Aerobic exercise capacity averaged at 32 ± 21 Watt (range 4–76 Watt) representing 29.8% of the maximum exercise intensity reached. PG before and after CPX was 9.3 ± 2.2 and 7.6 ± 1.7 mmol/L, respectively (p < 0.0001). PG was further decreased significantly at one and two h after exercise to 7.5 ± 1.6 mmol/L and 6.0 ± 1.0 mmol/L, respectively (p < 0.0001 for both as compared to PG before CPX). (4) Conclusions: Aerobic exercise capacity is very low in patients with CAD and T2DM. Exercise at aerobic intensity allowed for significant reduction of plasma glucose. Individual and effective aerobic exercise prescription is possible by CPX.(1) Background: Physical activity is recommended in patients with type 2 diabetes mellitus (T2DM) and coronary artery disease (CAD) to reduce hyperglycemia and cardiovascular risk. Effective aerobic exercise intensity, however, is not well defined. (2) Methods: 60 consecutive patients performed cardiopulmonary exercise testing (CPX) of 30 min duration targeting a respiratory exchange ratio (RER) between 0.85 and 0.95, being strictly aerobic. Plasma glucose (PG) was measured before and after CPX as well as one and two h after exercise. Maximum exercise intensity was evaluated using a standard bicycle exercise test. (3) Results: 50 patients completed the protocol  30.5 ± 4.9 kg/m According to guidelines and position statements, patients with type 2 diabetes mellitus (T2DM) should be advised to be physically active in order to decrease both hyperglycemia and their cardiovascular risk ,2,3. In In a pilot study, 10 patients with T2DM and concomitant CAD exhibited a very low aerobic exercise capacity of 29 ± 9 Watt (range between 10 and 45 Watt) as measured by cardiopulmonary exercise testing (CPX) . PhysicaPatients referred to CR after a recent acute coronary syndrome (ACS) or with stable CAD were included. In all patients, CAD was diagnosed by coronary angiography and T2DM was diagnosed using standard criteria . During 2, CO2 analysers were calibrated before each exercise session. Gases were analysed breath by breath . After adaptation to the mask, parameters at rest were determined followed by a period of two min of unloaded cycling  using the same stationary cycle ergometer. Workload was increased in two min stages and standard criteria for the cessation of the exercise test were used. At the beginning of CR, left ventricular ejection fraction (LVEF) [%] was measured by echocardiography using standard methods.p values. The relationship between aerobic exercise capacity [Watt], maximum exercise intensity [Watt] and left ventricular ejection fraction [%] was analyzed using Spearman correlations with two-sided p values and described by linear regression models. To account for the multiple testing of four hypotheses, the global significance level of 0.05 was corrected, so that p values < 0.05/5 are considered statistically significant. Subgroup analysis was not performed due to small numbers.Data in the entire group as well as in relevant subgroups are given as described by mean values ± standard deviations as well as median and range. Comparisons between plasma glucose values before and after exercise, after one and after two h were made using Wilcoxon tests reporting two-sided n = 4) and hypertension (n = 3). In two patients, it was not possible to hold RER constant between 0.85 and 0.95 and one patient became anaerobic with an RER > 1.00 despite maximum reduction of exercise intensity. These patients were excluded from statistical analysis. The study protocol could be completed in 50 patients, 10 female, 40 male (Inclusion criteria were met by 60 consecutive patients. CPX had to be discontinued prematurely in 10 patients (16.7%) due to hyperventilation  and GLP1 agonists (n = 3), each in combination with metformin). No patient was medicated with sulfonylurea. 14 out of 15 patients with newly diagnosed T2DM wanted to start with lifestyle interventions before beginning a pharmacological treatment.Primary cardiac diagnosis was ACS in 40 out of 50 patients (80%) and 10 patients exhibited stable CAD. Coronary intervention with stent (PCI) was performed in 45 of 50 patients (90%). All patients were medicated with platelet inhibition and five patients had additional oral anticoagulation. Patients received statins (86%), ezetimibe (13%), RAAS-Inhibition (92%) and beta blockers (86%), respectively. In 35 patients (70%) T2DM was known between 2 and 20 years (median 5 years) before participation in CR. In 15 patients T2DM was newly diagnosed during CR. Patients with known T2DM had pharmacological treatment, mostly by metformin (94%). Dual combination of anti-diabetic drugs was given to 28% of these patients and 10% received three antihyperglycemic drugs (SGLT2 inhibitors (n = 25), muscular pain (n = 1), burning feet (n = 1) and headache without hypertension (n = 1). Aerobic exercise capacity averaged at 31.8 ± 20.9 Watt (range 4–76 Watt). Further exercise parameters obtained during CPX are displayed in p = 0.71) but with maximum exercise intensity (Watt) , x = maximum exercise intensity (Watt)).Aerobic exercise (CPX) could be performed for 30 min duration without any problems in 22 of 50 patients (44%). In 28 patients (56%), CPX had to be terminated early due to muscular fatigue (In 49 out of 50 patients information about smoking could be retrieved retrospectively from our archives. Only eight patients (16%) had no history of smoking. In 41 patients (84%), smoking burden ranged between 2.5 and 50 packyears (median 30 packyears). 21 out of 41 patients (51%) stopped smoking on the occasion of this cardiac event, and 19 patients (46%) had stopped smoking earlier, between 1.5 and 40 years ago (median 13 years). Only one patient kept smoking. Aerobic exercise capacity was not influenced by any of these variables.2), larger waist (123 ± 15 vs. 110.4 ± 10 cm) and a higher HbA1c (7.2 ± 0.8 vs. 6.8 ± 0.7%). In addition, they exhibited a lower aerobic exercise capacity (22 ± 16 vs. 34 ± 21 Watt) and a shorter aerobic exercise duration (22.7 ± 6 vs. 26.7 ± 4 min).Plasma glucose values (PG) are displayed in Despite a IA  recommendation in all guidelines and position statements ,2,3, impEspecially, the request to exercise at greater intensities ,4,5 mighIn this study, the individual aerobic exercise intensity was evaluated by CPX in a larger cohort of 50 consecutive patients with T2DM and CAD. Average aerobic exercise capacity was very low at 32 ± 21 Watt  was similar to the patients studied here  in the study of Jarvie et al. [2peak of 18.8 ± 5.0 mL/kg/min [2peak was even further decreased in our earlier cohort, to an average of 15.9 ± 2.8 mL/min/kg [There is one study evaluating a similar cohort of patients with T2DM and prevalent CAD or at high risk for cardiovascular disease . This coied here  and in oL/kg/min , VO2peakL/min/kg . These dL/min/kg  or at hi2) with normal glucose metabolism (46%), impaired glucose tolerance (22%) and without insulin dependent diabetes mellitus (32%), the estimated energy expenditure (EEE) was assessed by structured interviews, and insulin sensitivity was measured [In 1467 participants  and high exercise intensity  were compared in a randomized trial [max), and high intensity exercise was performed at 90% of HRmax. After 16 weeks of training, fasting PG was significantly reduced by high exercise intensity  whereas moderate intensity did not reduce fasting PG (6.1 ± 0.5 vs. 6.5 ± 0.6). A positive two-h post load PG ≥ 11.1 mmol/L, however, was reduced from 7 out of 8 (87.5%) to 3 of 8 patients (37.5%) by moderate exercise intensity as compared with 8 of 11 (72.7%) to 4 of 11 patients (36.4%) by high intensity exercise training [2peak 34 to 36 mL/min/kg) as compared to the patients in our study (VO2peak 15.9 mL/min/kg), an absolute reduction of the fasting PG of only 0.03 mmol/L and a lower normalization rate of the two-h post load PG by high intensity (36% points), as compared to moderate intensity exercise training (50% points), does not justify the general recommendation of greater exercise intensities to yield greater benefits in HbA1c in patients with T2DM.In 19 patients with metabolic syndrome and T2DM, moderate exercise intensity  to a larger extent than did exercise volume . However, only one study included an unequivocally vigorous exercise intervention at 75% VO2peak. Most of the studies exercised the patients between 50 and 65% of VO2peak which is within the range detected as optimal in our study  is within the range of other studies [In 17% of patients, the method used was not applicable. Hence, CPX is not suitable for detecting the individual aerobic exercise intensity in all patients and measuring lactate might be an alternative in these individuals. We did not use the Borg scale to measure perceived exertion. Thus, our results cannot be compared to other studies using this valuable tool. Even though the number of patients studied ( studies ,21, it sOptimal exercise intensity for regular physical activity is not well defined in patients with T2DM. Effective intensity might even be different according to whether exercise is prescribed as an antihypertensive agent, to control hyperglycemia, to reduce body-weight or to increase physical fitness. In addition, exercise intensity should be tailored to the individual patient as high-intensity physical activity is able to prevent metabolic syndrome in healthy elderly people  and to r


Elevated plasma triglycerides are a risk factor for coronary artery disease, which is the leading cause of death worldwide. Lipoprotein lipase (LPL) reduces triglycerides in the blood by hydrolyzing them from triglyceride-rich lipoproteins to release free fatty acids. LPL activity is regulated in a nutritionally responsive manner by macromolecular inhibitors including angiopoietin-like proteins 3 and 4 (ANGPTL3 and ANGPTL4). However, the mechanism by which ANGPTL3 inhibits LPL is unclear, in part due to challenges in obtaining pure protein for study. We used a new purification protocol for the N-terminal domain of ANGPTL3, removing a DNA contaminant, and found DNA-free ANGPTL3 showed enhanced inhibition of LPL. Structural analysis showed that ANGPTL3 formed elongated, flexible trimers and hexamers that did not interconvert. ANGPTL4 formed only elongated flexible trimers. We compared the inhibition of ANGPTL3 and ANGPTL4 using human very-low-density lipoproteins as a substrate and found both were noncompetitive inhibitors. The inhibition constants for the trimeric ANGPTL3 (7.5 ± 0.7 nM) and ANGPTL4 (3.6 ± 1.0 nM) were only 2-fold different. Heparin has previously been reported to interfere with ANGPTL3 binding to LPL, so we questioned if the negatively charged heparin was acting in a similar fashion to the DNA contaminant. We found that ANGPTL3 inhibition is abolished by binding to low-molecular-weight heparin, whereas ANGPTL4 inhibition is not. Our data show new similarities and differences in how ANGPTL3 and ANGPTL4 regulate LPL and opens new avenues of investigating the effect of heparin on LPL inhibition by ANGPTL3. LPL activity is differentially regulated in adipose the body . Specifithe body , 3. Convthe body . Each prthe body . Upon fathe body . ANGPTL8the body ; it mustthe body , 10. Recthe body .Genome-wide association studies in humans have identified robust links between lipid profiles and variants in ANGPTL3, ANGPTL4, and ANGPTL8. Mutations resulting in the loss of ANGPTL3 function are associated with lower plasma triglycerides and protection from coronary artery disease (CAD) , 13, 14.in vitro . Using small-angle X-ray scattering (SAXS) we found both ANGPTL3 and ANGPTL4 adopt elongated and flexible structures in solution. We assessed the mechanism of LPL inhibition by both ANGPTL3 and ANGPTL4 on human very-low-density lipoprotein (VLDL) using an enzyme-coupled reaction to report on real-time liberation of free fatty acids. This revealed both ANGPTL3 and ANGPTL4 are noncompetitive inhibitors. We also investigated the different effects heparin has on ANGPTL3 and ANGPTL4 inhibition. These data provide new insight into how these two inhibitors influence LPL activity.in vitro studies, ANGPTL3 was shown to be a less potent inhibitor of LPL than ANGPTL4 (Escherichia coli and purified using nickel chromatography (A) copurified with a significant nucleic acid contamination, as observed by absorbance at 254 nm (B). The contaminating nucleic acid was sensitive to DNase treatment, but resistant to RNase treatment, indicating that ANGPTL3 was bound to DNA (C). We also observed two peaks in the included volume of the SEC that contained ANGPTL3 (B). We pooled and concentrated the ANGPTL3 from each peak separately and assessed in vitro LPL inhibition using human VLDL as a substrate. These experiments showed that ANGPTL3 peak 1 had a significantly lower LPL inhibition activity than peak 2 (D). In order to remove the contaminating DNA from the ANGPTL3 preparation, we added an additional purification step, MonoQ anion-exchange chromatography, which led to a significant reduction in DNA and cleaner ANGPTL3 as observed from the 254 nm trace from the subsequent SEC (E). Of interest, DNA-free ANGPTL3 still eluted in two distinct peaks from the SEC after this additional anion-exchange step. Both peak 1 and peak 2 contained >95% pure ANGPTL3 (F). Next, ANGPTL3 from peaks 1 and 2 were tested for inhibition of LPL acting on VLDL. These assays showed that protein from both peaks were now equally as effective at inhibiting LPL (G). Thus, removal of DNA from ANGPTL3 increased its potency as an LPL inhibitor.In previous  ANGPTL4 , 25, 26.tography , 25. We tography . Unexpectography A copurift 254 nm B. The cod to DNA C. We als ANGPTL3 B. We poon peak 2 D. In orduent SEC E. Of int ANGPTL3 F. Next, ting LPL G. Thus, A). The second peak had a molecular weight of 76 kDa, which corresponds to an ANGPTL3 trimer . It is intriguing that we also found that the ANGPTL3 hexamers and trimers do not interconvert. When we reinjected each separately concentrated peak onto SEC, we observed only a single peak on the second SEC run (A and B), indicating that ANGPLT3 hexamers do not break down into trimers and trimers do not assemble into hexamers. It should also be noted that N-terminal ANGPTL3 does not have any cysteine residues, which indicates disulfides cannot account for the oligomerization. To further investigate the oligomeric state, we subjected ANGPTL3 to a denaturing purification and then refolded the protein and compared the elution from SEC with that of natively purified ANGPTL3. This revealed that following refolding ANGPTL3 forms trimers .We also performed SEC-MALS on N-terminal ANGPTL4 and found ANGPTL4 elutes in a single peak with a molecular weight of 46 kDa, which corresponds to a trimer of ANGPTL4  at a fixed amount of VLDL triglycerides (A–C). This revealed that, when assayed on 10 nM LPL, the ANGPTL3 trimer (5.8 ± 0.2 nM) and ANGPTL4 trimer (4.8 ± 0.2 nM) share similar inhibition abilities, whereas the ANGPTL3 hexamer is a less potent inhibitor (14.4 ± 0.4 nM).We next analyzed ANGPTL3 and ANGPTL4 inhibition of LPL acting on one of its physiological substrates, VLDL. To do so, we adapted a previously published  method tycerides , A–C. Th50 data to guide design of assays varying both the inhibitor and VLDL concentration to collect a series of Michaelis–Menten curves for each inhibitor. We individually fit each Michaelis–Menten curve (D–F) and further graphed the data by creating reciprocal plots, slope replots, and fractional velocity plots . Both ANGPTL3 trimer (7.5 ± 0.7 nM) and ANGPTL4 (3.6 ± 1.0 nM) Ki’s were more potent; however, they differed more than as calculated with the IC50. ANGPTL4 was twice as potent as the ANGPTL3 trimer, although both are effective inhibitors.We used the ICen curve , D–F andty plots . This alty plots , 35. Folrac-glycero-3-glutaric acid 6′-methylresorufin ester (DGGR) as an LPL substrate demonstrated that ANGPTL3 inhibition of LPL is blocked by the presence of heparin, whereas ANGPTL4 inhibition is not (A and B). By contrast, ANGPTL4 showed no impairment of inhibition from 6-mer or 12-mer heparin (C and D). ANGPTL3 activity was also tested in the presence of mixed-molecular-weight heparin, and it significantly decreased ANGPTL3 inhibition similar to the 12-mer heparin . ANGPTL4 has previously been shown to bind unfractionated heparin .We hypothesized that the lack of ANGPTL3 inhibition in the presence of 12-mer was the result of heparin binding to ANGPTL3 and blocking inhibition by occluding ANGPTL3’s LPL-binding interface, rather than blocking the ANGPTL3-binding interface on LPL, as had previously been hypothesized . To confFig. 5 heparin , and herOur comparison of ANGPTL3 with ANGPTL4 has clarified many puzzling aspects of ANGPTL3 and ANGPTL4 inhibition of LPL. Our discovery that ANGPTL3 copurifies with a DNA contamination could explain the differences in the reported potency of ANGPTL3 , 25, 26.in vivo ANGPTL3/ANGPTL8 complexes that were identified by mass spectrometry to exist at a 3:1 ratio  used to determine the type of inhibition. Previously, we have shown that ANGPTL4 is a noncompetitive inhibitor using DGGR as a substrate . Fluoresmax with increasing inhibitor concentrations and no change in Km with inhibitor concentration . No. Nomax wA and B). We also found that ANGPTL4 bound both 6-mer and 12-mer heparin, but neither affected its inhibition of LPL (C and D). In the future, it would be interesting to explore if the presence of GPIHBP1 bound to LPL can also affect inhibitor binding in the presence of heparin. ANGPTL3’s loss of activity upon binding to heparin opens up new possible ways to block ANGPTL3 activity in vivo.A previously known difference between ANGPTL3 and ANGPTL4 is their interaction with heparin. N-terminal ANGPTL3 possesses a heparin-binding site adjacent to its LPL inhibitory domain, whereas N-terminal ANGPTL4 binds heparin at an unidentified site , 37. It n of LPL , C and DThis work presents new insights into the structure and LPL inhibition of ANGPTL3 and ANGPTL4. It is clear that ANGPTL3 and ANGPTL4 share many global similarities, including elongated structures, trimeric oligomers, and noncompetitive inhibition. We have also identified an important difference, which is that the binding of ANGPTL3 to 12-mer heparin can block LPL inhibition. This knowledge will help in the development of effective therapeutics to enhance LPL activity.The N-terminal domain of human ANGPTL3 (UniProt accession ID Q9Y5C1), encompassing residues S17 to R224, was cloned C terminally to a hexa-histidine tag and a TEV cleavage site. The resulting molecular weight of the purified protein was 26.8 kDa. The sequence was placed into a pETDuET vector (Novagen) at the multiple cloning site 1 (MCS1) (pETDuET-ANGPTL3). For human tissue culture expression, ANGPTL3 1 to 224 with a C-terminal hexa-histidine tag was placed in the pCDNA5-FRT-MCS expression vector.g for 30 min at 4 °C. Cells were resuspended in Binding Buffer  on ice and flash frozen. Cells were thawed, and all subsequent steps were performed at 4 °C. Cells were lysed using an Emulsiflex C3 high-pressure homogenizer; then the lysate was clarified by centrifugation at 27,000g for 70 min. The supernatant was filtered with a 0.2-μm filter and applied to a Ni-NTA resin column equilibrated in Binding Buffer. Following binding, the column was washed in Binding Buffer followed by Wash Buffer 1 , and Wash Buffer 2 . Protein was eluted using Elution Buffer . Fractions containing ANGPTL3 were pooled and dialyzed overnight into either MonoQ Buffer A  or SEC Buffer . For purifications using anion-exchange MonoQ chromatography, samples were loaded onto a MonoQ column using an AKTA explorer system. Protein was eluted via a linear gradient using MonoQ Buffer B . Fractions containing ANGPTL3 were pooled and dialyzed overnight into SEC Buffer. SEC was performed using either a Superdex S-200 or Sephacryl S-300 column. The S-300 column provided more separation between the excluded volume and the first peak of ANGPTL3, although ANGPTL3 peak 1 and peak 2 were in the included volume of both columns. ANGPTL3 peak 1 and peak 2 were pooled separately and concentrated using 10 kDa cutoff Millipore filters. The final protein concentrations were determined by bicinchoninic acid assay, and DNA contamination was assessed by the 260:280 nm ratio with a Thermo Nanodrop.pETDuET-ANGPTL3 was transformed into Bl21 (DE3) RIPL cells and grown in LB at 37 °C to an optical density of 0.6 at 600 nm. Protein expression was induced using 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and the cells were grown for 3 h at 37 °C before harvesting by centrifugation at 6700g for 70 min at 22 °C. The supernatant was filtered with a 0.2-μm filter and applied to a Ni-NTA resin column equilibrated in Denaturing Binding Buffer . A gradient mixer was used to rinse the column in a linear gradient from 100% Denaturing Binding Buffer to 100% Binding Buffer to refold the protein. The refolded ANGPTL3 was then eluted from the column as detailed above and assessed by SEC using a Superdex S-200 Increase column.For the ANGPTL3 denaturing purification, cells were grown as above and lysed by incubation for 1 h at 22 °C in 6 M guanidine hydrochloride (GuHCl). The lysate was clarified by centrifugation at 27,000E. coli expression.ANGPTL3 was stably integrated into Flp-In T-REx-293 human embryonic kidney cells (HEK-293) using the ANGPTL3 pCDNA5-FRT-MCS vector. Cells were maintained in Gibco DMEM with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% L-glutamine. Expression of ANGPTL3 was induced by the addition of expression media comprising Gibco Dulbecco's modified Eagle's medium with 1% FBS, 1% penicillin/streptomycin, 1% L-glutamine, and 2 μg/ml tetracycline. The expression medium was collected every 24 h and replaced with new expression medium for 5 days. The medium was flash frozen and stored at −80 °C. For purification, the medium was thawed, pooled, filtered, and bound to Ni Sepharose Excel beads at 4 °C. ANGPTL3 was then purified as detailed for N-terminal ANGPTL4 (UniProt accession ID Q9BY76) was cloned and purified as reported , 38. BovSEC-MALS was performed using a 24-ml Sephadex S-200 column connected to a Wyatt DAWN HELEOS II light scattering instrument interfaced to an Agilent 1260 Infinity FPLC System, Wyatt T-rEX refractometer, and Wyatt dynamic light scattering module. For both ANGPTL3 and ANGPTL4 the S-200 column was equilibrated with buffer  prior to injecting 90 μl of sample on to the column at 0.5 ml/min. SEC-MALS experiments were performed twice and calibration was confirmed with a bovine serum albumin (BSA) standard. Data were analyzed using the Wyatt ASTRA software package.−1. Each exposure frame was 3 s. SEC-SAXS curves were initially analyzed by the SIBYLS beamline staff using ScÅtter   in ChicaAW 1.4.0 . Buffer The ATSAS software package was used for SAXS data analysis . PRIMUS 2HPO4, 0.36 mM KH2PO4) and 2% FBS. Human VLDL  was used as a natural substrate, and the liberation of free fatty acid was detected using an enzyme-coupled reaction combined with a fluorescent reporter, Amplex UltraRed (amplex) .Activity assays were performed using a final concentration of 10 nM bovine LPL. LPL was incubated with ANGPTL3 or ANGPTL4 for 10 min at 22 °C in phosphate buffered saline (PBS) with FBS prior to adding substrate for a final concentration in the well of 0.2× PBS , 0.1 U/ml Acetyl-CoA synthetase (ACS), 6 U/ml horseradish peroxidase, 5 U/ml Acetyl-CoA oxidase (ACO), 0.2 mg/ml fatty acid–free BSA, 0.05 mM amplex, and 200 μg/ml triglycerides in human VLDL. For Michaelis–Menten curves, the amount of VLDL was varied by serial dilution. For assays including heparin, 2 nM 6-mer, 12-mer, or mixed-molecular-weight heparin was incubated with the LPL/ANGPTL mix for 10 min. Assays were conducted in a black-walled, 96-well plate. Immediately following the addition of the substrate, fluorescence was monitored using a M5 Spectramax plate reader at 37 °C, excitation at 529 nm and emission at 600 nm, with a 590-nm cutoff filter. The initial rate was determined from the first 180 s of the reaction. Three biological replicates were conducted for each assay. Data were graphed in KaleidaGraph, and significance was assessed using a two-tailed Student’s t test. Individual Michaelis–Menten curves were fit in KaleidaGraph. Global noncompetitive fitting was performed in Mathematica.The enzyme-coupled reaction is based on a colorimetric nonesterified fatty acid (NEFA) assay that we have previously used . The comet al. , and we t test.Activity assays were performed using a final concentration of 2.5 nM bLPL diluted in LPL assay buffer, which has a final concentration of 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.2% fatty acid–free BSA, and 1 mM sodium deoxycholate. ANGPTL3 or ANGPTL4 were diluted to the necessary concentrations using SEC Buffer. LPL was mixed with ANGPTL and incubated in a black-walled, 96-well plate at 22 °C for 10 min. Fluorescent substrate 1,2-di-O-lauryl-rac-glycero-3-glutaric acid-(6’-methylresofurin) ester (DGGR) (Sigma) in 0.01% Anzergent 3 to 16 (Anatrace) was added immediately before assaying activity at 37 °C as described . Data weHeparin 6-mer and 12-mer were synthesized according to a published chemoenzymatic method . More inFirst, 5 pmol of each protein (furin-resistant hLPL or ANGPTL3 hexamer or ANGPTL4) was incubated with 275 pmol of each biotinylated heparin (6-mer or 12-mer) in pull-down buffer . As a negative control, each protein was mixed with an equal volume of pull-down buffer instead of heparin. The samples were rotated at 4 °C for 30 min. Each sample was mixed with cleaned Streptavidin M-280 Dynabeads (Invitrogen). Samples with beads were rotated at 4 °C for 30 min. The beads were pulled down using a magnet, and the supernatant was removed by pipet. The beads were then washed with pull-down buffer three times. After removing the final supernatant, the beads were resuspended in 10% SDS to denature the protein and heated to 95 °C. The reactions were analyzed using Western blot. Proteins were separated by 14% SDS-PAGE, transferred to 0.22-μm polyvinylidene difluoride membrane, and blocked with 5% non-fat milk in TBS-T . LPL and ANGPTL3 were probed with a mouse anti-histidine tag antibody (Bio-Rad) using a 1:5000 dilution, and protein was detected with a horseradish peroxidase–conjugated goat anti-mouse antibody (Southern Biotech) using a 1:5000 dilution. ANGPTL4 was probed with a polyclonal rabbit anti-ANGPTL4 antibody (BioVendor) using a 1:5000 dilution and detected with a horseradish peroxidase–conjugated donkey anti-rabbit antibody (Southern Biotech) using a 1:5000 dilution. Western blots were developed as previously described .Data for SAXS structural studies were deposited in the SASBDB under ascension codes SASBJK8, SASDJL8, and SASDJM8.Human ANGPTL3 - UniProt accession ID Q9Y5C1Human ANGPTL4 - UniProt accession ID Q9BY76Bovine LPL - UniProt accession ID P11151Human LPL - UniProt accession ID P06858The authors declare that they have no conflicts of interest with the contents of this article.


Klebsiella pneumoniae ST258 (CRKP-ST258) are a global concern due to their rapid dissemination, high lethality, antibiotic resistance and resistance to components of the immune response, such as neutrophils. Neutrophils are major host mediators, able to kill well-studied and antibiotic-sensitive laboratory reference strains of K. pneumoniae. However, CRKP-ST258 are able to evade neutrophil phagocytic killing, persisting longer in the host despite robust neutrophil recruitment. Here, we show that neutrophils are unable to clear a CRKP-ST258 isolate (KP35). Compared to the response elicited by a prototypic K. pneumoniae ATCC 43816 (KPPR1), the neutrophil intracellular response against KP35 is characterized by equivalent production of reactive oxygen species (ROS) and myeloperoxidase content, but impaired phagosomal acidification. Our results ruled out that this phenomenon is due to a phagocytosis defect, as we observed similar efficiency of phagocytosis by neutrophils infected with KP35 or KPPR1. Genomic analysis of the cps loci of KPPR1 and KP35 suggest that the capsule composition of KP35 explain the high phagocytosis efficiency by neutrophils. Consistent with other reports, we show that KP35 did not induce DNA release by neutrophils and KPPR1 only induced it at 3 h, when most of the bacteria have already been cleared. l-arginine metabolism has been identified as an important modulator of the host immune response and positively regulate T cells, macrophages and neutrophils in response to microbes. Our data show that l-arginine supplementation improved phagosome acidification, increased ROS production and enhanced nitric oxide consumption by neutrophils in response to KP35. The enhanced intracellular response observed after l-arginine supplementation ultimately improved KP35 clearance in vitro. KP35 was able to dysregulate the intracellular microbicidal machinery of neutrophils to survive in the intracellular environment. This process, however, can be reversed after l-arginine supplementation.Carbapenem-resistant  Klebsiella pneumoniae (KP) is an extremely versatile bacterium able to colonize different environments including different kinds of soil, water courses, medical devices and mucosal surfaces  locus  locus ; and a ds) locus . These tl-arginine through the activity of Arginase-1 and iNOS  , we charand iNOS . The rolocytosis , anotherumigatus .l-arginine.In the present report we provide evidence that KP35, a representative clinical isolate of CRKP-ST258, evade the neutrophil intracellular response by preventing the normal activity of the intracellular machinery after phagocytosis, a process that can be reversed through the supplementation of All experimental protocols were reviewed and approved by the Scientific Ethical Committee for Animal and Environment Care  of the Pontificia Universidad Católica de Chile (protocol numbers 150721004 and 150721005).2+ and Mg2+ supplemented with 0.1% gelatin (Sigma- Aldrich G7765). To evaluate bacterial killing, neutrophils (1 × 105 cells/200 µl) were incubated with opsonized KPPR1 or KP35 (MOI = 1) for 120 min with slow rotation at 37°C. After 120 min, each tube was placed at 4°C to stop the reaction and the total viable cells were counted in a conventional hemocytometer with trypan-blue exclusion. Cells were lysed with 1% saponin (Sigma-Aldrich 47036) and bacterial viability was determined by serial dilutions.Bone marrow (BM) from femurs and tibias of C57BL6/J mice were flushed with RPMI 1640 medium supplemented with 10% FBS and EDTA 2 mM . Erythrocytes were osmotically lysed using a solution of NaCl 0.2% and NaCl 1.6%. Once BM cells were isolated, neutrophils were purified by magnetic negative neutrophil isolation kit for mouse (MACS Miltenyi Biotec) according to manufacturer instructions. Next, neutrophils were counted, and resuspended in HBSS containing Ca5 cells/200 µl) were infected with opsonized KPPR1 and KP35 (MOI = 1) for 10 or 30 min with slow rotation at 37°C. After 10 or 30 min each tube was placed at 4°C. Then, to separate extracellular and intracellular bacteria, we followed a two-step colony forming assay  for 30 min on ice. Then, neutrophils (1 × 105 cells/200 µl) were infected with FITC labeled KPPR1 or KP35 (MOIs = 10 and 50) in rotation at 37°C, 5% CO2 for 10 min. Samples were fixed with 2% PFA and analyzed in a BD FACSCanto II at Columbia University Medical Center. Trypan blue 0.4% was added to each sample to quench extracellular FITC fluorescence.KPPR1 and KP35 were stained with FITC (0.25 mg/ml) in staining buffer (50 mM NaHCO5 isolated BM neutrophils were resuspended in HBSS Ca2+ Mg2+ with 0.1% gelatin and infected with KP35 or KPPR1 (MOI = 10) for 30 min in slow rotation at 37°C, 5% CO2. At 30 min, neutrophils were centrifuged at 1,400 rpm for 7 min and fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH = 7.2). Once fixed, samples were prepared and analyzed in a Philips Tecnai 12 (Biotwin) Transmission microscope with the help of the Advanced Microscopy Unit of Pontificia Universidad Católica of Chile.5 × 10cps locus from KP35  were obtained from GenBank. KP35 contigs were ordered in Mauve v.2015026 build 10 (https://rast.nmpdr.org/) (cps genes were determined using Kaptive (https://kaptive-web.erc.monash.edu/) (The genomes of KP NJST258_1 and KPPR1 (Accession numbers: NZ_CP006923.1 and NZ_CP009208.1), and the contigs containing the build 10  and annodr.org/) . The capsh.edu/) . Finallysh.edu/)  using BL5/300 µl) were stimulated with KPPR1 and KP35  for 10 and 180 min at 37°C with 5% CO2. Then, culture supernatant was collected, and extracellular DNA was measured using Quant-iT PicoGreen dsDNA (Invitrogen), following the manufacturer’s instructions.BM neutrophils (1 × 102+ Mg2+ and then separated in three different groups: neutrophils incubated with LysoTracker Red DND-99 (50 nM) (Invitrogen L7528); neutrophils incubated with LysoTracker Red DND-99, with the acidification inhibitor chloroquine diphosphate (100 µM) (Sigma-Aldrich C6628); and neutrophils alone. Cells were incubated for 30 min at 37°C, 5% CO2. After incubation, 1 × 105 Neutrophils were infected with KP35 or KPPR1 (MOI = 10) in a final volume of 200 µl for 60 min at slow rotation at 37°C, 5% CO2. After the time of infection, neutrophils were fixed with 500 µl of 2% paraformaldehyde at room temperature, in darkness, for 15 min. After fixation, cells were washed twice with sterile 1× PBS and acidification fluorescence was read in the flow cytometer in the PE channel . Analyses were performed in a BD LSR FORTESSA X-20 at the Pontificia Universidad Católica de Chile. All data were analyzed using FlowJo vX.0.7BM neutrophils were resuspended in HBSS Cal-arginine (Sigma-Aldrich W381918) for 30 min prior KP infection. KP35 killing and phagosome acidification were evaluated as described above. To evaluate a possible impact on KP35 growth in presence of l-arginine, KP35 was growth in a nutrient rich medium (LB) and a nutrient poor medium (HBSS) non-supplemented and supplemented with l-arginine (100 mg/L and 200 mg/L), and OD600 was measured every 20 min during 160 min.Murine neutrophils were supplemented with 200 mg/L of 5 cells) were incubated in 96-well black plate treated with 0.01% poly-l-lysine (Sigma Aldrich P4707) in presence of the general oxidative stress indicator Carboxy-H2DCFDA (Thermo Fisher C400), 10 µM, for 60 min at 37°C. Then, neutrophils were washed to remove excess probe and infected with opsonized KPPR1, KP35 and Staphylococcus aureus USA300 (positive control) (MOI = 1) and centrifuged at 524g for 8 min at 4°C to synchronize phagocytosis  were incubated in 96-well black plate treated with 0.01% poly-l-lysine (Sigma Aldrich P4707) in presence of the NO substrate DAF-FM Diacetate (Thermo Fisher D23844), 10 µM, for 60 min at 37°C. Next, neutrophils were washed to remove excess probe and incubated for 30 min. Then, cells were infected with opsonized KPPR1, KP35 and S. aureus USA300 (MOI = 1) and centrifuged at 524 g for 8 min at 4°C to synchronize phagocytosis (BM Neutrophils (1 × 10ocytosis . After c5 isolated BM neutrophils were resuspended in HBSS Ca2+ Mg2+ with 0.1% gelatin and infected with KP35 or KPPR1 (MOI = 1) for 30 min in slow rotation at 37°C. Cell culture supernatant was then collected after spinning at 1200 rpm for 10 min. The myeloperoxidase content was then measured using the Neutrophil Myeloperoxidase Activity Assay Kit  according to the manufacturer’s instructions.5 × 10Supernatants from uninfected neutrophils, neutrophils infected with KPPR1 and neutrophils infected with KP35 were collected after 30 min and 60 min post infection. Production of neutrophil-relevant cytokines such as G-CSF, IL-1α, IL-1β, IL-10, TNF-α, and IL-12p70 was quantified using a 31-plex mouse discovery assay (Eve Technologies).l-arginine supplementation, ROS and NO production, and KP35 growth in the presence and absence of l-Arginine were compared with a two-way ANOVA test followed by a Tukey’s test for multiple comparisons. KP phagocytosis by flow cytometry, DNA release, phagosome acidification in l-arginine supplemented neutrophils, MPO production and cytokine production were compared through a one-way ANOVA test followed by a Tukey’s test for multiple comparisons. All comparisons were performed using the GraphPad PRISM 8.2.0 software for Macintosh.Single comparisons of bacterial killing at 2 h and phagocytosis at 10 and 30 min by differential centrifugation were analyzed using a Student’s t-test. Neutrophil survival, phagosome acidification of neutrophils infected with KP35 and KPPR1; KP35 killing after K. pneumoniae ATCC 43816), a classical KP strain that is easily killed by neutrophils  , which encode the core proteins involved in regulation, synthesis and export; and the central variable region that contains genes encoding capsule-specific glycosyl-transferases and the core assembly proteins Wzx and Wzy  that encodes a different repertoire of glycosyl-transferases compared with NJST258_1 (clade II cps locus wzi154/KL107), including the glycosyl-transferase responsible for initiating the capsule biosynthesis. Whereas KP35 harbors wcaJ — which encodes an undecaprenyl-phosphate glucose-1-P transferase — NJST258_1 lacks this gene and carries wbaP, encoding an undecaprenyl-phosphate galactose-1-P transferase. Interestingly, KPPR1, which is phagocytosed by neutrophils as well as KP35, also harbors the wcaJ gene . Our data show that neither KPPR1 nor KP35 induced DNA release to the supernatant at 10 min  in response to phagocytosed bacteria. We measured phagosome acidification in neutrophils infected with KPPR1 or KP35 in the presence or absence of chloroquine, an inhibitor of lysosomal acidification  production by neutrophils in response to KPPR1 and KP35. Compared with uninfected neutrophils, KPPR1 and KP35 induced equivalent amounts of ROS l-arginine supplementation could improve the neutrophil response against KP35. Our data show that l-arginine supplementation (200 mg/L) enhanced the neutrophil intracellular response against KP35. l-arginine supplementation improved phagosome acidification of neutrophils infected with KP35 after 30 min  and in HBSS (a poor-nutrient buffer) in the presence and absence of l-arginine. In both cases, l-arginine did not exert a deleterious effect in KP35 growth and even enhanced KP35 grow in HBSS  respectively are major initiating events in neutrophil phagocytic killing . HOCl geV-ATPase  results a pH = 6 . These ca pH = 6 . The imputrophil . Our datytoplasm , an impoytoplasm  and giveytoplasm  and the in vivo. Neutrophil depletion impairs lung CRKP-ST258 clearance  was enough to improve KP35 killing after 2 h and to increase phagosome acidification, ROS production and NO consumption during the first 30 min. l-arginine is the substrate of inducible nitric oxide synthase (iNOS), which catalyzes the production of nitric oxide (NO), an important molecule involved in the respiratory burst and an active substrate of reactive nitrogen species (RNS), important antimicrobial factors produced during phagocytic killing in neutrophils (2- are the substrates of peroxynitrite (ONOO−) which is a highly antimicrobial molecule , the Millennium Institute on Immunology and Immunotherapy (ICN09_016), the Comisión Nacional de Investigación Científica y Tecnológica de Chile (grant no. 21140214), and NIH K08 HL138289.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.


Precision therapy for lung cancer requires comprehensive genomic analyses. Specific effects of targeted therapies have been reported in Asia populations, including Taiwanese, but genomic studies have rarely been performed in these populations.We enrolled 72 patients with non-small cell lung cancer, of whom 61 had adenocarcinoma, 10 had squamous cell carcinoma, and 1 had combined adenocarcinoma and squamous cell carcinoma. Whole-exome or targeted gene sequencing was performed. To identify trunk mutations, we performed whole-exome sequencing in two tumor regions in four patients.EGFR, PIK3CA, KRAS, CTNNB1, and MET were identified in 34 of the 72 tumors evaluated (47.22%). A comparison with the Cancer Genome Atlas dataset showed that EGFR was mutated at a much higher frequency in our cohort than in Caucasians, whereas KRAS and TP53 mutations were found in only 5.56% and 25% of our Taiwanese patients, respectively. We also identified new mutations in ARID1A, ARID2, CDK12, CHEK2, GNAS, H3F3A, KDM6A, KMT2C, NOTCH1, RB1, RBM10, RUNX1, SETD2, SF3B1, SMARCA4, THRAP3, TP53, and ZMYM2. Moreover, all ClinVar pathogenic variants were trunk mutations present in two regions of a tumor. RNA sequencing revealed that the trunk or branch genes were expressed at similar levels among different tumor regions.Nineteen known driver mutations in We identified novel variants potentially associated with lung cancer tumorigenesis. The specific mutation pattern in Taiwanese patients with non-small cell lung cancer may influence targeted therapies. Lung cancer is the leading cause of cancer-associated mortality worldwide. An estimated 2.09 million new lung cancer cases and 1.76 million lung cancer-associated mortalities were reported in the GLOBOCAN 2018 database . TobaccoTP53, KRAS, EGFR, NF1, BRAF, MET, and RIT. Pathway alterations in ADC are involved in RTK/RAS/RAF, mTOR, JAK-STAT, DNA repair, cell regulation, and epigenetic deregulation. Mutations in TP53, CDKN2A, PIK3CA, NFE2L2, KEAP1, CUL3, PTEN, NF1, NOTCH1, 2 and 3, DDR2, and EGFR genes are frequently observed in SqCC. Pathway alterations in SqCC involve squamous differentiation, the oxidative stress response, PIK3CA, DNA repair, cell cycle regulation, and epigenetic deregulation [EGFR and BRAF mutations and ALK and ROS1 rearrangements. Molecular genotyping is now routine in ADC. In SqCC, targeted agents are largely ineffective, and many targeted therapies are currently undergoing clinical trials [Rapid advancements in next-generation sequencing technology and a better understanding of cancer biology have provided unprecedented opportunities to characterize the genome of human tumors including lung cancer. The Cancer Genome Atlas (TCGA) lung cancer working group has profiled and analyzed 230 ADC and 178 SqCC specimens to identify molecular aberrations at the DNA, RNA, protein, and epigenetic levels , 10. In gulation . ADC andgulation , 13. In l trials .To develop a more comprehensive genomic picture of NSCLC, we performed whole-exome sequencing (WES) or targeted gene sequencing (TGS) in 72 Taiwanese patients with NSCLC. In addition, we compared the results with the TCGA NSCLC dataset, which involves mainly Western populations. We also investigated the associations between genetic alterations and clinicopathological features.A total of 61 fresh-frozen and 11 formalin-fixed paraffin-embedded (FFPE) specimens were obtained from 72 Taiwanese patients with lung cancer who underwent surgical resection from May 2007 to April 2019 at the China Medical University Hospital. The 72 lung tumors comprised 61 ADCs, 10 SqCCs, and 1 combined ADC and SqCC. DNA from frozen tissues and FFPE specimens was extracted using the QIAamp® DNA Micro Kit  and QIAamp® DNA FFPE Tissue Kit (Qiagen) according to the manufacturer’s instructions. The DNA concentration was quantified using the NanoDrop1000 spectrophotometer  and a Qubit Fluorometer .TGS was performed using the Qiagen platform with a panel that included either 275 (cat. no. DHS-3501Z) or 72 genes (cat. no. DHS-005Z). DNA libraries were prepared using components from the QIAseq Targeted DNA Panel Kit (Qiagen) and QIAseq Targeted DNA Panel Human Lung Cancer Panel (Qiagen). Briefly, 80 ng DNA was enzymatically fragmented and end-repaired in a 25-μl reaction volume containing 2.5 μl 10 × fragmentation buffer and 5 μl fragmentation enzyme mix. The reaction was carried out at 4 °C for 1 min, 32 °C for 24 min, and 65 °C for 30 min. Next, 10 μl 5 × ligation buffer, 5 μl DNA ligase, and 2.8 μl 25 μM barcoded adapters were added along with enough water to reach a reaction volume of 50 μl. Reaction tubes were then incubated at 20 °C for 15 min. To ensure complete removal of free barcoded adapters, each reaction was purified using 1.4 × (or 1.0 ×) QIAseq beads for two rounds. The purified DNA was then mixed in a 20-μl reaction volume with 10 nM each target primer, 400 nM IL-Forward primer, 1 × TEPCR buffer, and 0.8 μl HotStarTaq DNA polymerase. The PCR protocol was as follows: 95 °C for 13 min; 98 °C for 2 min; six cycles of 98 °C for 15 s and 65 °C for 15 min; and 72 °C for 5 min. Each reaction was cleaned once using 1.4 × (or 1.0 ×) QIAseq beads to remove unused primers. Enriched DNA was combined with 400 nM IL-Index primers, 1 × UPCR buffer, and 1 μl HotStarTaq DNA polymerase in a volume of 20 μl. The universal PCR conditions were as follows: 95 °C for 13 min; 98 °C for 2 min; 20 cycles of 98 °C for 15 s and 60 °C for 2 min; and 72 °C for 5 min. The DNA library was purified once using 1.4 × (or 1.0 ×) QIAseq beads and quantified using Qubit Fluorometric Quantitation . Libraries were sequenced on Illumina NextSeq  according to the manufacturer’s instructions . TGS analysis was described in detail in our previous work [A total of 50 ng DNA (based on Qubit quantification) was tagmented by a transposome, followed by clean-up and amplification of the tagmented DNA. A 200–400 bp band was selected, and exome capture was performed using the Nextera Exome Library Preparation Kit (Illumina). The DNA library was quantified using the Qubit 3.0 Fluorometer (Invitrogen) and Agilent 4200 Bioanalyzer . Samples were subjected to paired-end sequencing using the Illumina NovaSeq 6000 platform with a 150-bp read length. WES analysis has been described in detail in our previous work .® RNA Kit  following the manufacturer’s instructions. The quality, quantity, and integrity of the total RNA were evaluated using the NanoDrop1000 spectrophotometer and Bioanalyzer 2100 (Agilent Technologies). Samples with an RNA integrity number > 6.0 were used for RNA-seq. An mRNA-focused, barcoded library was generated using the TruSeq strand mRNA Library Preparation Kit (Illumina). The libraries were sequenced on the Illumina Nova Seq 6000 instrument (Illumina), using 2 × 151-bp paired-end sequencing flow cells following the manufacturer’s instructions.Total RNA was extracted from clinical tissue samples using the NucleoSpinhttps://software.broadinstitute.org/morpheus), and the correlation coefficients were visualized using Seaborn, a Python data visualization library (https://github.com/mwaskom/seaborn).Illumina bcl2fastq Conversion Software v2.20.0.422) was utilized to convert raw sequencing data to fastq format (Illumina). Trimmomatic PE (v0.39) was applied to control the read quality and remove sequencing adapters . Reads w.0.422 waAll statistical analyses were performed using SPSS software ver. 22.0. Chi-squared or Fisher’s exact tests were used to compare two categorical variables. Survival analysis was performed using Kaplan–Meier survival plot and log-rank test. A p-value less than 0.05 was considered statistically significant.The patients in this study comprised 39 males and 33 females, with a mean age of 62.35 years, of whom 61 had ADC, 10 had SqCC, and 1 had combined ADC and SqCC. Stage I disease was identified in 38 patients, stage II in 12 patients, stage III in 10 patients, and stage IV in 6 patients , and PIK3CA . Besides EGFR and PIK3CA, other known mutations were detected in KRAS , which have all been reported as driver mutations in lung cancer. In addition, four samples carried known activating mutations in the well-known oncogenes CTNNB1  and MET . Overall, 34 specimens harbored driver mutations in five cancer genes , which are canonical driver mutations . TP53 was the most frequently mutated gene after EGFR  .Among the 72 samples, 32 contained driver mutations in well-known cancer genes in NSCLC, such as EGFR , KRAS , and TP53 . A full comparison of the frequencies of selected gene alterations between the two cohorts is depicted in Fig. To compare the frequency of driver mutations of NSCLC between Taiwanese and Caucasian patients, we obtained all available lung cancer cases (560 ADC and 489 SqCC) from the TCGA dataset. Notable differences from the TCGA data included the frequencues of mutations in EGFR , ERBB2 , KRAS , MET , and NTRK1 .Based on the latest NSCLC guidelines published by the National Comprehensive Cancer Network, clinically relevant genomic alterations were identified in 34 47.22%) patients , and one patient with good survival had an AR non-frameshift insertion  and a FBXW7 non-frameshift deletion , and another patient with good survival had a CTNNB1  mutation.Among the 26 patients with EGFR mutation rate was significantly higher in patients with ADC than in those with SqCC . Moreover, the EGFR mutation rate was significantly higher in patients without smoking than in those with smoking . No association was found between the EGFR mutation status and sex, age, or tumor stage of the patients. In contrast, the PIK3CA and TP53 mutation rates were significantly higher in patients with SqCC than in those with ADC . Furthermore, the TP53 mutation rate was significantly higher in patients with smoking than in those without smoking . No association was found between KRAS or CTNNB1 mutations and any clinicopathological characteristic.Correlations between the genotypes and clinicopathological characteristics are listed in Table PIK3CA mutation was a prognostic of worse overall survival  occurred much more frequently than branch mutations (median: 0.12–0.15%) , 1 (MYD88), 4 , and 2 (KLF5 and CDKN2A), respectively   in a patient with SqCC. cancers , and app [ARID1A . In thisKDM6A is located on chromosome Xp11 and encodes a tetratricopeptide repeat protein. The protein contains a Jumonji C domain and catalyzes the demethylation of tri/dimethylated histone H3. KDM6A is a tumor suppressor gene in different cancers, including lung SqCC [KDM6A mutation (p.S314X) in a patient with SqCC.ung SqCC . In thisNOTCH1 is reported to have a bimodal role as a tumor suppressor and an oncogene in several cancers [NOTCH1 mutation (p.E2534K) in a patient with SqCC.NOTCH1, a member of the NOTCH protein family, contains an extracellular domain consisting of multiple epidermal growth factor-like repeats (36) and an intracellular domain consisting of multiple domain types.  cancers , 25. In RB1 encodes a negative regulator of the cell cycle protein and was the first tumor suppressor gene identified. Its tumor suppressive function is due to inhibition of the transcription factor E2F1. Any defect in the RB1 gene causes cells to transition from the G1 to S-phase of the cell cycle [RB1 is inactivated in a wide range of cancers, including lung ADC and SqCC. In this study, we identified a novel RB1 mutation (p.I848fs) in a patient with ADC. The I848fs mutation occurs in the C domain, which mediates the interaction with E4F1 [ll cycle . RB1 is ith E4F1 .RBM10, a tumor suppressor gene [RBM10 mutation (p.A410fs) in a patient with ADC.sor gene , 29, encsor gene . In thisSETD2 encodes a protein that interacts with huntingtin. The protein is a histone methyltransferase responsible for the tri-methylation of lysine 36 on histone H3 (H3K36me3), using H3K36me2 as a substrate [SETD2 is a tumor suppressor gene expressed in different cancer types [SETD2 mutation (p.L1525P), present in the AWS domain, in a patient with SqCC.ubstrate . SETD2 ier types . In thisSMARCA4, also known as BRG1, encodes a member of the SWI/SNF family, which possesses helicase and ATPase activities. Human SWI/SNF enzyme subunits are mutated in approximately 20% of cancers [SMARCA4 is possibly tumor supressive in lung ADC. In contrast, SMARCA4 may also be an oncogene in liver hepatocellular carcinoma, lower-grade glioma, and pan-cancer [SMARCA4 mutation (p.Q570fs) in a patient with ADC. cancers . SMARCA4EGFR mutant TP53 wildtype cohort, 86.88 ± 15.41 months for the EGFR/TP53-mutant cohort; p = 0.839 , 114.50 ± 2.50 months for the EGFR/TP53-mutant cohort (n = 3); p = 0.216 (Additional file EGFR/TP53-mutant lung cancers. Our cohort contained eight patients with co-existing EGFR/TP53-alterations. We compared the mutational signatures between the patients with a good and those with a poor survival outcome. The presence of a co-existing MYC alteration was associated with worse survival in patients with EGFR/TP53-mutant lung ADCs. Moreover, a co-existing AR, FBXW7, or CTNNB1 alteration was associated with better survival in patients with EGFR/TP53-mutant lung ADCs. In our study, a relatively small number of patients with co-existing EGFR/TP53 alterations was identified.Recently, Skoulidis et al. reported that co-occurring genomic alterations affect the response of NSCLC to anticancer therapies . The meaIntratumoral heterogeneity presents a major challenge in precision cancer therapy because it can lead to underestimation of the tumor genomic landscape when based on single tumor biopsy samples, and this might contribute to drug resistance and treatment failure . All knoIn summary, we identified genomic aberrations underlying NSCLC in a Taiwanese population. Our study provides putative biomarkers for prognostic prediction in lung cancer. Further research is required to elucidate the functions of these genes and their pathways.Additional file 1: Table S1. Mutations in five cancer genes in the 72 patients. Table S2. Comparison of driver gene mutations of lung cancer between Taiwanese patients and the Caucasian cohort in TCGA dataset. Table S3. List of trunk mutations of ClinVar pathogenic variants in four paired samples. Table S4. List of trunk and branch mutations of prediction pathogenic variants in four paired samples. Table S5. List of differentially expressed genes with a fold change in expression > 4 in four paired samples. Table S6. Novel mutations in cancer driver genes.Additional file 2: Figure S1. Kaplan–Meier survival curve of patients with EGFR, KRAS, TP53, and CTNNB1 mutations. Figure S2. Kaplan–Meier survival curve of 18 patients with EGFR mutation TP53 wild-type versus 8 patients with EGFR/TP53 mutations. Figure S3. Kaplan–Meier survival curve of 8 patients with EGFR mutation TP53 wild-type versus 3 patients with EGFR/TP53 mutations after EGFR-TKI treatment.


A flash chromatography method was developed and optimized  using a binary gradient of hexane and acetone mobile phase and silica gel stationary phase to separate and isolate F2G and FG. The optimized parameters afforded F2G  and FG  from 3.0 g of the transesterification precipitate, n = 10 trials. Overall, all flash chromatography separations combined, F2G  and FG  were isolated in a combined yield of 48.5 g (51.4%), relative to the 96.4 g of transesterification precipitate collected. The optimized flash chromatography method was a necessary improvement over previously reported preparative HPLC and column chromatography methods used to purify milligram to low gram quantities of F2G and FG to be able to process ~100 g of material in a timely, efficient manner.Ethyl ferulate was transesterified with Enova Oil (a soy-based vegetable oil containing 80–85% diacylglycerol) using Novozym 435 at 60 °C. The resultant feruloylated vegetable oil reaction product produced a precipitate  after 7 d of standing at room temperature. Preliminary characterization of the precipitate identified the natural phenylpropenoids 1,3-diferuloyl- The phenylpropenoid, ferulic acid, is a 4-hydroxy-3-methoxy-substituted cinnamic acid and is a nearly ubiquitous component throughout the higher plant kingdom, and as such is a common dietary component . In plan2G  , major ion 465.0 m/z, and the major ion 266.9 [M − H]−, respectively, and by 1H NMR , relative to the initial 100 mg of raw precipitate. This was almost exactly a 4:1 F2G:FG ratio isolated from the raw precipitate.y 1H NMR , confirmIt should be noted that adjusting the flow rate from the manufacturer’s specified flow rate for a RediSep column  affected2G and FG using 2G and FG. Separation of 1, 3, 5, 7, and 10 g loads were attempted with a 120 g RediSep Separation Column  were attempted using a larger (330 g) RediSep Separation Column.Having shown that small samples (100 mg) of raw precipitate could be separated to isolate Fn Column , Run 3–9n Column , using an Column , Run 1, oad runs . Based ople load , Run 5 wple load , Run 4 on column , Run 6. n column , Run 7, n column , Run 3–6n Column , Run 8 sple load , Run 9 d2G and FG, producing isolated yields similar to the 5 and 7 g sample loads separated with the 120 g Separation Column  using a 0–80% acetone in hexane gradient over 15 CV  relative to the 3 g sample. The RediSep Load columns were aspirated to dryness after every use and were used up to seven times without noticeable deleterious effects. The RediSep Separation Columns that were stored in hexane in between separation runs could be used up to three times before the F2G and FG did not separate under a given run condition. When the RediSep Load and Separation Columns were aspirated to dryness and stored dry in between runs the columns could be reused up to seven times before the F2G and FG were observed to not separate under a given run condition. While the RediSep columns used herein are considered disposable by the manufacturer it was in the interest of cost that the columns were reused as often as possible.After considering the paration , Run 5 w2G and FG isolated from all flash chromatography runs, F2G and FG were isolated in a mass yields of 39.1 and 9.4 g, respectively , from the 96.4 g of raw precipitate filtered from the feruloylated Enova Oil. Previously described separation methods  to isolate F2G, FG, and similar phenylpropenoid glycerols as natural products from plant sources could only accommodate ~0.1–0.2 g of material [2G or FG [2G was previously reported in ~10 g yield as a precipitate collected from a similar lipase-catalyzed transesterification of ethyl ferulate with deacylated soybean oil; however, the purification was reported as a diethyl ether wash of the raw precipitate collected without FG being identified as a component [2G and FG are sparingly soluble in diethyl ether, it is presumed that the FG was lost in the diethyl ether washes. Comparatively, the flash chromatography method reported herein can purify up to 2.0–3.0 g of F2G and FG per run in about an hour and was a necessary improvement over previously reported methods to be able to process ~95 g of raw material containing a mixture of F2G and FG in a timely and convenient manner.Overall, combining the Fively 48. g total,2G or FG . FG has 2G or FG ,20 or pu2G or FG . The synomponent . Since b2G and FG have been previously reported to be synthesized or isolated as colorless to yellow, oily liquids [2G has also been reported to have been isolated as a white solid [2G synthesized from the lipase-catalyzed transesterification of Enova Oil with ethyl ferulate and isolated by flash chromatography was a brittle, crystalline, white solid, and FG was isolated as a clear, light-yellow oil ,2 positional isomer consists of feruloyl moieties covalently attached to an exterior α- and the interior β-carbon of the glycerol molecule. The two F2G positional isomers result in distinctly different 1H NMR frequency splitting patterns. The F2G sn-1,3 isomer would be expected to exhibit a symmetric, α-, β-proton splitting pattern in a frequency ratio of 4:1 as found in 2G isolated by flash chromatography was the sn-1,3 positional isomer. Similarly, FG can consist of the sn-1(3) and the sn-2 positional isomers where the feruloyl moiety is covalently attached to the α- or β-carbon, respectively. The FG sn-2 positional isomer would be predicted to exhibit a symmetrical α-, β-proton splitting pattern in a frequency ratio of 4:1 with the β-proton frequency being at 4.9–5.1 ppm as would be expected for an esterified β-carbon [sn-1(3) positional isomer. Acyl groups can migrate along the glycerol backbone, exchanging between the sn-1, -2, and -3 positions and is observed in mono- and diacylglycerols of which the kinetic rates of migration are dependent on time, temperature, presence of solvent, and acyl moiety [2G and FG isolated by flash chromatography and stored at both room temperature and refrigerated exhibited no observable acyl migration as determined by 1H NMR over four weeks of storage, which was consistent with the lack of acyl migration found in sn-2 monoacylglycerols at ≤25 °C [Theoretically, Fβ-carbon ,33,34. Il moiety ,36,37,382G and FG possess 4-hydrox-3-methoxycinnamoyl moieties , UVB (290–320 nm), UVA II (321–340 nm), and UVA I (341–400 nm) [2G and FG possessed a λmax 321 in acetonitrile solutions (max 328 in ethanol (data not shown), their strongest absorbances were in the UVA II region and extended into the UVB region. F2G and FG did not strongly absorb in the UVA I region. The UV absorbance spectra and λmax of F2G and FG were very similar to that of feruloylated soybean oil reported in ethanol and acetonitrile solutions [2G (10–50 µM) and FG (10–100 µM) were determined in ethanol  . F2G andolutions  and a λmolutions . The ext ethanol  and acet ethanol  as the sectively . Althougolutions ,46,47. R2G was determined by DSC and their thermograms are shown in onset = 118.07 °C and melting peak at Tpeak = 124.48 °C (ΔHmelt = 143.6 J g−1). As expected, the additional ferulate group increased the melting point of the material relative to FG as shown by the F2G thermogram that had an onset temperature of Tonset = 144.42 °C and an endothermic melting transition at Tpeak = 147.94 °C (ΔHmelt = 107.3 J g−1). Interestingly, the cooling cycle showed no endothermic transitions indicating recrystallization of the materials and the subsequent rescan of both FG and F2G only showed a small endothermic event at 7.8 °C and 42.8 °C, respectively, suggesting a glass transition of an amorphous material. From the rescan it is clear the crystallinity of both these compounds was lost due to the thermal processing of the samples. To further examine these samples, the FG and F2G DSC samples, having been exposed to 160 °C, were examined by 1H NMR and the spectra (not shown) demonstrated that acyl migration had occurred in both FG and F2G to form the sn-2 and sn-1,2 positional isomers, respectively. The presence of acyl migration products within FG and F2G likely precluded crystallization of the compounds.The thermal behavior of FG and F2G and FG that were obtained as a byproduct mixture from the lipase-catalyzed transesterification of ethyl ferulate and commercial vegetable oil, Enova Oil (≥80% DAG). Over 95.0 g of the crude byproduct mixture was processed by flash chromatography to yield 39.1 g of F2G and 9.4 g of FG. This method was an improvement from the preparative HPLC and column chromatography methods reported to purify milligram quantities of F2G and FG mixtures or used to individually isolate small gram quantities of F2G or FG to date. F2G and FG were isolated as the sn-1,3 and sn-1 positional isomers, respectively, and did not exhibit acyl migration during the flash chromatography purifications or during storage. F2G and FG possessed UV-absorbing characteristics, λmax 328 nm and ε 40,000 ± 60 and 19,200 ± 154 M−1 cm−1, respectively, in ethanol solutions, making them suitable as fungible replacements for commercial UVB absorbers or UVA II-absorbing enhancers when used with commercial UVB- and UVA-absorbing compounds.A flash chromatography method using a binary gradient of hexane and acetone was optimized for the separation of the natural compounds F


D = 53.23 ± 4.5 nM, which is 1.5 times lower than that obtained for 4 × LPG under similar experimental conditions. Circular dichroism (CD) spectroscopy and fluorescence spectroscopy studies indicated that the SBP degree of oligomerization has only a small effect on the secondary structure (the linker unravels the beginning of the protein G sequence) and chemical stability of the parent protein G. However, based on quartz crystal microbalance with dissipation monitoring (QCM-D), oligomerization is an important parameter for a strong and stable binding to silica. The replacement of three sequence repeats by a (GGGGS)12 glycine-rich spacer indicated that the overall length rather than the SBP oligomerization mediated the effective binding to silica.The bifunctional linker-protein G (LPG) fusion protein comprises a peptide (linker) sequence and a truncated form of Streptococcus strain G148 protein G (protein G). The linker represents a multimeric solid-binding peptide (SBP) comprising 4 × 21-amino acid sequence repeats that display high binding affinity towards silica-based materials. In this study, several truncated derivatives were investigated to determine the effect of the SBP oligomerization on the silica binding function of LPG . Various biophysical characterization techniques were used to quantify and compare the truncated derivatives against 4 × LPG and protein G without linker (PG). The derivative containing two sequence repeats (2 × LPG) showed minimal binding to silica, while the truncated derivative with only a single sequence (1 × LPG) displayed no binding. The derivative containing three sequence repeats (3 × LPG) was able to bind to silica with a binding affinity of K Solid-binding peptides (SBPs) are a group of short amino acid sequences that specially bind to the surfaces of various inorganic materials including minerals, semiconductors, and polymers. In doing so, they are able to mediate the simple and controlled attachment of biomolecules onto solid surfaces, conferring biological functionality. SBPs can also act as functionalizing agents for binding and linking biomolecules to solid nanostructures ,2. MolecProper orientation is usually crucial to preserve the bioactivity, as well as the avidity, of biomolecules. Care et al. demonstrated the oriented immobilization of antibodies onto silica-coated magnetic particles using a bifunctional linker-protein G (LPG) fusion protein, which contains a silica-binding peptide and an antibody-binding region . The binD = 0.69 µM) to zinc oxide nanoparticles than the single peptide domain (KD = 1.5 µM). These findings implied that there might be a direct relationship between the binding avidity and the number of repeated sequences for some SBPs.The interactions between SBPs and their substrates rely on the affinity that particular chemical groups within amino acid residues have for solid surfaces. For example, SBPs that bind to metals predominantly contain hydrophobic and hydroxyl-containing polar residues , while tStreptococcus protein G antibody-binding protein (PG) with an altered N-terminal domain to a multimeric linker made of a 4 × 21-amino acid sequence repeat  was initially reported by Sunna et al.  and was e repeat  1] that thatStre2) purchased from ATA Scientific . Pefabloc, lysozyme, and Benzonase nuclease were purchased from Sigma-Aldrich . Human serum was obtained from Sigma-Aldrich , while the humanized anti-HER2 monoclonal antibody trastuzumab was purchased from Jomar Life Research . The human HER2/ErbB2 protein (His-Tag) was purchased from Sino Biological Inc. . All biological assays were performed at room temperature with standard phosphate-buffered saline (1 × PBS) at pH 7.4 containing 10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, and 2.7 mM KCl . All other chemicals were purchased from Sigma-Aldrich  unless otherwise stated.The silica binding studies were performed using silica-coated quartz crystals (QSensor QSX 303 SiOE. coli Tuner (DE3) cells (Novagen) harbouring the expression plasmid. The culture was incubated at 37 °C with continuous shaking (250 rpm) until the A600 was approximately 0.7–1.0. The incubation temperature was reduced to 20 °C and protein synthesis was induced by the addition of 0.2 mM isopropyl β-D-thiogalactoside (IPTG). Cells were harvested after 3–4 h induction by centrifugation for 15 min at 10,000× g and 4 °C and were stored at −20 °C.The expression plasmids  used to produce the recombinant proteins were previously reported by [g and 4 °C. The supernatant obtained was first filtered through a 0.45 µm and then 0.22 µm sterile filter and stored at 4 °C.The cells were resuspended in ice-cold lysis buffer , supplemented with 4 mM of the Pefabloc serine protease inhibitor and 1.5 mg lysozyme. They were ruptured by three passages through a French pressure cell . After cell rupture, 50 units of Benzonase nuclease and 4 mM Pefabloc were added. The sample was incubated on ice for 20 min. The debris was removed by centrifugation for 30 min at 20,000× All purifications were performed using the Äkta™ start chromatography system . For purification, each truncated derivative soluble extract  was loaded onto a 5 mL HiTrap Q anion exchanger column  previously equilibrated with 25 mM Tris–HCl, pH 8.0, supplemented with 100 mM NaCl. The column was washed extensively with the same buffer. Under these conditions, all truncated derivatives eluted at 200 mM NaCl. The eluted protein samples were concentrated using an Amicon Ultra-15 centrifugal filter  followed by buffer exchange with a PD-10 desalting column . In addition, 1 × LPG was buffer exchanged into 20 mM Bis-Tris (bis-(2-hydroxyethyl)-amino-tris(hydroxymethyl)-methane) buffer pH 6.0. This fraction was applied again onto the 5 mL HiTrap Q column  previously equilibrated with 20 mM Bis-Tris buffer pH 6.0. The column was washed extensively with the same buffer and the 1 × LPG recombinant protein was eluted at 200 mM NaCl. 2 × LPG and 3 × LPG were buffer exchanged into 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer pH 8.0. The fractions were applied to a 5 mL HiTrap SP cation exchanger column previously equilibrated with 50 mM HEPES buffer pH 8.0. The column was washed extensively with the same buffer. Under these conditions, 2 × LPG eluted at 50 mM NaCl, while 3 × LPG eluted at 100 mM NaCl.® TGX™ Precast Protein Gels  by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) and stained with Coomassie Brilliant Blue. Individual fractions were concentrated using an Amicon Ultra-15 centrifugal filter and all final samples were stored in 1 × PBS buffer (after fresh addition of Pefabloc) at –80 °C.Fractions containing the truncated derivatives were identified on 4–15% Mini-PROTEANThe final protein concentration was measured using the Micro BCA protein assay kit  according to the manufacturer’s instructions.n linker sequence. This replacement was important to determine whether the oligomer or simply the distance from the PG was required for efficient binding to silica. The PG sequence was ligated into plasmid pET44a (+) via NheI/BamHI restriction sites to obtain the plasmid pET44-PG. A gene sequence containing one original SBP sequence from LPG followed by the synthetic linker sequence (GGGGS)12 was synthesized by Invitrogen GeneArt Gene Synthesis  and ligated into plasmid pET44-PG via NdeI/NheI restriction sites to assemble the expression plasmid pLink1 × (GGGGS)12-PG. E. coli α-Select  was used as a host for general gene cloning and vector storage, and E. coli Tuner (DE3) cells were used for recombinant protein expression.Three sequence repeats from the 4 × LPG peptide linker were replaced by a synthetic (GGGGS)12-PG was produced and the soluble protein fraction was obtained as described for the truncated derivatives. For purification of Link1 × (GGGGS)12-PG, the soluble extract was loaded onto a 5 mL HiTrap Q anion exchanger column previously equilibrated with 25 mM Tris–HCl, pH 8.0, supplemented with 100 mM NaCl. The column was washed extensively with the same buffer. Under these conditions, the Link1 × (GGGGS)12-PG did not bind to the column and was found in the unbound fraction. The Link1 × (GGGGS)12-PG was concentrated using an Amicon Ultra-15 centrifugal filter (10 kDa cut-off) followed by buffer exchange with a PD-10 desalting column and 50 mM MES (2-(N-morpholino) ethanesulfonic acid) buffer pH 6.0. This fraction was applied to a 5 mL HiTrap SP cation exchanger column previously equilibrated with 50 mM MES buffer pH 6.0. The column was washed extensively with the same buffer and the Link1 × (GGGGS)12-PG was eluted at 300 mM NaCl. Fractions containing the Link1 × (GGGGS)12-PG were identified by SDS-PAGE as described above. Fractions containing the purified Link1 × (GGGGS)12-PG were pooled and concentrated using an Amicon Ultra-15 centrifugal filter. Samples were stored in 50 mM MES buffer pH 6.0 at –20 °C after sterile filtration through a 0.22 µm filter.Link1 × (GGGGS)12-PG to silica was determined as follows: 5 mg silica  was washed three times with washing buffer . Soluble protein in a final volume of 100 µL was mixed with silica and incubated by rotation at room temperature for 1 h. The unbound fraction was removed after centrifugation at 14,000× g for 20 s. The silica pellet was washed three times by vortexing with 100 µL of 100 mM Tris–HCl buffer, pH 8.0. Finally, the silica-bound protein was eluted after addition of 100 µL of SDS PAGE-loading buffer and incubation at 99 °C for 10 min (with short mixing every 2 min). Fractions containing the Link1 × (GGGGS)12-PG protein were identified by SDS-PAGE as described above.The binding of 4 × LPG and the truncated derivatives used in this study  to silica was previously reported by [http://rsb.info.nih.gov/ij/index.html).The percentage of protein in the different fractions was semi-quantified by analysing the intensity of bands of digital images from SDS-PAGE with ImageJ software  (−1 cm−1 dm3.CD spectra were collected at room temperature (approximately 25 °C) on a JASCO J-1500 spectrophotometer . CD data were collected in water in order to avoid any interference or noise below 200 nm as PBS absorbs strongly at wavelengths below 200 nm. All protein samples were buffer exchanged to Milli-Q water using Amicon Ultra-10K 0.5 mL centrifugal filters and were then further diluted in Milli-Q water to a final concentration of 0.1 mg/mL (as determined by the Micro BCA protein assay). Wavelength scans were performed between 180 and 350 nm in a rectangular, 1 mm pathlength quartz cuvette . For each sample, 3 accumulations were recorded using a 2 nm bandwidth, a scan speed of 100 nm/min, and a digital integration time (D.I.T.) of 2 s. The data are reported in terms of molar extinction coefficient (Δɛ), expressed in molCircular dichroism spectra were analysed using a graphical user interface (GUI) version  is provided in It was visually apparent (from the shift in the negative maximum from 208 nm towards 200 nm) in the spectra that addition of longer peptides increased the unfolded percentages of the proteins. However, the fitting method was not selecting the random coil member of the reference set, due to it not being one of its best matching units. We, therefore, manually subtracted different fractions of the random coil spectrum for the SufI-KK peptide MSLSKKQFIQASGIALCAGAVPLKASA  and reraProtein samples were dissolved to a final concentration of 2 µM in 1 × PBS containing various concentrations (0–7 M) of guanidinium hydrochloride (GdnHCl). All measurements were recorded at room temperature (approximately 25 °C) in a micro-volume fluorescence cuvette with a 3 mm pathlength  using a JASCO FP-8500 Spectrofluorometer . Fluorescence emission spectra were collected between 300 and 550 nm using a 295 nm excitation wavelength, 2.5 nm excitation bandwidth, 5 nm emission bandwidth, and scan speed of 100 nm/min.2-coated sensors. Sensors were cleaned before use with 2% sodium dodecyl sulphate (SDS) for 30 min at room temperature, washed thoroughly with Milli-Q water, and dried with nitrogen gas prior to use. The temperature of the QCM-D was maintained at 22 ± 0.1 °C and the flow rate was set to 150 µL/min throughout all experiments. Sensor crystals were exposed to 1 × PBS, and stable frequency (f) and dissipation (D) measurements were established. Test protein solutions were prepared in 1 × PBS buffer pH 7.4 at room temperature at concentrations of 3.27–654 nM and exposed to the surfaces for 900–15,000 s and then rinsed with 1 × PBS until consistent f and D measurements were obtained. In addition, the ability of an antibody and antigen complex to bind to 3 × LPG immobilized on silica was explored. Sensor crystals were exposed to 1 × PBS, and stable f and D measurements were established. 3 × LPG was exposed to silica at a concentration of 3.27–654 nM for 900–15,000 s, and the sensors were then rinsed with 1 × PBS until consistent f and D measurements were obtained. To eliminate the risk of non-specific binding, the free binding sites on the crystal surface were blocked by injecting 1 mg/mL BSA (in 1 × PBS) followed by rinses by rinsing with 1 × PBS until stable f and D measurements were obtained. The humanized anti-HER2 monoclonal antibody, trastuzumab, was then injected into the system at a final concentration of 1 µg/mL until a stable response was observed followed by rinses by rinsing with 1 × PBS until stable f and D measurements were obtained. The final step included the injection of HER2 antigen at a concentration of 1 µg/mL until equilibrium was reached.The adsorption of the proteins  onto silica was monitored on a 4-channel QSense Analyzer QCM-D  using SiOf (5 MHz), as well as the third (15 MHz) to eleventh (55 MHz) overtones. All solutions were passed through a 0.22 μm filter and degassed at room temperature before use. The adsorbed mass is proportional to the change in f (∆f) via the Sauerbrey equation only when the adsorbed layer is rigid, uniformly distributed on the sensor surface, and the adsorbed mass is small compared to the mass of the crystal [6/10 Hz. Under these conditions, the Voigt viscoelastic model is used to estimate the adsorbed protein layer mass and thickness from f and D measurements at multiple harmonics, assuming an adsorbed protein layer density of 1000 g/m3 [f and ∆D versus time graphs or Df plots (∆D versus ∆f) for the third overtone.A minimum of three adsorption curves were recorded for each test protein at each concentration, which were measured at the fundamental  crystal ,32. Howe000 g/m3 ,34,35. TThe bioactivity of a biomolecule is dependent on its secondary structure. CD spectroscopy was used to determine the effect of different truncations of the linker sequence on the overall secondary structure of PG. A concentration of 0.1 mg/mL  was used to measure the far-UV spectra of the truncated derivatives . As shown in We, therefore, subtracted the CD of different fractions of a random coil polypeptide, selected the fraction that had the best SOMSpec predicted spectrum, and determined the secondary structure contents. The relative stability of all truncated derivatives was compared in the presence of the chemical denaturant GdnHCl using fluorescence spectroscopy. PG includes eight tyrosine and three tryptophan residues in the core of the protein. Accordingly, intrinsic tryptophan fluorescence (ITF) experiments were performed at an excitation of 295 nm to eliminate the contribution of tyrosine to the measured fluorescence. The linker sequence does not contain any aromatic residues, so it does not contribute to the overall fluorescence. The unfolding of 1 × LPG, 2 × LPG, 3 × LPG, and 4 × LPG was monitored by measuring the shift in the maximum tryptophan fluorescence emission with an increasing concentration of GdnHCl, and the ratio of the fluorescence intensities at 330 and 360 nm was plotted against increasing denaturant concentration .Assuming the protein denaturation to be a two-state process, GraphPad Prism 7.0  was used to non-linearly fit the data. The concentration of GdnHCl required for 50% unfolding of the core protein where the tryptophans are located was determined to be 3.4 ± 0.2 M (95% CI), 3.4 ± 0.1 (95% CI), and 3.4 ± 0.2 (95% CI) for 1 × LPG, 2 × LPG, and 3 × LPG, respectively. The concentrations for PG and 4 × LPG were previously reported as 3.4 ± 0.2 M and 3.6 ± 0.1 M, respectively . Thus, tf vs. time sensorgram, f and D measurements observed (D) of 53 ± 5 nM, when fitted using the Langmuir isotherm , indicating an overall lower binding affinity of the 3 × LPG truncated derivative. Based on these data, a general trend of increased binding to the silica surface was observed with linker oligomerization.The binding affinity of the various truncated derivatives to a silica surface was determined using QCM-D . The binobserved c,d and aisotherm . This KDf plot generated from the QCM-D data for 3 × LPG binding to silica 12 [12-PG recombinantly in E. coli. The purified Link1 × (GGGGS)12-PG has the same pI (4.69), overall charge at pH 7.0 (–13.8), and very similar grand average hydropathicity (–0.64) as 1 × LPG 12-PG) is enough to restore the binding affinity towards silica almost to the same level as 4 × LPG [Although the linker sequence is important for the binding of 3 × LPG and 4 × LPG to silica, it is not clear whether 4 × LPG binds better simply because it has a repeat of the binding peptide or simply because it is longer. GGGGS)12  12-PG, which seems to function as an analogue of 4 × LPG but carries only a single sequence of the silica-binding peptide rather than four repeats. This (GGGGS)12 spacer addition was enough to convert 1 × LPG, which displayed negligible binding to silica in the silica binding assays [The introduction of (GGGGS)g assays . Over 76g assays  and confg assays . Thus, t12 fused between the single linker sequence and PG (1 × LPG) to create a fusion protein of the same length as 4 × LPG with 1×L available for surface binding, Link1×(GGGGS)12-PG, restored the silica binding property of the truncated derivative 1 × LPG. The CD data, which showed that the linkers, as well as some of the N-terminus residues, were unfolded, support the conclusion that a key role of the middle linkers is simply to let the “fishing hook” of the final silica binding peptide meet the surface. These preliminary qualitative data provide further motivation to investigate not only oligomerization but also the length of the linker as potentially important parameters for strong silica binding. The results presented here also suggest that SBP oligomerization or incorporation of flexible interdomain linkers like (GGGGS)n can be used to tailor the adsorption and affinity of a given sequence to a solid material. This provides an additional manipulation tool for future applications requiring surface-specific SBPs that act as molecular linkers and assemblers.There is increased evidence of the contribution of mineral-templated self-assembling systems, including biomolecular self-assemblies of peptides, on the origins of life on Earth and the complexity of early livings systems . Recentl