However, the state of the Arctic cryosphere during the Pliocene remains poorly constrained. For the first time we combine outputs from a climate model with a thermodynamic iceberg model to simulate likely source regions for ice‐rafted debris (IRD) found in the Nordic Seas from Marine Isotope Stage M2 to the mid‐Piacenzian Warm Period and what this implies about the nature of the Arctic cryosphere at this time. We compare the fraction of melt given by the model scenarios with IRD data from four Ocean Drilling Program sites in the Nordic Seas. Sites 911A, 909C, and 907A show a persistent occurrence of IRD that model results suggest is consistent with permanent ice on Svalbard. Our results indicate that icebergs sourced from the east coast of Greenland do not reach the Nordic Seas sites during the warm Late Pliocene but instead travel south into the North Atlantic. In conclusion, we suggest a continuous occurrence of marine‐terminating glaciers on Svalbard and on East Greenland (due to the elevation of the East Greenland Mountains during the Late Pliocene). The study has highlighted the usefulness of coupled climate model‐iceberg trajectory modeling for understanding ice sheet behavior when proximal geological records for Pliocene ice presence or absence are absent or are inconclusive.The Arctic cryosphere is changing and making a significant contribution to sea level rise. The Late Pliocene had similar CO In the Nordic Seas, evidence suggests icebergs have persisted throughout the Late PlioceneA coupled climate model‐iceberg trajectory model can prove useful for understanding paleo‐ice sheet behaviorIce appears to have persisted on Svalbard and East Greenland throughout the Late Pliocene In the mPWP3.1.2STD scenario relative to the preindustrial shows a 3 to 4°C mean annual warming in the Norwegian Sea; however, along the western boundary with the Barents Sea this is approximately 1°C cooler  warmer.In the modeled M2 scenario the Norwegian Sea varies from 0 to 5°C cooler than preindustrial. The mean annual temperature is coldest closest to land and warms by 3 to 4°C in the center of the Nordic Seas. Around Iceland the mean annual SST is 1°C cooler on the southeastern coast and 8°C cooler on the northwestern coast. Along the coast of Greenland the difference in temperature from present is minimal (−1°C) north of approximately 70°N but increases to −3°C through the Denmark Strait and south to Cape Farewell. The SST anomaly for the mPWP3.1.3STD scenario, although the pattern is similar, the winds from Norway and those moving from the Greenland interior are weaker than those of the M2 scenario. In the mPWPALT scenario winds are similar but stronger moving southwest out of the Arctic Ocean.In the M2 scenario winds travel north from the UK but turn westward when they meet winds traveling westward across the Nordic Seas from Norway  SAT anomaly along the west Barents margin. North of Iceland cooler than present SATs exist (0 to 3°C), but along the east coast of Greenland SATs reach a high of 15°C warmer than present.During the M2 scenario, the anomaly of warm mean annual surface atmospheric temperatures (SATs) seen along the Norwegian coast are replaced with cooler temperatures of 0 to −10°C along the southern coast of Norway, dropping to −30°C toward the Arctic Ocean. In the Greenland interior, the mean annual SAT anomaly drops to −45°C. In the mPWP3.1.4STD scenario, summer sea ice is contained within the central Arctic Ocean area. However, during the winter, the sea ice extends to the Russian coastline, Canadian archipelago, northern Scandinavia, and NSVAL in the Arctic Ocean and down the east Greenland coast to Scoresby Sund. The Baffin Sea west of Greenland has sea ice from Nares Strait to Disko Bugt. In the mPWPALT scenario, the extent is similar; however, the percentage concentration of sea ice in the Nordic Seas is much less.The extent and percentage concentration of sea ice cover varies between seasons with the summer minimum (September) and winter maximum (March) averages shown in Figure 3.2STD, and mPWPALT) from each of the seeding locations are grouped  show very different patterns between M2 and mPWP scenarios. During the M2 Figure , icebergo Figure , modeled3.2.2STD and mPWPALT scenarios, icebergs do not reach across to the European coastline in the North Atlantic but rather melt in the Labrador Sea and northwestern Atlantic region. The Iceland Plateau does not appear to receive icebergs from the coast of Greenland during the mPWPSTD scenario, but the mPWPALT scenario does show icebergs reaching the location of 907A.In all the scenarios, modeled iceberg trajectories flow south in the EGC. In the M2 scenario, icebergs reach a more easterly extent in the Nordic Seas and in the North Atlantic Figure . During 3.2.3STD scenario  have very similar patterns during both the M2 and the mPWPo Figure , icebergo Figure , the ice3.3Figure ODP Hole 911A. Both the cold M2 and warm mPWPSTD scenarios show modeled iceberg trajectories traversing this site. The seeding locations for these icebergs are only from the margins of Svalbard. Neither icebergs from the Arctic coastline nor from further south in the Nordic Seas are able to reach the site. In the mPWPALT scenario, however, icebergs from VORP, BEAR, and SVAL reach this site.ODP Hole 909C. Iceberg trajectories during the M2 scenario from seeding locations on Svalbard (NSVAL and SVAL) are the only ones modeled to reach this site. During the mPWPSTD scenario, icebergs from the seeding locations of Bear Island and Svalbard (BEAR and SVAL) reach this site. In the mPWPALT scenario, sites from Norway  and SVAL reach this site.ODP Hole 907A. Iceberg trajectories reaching this site during the cold M2 scenario come from seeding locations in the Arctic Ocean , east Greenland coast , Svalbard and western Barents Sea (SVAL and BEAR), and off the coast of Norway (NORC and VORP). Sites from around Iceland and Scoresby Sund  do not appear to reach ODP Hole 907A. During the mPWPSTD scenario, only icebergs modeled from three locations (Norwegian Channel (NORC), west Norwegian coast (VORP) and Bear Island (BEAR)) reach ODP Hole 907A. However, in the mPWPALT scenario, icebergs from Arctic locations (SVAL and NSVAL2), east coast Greenland , east Iceland (EICE), and Nordic locations  all reach 907A.ODP Hole 642B. Icebergs from most seeding locations are unable to reach this site as it is covered by the strong northward flowing Norwegian Current. However, in the M2 scenario, modeled iceberg trajectories that can reach this site come from the Vøring Plateau and Bear Island, while in both the mPWPSTD and mPWPALT climate scenarios only Norwegian seeding locations NORC and VORP reach this site.3.4Figure The new occurrence data presented in this study support the presence of IRD throughout the Late Pliocene interval. In addition, the occurrence data documents a consistent presence of IRD at ODP Hole 909C Figure .4The results of this paper have combined modeled iceberg trajectories and the compilation of IRD data to provide further detail of circum‐Nordic Seas ice masses during the mPWP and the MIS M2.Modeled iceberg trajectories reaching Hole 911A during the M2 scenario are shown to have come from NSVAL. Modeled arctic icebergs  also come very close and so are plausible, if less likely, sources. Modeled icebergs from the southwest of Svalbard (SVAL) do not appear to cross Hole 911A during the M2 scenario which are moved westward in the stronger cyclonic gyre created by the subaerial Barents Sea Figure . It is aSTD scenario, modeled icebergs seeded from SVAL reached Hole 911A  and northern Svalbard (NSVAL) in the Arctic Ocean reach Hole 909C Figure . IcebergSTD scenario, modeled iceberg trajectories from SVAL still reach this site, as well as those seeded from Bear Island in the Barents Sea  appear to reach this site , Bear Island (BEAR), Svalbard (SVAL and NSVAL), the Arctic  and northeast Greenland  all reach ODP Hole 907A Figure  aided byIn the IRD record from Hole 907A, an increase is clearly seen during the cold MIS M2 Figure . The modSTD scenario, only modeled icebergs from Norway , where no ice would be expected during the warmest phases of the Pliocene  are the only ones modeled which produce icebergs reaching Hole 642B Figure . During STD scenario come from Norway (NORC and VORP; Figure ALT scenario shows NORC and VORP, again, to be the only two seeding locations with iceberg trajectories reaching Hole 642B.Modeled iceberg trajectories reaching this site during the mPWPThe IRD records for Hole 642B show variability in the amount of icebergs reaching this site. Contrasting the observations from the other sites, the MIS M2 is not characterized by a larger IRD peak for Hole 642B Figure . A minorThe lack of any modeled meltwater fraction at Hole 642B from Arctic, Greenlandic or Icelandic source regions is also in line with the lower count values of IRD found in the IRD data records.The modeled iceberg trajectory results show Norway as a potential source, which is not easily reconcilable with evidence from paleoenvironmental reconstruction.Pollen records from Hole 642B suggest cool temperate forest existed during the mPWP on the coast of Norway (Panitz et al., At face value, our modeling results agree that iceberg trajectories from Greenland or Svalbard do not cross Hole 642B. While our model results agree with the Jansen and Sjøholm  assessmeThis research has highlighted the need for further study of these sites as many questions remain outstanding. How does IRD reach Hole 642B on the Vøring Plateau during a warmer climate than present, when at present there are no icebergs? Where does the IRD come from that reaches Hole 907A? Is the IRD at Sites 909C and 911A deposited by icebergs or sea ice? The research done here relies on the assumptions that the ocean circulation in the model framework is correct and that IRD is transported to a site by icebergs and sea ice only. It also assumes that SSTs in the Nordic Seas are warmer than present (Bachem et al., 5Iceberg modeling and IRD data have, for the first time, been combined to provide clues to the extent of the ice margins surrounding the Nordic Seas. This novel combination shows the potential source locations from which icebergs reach ODP sites around the Nordic Seas. ODP sites around Svalbard receive icebergs throughout the mPWP. These IRD records are likely to be primarily sourced from Svalbard itself, although other Arctic icebergs may contribute.ALT climate scenarios, Hole 907A receives icebergs that are entrained in the eastern margins of the EGC. However, in the mPWPSTD scenario the current does not deliver modeled icebergs to Hole 907A. This means that only seeding locations from Norway reach this site which seems unlikely to be ice covered during the warmest parts of the Pliocene. This suggests that the changes in circulation associated with simulations incorporating altered paleogeography are important for the delivery of icebergs to 907A. The greater number of seeding locations with trajectories modeled to reach Hole 907A supports the evidence of higher amounts of IRD at this hole in contrast to that of Hole 642B. Under the mPWPALT scenario, icebergs from Svalbard, northern Greenland, and the east coast of Iceland are plausible IRD sources. Of these sources only northern Greenland is maintained in the M2 scenario. Abundance of IRD at Hole 642B is lower prior to the MIS M2 glacial period and at the MIS M1 warm peak. Combined with the Svalbard records, this suggests a consistent Svalbard source with northern Greenland icebergs swamping these from the MIS M2, apart from the very warmest period. Hole 642B receives icebergs only from the coast of Norway in the M2, mPWPSTD, and mPWPALT scenarios. No other seeding locations have modeled icebergs which reach this site during the warmer‐than‐modern mPWP, meaning the enigma of the persistent IRD off the coast of Norway remains, since a Norwegian source is inconsistent with present understanding of the Late Pliocene Norwegian climate.In the M2 and mPWPOverall, combining IRD records and iceberg modeling suggests that ice persists on Svalbard throughout the climate changes of the mPWP. It also suggests that ice is present on the northeast Greenland coast for much of the mPWP but that these locations do not produce icebergs or, at least, not sufficiently large enough icebergs, to reach the ODP Sites before melting during the warmest periods. There are several locations from which icebergs are seeded which reach each location in different climate scenarios. The altered paleogeography of Hill  is impor


Gracilariopsis lemaneiformis. In the present study, two novel angiotensin-converting enzyme (ACE) inhibitory peptides, FQIN [M(O)] CILR, and TGAPCR, were screened and identified from G. lemaneiformis protein hydrolysates by LC-MS/MS. The IC50 values of FQIN [M(O)] CILR and TGAPCR were 9.64 ± 0.36 μM and 23.94 ± 0.82 μM, respectively. In the stability study, both peptides showed stabilities of pH, temperature, simulated gastrointestinal digestion, and ACE hydrolysis. The Lineweaver–Burk plot showed that the two peptides were noncompetitive inhibitors of ACE. Molecular docking simulated the intermolecular interactions of two peptides and ACE, and the two peptides formed hydrogen bonds with the active pockets of ACE. However, FQIN [M(O)] CILR was more closely linked to the active pockets of ACE, thereby exerting better ACE inhibition. Spontaneously hypertensive rats (SHRs) were studied with an oral dose of 10 mg/kg body weight. Both peptides reduced systolic blood pressure (SBP) and diastolic blood pressure (DBP) in SHRs, of which FQIN [M(O)] CILR was able to reduce the systolic blood pressure by 34 mmHg (SBP) (p < 0.05). Therefore, FQIN [M(O)] CILR was an excellent ACE inhibitory peptide.A variety of biologically active products have been isolated from  Ca2+ stimulates the nitric oxide synthase (eNOS) reaction to produce NO, which results in vasodilation and reduced blood pressure  CILR and TGAPCR. The result shows that the peptides are two novel ACE inhibitors with the IC50 value of 9.64 ± 0.36 μM and 23.94 ± 0.82 μM, respectively.The physicochemical properties of the peptide were obtained by online database search. The pH and thermal stability of the peptides were measured. The results show that the peptides could maintain their ACE inhibitory activity after pH and heat treatment a,b. BothThe stability of the peptides in the simulated gastric juice and intestinal fluid is shown in −1·mL·min) decreases as the concentration of inhibitory peptide increases. This is due to the reduction of the apparent catalytic efficiency of ACE, which leads to the decrease of the enzymatic reaction efficiency  CILR and TGAPCR are noncompetitive inhibitors of ACE. ficiency . At the The Ki of FQIN [M(O)] CILR and TGAPCR are 0.71 ± 0.04 mM and 0.86 ± 0.06 mM, respectively. The Ki value indicates the affinity of ACE inhibitory peptide to ACE. The lower the inhibition constant (Ki), the higher the affinity of the inhibitors to ACE. The results show that FQIN [M(O)] CILR can produce better inhibitory activity than TGAPCR.−1 and 105.509 kcal·mol−1, respectively. The -CDOCKER ENERGY and -CDOCKER INTERACTION ENERGY of TGAPCR are 90.4226 kcal·mol−1 and 105.509 kcal·mol−1, respectively (CDOCKER was used to discover the ligand−receptor interaction mechanism. The -CDOCKER ENERGY and -CDOCKER INTERACTION ENERGY of FQIN [M(O)] CILR are 158.117 kcal·molectively . Both peThere are three main active site pockets in the ACE molecule. S1 pocket has three residues, Ala354, Glu384, and Tyr523. S2 pocket comprises of Gln281, His353, Lys511, His513, and Tyr520. S1′pocket includes residue Glu162 . Inhibitp < 0.05), with the largest decrease of SBP from 204 to 170 mmHg occurring at 2 h. The SBP then began to recover and maintained a level of 200 mmHg at 8 h (The in vivo antihypertensive activity of peptides was assessed by SHRs with a gavage dose of 10 mg/mL. Both FQIN [M(O)] CILR and TGAPCR decreased the SBP of SHRs. FQIN [M(O)] CILR significantly decreased the SBP between 2 to 4 h (g at 8 h a. It wasp < 0.05), then restore to its original level at 8 h. TGAPCR could decrease DPH to 120 mmHg at 1 h and maintain the DPH until 4 h. TGAPCR can maintain low DBP for a longer period of time compared to FQIN [M(O)] CILR. In general, FQIN [M(O)] CILR has a better antihypertensive effect in SHRs than TGAPCR.In addition, both FQIN [M(O)] CILR and TGAPCR could also affect DBP b. FQIN  CILR has poor water solubility. However, in our study, we found that the FQIN [M(O)] CILR had good water solubility when the concentration was lower than 10 mg/mL. The solubility gradually decreased when the concentration was above 10 mg/mL. The hydrophobicity of FQIN [M(O)] CILR is related to the presence of hydrophobic amino acids, such as Phe, Leu, Ile, and Met. The hydrophobicity of FQIN [M(O)] CILR may facilitate the binding of peptides to the hydrophobic active center of ACE, in turn, produce better inhibitory activity.Small molecule peptides can cross the intestinal wall and enter the blood circulation. Prior to this, peptides must resist the gastrointestinal digestion and maintain their integrity, otherwise their biological activity may be activated or inactivated . Our resAngiotensin I-converting enzyme is a carboxypeptidase with very broad substrate specificity . Some ACThere are multiple inhibition patterns of ACE inhibitory peptides: competitive inhibition, noncompetitive inhibition, and mixed-competitive inhibition . The res2O molecule  CILR and TGAPCR not only showed strong ACE inhibitory activity in vitro but also showed strong antihypertensive effects in SHRs. This study suggested that these two natural peptides have the potential to be developed for both antihypertensive drugs and health care products.FQIN [M(O)] CILR and TGAPCR are two novel ACE inhibitory peptides, isolated from the trypsin hydrolysate of ACE (from rabbit lung), hippuryl-histidyl-leucine (HHL), high-performance liquid chromatography (HPLC)-grade acetonitrile (ACN), trifluoroaceticacid (TFA) and porcine pancreatin (8× USP) were purchased from Sigma-Aldrich Co. . Trypsin , pepsin , and captopril (>99% purity) were obtained from Solarbio . All other chemical reagents were of analytical grade.G. lemaneiformis was hydrolyzed with 2% trypsin  for 2 h. The 80% alcohol was used to precipitate the impurities in the hydrolysates. The supernatant was concentrated to remove ethanol. Next, the concentrated supernatant was fractionated by ultrafiltration  and the fraction (<3 KDa) was collected and freeze-dried before storage at −80 °C.m/z were selected to obtain MS data. Peptide sequences were determined based on the MS/MS spectra and Mascot 2.2  searches of dataset of G. lemaneiformis .Peptide sequences were determined according to Kai Lin . IdentifThe inhibitory peptides were chemically synthesized in Shanghai Qiangyao Biotechnology Co., Ltd. . The peptides were synthesized using the Fmoc solid-phase method. To increase the stability of FQINMCILR, Methionine is selectively modified to methionine sulfoxide (FQIN [M(O)] CILR). The purity of the two peptides is 97.85% and 97.59%.inhibitor is the relative area of the hippuric acid (HA) peak obtained from the reaction of ACE and HHL with inhibitor. Acontrol is the relative area of the hippuric acid (HA) peak obtained from the reaction of ACE and HHL without inhibitor. IC50 is defined as the concentration of peptides that can inhibit half of the ACE activity.ACE inhibition activity was assayed according to Cushman and Cheung, with some modification . ACE washttp://crdd.osdd.net/raghava/toxinpred/). The online Innovagen server was used to evaluate the solubility of the peptide (www.innovagen.com/proteomics-tools).The properties of ACE inhibitory peptides are important for future research. These parameters can be retrieved from the online database. The ToxinPred server can analyze toxicity of the peptides (The synthetic peptide solutions (0.5 mg/mL) were incubated at different temperatures  for 2 h. Next, 20 μL of peptide was used to assay the ACE inhibitory activity using the above method .The synthetic peptide solutions (0.5 mg/mL) were incubated at different pH levels  for 2 h, and then solutions were neutralized to pH 7.0 . Then, 2Gastrointestinal stability of peptides was evaluated in vitro ,47. PeptThe stability of peptides against ACE was assayed according to Salampessy. J and Fujita. H ,49. 30 μX-axis of the plot, of which the Y-axis displayed the slopes of Lineweaver-Burk line and the X-axis indicted peptides concentrations  CILR and TGAPCR can reduce blood pressure of SHRs. Therefore, these peptides have the potential to treat hypertension, while G. lemaneiformis can be developed as an antihypertensive food.Two ACE inhibitory peptides, identified as FQIN [M(O)] CILR and TGAPCR from


A prospective single-center study.Assess to what degree contamination of pedicle screws occur in standard intraoperative practice and if use of an impermeable guard could mitigate or reduce such an occurrence.Two groups of sterile prepackaged pedicle screws, one with an intraoperative guard (group 1) and the other without such a guard (group 2), each consisting of 5 samples distributed over 3 time points, were loaded onto the insertion device by the scrub tech and left on the sterile table. Approximately 20 minutes later, the lead surgeon who had just finished preparing the surgical site touches the pedicle screw. Then instead of implantation it was transferred to a sterile container using fresh clean gloves for bacterial and gene analysis. Guarded screw implies that even after unwrapping from the package, the screw carries an impermeable barrier along its entire length, which is only removed seconds prior to implantation.5 to 107 (colony forming units/implant) with bacterial genus mostly consisting of Staphylococcus and Micrococcus, the 2 most common genera found in surgical site infection reports. The common species among them were Staphylococcus epidermis, Staphylococcus aureus, Micrococcus luteus, and Staphylococcus pettenkoferi, whereas the guarded pedicle screws showed no bioburden.The standard unguarded pedicle screws presented bioburden in the range of 10Shielding the pedicle screws intraoperatively using a guard provides a superior level of asepsis than currently practiced. All unshielded pedicles screws were carrying bioburden of virulent bacterial species, which provides an opportunity for the development of postoperative infections. Researchers have already detected colony-forming units (CFUs) of bacteria on exposed sterile implants and the gloves from the surgeon, scrub nurse, and assistants. Furthermore, they have also found significant reduction in surgical site infection (SSI) rates in spine surgery due to stand-alone measures, like glove change before touching the implants.313 In addition, recent literature is growing with studies that look at process variables in the operating room (OR), for example, keeping implants covered until the immediate time of use, reducing OR traffic, avoiding reprocessing of implants , and to avoid touching the implants altogether. Last, contemporary fields like plastic surgery and general surgery have already adapted such practices, through the use of Keller funnels, and wound protectors, respectively, which prevent cross-contamination between the wound, gloves, and the implants (irrigation fluid for the latter).1215 The purpose of this study was 2-fold, to evaluate the bioburden and the species of bacteria present on each pedicle screw being implanted, and the efficacy of an intraoperative guard in reducing such occurrences.Spinal infections continue to be a significant problem both clinically for patients and socioeconomically in terms of growing health care costs. The average hospitalization cost resulting from such infections is $63 000 per case at an average frequency of 5.5% of cases (built in cost of $3465 for every surgery).No institutional review board approval was needed for this study. The study consisted of 2 groups of sterile prepackaged pedicle screws, one with an intraoperative guard (group 1) and the other without such a guard (group 2). Each group consisted of 5 samples distributed over 3 time points , and each was performed in a different OR, with a different surgeon and surgical staffs . During 5 to 107 per sample. The colonies continued to grow until confluency was reached. No CFU growth occurred in plates extracted from group 1 for the entire duration of the incubation period  and the treatment (guarded) groups are single-use, presterilized pedicle screws, and therefore, the preoperative preventative measure was in place for both the groups. The current study focusses on the second step toward infection or inflammatory risk prevention, that is, the intraoperative preventative measure.Recent studies looking at implant cleanliness to reduce risk of infection or inflammatory responses are categorized into (1) preoperative preventative measure and (2) intraoperative preventative measure. Preoperative preventative measure focuses on providing the cleanest and sterile implant, free of contaminants  to the OR. Whereas intraoperative preventative measure focuses on avoiding contamination of the clean and sterile implant, provided to the OR, during the surgery. An example of preoperative preventative measure is the study done by Litrico et al,16 The virulence is the microorganism’s ability to infect the host. Although many bacterial species have been identified to cause SSI, the most common ones, Staphylococcus epidermidis and Staphylococcus aureus, are always present at the vicinity as part of a patient’s own flora. In addition, they have the potential to form biofilms, secluding itself from macrophages or other immune responses at the host site.1719 The host sites in spine surgery are the pedicles of the vertebrae. This in combination with availability of metal surface (pedicle screws) provides a conducive environment for the bacteria to grow. Besides clinical signs of infection, evidences have also shown occurrence of occult infection, which manifests as hardware loosening or dislodgement, thus requiring a revision surgery.21 Last, the dose dictates how much bacterial bioburden the “sterile” implant carries, after handing and at implantation. This study characterizes the dose and the virulence, that is, type and quantity of bacteria species present, carried through a pedicle screw during spine surgery, alongside establishing an efficient method to mitigate it.The study presents a pivotal single-center data on a stand-alone method of preventing microbial dose delivery through pedicle screws. The binary nature of the data across all the sample pairs highlights its clinical significance, that is, in terms of dosage, presence and absence of guard is equivalent to presence or absence of microbial contaminants. Four key factors that define the pathogenesis of SSI are the virulence, host site, immunity, and dosage.22 Therefore, any practice that could cost-efficiently reduce the current bioburden beginning transferred to the patients would be desirable. Such a practice has been common in breast augmentation surgeries, where the surgeons use a sterile guard called Keller funnel to handle the breast implants.14 Similarly, wound protectors have become the standard of practice in general surgery to avoid contamination of the wound irrigation fluid from the wound edges.15 The premise of both these practices includes provision of an impermeable guard. This guard, in essence, avoids contact between the contaminated object and the liquid or implant being delivered into the surgical site. Rehman et al have recently shown a significant reduction in SSI rate by avoiding handling the screw with blood-stained gloves.12 In theory, adoption of this practice would require universal education and is dependent on the consistency and compliance of every individual surgeon. Even then, a pedicle screw is held by a scrub tech during unwrapping and attachment to an insertion device, followed by its placement next to other dirty surgical instruments. In contrast, the current study evaluates a guard that provides uninterrupted protection from all the aforementioned elements. Therefore, based on literature, contemporary practices, and the data from the current study, it seems rational to hypothesize that such a guard could mitigate undue infections, and thus reduce the rate of SSI, post spinal fusion.Aseptic surgical techniques rely on methods that reduce or at least avoid transfer of contamination, a majority of which are microbial organisms and not visible to naked eyes. Over the past decade, the use of antibiotics, both local and systematic, have increased enormously. As a by-product this has led to the increase in the number and type of resistant bacterial species, to an extent that the World Health Organization ranked this concern next to issues like terrorism and global warming.The results of this study provide preliminary clinical evidence that further improvement in asepsis is possible through simple cost-efficient innovations in intraoperative practices. The method proposed involves the use of an intraoperative guard, which shields the pedicle screws, until implantation, without affecting other preparatory process like unwrapping, attaching to the instrument, and so on.


Flavonoids were most effective in inhibition against CYP2A6 and CYP2A13, followed by thiophenes, and hirsutinolides. Hirsutinolides and thiophenes exhibited mechanism-based inhibition and in irreversible mode against both enzymes. The inactivation kinetic KI values of hirsutinolides against CYP2A6 and CYP2A13 were 5.32–15.4 and 0.92–8.67 µM, respectively, while those of thiophenes were 0.11–1.01 and 0.67–0.97 µM, respectively.The human liver cytochrome P450 (CYP) 2A6 and the respiratory CYP2A13 enzymes play role in nicotine metabolism and activation of tobacco-specific nitrosamine carcinogens. Inhibition of both enzymes could offer a strategy for smoking abstinence and decreased risks of respiratory diseases and lung cancer. In this study, activity-guided isolation identified four flavonoids  Tobacco smoking can be the main cause of adverse human health effects and many tobacco-related diseases, including respiratory diseases and lung cancer, which is a leading cause of cancer death worldwide. The use of tobacco products continues to be widespread and might be responsible for approximately six million deaths in the world each year, as estimated by the WHO,,In human, the liver heme-containing cytochrome P450 (CYP) 2A6 and the respiratory tract-expressed CYP2A13 play role in nicotine metabolism and they mediate metabolic activation of NNK to exhibit carcinogenic potentialVernonia cinerea against CYP2A6 and monoamine oxidases that are therapeutic targets for the abstinence of nicotine addictionV. cinerea for smoking cessation, possibly via maintaining blood nicotine and dopamine levelsV. cinerea (Asteraceae) is a perennial herbaceous medicinal plant in the Asteraceae family and is found distributed in Asia, India and Africa. It has been used for various remedies, such as fever, cold, sore throat, malaria, asthma, bronchitis,V. cinerea comprises of compounds that exhibit inhibitory effects against CYP2A13 has not been explored and it should be beneficial to gain an understanding of inhibitory roles of V. cinerea compounds against both CYP2A6 and CYP2A13 enzymes. Moreover, our prior search for naturally occurring plant compounds that could inhibit CYP2As has revealed that Pluchea indica possesses promising inhibitory effects against CYP2As enzymes. P. indica (L.) Less (Asteraceae) is a shrub found widely distributed in Asia. It has been used in traditional medicine for treating respiratory diseases, including sore throat and tuberculosis, and its leaves have been reported pharmacological activities such as antituberculosis, anti-inflammatory and antiproliferative effects on nasopharyngeal cancer cellsWe previously reported inhibition effects of flavonoids and sesquiterpene lactones isolated from 1), luteolin (2), quercetin (4), from both V. cinerea and P. indica and chrysoeriol (3) from V. cinerea), four hirsutinolide-type sesquiterpene lactones 5–8 from V. cinerea, and three acetylenic thiophenes 9–11 from P. indica . Kinetics and modes of inhibition of purified compounds were determined. Mechanism-based inhibition presented by concentration-, time- and NADPH-dependent inhibition of compounds was also examined. Investigation of the three different chemical structural types of compounds might also give insights into the nature of inhibitory compounds against human CYP2As enzymes.In this study, four flavonoids (apigenin (S)-nicotine, glutathione (GSH), catalase, semicarbazide, potassium ferricyanide and coumarin were purchased from Sigma-Aldrich . Silica gel (230–400 mesh), pre-coated Kiesel gel 60 F254 thin-layer chromatography (TLC) sheets and Sephadex LH-20 gel were obtained from Merck . AR-grade ethanol (EtOH), methanol, n-hexane, ethyl acetate (EtOAc), diethyl ether and HPLC-grade acetonitrile (ACN) were supplied by RCI Labscan .Nicotinamide adenosine diphosphate reduced form (NADPH), methoxsalen, . The EtOH crude extract and partitioned hexane and EtOAc extracts of V. cinerea were prepared and subjected to in vitro inhibition assay using 10 µg/ml each as described1) and 6 . Fractions 7–9 (diethyl ether: EtOAc =30–60: 70–40) were combined and further purified using silica gel chromatography to yield seven subfractions, followed by sephadex LH-20 gel chromatography and HPLC using conditions previously described4) in subfraction 7.7) and four hirsutinolide-type sesquiterpene lactones (8α-(2-methylacryloyloxy)-hirsutinolide-13-O-acetate (5), 8α-tigloyloxyhirsutinolide-13-O-acetate (6) in subfraction 7.4, 8α-(4-hydroxymethacryloyloxy)-hirsutinolide-13-O-acetate (7) and 8α-(4-hydroxytigloyloxy)-hirsutinolide-13-O-acetate (8) in subfraction 7.5) comprised inhibitory activity against CYP2A13. These compounds also previously exhibited inhibition effect on CYP2A6 enzymeAerial parts of P. indica were purchased from the Community Enterprise, Bon subdistrict, Khlung, Chantaburi province, Thailand and identified by Dr. Benchawan Chewprecha, Department of Biology, Faculty of Science, Burapha University, Thailand. A voucher specimen (BCSK-003) was deposited at the Faculty of Science, Burapha University. The dried and ground material (1 kg) was macerated in 95% EtOH three times at room temperature to yield 60 g of EtOH extract and partitioned with hexane and EtOAc, yielding hexane (17 g), EtOAc (15 g) and aqueous (28 g) soluble extracts.The aerial parts of 4, 5.5 mg), luteolin  and apigenin , respectively.The active components in EtOAc extract were separated by silica gel column chromatography, followed by successive elution with EtOAc: diethyl ether (0:100 to 100:0), and a final elution with methanol to afford eight fractions. Fractions 4 (1.5 g) and 5 (0.9 g) containing at least 50% inhibition effects on both enzymes at 10 µg/ml were subjected to HPLC analysis, using semipreparative RP-18 column . The solvent system-contained ACN and water and was set as follows: 0 min, 30% v/v ACN in water; 0–5 min, a linear gradient of 30–40% ACN; 5–10 min, 40–50% ACN; 10–15 min, 50% ACN; 15–20 min, 50–100% ACN; 20–25 min, 100% ACN; 25–30 min, cycled back to 30% ACN, with total running time of 35 min and flow rate at 1.5 ml/min. Three flavonoids eluted at 8.7, 9.7, 11.48 min were identified as quercetin , compounds in fractions 4 and 5 that were eluted at 16.36 min and 13.1 min were 2-(prop-1-inyl)-5- thiophene  and 2-(prop-1-inyl)-5- thiophene , respectively. The chemical structures of compounds 9–11 were elucidated based on UV spectra determined on HPLC, electrospray ionisation source equipped with tandem mass spectrometer (ESI-MS/MS) and nuclear magnetic resonance  spectra analysis, compared to those of previous reports,Hexane extract was subjected to silica gel column chromatography and successively eluted with a solvent system containing hexane: EtOAc (100:0 to 0:100), and a final elution was done with methanol to resolve seven fractions. The yields obtained from fractions 1–7 were 0.38, 2.55, 4.25, 6.39, 1.75, 1.19 and 0.45 g, respectively. Fractions 2–4 were further separated by sephadex LH-20 gel column chromatography and eluted with chloroform: methanol (2:1) to afford five fractions. Fractions 2 (6.46 mg), 4 (8.84 mg) and 5 (15.79 mg) containing inhibitory activity toward both enzymes were proceeded to the final purification step by HPLC, with stepwise elution utilising solvent system containing ACN and water (flow rate at 1.5 ml/min) that was set as follows: 0 min, 25% v/v ACN/water, a linear gradient of 25–100% ACN; 0–20 min,100% ACN; 25–30 min, 100–25% ACN; 30–35 min, a final equilibration at 25% ACN and with a total running time of 35 min. The compound in fraction 2 eluted at 22.0 min on HPLC was identified as 2--5-(4-acetoxy-3-hydroxybuta-1-yn-1-yl) thiophene , was performed at room temperature as describedex = 355 nm, λem = 460 nm. Inhibition effects were determined by incubation with extracts, fractions or purified compounds with coumarin substrate in the reaction mixture and calculated as per cent relative inhibition compared with vehicle control reaction50 values were calculated using GraphPad Prism 5 . Inhibitory effect of plant extracts, fractions, subfractions and constituents was determined by inhibition assay on CYP2A6- and CYP2A13-mediated coumarin 7-hydroxylation. The recombinant human CYP2A6, CYP2A13 and rat NADPH-dependent cytochrome P450 oxidoreductase (CYPOR) proteins were expressed, purified, according to the methods previously describedKi) were determined by performing inhibition with coumarin substrate (0–40 µM) and with different concentrations of each test compound (0–100 µM). Compounds that primarily showed an increased inhibition under 10-min pre-incubation condition in the presence of NADPH compared to the absence of NADPH were further explored for time- and NADPH-dependent inhibition that defined a mechanism-based inhibition (MBI). MBI requires NADPH and time to generate reactive intermediate as inhibitor and ultimately inactivates the enzyme. MBI was evaluated by pre-incubation with each test inhibitor in the reaction at different time periods  in the presence of NADPH for time-dependent assay, as describedKI and Kinact, were obtained from double reciprocal plots of kobs versus inhibitor concentrations by linear regression analysis on GraphPad Prism 5. Methoxsalen was used as a control mechanism-based inhibitor for time- and NADPH-dependent inhibition assays. Effects of trapping agents , 70 µM potassium ferricyanide and dialysis were performed as previously described with compounds that exhibited MBI patternMode of inhibition and inhibition kinetics . Results with p ≤ 0.05 were considered being significantly different.Data shown in V. cinerea exhibiting inhibition against the recombinant CYP2A13 were examined using enzymatic inhibition assays and their IC50 values were compared to those previously reported for the recombinant human CYP2A6 (11). Extracts and constituents of P. indica were also investigated inhibition activities against CYP2A6 and CYP2A13 enzymes. Upon compound purification from both plant extracts using activity-guided isolation, we obtained compounds that could be grouped into flavonoids, hirsutinolide-type sesquiterpene lactones and acetylenic thiophenes. In V. cinerea, we found flavones 1–3 , one flavonol  and hirsutinolides 5–8 as active components in inhibition against both enzymes, while flavonoids apigenin, luteolin, quercetin and acetylenic thiophenes 9–11 were found active in P. indica. With the purification conditions described, all active components in V. cinerea resided in the EtOAc extract and there was mostly chlorophyll found in hexane extract that elicited inhibition on CYP2A13. This was also previously found for the inhibition of CYP2A6 (11). The partially purified chlorophyl in V. cinerea hexane extract could inhibit CYP2A6  and CYP2A13 . In P. indica, flavonoids 1, 2, 4 were found in EtOAc extract and thiophenes 9–11 were in hexane extract.In this study, extracts and constituents of 50 values, modes of inhibition and kinetic parameters were determined for each of the purified compounds and are displayed in 1–11 displayed higher inhibition effects on CYP2A13 than CYP2A6, especially flavonoids 1–4 possessed approximately 1.4- to 18-fold lower IC50 values against CYP2A13 than CYP2A6  was less active in inhibition on both enzymes than flavones apigenin (1) and luteolin (2). The methoxylated flavone, chrysoeriol (3), was similarly effective as the flavone 1, 2 in inhibition of CYP2A6 but was poorer for CYP2A13 inhibition. Similarly, previous study observed that most flavones tested comprised high inhibitory activity towards CYP2A6 and CYP2A13, with lesser activity found for flavonolsAmong flavonoids displayed in 1–11 in the presence of NADPH to primarily test for time-dependent inhibition (TDI) indicated that hirsutinolides 5–8 and thiophenes 9–11 revealed an increased inhibition towards both enzymes compared to coincubation condition (5–11 further exhibited TDI and NADPH-dependent inhibition (unreported data), indicating that inhibition of hirsutinolides 5–8 was MBI towards CYP2A13, similar to the previous report on CYP2A6 (11), and thiophenes 9–11 were mechanism-based inhibitors of both enzymes . The Ki values were determined for all purified compounds and showed that the values were in similar trend as their IC50 values against both enzymes  in the inhibition reaction mixture were performed. As shown in 5–8, similar to effects previously observed on CYP2A6 (11). Similarly, mechanism-based inactivation of both CYP2A6 and CYP2A13 by thiophenes 9–11 could not be reversed  for natural plant compounds have been reported, for example, the values of rhinacanthin-A, rhinacanthin-B, rhincanthin-C, decursinol angelate and selegiline, were 0.69, 0.44, 0.97, 2.42 and 15.6 µM, respectively, against CYP2A6,,KI values for phenylpropyl isothiocyanate, rhinacanthin-A, rhinacanthin-B, rhinacanthin-C, phenylhexyl isothiocyanate and tert-butyl isothiocyanate against CYP2A13 were 0.14, 0.42, 0.16, 1.68, 1.1 and 4.3 µM, respectively,9–11  could bind CYP2As comparable to rhinacanthin-B and rhinacanthin-C and were more potent than decursinol angelate and selegiline. Moreover, the major flavonoid luteolin, hirsutinolide 6 (in V. cinerea) and thiophene 11 (in P. indica) showed inhibition on CYP2A6- and CYP2A13-mediated nicotine metabolisms , implicating that the active compounds 1–11 in both plants might also inhibit nicotine metabolism catalysed by both enzymes. Furthermore, the methoxylated flavone chrysoeriol 3 expressed potent inhibition on both CYP2As and MAO-B, with Ki values within 1 µM (11 and results reported herein), and thiophenes 9–11 were irreversible inhibitors of both CYP2As. These compounds might possibly serve as potential lead compounds for the development of chemopreventive agents for cigarette smokers. However, whether they could selectively inhibit CYP2A6 and CYP2A13 requires further investigation. It has been reported that chronically exposure to nicotine could cause proinflammatory effect on neutrophils and oxidative damage in lung of the experimental ratP. indica and V. cinerea might also be effective in the alleviation of oxidative damage in lung caused by nicotine exposure as they have been reported comprising anti-oxidative and anti-inflammatory activities,V. cinerea and P. indica and the composite of these compounds or extracts of these two medicinal plants might have implications for smoking cessation and reduced risks of respiratory diseases and lung cancer.Comparing among the three groups of compound structure, judging from ICf CYP2As . Similarn CYP2A6  and 2, p


The purpose of the study was to investigate cross-language effects in verbal fluency tasks where participants name in English as many exemplars of a target as they can in one minute. A series of multiple regression models were used that employed predictors such as self-rated proficiency in English, self-rated proficiency in a language other than English, a picture naming task used to measure productive vocabulary, the percentage of English use, and the frequency of language switching. The main findings showed that self-rated proficiency in the non-English language accounted for unique variance in verbal fluency that was not accounted for directly by self-rated proficiency in English. This outcome is consistent with cross-language interference, but is also consistent with an account that assumes bilingual disadvantages in verbal fluency and picture naming are due to bilinguals having weaker links between semantic concepts and their phonological form. The present study is also discussed in terms of a broader framework that questions whether domain-general inhibition exists and also whether it plays an important role in bilingual language control. Both category fluency  and letter fluency  were tested in English. Like the vast majority of studies  language should predict unique variance in verbal fluency performance apart from that accounted for by the degree of proficiency in English.The weaker links account of bilingual disadvantages in picture naming and verbal fluency would make the same prediction, namely, that L2 proficiency has a negative effect on L1 verbal fluency and accounts for unique variance beyond that accounted for directly by L1 proficiency. The weaker links account relies on the cumulative history of language use prior to a verbal fluency test. That is, using L2 steals time from using L1 and results in L1 links that are weaker in bilinguals compared to monolinguals. Furthermore, as L2 use increases (and L2 proficiency improves) the strength of the L1 links will lag further behind those for monolinguals. Thus, the weaker links perspective also predicts that increases in L2 proficiency will be associated with decreases in L1 verbal fluency—not because activated L2 representations compete, but because L2 practice comes at the expense of more L1 use.A problem with testing this prediction is how to obtain a pure measure of lexical knowledge in both English and the Other language that is not influenced by additional factors. To advance the argument, assume, for now, that self-ratings by bilinguals for general proficiency in English and their Other language provided reasonably valid measures of lexical knowledge   correlatAlthough bilingualism researchers routinely use self-ratings of proficiency in understanding, speaking, and reading  these seThe current study reports new analyses of an existing dataset and, consequently, many additional details are presented in the original study .Participants in  were 236In the verbal fluency tasks, participants were asked to produce as many words as they could in one minute that followed the prescribed rule. There were two trials of single-letter fluency  and two trials of single-category fluency . The target categories were selected on the basis of previous work with the intent to have targets of comparable and intermediate difficulty. They were not explicitly told to respond in English, but all participants did so. Between trials, bilinguals would occasionally mention that they thought of the name of a vegetable or instrument in their other language, but did not know it or could not think of the word for it in English.For completeness, we show the mean number of correct responses, the number of intrusions from an incorrect category, and the mean number of repeated responses for each of four verbal fluency tasks given to each participant. Our analyses focus on the single-category and single-letter trials. Inspection of t(127) = 1.02, p = 0.311. Thus, in all subsequent analyses any reference to verbal fluency refers to the composite mean based on the two categories (instruments and vegetables) and the two letters (“F” and “A”).The distinction between category- and letter-fluency has been of theoretical interest because of the twin hypotheses that there is a bilingual advantage in executive functioning (EF) and that letter-fluency may require more EF. If both hypotheses were correct, then any bilingual disadvantage in category fluency might be reduced in the letter-fluency task because bilinguals could recruit their superior EF to close or eliminate the performance gap. Although some earlier studies reported a Group  by Task (category fluency versus letter fluency) interaction with just such a pattern, Paap et al.’s review  showed tThe primary hypothesis of this study is that Other language proficiency can account for unique variance in an English-language test of verbal fluency when English language proficiency is included in the regression model. Model 1. English Proficiency and Other Proficiency are the two predictors. Tested as a single bivariate correlation, it is not surprising that self-rated proficiency in English predicts the number of correct responses in a test of English verbal fluency, r(128) = +0.27, p = 0.001. In regression Model 1, the two predictors combined to account for about 16.4% of variance in the composite verbal fluency scores, R = 0.406, p < 0.001). The standardized coefficients showed that both were significant predictors, β = +0.18, p = 0.033 for English fluency and β = −0.32, p <0.001 for Other Proficiency. As predicted, the correlation for the test language (English) was positive, but negative for the competing language (Other). Observing a significant negative effect of Other Proficiency is consistent with the hypothesis that there is cross-language interference. Impressively, it is numerically more potent than proficiency in the test language. Given that English Proficiency and Other Proficiency are correlated with one another, r(128) = −0.27, p = 0.001, it is prudent to assess collinearity. Following the guidelines provided by Field [Model 2. Many of our bilinguals  do not write or read their non-English language. In our past publications, we have considered proficiency in spoken language and have ignored reading. In this past work, if we had included reading and writing in our measure of other-language proficiency, then some native and highly fluent speakers would have mediocre proficiency scores even though they were native speakers and listeners. Thus, the analyses in the original paper [r(125) = +0.57, p < 0.001. However, does Other Proficiency still account for unique variance in verbal fluency when both written and spoken proficiency are included in the model? Yes, self-related proficiency in the Other language accounts for unique variance in English verbal fluency even when both spoken English proficiency and reading English proficiency are included in the model, β= −0.31, p < 0.001. Thus, Model 2 (like Model 1) is consistent with the hypothesis that cross-language effects play a significant role in verbal fluency.al paper  and in MModel 3. Researchers in bilingual language control have emphasized the importance of several facets of current use and these include the relative use of each language and the frequency of language switching [p = 0.117 and for Frequency-of-Language Switching, β= +0.22, p = 0.09. Again, the most important result is that Other Proficiency accounts for unique variance even when English Proficiency, percentage of current English use, and frequency-of-switching are taken into account, β= −0.35, p = 0.004.witching ,7. To thModel 4. The original dataset includes another measure that is arguably more directly related to English vocabulary knowledge and that is the number of correct responses on the Multilingual Naming Task (MINT) [p < 0.001. Furthermore, Other Proficiency does not account for any unique variance not accounted for by MINT, β= −0.10, p = 0.206.k (MINT) . Model 4Model 5. Model 4 potentially upsets the apple cart. It clearly calls into question the conclusion that the earlier models implicate cross-language interference as Other Proficiency does not account for unique variance when MINT scores are the other predictor. This might be expected if MINT scores were a more valid measure of vocabulary knowledge than self-ratings of general English proficiency. On the surface, this seems reasonable as self-ratings are often viewed as subjective, fuzzy, and open to bias. Furthermore, picture naming (MINT) is an objective measure that enjoys face validity as a measure of productive vocabulary. Perhaps the independent contribution of Other Proficiency observed with Models 1 to 3 was enabled by a mediocre measure of English vocabulary, namely, self-rated English Proficiency.p < 0.001.Although the preceding argument seems quite plausible, it fails to consider a potentially critical aspect of picture naming , namely that picture naming may also be influenced by cross-language interference. If MINT performance already reflects cross-language interference, then there would be little or no unique variance for Other proficiency to account for when predicting verbal fluency. If this is the case, then Other Proficiency should strongly predict MINT scores when they trade places compared to Model 4. Thus, Model 5 used English Proficiency and Other Proficiency as predictors, but uses MINT scores as the outcome variable. The key result is that Other Proficiency accounts for unique variance in MINT scores, β= −0.33, The first three regression models established that Other proficiency accounts for unique variance in English verbal fluency beyond that accounted for by English proficiency. This implicates a cross-language effect in verbal fluency in a single-language context. However, Model 4 replaced self-rated English proficiency with MINT English scores and the inclusion of Other proficiency no longer improved the model. If the outcome of Model 4 was simply due to MINT being a more reliable measure of English proficiency compared to self-ratings, then this would undermine the evidence for cross-language effects obtained in Models 1 to 3. Alternatively, we suggested that MINT may not be a more reliable measure, but rather one that is, itself, influenced by cross language interference. If MINT scores are a composite of benefits from greater English proficiency and cost from greater Other proficiency, then self-rated Other proficiency is less likely to account for additional unique variance in English verbal fluency. The coherence of this account rests on the assumption that self-rated English proficiency is less effected by Other proficiency (and hence Other proficiency can scoop up variance unaccounted for by English proficiency) but that MINT scores are influenced by Other proficiency. We consider next the plausibility of this account by speculating on how bilinguals self-rate their proficiencies and linking these speculations to work on self-ratings recently published by Tomoschuk and colleagues .The subjective experience of proficiency or fluency for a bilingual could rely on three different frames of reference. One framework for a bilingual is to compare fluency in one language to that of the other. Tomoschuk et al. tap into this type of comparison when they explicitly ask bilinguals to rate the degree to which one of their languages is stronger and conclude that these dominance ratings are more closely aligned with their MINT scores than numerically rating individual languages on a seven-point scale. Thus, in order to explain the difference between absolute ratings of proficiency and dominance ratings, one must conclude that self-ratings do not heavily rely on the relative comparison of fluency in one language compared to the other.bilinguals who have English as a native language”“My dominant language is English and I am very fluent AND just as good as other . However, we know that, on average, there are bilingual disadvantages compared to monolinguals in verbal fluency and picture naming. Thus, self-reflecting on comparisons to other bilinguals will lead to an overestimation of proficiency compared to MINT. We suggest that the overestimate is caused by MINT’s sensitivity to cross-language effects and the absence of such effects when other bilinguals serve as the frame of reference for self-ratings. This agrees with Tomoschuk et al.‘s assertion that “One possible explanation for this pattern of results is that different participants have different frames of reference that they use to evaluate their language proficiency” p. 532.A second source of information about proficiency would be a bilingual’s perception of their proficiency relative to others. If the others are also bilinguals, the effects of cross-language interference may be neutralized. For example, the feeling that “My dominant language is Chinese and I am very fluent, but not quite as good as my peers who speak only Chinese.” Considering this evidence should reduce the tendency to overestimate Chinese proficiency. In support of this argument, Tomoschuk et al. show striking differences between Chinese–English bilinguals raised in the USA compared to a group who immigrated to the USA relatively recently. The recently immigrated group  self-rated their Chinese fluency in closer alignment to their MINT scores, in other words, they had lower self-ratings. Tomoschuk et al. suggest that recently immigrated speakers are comparing themselves to family and friends in China, many of whom are native eiChinese speaking monolinguals who, by definition, have not been adversely influenced by acquiring or using an L2.Only if bilinguals have rich opportunities to compare their performance in their dominant language to the performance of monolingual speakers of that language would they have evidence that their proficiency was, on average, less than monolinguals. For example, from the perspective of a bilingual: unique variance in verbal fluency for L2 proficiency to account for. Because the evidence drawn from Tomoschuk et al. is somewhat circumstantial, we emphasize that we are speculating, not claiming, that the self-ratings of our bilinguals are dominated by adopting a frame-of-reference of other bilinguals. It may be worthwhile to explore the possibility that bilinguals could be instructed to use the three different frames of reference and generate reliability different self-ratings.A conjecture offered here is that self-rated English proficiency in a bilingual is a weighted average of: (1) comparing my languages to each other, (2) comparing my proficiency to that of other bilinguals, and (3) comparing my proficiency to that of monolinguals. The first two will lead to an overestimation of MINT scores that are caused by failing to take adverse cross-language effects into account. This provides a valid interpretation for Model 1: Other proficiency predicts unique variance in English verbal fluency beyond that directly accounted for by English proficiency. This would not be the case if self-ratings of English proficiency fully reflected the adverse effects accruing from an L2. If so, there would be no One account of the bilingual disadvantage in verbal fluency is cross-language interference. This is intuitive if one thinks of Model 1 as reflecting online causal relationships during a verbal fluency task. That is, what happens when a bilingual is asked to generate names of vegetables? At some point, attention may gravitate toward the concept for CUCUMBER, but the word form “pepino” may compete with the word form “cucumber” and render “cucumber” inaccessible. Thinking of Model 1 as an online model of verbal fluency, English Proficiency reflects the strength of English word forms and Other Proficiency the strength of competing words from the non-target lexicon. Thus, cross-language interference will increase as Other Proficiency increases.In contrast, Model 1 can be thought of in terms of the lexical structures that have been acquired over the bilingual’s lifetime and not-so-much as a description of online processing. This opens the door to explanations of the bilingual disadvantage in verbal fluency that appeal to weaker links rather than interference. Model 1 showed that Other Proficiency degrades verbal fluency. Although this could be due to interference, the “damage” could have been caused by past language experience. That is, if a bilingual is a native speaker of Spanish, acquires English when she starts school, and uses mostly Spanish at home and with friends, then she accrues far less experience in traversing links like the one from CUCUMBER to “cucumber” compared to a monolingual English-speaking peer. The weaker link alone may render “cucumber” inaccessible without suffering direct inhibition or suppression at the hands of “pepino”. The relationship between English Proficiency and Other Proficiency may reveal the importance of the history between the two measures rather than their interaction at the time of testing. That is, Other Proficiency is associated with lower verbal fluency not because it interferes more with finding correct English words, but because those individuals with higher proficiency, in say Spanish, have been practicing Spanish at the expense of English and, consequently, have forged weaker English links. Thus, although Model 1 shows that Other Proficiency accounts for unique variance in English verbal fluency apart from that directly accounted for by English Proficiency, there is nothing in the data considered to this point that rules out an explanation based on weaker links.The most direct evidence supporting the weaker links interpretation is that the bilingual disadvantage in picture naming is greater when producing low-frequency than high-frequency picture names . As niceHowever, in contrast to the above, when translation equivalents (that are not cognates) exist in both lexicons, low-frequency words may be more vulnerable to interference than high-frequency words. For example, an English dominant bilingual growing up in the self-proclaimed artichoke capital of the world  may know “alchachofa” but suffer intense interference because “artichoke” has very high frequency. All things considered, it is not clear (at least to us) that cross-language interference in picture naming  should be more prevalent for high-frequency picture names compared to low-frequency, especially when a non-target competitor enjoys a frequency advantage over the target. It appears that both weaker links and cross-language interference continue to compete as explanations for the bilingual disadvantage in verbal fluency. Of course, they could both contribute to the overall effect.Given its extraordinary influence in bilingualism research, it is worthwhile to consider our results in the context of Green’s Inhibitory Control Model (ICM) . The ICMHowever, the ICM makes an interesting and more specific assumption, namely, that the actual inhibition is not applied until competition for production occurs. Consider a picture-naming task where a pre-cue indicates whether the upcoming picture should be named in English or Spanish. When the pre-cue requires a switch, say from Spanish to English, the new schema (“name-in-English”) must be selected and this sets the stage for the global inhibition of all the lemmas with Spanish tags. However, the actual inhibition is not applied until: (1) the picture appears, (2) the participant initiates plans to name the picture, and (3) response competition is detected. For example, if the stimulus is a picture of a CAT, then the competition between the correct response “cat” and the competitor “gato” will (4) trigger inhibition of all Spanish lemmas in proportion to the degree that they were activated by the picture. Thus, “cat” would be inhibited more than unrelated English words like “table”. In summary, Green’s ICM assumes that competition between the lexicons is controlled via inhibition and that inhibition is reactive, that is, in proportion to the amount of competing activation that needs to be neutralized.What does the ICM predict for verbal fluency? If the task language is English and the memory search starts to hone in on a potential exemplar  then “cat” and “gato” will compete. If the task schema is “speak-English”, then all the Spanish lemmas will receive inhibition, but “gato” will receive the most because CAT has led to the coactivation of both “cat” and “gato”. However, the cross-language interference account of the bilingual disadvantage in verbal fluency relies on the inhibition flowing in the opposite direction, namely, from “gato” to “cat”. That is, might there be occasions when “gato” gets the jump on “cat” (despite the fact that the “speak-English” schema is selected) and suppresses “cat” to the point where it is inaccessible? Although it is easy to imagine that the initial competition delays the production of “cat”, it seems far less likely that “gato” can completely block the access of “cat”. Thus, we argue that the interference account of the bilingual disadvantage in verbal fluency is not very compelling within the framework of Green’s ICM.“stop thinking about inhibition” in that way. They required each participant to complete six tasks assumed to reflect Inhibition of Prepotent Responses and five assumed to reflect Resistance to Distraction . Rather than examining only the fit of various models, Bayesian hypothesis testing was also conducted. These additional tests showed that the data are ambiguous as to: (a) whether there is one inhibition factor or two and (b) if there are two factors, whether they are correlated or not. Another problem pointed out by Rey-Mermet et al., both in their data and in other latent-variable analyses of the inhibition construct, is that for each latent variable, the loading for one task tends to dominate the others. Consequently, each latent variable represents mainly the variance of one task with the remaining tasks burdened with high error variances. These problems led Rey-Mermet et al. to suggest that all latent-variable models of the inhibition construct have poor explanatory power. From their view of this evidence, Rey-Mermet et al. suggested that nonverbal interference tasks do not measure a common underlying latent variable associated with general inhibitory-control, but rather a highly task-specific ability to resolve the type of conflict instantiated in each task.Inhibition is usually viewed as a domain-general ability to suppress competing or irrelevant information during goal-directed behavior. Recently, Rey-Mermet, Gade, and Oberauer  have urgIn Opposition to Inhibition, provided alternatives to inhibition for several classic demonstrations of “inhibition” including inhibition of return (IOR), Stroop interference, and negative priming. This begs the question: Is inhibition needed to account for bilingual language control?If inhibition is task specific, as argued by Rey-Mermet et al. (2018), then there would be no basis for a bilingual advantage in domain-general inhibitory control  is always in one language and the lexical decision (the probe event) in the other language.The evidence for local inhibition accrues from the presence of negative priming (NP) and this reliably occurs when the prime event is in L1, the probe event in L2, and the prime distractor  becomes the probe target . Lexical decisions to cat are slower when CAT was the distractor compared to an unrelated control. To be more specific, in order for NP to occur (according to the interference account) CAT must be automatically activated despite the intention to ignore it. If CAT becomes sufficiently activated, then it will compete with the to-be-named target  and will trigger reactive inhibition of CAT to resolve the conflict. If the inhibition of CAT persists into the probe event and cat appears as the probe target, then the suppressed activation will result in a slower lexical decision compared to a control trial.Although inhibition is an intuitively appealing explanation for NP, it is one of several phenomena  that MacLeod and colleagues  showed cThe critical contrast in Neumann’s experiments is that NP occurs when the consistent order of tasks is L1 (naming) then L2 , but not the reverse. This is what would be predicted from the interference perspective if inhibition is reactive and in proportion to need. If an L1 distractor is harder to ignore (more automatic) than an L2 distractor and is more highly activated, then it will receive more inhibition. If the inhibition actually drives the activation of the distractor below the normal baseline, then it will take longer to make the lexical decision when that distractor becomes the probe target. Indeed, NP occurs in the L1-then-L2 order. However, if the order is L2-then-L1 the amount of inhibition applied to the distractor in the initial naming task should be less because less is needed. If the amount is insufficient to carry over into the probe task, then significant NP may not occur.This interference account of NP is usually presented as intuitive and fully following from Green’s ICM. As an aside, we suggest that in the absence of a computational model, there are implicit parameter decisions that are needed to predict the L1/L2 asymmetry and these may not enjoy high plausibility. To illustrate, consider the assumption that the amount of inhibition is in proportion to need. If this could be precisely titrated, then “need” might be defined as the amount of inhibition needed to return the distractor to the normal baseline. If L1 distractors are driven back down to baseline by substantial inhibition and L2 distractors driven to baseline by more moderate amount of inhibition, then it would not matter if the prime task used L1 or L2. In fact, there would be no negative priming because L1 distractors, L2 distractors, and control distractors would all be at baseline at the start of the probe trial. Thus, we suggest that an interference account of NP requires that inhibition is applied proportionately and beyond what is needed such that L1 distractors are driven below normal baseline and L2 distractors just to baseline. Thus, the interference account of NP may be more of a “just so” story than generally acknowledged.Neumann and colleagues assert that their between-language findings “undermine” episodic retrieval models, in part, because they do not predict the L1/L2 asymmetry in NP effects. This is true if you restrict the episodic retrieval models to a single parameter specifying whether the response codes either match or mismatch. However, just like interference models, there are many implicit assumptions that could be instantiated in different versions of a computational model. Consider for example the formation of an event file consisting of a stimulus and the response to that specific stimulus. What governs the probability of encoding and storing an episode or the strength of that memory trace? The distractors are located just above or below the targets and are likely to be encoded even if the participant’s intention is to ignore them. However, it would seem reasonable to propose that the likelihood or strength of distractor activation and its episodic trace would vary as a function of its salience and its propensity for automatic coding. In this framework, L1 distractors should be advantaged compared to L2 distractors just as L1 words generate more Stroop interference compared to L2 words. We suggest that it is not easy to judge which theory has the most parsimonious explanation for the observation that NP occurs in the L1-then-L2 order, but not the reverse.“a surprisingly elusive phenomenon” (p. 274) when primes are in the L2 and targets are in the L1 , but not NP. Based on translation priming in simpler tasks not involving distractors, PP would not have been predicted. As Grainger and colleagues  pointed see also ).Neumann and colleagues do not dwell much on why PP occurs in the L2-then-L1 order for their task, but rather seek to explain why PP disappears in the L1-then-L2 order. Their account invokes a second type of inhibitory control, namely, proactive suppression of the language used in the prime task. This follows from the assumption that the participant knows that the probe task will always be in the other language and that an excellent strategy would be, at the onset of the LD task, to globally inhibit the language used in the naming task. However, if complete and global suppression of the previously used language was possible, why would participants not engage in proactive inhibition in the L2-then-L1 order as well? If proactive inhibition was effortful, it would even require less effort to fully suppress L2.These questions aside and switching to the other perspective, it is clear that episodic retrieval models would not predict the absence of PP in the L1-then-L2 order based on the simple notion of response codes that either match or mismatch because they do match. However, perhaps it is the presence of PP in the L2-then-L1 order that deserves additional scrutiny given its rarity in other translation priming tasks. The fact that the probe task is LD may impose limits on the generality of the findings because the LD task is rather infamous for its vulnerability to strategic influences on the criteria used to classify the target string as a word or nonword ,25.In summary, the series of experiments by Neumann and colleagues offers a tantalizing pattern of results that follow from one instantiation of an interference account of bilingual language control. However, caution is recommended before assuming that the “inhibitory control” reflected in NP is an instance of domain-general inhibitory control. An individual differences examination would be useful ,27. We kr(122) = −0.072, p = 0.43. Thus, there is no evidence from this test that bilinguals with better inhibitory control (and presumably better able to inhibit the interference from the Other language) have better verbal fluency as one would expect if cross-language effects in verbal fluency were, indeed, caused by interference. Furthermore, it is worth noting that switch costs did significantly correlate with MINT scores, r(128) = −0.223, p = 0.01, but not with self-rated English proficiency, r(138) = −0.043, p = 0.62. This pattern is consistent with the hypotheses that inhibitory control affects performance in MINT, but does not influence self-ratings of English proficiency. These types of relationships could be productively studied in future work.An individual differences approach could also be used in a replication of the current study by including a measure of inhibitory control and then correlating that measure with verbal fluency. If a domain-general measure of inhibitory control predicted verbal fluency, this would support the interpretation that part of the bilingual disadvantage in verbal fluency was caused by interference from the other language. This is a challenging recommendation given the controversy regarding the construct of inhibition and claims that there may not be a domain-general inhibitory ability. Be that as it may, our original study did include three different cued switching tasks and the switch costs from those tasks correlated with each other, thus demonstrating convergent validity. Furthermore, switch costs are generally assumed to be caused, at least in part, by having to inhibit the task schema active on the previous trial on “switch trials” in contrast to “repeat trials”. In order to explore possible associations between measures of inhibitory control and verbal fluency, we correlated the switch costs in the color-shape switching task with total verbal fluency, but the correlation was not significant, There are small but fairly consistent bilingual disadvantages in picture naming and tests of verbal fluency. These disadvantages could be due to cross-language interference or weaker-links between semantic concepts and their phonological form. Both hypotheses lead to the prediction that the degree of proficiency in the nontarget language should be negatively correlated with and account for unique variance in verbal fluency performance apart from the direct effects of degree of proficiency in the target language. Although several regression models were presented that are consistent with this prediction, there are potential complications having to do with the measures of vocabulary knowledge and the degree to which different measures  are sensitive to adverse cross-language effects. In summary, the regression models showing that Other Proficiency is a potent predictor of verbal fluency cannot rule out the alternative explanation that the effect of Other Proficiency is simply due to past language usage that has led to weaker links in the test language.Considering explanations of the bilingual disadvantage in picture naming and verbal fluency that do not involve inhibition led to a general discussion of recent claims that inhibitory control may not be a domain-general cognitive ability and, even more radically, that inhibition may play no role in cognitive control (see  for a mo


Resistance of breast cancer to human epidermal growth factor receptor 2 (HER2) inhibitors involves reprogramming of the kinome through HER2/HER3 signaling via the activation of multiple tyrosine kinases and transcriptional upregulation. The heterogeneity of induced kinases prevents kinase targeting by a single kinase inhibitor and presents a major challenge to the treatment of therapeutically recalcitrant HER2-positive breast cancers (HER2+ BCs). As a result, there is a critical need for effective treatment that attacks the aberrant kinome activation associated with resistance to HER2-targeted therapy. Here, we describe a novel treatment strategy that targets cyclin-dependent kinase 7 (CDK7) in HER2 inhibitor-resistant (HER2iR) breast cancer. We show that both HER2 inhibitor-sensitive (HER2iS) and HER2iR breast cancer cell lines exhibit high sensitivity to THZ1, a newly identified covalent inhibitor of the transcription regulatory kinase CDK7. CDK7 promotes cell cycle progression through inhibition of transcription, rather than via direct phosphorylation of classical CDK targets. The transcriptional kinase activity of CDK7 is regulated by HER2, and by the receptor tyrosine kinases activated in response to HER2 inhibition, as well as by the downstream SHP2 and PI3K/AKT pathways. A low dose of THZ1 displayed potent synergy with the HER2 inhibitor lapatinib in HER2iR BC cells in vitro. Dual HER2 and CDK7 inhibition induced tumor regression in two HER2iR BC xenograft models in vivo. Our data support the utilization of CDK7 inhibition as an additional therapeutic avenue that blocks the activation of genes engaged by multiple HER2iR kinases. The human epidermal growth factor receptor 2  , 2 is coThe cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the well-known CDK-activating kinase (CAK), a kinase complex that catalyzes phosphorylation of the T-loops and thereby activating multiple cyclin-associated kinases including CDK1, CDK2, CDK4, and CDK6. CAK also complexes with the core human transcription factor II (TFIIH) basal transcription complex and activates RNA polymerase II (RNA Pol II) by serine phosphorylation of the repetitive C-terminal domain (CTD) of its largest subunit. This phosphorylation permits RNA Pol II escape from promoters, thus facilitating the subsequent elongation of the resulting transcripts . In addiWe first investigated whether CDK7 inhibition affected the viability of HER2+ tumors cells with differing ER and progesterone receptor (PR) status. Cell lines tested included two HER2 inhibitor-sensitive (HER2iS) cell lines (SKBR3 and BT474) and four HER2iR cell lines . BT474 and MDAMB453 cells were positive for both HER2 and either ER or PR , 22. TNBCDK7 regulates RNA Pol II-mediated transcriptional initiation and pausing, in addition to its effects on transcript elongation through its CAK activity working via other transcriptional CDKs –25. We oMMTV-rtTA-TetO HER2 transgenic mice [Based on the hypersensitivity of HER2+ BC cells to THZ1, we next investigated whether HER2 expression affects CDK7 activity tumor cells. Ectopic expression of human wild-type HER2 increased protein levels of both CDK7 and RNA Pol II, and phosphorylation of RNA Pol II CTD in immortalized human mammary epithelial (HMEC) cells Fig. . Consistnic mice  resultedGiven the role of CDK7 in phosphorylation of the RNA Pol II CTD at active genes , 23, 27,p < 0.05) were differently modulated by overexpression of HER2 in HMEC cells. Gene ontology analyses of these HER2 regulons showed significant enrichment in the pathways of cytokine–receptor interaction, cell adhesion molecules (CAMs) and NOD-like receptor signaling.  were differentially expressed in HEMC-PIK3CAH1047R cells or HEMC-SHP2E76A cells, respectively, compared with HEMC-pBABE cells. Activation of PI3K by PIK3CAH1047R enriched genes in the pathways of DNA replication, cellular senescence, cytokine–cytokine receptor interaction and PI3K-AKT signaling  and THZ1 (5 or 10 nM) individually or in combination for up to 2 weeks. Although treatment with THZ1 alone suppressed clonogenic growth, combined THZ1 and lapatinib treatment were more effective Fig. . QuantitWe next assessed the activities of CDK7/RNA Pol II complex in response to lapatinib and THZ1 alone or in combination in these three cell lines. Unlike in HER2iS cell line SKBR3 Fig. , lapatinLast, we tested if CDK7 could resensitize HER2iR breast cancer cells to lapatinib in vivo. NCI nude mice bearing palpable HCC1569 or HCC1954 xenografts were treated with lapatinib and THZ1 alone or in combination. Lapatinib alone resulted in marginal inhibition of tumor growth, whereas THZ1 alone significantly impaired tumor growth Fig. , b. CombTo evaluate signaling and pharmacodynamic responses of HCC1569 and HCC1954 xenografts during inhibitor treatment we isolated tumors 72 h after drug administration and analyzed molecular markers. Dual HER2 and CDK7 inhibition decreased RNA Pol II phosphorylation, suppressed cell proliferation, and induced apoptosis Fig. . These rHere we show that a CDK7 inhibitor, THZ1, strongly inhibited HER2+ BC cell growth and increased apoptosis, even in cancer cells that exhibited resistance to HER2-targeted therapy. Unexpectedly, CDK7 did not promote cell cycle progression directly through phosphorylation of CDKs and ER, indicating that CDK7 plays its primary role in transcription in these cells. Using HMEC cell models, we demonstrated that the transcriptional activity of CDK7 was modulated by HER2, other RTKs and their downstream PI3K and SHP2 signaling nodes. Our studies also demonstrated a novel codependency of HER2 and CDK7 that might be exploited in therapeutically recalcitrant HER2+ BC. Our results also suggest that patients with HER2+ BC may have a durable response to combined inhibition of HER2 and CDK7.Multiple kinases can contribute to escape from lapatinib-mediated growth inhibition, consistent with a shift in dependency to alternative signaling nodes in addition to HER2. This prevents their targeting by a single kinase inhibitor and underscores the difficulty in choosing the most effective kinase inhibitor combinations to treat HER2+ tumors. We approached this problem with the hypothesis that lapatinib would have a more durable effect in inhibiting HER2+ cell growth if we could block the signaling of multiple HER2iR kinases. We therefore targeted proteins involved in transcriptional regulation of RTKs that drive the signaling networks responsible for lapatinib resistance. CDK7 functions as a core transcriptional kinase that mediates the activity of multiple RTKs capable of generating resistance to HER2-targeted therapies. By inhibiting signaling of multiple RTKs through CDK7 inhibition we could achieve durable growth inhibition and potentially block the adaptive responses seen to a single RTK inhibitor.L858R [Del [TNBC and ER+ BC cells have been reported to respond to CDK7 inhibition through different mechanisms , 22. TNBL858R  and EGFR58R [Del , in our MEK1Q56P, which exhibits resistance to BRAF inhibitors [MEK1Q56P showed less response to THZ1 treatment compared with the parental cell A375 with BRAFV600E  and microarray, immunofluorescence, quantitative real-time PCR, mouse xenotransplantation experiments, histology and immunohistochemistry (IHC), and data statistical analyses.Supplementary InformationSupplementary Table 1Supplementary Table 2