar overall created the highest number of high quality planting spots. On the other hand; on very stony sites disc trenching has the advantage of producing more acceptable planting spots due to its continuous operational mode. In the results this is demonstrated by the fact that Bracke T26 produced highest number of planting spots on very stony sites.


The results were as follows: (1) The thickness reduction in glaciers would lead to a decrease in ice surface tension and basal pressure and friction at the bottom, and the resulting extensional and compressional flow played an important role in the variations in glacial velocity. (2) The force at the bottom of the glacier was key to maintaining the overall stress balance, and the glaciers that often melted and collapsed in bedrock were more easily destroyed by the overall force balance and increased change rate of glacial thaw. (3) Temperature changes at different altitudes affected the ice viscous force. The closer the ice surface temperature was to the melting point, the greater the influence of temperature changes on the ice viscous force and ice surface velocity. Finally, we used the RCP 4.8 and 8.5 climate models to simulate the changes in H8 over the next 40 years. The results showed that with some decreases in ice surface compression and tension, the gravity component changes caused by local topography begin to control the ice flow movement on the surface of glacier, and melting of the glacial surface will appear as an irregular change. The simulation results further confirmed that the fluctuation in glacial dynamic characteristics could be attributed to the change in the gravity component caused by ablation.Ice thickness has a great influence on glacial movement and ablation. Over the course of the change in thickness, area and external climate, the dynamic process of how glaciers change and whether a glacier’s changes in melting tend to be stable or irregular is a problem that needs to be studied in depth. In our study, the changes in the dynamic process of the No. 8 Glacier in Hei Valley (H8) under the conditions of different thicknesses in 1969 and 2009 were simulated based on the Full-Stokes code Elmer/Ice ( Therefore, researchers have observed many of the glacial changes in the Tianshan Mountains, including glacial area change8, volume change12, variation in mass balance15, runoff characteristics19 and so on. These studies reflect the changes in some factors associated with glacial melting; however, these studies do not clearly explain the state and potential trends of glaciers based on the mechanism of glacial movement. Because glacial dynamic characteristics exert an imperceptible influence on glacial processes, there is more contingency and complexity in the process. That is, glacial stress changes with temperature and thickness all the time, including changes in velocity, glacial melting and hydrological process caused by stress. However, changes in stress are difficult to observe directly. Obviously simple observations can no longer meet the needs of research.Understanding the potential impacts of climate on glaciers is significant in studying regional climate, water resources and predictions of future glacial changes24. Much of the negative mass balance is the result of climate change, but the creep of the glacier will also lengthen the ice flow and reduce the glacier’s thickness. Therefore, the dynamic process also affects the variation in mass balance to some extent25. Understanding the ice deformation mechanism of the main body of glaciers and its effect on glacial flow behaviour is the core of understanding the deformation characteristics of ice body units26. Deformation rates largely depend on extrinsic parameters, such as atmospheric temperature, surface pressure caused by precipitation or snowfall, and water content caused by ice ablation28. Moreover, the ice deformation rate will govern the overall flow and ablation behaviour of glaciers. Together, plastic deformation and stress lead to distortion and deformation of ice30.The force exerted on the ice body has a vital influence on the velocity and ablation of glaciers31, and there is a great lack of complex force analysis available for this area. In this study, we employed the incompressible fluid, momentum and continuity equations to simulate the glacial stress variation under conditions of two types of glacial morphology and analysed the reason for ice velocity and stress changes. Compared with previous studies, we adjusted the viscous parameter A, and very few adjustments were made to other environmental settings except glacial topography and boundary conditions. The diagnostic simulation presented the differences in stress on the surfaces and bottoms of glaciers in 1969 and 2009. The influences of both external and internal factors on glacial movement and ablation were analysed simultaneously. Finally, future change trends of glaciers was also discussed, including the effects of dynamic characteristics on ice flow velocity, force, englacial structure and hydrological processes. Our prognostic simulations verified the results of previous studies and future trends are presented.To simulate and analyse the relevant parameters, we used the Elmer/ice model in this study. Although there have been similar glacial dynamic simulation studies before, only a few researchers have performed glacial research using the two-dimensional ice flow model in the Tianshan Mountains32.The No. 8 glacier in the Hei Valley (H8)  . The Elmer software was developed in Center for Science (CSC) Ltd., Espoo, Finland, with worldwide collaborators. Considering all stress components, Elmer can solve the thermomechanically coupled problem, such as the Full-Stokes problem of flow35. The momentum and continuity equations and incompressible heat equations have been used to describe the ice velocity, temperature and stress. The specific equation descriptions are as follows.The numerical solutions of the Full-Stokes equations were obtained using the finite element method based on the code Elmer  is the fraction of liquid material as a function of temperature, and L is the latent heat. When using the enthalpy-temperature curve to compute an effective heat capacity, Eq. Phase change modelling is performed by modifying the definition of heat capacity according to whether a point in space is in a solid phase, liquid phase or in a “mushy” region. The choice of heat capacity within the intervals is explained in detail below. When the phase change occurs within a finite temperature range, enthalpy is needed when the solidification phase change model Elmer is used. The enthalpy is defined as follows:α and the external temperature, which can be written as follows:Text can be input as the geothermal and external atmospheric temperatures.For temperature, we can apply boundary conditions and specify temperature or heat flux. In the Dirichlet boundary condition (temperature is prescribed), the heat flux depends on the heat transfer coefficient The surface temperature is affected by the temperature gradient that varies with altitude. The subglacial boundary condition of the temperature simulation is the mean annual atmospheric temperature, while the bedrock boundary condition is a prescribed geothermal heat flux gradient. The formula is as follows:36. The compilation of heat flow data in China37 shows that the geothermal flux value near the glacier ranges from 40 to 50 mWm−2. Therefore, the geothermal flux value at the bedrock location was set to an average value (45 mWm−2) in our study.We set Due to the lack of relevant measured data, especially data on ice temperature and subglacial hydrological process data, it is difficult to determine the parameters that are more suitable for the study area. Therefore, most of the parameters are set to the most commonly used values. The specific parameter values are shown in Table In September 2009, ice thickness of the H8 glacier was investigated using a ground penetrating radar (GPR) . Simultaneously, a global positioning system (GPS) device  was used to position each profile and obtain surface elevation in 2009. At the same time, our study team also obtained some surface elevation points outside the GPR survey line. All GPS data were presented in the China Geodetic Coordinate System 2000 (CGCS2000) format. To compare these data with the surface elevation data in 1969, we used the Kriging method to interpolate all the data obtained by GPR and GPS into a contour interval of 5 m and generated a digital elevation model (DEM) in 2009 with a pixel resolution of 30 m.In our study, we used a digital topographic map of China of 1:50000 in 1969 measured by the Chinese Military Geodetic services. The topographic map was first scanned at 600 dpi, and then, the 20 m and 5 m contour intervals and determined point height were digitized to produce DEMs in 1969 with a 30 m cell size Fig. . The coo38. The magnitude of ice thickness fluctuation had little effect on the simulation results based on the ice thickness of the two phases, so we ignored the effect of the error caused by the transformed DEM data.To evaluate the error, we randomly selected five nonglacialized areas below 4500 m.a.s.l., all of which had slopes of less than 15°. The results showed that the average error was 6.3 m, which was equivalent to advanced spaceborne thermal emission and reflection radiometer (ASTER) data (6.24 m)The GPR data in 2009 were used to obtain the ice thickness data. We supposed that the bedrock elevation was changeless. The ice thickness in 1969 was provided by the DEM in 1969 minus the bedrock terrain in 2009. The surface and subglacial terrain of H8 is shown in Fig. We erected four ablation stakes near the GPR survey area Fig.  on Augus−1 39. This warming trend was close to the RCP 4.5 result.Because there was no weather station on H8, we used the annual mean temperature data from 1958 to 2006 recorded at the Urumqi weather station near H8. According to the principle of air temperature decrease by 0.6 °C for every 100 m of elevation gradient above sea level, we calculated that the average temperature of the Urumqi weather station near H8 was −4.1 °C and the temperature at the position of the terminus of H8 was −5.9 °C, while the temperature of the highest elevation in the GPR survey was −9.5 °C. The mean temperature trend rate of the 14 weather stations around the Tianshan Mountains was 0.34 °C (10a)The grid was divided into five layers in the simulated area, each of which used the same number of nodes to simulate the related dynamics characteristic Fig. . The griThe tests showed that suitable simulation results could be achieved in a 40 m resolution grid  to temperature changes.In this study, we replaced the change in mass balance in the future with the variation in average thickness from 1969 to 2009. Changes in glacial thickness at different altitudes in the future were expressed by the following formula:α value) at different altitudes from 1969 to 2009 was obtained by the thickness subtraction calculation. The calculation process and results were as follows: −1.25 ma−1, −1.18 ma−1, −1.05 ma−1, −0.92 ma−1, and −0.71 ma−1 were obtained from 3394 minus 3594, 3594 minus 3694, 3694 minus 3794, 3794 minus 3894, and 3894 minus 4000 m.a.s.l., respectively. In this case, the initial surface velocity referred to the simulated velocity in this study. In terms of the relationship between the thickness variation and atmospheric temperature, from 1969 to 2009, when the atmosphere temperature increased by 1 °C, the average thickness decreased by 36.03 m. The variation in thickness was distributed in different elevation zones in the same proportion. Simultaneously, we used the RCP 4.8 and RCP 8.5 climate models to obtain the future atmospheric temperature changes  flotation thickness59. Forward ice flow is also the reason for the rapid regression of small scale glaciers61.Generally, the variation in observation velocity reflects the extent of internal stress variation. First, the decrease in thickness means that the horizontal driving force produced by the gravity of the glaciers is reducedMoreover, the gravity component of the surface ice flow could be increased by the increasing surface gradient, while the local topographic changes could only change the velocity of the protruding part but had little effect on the velocity of the deep ice layer. Simultaneously, the fluctuation in surface topography increases under the influence of further erosion by melt water, and finally, the ice surface topography shows a serac morphology. The simulation results show the influence of stress on the surface topography.62. In some main glacial branches, the ice velocity on the surface reaches a peak from late winter to midsummer and reaches the minimum between late summer and fall. The near-front velocity of the ice reached up to 14 mday−1 in May and the lowest speed of 1 mday−1 in October63. Although there also exists a close relationship between the basal water pressure and surface velocity64, the contribution of temperature to the increase in velocity accounts for a considerable proportion. Figure −1 from 1969 to 2009, while the maximum thickness decreased from 280 m to 210 m, indicating that there were other factors that could compensate for the acceleration loss caused by the gravity component. Although the velocity was the result of the combined interaction of thickness and ice temperature, the effect of the two on the velocity was the opposite. Thus, the flow velocity changed little because of the counteracting effect with the decrease in ice thickness and the increase in ice temperature at the same time. In addition, Howat et al. inferred that terminal retreat can also contribute to surface flow by weakening the resistance stress65, mainly by improving the ice flow velocity downstream. Compared to the results of the XD Glacier study in the Qinghai-Tibet Plateau using the Elmer/ice model48, according to the geothermal catalogue, the same geothermal value is used at the bottom, but only the difference in surface temperature leads to the maximum ice velocity of H8 being nearly five times greater than that of XD.Because the geothermal energy at the bottom was determined, there was no disturbance to the simulation results. The fluctuation in the simulation results mainly came from the influence of atmospheric temperature. Equation To study the influence of ice temperature on ice flow movement, we selected two ice profiles of 180 m and 80 m in 2009 to simulate the sensitivity of ice flow velocity to temperature, which was mainly to simulate the influence of atmospheric temperature on the velocity when the temperature increases or decreases by 1 °C.The final steady-state simulation results are shown in Fig. Due to the low resolution of the model, the surface ice did not show obvious characteristics of the active layer temperature Fig. , but the67. The occurrence of englacial drainages initiated by hydrofracturing in diverse glaciological regimes suggests that it is a very widespread process. As long as the high meltwater supply coincides with ice subjected to sufficiently large tensile stresses, drainage will occur from the surface to the bed68. Figure 71.As far as we know, stress can change the englacial structure, distribution of cracks and drainage systemsDue to the effect of changes in glacial morphology on stress balance, especially by both upstream compressive force and downstream tensile force, the different viscous forces of the ice body lead to complex interleaving of transverse stress and longitudinal stress. These irregular forces lead to more englacial crevasses and channels, thus accelerating the melting and retreat of glaciers.34. When ice flow at the bottom suffers differential stress from different directions, the ice is more likely to break and distort, and ablation intensity increases73. Simultaneously, local heterogeneity, such as the presence of cavities, may concentrate stress at much higher values34. The erosion rate is commonly assumed to be on a scale of base sliding velocity or ice discharge75. With the development of englacial melting holes, supraglacial melt water is able to rapidly reach the bed at elevations below equilibrium line via connections among moulins and crevasses76. The locally closed melt water promotes basal sliding, which can also result in more basal melt through frictional heating77. In our study, the simulated stress at the bottom in 2009 was greater than that in 1969, which may be related to the elastic - viscous deformation of ice caused by the temperature increase set in the models.The basal stress is an important underlying factor for scaling glacial erosion79 and transient climate forcing80. Therefore, the ice flow at the bottom is the most prone to collapse in vulnerable locations. With the change in glacial morphology, the deformation or collapse will be prompted by the changes in stress in different directions at the location where the stress is greater. More importantly, a climatic response will occur first. Thus, our research provides a good method for judging vulnerable glacial locations and predicting future trends under different climatic conditions.The simulation results of the bottom stress revealed that the maximum stress distribution was maintained on the bedrock for most of the time, especially the deviation stress in the gravity direction. Thus, the ice flow at the bottom is simultaneously affected by two factors: tectonic forcing82. However, there are significant differences of individual glaciers in response to climate changes83, as our study in H8 reflects this characteristic. Our simulation results showed that with the decrease in glacial thickness, all of the stress decreases. First, the decrease in ice thickness will reduce the ice velocity and ice supply from upstream to downstream. The high summer temperatures can accelerate ice velocity. The high summer temperatures can also increase ice flux movement and promote glacial area in the low and middle altitudes. Moreover, slowing of the average glacial velocity and increased thinning of glacial thickness jointly promote the retreat of glaciers in the Tianshan Mountains, especially small scale glaciers with slow velocity and shrinkage rate, which are more likely to show accelerated retreat in the absence of ice supply from upstream. Some studies have shown that there is an increasing trend in glacial area under the background of climate warming5. The viscoplastic deformation of glaciers under the influence of high temperature may lead to an increase in glacial area. Our study also indicated that an increase in area may not mean an increase in mass balance but may indicate an increase in ice velocity caused by warming.Previous studies have shown that most glaciers in the Tianshan Mountains present generally negative trends over the past half century, in which the increase in the mean temperature in summer is the main cause of glacial shrinkageIn this study, we simulated the ice velocity, temperature and deviation stress under glacial thickness conditions in 1969 and 2009 and analysed the influence of climate and thickness change on glaciers due to stress variations in the future. The diagnostic simulation showed that the ice flow velocity was mainly influenced by the combined action of the gravity component and ice viscous force, and the homogeneity of the regional force was the reason for the complexity in glacial movement. The atmospheric temperature variations led to ablation and caused differences in ice temperatures to shape the differential forces of the ice body, which resulted in a complex balance of the subglacial and englacial compressive and tensile stresses. With the decrease in thickness and surface velocity, coupled with the spatial difference in ablation and difference in surface velocity, the difference in mass balance was magnified. The formation of icebergs implies the intensification of this trend. With the change in stress and ice flow structure caused by ablation, the location of ice flow at the bottom will more likely facilitate the occurrence of various hydrological processes. Sliding-induced ablation of the basement and strain softening of basal ice will promote the acceleration of ice flow. The prognostic simulation showed the variation tendency of thickness and area. The results suggested that the abnormal surface ablation caused by atmospheric temperature and ice thickness is related to the variation in surface velocity and ice flow viscoplasticity.The entire study shows that the difference in regional force caused by accumulated ablation and ice flow is the cause of the complicated glacial movement. The short-term area increase may not indicate an increase in mass balance, but it may be an increase in ice velocity caused by warming. The Elmer/Ice model is suitable for the simulation of glacial dynamics in areas of complex terrain and morphology. This model has good application prospects in cryospheric research. Combined with other cryospheric studies, the application of the Elmer/Ice model can explain more glacial motion mechanisms.


TGR5 (Gpbar1) is a G protein-coupled receptor responsive to bile acids (BAs), which is expressed in different non-parenchymal cells of the liver, including biliary epithelial cells, liver-resident macrophages, sinusoidal endothelial cells (LSECs), and activated hepatic stellate cells (HSCs). Mice with targeted deletion of TGR5 are more susceptible towards cholestatic liver injury induced by cholic acid-feeding and bile duct ligation, resulting in a reduced proliferative response and increased liver injury. Conjugated lithocholic acid (LCA) represents the most potent TGR5 BA ligand and LCA-feeding has been used as a model to rapidly induce severe cholestatic liver injury in mice. Thus, TGR5 knockout (KO) mice and wildtype (WT) littermates were fed a diet supplemented with 1% LCA for 84 h. Liver injury and gene expression changes induced by the LCA diet revealed an enrichment of pathways associated with inflammation, proliferation, and matrix remodeling. Knockout of TGR5 in mice caused upregulation of endothelin-1 (ET-1) expression in the livers. Analysis of TGR5-dependent ET-1 signaling in isolated LSECs and HSCs demonstrated that TGR5 activation reduces ET-1 expression and secretion from LSECs and triggers internalization of the ET-1 receptor in HSCs, dampening ET-1 responsiveness. Thus, we identified two independent mechanisms by which TGR5 inhibits ET-1 signaling and modulates portal pressure. TGR5 Ago (n = 7) 1.0 vs. 0.73 ± 0.08 ; DMSO vs. forskolin (n = 6) 1.0 vs. 0.58 ± 0.12 ) (n = 10) 1.0 vs. 0.80 ± 0.05 ; DMSO vs. TGR5 Ago (n = 9) 1.0 vs. 0.67 ± 0.12 ; DMSO vs. forskolin (n = 5) 1.0 vs. 0.54 ± 0.11 ). Thus, stimulation of TGR5 not only reduces ET-1 mRNA expression, but also ET-1 secretion from LSECs.ET-1 in liver is mainly secreted by LSECs under physiological conditions . We have< 0.05)) B. We hav< 0.05)) C  towards its ligand on activated HSCs through internalization. Thus, the ETAR’s responsiveness is being shifted from picomolar to nanomolar concentrations [AR. Immunofluorescence staining localized the ETAR within the plasma membrane of HSCs, as demonstrated by colocalization with the plasma membrane marker wheat germ agglutinin (WGA)  0.37 ± 0.06 Pearson’s correlation coefficient vs. 0.11 ± 0.04, 3.4-fold decrease, p < 0.01).Activated HSCs are able to contract, leading to an increase in portal pressure due to their perivascular location in the space of Disse . ET-1 hatrations . Since Tin (WGA) B. Treatmreceptor C (DMSO v20 vs. 1.42 ± 0.18 cmH20 (1.7-fold decrease); DMSO vs. TGR5 Ago/+5 nM ET-1 2.9 ± 0.18 cmH20 vs. 1.89 ± 0.28 cmH20 (1.5-fold decrease), DMSO vs. TGR5 Ago/+15 nM ET-1 3.14 ± 0.15 cmH20 vs. 2.20 ± 0.3 cmH20 (1.4-fold decrease), (n = 5–6), p < 0.05)  A.4)-mediated fibrosis [Liver injury of various etiologies triggers morphological and functional changes within hepatic sinusoids, resulting in development of fibrosis and portal hypertension ,43,44. Dfibrosis ,49. Whilfibrosis ,51. To wfibrosis . HepaticAR. These two mechanisms may act synergistically to reduce ET-1 signaling within the sinusoid , a potent vasodilator [2S and NO) and inhibit expression and secretion of the potent vasoconstrictor ET-1 from LSECs [AR [AR from the plasma membrane, explaining the reduced ET-1-dependent contractility of activated HSCs in the presence of a TGR5 agonist in vitro. In summary, TGR5 activation reduces hepatic vascular resistance through several mechanisms, both in LSECs and in HSCs, that act synergistically. Through modulation of PDGFRβ expression, it may also contribute to the response of HSCs to microvascular thrombosis and platelet-derived signals. A recent study explored the effect of the non-bile acid TGR5 ligand oleanolic acid in a rat model of liver fibrosis development [Moreover, expression of PDGFRα/β, TGF-β1 and ET-1, which are related to HSC activation, development of fibrosis, and portal hypertension, were upregulated in both genotypes in response to LCA. However, mRNA levels were significantly higher in livers of TGR5 KO mice as compared to liver tissue from WT littermates. While periportal fibrosis, as measured by Sirius red staining and hydroxyproline content, was not significantly increased in either genotype as early as 3.5 days after starting the LCA feeding, expression of collagen was significantly upregulated in both genotypes, suggesting initiation of fibrogenesis in line with previous data . Since Tsinusoid B. In ratelopment , suggestodilator ,54. Amonodilator . Taken tom LSECs ,46,53,54om LSECs ,56. Furtom LSECs . MoreoveSECs [AR . Using aelopment . Treatmeelopment . It was elopment . Thus, selopment . Furtherelopment . Since Felopment , it is h


Background: Cervical pedicle screws are advantageous in their biomechanical stability within cervical and cervicothoracic constructs. The seventh cervical vertebra contains relatively large pedicles and has a low incidence of vertebral artery localization within the transverse foramina. The freehand technique of pedicle screw insertion is advantageous in decreasing intraoperative radiation exposure both to the patient and surgeon. In this study, we investigated the safety and accuracy of C7 pedicle screw placement at our institution utilizing an anatomic freehand technique.Methods and Materials: A retrospective study was performed, and 20 patients were identified who met the inclusion criteria over a five-year period (2013-2018). The C7 pedicle screw placement capability and accuracy were recorded. Accuracy was graded based upon postoperative imaging on a Grade 0-3 scale for breach assessment. Any neurologic complications related to screw placement were also recorded.Results: Successful pedicle screw placement occurred in 90% of attempts (36/40). The overall screw accuracy rate was 89% (32/36). There were four minor breaches (Grade 1) identified on CT, without neurologic complications. The fusion rate in our cohort for patients with follow up greater than eight months was 100%.Conclusions: In our patient series, the freehand technique of C7 pedicle screw placement utilizing a small laminotomy with direct pedicle palpation appears to be a safe and accurate method for screw placement, and provides adequate biomechanical stability for cervical and cervicothoracic construct fusion. Fixation options for the subaxial cervical vertebrae include lateral mass, translaminar, transfacet, and pedicle screws . PedicleWith IRB approval, a retrospective review was conducted by two independent reviewers  of all patients undergoing cervical or cervicothoracic fusion by a single surgeon (senior author) at our institution and it was performed over a five-year period (2013-2018). Our inclusion criteria for the study were: adult patients (18 years or older) undergoing posterior cervical or cervicothoracic fusion for any indication that included the C7 level using freehand technique and postoperative X-ray or CT, and any intraoperative attempt to place C7 pedicle screws. Exclusion criteria were patients with previous C7 instrumentation undergoing revision surgery, patients who did not have an intraoperative attempt at C7 pedicle screw placement, and instrumentation using navigation or intraoperative fluoroscopy during screw placement.Primary outcomes were C7 screw placement capacity, accuracy, and any neurologic complications related to screw placement. Secondary outcome measurement was fusion rates for patients with at least eight months of follow up. Fusion was defined as bony bridging between adjacent vertebral segments throughout the entirety of the construct identified on CT or plain film. Accuracy was graded for patients by analysis of postoperative X-ray or CT. For patients with a postoperative CT, accuracy was graded upon previously published scales : for patDetails of the utilized operative technique for freehand C7 pedicle screw placement are as follows. A preoperative MRI is obtained on all preoperative patients for surgical planning. The vertebral artery can be easily identified on T2 sequences to determine its course in relation to the C7 pedicle. If a transverse foramen exists at C7 and includes the vertebral artery, then pedicle screws are not placed to avoid neurovascular complications. After complete exposure of the posterior elements of the levels desired to be instrumented, the C7 lateral mass is identified. If the construct is planned to cross the cervicothoracic junction, the thoracic screws are placed first. This aids in starting point planning for the C7 pedicle screw in order to successfully place the rod into the polyaxial heads across the junction. After the thoracic screws are placed, a small laminotomy is performed at the superior border of the C7 lamina  was 89% (32/36). There were three patients with four screws with identifiable minor (<2 mm) breaches on CT. Patient #3 had slight bicortical purchase of the left screw. Patient #4 had a small medial breach of the right screw in the proximal portion of the pedicle. Patient #16 had mild anterior bicortical purchase of the right screw, and a small lateral breach on the left screw. None of these patients had neurologic symptoms or complications related to these minor breaches. Fusion rates for patients with at least eight months of follow up was 100% (n = 12). Eight patients received CT evaluation and four received X-ray evaluation of the construct. Pedicle screw insertion at C7 is a well-published technique to enhance the biomechanical stability of cervical and cervicothoracic constructs , 15-16. In our study, we did not observe any complications relating to pedicle screw placement. For the 11 patients with postoperative CT, three patients exhibited minor (Grade 1) breaches. Two were bicortically placed anteriorly, one lateral, and one medial . For patients with a postoperative plain film, there were no observed breaches based on parameters of radiographic evaluation . Our accThere were 12 patients in our series that had at least eight months of follow up with imaging to assess fusion. All patients met the criteria for construct fusion. This was not compared with a control, so it is difficult to determine whether there is a significant difference in fusion rates for our cohort with C7 pedicle screws compared with other construct designs. Posterior cervical constructs have a high fusion rate in general as reported in the existing literature . As the There are several limitations to our study. The first is the retrospective method for data acquisition. The second is the heterogenous nature of determining breach rates with CT and plain film. It is likely that very minor breaches (Grade 1) were not able to be identified on plain film evaluation that may have been observed on CT due to resolution differences between the two modalities, possibly creating false negative results. With respect to clinical significance, the Grade 1 breaches observed on CT in our patient cohort did not contact neurovascular structures or produce any patient symptoms. The consequences of minor breaches in pedicle screw placement have been investigated and established in previous publications , 14, 19.Freehand pedicle screw placement at C7 is a safe and viable option for fixation of posterior cervical constructs at the cervicothoracic junction. In the widespread advent of new technologies for instrumentation such as robotic systems and optical navigation, the surgical anatomy of proper C7 pedicle screw placement remains an important and relevant concept to ensure accurate instrumentation independent of technologic availability.