When activated by tumour antigen-specific IgEs, monocytes may contribute to the restriction of cancer growth in animal models of cancer. In this study, we investigated the effects of IgE stimulation on the activation state of human monocytes from healthy subjects and from patients with cancer. Cross-linking of cognate Fc receptors by IgE on human monocytes potentiated: (a) upregulation of activatory and down regulation of regulatory monocyte cell surface markers; (b) phosphorylation of intracellular protein kinases in monocytes previously described to be downstream of the mast cell and basophil FcεRI signalling pathway; (c) ovarian cancer patient monocyte-mediated cytotoxic killing of tumour cells and release of pro-inflammatory mediators, potentially associated with favourable patient survival. IgE can therefore activate human monocytes to acquire a pro-inflammatory phenotype capable of mediating effector functions against tumour cells. This may contribute to the mechanism of cancer immunotherapy using IgE antibodies.IgE contributes to host-protective functions in parasitic and bacterial infections, often by monocyte and macrophage recruitment. We previously reported that monocytes contribute to tumour antigen-specific IgE-mediated tumour growth restriction in rodent models. Here, we investigate the impact of IgE stimulation on monocyte response, cellular signalling, secretory and tumour killing functions. IgE cross-linking on human monocytes with polyclonal antibodies to mimic formation of immune complexes induced upregulation of co-stimulatory , and reduced expression of regulatory  monocyte markers. Cross-linking and tumour antigen-specific IgE antibody-dependent cellular cytotoxicity (ADCC) of cancer cells by cancer patient-derived monocytes triggered release of pro-inflammatory mediators . High intratumoural gene expression of these mediators was associated with favourable five-year overall survival in ovarian cancer. IgE cross-linking of trimeric FcεRI on monocytes stimulated the phosphorylation of intracellular protein kinases widely reported to be downstream of mast cell and basophil tetrameric FcεRI signalling. These included recently-identified FcεRI pathway kinases Fgr, STAT5, Yes and Lck, which we now associate with monocytes. Overall, anti-tumour IgE can potentiate pro-inflammatory signals, and prime tumour cell killing by human monocytes. These findings will inform the development of IgE monoclonal antibody therapies for cancer. Monocytes engender multiple roles in homeostatic and disease processes, including in tissue repair , allergiWhile IgG is a well-established antibody modality for the treatment of cancer, IgE has not been used for cancer therapy until now, with the first clinical trial of an IgE class therapeutic candidate for oncology nearing completion . SeveralSeveral studies have demonstrated that when designed to recognize tumour-associated antigens, IgE antibodies can activate immune cells, such as monocytes ,15 and mHere, we hypothesised that IgE may engender fundamental changes in monocytes’ activation state, their signalling and secreted immune mediator network. To evaluate the immunological environment associated with IgE-dependent monocyte-mediated functions, we investigated the cytokine and chemokine milieu, signalling pathways and anti-tumour functions triggered by IgE stimulation of human monocytes derived from healthy subjects and from patients with ovarian cancer.Human peripheral blood monocytes (CD14+CD56-CD3-CD19-) were identified . Due to We evaluated whether IgE cross-linking on human monocytes could influence cell surface expression of markers of classical and alternative immune activation C. MonocyThese findings suggest that IgE cross-linking may skew monocytes towards a pro-inflammatory and activated state.IgE treatment has been associated with pro-inflammatory mediator release in the tumour microenvironment in rodent models . Thus, wTogether, these findings suggest that IgE stimulation on human monocytes triggered release of pro-inflammatory mediators  B.IgE engagement and FcεRI downstream signalling have been widely studied in mast cells ,27, but Overall, IgE cross-linking on monocytes triggered increased phosphorylation of protein kinases, already shown to be downstream of FcεRI in mast cells and basophils, but also of recently-identified kinases such as Fgr, STAT5, Yes, and Lck, which we now associate with FcεRI pathway signalling in monocytes.n = 34) and patients (n = 110)  A. A smaln = 110) .We next evaluated whether circulating monocytes from patients and healthy subjects could mediate ex vivo tumour cell killing by IgE, namely MOv18 IgE or non-specific NIP IgE isotype control. Primary monocytes from healthy volunteers and ovarian cancer patients mediated ADCC of IGROV1 cells  B. In supTherefore, monocytes from both healthy subjects and cancer patients can be activated by tumour antigen-specific IgE to kill tumour cells and to secrete pro-inflammatory mediators.Upregulation of TNFα, MCP-1 and IL-10 has been consistently observed in this study and in previous in vitro and in vivo models of cancer following IgE immunotherapy . We therCross-linking IgE antibodies of various antigen specificities on the surface of human monocytic U937 cells resulted in up to three-fold increase in TNFα expression. However, IgE cross-linking alone did not trigger MCP-1 or IL-10 upregulation A. When mTogether, these data suggest that a TNFα-MCP-1-IL-10 axis is potentiated by IgE engagement on monocytes.p = 0.04; HR = 0.68, p = 0.00013, respectively)  was associated with better patient survival in comparison to patients with low intratumoural gene expression of these mediators  A. Consisctively) B. This swww.clinicaltrials.gov) recently reported promising interim data in patients with ovarian cancer [IgE antibodies directed against cancer-associated antigens have been shown to restrict tumour growth in several in vivo cancer models ,22,23,34n cancer . IgE is n cancer ,14,35. HWe studied the effects of IgE stimulation on the surface of primary human monocytes by cross-linking FcεRs either partially engaged with endogenous IgE or saturated by addition of exogenous MOv18 IgE to mimic antibody-immune complex formation. IgE cross-linking induced a phenotypic shift in monocyte surface marker expression suggestive of pro-inflammatory immune activation. This included a significant upregulation of co-stimulatory molecules, CD40, CD80 and CD86 . These mThis activatory surface marker signature induced by IgE cross-linking was accompanied by pro-inflammatory cytokine and chemokine secretion. TNFα, MCP-1 and IL-10, which we previously reported to be enhanced in the tumour microenvironment following systemic IgE treatment in vivo , were seWe also found that saturation of FcεRs by IgE did not induce higher levels of cytokine and chemokine release or enhanced FcεRI pathway signalling in comparison to cross-linking FcεRs partly-occupied by endogenous IgE. This observation is consistent with our previous findings showing maximal activation of human macrophages by IgE stimulation even in the absence of FcεR saturation . Cross-lWe investigated the downstream signalling effects of monocyte activation by IgE cross-linking of the trimeric FcεRI. FcεR signalling in monocytes is not well-studied while IgE-mediated activation of the tetrameric FcεRI αβγ2 on the mast cell and basophil surface has been extensively researched. Monocytes express low levels of the trimeric FcεRI, αγ2. This isoform lacks the β-chain, reported to be responsible for enhanced downstream signalling and activation in mast cells ,19,50. ADespite a slight decrease in FcεRI-expressing monocyte populations in patients compared to healthy subjects A, MOv18 In ex vivo functional studies, we aimed to understand the conditions required for the production of TNFα, MCP-1 and IL-10, consistently detected in IgE stimulated and ADCC supernatants. Cross-linking of IgE antibodies of different antigen specificity on monocytic cell surface triggered upregulation of TNFα A, consisOur study therefore provides evidence that IgE stimulates human monocytes towards a pro-inflammatory state and triggers immune mediator cascades and classical immune signalling pathways. These attributes can be directed against cancer cells and may contribute to the promise of IgE as a new candidate class for cancer immunotherapy.Venous blood samples were collected from healthy subjects and patients with ovarian cancer in K2EDTA tubes with informed written consent, in accordance with the Helsinki Declaration. The study design was approved by the Guy’s Research Ethics Committee (reference 09/H0804/45), Guy’s and St. Thomas’ NHS Foundation Trust. Peripheral blood samples were also purchased from the UK National Health Service (NHS) Blood and Transplant (BT) system from anonymous donor leukocyte cones. Monocyte isolation and flow cytometric analyses are described in 6 cells/mL with 5 µg/mL IgE, or media control, for 45 min at 37 °C. Following washing, cross-linking was stimulated with 5 µg/mL polyclonal goat anti-human IgE at 37 °C. For analysis of protein kinase phosphorylation profiles, cross-linking for 5 min was followed by immediate wash and cell lysis. For flow cytometric surface cell marker expression and soluble mediator analyses, cells were washed after 45 min cross-linking, re-suspended in fresh media (supplemented with 20 ng/mL M-CSF for surface cell marker expression), and incubated for 24 h at 37 °C. A Magnetic Luminex Performance Assay  was performed to detect chemokine and cytokine release in cell culture supernatants, using a Luminex® FlexMap 3D® analyser. Serum IgE levels were analysed by Viapath Analytics (UK). Analysis of TNFα, MCP-1 and IL-10 mRNA expression (qPCR) and secretion (ELISA) assays using cell lines are described in Primary monocytes were incubated at 1 × 10The Proteome Profiler Human Phospho-Kinase Array  was performed according to manufacturer’s instructions. Densitometric quantification was conducted using ImageJ software .p < 0.05) upon IgE cross-linking compared to the baseline condition (untreated (endogenous IgE) vs. anti-IgE (endogenous IgE + anti-IgE); MOv18 IgE vs. MOv18 IgE + anti-IgE) were mapped based on previously reported data, new findings in this study, and the FcεRI signalling pathway as described in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [p-value cut-off of 0.01 and q-value 0.2. To derive immunologically-relevant pathways, enriched pathways were curated to include a minimum gene set size of 2, maximum gene set of 134 genes and a ratio of differentially phosphorylated genes to total genes in the pathway equal to 0.029 or higher.Protein kinases that displayed significantly modulated phosphorylation  as described previously [t-test or a one-way ANOVA with Tukey’s post-test were used to determine statistical significance of the data.All statistical analyses were performed using GraphPadTM Prism Software (version 8.0). Error bars represent standard error of mean (SEM). Clinical associations of intratumoural gene expression of mediators were assessed using publicly-available data in Kaplan–Meier plotter (eviously , betweenIgE can activate human monocytes to acquire a pro-inflammatory phenotype capable of mediating effector functions against tumour cells through monocyte-specific FcεRI intracellular pathway signalling. These insights deepen our understanding on the mechanism of IgE-based cancer immunotherapy and may further contribute to the development of novel treatments for solid tumours.


Satellite altimeters can be used to derive long-term and large-scale sea ice thickness changes. Sea ice thickness retrieval is based on measurements of freeboard, and the conversion of freeboard to thickness requires knowledge of the snow depth and snow, sea ice, and sea water densities. However, these parameters are difficult to be observed concurrently with altimeter measurements. The uncertainties in these parameters inevitably cause uncertainties in sea ice thickness estimations. This paper introduces a new method based on least squares adjustment (LSA) to estimate Arctic sea ice thickness with CryoSat-2 measurements. A model between the sea ice freeboard and thickness is established within a 5 km × 5 km grid, and the model coefficients and sea ice thickness are calculated using the LSA method. Based on the newly developed method, we are able to derive estimates of the Arctic sea ice thickness for 2010 through 2019 using CryoSat-2 altimetry data. Spatial and temporal variations of the Arctic sea ice thickness are analyzed, and comparisons between sea ice thickness estimates using the LSA method and three CryoSat-2 sea ice thickness products (Alfred Wegener Institute (AWI), Centre for Polar Observation and Modelling (CPOM), and NASA Goddard Space Flight Centre (GSFC)) are performed for the 2018–2019 Arctic sea ice growth season. The overall differences of sea ice thickness estimated in this study between AWI, CPOM, and GSFC are 0.025 ± 0.640 m, 0.143 ± 0.640 m, and −0.274 ± 0.628 m, respectively. Large differences between the LSA and three products tend to appear in areas covered with thin ice due to the limited accuracy of CryoSat-2 over thin ice. Spatiotemporally coincident Operation IceBridge (OIB) thickness values are also used for validation. Good agreement with a difference of 0.065 ± 0.187 m is found between our estimates and the OIB results. Sea ice forms an interface between the upper ocean and the atmosphere and plays an important role in the Earth’s climate and environmental system through modifying the radiative and energy balance and modulating the exchange of heat and moisture between the ocean and atmosphere . Screen Thickness is one of the most important sea ice parameters because its changes during the seasonal cycle may cause significant impacts on global change ,4,5. Sea3 was used, Kern et al. [3, while Laxon et al. [3 for FYI and 882 kg/m3 for multiyear ice (MYI). The range of sea ice density values from MYI is particularly large at 43 kg/m3, which can cause a bias of 1.1 m in sea ice thickness estimation [Satellite altimeters are widely used to retrieve hemispheric estimates of sea ice thickness ,10,11,12n et al.  used a vtimation .The selection of snow depth and sea ice density plays important roles in the retrieval of sea ice thickness, and it also has implications for the uncertainty in temporal trends in Arctic sea ice thickness estimates . The focCryoSat-2 was launched in 2010 with the purpose of observing Earth’s cryosphere. CryoSat-2 carries a newly developed radar altimeter named the synthetic aperture radar (SAR) interferometric radar altimeter (SIRAL) [We use CryoSat-2 Baseline-C L1b and L2 data available from the European Space Agency (ESA) to estimate the Arctic sea ice thickness. The L1b data contain the multi-looked echo power, measurement time, geolocation, satellite altitude, surface type flag, and other instrumental measurements. The product includes additional information, such as geophysical and tidal corrections and quality flags. The L2 data include surface elevation estimates.Recently publicly available sea ice thickness products based on CryoSat-2 have been provided by institutions, including the Centre for Polar Observation and Modelling (CPOM) ,21, the CPOM provides monthly Arctic sea ice thickness product on a 5 km grid with CryoSat-2 altimetry data. CPOM also provides near-real-time Arctic sea ice thickness estimates based on fast-delivery CryoSat-2 data . AWI proOperation IceBridge (OIB) is a NASA program of airborne remote sensing measurements designed to fill the gap in measurements between ICESat and ICESat-2. OIB can obtain sea ice thickness estimates by onboard instruments including the airborne topographic mapper and snow radar. Here, we employ the OIB sea ice thickness datasets available at the National Snow and Ice Data Center (NSIDC)  to validBefore performing the sea ice freeboard calculation, measurements of the leads and ice should be discriminated. Leads are openings in the ice that appear even in regions covered by thick ice . In publh is the surface elevation and h together with The radar measurements are processed for each CryoSat-2 orbit. First, geoid undulations and the mean sea surface height (MSS) are removed by subtracting the MSS height ( follows :(2)hfb=hThe local instantaneous sea surface height anomaly  :(3)hsi=ρtions. 3 ,30,31,32ns. 3 [3 ,31,33,34 3 [3 [3  and 320 [3 [3 [3 ,31,33 ar[3 [3 [3 ,36 use dTo minimize the uncertainty from the input parameters  can be rewritten as follows:We can retrieve the model coefficients  method ,39, whicPrior values for To determine the eight unknown parameters, at least eight observations are needed in each grid cell. The numbers of observations that fall in each grid cell are calculated. Based on the LSA method, we are able to derive estimates of the Arctic sea ice thickness for November 2010 through April 2019 using CryoSat-2 altimetry data. In this paper, the Arctic sea ice region is defined as latitudes above and including 65° N. We do not calculate the sea ice thickness in the months of May to September due to melt ponds. This is because during the melt season, it is difficult to distinguish between measurements from leads and ice floes due to melt ponds forming on the ice that cause specular return echoes ,40. Spec3). 3, while the thick ice density ranges from 880 to 885 kg/m3.We can also derive sea ice density from The sea ice thickness results are presented in seven regions covering the Arctic Basin. The region-dividing criterion is based on Ye et al. . Region During the 2018–2019 sea ice growth season, the thickness growth rate of the entire Arctic Basin is approximately 13 cm per month. In October, the sea ice thickness is the smallest, with an average value of 1.315 m, and a few thick ice packs can be seen off the northern coast of the Canadian Archipelago. Afterward, the sea ice thickness increases over the course of the sea ice growth season. In November, more thick ice appears in region 1 and north of region 7, and thin ice begins to appear in the south of regions 2, 3, 4, and 6. In December, the ice thickness growth rate is small (6.4 cm per month), and more thin ice appears in the southwest of region 2. In January, more thick ice occurs in region 1. In February, the ice thickness experiences the largest growth rate (21.2 cm per month). In March, the thick ice area continues to expand. In April, the ice growth rate decreases to 6.5 cm per month, but more thick ice appears in the eastern part of region 1.Region 1 features the largest mean ice thickness (1.775 m) and the second highest growth rate (13.9 cm per month) during the 2018–2019 sea ice growth season. The largest ice growth rate in region 1 occurs in November (32.9 cm per month). Region 2 features the highest ice growth rate (14.5 cm per month) and the second largest mean ice thickness (0.825 m). In region 2, the ice growth rate from October to December reaches 20.7 cm per month, which is much larger than the rate during the following months (11.7 cm per month). The ice thickness growth rates in region 3 and region 6 are very close. Region 4 has the smallest mean ice thickness (0.178 m), and region 5 has the lowest ice growth rate (3.4 cm per month). The ice thickness in regions 3, 4, 5, 6, and 7 decreases in April, while it continues to grow in the other regions. Owing to the growth in region 1 and region 2, the mean ice thickness in the entire Arctic Basin shows a small increase in April.Monthly average thickness variations of the Arctic sea ice derived with the LSA method are shown in Comparisons between sea ice thickness estimates using the LSA method and three CryoSat-2 ice thickness products  are performed for the 2018–2019 Arctic sea ice growth season. The average bias between the LSA and AWI in the entire Arctic Basin during the growth season is 0.025 ± 0.640 m. In October, the AWI product does not provide any ice thickness estimates for region 4. The mean thickness of the LSA in the entire basin is larger than that of the AWI for November through March but smaller than that of the AWI for October and April. The maximum bias (0.094 ± 0.480 m) of the two products occurs in January, and the minimum bias (0.003 ± 0.594 m) occurs in November. In regions 1, 5, and 7, the mean thickness of the LSA is smaller than that of the AWI. The bias between the LSA and AWI is less than 0.1 m in region 1 and 2. In region 1, the difference between the two products is −0.080 ± 0.494 m from 2018 to 2019. The largest mean difference in region 1 occurs in April. The bias in the western part of region 1 is positive, while it is negative in the eastern part of region 1. Region 2 features the lowest bias (0.013 ± 0.399) of the seven regions, while region 5 has the largest bias (−0.285 ± 1.048 m). In regions 3 and 4, most of the bias is positive. The bias in region 6 during October reaches −0.442 ± 1.152 m because of the limited ice in this area. The bias in regions 6 and 7 is reduced as the growth season progresses.The overall mean difference between the LSA and CPOM is −0.143 ± 0.640 m. The largest bias (−0.173 ± 0.691 m) of the two products occurs in March 2019. Region 5 features the largest thickness bias (−0.270 ± 1.017 m) from the LSA and CPOM for the seven regions, which is consistent with the bias of the LSA and AWI. In region 1, the difference between the two products is −0.197 ± 0.525 m from 2018 to 2019. The largest mean difference in region 1 occurs in March. The bias in region 1 is positive in October and negative from November to April. The largest difference in region 2 occurs in October, and the difference is reduced as the growth season progresses. In region 3, the largest difference occurs in April. Large differences occur in southern offshore areas of region 3 during March and April. The mean ice thickness of regions 4, 5, and 6 in October is less than 0.1 m, and the difference between the LSA and CPOM is extremely large in the three regions.The GSFC product does not provide thickness estimates in regions 5 and 6. The difference between the LSA and GSFC is −0.274 ± 0.628 m. The bias for the entire basin of the two products reaches a maximum (−0.531 ± 0.577 m) in October, reduces as the growth season progresses, and reaches a minimum (−0.118 ± 0.687 m) in April. Region 7 shows the largest bias (−0.516 ± 0.864 m) of the seven regions, while region 2 features the lowest bias (−0.167 ± 0.423 m). The bias in region 1 is −0.345 ± 0.757 m during the entire season. In October, most of the bias in region 1 is negative, whereas more positive bias appears as the growth seasons progresses, especially in the western part of region 1. This phenomenon can also be found in regions 2 and 3. Limited ice is found in region 4 during October, and the bias is extremely large (−2.144 ± 0.005 m).From the comparison, we can find that large biases tend to occur in areas covered with thin ice, such as the margin of the Arctic Basin. Moreover, regions 4 and 5 have the lowest sea ice thickness and feature the largest bias in the comparison, which is mainly because of the limited accuracy of CryoSat-2 and other satellite altimeters for observing thin ice.To assess our sea ice thickness estimates from the LSA method, we compare our results, as well as the three products, with the OIB sea ice thickness products. As shown in The overlapping parts are divided into 628 segments every 5 km, and the mean sea ice thicknesses for the LSA and OIB are calculated for each segment. While comparing with the three sea ice thickness products, the OIB thickness is averaged within a grid cell. In this study, we develop a new method to estimate sea ice thickness from CryoSat-2 measurements without prior knowledge of parameters, including the snow depth, snow density, sea ice density, sea water density, and radar penetration factor. A quadratic model between the sea ice freeboard and thickness is established within a 5 km × 5 km grid. The model coefficients and the sea ice thickness are calculated using the LSA method. Our method can minimize the uncertainty in the conversion from freeboard to thickness.Based on the new method, we derive estimates of the Arctic sea ice thickness from 2010 to 2019. Seasonal and annual variations of Arctic sea ice thickness are discussed. The total Arctic sea ice thickness increases each month over a given growth season from October until March or April, which is generally true for the thickness of FYI and MYI but punctuated with more variability from month to month. The seasonal and annual variations of FYI thickness are closer to the variations of total sea ice thickness than those of MYI, indicating that FYI is the dominant ice type in the Arctic sea ice cover.To validate our estimates, we compare our results with three CryoSat-2 sea ice thickness products: AWI, CPOM, and GSFC. The comparison shows an overall difference of 0.025 ± 0.640 m for the LSA versus AWI, 0.143 ± 0.640 m for the LSA versus CPOM, and −0.274 ± 0.628 m for the LSA versus GSFC. Large differences between the LSA and the three products tend to appear in areas covered with thin ice, which indicates the limited accuracy of CryoSat-2 over thin ice. We also validated our thickness results with spatiotemporally coincident OIB thicknesses. Our results show good agreement with the OIB thickness, with a correlation coefficient of 0.935 and a difference of 0.065 ± 0.187 m.


Saccharomyces cerevisiae, is summarized. A special focus is laid upon the regulation and the localization of these enzymes. Furthermore, research needs for a deeper insight into the high complexity of phosphatidic acid biosynthesis and consequently the entire lipid metabolic network is presented.Phosphatidic acid biosynthesis represents the initial part of de novo formation of all glycerophospholipids (membrane lipids) as well as triacylglycerols (storage lipids), and is thus the centerpiece of glycerolipid metabolism. The universal route of phosphatidic acid biosynthesis starts from the precursor glycerol-3-phosphate and comprises two consecutive acylation reactions which are catalyzed by a glycerol-3-phosphate acyltransferase and a 1-acyl glycerol-3-phosphate acyltransferase. In addition, yeast and mammals harbor a set of enzymes which can synthesize phosphatidic acid from the precursor dihydroxyacetone phosphate. In the present review our current knowledge about enzymes contributing to phosphatidic acid biosynthesis in the invaluable model organism yeast,  In this yeast two GPATs with overlapping function are expressed which are named Sct1 and Gpt2 . Topformants . Furtherformants . Strikinwww.yeastgenome.com). Interestingly, three of the phosphorylation sites located to the C-terminus (S664/S668/S671) form a motif which is highly conserved among GPATs from different yeast species . Th. Th33]. www.yeastgenome.org). Since ubiquitin marks a protein for degradation, possibly the contribution of Slc1 to lipid metabolism is regulated via its protein abundance.A common means to regulate the activity of an enzyme is via a change of its phosphorylation status. This kind of regulation may also be true for Slc1, because this AGPAT has been reported as phospho-protein . Further3.2slc1Δ mutant and control revealed not only that Slc1 is dually localized to lipid droplets and the ER, but also that the latter organelle harbors at least one additional AGPAT . Be. Be47]. www.yeastgenome.org). One challenging task of the future will be to confirm the correctness of this information and to determine the physiological role of these post-translational modifications.To date, the question as to the regulation of Ale1 has not been studied in detail. However, global analysis revealed that Ale1 is a phosphoprotein which is modified by phosphorylation at several sites  which is involved in catalysis the conserved histidine is replaced by a cysteine. The official name Loa1 stands for lyso-PA:oleoyl-CoA acyltransferase 1 and already points to the enzyme’s strong preference for the substrates lyso-PA and oleoyl-CoA . Pr. Pr21]. ing LOA1 .www.yeastgenome.org). However, this enzyme is modified by ubiquitin (www.yeastgenome.org), which suggests that the contribution of Loa1 to cell metabolism may be regulated via this protein modification.To date, no phosphorylation sites have been reported for Loa1  out of the four characteristic motifs of this protein family. Interestingly, in contrast to most GPATs/AGPATs, in Ict1 the HX4D motif is found in the C-terminal portion of the protein instead of in the N-terminal one. This finding may indicate that Ict1 is a remnant of a former full size GPAT/AGPAT. Ict1 seems to play a specific role in PA biosynthesis in cells challenged with the organic solvent iso-octanol . Th. Th23]. www.yeastgenome.com). In addition, several lysines of Ayr1 have been reported to be modified by ubiquitin. Since the protein amount of Ayr1 increases during growth [The question as to the regulation of Ayr1 has not been studied yet. However, the contribution of this enzyme to PA biosynthesis may be regulated by phosphorylation, because high throughput experiments identified Ayr1 as target of a protein kinase. The detected phosphorylation site is a serine, which is located to the C-terminus of the reductase (g growth , this poA short summary of characteristics of the enzymes involved in PA biosynthesis is provided in 5Biosynthesis of PA is prerequisite for the de novo formation of all glycerolipids, i.e., glycerophospholipids and triacylglycerols. As outlined above, to date all major as well as several minor enzymes contributing to PA biosynthesis have been identified at the molecular level. However, our knowledge about the regulation of these enzymes, which is vital for understanding the high complexity of the lipid metabolic network, is still very limited. Thus, one focus of future studies should be laid upon elucidating regulatory aspects of PA biosynthesis. High throughput experiments investigating post-translational modifications revealed that most enzymes involved in PA biosynthesis are modified by phosphorylation. A change in the phosphorylation status of an enzyme regulates its contribution to metabolism for instance by affecting the enzyme activity, protein stability, and/or localization. Presently, only the significance of one single conserved phosphorylation motif of the major GPAT Gpt2 has been studied in detail. This study showed that phosphorylation/de-phosphorylation at this motif affects the enzyme’s activity  see Sec.. HoweveInterestingly, not only Gpt2 is dually localized to lipid droplets and the ER, but also the AGPATs Slc1 and Loa1, as well as the 1-acyl dihydroxyacetone phosphate reductase Ayr1 ,23,33. TAnother interesting topic which should be addressed in the future concerns the association of PA synthesizing enzymes with lipid droplets. For the ER associated portion of Gpt2 it has been shown that this GPAT contains 6 transmembrane domains . SimilarLast but not least, also minor enzymes contributing to PA biosynthesis should not be lost out of focus, because the knowledge about these enzymes, their features and regulation will be similarly important to complement our current picture of PA biosynthesis. Collectively, by shedding light on the entire set of enzymes involved in PA biosynthesis, their regulation, their structural features, but also their interplay, we will be able to contribute important information toward a better understanding of PA biosynthesis, a centerpiece of the overall complex network of lipid metabolism.


The cell wall sensor Wsc1 belongs to a small family of transmembrane proteins, which are crucial to sustain cell integrity in yeast and other fungi. Wsc1 acts as a mechanosensor of the cell wall integrity (CWI) signal transduction pathway which responds to external stresses. Here we report on the purification of Wsc1 by its trapping in water-soluble polymer-stabilized lipid nanoparticles, obtained with an amphipathic styrene-maleic acid (SMA) copolymer. The latter was employed to transfer tagged sensors from their native yeast membranes into SMA/lipid particles (SMALPs), which allows their purification in a functional state, i.e., avoiding denaturation. The SMALPs composition was characterized by fluorescence correlation spectroscopy, followed by two-dimensional image acquisition from single particle transmission electron microscopy to build a three-dimensional model of the sensor. The latter confirms that Wsc1 consists of a large extracellular domain connected to a smaller intracellular part by a single transmembrane domain, which is embedded within the hydrophobic moiety of the lipid bilayer. The successful extraction of a sensor from the yeast plasma membrane by a detergent-free procedure into a native-like membrane environment provides new prospects for in vitro structural and functional studies of yeast plasma proteins which are likely to be applicable to other fungi, including plant and human pathogens. Saccharomyces cerevisiae [S. cerevisiae, Wsc1 has best been studied both by genetic and biophysical methods . Taken revisiae ,5. Amongiewed in ). Atomiciewed in . AFM stuiewed in ,5). Quaniewed in .In all studies performed so far, Wsc1 functions were assessed either in living yeast cells ,9,10 or NpSRII/NpHtrII) into a 3:1 SMA copolymer, with a photoreceptor: transducer stoichiometry of 2:2, from Escherichia coli polar lipid liposomes [To overcome these technical barriers, novel membrane mimetic systems for MPs on the nanometer scale have been developed, which avoid the use of detergents . Amphipaiposomes . Detergeiposomes ,26,27,28iposomes .An extensive set of biophysical methods is compatible with the SMA lipid particle (SMALP) system . Thus, tIn this work, we applied the 3:1 SMA copolymer to isolate Wsc1-GFP (green fluorescent protein) fusions into SMALPs directly from native yeast membranes. We purified Wsc1-GFP-containing SMALPs by affinity chromatography and characterized SMALP preparations using dynamic light scattering (DLS), fluorescence correlation spectroscopy (FCS) and single particle transmission electron microscopy (TEM). This serves as a proof-of-principle for further structural and functional studies of Wsc1 and other fungal MPs in vitro.S. cerevisiae strain HOD356-1A  was obtained in this work as a source of the Wsc1 sensor. It was constructed in the background of the HD56-5A strain by homologous recombination with PCR-generated tags at the native WSC1 locus, based on constructs used in previous works [Growth media and genetic manipulations followed previously described standard protocols . The S. us works ,8,10. NoWSC1 gene was expressed under the control of its native promoter. Yeast cells were grown to late logarithmic phase (~5 × 107 cells/mL) in 50 mL of rich medium  with 2% w/v glucose as a carbon source (YEPD) at 30 °C with constant rotary shaking at 180 rpm.The Arthrobacter luteus provided by MP Biomedicals, Eschwege/Germany; applied from a stock solution of 10 mg/mL in sterile water and stored at −20 °C) were added and the suspension was incubated for 30 min at 37 °C. After centrifugation , pellets containing the spheroplasts were either processed immediately or flash frozen and kept at −80 °C until further use.Yeast cells were harvested from 10 mL aliquots of the overnight culture by centrifugation to obtain approximately130 mg of wet cells washed once with 3 mL of sterile water and transferred to an Eppendorf tube. The wet cell pellet after centrifugation was resuspended in 0.5 mL of spheroplast buffer . To digest the cell wall glucans, 2 µL Zymolyase 20T , the resulting pellet was resuspended at 80 mg/mL of wet cells in buffer A and directly used for protein extraction by SMA or flash frozen and kept at −80 °C until further use.Spheroplasts were washed twice and resuspended at 20 mg/mL of wet cells in buffer A: 10 mM Tris-HCl, pH 8.0, 150 mM NaCl supplemented with one tablet per 50 mL of protease inhibitor cocktail . The cell suspension was sonicated on ice . After centrifugation  solution of SMA, which was extensively dialyzed against buffer A, was used for the membrane solubilization.The styrene maleic acid (SMA) copolymer with a styrene-to-maleic acid molar ratio of 3:1  was kindly provided as a gift by Cray Valley . A 5% (w/v) solution of SMA copolymer in buffer A was added dropwise to the membrane suspension to get a final cell-to-SMA weight ratio of 1:2.5. The suspension was incubated for 30 min at RT and then for 16 hrs at 4 °C. All incubations were performed with gentle shaking. The suspension was then centrifuged for 20 min at 134,000× g at 4 °C. Before the affinity purification, aliquots of the supernatant were taken for Dynamic Light Scattering and Fluorescence Correlation Spectroscopy analyses. The pellet and the supernatant were also analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The supernatant was subsequently purified on affinity resin.A 5% (Anti-HA (hemagglutinin) agarose  was pre-equilibrated with PBS buffer. SMA-solubilized membranes were added to the resin and incubated overnight at 4 °C with gentle mixing. The resin was then pelleted by brief centrifugation, and the supernatant (column flow through) was discarded. The resin was washed with 40 column volumes of PBS buffer and protein was eluted with four column volumes of the same buffer, supplemented with 100 µg/mL HA peptide .All stages of cell fractionation, SMA solubilization and affinity purification were applied to 12% SDS-PAGE. For immunodetection proteins from gels were transferred onto a 0.2 µm nitrocellulose membrane , blocked with 5% non-fat milk and probed with rabbit polyclonal HA-antiserum  as primary antibodies. The secondary antibodies were anti-rabbit (H+L) HRP-conjugated . Chemiluminescent signals were developed with ECL SuperSignal West Pico substrate  and registered on a ChemiDoc XRS+ imager with ImageLab software .Dynamic light scattering (DLS) measurements were performed on a Zetasizer Nano ZS  at 550 nm and 25 °C. Data represent the average of three sets of 14 runs of 10 s each. The particle size distribution was obtained by using the ZETASIZER software package v7.02. under the assumption that the analyzed particles would have a spherical shape.2/s in water at 25 °C [BT/(6πη(T)r). Viscosity was estimated by η(T) = a∙exp(−b/(T−c)) with a = 2.414∙10−5 Pas, b = 247.8 K, and c = 140 K (Picoquant application note). Calibration and SMALP measurements at different temperatures were corrected by these equations. The final hydrodynamic radius was estimated by the measured diffusion coefficient and the Stokes–Einstein equation.Fluorescence Correlation Spectroscopy (FCS) was conducted on a confocal laser scanning microscope  equipped with a FLIM/FCS upgrade kit . For excitation of GFP, a picosecond pulsed 485 nm laser diode  at a repetition rate of 20–40 MHz was used. Fluorescence was detected by a single photon avalanche detector (Picoquant) using a bandpass filter from 500–550 nm . All measurements were performed in a 100 µL droplet placed on a high-precision coverslip at room temperature (24–26 °C). Exact temperature at sample position was determined for each measurement. Fluorescence was collected with a 60× water immersion objective  at 20 µm above coverslip inside the droplet. Autocorrelation functions (ACF) were analyzed by the software SymPhoTime 64 . This software allowed for correction of background and afterpulsing artefacts using fluorescence lifetime information. A diffusion model assuming a single population of particles showing three-dimensional free diffusion and considering triplet state blinking of GFP was fitted to the resulting ACFs . FCS fitat 25 °C . DiffusiCopper grids (300 mesh formvar/carbon-coated)  were hydrophilized by glow discharge  with a Pelco EasyGlow apparatus . Fresh protein samples (3 µL) were placed onto the grid and incubated at room temperature (RT) for 30 s. Excess of the sample was removed with filter paper. Grids were stained twice, using 1% aquatic uranyl acetate solution for 30 s at RT, and air-dried.Micrographs were acquired using an analytical transmission electron microscope Jem-2100  equipped with a 2K × 2K CCD camera Ultrascan 1000XP . The microscope operated at 200 kV in a low dose mode, with a magnification ×40,000 (2.5 Å/pix) and defocus 0.5–1.9 µm. Images were acquired automatically with SerialEM3.8.0 software .WSC domain (Protein Data Bank ID: 5fws) were fit into the density map using UCSF Chimera [To obtain the 2D projections of purified Wsc1 protein molecules, 107712 particles were selected automatically from the 870 EM images using crYOLO neural network . Particl Chimera .https://blast.ncbi.nlm.nih.gov/Blast.cgi [https://www.rcsb.org/ [https://prosite.expasy.org/ [For modelling the 3D structure of WSC1, we searched for its structural homologs by performing searches with the Position-Specific Iterative Basic Local Alignment Search Tool (PSI-BLAST) (last.cgi ) againstcsb.org/ ). The vaasy.org/ ).WSC1 gene tagged at its native locus with the coding sequence for the green fluorescent protein (GFP) to produce a C-terminally tagged, fully functional sensor fusion protein [In previous works we employed a yeast strain carrying a  protein . While p protein ; note thIn order to purify the tagged Wsc1 protein for further characterization, the general scheme depicted in Western blotting and immunological detection b verifieDynamic light scattering (DLS) was employed to analyze the size distribution of the SMA copolymer particles in the supernatant fraction obtained after membrane solubilization (further referred to as soluble membrane fraction). These data confirmed the presence of monodisperse nano-sized particles a. Suppor2/s (± standard error). The average hydrodynamic particle radius, r, was 4.9 ± 0.3 nm and the Wsc1-GFP concentration was approximately 30 nM. We took these data as strong evidence that the Wsc1-GFP protein was indeed present in the SMALP preparation after membrane solubilization, basically carrying a single sensor molecule per particle. The latter assumption was confirmed by the TEM pictures taken of the purified SMALPs as described below. Moreover, the FCS values obtained for the size of the nanoparticles correlated well with the DLS data discussed above.These SMALPs were then subjected to fluorescence correlation spectroscopy (FCS), which allows the robust determination of diffusion constants at nanomolar concentrations following fluorescent labels, in order to determine their content of Wsc1-GFP. First, the identity of the GFP signals was verified by determination of the fluorescence emission spectrum. From the diffusion constants obtained in the FCS, hydrodynamic radii were estimated using the Stokes-Einstein equation, assuming a spherical shape of the particles. Samples were then subjected to robust FCS measurements based on their GFP fluorescence, using appropriate dilutions to nanomolar concentrations if necessary. Typical FCS autocorrelation results are shown in 2/s as compared to 53.0 ± 3.0 µm2/s, respectively. Likewise, the corresponding hydrodynamic radius, r, of 4.9 ± 0.3 nm remained unaltered. This confirmed that affinity purification did not affect the physicochemical properties of the SMALP-embedded sensor.We assumed that the soluble membrane fraction, i.e., the supernatant fraction obtained after membrane solubilization by incubation with the SMA copolymer, contained both SMALPs loaded with Wsc1-GFP and sensor-free SMALPs carrying other yeast membrane proteins. The fraction containing the GFP-tagged sensor was further purified by application of the supernatant to an anti-HA affinity column. Similar to the soluble membrane fraction preparation shown in Finally, affinity-purified SMALPs loaded with Wsc1-GFP were analyzed by negative-stain transmission electron microscopy (nsTEM). As shown in the exemplary image in A PSI-BLAST search immediately revealed sequence similarity between residues 28–80 of the mature Wsc1 protein and a sequence stretch of the human KREMEN1 receptor. All eight functionally important cysteine residues matched  displays short, disordered α-helices and β-strands . It shouSince the position of the membrane-crossing α-helix could only be identified in images with the extended conformation c,d, onlyThe size of the third, bulkier domain fits the short cytosolic sequence of Wsc1 fused to the GFP ß-barrel. The crystal structure of GFP  was thusS. cerevisiae. Wsc1 is a major player in the activation of the cell wall integrity (CWI) pathway, which regulates cell wall remodeling during periods of extracellular stress, mating and normal cell growth [Recently, the amphipathic styrene-maleic acid (SMA) and SMA-related copolymers became a promising tool for membrane protein purification. Here, we have reported the application of SMA in the solubilization of the membrane spanning sensor protein Wsc1 as an important constituent of the native plasma membrane from the model yeast l growth . We tookl growth ,46 and eThe model is consistent with a tripartite organization of Wsc1, previously suggested based on its primary sequence and mechanical features revealed by single-molecule atomic force microscopy . These We assume that the shrinking and reduction of the external length to about 6–7 nm, as observed for the EM images, is a manifestation of an entropically collapsed structure of the nanospring, which, otherwise, could be considerably stretched by interaction of the N-terminal WSC-domain with the cell wall in living yeast cells under standard conditions, and even more though upon perturbances of the cell surface . As statThe cytosolic tail of Wsc1 is of utmost importance in signal transduction to the CWI pathway. It has been shown to interact with the guanine nucleotide exchange factor (GEF) Rom2 triggering the downstream signaling cascade . InteresIn our 3D reconstruction, the cytosolic part probably best reflects the natural conformation in living yeast cells, as it is in the natural aqueous environment and relatively short. As evident from This work provides interesting insights into the structure of the Wsc1 sensor and confirms the importance of studying TM receptors and sensors in lipid-based, native-like environments. For instance, the monodisperse purified SMALPs containing the sensor had an average diameter of only 10 nm, accommodating only single sensor molecules. However, Wsc1 has been shown to cluster in large complexes within the plasma membrane of living yeast cells and was suggested to form at least dimers, both homodimers and heterodimers with other Wsc-type sensors, as deduced from bimolecular fluorescence interactions . An extrDiisobutylene/maleic acid (DIBMA) copolymers, which differ from SMA copolymers in the non-aromatic nature of their hydrophobic groups, can also solubilize membrane proteins directly from a lipid bilayer. However, the size of DIBMA/lipid particles (DIBMALPs) can be varied in a broader range than that of SMALPs, thus reaching dimensions of up to 50 nm ,65,66. IS. cerevisiae, but also of pathogenic yeasts and other fungi. Choosing essential members of such membrane proteins to study their 3D structure could thus provide a new angle for the design of antifungal agents, urgently needed in medicine and agriculture.Nevertheless, this work demonstrated the applicability of a detergent-free extraction and purification procedure, which is suitable not only for other membrane proteins of


Introduction: It is well known that the main segments of spinal fracture is thoracolumbar (T11-L11). Therefore, in addition to the lumbar, the lower thoracic vertebra (T9-T12) often has the clinical needs of implantation of cortical bone trajectory (CBT) screws. However, the anatomic parameters of the lower thoracic vertebrae are quite different from those of the lumbar vertebrae, which means that if CBT screws are to be implanted in the lower thoracic vertebrae, the selection of the screw entry point, the length, diameter, angle and path of the screws in each segment need to be redefined. Methods In this part, 3-dimensional finite element model was established to analyze the stress and fixation efficiency of CBT screws in thoracic vertebrae after 5000 times of fatigue loading of normal model and osteoporosis model. Discussion If the outcomes indicate the trial is feasible and there is evidence to provide some basic anatomical parameters for CBT screw implantation in the lower thoracic spine, so that the ideal insertion point, length, diameter, and angle of CBT screw in different segments of the lower thoracic spine were determined.Trial Registration Chinese Clinical Trial Registry, ChiCTR1900026915.Registered on September 26, 2019. Among all segments of the spine, the thoracolumbar segment (T11-L1) is the most prone area for spine fracture due to its high mobility.–3With the acceleration of the aging process and the increase of the number of patients with primary osteoporosis, there will be more and more patients suffering from it in the future.–6At present, thoracolumbar fracture is still the main type of posterior surgery, while the classic pedicle screw fixation technology plays an absolutely dominant role in the selection of posterior fixation. The technique of posterior transpedicular fixation of spine was used in clinic in 1970 s. At first, it was mainly used in the treatment of lumbar fracture. In view of its good fixation effect, it was gradually used in the reconstruction and fixation of various thoracic and cervical vertebrae. In the past decade, the technology has developed rapidly and has become the main means of spinal fixation.–8However, since the pedicle screw implantation path is from the posterior of the vertebral arch into the vertebral body, although three column fixation can be achieved from the posterior to the anterior, the screw path characteristics determine that the screw only has a good holding force in the pedicle with a limited length, while the vertebral body where the screw is implanted is mostly cancellous bone, so the holding force of the screw is relatively limited, and it is especially true for patients with osteoporosis.–12 Zindrick et al showed that increasing the length and diameter of the screw can indeed increase the biomechanical strength of the screw. However, he also stressed that increasing the length and diameter of the screw during the operation will inevitably lead to the risk of damaging the pedicle and the anterior and posterior walls of the vertebral body, and even the potential risk of damaging nerves and blood vessels. Another method which is widely used to increase the strength of pin bone interface fixation in patients with osteoporosis is to strengthen the nail way by using bone cement technology. Although bone cement strengthening technology can immediately increase the instant pull-out force of pedicle screws, the periodic anti flexion effect of screws after bone cement strengthening does not increase, and there is a risk of thermal injury and leakage of bone leading to nerve injury in the use process, and there is also a risk of pulmonary embolism in the operation process.–14 In addition, some scholars have improved the design and implant technology of pedicle screw, and want to increase the screw holding force of osteoporosis patients by these methods, such as the design and application of porous screw and expansion screw, but the effect of these changes in osteoporosis patients is not satisfactory. The improvement of screw design and the use of strengthening materials are only focused on the improvement of traditional pedicle screw design and the change of screw placement technology, and these improved methods have not achieved good results.–16In the fixation of patients with severe osteoporosis, how to increase the holding power of screws is still a challenge for spinal surgeons. It has been reported that the length, diameter and thread design of screws can affect the biomechanical strength of screw fixation.In 2009, SANTONI et al put forward the internal fixation technology of cortical bone trajectory (CBT) through the improvement of screw implantation path and screw pathway. The screws used in CBT screw internal fixation technology are smaller in diameter and shorter in length than the traditional screws, but the thread arrangement is tighter, and fully contact with cortical bone concentration area, which can increase the strength of bone screw interface. Compared with the common pedicle screw, CBT screw has 4 significant advantages:(1)The screw has four points of cortical fixation: lamina, vertebral body, internal, and external walls of pedicle, which makes the screw travel a long distance in cortical bone, and can significantly improve its holding power.(2)In sagittal position, the screw is shaped from tail to head, and in transverse position, it is shaped from inside to outside, thus reducing the risk of nerve root injury and penetrating the inner wall of pedicle see Fig. .(3)The insertion point of screw is closer to the midline of vertebral lamina, which reduces the stripping of paravertebral muscles. Therefore, CBT screw technology is an excellent choice for patients with osteoporosis and screw failure requiring revision surgery. At the same time, CBT screw is a minimally invasive technology, which conforms to the development direction of spine surgery. Matsukawa et al evaluated the torque of the 2 screw techniques through in vivo experiments. The results showed that the torque of lumbar CBT screw fixation was 1.7 times higher than that of traditional pedicle screw. Gonchar et al prospectively compared the effect of lumbar CBT screw and percutaneous pedicle screw in posterior lumbar fusion, pointed out the occurrence rate of screw loosening and orthopedic maintenance effect of lumbar CBT screw technique. Results: the trauma caused by operation was better than that by percutaneous pedicle screw. Ueno et al thought that the difference of fixation strength of screws to vertebral body was mainly caused by different screw placement channels. They found that there was no significant difference in stiffness between 2 types of screws when using the same screw placement channel. However, no matter what type of screw was used in different screw placement channels, CBT technology had higher screw placement and pull-out force.Zhang et al compared the traditional pedicle screw with the cortical bone channel screw, and found that the single axial pull-out force of CBT was 30% higher than that of the traditional pedicle screw.–23 However, the occurrence area of spine fracture is mostly in the thoracolumbar (T11-L1), which means that in the lower thoracic vertebra (T9-T12) patients with severe osteoporosis or revision surgery, CBT screws need to be inserted. However, there are great differences between the anatomical structure of the lower thoracic vertebrae and the lumbar vertebrae, and there are few studies on CBT screws of the lower thoracic vertebrae at home and abroad. Therefore, is it suitable to implant CBT screws in the lower thoracic vertebra of Chinese? We do not know the ideal point, the ideal path, the diameter range, the angle, the mechanical efficiency and the clinical reliability.At present, the basic and clinical research of CBT screw is mainly limited to the lumbar vertebra, and there is no relevant data research in China.–23This trial aims at the main clinical problems of spinal surgery, that is, the fixation efficiency of traditional pedicle screw may be insufficient in the case of osteoporosis thoracolumbar fracture, and there is a clinical demand for CBT screw implantation in the lower thoracic vertebra. Although CBT screw has a certain mechanical advantage for the fixation efficiency of osteoporosis patients, at present, the relevant research at home and abroad is mainly limited to the lumbar spine, and there are few reports about the clinical anatomy parameters, fixation efficiency and in vivo work of CBT screw in the lower thoracic spine in China.2This study will improve the clinical anatomy and biomechanical study of CBT screw in the lower thoracic vertebrae (T9-T12) of different groups in China, and provide the corresponding theoretical basis and basis for its clinical application. It provides a new choice for patients with osteoporosis who may need fixation in thoracolumbar region.33.1The computed tomography (CT) images of the lower thoracic vertebrae (T9-T12) of 40 male and 40 female normal adults in China were collected. The trial was examined and approved by the Ethics Committee of the Hospital of Chengdu University of traditional Chinese medicine. No. nt-63543.2The age of applicants is between 20 to 60 years old in good health and they are good at spirit and intelligence. They obey the arrangement of the research group, accept the treatment plan designed by the research group and sign the informed consent;3.3Participants suffered from severe spinal degeneration or severe irreversible damage of multiple spinal columns such as spinal tuberculosis and tumor.3.43.4.1Simulate the routing of the screw with diameter of 3.0 mm on the lateral image of the vertebral body. The screw passes through the middle point of the narrowest point of the pedicle height from the bottom to the back one thirds of the junction area of the superior endplate of the vertebral body, and make a oblique upward transverse section along the bilateral axis. The intersection line between the section and the posterior lamina of the vertebral body is the transverse axis of the screw see Fig. .3.4.2Simulate the routing of the screw with diameter of 3.0 mm on the above oblique cross-section. The screw passes through the middle point of the narrowest part of the pedicle from the inside to the outside and ends at the superior part of the vertebral body or the lateral cortex. Make a sagittal section along this axis. The intersection of this section and the posterior vertebral plate is the longitudinal axis of the screw  in 40 men and 40 women, and the relationship between the ideal insertion point and the surrounding anatomical structure, so as to determine the ideal insertion point of CBT screws in different segments of the lower thoracic vertebra . After the mesh was divided, the 3-dimensional finite element model of the normal lower thoracic vertebrae and the osteoporosis lower thoracic vertebrae were established by ansys15.0 finite element software (ANSYS). In ansys15.0, the 3-dimensional finite element model of the lower thoracic vertebrae of healthy adults and the 3-dimensional finite element model of the lower thoracic vertebrae of osteoporosis were tested according to the experimental flow chart shown in Table 3.6.3The investigators and research assistants in the study team will collect the data and enter them directly into the electronic-Case Report Form. The data will be securely stored, with specific access rights granted to members of the study team according to their role in the study. Participants, healthcare professionals, the public, and other relevant groups could get the results of the trial as soon as we completes the trial and publishes our achievements.3.7Any observed side effects related to radiation exposure will be documented throughout the trial period and reported to the Sponsor without delay. These data will be provided for periodical review by the Data and Safety Monitoring Board.4Based on the previous research in the field of clinical applied anatomy and medical biomechanics, this trial measures the anatomical data related to the CBT screws of the lower thoracic spine of Chinese men and women, improves and optimizes the screw placement path and method of the CBT screws of the lower thoracic spine of Chinese people, and objectively analyzes the fixation technology of the CBT screws of the lower thoracic spine in the normal vertebral body and the clinical application value of osteoporotic vertebral body.Its innovation lies in that this anatomical measurement is made in accordance with the actual direction of the screw, no matter the transverse section or the sagittal section. The measured screw length, width and angle are all actual values, which is more accurate and reliable. This method avoids the problem that the measured length of the nailway is not its real length, but the projection length of the nailway in the horizontal plane or the sagittal plane caused by the previous study of the measurement of the nailway.This study also provides a technical choice for patients with osteoporosis who need posterior fixation of the lower thoracic spine, and makes a detailed anatomical, biomechanical and in vivo simulation study on this technical choice.


Fractures of the distal radius are a common orthopaedic presentation in Irish emergency departments. As a nation, Irish people tend to ice-skate seasonally with a peak of interest seen during the Winter months in temporary ice-rinks. This case series describes winter ice-skating as a significant cause of wrist fractures in the younger patient, including five cases of distal radius fractures, four of which ultimately required internal fixation, under general anaesthesia, over a single weekend in the month of December. Despite all five patients being amateur ice-skaters, all denied ever having taken ice-skating lessons. This demonstrates the dangers of wrist trauma in the inexperienced or beginner ice-skaters on temporary ice-rinks; the seasonal morbidity suffered as a result. Fractures of the distal radius are common presentations in the western world, accounting for over 16% of orthopaedic trauma presentations in emergency departments . HoweverDue to Ireland’s cool temperate oceanic climate, participation in ice-skating is typically only facilitated using non-natural ice-skating rinks. Therefore, peaks of interest in ice-skating are seen in Ireland during the Christmas period with seasonal ranks opening on a temporary basis in December nationwide. Despite the significant morbidity associated with this activity, many amateur Irish ice-skaters elect to ice-skate without having undergone lessons leading to a significant increase in ice-skating related injuries presenting to Irish emergency departments . This caCase 1A 40-year-old right-hand-dominant lady presented to the emergency department following a fall on an outstretched hand (FOOSH) injury whilst ice-skating. She suffered an extra-articular distal radius fracture with dorsal comminution and angulation to her non-dominant left hand Figure . FollowiCase 2A 53-year-old right-hand-dominant lady presented to the emergency department following a fall backwards whilst holding hands with her friend while ice-skating. She suffered an extra-articular distal radius fracture with dorsal comminution and angulation to her dominant right hand; this had been the contra-lateral hand to that held whilst ice-skating. Following the reduction in the emergency department, she was treated with open reduction and internal fixation using an Angle Stable Distal Radial Plate System .Case 3A 36-year-old left-hand-dominant female teacher presented to the emergency department following a fall during her first-time ice-skating. She suffered an intra-articular distal radius fracture with dorsal angulation and severe comminution to her dominant left hand. She underwent open reduction and internal fixation using a Variable Angle LCP Two-Column Volar Distal Radius Plate 2.4; she remained off work during the holidays.Case 4A 41-year-old right-hand-dominant lady presented to the emergency department after a FOOSH injury whilst ice-skating. She suffered an intra-articular left distal radius fracture involving a significant portion of the radial styloid. She underwent closed reduction using two 1.6 mm Krischner wires (k-wires). She was neurovascularly intact post-operatively. Her k-wires were removed in the outpatient department at six-weeks with satisfactory fixation.Case 5A 37-year-old right-hand-dominant lady presented to the emergency department after falling backwards onto an out-stretched left hand whilst ice-skating. This resulted in an extra-articular left distal radius fracture with dorsal comminution. She was neurovascularly intact. This lady was treated conservatively using a molded cast and followed up in the outpatient fracture clinic thereafter.Distal radius fractures are reported to occur more commonly in elderly, osteoporotic patients . Our casAs a nation, ice-skating remains a novelty to the Irish population, which is commonly enjoyed during the Christmas period . Ice-skaSignificant trauma can occur as a result of ice-skating and serious injuries  related to this activity tend to occur in beginner ice-skaters . DespiteThis case series demonstrates the risk distal radius fractures in light of our desire to enjoy the hazardous activity of ice-skating on the annual trip to the temporary ice-rink. Furthermore, acknowledgement must be given to the fact that ice-skating lessons may need to be more widely available to newcomers and amateur ice-skaters. This, alongside significant public education, may play a role in the future to reduce the burden on our emergency departments.


The modelled bio-optical relationships were applied to hyperspectral imagery captured both in situ (using an under-ice sliding platform) and ex situ (on the extracted cores) to quantitatively map Chl a in mg m−2 at high-resolution (≤ 2.4 mm). The optical quantification of Chl a on a per-pixel basis represents a step-change in characterising microspatial variation in the distribution of ice-associated algae. This study highlights the need to increase the resolution at which we monitor under-ice biophysical systems, and the emerging capability of hyperspectral imaging technologies to deliver on this research goal.Ice-associated microalgae make a significant seasonal contribution to primary production and biogeochemical cycling in polar regions. However, the distribution of algal cells is driven by strong physicochemical gradients which lead to a degree of microspatial variability in the microbial biomass that is significant, but difficult to quantify. We address this methodological gap by employing a field-deployable hyperspectral scanning and photogrammetric approach to study sea-ice cores. The optical set-up facilitated unsupervised mapping of the vertical and horizontal distribution of phototrophic biomass in sea-ice cores at mm-scale resolution (using chlorophyll  Phototrophic ice algae dominate ice-associated biomass and contribute significantly to the overall primary production of ice-covered waters and serve as critical food source for marine herbivores5. The dynamic and multiphase nature of sea ice imposes strong horizontal and vertical gradients in temperature, salinity, porosity and light transmittance7, all of which influence the in situ distribution of algal cells8. Biological properties of ice algal communities such as abundance, species composition and photosynthetic rates are thus extremely variable over time and at spatial scales from millimeters to kilometers10. While most of the algal biomass is concentrated at the ice-water interface, microbes are also present in the interior of the ice matrix, which adds to the biocomplexity of the system8. Measurement capability to determine the mm-scale spatio-temporal distribution of algal biomass in sea ice is currently lacking, thereby limiting a mechanistic understanding of its environmental drivers which is needed to inform predictive modelling and strategic sampling.Sea ice is a porous multiphase medium whose interstitial environment is inhabited by diverse phototrophic and heterotrophic microbial communities12. There is now evidence that variation in under-ice biophysical properties can range from the microscale (0.001 m2) to the mesoscale (10 m2)15, and this cannot be satisfactorily resolved using point-based sampling methods17. Traditional and emerging sea-ice field sampling methods include ice coring18, under-ice bio-optical sensing techniques via L-shaped deployment arms21 or unmanned underwater vehicles (UUVs)23. The use of under-ice optical sensing from UUVs has extended the spatial coverage of algal surveys , but resolutions still remain coarse as a result of large footprints of underwater radiance (or irradiance) sensors22. This shortcoming demonstrates a gap in field-sampling techniques that permit the quantification of horizontal and vertical distributions and temporal dynamics of ice algae biomass at relevant spatial scales.In particular there is the lack of efficient methods capable of non-invasively tracking algal biomass across different scales, both vertically and horizontally, and concurrently with its physical drivers25. HI can be used to quantify biogeochemical properties of a target in each spectrally-resolved pixel within an image28. In situ HI has revolutionized the scales of observation on both terrestrial31 and marine34 ecosystems. HI of extracted samples (ex situ) can be used to better understand the target spectral behaviour and to create a baseline measurement with the data acquired in situ. This enables detailed understanding of a particular target’s interaction with light but also allows us to capture dimensions and dynamics that are not visible from the in situ surface perspective . Some recent examples of ex situ applications include scanning of glacial ice cores to detect chemical impurities35, scanning of soil cores to map fine-scale organic carbon hotspots36, and scanning of sediments to determine pigment concentrations in microbial phototrophs38.Underwater Hyperspectral Imaging (HI) is one method that could deliver a methodological turning-point for quantitative mapping of fine-scale sea-ice biophysical conditions that is relevant to larger scale analyses24 and in situ under Antarctic fast ice25. In this study, we advance the use of HI technology to map the fine-scale vertical and horizontal distribution of sea-ice algae through optical quantification of the photosynthetic pigment chlorophyll a (Chl a). Traditional and novel spectral indices, established from transmittance measurements of ice-core sections, were developed and correlated with fluorometrically derived Chl a values from the ice cores. The retrieved bio-optical regression models were then applied to hyperspectral imagery acquired both in situ, using an under-ice HI scanning platform, and ex situ to the extracted ice cores. The resulting maps provide the first spatially explicit quantitative estimates of Chl a concentration, illustrating that HI is a critical developmental step in our capacity to make scalable under-ice observations for sea-ice algae.We have recently demonstrated how HI can qualitatively capture biomass variability at the sub-mm spatial resolution in both artificial laboratory iceA field camp was established at Cape Evans, Antarctica , from the 14th of November to the 5th of December 2018. The sea ice across the study area had a thickness of 180 ± 1 cm, except for occasional ridged or cracked areas. The distinct under-ice biophysical environment was visually explored using a Seabotix LBV-300 remotely operated vehicle (ROV) . Sea-ice surface conditions in the area were typically snow-free apart from patches of 0.5–1 cm hard old snow layers or 1 to 5 cm compacted snow reliefs throughout the 21-day study period.Forty-two ice cores were extracted using a Kovacs Mark V ice corer  between the 19th of November to the 2nd of December. Of these, 22 were taken from bare ice (snow free) areas, 12 from the area with 0.5–1 cm thin snow cover, 7 cores on the 1–5 cm snow drifts, and 1 core from an area with a 10 cm snow patch. After retrieval, the bottom (~ 60 cm) of each core was immediately cut off and placed into a black plastic bag to protect it from sunlight and promptly taken into a darkened field laboratory for image acquisition and biological processing.2 area)  digital photogrammetry on selected ice core bottoms 0.015 m5 m2 area43. We devised an optical configuration to measure transmitted radiation along the vertical axis of the bottom 9 cm, and horizontally on 3 cm thick sections, of the ice cores at high spectral (1.7 nm) and spatial (≤ 0.07 cm) resolution  following standard radiometric correction procedures26. All horizontal and vertical imagery of cores were manually masked to ensure that only pixels within the ice-core surface were analysed. Spectral sub-setting was applied to keep only photosynthetically active radiation (PAR) (between 400 and 700 nm), which resulted in 179 spectral bands for the core sections imagery and 89 bands for the in situ imagery. This allowed us to focus on Chl a absorption features, which improved processing time and reduced noise interference outside of this range.The image pre-processing and exploration workflow is illustrated in Fig. 24 and in situ25. Mean-centred PCA was here employed on the pre-processed imagery of both vertical and horizontal ice-core sections  has been used in HI studies of sea ice to capture per-pixel fine-scale spatial variability of the first two principal components (PCs) scores embodying light intensity variability and biomass proxies in both laboratory artificial sea iceTransmitted radiance, mentclass2pt{minimThis provides directional transmittance spectra, T(λ), in each pixel of the core surface area Fig. a.For the image of the in situ block B area, the downwelling radiance spectrum 45 with a polynomial order of 1–3 , and window length of 9 bands  were converted to areal concentrations (mg m−2) utilizing surface area of the 14 cm diameter core (0.015 m2). Sea-ice algae species composition was determined by standard light microscopy (400 × magnification) of melted ice-core samples.After hyperspectral scanning of the ice-core sections, the samples were left to melt in the dark at 4 °C. After complete melting, the final melt volume was gently homogenized, and 50 mL sub-samples were filtered onto Whatman GF/F filters. The filters were then placed in ethanol for 24 h extraction of Chl a values. The aim was to retrieve regression models that could be applied on a per pixel basis on the pre-processed pseudo-transmittance imagery. In order to retrieve an index value for each core section, two approaches were available: calculate the spectral index (1) from the mean transmittance spectrum of all pixels of the scanned core or (2) from each pixel spectrum in the pre-processed imagery of the core and then calculate the mean  is transmittance at two selected wavelengths λ1 and λ2. Optimal NDI wavelength were selected by calculating NDIs for all possible wavelength combinations, correlating them with Chl a values and plotting them onto a Pearson correlation surface20. The two best NDI wavelength combinations were selected based on the following criteria: a good Pearson correlation coefficient (p > 0.7 or p <  − 0.7), a minimal separation of wavelengths to avoid autocorrelation (> 12 nm ), and different spectral regions that include at least a band from the Chl a absorption maxima .Sea-ice bio-optical studies have mostly relied on Normalized Difference Indices (NDIs) to relate under-ice transmitted spectra to Chl 650–700)48, the area under the curve normalised to constant band depth (ANCB650–700) and area under the curve normalised to maximum band depth (ANMB650–700) which were all calculated from the continuum-removed transmittance spectrum between 650 and 700 nm49. Continuum removal transformation on the spectrum was used to enhance and standardize the specific absorption features of biochemical constituents50. The range 650–700 nm was chosen to include the most sensitive area to the secondary in vivo Chl a absorption maximum as seen from the transmittance plots ) to deal with the high range of values measured and with the high variance at high Chl a values (heteroscedasticity). This transformation is a common approach in sea-ice bio-optical model development and allows for direct comparison across different studies developing indices for under-ice biomass mapping48. The log-linear regression model takes then the following form:Linear regression analysis was employed to derive bio-optical relationships between integrated Chl 2) for each model. To account for underestimation of the prediction power of the model by the calibration (or training) error, we include adjusted criteria such as the adjusted R2 and the Akaike Information Criterion (AIC)53. For unbiased and reliable model estimation, we performed a tenfold cross-validation (CV)52, for which the data were subset into 10 different random folds. The fitting of the model and the error calculations were then repeated 10 times, one for each subset. Each time, nine folds (or subsets) of the data were combined to produce a regression model, and then tested to the 10th holdout data fold.The log-linear regressions were performed for each of the spectral indices and the regressions evaluated through root mean square error (RMSE) and the coefficient of determination  and by randomly drawing 10,000  points (pixels) from the block B map. Microscale variability was highlighted by computing the gradient magnitude for every pixel in the image through a Prewitt filter operator. This edge detection filter calculates the maximum rate of change for each pixel in the image in relation to its surrounding pixels.Based on the results of the statistical analyses, we selected the best performing model and applied it on a per pixel basis to a set of selected transmittance images of horizontal core sections Fig. . The reg55. Overall, for the 42 horizontal bottom core Sects. (0– to 3 cm), we observed a mean Chl a of 18.74 ± 18.04 mg m-2 (range 1.1–117.5 mg m−2), in accordance with maximum algal biomass ranges reported for bottom ice in McMurdo Sound2. The six horizontal mid-core Sects. (3– to 6 cm) yielded a mean Chl a of 0.61 ± 0.4 mg m−2 (range 0.13–1.2 mg m−2). Finally, for the six horizontal top core sections (6–9 cm), the mean Chl a was 0.64 ± 0.48 mg m−2 (range 0.14–1.35 mg m−2)  of the skeletal layer  are available at this scale for validation, PC2 was able to illustrate a limited range of physical features through the ice algal biomass associated with them. We acknowledge that the sea ice analysed in this study was characterized by a relatively simple composition of organic material, mostly microbial derived, and relatively simple translucent and columnar texture common for land-fast ice. Therefore, PC analysis was able to separate the variability in the transmitted light field from the variability due to Chl a absorption. Sea-ice textural and structural properties can however vary considerably depending on the growth regime6. Consequently, separation through PCA might not be as straightforward in ice cores with diverse sea ice textural mixtures , or with highly variable sea-ice biogeochemical compositions .The PC2 score maps of the vertical sections showed the fine-scale vertical distribution of ice algal biomass Fig. . As expeyer Fig. a. Additiyer Fig. a displayyer Fig. c. This pyer Fig. d,f,g on 61 and how established bottom ice algae respond to bottom ice growth and ablation. Combining these images with high resolution spatio-temporal temperature and salinity data, will permit for habitable space to be examined alongside nutrient fluxes. This will allow us to better understand how they impact on the vertical variability of algae distribution throughout the sea-ice season62. The methodology could also be applied to explore how ice algae directly respond to changes in their environment through vertical migration following either self-shading or unfavourable light conditions63. Capturing such fine-scale patterns using this HI method is a more efficient and quantitatively accurate than cutting ice cores using conventional methods, e.g. sawing. It also permits the extension of the imaging area to larger and more customizable ice sections compared to pulse-amplitude-modulation (PAM) fluorescence imaging techniques64.Unsupervised exploration with PCA opens the potential for investigating complex vertical biophysical patterns and dynamics. For example, understanding how algae migrate through the ice whilst it is being formed and grows25. The ice core scanning method presented here permits the investigation of bio-optical relationships between traditional and alternative spectral indices  against extracted Chl a values.Previous HI studies in sea ice have focused on assessing HI suitability for ice algal habitat mapping but lacked the availability of quantitative relationships applicable to our particular sensor configuration and study environment20. The relationships between tested spectral indices and log(Chl a [mg m−2]) are shown in Fig. The selected optimal NDI wavelength combinations based on NDI Pearson’s correlation surface and selections criteria were NDI 587:621) and NDI (517:449) , ANCB650–700, ANMB650–700 and LAUC650–700 indices performed considerably better than both NDIs and AUC650–700 for our study case  from ice-algal assemblages. Despite being able to produce good correlations, both NDIs and AUC650–700 seemed to suffer considerably from index saturation at the medium to high biomass values relating mainly to the bottom core sections that dominated the dataset (> 1 log[Chl a mg m−2]). The ANMB650–700 and ANCB650–700 indices performed relatively better , can change the optical pathway of light considerably9. This has an impact on the retrieved model coefficients thus affecting the robustness of model to be applied onto new datasets48. For nearby sites, recent studies have struggled to formulate reliable bio-optical regression models21. This was attributed mostly to the presence of platelet ice, which results in considerable biomass losses during sampling and consequently narrow biomass variability range. Another reason could be attributed to the particularly high biomass concentrations found in the fast-ice of McMurdo Sound2, because high biomass concentrations can negatively affect linear relationships through the saturation of various vegetation spectral indices67.In recent years, bio-optical algorithms capable of mapping biomass under-ice have been derived from cosine corrected irradiance sensors deployed via L-arms followed by the extraction of overlapping core samples to produce series of regression points21 and the light-sample collection procedures were also noticeably different. HI on extracted ice cores poses a distinct advantage in that the spectral data specific for Chl a is extracted in full. The differences between the in situ spectral measurements and the standard biological processing that are caused through to the process of coring, brine drainage or platelet slough-off result therefore minimized. Another advantage of the spatial resolution of the system is that we can precisely contour relevant per-pixel radiance and operate within the exact surface area that is being sampled for Chl a extraction.Through the development of an ice-core HI approach and alternative spectral indices, we were able to compensate for some of the caveats relating to sample collection and model development. This core scanning method also allowed the elaboration of relationships that are more suitable to be applied to imagery from high-resolution HI sensors. Results cannot be compared with previous studies in the same area using L-arms, as sea-ice conditions were drastically differenta at scales closer to the optical resolution of HI scanning systems so that small features can be referenced in the HI products. In our case, the variance in Chl a samples was reduced as we averaged over the entire ice core surface of 14 cm-diameter which portrayed high biomass gradients within single ice core surfaces  characterize the remarkable patchiness of sea-ice algal biomass at the micro and macro scale  and core 24 (23.12 mg m−2). Variogram analyses revealed a Chl a autocorrelation length scale of about 12 cm  and stark gradients occurring at the mm scale. The gradient intensity map shown in Fig. a abundance gradients, with a mean of 3.3 ± 2.3 mg m−2 mm−1 and reaching up to a maximum of 18 mg m−2 mm−1  were shown to lead the spatial biocomplexity portraying stark small-scale biomass gradients  production affecting the sea-ice microstructure76. Grazing from pelagic herbivores can also influence patchiness and distribution although little is known about their quantitative influence at any scale. Time-lapse approaches coupling under-ice HI and RGB systems25, integrated with ice core scanning, could further help assessing grazer-biomass interactions at relevant spatial scales. In addition, through concomitant monitoring of both sea-ice microstructural properties, biomass dynamics and their environmental drivers over time, the method could further support and add to the parameterization of sea-ice biogeochemical and ecological models aimed at regional scale analyses.Although still under development, we underline that HI approaches present major opportunities for microspatial mapping of ice algal biomass in situ, and ex situ across different vertical and horizontal ice core sections. We further advocate the potential for the methodology, coupled with RGB imaging, to be mounted onto UUVs or stationary monitoring stations to drastically increase the spatial and temporal mapping capability of under-ice biophysical dynamics quantitativelySupplementary Information.


Basophils are rare granulocytes and dysregulated functions of these cells are associated with several atopic and non-atopic allergic diseases of skin, respiratory system and gastrointestinal tract. Both cytokines and immunoglobulin E (IgE) are implicated in mediating the basophil activation and pathogenesis of these disorders. Several reports have shown that healthy individuals, and patients with allergic disorders display IgG autoantibodies to IgE and hence functional characterization of these anti-IgE IgG autoantibodies is critical. In general, anti-IgE IgG autoantibodies modulate basophil activation irrespective of allergen specificity by interacting with constant domains of IgE. Therefore, an ideal solution to prove the functions of such anti-IgE IgG autoantibodies would be to completely eliminate type I high affinity immunoglobulin E receptor (FcɛRI)-bound IgE from the surface of basophils and to demonstrate in an unequivocal manner the role of anti-IgE IgG autoantibodies. In line with previous reports, our data show that FcɛRI on peripheral blood basophils are almost saturated with IgE. Further, acetic acid buffer (pH 4) efficiently removes these FcɛRI-bound IgE. Although immediately following acetic acid-elution of IgE had no repercussion on the viability of basophils, following 24 h culture with interleukin-3 (IL-3), the viability and yield of basophils were drastically reduced in acid-treated cells and had repercussion on the induction of activation markers. Lactic acid treatment on the other hand though had no adverse effects on the viability of basophils and IL-3-induced activation, it removed only a small fraction of the cell surface bound IgE. Thus, our results show that acid buffers could be used for the elution of FcɛRI-bound IgE on the basophil surface for the biochemical characterization of IgE antibodies or for the immediate use of basophils to determine their sensitivity to undergo degranulation by specific allergens. However, these methods are not utile for the functional assays of basophils that require longer duration of culture and entire removal of surface IgE to validate the role of anti-IgE IgG autoantibodies that interact with FcɛRI-bound IgE irrespective of allergen specificity. Basophils express diverse receptors such as type I high affinity immunoglobulin E receptor (FcεRI) that binds to IgE, toll-like receptors that recognize pathogen-associated molecular patterns (PAMPs), and cytokine receptors including the receptors for interleukin (IL)-3, IL-33 and thymic stromal lymphopoietin (TSLP) to sense activation signals derived from the varied sources [Basophils are rare granulocytes and have various functions in the orientation of immune responses by regulating the macrophage polarization, supporting the CD4 sources ,2. Activ sources ,6,7,8. BIgE is the least represented immunoglobulin class. With a half-life of 1.5 days, IgE accounts for nearly 0.05% of total immunoglobulins in the body . DespiteSeveral reports over the years have shown that patients with allergic disorders display IgG autoantibodies to IgE, which either activate IgE-sensitised basophils (agonistic anti-IgE IgG autoantibodies) or inhibit allergen-induced basophil activation (antagonistic anti-IgE IgG autoantibodies) in part by blocking the binding of specific allergen to IgE ,14,15. IFunctional characterization of anti-IgE IgG antibodies-induced FcɛRI-IgE signalling events in the context of basophil functions is critical for dissecting the role of these anti-IgE IgG autoantibodies in pathophysiologies. Various lines of evidences now suggest that anti-IgE IgG autoantibodies modulate human basophil activation and function not by interacting with variable region of IgE rather by recognizing constant domains of heavy chains of IgE, in particular Cε1, Cε2 or Cε4 domains ,19,21. TPrevious studies have attempted to elute IgE from the surface of basophils by brief treatment of cells with either 0.01 M lactic acid-containing buffer (pH 3.9) or acetic acid buffer (pH 2.5 to 4) ,26,27,28The viability of basophils following isolation from the buffy bags of healthy donors was in the range of 98–99% as analyzed by fixable viable dye (FVD) staining A. PrevioWe analyzed the viability of basophils immediately following acetic acid buffer (pH 4) treatment by staining with FVD. We did not observe major changes in the viability of acid-treated basophils compared to phosphate-buffered saline (PBS)-treated cells A.We then assessed the efficacy of stripping of basophil surface-bound IgE. Interestingly, treatment of cells with acetic acid buffer (pH 4) led to almost complete stripping of IgE from the basophil surface B. Over 9IL-3 plays an important role in the biology of basophils ,33,34,35Therefore, as a first step, we cultured PBS or acetic acid buffer (pH 4)-treated basophils in IL-3 for 24 h. Analyses of basophils by flow cytometry revealed that PBS-treated basophils remain highly viable after 24 h culture and we observed only minor changes in the cell yield A. In addn = 4 donors) -treated basophils. Based on the forward and side scatter pattern of cells, the percentage of remaining basophils in P1 gate relative to PBS-treated cells was in the range of 8.2 ± 4.9% ( donors) A,B. This donors) C,D. ThesWe then wondered if pH of the acetic acid buffer close to neutral would overcome this problem and hence treated the basophils with acetic acid buffer (pH 6). Although the percentage of remaining basophils in P1 gate following 24 h culture was considerably improved A, aceticSeveral studies have performed elution of IgE from the basophil surface by using 0.01 M lactic acid pH 3.9 ,26,27,28Analyses of viability of basophils immediately following lactic acid treatment confirmed that lactic acid did not affect the viability of basophils A. FurtheThe distinct effects of acetic acid buffer (pH 4) and lactic acid buffer treatments on the yield of basophils cultured for 24 h in the presence of IL-3 prompted us to investigate the viability of cells by Annexin V and propidium iodide (PI) staining. Basophils cultured in the medium alone without supplementation of IL-3 showed high levels of Annexin V positivity A,B. IL-3We wondered whether inability of IL-3 to rescue acetic acid buffer (pH 4)-treated basophils was due to the loss of IL-3 receptor following acid treatment. Therefore, we analyzed CD123 (IL-3Rα) expression on the basophils immediately following treatment of cells with PBS, lactic acid or acetic acid buffer (pH 4). We observed that irrespective of the treatments, basophils retained similar intensity of CD123 expression C,D thus Our data show that FcɛRI on peripheral blood basophils are almost saturated with IgE. Hence, the addition of exogenous IgE did not considerably alter the intensity of IgE on the surface. Further, acetic acid buffer (pH 4) efficiently removes these FcɛRI-bound IgE, while buffer with pH 6 was ineffective. Acetic acid buffer (pH 4)-elution of IgE had no immediate repercussion on the viability of basophils but following 24 h culture with IL-3, the yield and viability of basophils was drastically reduced and had repercussion on the induction of activation markers. Lactic acid treatment on the other hand though had no adverse effects on the viability of basophils and IL-3 induced activation, it removed only a small fraction of the cell surface bound IgE. Thus, our results show that although acid treatments could be used for the elution of FcɛRI-bound IgE on the basophil surface for the biochemical characterization of IgE antibodies or for the immediate use of basophils to determine their sensitivity to undergo degranulation by specific allergens, these methods are not utile for the functional assays of basophils that require longer duration of culture and entire removal of surface IgE.Monomeric IgE has been shown to support the survival of mast cells in vitro under growth-factor deprived conditions . HoweverIn line with our current data, previous report has revealed that following exposure of basophils to acetic acid buffer pH 3.7 to elute surface bound IgE, lost the ability to undergo passive sensitization . Even, lIn view of these shortcomings with acid-based stripping of IgE, we believe that this method could not be used for deciphering the functions of anti-IgE autoantibodies that are found in the healthy individuals and in several pathologies including atopic and non-atopic asthma, chronic spontaneous urticaria, atopic dermatitis and others ,48,49,50Basophils were isolated from the buffy coats of healthy donors by using Basophil Isolation Kit II  and autoMACS (Miltenyi Biotec) . Buffy b6 cells/well per 200 µL) in 96-well U-bottomed plate along with IL-3 (100 ng/0.5 million cells) for 24 h.Basophils were incubated on ice for 5 min in phosphate buffered saline (PBS) (control) or in ice-cold acetic acid buffer pH 4 ) or for 0.5 min in ice-cold lactic acid buffer pH 3.9  ,27,28,54Basophils were incubated with recombinant human IgE  for 30 min at 4 °C. Following washing, cells were stained with fluorochrome-conjugated anti-IgE antibodies for the analyses by flow cytometry.Basophils were analyzed for the expression of FcεRIa , CD69 , CD123 , ST2  and surface intensity of IgE  by using LSR II flow cytometer (BD Biosciences) and data were analyzed by BD FACSDiva v8.0.1 (BD Biosciences) and FlowJo v10 softwares . The viability of basophils following isolation was determined by FVD staining. Following culture of basophils for 24 h, viability was analyzed by staining with Annexin V  and propidium iodide .t-test or one-way ANOVA with Tukey’s multiple comparison test were used for the determination of statistical significance as indicated in the figure legends.Prism 6 GraphPad Software  was used for the statistical analyses. Two-tailed Students’ Acetic acid buffer (pH 4) efficiently removes FcɛRI-bound IgE on the basophils. Acetic acid buffer (pH 4)-elution of IgE had no immediate repercussion on the viability of basophils but following 24 h culture with IL-3, the yield and viability of basophils was drastically reduced and had repercussion on the induction of activation markers. Lactic acid treatment on the other hand though had no adverse effects on the viability of basophils and IL-3 induced activation, it removed only a small fraction of the cell surface bound IgE. Thus, acid treatments could be used for the elution of FcɛRI-bound IgE on the basophil surface for the biochemical characterization of IgE antibodies or for the immediate use of basophils to determine their sensitivity to undergo degranulation by specific allergens. However, these methods are not utile for the functional assays of basophils that require longer duration of culture and entire removal of surface IgE.