Decades of cardiovascular research have shown that variable and flexible levels of protein phosphorylation are necessary to maintain cardiac function. A delicate balance between phosphorylated and dephosphorylated states of proteins is guaranteed by a complex interplay of protein kinases (PKs) and phosphatases. Serine/threonine phosphatases, in particular members of the protein phosphatase (PP) family govern dephosphorylation of the majority of these cardiac proteins. Recent findings have however shown that PPs do not only dephosphorylate previously phosphorylated proteins as a passive control mechanism but are capable to actively control PK activity via different direct and indirect signaling pathways. These control mechanisms can take place on (epi-)genetic, (post-)transcriptional, and (post-)translational levels. In addition PPs themselves are targets of a plethora of proteinaceous interaction partner regulating their endogenous activity, thus adding another level of complexity and feedback control toward this system. Finally, novel approaches are underway to achieve spatiotemporal pharmacologic control of PPs which in turn can be used to fine-tune misleaded PK activity in heart disease. Taken together, this review comprehensively summarizes the major aspects of PP-mediated PK regulation and discusses the subsequent consequences of deregulated PP activity for cardiovascular diseases in depth. Myriads of studies starting from the late 1930s have unequivocally shown that protein kinases (PKs) are essential for cellular homeostasis not only in the heart but in virtually any tissue of the body . We haveProtein phosphatases can be divided into three different subgroups, namely serine-threonine, tyrosine, and dual-specific phosphatases depending on their endogenous phosphorylation substrate . Thereby1–3) and PPP1CC (PP1γ1/2). PP1γ2 seems to be the only tissue-specific expressed PP1 (brain and testis) while all other isoforms are expressed ubiquitously  which seem to have distinct subcellular functions as suggested by initial studies using isoform specific knock-down and knock-out studies . Neverthuitously . Multipluitously .The composition of the PP2A holo-enzyme is even more complex. It can be either a heterodimer consisting of the catalytic (PP2A-Cα or Cβ) and the structural scaffold (PP2A-Aα or Aβ) or a trimer consisting of the catalytic, the structural scaffold and another regulatory subunit PPP2RX; . The imp2+ and phosphorylation-dependent signaling pathways. Initially mainly studies identified calcineurin as a regulator of Ca2+ mediated gene transcription during cardiac remodeling, but recently calcineurin was also shown to directly act as an essential enzyme for reversible cardiac protein phosphorylation, e.g., at the cardiac L-Type Ca2+-channel (2+-binding subunit (CNBα/β) and sometimes another regulatory protein, e.g., AKAP or Cain  as an essential cardiac PP which links Ca-channel . Heart-r-channel . Again,  or Cain .After clarifying the functional impact and physiological importance of PPs in the heart, we will now move on to discuss which cardiac proteins are targets of reversible protein phosphorylation by PKs and PPs and how this may relate to CVD pathology.One of the hallmarks of CVDs is altered phosphorylation of cardiac proteins . TherebyV1.2) and the Na+/K+-ATPase dependent substrates in cardiac cells. Phosphorylation at the α-Subunit of CaV1.2 takes place on multiple residues including Ser1512 and Ser1570 (CaMKII) as well as Ser1866 and Ser1928  or PR59 subunits, while the mechanism of regulation of PP1 is still unknown  or adrenoceptor induced (PKA) CaV1.2 phosphorylation  residing proteins ryanodine receptor type-2 (RyR2) and phospholamban (PLB) in this context. Consequently, tremendous efforts have been undertaken to identify the exact contribution of kinases and phosphatases toward deregulated phosphorylation of the latter proteins. While the large intracellular C-terminus of RyR2 contains multiple putative phosphorylation sites, only three of them have been extensively studied until today. Although still under debate, it seems as if Ser2808 and Ser2030 are the major PKA and Ser2814 is the major CaMKII phosphorylated site (Disturbed Cated site . Dephospted site . There ated site .2+-ATPase (SERCA2a). A small contribution has been also shown for PP2A, but this relates to less than 30% of the dephosphorylation events at positions Ser16 (PKA-controlled) and Thr17  or the Inhibitor of the Troponin complex, Troponin-I . I-1 fulfills the function as a central signaling hub in feedback control of phosphatase activity and is thus topic of a number of reviews itself , 2011. IMuch less is known about reversible phosphorylation of phosphodiesterases, another important players of the adrenergic response. PP2A could be shown as a negative regulator of PDE4D3 activity in a complex with mAKAP, RyR2 and PKA . In thisSeveral studies have implicated localization and activity of phosphatases in different compartment of the nucleus including chromatin bound fractions . There i2+/calmodulin, they are dephosphorylated, leading to their nuclear translocation, initiation of transcription and induction of cardiac hypertrophy as well as electrical remodeling  activate PP2B in CVD (Nuclear factors of activated T-cells (NFATs) are a family of transcription factors in which the nuclear localization signals (NLS) are masked by multiple phosphorylations in their serine-rich domains. Upon activation of PP2B by Camodeling . A potenmodeling . The impmodeling . Moreovemodeling  as well modeling . Since cB in CVD .Cardiac electrical excitation is mainly conducted from cell to cell by connexins. In heart especially connexins 43 and 45 fulfill an important function for conduction and increased phosphatase activity of either PP1 or PP2A was shown to induce conduction slowing possibly due to influence on protein stability and nearby channel activation . DecreasAlthough being the first PP substrates studied in terms of regulation and mechanism, the knowledge about functional impact of phosphatases on proteins of the glycogen metabolism has been underestimated for cardiac function a long time. However, recent milestone publications have led to a revival of the study of metabolic pathways and their contribution toward CVDs . The mosWhile this chapter indicated the widespread target portfolio of PPs in the heart, the molecular regulation of PPs themselves is at least equally important for cardiac physiology and manifestation of CVDs. Therefore we are going to review the multiple checkpoints of PP regulation in the heart in the light of PP-dependent PK activity regulation.PPs themselves can be regulated at multiple checkpoints. We will first discuss and highlight some of this regulative mechanisms and subsequently summarize the consequences of deregulated phosphatase activity in CVDs.Drosophila or mice give rise to apparently different phenotypes . However, most of the known examples in the heart have revealed control of PP1, PP2A, and PP2B regulatory subunit rather than catalytic subunit expression by miRs so far. Prominent examples are, e.g., miR-1 and miR-133 control of RyR2/PP2A mediated arrhythmogenesis, miR-34-a control of PPP1R10 during cardiac aging and contraction, as well as miR-212/132 control of calcineurin activity during cardiac hypertrophy . It is aThe most intensive studied way of PP activity control is, however, post-translational PP activity control, mediated either by a regulatory or an inhibitory subunit. Recent studies have suggested that the number of PIPs is bigger than the number of PP, leading to a situation of competition between the different regulatory and inhibitory subunits for the catalytic subunits of PPs . Geneticvice versa for different CVDs.One of the best studied examples of PP-inhibitory subunits in the heart is the I-1. Originally identified in rabbit skeletal muscle, it’s expression has now been verified in nearly every tissue with highest expression in skeletal muscle, adipocytes, kidney and liver and brain . Interes2+ levels, may contribute to the decrease in I-1 phosphorylation described above  or PLB (at Thr17) were in turn hyper-phosphorylated arguing for rather subcellular than general upregulation of PP activity  at the cellular level  and functional effects (loss of inotropic response to dobutamine) of chronic β-adrenoceptor stimulation via isoprenaline infusions with minipumps . In addiin vitro co-immunoprecipitation studies using overexpressed GST-RyR2 fusion proteins  of spinophilin is necessary and sufficient for complete interaction with PP1 . However2+ amplitude as well as maximal rate of Ca2+ rise during systole, decreased shortening amplitude and maximal rate of shortening, while β-adrenergic stimulation remained intact phosphorylation . In ordein vitro and in vivo. Interestingly, dephosphorylation activity strongly depends on oxidative state, which is mechanistically mediated via the neighboring cysteine 199  which bind to the active site of the enzyme, thus blocking access for their endogenous substrates. Due to the very high structural conservation in PP1’s catalytic site with that of PP2A, it has remained difficult to specifically target the active site of either phosphatase . Neverthrocesses . Even berocesses . The userocesses . Due to rocesses . Concretrocesses . Moreoverocesses . Thus, cvice versa: in order to increase potency of currently used drugs, exact knowledge about the contribution of kinases and phosphatases during reversible protein phosphorylation of a specific substrate may help to develop novel and successful combination therapies—similar to the success story of LCZ-696/Entresto (Last but not least we propose two novel strategies to further enhance the feasibility of PP targeting drugs in CVDs: 1. Engaging subcellular and isotype-specific control of PPs: a recent review has highlighted the isotype-specific interaction of PP1 with different regulatory subunits and inhibitors. Combined with compartment-specific PP1 targeting this knowledge might lead to the development of really specific PP targeting drugs. 2. Combination of PK inhibitor and Phosphatase activating drugs and Entresto .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The authors would like to clarify that some of the blots previously depicted in The correct blots for The authors have provided new versions of Figs The text in the Results sections titled “The effects of acute Herceptin treatment on HER3 and PKB phosphorylation” and “The discordant effects of Herceptin on ERK and PKB pathways” has been edited to accommodate the removal of panel E in The corrected text, Figs We found that the downregulation of HER2 receptors was detectable after Herceptin treatment in SKBR3 and BT474 cells, and was associated with an increase in HER2 phosphorylation . Lee-HoeWe analysed the effects of Herceptin on the downstream signalling pathways in HER2 positive breast cancer cells. It was found that the effect of acute Herceptin treatment on phosphorylation of PKB and ERK1/2 was not concordant Figs , in contAcute Herceptin exposure increased EGFR/HER2 and HER2/HER4 dimerisation, correlating with an increase in ERK phosphorylation . In contTherefore, Herceptin treatment decreased PKB phosphorylation due to a decrease in HER3 phosphorylation induced by HER2 downregulation. However, one hour of Herceptin treatment increased ERK phosphorylation as a result of ligand-dependent EGFR and HER4 activation.We found that although Herceptin downregulated HER2 receptors, the remaining cells had persistent and increased HER2 phosphorylation in both SKBR3 and BT474 cells . Since HWe postulated that the increased dimerisation of EGFR, HER3 and HER4 with HER2 was due to activation by their respective ligands. We proceeded to assess the levels of endogenous ligands, using heregulin (ligand for HER3 and HER4) and betacellulin (ligand for EGFR and HER4) as examples. Herceptin treated cells were lysed and endogenous ligand levels were detected using ELISA. We found that Herceptin induced a statistically significant upregulation of heregulin and betacellulin (p = 0.0152 and p = 0.0286 respectively) after 1 hour of Herceptin treatment compared to untreated cells in both SKBR3 and BT474 cells (data on BT474 cells are shown in Thus, Herceptin increased the dimerisation of EGFR, HER3 and HER4 with HER2 as a result of the activation by their ligands.


In skeletal muscle cells, GLUT1 is responsible for a large portion of basal uptake of glucose and dehydroascorbic acid, both of which play roles in antioxidant defense. We hypothesized that conditions that would decrease GLUT1-mediated transport would cause increased reactive oxygen species (ROS) levels in L6 myoblasts, while conditions that would increase GLUT1-mediated transport would result in decreased ROS levels. We found that the GLUT1 inhibitors fasentin and phloretin increased the ROS levels induced by antimycin A and the superoxide generator pyrogallol. However, indinavir, which inhibits GLUT4 but not GLUT1, had no effect on ROS levels. Ataxia telangiectasia mutated (ATM) inhibitors and activators, previously shown to inhibit and augment GLUT1-mediated transport, increased and decreased ROS levels, respectively. Mutation of an ATM target site on GLUT1 (GLUT1-S490A) increased ROS levels and prevented the ROS-lowering effect of the ATM activator doxorubicin. In contrast, expression of GLUT1-S490D lowered ROS levels during challenge with pyrogallol, prevented an increase in ROS when ATM was inhibited, and prevented the pyrogallol-induced decrease in insulin signaling and insulin-stimulated glucose transport. Taken together, the data suggest that GLUT1 plays a role in regulation of ROS and could contribute to maintenance of insulin action in the presence of ROS. •Inhibition of GLUT1, but not inhibition of GLUT4, increases ROS levels in myoblasts.•Mutation of an ATM target site on GLUT1 to alanine (GLUT1-S490A) increases ROS.•The ATM activator doxorubicin decreases ROS except in cells that express GLUT1-S490A.•Inhibition of ATM increases ROS except in cells transfected with GLUT1S490D.•Expression of GLUT1-S490D protects cells against ROS-mediated insulin resistance. Reactive oxygen species (ROS) are a byproduct of metabolism, being produced, for example, during electron transfer in mitochondrial respiration . ROS canGlucose transporter 1 (GLUT1) transports glucose  and dehyThe objective of the present study was to determine whether GLUT1 contributes to regulation of ROS levels in myoblasts. Here we show that inhibition of GLUT1, but not GLUT4, increases ROS. Additionally, activation of ATM decreases ROS, except in cells expressing a GLUT1 serine•alanine mutation at an ATM target site, S490. Further, inhibition of ATM increases ROS, except in cells expressing GLUT1 with an ATM target site mutated to aspartate (S490D). Finally, we found that expression of GLUT1-S490D protects against ROS-induced insulin resistance.Phosphate buffered saline (PBS), trypsin, Dulbeccotm)s modified Eagletm)s medium (DMEM), antimycin A, phloretin, and fasentin were acquired from Sigma Aldrich . The general ROS and superoxide specific fluorescent probes, CM-H2DCFDA (DCFDA) and Mitosox Red, respectively, were purchased from Life Technologies, Invitrogen . Doxorubicin was purchased from Sigma-Aldrich . The ATM inhibitor KU55933 (KU)  was a gi2.L6 myoblasts were bought from the American Type Culture Collection . DMEM containing 10% FetalPlex  and 1% antibiotic•antimycotic solution were used to culture the L6 myoblasts at 37 °C in 5% COThe FLAG-GLUT1 construct , contain4, and 1 mM CaCl2) to obtain a baseline fluorescence reading. DCFDA has an excitation/emission of 485/528 nm. MitoSox Red has an excitation/emission of 485/590 nm. DCFDA is a general ROS probe [c reductase in the electron transport chain [L6 myoblasts were loaded with fluorescent probes (primarily 10 α/4M DCFDA but for one experiment 5 α/4M Mitosox Red in serum free DMEM) for 30•60 min, washed with PBS to clear away phenol red, and incubated in HEPES buffered saline  then subjected to either a Western blot analysis or glucose transport assay (both described below).L6 myoblasts transiently transfected with FLAG-GLUT1 constructs and incubated as explained above were subjected to Western blot analysis as described previously .L6 myoblasts, treated as explained in insulin action experiments above, were exposed to 2-deoxy-glucose (2DG) transport media  for 10 min. An ice-cold 0.9% saline solution was used to wash the cells. The cells were then incubated with lysis buffer (0.2% SDS and 0.2 N NaOH) for 30 min and processed for scintillation counting as described previously . A BCA ap < 0.05 was used to analyze the data. The software used was SPSS Statistics 21.0 .Univariate one-way analysis of variance (ANOVA) with a significance determined as p < 0.05) (p < 0.05)  A and B.  < 0.05) C and D. To determine whether GLUT4 plays a role in regulation of ROS in muscle cells, DCFDA loaded L6 myoblasts were incubated in the presence or absence of 100 µM indinavir, which inhibits GLUT4 but not GLUT1, and then oxidative stress was induced by Ant-A or PG. From 4 min post-induction and beyond, indinavir treated muscles displayed no significant change in Ant-A or PG-induced ROS levels from control muscle cells E and F. GLUT1 has a cytosolic N-terminus, 12 membrane-spanning helices, and a cytosolic 42 amino acid C-terminus . The c-tp < 0.05) and enhanced basal Mitosox Red signal   A. Fluorop < 0.05) (p < 0.05) (p < 0.05) (p < 0.05) (There was nearly a ten-fold increase in Ant-A-induced ROS levels ( < 0.05) B, and a  < 0.05) C. GLUT1- < 0.05) D, and bo < 0.05) E and F. p < 0.05) (p < 0.05) (Previous work has shown that GLUT1-mediated glucose and DHA transport is regulated by ATM . To deci < 0.05) A and B.  < 0.05) C and D.p < 0.05, To determine if these ATM effects on regulation of ROS levels might involve a direct effect on GLUT1, transiently transfected FLAG-GLUT1 L6 myoblasts expressing wild-type GLUT1 or GLUT1-S490A were loaded with DCFDA, incubated in media plus and minus 10 α/4M KU (an ATM inhibitor), and then exposed to superoxide generator, pyrogallol (50 α/4M). Cells expressing wild-type GLUT1 and treated with KU displayed a 28% increase in PG-induced ROS levels (p < 0.05) (p < 0.05)  A and B.  < 0.05) C. Furthe < 0.05) C. These 2O2 [p < 0.05) (p < 0.05) (p < 0.05) (p < 0.05)  A and B.  < 0.05) A. Likewi < 0.05) B. Thus,  < 0.05) A and B.  < 0.05) C. ExpresThe findings presented in this study show that GLUT1 plays a role in regulation of ROS levels in muscle cells. For example, inhibition of GLUT1-but not inhibition of GLUT4-increased ROS levels caused by challenge with pyrogallol or antimycin A. Site mutation of GLUT1-S490, a known ATM phosphorylation target, to alanine (S490A) augmented ROS levels; in contrast, mutation of this ATM target site to aspartic acid (S490D) decreased ROS levels. Inhibition of ATM increased ROS, but not in cells expressing GLUT1 mutated to mimic phosphorylation at an ATM target site (S490D). Conversely, the ATM activator doxorubicin decreased ROS, but not in myoblasts expressing GLUT1-S490A. Finally, insulin resistance caused by a superoxide generator was prevented by expression of GLUT1-S490D. Together, the data suggest the novel idea that GLUT1 plays a role in antioxidant defense and also functions to maintain insulin action in the face of oxidative stress.Insulin exerts its effects of lowering blood glucose levels by activating a signaling cascade that results in the translocation of GLUT4 . IRS-1 uATM is well known for its nuclear role of sensing and commencing the signaling for DNA damage and repair . Double-Antioxidant defense proteins include single molecule antioxidants as well as intricate antioxidant enzyme systems . AscorbiIn summary, a role of GLUT1 in regulation of ROS levels has been elucidated by means of GLUT1 inhibitors that increase ROS and expression of GLUT1 with mutations at an ATM target site that prevent (S490A) or mimic (S490D) phosphorylation and increase or decrease ROS, respectively. Notably, the ATM activator doxorubicin decreases ROS except in cells that express GLUT1-S490A, while inhibition of ATM increases ROS except in cells transfected with GLUT1S490D. Furthermore, expression of GLUT1-S490D protects cells against ROS-mediated insulin resistance. Thus, the findings suggest the novel idea that GLUT1 plays roles in regulation of ROS and maintenance of insulin action under conditions of oxidative stress.No conflicts of interest exist for any of the authors.


Transpedicular screws may cause damage to the cartilage in the neural arch of the vertebra, and give continuous pressure to the skeleton besides the vertebral body. The aim of this study is to examine the morphological change of the vertebral body at fixation sites and development of the vertebral body after fixation.A piglet model was used to study the influence of transpedicular screw fixation on spine development. Transpedicular screw fixation was adjusted to meet specific requirements of surgery on piglet. The screws and plates were placed at L1–L3 vertebral plates via routine surgical approach. Scoliosis and kyphosis Cobb angles were measured.Anatomical characteristics of 6-week-old piglets fit the transpedicular screw system, and can meet the requirements of related studies. Transpedicular screw fixation system has no significant influence on the development of canalis vertebralis. Fixation did not cause developmental stenosis of canalis vertebralis and damage to spinal cord or nerve root. However, transpedicular screw fixation significantly impacted the development of the spine: it shortened the spine by curtailing the length of the vertebral body and intervertebral space. Our results also suggested that slow growth of epiphyseal plate may contribute to the shortening of the vertebral body.Transpedicular screw fixation system is beneficial for fixation of the developing spine. It may not cause scoliosis but could lead to change of cervical curvature. Transpedicular screw fixation was developed by Roy-Camille in the 1970s –4.Spinal deformity is a common disease with high frequency in adolescences . As the Since spines of children are still in development, transpedicular screw fixation may slightly impact the development of spines. Jeszenszky et al.  demonstrIn this study, a piglet model was used to study the influence of transpedicular screw fixation on spine development. Transpedicular screw fixation was adjusted to meet specific requirements of operating on piglet. Animals were examined by CT and X-ray before and after surgery. The screws and plates were placed at L1–L3 vertebral plates through routine surgical approach. Data from this study suggested that transpedicular screw fixation system may be beneficial for fixation of developing spine, while not causing scoliosis.The use of piglets in this study is reviewed and approved by the ethical committee of General Hospital of People’s Liberty Army. Piglets  were used in this study. All animals passed the investigation by National Animal Quarantine Center, and the possibility of tumor and deformity of spine were ruled out by spiral computed tomography (CT) and X-ray.A cohort of 23 piglets were randomized into four groups: group A included three subjects as control, without any treatment; group B included three subjects as control, with vertebral lamina revelation (L1–L3); group C included five subjects as control, with six screws bilaterally placed at L1, L2, and L3 in each pig. All screws, which penetrated through cartilages in the neural arch of the vertebra, were left within pedicles; and group D included 12 subjects as treatment, with six screws bilaterally placed at L1, L2, and L3 in each pig. All screws were fixated with titanium plates.All screws used in this study are made of titanium . The dimensions of screws are 3.5 mm in diameter and 20 mm in length.Preparation of pigs before surgery included the following: (1) spiral CT  and X-ray  investigation. (2) Preoperative fasting for 24 h. (3) Preoperative preserved skin: skins were shaved in corresponding areas (approximately 25 cm × 15 cm) then cleaned with soap water.Anesthesia: A mixture of 0.25 mL/kg Sumianxin and 18 mg/kg ketamine was used for anesthesia. The operation started as soon as corneal reflection disappeared. Depending on animal conditions, an additional anesthesia  might be used.Positioning: Animals were at prone position with four limbs fixed.Operation procedure: Skins at the incision site were sterilized with iodophor, and incised at the center of the back. The incision started from T14 to L4. Skins and subcutaneous tissues were cut layer by layer. Sub-periosteal dissection was conducted bilaterally, and paravertebral muscles were pulled aside. The sides of L1–L3 were revealed to the exterior side of zygopophysis, and the root of processus transversus were exposed.Group B: After revelation, the incision sites were washed, sterilized with 70 % ethanol (volume/volume), and the wound was sutured. During the operation, approximately 15 mL of blood flowed out. The average time for surgery is about 1.5 h.Group C: After revelation, a total of six screws were bilaterally placed into L1–L3. The average sagittal angle of L1–L3 pedicle and vertebral body is 42.5°. Thus, the screw sites were the central point on the root of processus transversus, with a separation angle of 40° between screws and vertical plane of spine . After fixation, the incision sites were washed, placed with drainage strip, sterilized with ethanol, and sutured. During the operation, approximately 20 to 30 mL of blood flowed out. The average time for surgery was about 3 h.Group D: After revelation, a total of six screws were bilaterally placed into L1–L3. The screw sites were the central point on the root of processus transversus, with a separation angle of 40° between screws and vertical plane of spine . After the screws were placed, they were fixed with titanium plates. After fixation, the incision sites were washed, placed with drainage strip, sterilized with ethanol, and sutured. During the operation, approximately 20 to 30 mL of blood flowed out. The average time for surgery was about 3 h.Post-surgery procedures included the following: (1) sauteralgyl (50 mg) was administered through intramuscular injection. (2) Penicillin (800 K units) was administered through intramuscular injection, three times per day and for three consecutive days after surgery. (3) X-ray and spiral CT were used at different time points . (4) Twelve days after the operation, stitches were removed. All animals were sacrificed 3 months after the operation.X-ray and spiral CT were used for measurements : (1) area, transverse diameter, and sagittal diameter of the vertebral canal Fig. ; (2) traThe area of the vertebral canal was automatically measured by the software after drawing out its curve. The planes with the smallest area were chosen for further measurements of transverse diameter (the widest) and sagittal diameter of the vertebral canal, vertebral transverse diameter (the widest) and sagittal diameter, heights of the vertebral body, and spine lengths of L1–L3 .Three months after the operation, the average weight was 40.4 ± 8.0 kg, while the average body length was 88.7 ± 7.3 cm.Due to overdosing in anesthesia (death from suffocation by glossocoma blocking the respiratory tract), one piglet in group D died before the operation and another died during the operation. In group C, infection occurred in two piglets after operation. The bacteria were identified as Area, transverse diameter, and sagittal diameter of the vertebral canal were measured. Also, transverse diameter, sagittal diameter, and height of the vertebral body were measured. In addition, spine lengths of L1–L3 were observed Tables , and 3.Tp > 0.05).Right after the operation, there were significant differences in area, transverse diameter, and sagittal diameter of the vertebral canal; transverse diameter, sagittal diameter, and height of the vertebral body; and spine length of L1–L3 between groups . After implantation of transpedicular screw fixation system, area, transverse diameter, and sagittal diameter of the vertebral canal and transverse diameter, sagittal diameter, and height of the vertebral body did not decrease significantly.There were no significant differences in area, transverse diameter, and sagittal diameter of the vertebral canal and transverse diameter, sagittal diameter, and height of the vertebral body between groups (p < 0.05) while no significant difference within groups A, B, and C (p > 0.05). After implantation of transpedicular screws, heights of the vertebral body and spine lengths of L1–L3 did not change significantly. However, after fixation, heights of the vertebral body and spine lengths of L1–L3 significantly decreased.There were significant differences in heights of the vertebral body and lengths of L1–L3 between groups A, B, C, and D (p > 0.05). However, the vertebral body of L2 is significantly shorter than that in L1 and L3 (p < 0.05).There were no significant differences between heights of the vertebral body of L1 and L3 in group D  the vertebral canal of piglets (6 weeks old) was mostly developed in terms of morphology. (2) Three months after the operation, transverse diameter and sagittal diameter of the vertebral canal in groups A, B, and C increased only 2.0 and 2.3 mm on average, respectively. The external diameter of screws was only 3.5 mm, approximately half of the diameter of pedicles. Also, the internal diameter of screws was only 2.5 mm, posing little damage to NCC. (3) As the spine of piglets develops, the diameter and length of screw decreased relatively, providing less holding strength. Thus, the impacts of screws further decreased. (4) During the development of the vertebral canal, the relative position of screws and spine changed. The distance between screws and pallium in the front area of the vertebral body 3 months after the operation and right after the operation was compared using the X-ray results. The fixation effects further decreased while the spine developed continuously.Based on these four reasons, transpedicular screw fixation had limited impacts on sagittal diameter of the vertebral canal. In this study, no transversal junctions were used for spine fixation. Therefore, the transversal development of the vertebral canal was not hindered.To animals with developing spines, transpedicular screw fixation can affect the development of the vertebral body and intervertebral cartilages, when providing firm holding strength. Transpedicular screw fixation can limit the development of the vertebral body and also leads to dysplasia of intervertebral cartilages. Since the limitation of fixation system, the vertical growth of the vertebral bodies was hindered within the limited heights of intervertebral cartilages. Therefore, intervertebral space at fixation sites showed stenosis to different extents. Moreover, from the X-ray image of the lumbar spine, it was almost impossible to observe the intervertebral space 3 months after the operation. Based on these analyses, transpedicular screw fixation controlled the spine development through limiting the vertical growth of the vertebral body. If the direction of gravity was taken into account, the limitation of transpedicular screw on vertical growth of the vertebral body could be more obvious, since the direction of gravity is different in humans comparing to pigs. On the other hand, the transpedicular screw combined with plate may give enough support to counteract gravity; thus, the limitation of transpedicular screw on growth of the vertebral body may not be strengthened by gravity. It is hard to predict the influence of gravity on growth of the vertebral body with the data we currently have. To answer this question, further studies must be conducted.However, our data also showed that the growth of the vertebral body at fixation sites, especially L2, did not completely cease. This phenomenon might be explained by the following reasons: (1) the relative length of screws decreased as the spine developed, resulting in less holding strength to the spine; (2) due to the vertical growth of spines, screws showed cutting function within sclerotin; (3) due to the continuous stress from transpedicular screw fixation, the connections between screws and titanium plates deformed. Therefore, based on the aforementioned reasons, transpedicular screw fixation could not fully limit the development of spines.Through sagittal sections of lumbar spine samples, the changes in intervertebral cartilages were observed in this study. It was demonstrated that chondrocytes in epiphysis were sensitive to mechanical pressure . Due to This study investigated the effects of transpedicular screw fixation on spine development in 20 piglets, and laid a solid foundation for its clinical application in treating children’s spinal diseases. Admittedly, this work is still a pilot study due to a small number of subjects.Transpedicular screw fixation system had no significant influence on canalis vertebralis development, and neither did it cause developmental stenosis of canalis vertebralis nor damage of spinal cord and nerve root. Transpedicular screw fixation system could significantly shorten the spine by shortening the length of the vertebral body and intervertebral space. It was shown that slow growth of epiphyseal plate could contribute to the shorter length of the vertebral body. Transpedicular screw fixation system is beneficial for the development of the spine. It will not cause scoliosis but can lead to change of cervical curvature.


N-glycosylation of the major plasma glycoproteins alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-glycoprotein 1, ceruloplasmin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, vitronectin, and zinc-alpha-2-glycoprotein. In addition, the less abundant immunoglobulins D and E are included because of their major relevance in immunology and biopharmaceutical research. Where available, the glycosylation is described in a site-specific manner. In the discussion, we put the glycosylation of individual proteins into perspective and speculate how the individual proteins may contribute to a total plasma N-glycosylation profile determined at the released glycan level.Glycosylation is the most abundant and complex protein modification, and can have a profound structural and functional effect on the conjugate. The oligosaccharide fraction is recognized to be involved in multiple biological processes, and to affect proteins physical properties, and has consequentially been labeled a critical quality attribute of biopharmaceuticals. Additionally, due to recent advances in analytical methods and analysis software, glycosylation is targeted in the search for disease biomarkers for early diagnosis and patient stratification. Biofluids such as saliva, serum or plasma are of great use in this regard, as they are easily accessible and can provide relevant glycosylation information. Thus, as the assessment of protein glycosylation is becoming a major element in clinical and biopharmaceutical research, this review aims to convey the current state of knowledge on the The online version of this article (doi:10.1007/s10719-015-9626-2) contains supplementary material, which is available to authorized users. Protein glycosylation is recognized to be involved in a multitude of biological processes such as receptor interaction, immune response, protein secretion and transport –6. In adProtein glycosylation can differ between persons, but is remarkably stable per individual . It is oAt the same time, specific studies based on smaller sample sets have revealed changes of glycosylation in various diseases, inflammatory states, congenital disorders of glycosylation (CDGs), but also throughout pregnancy where an increase in galactosylation and sialylation, as well as a decrease in bisection was reported –28. In aN-glycosylation (TPNG) of a clinical cohort at the released glycan level, it is not directly apparent whether an observed change originates from a change in relative protein abundance, in the relative glycoforms of a specific protein, or whether it reflects a general regulatory effect influencing the glycosylation of many different glycoproteins. We expect that a better understanding of the glycosylation of individual proteins of human plasma will help to put total plasma N-glycomic changes into perspective.Recent advances in chromatographic separation, mass spectrometry, robotization and automated data processing allow the rapid analysis of glycosylation, and facilitate the development of novel biomarkers –39. WhilN-glycosylation originates from 2008 [As the previous review on plasma protein rom 2008 , we hererom 2008 –43.N-glycosylation of the proteins is reported both on a general level and, where available, with site specific information about glycan composition, glycan structure and occupancy. The information is condensed in Table Furthermore, we tried to limit our review to human plasma and serum but we also reported findings coming from other biological fluids when information complementary information could be added. The N-acetylglucosamine, G for galactoses and S for sialic acids. The number directly after the letter indicates the quantity of the specific features and the number in parenthesis its linkage. UniProt numbering was used for sequence and site identification.Throughout the text, Oxford nomenclature has been used to annotate individual glycan structures or compositions with A giving the number of antennae, F for the fucose (location specific), B for bisecting ORM1 and ORM2 respectively), differing in 22 amino acids [Alpha-1-acid glycoprotein (AGP), also known as orosmucoid-1, is a 201 amino acid glycoprotein, which includes an 18 amino acid signal peptide. The molecular weight of the bare protein is 23.5 kDa, but the carbohydrate content leads to observed masses around 41–43 kDa . Two isono acids . The prono acids , 205. ThThe main functions of AGP are acute phase negative modulation of the complement system and transport of lipophilic compounds, both of these heavily modulated by the glycosylation of the protein , 207. ThN-linked glycosylation sites, namely Asn33, Asn56, Asn72, Asn93 and Asn103. Overall, the glycosylation was determined to mainly consist of fully sialylated tri- and tetraantennary structures, with potential antennary fucosylation in the form of sialyl-Lewis X [AGP has five -Lewis X , 45. Sit-Lewis X . Asn33 m-Lewis X   increase significantly , 47–49, Alpha-1-antitrypsin (AAT), also known as alpha-1-protease inhibitor, alpha-1-antiproteinase or serpin A1, consists of 418 amino acids  with an apparent mass of 51 kDa (including glycosylation). It is mainly produced in the liver by hepatocytes, but is also synthesized in monocytes, intestinal epithelial cells, and in the cornea , 208–211AAT inhibits a wide range of serine proteases, protecting tissues from enzymatic attacks . NeutropN-glycosylation sites have been identified on AAT, located at Asn70, Asn107 and Asn271 [Three d Asn271 –54. MALDd Asn271 .N-acetylglucosamine of the α1-3-arm [LC-MS/MS analysis on tryptic glycopeptides treated with various specific exoglycosidases enabled a precise determination of the glycosylation in a site-specific manner . Asn70 aα1-3-arm .i.e. non-, mono- and diglycosylation across the three sites. A clear pattern could be found for which sites were occupied, as only Asn70 was occupied in the monoglycosylated form, and Asn70 and Asn271 were occupied in the diglycosylated isoform [In a non-site-specific study, the glycosylation of AAT has been associated with physiological parameters such as BMI, cholesterol, glucose and insulin level. The same study showed that the changes in glycosylation could be found related to age and sex . Further isoform .Alpha-1B-glycoprotein (A1BG) is a 474 amino acid polypeptide with an apparent mass of 63 kDa (including glycosylation) . The proN-glycosylation consensus motif (Asn-X-Ser/Thr) has been found at four locations Asn44, Asn179, Asn363, and Asn371 [In A1BG, the d Asn371 . The occd Asn371 , 60. In d Asn371 . Also, wd Asn371 . Little Alpha-2-HS-glycoprotein (A2HSG), also known as fetuin-A, alpha-2-Z-globulin, ba-alpha-2-glycoprotein and alpha-2-Heremans-Schmid-glycoprotein, is a 367 amino acids , 51–67 kDa glycoprotein , 64, 228Increased levels of A2HSG are associated with obesity and type 2 diabetes mellitus . On the N-glycosylation sites at Asn156 and Asn176, as well as two O-glycosylation sites at Thr256 and Thr270 [O-glycan on Ser346, and no N-glycans [N-acetylglucosamines, pointing towards two diantennary N-glycans in addition to the O-glycosylation [The A-chain of A2HSG contains two d Thr270 . The B-c-glycans . Exoglyc-glycans . Sequentsylation . LC-ESI-sylation . Furthersylation , 67.Differential abundance of Asn156 glycopeptides has been shown in pancreatic cancer and pancreatitis, with increased levels of fully sialylated triantennary glycans with or without fucose, and a decrease in the A2G2S2 structure in pancreatitis .Alpha-2-macroglobulin , also known as C3 or PZP-like alpha-2-macroglobulin domain-containing protein 5, is a 1474 amino acid  720 kDa (glycosylated) glycoprotein consisting of four similar 180 kDa subunits (160 kDa without glycosylation) which are linked by disulfide bridges . It is pN-glycosylation sites have been identified on each alpha2M subunit at Asn55, Asn70, Asn247, Asn396, Asn410, Asn869, Asn991 and Asn1424 [Eight  Asn1424 , 69–71.  Asn1424 . In addi Asn1424 .N-glycosylation sites mainly contain complex type glycans and glycoproteomic analysis suggests that the core-fucosylated species are present to at least some degree at specific sites Asn55 and Asn1424 [Interestingly, the high-mannose type glycans have been shown to specifically occur at Asn869 with a relative abundance of approximately 70 %, the other 30 % being FA1G1S1 . This hi Asn1424 .Changes in the glycosylation of alpha2M have been associated with autoimmune diseases and cancer. Site occupancy in particular has been linked with systemic lupus erythematosus, while a compositional change has been described in multiple sclerosis , 74.SERPINC1 gene, is a single chain 464 amino acid  protein of approximately 58 kDa, of which 17 % are carbohydrates [Antithrombin-III , encoded by the hydrates –242. It hydrates . The prohydrates . Antithrhydrates . Its funhydrates , 246. Sehydrates .N-glycosylation sites: Asn128, Asn167, Asn187 and Asn224. The α form is fully glycosylated, while the β form is not glycosylated at Asn167 [The sequence of antithrombin shows four potential t Asn167 , 78. Thet Asn167 –81.The glycans present on AT-III are mainly of the diantennary complex type without core fucose, bearing one (0–30 %) or two (70–100 %) α2-6-linked sialic acids, as it was established using chemical and enzymatic methods , 83. UsiA mutation associated with type II antithrombin deficiency (K241E), although not adjacent to a glycosylation consensus sequence, was found to result in decreased heparin binding due to the presence of core fucose .N-glycosylation site located at Asn263  [Apolipoprotein (Apo) B-100 is a 550 kDa 4560 amino acid protein  found in low density and very low density lipoproteins (LDL and VLDL) , 249. A glycans) , 258.APOB100 and MTP  gene defects are associated with metabolic disorders like abetalipoproteinaemia, hypobetalipoproteinemia and hypercholesterolemia [In coronary heart disease, the ratio of LDL-Apo A/B-100 is used for estimating the risk of acute myocardial infarction . Apo B-1erolemia –261.N-glycosylation sites located at Asn34, Asn185, Asn983, Asn1368, Asn1377, Asn1523, Asn2239, Asn2560, Asn2779, Asn2982, Asn3101, Asn3224, Asn3336, Asn3358, Asn3411, Asn3465, Asn3895, Asn4237 and Asn4431. Of these, 17 are reported occupied by diantennary complex type glycans, as well as by high mannose and hybrid type structures [Apo B-100 is highly glycosylated, and contains 19 potential ructures . LC-fluoructures . In addiructures .N-glycosylation site to the receptor of the LDL-binding site shows degrees of fucosylation. Asn34 and Asn2560 are not reported to be glycosylated [A site specific analysis of the glycosylation has been performed by LC-ESI-MS(/MS) on tryptic and chymotryptic glycopeptides . It was osylated .The role of the glycans structures in LDL and/or Apo B-100 has been examined in several studies but their exact function is still unknown, although its degree of sialylation might serve the atherogenic properties of LDLs –90.Apolipoprotein D (Apo D), also referred to as thin line polypeptide, is a small glycoprotein of 189 amino acids , with a molecular weight varying between 19 and 32 kDa depending on its glycosylation , 263. WhApo D can form complexes with lecithin cholesterol acyltransferase and is implicated in the transport and transformation of lipids , 268–270N-glycans ranging from diantennary to tetraantennary structures, with potential elongation of the antennae in the form of N-acetyllactosamine (LacNAc) repeats [Two glycosylation sites have been reported and confirmed for Apo D, namely Asn65 and Asn98 , 60, 91. repeats , 91, 92. repeats . StudiesApolipoprotein F (Apo F), also called lipid transfer inhibitor protein (LTIP), is a glycoprotein with an apparent mass of 29 kDa. The polypeptide chain of 326 amino acids is processed, with the first 165 amino acids being the signal peptide and the propeptide, resulting in a theoretical mass of 17.4 kDa for the peptide backbone of the mature protein . Apo F iN-glycosylation sites of Apo F are located at Asn118, Asn139 and Asn267, as well as one O-glycosylation site at Thr291 [N-glycans [O-glycanase has also shown the presence of O-glycans on the protein, it is unclear whether the sialylation arises from the N- or O-glycosylation [O-glycans on Thr291 are reported to be of core 1 or 8 type [Three potential t Thr291 , 95. Asn-glycans . Howeversylation . In CSF,r 8 type . No disei.e. open or closed, which is proposed to be dependent on the glycosylation [Beta-2-glycoprotein 1 (B2GPI) is also called apolipoprotein H, APC inhibitor, activated protein C-binding protein, and anticardiolipin cofactor. It is a 50 kDa (including around 19 % carbohydrate content) 345 amino acid single polypeptide chain  belonging to the complement control protein (CCP) superfamily . It conssylation , 99, 286The serum level of B2GPI increases with age, and is reduced during pregnancy and for patients suffering from stroke and myocardial infarctions , 287. AdN-glycosylation sites at Asn162, Asn183, Asn193 and Asn253, as well as an O-glycosylation site at Thr149 [N-glycosylation sites have been confirmed by crystallography (finding attached N-acetylglucosamines and mannoses) as well as by deglycosylated Lys-C peptide reverse phase (RP)-LC-MS after lectin capture [B2GPI possesses four theoretical t Thr149 , 101. Th capture , 101. Ge capture . Site-sp capture .N-glycosylation sites (Asn183 and Asn253) di- and triantennary glycans are expected as well, given the 19 % of the total protein weight being attributed to the carbohydrate content [Glycopeptide LC-ESI-quadrupole (Q)-TOF-MS revealed the glycosylation of Asn162 to be 67 % diantennary disialylated (A2G2S2) and 22 % triantennary trisialylated (A3G3S3), minor species including the di- and triantennary species lacking one sialic acid (5 and 3 % respectively) . The Asn content .Patients suffering from antiphospholipid syndrome (APS) showed a decrease in the amount triantennary sialylated glycans, and thus a relative increase in diantennary fully sialylated ones. This effect was particularly pronounced for Asn162 .2+ to Fe3+ without releasing radical oxygen species, while also facilitating iron transport across the cell membrane [Ceruloplasmin (CP), also called ferroxidase, is a 132 kDa (120 kDa without glycosylation) 1065 amino acid  glycoprotein synthesized by the liver . It consmembrane .N-glycosylation sites Asn138, Asn227, Asn358, Asn397, Asn588, Asn762 and Asn926, four  are confirmed to be glycosylated [Of the seven potential CP osylated . The remosylated . NMR speosylated .N-glycosylation sites, tryptic glycopeptide LC-ESI-MS(/MS) was used to study the site-specific glycosylation on a compositional level, and relatively similar ratios of di- and triantennary glycan species were found across the sites [For the confirmed he sites . Asn138 he sites . No info2β2γ2 hexamer linked by disulfide bonds [Fibrinogen is a 340 kDa glycoprotein that is synthesized in the liver by hepatocytes, and plays a key role in blood clotting , 291. Thde bonds , 293. Inde bonds , 293–296Fibrinogen is cleaved by thrombin into fibrin, one of the essential components of blood clots after injury , 297. FuN-glycosylated, even though it harbors two potential N-glycosylation sites at Asn453 and Asn686. The β- and γ-chain are N-glycosylated at Asn394 and Asn78, respectively [N-acetylglucosamine and core fucosylation are found in minor quantities [N-glycan profiles revealed that fibrinogen could contribute for 22 % to the total intensity of the diantennary monosialylated structures (A2G2S1) [The α-chain of fibrinogen is not ectively –108. By ectively , 110. BiN-glycosylation, all fibrinogen chains may carry O-glycans [Site-specific analysis showed diantennary glycans with zero, one or two sialic acids on Asn394 (β-chain) and Asn78 (γ-chain) . The gly-glycans .N-glycosylation site [The general degree of sialylation may be influencing the solubility of fibrinogen, and thereby play a crucial role in blood clotting processes resulting in different fiber structures. –115. In ion site . Patiention site , 117.1 having this subsequence once while α2 has it twice, resulting in α chains of 83 or 142 amino acids with a respective molecular mass of 9 and 16 kDa. The 40 kDa β chain is made of 245 amino acids originating from the sequence Ile162-Asn406 [1 and α2) with β chain(s) creates the polymorphism observed in Hp. There are three major Hp phenotypes called Hp1-1, Hp2-1 and Hp2-2. They have a configuration of (α1β)2, (α1β)2 + (α2β)n = 0, 1, 2, … and (α2β)n = 3, 4, 5, …, respectively, which are observed at different ratios among ethnicities [Haptoglobin (Hp) is a 406 amino acid  acute-phase glycoprotein with a peptide backbone of 45 kDa. It is synthesized in the liver by hepatocytes as a single polypeptide chain and is also found in skin , 305. Du2-Asn406 , 307. Thnicities , 308–310nicities . Elevatenicities , 312. Itnicities . The halThe major function of Hp is to protect tissues from oxidative damage by capturing hemoglobin , 313. ItN-glycosylation sites have been identified on the β-chain of Hp, located at Asn184, Asn207, Asn211 and Asn241 [N-glycans [Four d Asn241 –121. Ana-glycans , 120. ThTwo recent studies showed some discrepancies in the relative abundances for the identified sites. For example, Asn184 was found to contain mainly diantennary species with two sialic acids , followed by diantennary monosialylated  and triantennary disialylated  glycans. A low percentage of fucosylation was identified  , 120.N-glycosylation sites (Asn207 and Asn211) on the same peptide backbone that showed 7 different combinations. The major combinations were 1) one diantennary fully sialylated (A2G2S2) and one triantennary disialylated , 2) two diantennary disialylated glycans , and 3) one diantennary fully sialylated (A2G2S2) and one triantennary disialylated and fucosylated . The combination of a diantennary monosialylated (A2G2S1) with a diantennary disialylated glycan (A2G2S2) accounted for 6 %, the diantennary and triantennary fully sialylated species (A2G2S2 and A3G3S3) for 5 %, and the remaining combinations accounted for approximately 1 % in total [A possible reason for discrepancies at Asn207/Asn211 is that the first study did not differentiate the two in total . The secin total .The two studies report that Asn241 carries mainly diantennary glycans, A2G2S2 being the most abundant variant with 87 and 47 % , followed by A2G2S1 (4 and 26 %), A3G3S1 (n.d. and 10 %), A3G3S2 (6 and 8 %), A3G3S3 (n.d. and 4 %) and A2FG2S1 (<1 and 2 %). Low levels of tetraantennary species have been detected as well, with and without fucosylation varying from mono- to tetrasialylated , 120.N-glycosylation sites Asn207 and Asn211 [Both studies evaluated the glycosylation of Hp in patients with liver cirrhosis (LCH) and hepatocellular carcinoma (HCC). No difference in site occupancy could be observed between healthy and disease, but the number of detected glycoforms was increased  . Increasd Asn211 . Those cd Asn211 .N-glycosylation site Asn184 [Furthermore, core-fucosylation was identified on the e Asn184 . Diantene Asn184 , 124. Ane Asn184 , 126.It is interesting to see that two studies from the same year report different glycosylation patterns for Hp , 120. ThHemopexin (HPX), also known as beta-1B-glycoprotein, is a 462 amino acid  single polypeptide chain plasma glycoprotein with a peptide backbone of 51 kDa and an apparent mass ranging from 57 to 80 kDa depending on its glycosylation –317. TheN-glycosylation sites located at Asn64, Asn187, Asn240, Asn246 and Asn453 [N-glycosylation consists mainly of diantennary structures with high levels of galactosylation, while low levels of triantennary and fucosylated structures have also been reported [N-glycosylation sites Asn64, Asn187 and Asn453, each of them showing similar ratios of glycoforms  [O-glycosylation sites (Thr24 and Thr29) one of which is located on the N-terminal threonine , and a potential minor O-glycosylation in the Ser30-Thr40 region [HPX contains five confirmed d Asn453 , 127–130reported , 131–133reported . Combiniuctures) , 121. Asuctures) . Notably2+ [Histidine-rich glycoprotein (HRG), also called histidine-proline-rich glycoprotein (HPRG), has an apparent molecular mass of 72 kDa (peptide backbone of 60 kDa) and consists of 525 amino acid  , 321. Th2+ . It is a2+ .N-glycosylation sites have been confirmed by glycoproteomic analysis, located at Asn63, Asn125 and Asn344 [N-glycanase treatment revealed a mass difference of 2 kDa attributed to the new Asn202 carbohydrate compared to the unmodified form of HRG [HRG is expected to have a large degree of glycosylation, as 14 % of the protein weight (around 10 kDa) has been attributed to the oligosaccharide portion . Three Nd Asn344 , 70. Anom of HRG . The seqm of HRG , 138. WiKininogen-1, also called alpha-2-thiol proteinase inhibitor, Fitzgerald factor, high-molecular-weight kininogen (HMWK) or Williams-Fitzgerald-Flaujeac factor, has a single polypeptide chain of 644 amino acid  and approximates 114 kDa apparent molecular mass  . KininogN-linked glycosylation sites at Asn48, Asn169, Asn205, Asn294  and eight O-linked glycans at sites Thr401, Thr533, Thr542, Thr546, Thr557, Thr571, Ser577 and Thr628 [n after capturing with L. culinaris lectin [N- and O-glycan structures, as the observed protein mass is more than 40 kDa higher than the mass calculated from the amino acid sequence. Kininogen glycosylation changes due to diseases have not yet been described.Kininogen has four d Thr628 , 139. Whs lectin . In addis lectin . KininogSerotransferrin (STF), also known as transferrin, β1 metal binding globulin or siderophilin, is a 698 amino acid protein  with a molecular mass of approximately 77 kDa (without glycosylation) . The pro3+ ions and transport those throughout the body, avoiding the toxicity of free radical formation that may be caused by free Fe3+ ions [e.g. Tf-ADR versus HeLa, HL-60 and H-MESO-1 cell lines) [STF is an iron binding protein and it regulates iron levels in biological fluids. It can bind two Fee3+ ions . Iron ise3+ ions . Severale3+ ions , 342. Ine3+ ions . In infle3+ ions . The proe3+ ions . The proe3+ ions . Additiol lines) , 345.N-glycosylation sites located at Asn432 and Asn630 and a potential minor site at Asn491 (Asn-X-Cys) [STF has two n-X-Cys) , 144. Arn-X-Cys) . Lectin n-X-Cys) . The glyn-X-Cys) , 144. Thn-X-Cys) , 144.N-glycosylation has been investigated in other biologic fluids like CSF, where similar structures have been found along with some disease-related ones [The glycans present on Asn432 are A2G2S2 (93.5 %), A3G3S2 (2.5 %), A2G2S1 (2.4 %) and A2FG2S2 (1.6 %), while Asn630 contains A2G2S2 85.9 %), FA2G2S2 (6.9 %), A2FG2S2 (2.8 %), A2G2S1 (2.2 %), A3G3S2 (1.0 %), as well as some lower abundant species with increased fucosylation , 144. Th5.9 %, FAted ones .N-glycosylation related CDGs while apolipoprotein C-III is the protein of choice for the test of O-glycosylation related CDGs [The glycosylation of STF has shown to be different across fluids and phenotypes with the abundance of A2G2S2 being significantly reduced in human amniotic fluid (55 %) or in the plasma of hepatoma patients (37–63 %) , 146. Thted CDGs , 150. Inted CDGs , 151.Vitronectin (VN), also called S-protein, serum spreading factor, or V75, is a 459 amino acid 52.4 kDa member of the pexin family and of the adhesive glycoproteins group –349. TheVN is an adhesive glycoprotein, and shows a role in blood coagulation, extracellular matrix binding, regulation of cell adhesion and spreading, and innate immunity , 347. ItN-glycosylation sites have been identified in VN at Asn86, Asn169 and Asn242 by LC-MS(/MS) [N-acetyllactosamine of the α1-3-arm of triantennary structures. Core fucosylation of vitronectin accounts for 7.9 % [Three -MS(/MS) , 70. Wit-MS(/MS) . Sialic or 7.9 % .N-glycans , with possible core fucose on the fully sialylated variant . In addition, triantennary fully sialylated structures have been detected with and without fucose  [When looking at the glycosylation in a site-specific manner by trypsin digestion and LC-ESI-MS(/MS) analysis, Asn86 shows mainly A2G2S2 species (45 %), as well as A3G3S3 (33 %) and A3FG3S3 (20 %) . At Asn13, 10 %) .Core fucosylation of VN has been reported at Asn242 in healthy individuals and on Asn86 in HCC patients . Hybrid ZAG which is the short name of its AZGP1 gene), also abbreviated Zn-alpha-2-glycoprotein or Zn-alpha-2-GP, is a 41 kDa glycoprotein (15 % of the mass being carbohydrate) comprising a single 298 amino acid chain , with two intra-chain disulfide bridges [Zinc-alpha-2-glycoprotein  spectroscopy has revealed the major N-glycan structure to be diantennary and disialylated A2(2)G2(4)S2(6) [N-glycans, and the sialylation thereof, has been verified by proteomic experiments, but to date no extensive study has been made on its glycan microheterogeneity [Four d Asn259 , 157. Fo(4)S2(6) . For Asnogeneity , 158. Asogeneity . No infoL or VL, respectively) and three or more constant domains on the heavy chain (CHn), or one on the light chain (CL). Furthermore, immunoglobulins can be subdivided into a fragment antigen-binding (Fab) and a fragment crystallizable (Fc) portion. The Fab domain consists of the VH and VL domains and the adjacent N-terminal constant CH1/CL domain. The Fc domain is built up of the remainder of the heavy chains. Immunoglobulins thus contain two Fab domains per Fc domain. Each immunoglobulin has its own specific heavy chains  which are joined by one or more disulfide bridges. The light chain can occur in two variants (λ and κ) that are shared by all immunoglobulins. Some immunoglobulins additionally contain a flexible hinge region between the CH1 and CH2 domains . The remaining immunoglobulins (IgE and IgM) have a rigid Ig domain instead of a hinge region. Immunoglobulin N-linked glycosylation occurs mostly on the heavy chains, accounting for between 2 and 14 % of the protein weight. However, the light chain can also contain N- and O-linked glycans [Immunoglobulins (Igs) are a major component of the adaptive immune system . There a glycans .O-glycosylated [Immunoglobulin alpha (IgA) is an antibody that exists in two subclasses (IgA1 and IgA2), and in both mono- and dimeric form. Compared to IgA2, IgA1 contains a 13 amino acid extended hinge region, which is heavily osylated , 357. Seosylated . Secretoosylated , 358. Seosylated . The preN-glycosylated at Asn144 (IgA1) and Asn131 (IgA2) in the CH2 domain as well as at Asn340 (IgA1) and Asn327 (IgA2) and in the tail piece domain [N-glycosylation sites at Asn47 of the CH1 domain and at Asn205 of the CH2 domain [IgA is e domain . IgA2 co2 domain . While gN-glycans present on IgA1 as detected by hydrophilic interaction liquid chromatography (HILIC)-HPLC with exoglycosidase digestion are diantennary disialylated , diantennary monosialylated  and fucosylated diantennary bisected disialylated  [The main 2, 14 %) . The amo2, 14 %) . A site-2, 14 %) . Further2, 14 %) .N-glycosylation of IgA in diseases is not well understood. The glycan might have an influence on the binding of IgA to the FcαR receptor, although this finding was not validated in a later study [The role of er study , 164. Bier study . In addier study .Immunoglobulin delta (IgD) has, compared to the other immunoglobulins, a rather long hinge region of 64 amino acids resulting in a total apparent mass of 175 kDa. The average concentration of IgD in plasma is 0.03 mg/mL, but it can range from <0.003 to 0.4 mg/mL without any clear sex or age dependence –361. ComN-glycosylation sites have been identified on the heavy chain of IgD, located at Asn225, Asn316 and Asn367, as well as seven O-glycosylation sites at Ser109, Ser110, Thr113, Thr126, Thr127, Thr131 and Thr132 [N-glycans revealed a mixture of high mannose and complex type glycosylation. The total pool contained 35 % core fucosylation, <1 % terminal N-acetylglucosamine, 33 % bisection, 20 % terminal galactosylation and 31.5 % monosialylation and 21.5 % disialylation . The most abundant glycoforms detected were Man8 (14.4 %), Man9 (13.5 %), FA2G2S2 (7.6 %), FA2G2S1 (7.3 %), FA2BG2S2 (6.5 %) and A2G2S1 (6.1 %). Interestingly, monoglucosylated Man8 and Man9  were observed as well [Three d Thr132 –168. HIL as well . The hig as well .Immunoglobulin epsilon (IgE) is a 188 kDa antibody lacking a hinge region, but that instead contains an extra C domain in its heavy chain . The proN-glycosylation sites, located at Asn21, Asn49, Asn99, Asn146, Asn252 and Asn275 [N-acetylglucosamine . In addition, 14.2 % of the glycan pool proved to be high-mannose type (mainly Man5) [Carbohydrates form 12 % of the IgE molecular mass, making it the most glycosylated antibody in plasma . The prod Asn275 . An addid Asn275 . HILIC-Hd Asn275 . These gly Man5) .By performing site-specific analysis by LC-MS/MS of tryptic glycopeptides, it was found that Asn21 mainly contains FA2G2S1 (30 %), FA2BG2S1 (30 %), FA2G2S2 (15 %) and FA2BG2S2 (10 %), while Asn49 is occupied by FA2G2S2 (30 %), FA2G2S1 (18 %), FA2BG2S2 (15 %), and FA2BG2S1 (15 %) . Asn99 cIn the same study, IgE from hyperimmune donors was seen to be similarly glycosylated as normal IgE, while IgE derived from monoclonal myelomas showed the loss of bisection and a drastic appearance of triantennary structures (up to 50 % per site) . There iL) as well as a constant (CL) domain. The heavy chain contains four domains with one domain which comprises the variable region (HL) followed by three constant domains: CH1, CH2 and CH3. Each light chain is paired with the HL and CH1 domain of the heavy chain to form a Fab portion, whilst CH2 and CH3 domains of the two heavy chains together form the Fc portion. Between the two Fab portions and the Fc portion a flexible hinge region is positioned, which makes it possible for the two Fab arms to move individually [Immunoglobulin gamma (IgG) is a glycoprotein with a total molecular mass of approximately 150 kDa , 177. Thvidually , 364. Thvidually , 370. Bavidually , 372. NoDuring a secondary immune response IgG is secreted in high amounts by B cells . In healThe majority of therapeutic antibodies is derived from IgG1, where the glycosylation plays an important role for their function , 377–380N-glycosylation site in the CH2 domain at Asn297. Notably, this site may have a different number for different IgG subclasses and variants [Glycosylation can occur on both the Fc and Fab portions of the IgG molecules . The Fc variants .N-glycosylation site may be found at Asn322 of IgG3 although no occupation has yet been described [N-glycosylated in 15–25 % of the cases [Another possible escribed . The Fabhe cases .i.e. FA2G1 (31 %), FA2G2 (23 %), FA2G2S1(6) (13 %), FA2 (10 %) and FA2BG1 (5 %) [The overall glycosylation of IgG has been the subject of many studies using a variety of different methods . In a reG1 (5 %) . Only a G1 (5 %) . These fG1 (5 %) . Next toG1 (5 %) . The Fc G1 (5 %) .O-glycosylation of IgG3 in the hinge region, revealing that the threonine sites (T) in the three repeated peptide sequences (CPRCPEPKSCDTPPP) are partially occupied with core 1-type O-glycans [Affinity capturing followed by LC-MS with CID and electron-transfer dissociation (ETD) fragmentation of tryptic IgG glycopeptides revealed that the various IgG subclasses are similarly glycosylated, but with some notable differences , 183. Ig-glycans .A vast body of literature exists describing disease-associated changes of IgG glycosylation, as well as the regulation and immunological effects of such glycosylation changes. In the following, only a very concise view of this field will be given, and we would like to refer the interested reader to more specialized reviews , 186.e.g. the lectin pathway of the complement system [ϒRIIIa receptor [ϒRIIIa receptor, as a consequence the activity of ADCC is decreased and anti-inflammatory effects are enhanced, although this only appears to be the case for α2-6-linked sialylation [IgG glycosylation has been strongly associated with age, with a negative correlation between age and galactosylation , 188. Igt system . Increast system –193. Sevreceptor , 195. Threceptor , 195. Thlylation , 197. Inlylation , 198–200lylation .Immunoglobulin mu (IgM) is a 970 kDa (in pentameric form) antibody, consisting of five 190 kDa subunits (of 452 amino acids) , 381. ThIgM antibodies are an early and main activator of the classical complement pathway , but alsN-glycosylation sites at Asn46, Asn209, Asn272, Asn279 and Asn439, of which Asn439 is only 17 % occupied [N-glycosylation to be mainly diantennary with core fucosylation, either with- or without bisecting N-acetylglucosamine , followed by the oligomannose compositions Man6 and Man5  [N-glycans [IgM contains occupied , 202. HIctively) , 203. In-glycans , 203.N-glycosylation of 24 major plasma glycoproteins. It has been shown for many of these proteins that glycosylation changes are implicated in serious pathological states such as cancers, autoimmune diseases and CDG and that their glycosylation pattern could be used as biomarkers, prognostic tools or even as anchor points for targeted treatments [N-glycome and allow the screening of large cohorts in reasonable times, thus enhancing the possibilities to search for putative biomarkers and predictions tools [Here we present an overview of the eatments , 389. Thns tools , 390–392The information contained in this review stems from many sources and methodologies. When exploring protein glycosylation to its full complexity, this comprises the occupancy per site and the relative abundances of glycoforms present per site, as well as information on linkages and isomer distribution. No single analysis method is capable of providing all this information in a comprehensive manner, let alone on a complex sample such as human plasma. Consequently, different levels of detail are available with respect to the glycosylation of the major glycoproteins that make up human plasma. Analysis methods encountered in this review are very diverse, including NMR, lectin capturing, LC-fluorescence, as well as several mass spectrometric methods , 393. NMN-glycome, the most abundant species for most sites having full coverage of all their antennae. Next to receptor interaction and charge induction, the terminal sialic acids are known to play a large role in determining the half-life of proteins, and less than full sialylation would lead to hepatic clearance via the asialoglycoprotein receptor [Well-studied proteins covered in this review include alpha-1-acid glycoprotein, alpha-1-antitrypsin, haptoglobin, serotransferrin, vitronectin, and IgG, while others such as histidine-rich glycoprotein and kininogen-1 still remain to be studied at the most basic level . Other candidates likely to contain the larger tri- and tetraantennary structures are kininogen-1 and histidine-rich glycoprotein , but this has not been confirmed yet, possibly due to the technical difficulty associated with the analysis of these glycosylations.Tri- and tetraantennary structures are found in lower abundance than the diantennary structures, and have some discerning features. Whereas diantennary glycans are mainly sialylated with an α2-6-linkage, for the triantennary species on average one in three sialic acids is α2-3-linked . FurtherN-glycome, a remarkable congruence is observed with a TPNG profile registered for N-glycans released from human plasma Below is the link to the electronic supplementary material.


The distribution of life in the continental subsurface is likely controlled by a range of physical and chemical factors. The fundamental requirements are for space to live, carbon for biomass and energy for metabolic activity. These are inter-related, such that adequate permeability is required to maintain a supply of nutrients, and facies interfaces invite colonization by juxtaposing porous habitats with nutrient-rich mudrocks. Viable communities extend to several kilometres depth, diminishing downwards with decreasing porosity. Carbon is contributed by recycling of organic matter originally fixed by photosynthesis, and chemoautotrophy using crustal carbon dioxide and methane. In the shallow crust, the recycled component predominates, as processed kerogen or hydrocarbons, but abiotic carbon sources may be significant in deeper, metamorphosed crust. Hydrogen to fuel chemosynthesis is available from radiolysis, mechanical deformation and mineral alteration. Activity in the subcontinental deep biosphere can be traced through the geological record back to the Precambrian. Before the colonization of the Earth's surface by land plants, a geologically recent event, subsurface life probably dominated the planet's biomass. In regions of thick ice sheets the base of the ice sheet, where liquid water is stable and a sediment layer is created by glacial erosion, can be regarded as a deep biosphere habitat. This environment may be rich in dissolved organic carbon and nutrients accumulated from dissolving ice, and from weathering of the bedrock and the sediment layer. Cores and water samples from beneath both the continents and the ocean floor yield microbial  populations, which microbiological and genetic studies show are distinct from those at the surface, and thus represent life from a separate habitat rather than a contamination ,2. The cediments . Despiteediments . This pa2.The fundamental requirements of life are liquid water, space in which to live, carbon and a suite of other nutrients to make and replenish biomass, energy for growth and maintenance, and protection from physical and chemical stressors . Whereas the inter-related requirements for space, energy and carbon are abundantly met by much of the Earth's surface, they can be severely limiting in the subsurface, with the result that biomass declines rapidly with depth in the uppermost kilometre . The varIn the continental subsurface, living space is provided by the porosity (void space) of rock, sediment and ice. In the basal layers of ice sheets several kilometres thick, living space is available in the thin veins of liquid water on ice-crystal boundaries . The uppIn rocks and sediments, the decay of porosity with increasing depth is on average approximately exponential, but varies strongly with the local rock or sediment type and geological history. Although clastic sedimentary rocks generally have higher primary porosities than crystalline rocks, poorly sorted or highly cemented sediments contain little porosity, whereas vesicular volcanic rocks are commonly highly porous. Igneous and metamorphic rocks also have higher compressive strengths and therefore retain their porosity at greater burial depths . Porosit2, methane, acetate and other molecules from which microbes can obtain carbon [2 and methane [2 also generate methane, notably the serpentinization of mafic and ultramafic igneous rocks, e.g. in ophiolites [Carbon, like nitrogen, phosphorus and a host of minor nutrients, is required for the construction and repair of microbial cells and spores. In the subsurface, carbon is contributed by the recycling of organic matter originally fixed by photosynthesis at the surface, and by crustal carbon dioxide and methane. In the shallow continental crust, the recycled component predominates, as kerogen (ancient organic matter), bitumen and dissolved organic carbon (DOC). Even deeply buried hydrocarbons continue to sustain life: onshore oil and gas reservoirs are commonly biodegraded and have yielded culturable anaerobic heterotrophs from several kilometres depth . Microorn carbon ,18. Howe methane ,20. Low-hiolites .Microbes obtain energy via redox reactions; in most metabolic systems, the transfer of electrons from reductants to oxidants is coupled to the shuttling of protons across a membrane, driving adenosine triphosphate synthesis. Although numerous redox couples are employed in the subsurface, many are in meagre supply and most yield little energy compared with photosynthesis and aerobic respiration. Energy limitation is consequently thought to be a widespread constraint on the productivity of the deep biosphere . It seem2, sulfate and nitrate, all of which fuel both heterotrophic and autotrophic metabolisms. Near the surface, molecular oxygen is rapidly consumed by the oxidation of organic matter, leaving anaerobic sulfate reduction, methanogenesis and acetogenesis to dominate in deeper environments. Electron acceptors indigenous to the subsurface include magmatic/hydrothermal CO2 and Mn(IV) and Fe(III) in minerals [2 as well as a range of electron donors  [2, CH4, −) derived from the mantle, from hydrothermal and metamorphic fluids, and from low-temperature water–rock reactions (notably serpentinization). Other mechanisms potentially capable of producing biologically significant concentrations of H2 in the subsurface include the radiolysis of pore water by radioactive decay, and ‘mechanoradical’ chemistry associated with earthquakes [Biologically important oxidants in the crust include atmospheric Ominerals ,23. The methane) ,18. Altethquakes ,25. DespMicrobes have also been shown to oxidize a range of divalent cations in rocks and minerals . The impThe requirements for carbon, energy and space are inter-related in this and numerous other ways. Burial depth strongly controls all three: as we have seen, carbon and energy are consumed in the shallow crust but potentially generated by other means at greater depths; likewise, the generation of secondary porosity by mineral dissolution may offset the decline in primary porosity caused by pressure compaction. Organic compounds provide chemical energy as well as carbon for biomass, and their supply and distribution, as well as those of other nutrients, depend on the amount and configuration of porosity. To allow water, cells and metabolites to circulate, pore spaces must be connected together laterally and vertically. The importance of adequate permeability is exemplified in a study by Hoehler & Jørgensen  which sh3.The deep biosphere represents a major component of life on Earth. It is, therefore, likely to have persisted through much of geological history, and indeed some workers propose that life had a thermophilic, subsurface origin. Several types of evidence support a long-lived deep biosphere, including carbonate concretions in mudrocks; sulfides precipitated in mudrocks, at mudrock–sandstone interfaces and at redox boundaries in sandstone aquifers; sulfides in serpentinites and vesicular basalts; bioalteration features in volcanic glass; and mineralized filaments in hydrothermal and other fracture systems. In several cases, this evidence extends back to a Precambrian record ,28,29. T4.4 per millilitre of water at 3.6 km depth [7 cells per millilitre in the ice and up to 1010 attached cells per cubic centimetre of silt [4 per millilitre under 300 m of ice [5 bacterial cells per millilitre [There is much evidence for microbial activity in subglacial settings ,36,37,38km depth . These mkm depth . Silty i of silt . These om of ice ; Skaftárllilitre . High cellilitre , New Zeallilitre , the Canllilitre , Svalbarllilitre , the Antllilitre  and the Most of this activity depends on resources derived from minerals and organic matter in sediments below the ice, including large sedimentary basins ,49. HighWhere ice has been progressively buried from the surface down to the base of an ice sheet, it carries components introduced at the surface. The environmental records contained within ice cores are evidence of this stored material. The entrained components become released on melting of the ice. These are dominated by wind-borne components. Dust  from abrasion of bedrock would in most cases be from sources within tens of kilometres. This could include nutrients depending on the nature of the bedrock, and also organic compounds . Finer a7 cells g−1 throughout may be reduced as more data become available, but the estimate for sub-ice life is high enough to suggest that during episodes of global glaciation, life in this setting could predominate if much of the subglacial environment remains unfrozen.An indication of the potential magnitude of the sub-ice and other polar biospheres is given by DOC levels in Antarctic media. DOC levels in Antarctic lakes, glacier ice and sea ice all range up to values exceeding 400 μM ,60. ThesThe special case of mechanical grinding of bedrock at the base of glaciers can accelerate the release of nutrients and energy sources, including iron, phosphorus and sulfides, through formation of fresh mineral surfaces with large surface area. Rock flour in particular presents very high surface area per unit mass. Subglacial microbial activity includes sulfide oxidation, Fe(II) oxidation, sulfate reduction, nitrate reduction, Fe(III) reduction, Mn(IV) reduction, aerobic respiration using oxygen from ice melt, and the release of carbon dioxide to weather silicates and carbonates ,35,49. SBelow both the West Antarctic Ice Sheet and Greenland Ice Sheet, there are proportionally large areas of high heat flow. This causes relatively high rates of basal ice melting ,75, whic5.The subsurface biosphere is, proportionally, a very significant component of life on Earth. Several factors contribute to this, including connected pore space down to several kilometres depth, widespread groundwater flow, heat-assisted mineral alteration which provides nutrients and energy, and a supply of carbon. The sub-ice biosphere takes advantage of high porosity and water flow, and the availability of nutrients/energy from fresh erosion products. Carbon is available from the activity of methanogens, methane released by crushing of bedrock and the direct access to sedimentary organic matter including DOC from melting ice. The crushing of bedrock should additionally yield hydrogen to allow the possibility of subglacial chemosynthesis. Future exploration should take us from speculation to quantification of these subsurface processes.