Fracture of Zotarolimus-Eluting Stent after Implantation

Drug-eluting stents were developed and approved for the reduction of in-stent restenosis. However, restenosis still occurs, and stent fracture is suggested as a cause of restenosis after implantation. Although sirolimus-eluting stents are considered to carry a high risk of fracture, the risk is also present with other drug-eluting stents. Herein, we report the case of a 78-year-old woman who received a zotarolimus-eluting stent for a bifurcation lesion of the left anterior descending coronary artery. Ten months later, she underwent coronary angiography due to angina. The angiogram revealed in-stent restenosis, with a grade IV stent fracture. After percutaneous coronary angioplasty, the patient''s clinical symptoms improved.Key words: Blood vessel prosthesis implantation/instrumentation, coronary restenosis/diagnosis/etiology/prevention & control, drug-eluting stents, drug implants/adverse effects, prosthesis failure, retreatment, stents/adverse effectsAlthough drug-eluting stents (DESs) reduce in-stent restenosis, important complications of coronary DESs are restenosis and thrombosis. Stent fractures have also been reported¹ and are considered to be possible causes of restenosis² within DESs. The sirolimus-eluting stent is reported to be prone to fracture.³ For example, there is the Cypher® stent (Cordis Corporation, a Johnson & Johnson company; Miami Lakes, Fla)—made of balloon-expandable stainless steel, a durable copolymer mixture of polyethylene-covinyl acetate and poly-u-butyl methacrylate, and sirolimus, which is a Gap 1 (G1) cell-cycle inhibitor.⁴ Other DESs are also considered to be at risk of fracture. One, the Endeavor® Sprint Zotarolimus-Eluting Coronary Stent System (Medtronic, Inc.; Minneapolis, Minn), uses a cobalt chromium stent platform; a durable, antithrombotic, phosphorylcholine-encapsulated coating; and another G1 cell-cycle inhibitor, zotarolimus.⁴ Here, we report the case of a woman who was treated with a zotarolimus-eluting stent that subsequently fractured.     

Covered-Stent Treatment of Coronary Aneurysm after Drug-Eluting Stent Placement: Case Report and Literature Review

Most commonly, coronary artery aneurysms are secondary to atherosclerosis, but cases have been reported in patients who have vasculitis or tissue disorders, and in patients who have undergone interventional procedures. However, over the past few years, an increasing number of cases of coronary artery aneurysms after drug-eluting stent implantation have been reported. The exact mechanism is unknown. Experimental animal studies have shown that both the active drug and the polymer coating, under certain circumstances, might cause progressive luminal dilation, positive vascular remodeling, and aneurysmal formation. Complications like rupture, thrombosis, embolization, myocardial infarction, and even sudden death have been reported. Treatment options vary from aggressive surgical ligation of the aneurysm, in union with distal bypass surgery, to percutaneous implantation of a covered stent or conservative medical management with continued antiplatelet therapy. Currently, there is no consensus on an ideal approach to treating coronary artery aneurysm after drug-eluting stent implantation. Polytetrafluoroethylene-covered stents, easy and rapid to deploy, have emerged as a newer option. We report a case of coronary artery aneurysm at the site of a previous drug-eluting stent. The lesion was successfully treated with a polytetrafluoroethylene-covered stent.Key words: Blood vessel prosthesis, coronary aneurysm/etiology/therapy, coronary disease/therapy, covered stents, dilatation, pathologic, drug delivery systems/adverse effects, drug-eluting stents/adverse effects, polytetrafluoroethylene, postoperative complications, sirolimus/administration & dosage, stents/adverse effectsAneurysmal dilation of the coronary arteries was first described by Bougon in 1812.¹ Most commonly, coronary artery aneurysms are secondary to atherosclerosis,² but cases have been reported in patients who have vasculitis (Kawasaki syndrome,³ for example) or tissue disorders (Ehlers-Danlos⁴ or Marfan syndrome,⁵ for example), and in patients who have undergone interventional procedures.^6,7 Over the past few years, an increasing number of case reports have described a growing incidence of coronary artery aneurysms after drug-eluting stent (DES) implantation.^8–11 Since 2003, when the U.S. Food and Drug Administration approved the 1st such stent, DESs have unequivocally demonstrated their superiority to bare-metal stents in regard to in-stent restenosis.^9–12 Nevertheless, safety concerns brought up from time to time—especially regarding the increased risk of late stent thrombosis¹³—have raised questions about the long-term safety of DES implantations.The exact mechanism of coronary artery aneurysmal formation after DES placement is unknown. Complications such as rupture,¹⁴ thrombosis,¹⁵ distal embolization,¹⁶ myocardial infarction,¹⁷ and even sudden death¹⁸ have been reported. Here we report a case of coronary artery aneurysm at the site of DES implantation, which we successfully treated with a polytetrafluoroethylene (PTFE)-covered stent. In addition, we present a review of the literature on the use of PTFE-covered stents in the repair of coronary artery aneurysms that have formed at the site of DES implantation.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Synergistic regulation of nonbinary molecular switches by protonation and light

We report a molecular switching ensemble whose states may be regulated in synergistic fashion by both protonation and photoirradiation. This allows hierarchical control in both a kinetic and thermodynamic sense. These pseudorotaxane-based molecular devices exploit the so-called Texas-sized molecular box (cyclo[2]-(2,6-di(1H-imidazol-1-yl)pyridine)[2](1,4-dimethylenebenzene); 1⁴⁺, studied as its tetrakis-PF₆ ⁻ salt) as the wheel component. Anions of azobenzene-4,4′-dicarboxylic acid (2H⁺•2) or 4,4′-stilbenedicarboxylic acid (2H⁺•3) serve as the threading rod elements. The various forms of 2 and 3 (neutral, monoprotonated, and diprotonated) interact differently with 1⁴⁺, as do the photoinduced cis or trans forms of these classic photoactive guests. The net result is a multimodal molecular switch that can be regulated in synergistic fashion through protonation/deprotonation and photoirradiation. The degree of guest protonation is the dominating control factor, with light acting as a secondary regulatory stimulus. The present dual input strategy provides a complement to more traditional orthogonal stimulus-based approaches to molecular switching and allows for the creation of nonbinary stimulus-responsive functional materials.

Multifactor regulation of biomolecular machines is essential to their ability to carry out various biological functions (1 –11). Construction of artificial molecular devices with multifactor regulation features may allow us to understand and simulate biological systems more effectively (12 –31). However, creating and controlling such synthetic constructs remains challenging (16, 32 –37). Most known systems involving multifactor regulation, including most so-called molecular switches and logic devices (38 –43), have been predicated on an orthogonal strategy wherein the different control factors that determine the distribution of accessible states do not affect one another (20, 44 –56). However, in principle, a greater level of control can be achieved by using two separate regulatory inputs that operate in synergistic fashion. Ideally, this could lead to hierarchical control where different states are specifically accessed by means of appropriately selected nonorthogonal inputs. However, to our knowledge, only a limited number of reports detailing controlled hierarchical systems have appeared (57). Furthermore, the balance between specific effects (e.g., kinetics vs. thermodynamics) under conditions of stimulus regulation is still far from fully understood (54). There is thus a need for new systems that can provide further insights into the underlying design determinants. Here we report a set of pseudorotaxane molecular shuttles that act as multimodal chemical switches subject to hierarchical control.     

Components of the Complete Blood Count as Risk Predictors for Coronary Heart Disease: In-Depth Review and Update

Atherosclerosis is an inflammatory disease, and several inflammatory biomarkers, such as C-reactive protein, have been used to predict the risk of coronary heart disease. High white blood cell count is a strong and independent predictor of coronary risk in patients of both sexes, with and without coronary heart disease. A high number of white blood cells and their subtypes (for example, neutrophils, monocytes, lymphocytes, and eosinophils) are associated with the presence of coronary heart disease, peripheral arterial disease, and stroke. The coronary heart disease risk ratios associated with a high white blood cell count are comparable to those of other inflammatory markers, including C-reactive protein. In addition, other components of the complete blood count, such as hematocrit and the erythrocyte sedimentation rate, also are associated with coronary heart disease, and the combination of the complete blood count with the white blood cell count can improve our ability to predict coronary heart disease risk. These tests are inexpensive, widely available, and easy to order and interpret. They merit further research.Key words: Atherosclerosis, biological markers/blood, complete blood count, coronary artery disease, coronary heart disease, inflammation, leukocyte count, white blood cell countCoronary heart disease (CHD) is the leading cause of death in the United States, and it is estimated that the prevalence of cardiovascular disease will increase by approximately 10% over the next 20 years.^1,2 During the past 2 decades, extensive research has established that atherosclerosis is an inflammatory disease,³ a finding that has offered new possibilities for predicting CHD risk. The presence of many types of inflammatory biomarkers, most notably high-sensitivity C-reactive protein (CRP), have been found to be consistent predictors of CHD events.⁴ Although an increasing number of novel inflammatory biomarkers are being studied in this context, many offer little improvement in the current risk-prediction models.⁵ Whereas measurement of CRP and lipoprotein-associated phospholipase A₂ (Lp-PLA₂) have shown promise as predictors of CHD events,⁵ most of the other newly introduced inflammatory risk markers are expensive to test, are not readily available, lack standardization, and have not been confirmed by multiple prospective studies.We have previously shown that a growing body of evidence supports the usefulness of the white blood cell (WBC) count as a predictor of future coronary events.⁶ Herein, we review the most recent data on the use of the WBC count and other components of the complete blood count (CBC) to predict CHD risk. An elevated WBC is a well-recognized indicator of inflammation.⁶ The total number of WBCs and each subtype (for example, neutrophils, monocytes, lymphocytes, and eosinophils) have been implicated as predictors of CHD.⁶ Nearly all of the cellular elements in the blood, including WBCs, red blood cells (RBCs), and platelets, are involved in the underlying pathogenesis of atherosclerosis.^3,6 These markers not only play a role in the development of CHD in asymptomatic patients, but they predict recurrent events and death in patients who already have CHD.^7,8 In addition to the cellular components, an elevated erythrocyte sedimentation rate (ESR) has been shown to be a weak prognostic factor in CHD patients.^9–11

Total White Blood Cell Count and Coronary Heart Disease

The relationship between white blood cell count and CHD was first suggested more than 80 years ago.¹² Results from multiple studies of patients with and without CHD at baseline have indicated that the WBC count can be used to predict the incidence of coronary events.^13–25 A high WBC count also has been shown to be predictive of future cardiovascular events in individuals who were disease free at baseline (Fig. 1 ^14–25).⁶ Multiple studies have shown that a high WBC count is associated with increased mortality rates in patients who present with unstable angina pectoris,^26,27 acute coronary syndromes (ACS),²⁸ and acute myocardial infarction (MI),^29–32 and in patients who undergo percutaneous coronary interventions (PCIs)³³ or coronary artery bypass grafting (CABG) (Fig. 2 ^{8,29,32,34–40}).^34–36,41 Moreover, elevated total WBC and neutrophil and monocyte counts are associated with cerebral ischemia and stroke.^42–45 In 2004, Grau and colleagues³⁸ showed that, in patients who had had an ischemic stroke or MI or who had peripheral arterial disease (PAD), there was a relationship between high WBC counts and the recurrence of vascular events.Open in a separate window Fig. 1 The 95% confidence interval and point estimate for coronary heart disease (CHD) incidence and CHD mortality in patients free of CHD. The solid vertical line denotes a relative risk of 1 (no effect); horizontal lines represent 95% confidence interval; and tick marks show point estimates for relative risk. Open in a separate window Fig. 2 The 95% confidence interval and point estimate for coronary heart disease death in patients with acute coronary syndromes, as determined on the basis of total white blood cell count. An increase in quartiles of WBC count from baseline over 24 hours after an MI episode has been correlated with an increase in all-cause death after an MI,⁴⁶ and a recent subanalysis of the Ongoing Tirofiban In Myocardial Infarction Evaluation (On-TIME) trial showed that an increase in WBC count (defined as WBC count after 6 or 24 hours minus the WBC count on admission) was a significant predictor of death after PCI.³⁹ In an analysis of 900 patients in the Stent Primary Angioplasty in Myocardial Infarction (Stent PAMI) trial, investigators found that an elevated WBC count upon hospital admission (highest tertile, ≥12) had a strong independent association with reinfarction at 1 year.⁴⁷ Moreover, in a subanalysis using data from the Atherosclerotic Risk in Communities (ARIC) and Cardiovascular Health Study that compared patients with and without stages 3 to 4 chronic kidney disease, investigators found that elevated inflammatory markers, including the WBC count, were associated with an increase in adverse cardiac events and death.²⁵ They also found that, although the risk is additive, the synergy between inflammation and chronic kidney disease was not statistically significant.²⁵ Finally, results from a meta-analysis of 5,337 CHD patients from 7 large studies showed that a high WBC count was associated with a risk ratio of 1.4 (95% CI, 1.3–1.5) for CHD.⁴⁸ This risk ratio is comparable to those seen with other novel inflammatory markers associated with atherosclerosis, including CRP.⁴⁹ Therefore, the relationship between white blood cell count and CHD is strong, consistent, dose-dependent, independent, biologically plausible, and coherent with the current paradigm of the inflammatory origin of atherosclerosis. The association between WBC count and CHD has been consistently observed in different populations with varying degrees of baseline risk—in asymptomatic individuals and symptomatic patients. The association between the WBC count and CHD appears to be independent of other traditional coronary risk factors, including smoking. Although smoking is associated with leukocytosis, studies have shown that the predictive value of a high WBC count for CHD is independent of whether patients are smokers.⁶

Differential White Blood Cell Count and Coronary Heart Disease

Elevated levels of almost all subtypes of WBCs, including eosinophils,^50,51 monocytes,^52–54 neutrophils,^38,55–60 and lymphocytes (an inverse relationship),^52,61–63 have been associated with increased risk of CHD (Fig. 3 ^{16,38,40,52,55,57,64}).⁶ In a large, disease-free patient cohort from the Adult Health Study (AHS) of Hiroshima and Nagasaki, results showed a relationship between the total WBC count, including the eosinophil, neutrophil, and monocyte counts, and the incidence of CHD.⁵⁰ Results from the Paris Prospective Study II showed an increased risk of CHD in patients with high monocyte counts.⁵² In the Caerphilly and Speedwell studies, increased coronary risk was associated with high neutrophil, eosinophil, lymphocyte, monocyte, or basophil counts.⁵⁵ A prospective study⁴⁰ of 1,037 patients who experienced an acute MI showed that elevated total WBC, monocyte, and neutrophil counts and low lymphocyte counts were independent predictors of all-cause death. The investigators in that study found that adding the neutrophil count to models of total WBC, monocyte, and lymphocyte counts improved the models'' predictive ability.⁴⁰ Furthermore, Papa and colleagues⁶⁵ found that higher neutrophil-lymphocyte counts in patients with angiographically documented coronary artery disease (CAD) increased the risk of cardiac death within a 36-month follow-up period.Open in a separate window Fig. 3 The 95% confidence interval and point estimate for coronary heart disease incidence and mortality rate in disease-free persons and in patients with acute coronary syndromes, as determined on the basis of white blood cell subtypes. In a large cross-sectional study of middle-aged, asymptomatic participants in the 1999–2002 National Health and Nutrition Examination Survey (NHANES), elevated monocyte and neutrophil counts, along with elevated CRP and fibrinogen levels, were associated with the presence of subclinical PAD.⁶⁴ In another study of patients with PAD, only an elevated neutrophil count was predictive of an increased risk of major adverse cardiovascular events or death within the study''s 20-month follow-up period.⁶⁶ However, a study using intravascular ultrasonography showed a direct relationship between the monocyte count and an increase in the volume of atherosclerotic plaque in the arteries of patients who have had an acute MI.⁶⁷ In addition, a higher prevalence of monocytosis has been reported in patients with angiographically documented CAD.⁵⁴ When the relationship between peripheral monocyte count and coronary vasospasm was examined in 180 patients with chest pain, an elevated monocyte count was strongly associated with coronary vasospasm.⁶⁸ A more recent (2008) prospective, observational study in asymptomatic adults showed that, of all the WBC subtypes, the monocyte count had the strongest independent relationship with CHD risk, as estimated by the Framingham and SCORE calculations.⁶⁹ Moreover, several studies have shown that the WBC^70,71 and, more specifically, the monocyte count are independent predictors of subclinical carotid plaque deposition and carotid intima medial thickening.^71,72 In a genetic analysis of the inflammatory role of eosinophils, Gudbjartsson and colleagues⁷³ undertook a genome-wide association scan of the blood of more than 9,000 Icelanders to determine the sequence variants affecting eosinophil counts and to evaluate the association with inflammatory disorders. The analysis revealed that a single nucleotide polymorphism at 12q24, in SH2B3, had a significant association (P < 0.002) with MI in their scan data set and was consistent with 6 replication sets of European ancestry, with an odds ratio of 1.13 (95% CI, 1.08–1.18).⁷³ Further studies are needed to evaluate the consistency of the relationship of WBC subtypes and cardiovascular risk. It should be noted that most of the above-mentioned studies have excluded subjects with hematologic disorders, such as chronic lymphocytic leukemia or aplastic disorders, and the relationship between WBCs and CHD requires separate investigation in those situations.

White Blood Cell Count versus High-Sensitivity C-Reactive Protein as Coronary Heart Disease Risk Markers

High-sensitivity C-reactive protein is increasingly used for coronary risk prediction in clinical settings. With the knowledge that neither CRP nor the WBC count is the “perfect” screening tool, it is noteworthy that the WBC count is comparable in efficacy to CRP for predicting CHD risk (Fig. 4 ^74–80). Both tests are nonspecific markers of systemic inflammation and have comparable power for predicting coronary events. Meta-analyses by Danesh and colleagues^48,49 found that the WBC had an odds ratio of 1.4 (95% CI, 1.3–1.5) and the CRP had an odds ratio of 1.45 (95% CI, 1.2–1.6).Open in a separate window Fig. 4 The 95% confidence interval and point estimate for coronary heart disease (CHD) event risk based on white blood cell count versus C-reactive protein. PAD = peripheral arterial disease Unfortunately, there are few head-to-head comparisons of WBC count with CRP for CHD risk prediction (Fig. 4). In a subset of the West of Scotland Coronary Prevention Study, univariate analysis showed that both the WBC count and the CRP were risk predictors of coronary events, with respective risk ratios of 1.15 (95% CI, 1.02–1.31) and 1.21 (95% CI, 1.06–1.31).⁷⁴ However, multivariate analysis showed that only Lp-Pla₂ had significant predictive ability for the risk of a coronary event, with a risk ratio of 1.18 (95% CI, 1.05–1.33).⁷⁴ In a study of the relationship between inflammation and PAD that used data from the 1999–2002 NHANES, the multivariate adjusted odds ratio of PAD was increased for the highest quartiles of both WBC count (OR, 1.67; 95% CI, 0.84–3.31) and CRP (OR, 2.14; 95% CI, 1.41–3.25), compared with the lowest quartiles.⁷⁵ These relations remained significant across different subgroups that were used to evaluate the influence of sex, obesity, and diabetes mellitus.⁷⁵ In a fully adjusted model from the Women''s Health Initiative Observational Study,⁷⁶ a comparison of WBC counts in the first and 4th quartiles showed that women in the 4th quartile were at higher risk for CHD events: the odds ratio was 2.36 (95% CI, 1.33–4.19), which was higher than the odds ratio of 1.95 (95% CI, 0.95–4.01) for women with elevated CRP levels. In that study, elevated WBC counts and CRP levels synergistically predicted CHD risk, with an odds ratio of 6.8 (95% CI, 2.7–16.9).⁷⁶ Another study, from the Centers for Disease Control and Prevention (CDC), evaluated 8,355 participants in the 1999–2002 NHANES for lifestyle and emerging risk factors that could contribute to participants'' Framingham risk score. Results of the study showed that an elevated WBC count (>7 ×10⁹/L, the population median) was associated with an odds ratio of 1.49 (95% CI, 1.32–1.67) of the highest 10-year CHD risk—as was an elevated CRP level, with an odds ratio of 2.10 (95% CI, 1.74–2.53).⁷⁷ In addition, in a study of outcomes after patients underwent stent placement for unprotected left main coronary artery stenosis, Palmerini and colleagues⁷⁸ found that WBC counts and CRP levels were comparable in predicting outcomes at 9 months.⁷⁸ Conversely, in a study of 270 patients with angiographically documented CAD, results showed that increases in the total WBC count and the neutrophil and eosinophil counts all were significantly associated not only with the presence of CAD but also with its severity, as determined by the number and extent of lesions, whereas levels of CRP and the ESR were not.⁷⁹ Similarly, in a study of patients undergoing diagnostic coronary angiography, the WBC count was independently associated with angiographically documented CAD and multivessel disease, whereas CRP values, tissue inhibitor of metalloproteinases-1 (TIMP-1), and the ESR were not.⁸¹ However, in a subanalysis of the On-TIME trial that evaluated early versus late administration of tirofiban during primary PCI for ST-elevation myocardial infarction (STEMI), results showed that the baseline CRP value was a significant predictor of re-infarction and 1-year death, with an odds ratio of 1.03 (95% CI, 1.01–1.05; P = 0.012), whereas the baseline WBC count was not significantly associated with worse outcomes, with an odds ratio of 1.09 (95% CI, 0.96–1.23; P = 0.202).⁸⁰ Kruk and colleagues⁸² also evaluated patients with STEMI who underwent primary PCI and found that elevated WBC counts and CRP values were independent predictors of in-hospital death.⁸² In a study of patients who presented at the emergency department with chest pain suggestive of ACS, both the WBC counts and the CRP values were associated with a diagnosis of ACS upon hospital discharge; however, multivariate analysis showed that only the WBC count was actually associated with the presence of ACS, with an odds ratio of 20.9 (95% CI, 3.7–19.5).⁸³ In a prospective cohort study of 128 patients with angina pectoris,⁸⁴ investigators found that WBC counts and CRP levels were significantly higher in patients with unstable angina pectoris than in patients with stable angina (P < 0.0001) and that the circulating level of CRP was strongly associated with the clinical setting of unstable angina, with an odds ratio of 1.56 (95% CI, 1.23–2.0).Consequently, it appears that both tests are useful in different populations and among men and women and that these tests have comparable weaknesses and strengths in predicting coronary risk. However, the test for WBC count is less expensive and more widely available than is the test for CRP. Obviously, a high WBC count, like other markers of risk such as high cholesterol level and CRP, is not synonymous with the presence of CHD and is one of multiple risk markers used to predict the risk of CHD. Similarly, many subjects with cardiovascular disease have normal WBC counts.

Effect of Statins on White Blood Cell Count

Statins are known to reduce cardiovascular events and related inflammatory markers,⁸⁵ but they also reduce the WBC count. In a study of 1,246 patients with angiographically documented CAD, the use of statins was associated not only with lower WBC counts, but also with reduced levels of CRP, fibrinogen, and von Willebrand factor.⁸⁶ Lower WBC counts and lower values of other inflammatory markers have also been seen in CHD patients who take statins, even in the absence of changes in total cholesterol, low-density-lipoprotein cholesterol (LDL-C), high-density-lipoprotein cholesterol (HDL-C), and triglyceride levels.⁸⁷ In the National Registry of Myocardial Infarction (NRMI)-4 study, the WBC count was an independent predictor of in-hospital death from acute MI and was lower in patients who were taking statins or aspirin.³⁷ Statin use is also associated with a decrease in the neutrophil count in patients with existing peripheral vascular disease.⁸⁸ In a 6-month prospective study of patients with stable CAD, pravastatin significantly lowered the WBC count (8.9% vs baseline; P < 0.01), and multivariate regression analysis showed that the change in WBC count was an independent predictor of coronary plaque regression (14% reduction vs baseline; P < 0.0001).⁸⁹ In the Long-Term Intervention with Pravastatin in Ischemic Disease (LIPID) study,⁹⁰ in patients with stable CAD, the baseline WBC count was associated with greater CHD death in patients who were randomly assigned to receive a placebo, but not in patients given pravastatin. The number of coronary deaths and other cardiac outcomes that were prevented in the statin arm of the study increased, as did baseline WBC quartiles. The WBC count was a stronger predictor of treatment benefit than was the ratio of total cholesterol to HDL-C. Moreover, after statin treatment, the WBC count decreased at 1-year follow-up and remained stable thereafter.⁹⁰

Beyond Leukocytes: Use of the Complete Blood Count to Predict Coronary Heart Disease Risk

Other components of the CBC, such as the RBC and platelet counts and hemoglobin and hematocrit values, also are associated with CHD and can be used in combination with the WBC count to predict coronary risk. Few studies, however, have examined the usefulness of combining multiple CBC elements to predict CHD risk. According to one such study by Ronnow and colleagues,⁹¹ a simple CBC-derived risk score comprising the hematocrit and WBC and platelet counts could be used to predict death in a meaningful way; but an expanded CBC-derived risk score comprising the hematocrit, mean corpuscular volume (MCV), red cell distribution width (RDW), mean corpuscular hemoglobin concentration, and platelet and WBC counts provided substantially greater predictive value. Predictive ability was further improved by adjustment for age, sex, and other traditional risk factors.⁹¹ In another study, the same group showed that in patients with angiographically documented CAD, the total WBC count was an independent predictor of death or MI.⁶² In these patients, good predictive power was provided by high neutrophil or low lymphocyte counts, but the greatest risk prediction was achieved by using the neutrophil-to-lymphocyte ratio.⁶² Gibson and colleagues⁹² found that, in patients undergoing CABG, the preoperative WBC count was not a predictor of death during their 3.6 years of follow-up, but that the neutrophil-lymphocyte count was a significant predictor of both cardiovascular and all-cause death, especially in the highest quartile (hazard ratio, 2.09; 95% CI, 1.54–2.84).⁹² In an extension of the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT), Spencer and colleagues⁹³ screened 383 patients with hypertension to determine whether there was a relationship between hematologic factors and global measures of cardiovascular risk. They found that elevated WBC counts and increased levels of fibrinogen and von Willebrand factor significantly correlated with a higher Framingham 10-year CHD risk score and were independent predictors of Framingham CHD risk.⁹³ A lower hematocrit significantly correlated with both the Pocock and Framingham 5-year cardiovascular risk scores.⁹³ Anderson and colleagues⁹⁴ evaluated the predictive ability of the CBC in a prospective cohort of 29,526 patients undergoing coronary angiography and found that the CBC score had discriminatory predictive ability for all-cause death at 30 days, with an area under the curve of 0.75. The age- and sex-adjusted CBC model had an area under the curve of 0.78. In addition, the CBC score showed strong predictive ability at 30 days and at 1, 5, and 10 years when a scalar risk score incorporating age and sex was used (linear trend, P < 0.001).⁹⁴

Red Blood Cell Count and Coronary Heart Disease Risk

Several factors related to RBCs are associated with CHD, including hemoglobin levels and the hematocrit, RDW, and ESR⁹⁵; however, there are not enough data to suggest an association between the RBC count and cardiovascular disease. Although polycythemia vera is associated with an increased risk of macrovascular events,⁹⁶ elevated RBC counts within the upper limits of normal have been reported to have only weak or no association with cardiovascular risk.⁹⁷

Hematocrit and Coronary Heart Disease Risk

Several studies have shown a relationship between the hematocrit and incident cardiovascular events in patients who have had an MI,^98,99 in those with PAD,¹⁰⁰ and in asymptomatic individuals.^101–109 However, results of some studies have not shown a significant relationship between hematocrit and CHD risk.^103,110,111 In fact, while most studies of different patient populations do show an association between increased hematocrit and increased risk of CHD, the observed risk ratios are generally low, and, therefore, the clinical usefulness of hematocrit alone is unclear.A meta-analysis of 19 prospective studies of hematocrit and CHD risk showed a pooled odds ratio of 1.16 (95% CI, 1.05–1.29) in disease-free subjects, and a risk ratio of 1.81 (95% CI, 1.19–2.76) in patients with vascular disease.⁹⁵ The same meta-analysis also dealt with the value of viscosity and the ESR for CHD risk prediction. The investigators reported a risk ratio of 1.57 (95% CI, 1.34–1.85) for the top tertile of plasma viscosity in population-based studies, and a risk ratio of 2.6 (95% CI, 1.64–4.12) for patients with vascular disease.⁹⁵ Results of a more recent study show that the hematocrit-to-blood viscosity (Hct-BV) ratio had significant negative correlation with the frequency of hospital admission and that a lower Hct-BV ratio was associated with a greater likelihood of cardiac death in CHD patients.¹¹² Uncertainty remains in regard to the effectiveness of blood transfusion in correcting a low hematocrit in ACS patients.¹¹³ Although an observational study by Wu and colleagues¹¹⁴ suggests that blood transfusion can improve short-term survival in patients who are anemic at presentation, Hebert and colleagues¹¹⁵ found no such benefit, while others^116–118 actually report a harmful effect. A retrospective cohort study (2006) suggests that higher mortality rates in women after CABG are due in part to the more common occurrence of blood transfusions in women, leading to immunomodulation and an increased risk of infection and death after bypass surgery.¹¹⁹ Results from a pooled study of ACS patients from 3 large international trials showed an increased mortality rate after blood transfusion in patients who developed moderate-to-severe bleeding, anemia, or both, during hospitalization.¹¹³ In another analysis involving a subset of 44,242 patients, investigators found an inverse relationship between in-hospital death and nadir hematocrit in non-STEMI patients.¹²⁰ They also found that transfusions were related to significantly increased in-hospital death in the highest hematocrit quartile (Hct >30).¹²⁰ Therefore, it appears that well-designed, randomized, controlled trials of transfusion strategies are needed to provide clear guidelines with regard to blood transfusion in ACS patients. Until then, a cautious approach is warranted in the use of blood transfusion in these patients.The RDW, a numerical measure of the variability of the size of circulating erythrocytes, is significantly associated with an increased risk of all-cause death, and specifically with death secondary to cardiovascular disease in cross-sectional studies of the population of the United States.^121,122 In addition, the RDW is an independent predictor of death in patients who have had previous MIs¹²³ or stroke¹²⁴ and in men referred for coronary angiography.¹²⁵

Erythrocyte Sedimentation Rate and Coronary Heart Disease Risk

Although the ESR varies among elderly patients, it has a positive correlation with several CHD risk factors, including age, sex, smoking, systolic blood pressure, total cholesterol levels, heart rate, body mass index, diabetes, alcohol consumption, and fibrinogen, hemoglobin, and albumin levels.^126–129 After multivariate adjustment, the ESR is an independent and strong short- and long-term predictor of CHD death.¹²⁸ In young subjects, a moderate but persistent elevation in the ESR has been associated with an increased risk of incident MI.¹³⁰ Other conditions associated with a persistently elevated ESR include chronic infectious states, renal failure, rheumatoid arthritis, and chronic bronchitis.¹³¹ In the Stockholm Prospective Study, there was a positive and independent relationship between the ESR and fatal MI in asymptomatic men and women,¹³² but in NHANES I, the ESR was a risk factor for fatal MI only in men.¹⁰ In the Reykjavik Study, the ESR was an independent long-term predictor of CHD and death due to stroke in both men and women.⁹ Another study found that the ESR was related to the extent of coronary atherosclerosis on angiography and was a predictor of cardiac death in men with ischemic heart disease.¹²⁹ Finally, a meta-analysis of 4 population-based studies showed that an ESR in the top third tertile yielded a risk ratio of 1.33 (95% CI, 1.15–1.54), compared with an ESR in the bottom tertile.⁹⁵

Platelets and Coronary Heart Disease Risk

High platelet counts—such as those seen with hematologic neoplasms—are clearly associated with an increased risk of thrombosis; however, the effect of increased platelet numbers that are still within physiologic ranges remains unclear. A limited number of studies have shown that high platelet counts and a rapid platelet-aggregation response are associated with increased long-term coronary death.¹³³ Conversely, a low platelet count in the presence of an increased mean platelet volume after ischemic cerebral infarction is also associated with increased mortality rate.¹³⁴ In a cross-sectional study of patients undergoing coronary angiography, the mean platelet volume was higher in MI patients than in control patients, and stable angina pectoris correlated with the extent of CHD.¹³⁵ Other studies suggest that platelet counts and aggregation that are within physiologic range are not related to CHD events.¹³⁶ Khandekar and colleagues¹³⁷ found a significant increase in platelet volume indices, mean platelet volume, and platelet distribution width in patients with unstable angina or acute MI, compared with stable CHD patients and healthy matched control patients.¹³⁷ It appears that the role of platelets in the pathogenesis of CHD is due mainly to their functional properties and their interaction with plasma and tissue factors; only a small proportion of their effect—if any—is related to their absolute number. The small number of related positive studies may be the result of a publication bias toward the withholding of negative findings.

Potential Mechanisms

The effect of leukocytosis on CHD can be explained by multiple mechanisms; however, the inflammatory basis of atherosclerosis remains the cornerstone of this relation. Atherosclerotic lesions are inflammatory in nature, occur predominantly in large- and medium-sized arteries, and develop early in life.³ Leukocytosis can be considered a marker of inflammatory changes in atherosclerotic lesions, because leukocytes play a key role in the initiation and progression of the disease. In terms of ACS pathophysiology, leukocytes themselves are directly responsible for myocardial injury. Leukocytes release cytokines, bringing about further macrophage recruitment and the proliferation of smooth muscle cells within the vascular wall. In addition, protease secretion leads to endothelial damage of the coronary vessels, exposing thrombogenic collagen and predisposing the vessels to thrombus formation. Phagocytes release myeloperoxidase, which generates reactive oxygen species that are involved in the generation and progression of atherosclerosis and that contribute to the development of plaque instability in acute MI.³¹ A 2007 study has linked high WBC counts to the presence of vulnerable plaque.¹³⁸ The investigators used optical coherence tomography to visualize lesions in patients undergoing coronary catheterization. An elevated baseline WBC count correlated with the macrophage density of the visualized plaque (r = 0.483; P = 0.001), and it was shown that patients with thin-cap fibroatheromas had higher WBC counts than did those without thin-cap fibroatheromas (11 vs 7.9; P = 0.007).¹³⁸ Other studies^139–148 have shown that leukocytes also play an important role in the pathogenesis of myocardial injury through multiple mechanisms, such as vessel-plugging, decreased perfusion, and abnormal WBC aggregation.Elevated WBC counts are associated with several coronary risk factors, including smoking, elevated serum triglyceride and cholesterol levels, clotting factors, body mass index, obesity, and diastolic blood pressure.^149–152 Leukocytosis also is associated with several disorders that characterize the metabolic syndrome^153,154 and with micro- and macrovascular complications in patients with diabetes.¹⁵⁵ The different mechanisms by which leukocytosis might affect CHD are listed in 30,75,139–146,148–152,156–161 TABLE I. Possible Mechanisms of Action of White Blood Cells in Coronary Heart Disease Open in a separate window Elevated RBC counts and hematocrit are associated with rheologic effects, such as increased viscosity, which is a risk factor for coronary events.¹⁶² The adenosine diphosphate released from the RBCs enhances platelet aggregation,¹⁶³ and the elevated hematocrit is associated with a concomitant increase in platelet adhesion.^162,164 Aggregation of RBCs is significantly higher in men with proven vascular disease.¹⁶⁵ In addition, RBC indices are associated with a number of CHD risk factors. For example, increased hemoglobin levels are associated with elevated serum cholesterol and triglyceride levels,^166,167 and it is believed that the lipid-rich membranes of RBCs contribute to atheroma formation in the coronary arteries.¹⁶⁸ In an elaborate set of studies in patients who died suddenly of cardiac causes, Kolodgie and colleagues¹⁶⁹ found that an accumulation of erythrocytic membranes within an atherosclerotic plaque increases the risk of plaque destabilization by contributing to the deposition of free cholesterol. A prospective study using data from the Nurses'' Health Study showed that a higher total trans-fatty acid content in the erythrocytes is associated with a significantly increased risk of CHD, with the highest quartile of trans-fatty acid content in erythrocytes having a relative risk of 2.7 (95% CI, 1.5–5; P < 0.01 for trend), compared with the lowest quartile.¹⁷⁰ Accordingly, Tziakas and colleagues¹⁷¹ showed that the cholesterol content of erythrocytic membranes was significantly higher in patients with ACS than in patients with stable angina. It is thought that the altered membranes might decrease the fluidity of the RBCs. The different mechanisms by which RBCs might affect CHD are summarized in 89,162–164,166–171 TABLE II. Possible Mechanisms of the Role of Red Blood Cells in Coronary Heart Disease Open in a separate window Inflammation and thrombosis are closely related.¹⁷² Platelets have clear roles in thrombosis and contribute to inflammation.^158,173,174 Under stress, activated platelets help neutrophils adhere to the subendothelial matrix. Chirkov and colleagues¹⁷⁵ have shown that there is increased platelet aggregability and resistance to nitric oxide in patients with stable angina pectoris and ACS, compared with patients without CHD (P < 0.01). Platelets also synthesize interleukin-1 beta (IL-1β), an important mediator of platelet-induced activation of the endothelial cells, which, in turn, induce chemokines that up-regulate the molecules that promote endothelial adhesion of neutrophils and monocytes.¹⁷⁶ This increased total platelet-monocyte binding has been shown in ACS patients.¹⁷⁷ In addition, patients with a larger territorial burden of atherosclerotic disease have shown greater P-selection expression, which promotes inflammation, atherogenesis, and thrombosis.¹⁷⁸ Activated platelets also have been implicated in the oxidative modification of LDL-C that can contribute to proliferation of smooth-muscle cells. Platelets are the source of 90% of the circulating CD40L, which has proatherogenic and prothrombotic functions and is a predictor of incident MI, stroke, and cardiovascular death.¹⁷⁹ The different mechanisms by which platelets might affect CHD are summarized in 158,172–179 TABLE III. Possible Mechanisms of the Role of Platelets in Coronary Heart Disease Open in a separate window

Conclusions

Elevated WBC counts, along with other components of the CBC, are associated with CHD morbidity and death. The tests are inexpensive and widely available and warrant further use for the purpose of coronary risk evaluation in the clinical and research settings. Comparative studies are needed of the WBC count and the CBC versus more novel inflammatory markers (such as CRP) for predicting cardiovascular disease risk. In addition to the absolute number of leukocytes and other blood cells, several indices of their activity, such as expression of different leukocytic proinflammatory genes, should also be tested in this context. Such analyses should take into account the intra-individual variability of WBC counts and subtypes, and also the known racial variances in WBC counts.The WBC count should be further tested as a surrogate marker of outcomes for evaluating the effect of statins and similar pharmacologic interventions. Results of a 2008 study showed that statins can prevent vascular events in patients with high baseline CRP and moderate levels of LDL-C.¹⁸⁰ Future studies and subgroup analyses are needed to determine whether patients with high WBC counts and moderate-to-low LDL-C levels (with either high or low CRP levels) are a new target population that could benefit from statin therapy. Elevated baseline WBC counts or no reduction in WBC counts (or related cell subtypes) during therapy might identify different response patterns and a need for more aggressive or combined therapy. Results from such trials could be used to develop clinical guidelines and to promote the use of these tests in the most appropriate manner.     

Thermodynamics of formation of coffinite,USiO4

Coffinite, USiO₄, is an important U(IV) mineral, but its thermodynamic properties are not well-constrained. In this work, two different coffinite samples were synthesized under hydrothermal conditions and purified from a mixture of products. The enthalpy of formation was obtained by high-temperature oxide melt solution calorimetry. Coffinite is energetically metastable with respect to a mixture of UO₂ (uraninite) and SiO₂ (quartz) by 25.6 ± 3.9 kJ/mol. Its standard enthalpy of formation from the elements at 25 °C is −1,970.0 ± 4.2 kJ/mol. Decomposition of the two samples was characterized by X-ray diffraction and by thermogravimetry and differential scanning calorimetry coupled with mass spectrometric analysis of evolved gases. Coffinite slowly decomposes to U₃O₈ and SiO₂ starting around 450 °C in air and thus has poor thermal stability in the ambient environment. The energetic metastability explains why coffinite cannot be synthesized directly from uraninite and quartz but can be made by low-temperature precipitation in aqueous and hydrothermal environments. These thermochemical constraints are in accord with observations of the occurrence of coffinite in nature and are relevant to spent nuclear fuel corrosion.In many countries with nuclear energy programs, spent nuclear fuel (SNF) and/or vitrified high-level radioactive waste will be disposed in an underground geological repository. Demonstrating the long-term (10⁶–10⁹ y) safety of such a repository system is a major challenge. The potential release of radionuclides into the environment strongly depends on the availability of water and the subsequent corrosion of the waste form as well as the formation of secondary phases, which control the radionuclide solubility. Coffinite (1), USiO₄, is expected to be an important alteration product of SNF in contact with silica-enriched groundwater under reducing conditions (2–8). It is also found, accompanied by thorium orthosilicate and uranothorite, in igneous and metamorphic rocks and ore minerals from uranium and thorium sedimentary deposits (2, 4, 5, 8–16). Under reducing conditions in the repository system, the uranium solubility (very low) in aqueous solutions is typically derived from the solubility product of UO₂. Stable U(IV) minerals, which could form as secondary phases, would impart lower uranium solubility to such systems. Thus, knowledge of coffinite thermodynamics is needed to constrain the solubility of U(IV) in natural environments and would be useful in repository assessment.In natural uranium deposits such as Oklo (Gabon) (4, 7, 11, 12, 14, 17, 18) and Cigar Lake (Canada) (5, 13, 15), coffinite has been suggested to coexist with uraninite, based on electron probe microanalysis (EPMA) (4, 5, 7, 11, 13, 17, 19, 20) and transmission electron microscopy (TEM) (8, 15). However, it is not clear whether such apparent replacement of uraninite by a coffinite-like phase is a direct solid-state process or occurs mediated by dissolution and reprecipitation.The precipitation of USiO₄ as a secondary phase should be favored in contact with silica-rich groundwater (21) [silica concentration >10⁻⁴ mol/L (22, 23)]. Natural coffinite samples are often fine-grained (4, 5, 8, 11, 13, 15, 24), due to the long exposure to alpha-decay event irradiation (4, 6, 25, 26) and are associated with other minerals and organic matter (6, 8, 12, 18, 27, 28). Hence the determination of accurate thermodynamic data from natural samples is not straightforward. However, the synthesis of pure coffinite also has challenges. It appears not to form by reacting the oxides under dry high-temperature conditions (24, 29). Synthesis from aqueous solutions usually produces UO₂ and amorphous SiO₂ impurities, with coffinite sometimes being only a minor phase (24, 30–35). It is not clear whether these difficulties arise from kinetic factors (slow reaction rates) or reflect intrinsic thermodynamic instability (33). Thus, there are only a few reported estimates of thermodynamic properties of coffinite (22, 36–40) and some of them are inconsistent. To resolve these uncertainties, we directly investigated the energetics of synthetic coffinite by high-temperature oxide melt solution calorimetry to obtain a reliable enthalpy of formation and explored its thermal decomposition.     

Fenton chemistry at aqueous interfaces

In a fundamental process throughout nature, reduced iron unleashes the oxidative power of hydrogen peroxide into reactive intermediates. However, notwithstanding much work, the mechanism by which Fe²⁺ catalyzes H₂O₂ oxidations and the identity of the participating intermediates remain controversial. Here we report the prompt formation of O=Fe^IVCl₃⁻ and chloride-bridged di-iron O=Fe^IV·Cl·Fe^IICl₄⁻ and O=Fe^IV·Cl·Fe^IIICl₅⁻ ferryl species, in addition to Fe^IIICl₄⁻, on the surface of aqueous FeCl₂ microjets exposed to gaseous H₂O₂ or O₃ beams for <50 μs. The unambiguous identification of such species in situ via online electrospray mass spectrometry let us investigate their individual dependences on Fe²⁺, H₂O₂, O₃, and H⁺ concentrations, and their responses to tert-butanol (an ·OH scavenger) and DMSO (an O-atom acceptor) cosolutes. We found that (i) mass spectra are not affected by excess tert-butanol, i.e., the detected species are primary products whose formation does not involve ·OH radicals, and (ii) the di-iron ferryls, but not O=Fe^IVCl₃⁻, can be fully quenched by DMSO under present conditions. We infer that interfacial Fe(H₂O)_n²⁺ ions react with H₂O₂ and O₃ >10³ times faster than Fe(H₂O)₆²⁺ in bulk water via a process that favors inner-sphere two-electron O-atom over outer-sphere one-electron transfers. The higher reactivity of di-iron ferryls vs. O=Fe^IVCl₃⁻ as O-atom donors implicates the electronic coupling of mixed-valence iron centers in the weakening of the Fe^IV–O bond in poly-iron ferryl species.High-valent Fe^IV=O (ferryl) species participate in a wide range of key chemical and biological oxidations (1–4). Such species, along with ·OH radicals, have long been deemed putative intermediates in the oxidation of Fe^II by H₂O₂ (Fenton’s reaction) (5, 6), O₃, or HOCl (7, 8). The widespread availability of Fe^II and peroxides in vivo (9–12), in natural waters and soils (13), and in the atmosphere (14–18) makes Fenton chemistry and Fe^IV=O groups ubiquitous features in diverse systems (19). A lingering issue regarding Fenton’s reaction is how the relative yields of ferryls vs. ·OH radicals depend on the medium. For example, by assuming unitary ·OH radical yields, some estimates suggest that Fenton’s reaction might account for ∼30% of the ·OH radical production in fog droplets (20). Conversely, if Fenton’s reaction mostly led to Fe^IV=O species, atmospheric chemistry models predict that their steady-state concentrations would be ∼10⁴ times larger than [·OH], thereby drastically affecting the rates and course of oxidative chemistry in such media (20). Fe^IV=O centers are responsible for the versatility of the family of cytochrome P450 enzymes in catalyzing the oxidative degradation of a vast range of xenobiotics in vivo (21–28), and the selective functionalization of saturated hydrocarbons (29). The bactericidal action of antibiotics has been linked to their ability to induce Fenton chemistry in vivo (9, 30–34). Oxidative damage from exogenous Fenton chemistry likely is responsible for acute and chronic pathologies of the respiratory tract (35–38).Despite its obvious importance, the mechanism of Fenton’s reaction is not fully understood. What is at stake is how the coordination sphere of Fe²⁺ (39–46) under specific conditions affects the competition between the one-electron transfer producing ·OH radicals (the Haber–Weiss mechanism) (47), reaction R1, and the two-electron oxidation via O-atom transfer (the Bray–Gorin mechanism) into Fe^IVO²⁺, reaction R2 (6, 23, 26, 27, 45, 48–51):Ozone reacts with Fe²⁺ via analogous pathways leading to (formally) the same intermediates, reactions R3a, R3b, and R4 (8, 49, 52, 53):At present, experimental evidence about these reactions is indirect, being largely based on the analysis of reaction products in bulk water in conjunction with various assumptions. Given the complex speciation of aqueous Fe²⁺/Fe³⁺ solutions, which includes diverse poly-iron species both as reagents and products, it is not surprising that classical studies based on the identification of reaction intermediates and products via UV-absorption spectra and the use of specific scavengers have fallen short of fully unraveling the mechanism of Fenton’s reaction. Herein we address these issues, focusing particularly on the critically important interfacial Fenton chemistry that takes place at boundaries between aqueous and hydrophobic media, such as those present in atmospheric clouds (16), living tissues, biomembranes, bio-microenvironments (38, 54, 55), and nanoparticles (56, 57).We exploited the high sensitivity, surface selectivity, and unambiguous identification capabilities of a newly developed instrument based on online electrospray mass spectrometry (ES-MS) (58–62) to identify the primary products of reactions R1–R4 on aqueous FeCl₂ microjets exposed to gaseous H₂O₂ and O₃ beams under ambient conditions [in N₂(g) at 1 atm at 293 ± 2 K]. Our experiments are conducted by intersecting the continuously refreshed, uncontaminated surfaces of free-flowing aqueous microjets with reactive gas beams for τ ∼10–50 μs, immediately followed (within 100 μs; see below) by in situ detection of primary interfacial anionic products and intermediates via ES-MS (Methods, SI Text, and Figs. S1 and S2). We have previously demonstrated that online mass spectrometric sampling of liquid microjets under ambient conditions is a surface-sensitive technique (58, 62–67).     

Elastic mucus strands impair mucociliary clearance in cystic fibrosis pigs

Defective mucociliary transport contributes to infections and inflammation that destroy cystic fibrosis (CF) lungs. To better understand mechanisms that impair mucociliary transport, we tracked insufflated microdisks with X-ray computed tomography in spontaneously breathing CF pigs. Aerosolized saline increased microdisk movement toward the larynx. Surprisingly, forward progression was repeatedly interrupted as microdisks abruptly recoiled. Thus, although saline increased motion, it had minimal effects on microdisk clearance. To test if abnormally elastic mucus strands were responsible, we aerosolized Tris-carboxyethyl phosphine (TCEP), a reducing agent that breaks disulfide bonds linking mucins. TCEP largely eliminated retrograde movement and increased microdisk clearance. Ex vivo studies confirmed that TCEP broke CF mucus strands, unleashing them from submucosal gland ducts, and cilia carried them rostrally. These findings emphasize the role of abnormally elastic submucosal gland mucus strands in impairing CF mucociliary clearance and suggest that disrupting mucus strands may improve mucociliary clearance in CF.

Mucociliary transport (MCT) is an innate defense mechanism that protects lungs from inhaled and aspirated material (1–3). MCT depends on mucus, which traps particulates and pathogens, and motile cilia, which protrude from epithelia lining the airways and by their beating propel mucus up the airways and out of the lung. In the cartilaginous airways of humans, pigs, and likely other large mammals, submucosal glands (SMGs) are required for normal MCT (4, 5). SMGs expand the number of fluid- and mucus-producing cells beyond that in surface epithelia and secrete much of the airway mucus (5–7). The major structural component of mucus is mucins, and SMGs produce the mucin MUC5B, which is key for MCT (8–10). SMGs secrete mucus under basal conditions, and when the respiratory tract is challenged, they respond to neurohumoral signals by secreting copious amounts of mucus (5, 11–13).Mucus emerges from SMG ducts onto the airway surface in the form of mucus strands (10, 14–18). Beating cilia pull the mucus strands; they stretch, eventually break, and are then swept upward toward the larynx (14–16). Elasticity dominates the biophysical properties of the mucus (19, 20). Reducing agents break the disulfide bonds that link mucins and thereby disrupt the integrity of mucus strands (14, 21–24). As a result, when reducing agents were applied to the airway surface liquid (ASL), the mucus emerging from SMG ducts immediately fragmented, failed to generate strands, and thereby prevented normal MCT (14). Thus, mucus strands secreted from SMGs make key contributions to MCT. Consistent with this conclusion, when pigs lack the SMGs that produce mucus strands, MCT is disrupted (4).MCT is defective in cystic fibrosis (CF), a life-threatening genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel (2, 25, 26). MCT is impaired, in part, by the altered biophysical properties of the mucus produced by CF SMGs (10, 15, 16, 20). Defective SMG mucus is apparent even before birth; histopathologic examination of lungs from human fetuses with CF revealed abnormal lamellated Periodic acid-Schiff positive material obstructing the lumen and ducts of airway SMGs (27, 28). Similar changes were observed in deceased infants with CF, although inflammation and infection occurring after birth may have contributed (17, 28–30). Studies in CF pigs and sheep immediately after birth also reveal mucus accumulation in SMG ducts and acini (10, 15, 16, 31). Mucus accumulation in SMGs also appears in CF ferrets and rats once they develop SMG glands postnatally (32, 33) and in older CF rabbits in Bowman glands of the olfactory epithelium (34). The abnormal mucus and impaired MCT contribute to the recurrent airway infections and inflammation in CF.Unlike mucus strands secreted from wild-type SMGs, mucus strands from CF SMGs often fail to break after emerging from their ducts. As a result, they accumulate and prevent particles from moving up the airways (10, 14–16). Defective HCO₃⁻ and Cl⁻ secretion in SMGs are responsible for the abnormal mucus strands (10, 15); without CFTR function, SMG liquid becomes abnormally acidic, and the protein concentration increases (20, 35). These abnormalities produce mucus with increased elasticity and tensile strength and reduced breakage when mucus strands are stretched (15, 20). In freshly excised non-CF airways, removing HCO₃⁻ and inhibiting Cl⁻ secretion with bumetanide reproduces CF-like conditions so that mucus strands emerging from SMGs often fail to break loose from the ducts and accumulate on the airway surface, preventing normal MCT (10, 15).The reducing reagent Tris-carboxyethyl phosphine (TCEP) breaks mucus strands (14, 21, 22). Because abnormal mucus strands impair MCT in CF, we asked if aerosolized TCEP would enhance MCT in CF pigs. The answer was of interest because TCEP has the opposite effect in non-CF pigs, decreasing MCT (14). We also wished to know the answer because reducing agents are being investigated as a potential therapy for CF (23, 24). However, before directly addressing this question, we needed to assess the effect of aerosolized saline, which is used to deliver the TCEP. We were encouraged to do that because in a previous in vivo study, we found that aerosolizing 0.5 mL of saline to non-CF pigs increased MCT (14). In other studies, isotonic physiologic salt solutions reversed the impairment in MCT induced by blocking Cl⁻ and HCO₃⁻ secretion in porcine tracheas (36), and in humans, inhalation of isotonic saline increased mucociliary clearance in normal and asthmatic lungs (37).Here, we took advantage of a model of CF in pigs, which like humans have abundant SMGs and replicate CF abnormalities. We also used a computed tomography (CT) imaging method with high spatial and temporal resolution that reveals the trajectories of individual microdisks as they are carried by mucus strands within intrapulmonary airways.     

DC-SIGN activation mediates the differential effects of SAP and CRP on the innate immune system and inhibits fibrosis in mice

Fibrosis is caused by scar tissue formation in internal organs and is associated with 45% of deaths in the United States. Two closely related human serum proteins, serum amyloid P (SAP) and C-reactive protein (CRP), strongly affect fibrosis. In multiple animal models, and in Phase 1 and Phase 2 clinical trials, SAP affects several aspects of the innate immune system to reduce fibrosis, whereas CRP appears to potentiate fibrosis. However, SAP and CRP bind the same Fcγ receptors (FcγR) with similar affinities, and why SAP and CRP have opposing effects is unknown. Here, we report that SAP but not CRP binds the receptor DC-SIGN (SIGN-R1) to affect the innate immune system, and that FcγR are not necessary for SAP function. A polycyclic aminothiazole DC-SIGN ligand and anti–DC-SIGN antibodies mimic SAP effects in vitro. In mice, the aminothiazole reduces neutrophil accumulation in a model of acute lung inflammation and, at 0.001 mg/kg, alleviates pulmonary fibrosis by increasing levels of the immunosuppressant IL-10. DC-SIGN (SIGN-R1) is present on mouse lung epithelial cells, and SAP and the aminothiazole potentiate IL-10 production from these cells. Our data suggest that SAP activates DC-SIGN to regulate the innate immune system differently from CRP, and that DC-SIGN is a target for antifibrotics.Fibrosing diseases such scleroderma, pulmonary fibrosis, and renal fibrosis are caused by aberrant scar tissue formation in internal organs and are associated with 45% of deaths in the United States (1). At a fibrotic lesion, monocytes leave the blood, enter the tissue, and differentiate into cells such as macrophages and fibrocytes (2). Fibrocytes and macrophages then secrete extracellular matrix (ECM) proteins, ECM modifying enzymes, and/or cytokines such as IL-4 to promote scar tissue formation and fibrosis (3, 4).Pentraxins are a family of highly conserved secreted proteins that have a profound effect on the development of fibrosis and the regulation of the innate immune system (5–7). The pentraxin serum amyloid P (SAP) reduces neutrophil activation and recruitment (8, 9), inhibits the differentiation of monocytes into fibroblast-like cells called fibrocytes (8, 10), and promotes IL-10–secreting macrophages (11–13). In animal models and two human trials (6, 14, 15), injections of SAP decrease fibrosis, indicating that SAP has a dominant effect on a disease that is mediated in part by the innate immune system. Conversely, the closely related pentraxin C-reactive protein (CRP) is proinflammatory and promotes fibrosis (5, 16). However, under some conditions, CRP decreases inflammation, indicating that much remains to be understood about this molecule (5, 17). Despite the strong effects of pentraxins on the innate immune system and fibrosis (5, 6), little is known about their mechanism of action. For instance, pentraxins such as SAP and CRP appear to bind the same Fcγ receptors (FcγR) with similar affinities (7, 8, 18), but they generally have opposite effects. What causes this functional difference is not known.Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN/CD209) is a C-type lectin found on dendritic cells, macrophages, and monocytes (19, 20). DC-SIGN mainly binds to mannosylated and fucosylated proteins (19, 20). Humans have DC-SIGN and L-SIGN, whereas mice have eight DC-SIGN orthologs called SIGN-R1–8 (21). SIGN-R1 most closely resembles DC-SIGN (21). DC-SIGN and SIGN-R1 also bind sialylated IgG (sIgG) (20). This interaction appears to be a protein:potein interaction and not a sialic acid:DC-SIGN interaction (22). Both sIgGs and SAP have α(2,6)-linked terminal sialic acids on the protein surface, and both sIgGs and SAP alleviate inflammation in mice (6, 20, 23).In this report, we show that in absence of all of the FcγR, neutrophils, monocytes, and macrophages still respond to SAP, indicating that SAP uses other receptors. For SAP, we show that one of the other receptors is DC-SIGN. We also found that anti–DC-SIGN antibodies and a small-molecule DC-SIGN ligand mimic the effects of SAP. The synthetic DC-SIGN ligand shows efficacy in murine models of acute lung inflammation and pulmonary fibrosis. In contrast to SAP, we find that CRP requires the FcγR to regulate neutrophils and IL-10 secretion from macrophages, but not to increase ICAM-I⁺ macrophages. This finding suggests that there are additional CRP receptors that regulate macrophage polarization. Our findings suggest the presence of a previously unidentified pentraxin target that accounts for the functional difference between SAP and CRP, and which might be useful as a therapeutic target to regulate the innate immune system and fibrosis.     

Symmetric activation and modulation of the human calcium-sensing receptor

The human extracellular calcium-sensing (CaS) receptor controls plasma Ca²⁺ levels and contributes to nutrient-dependent maintenance and metabolism of diverse organs. Allosteric modulation of the CaS receptor corrects disorders of calcium homeostasis. Here, we report the cryogenic-electron microscopy reconstructions of a near–full-length CaS receptor in the absence and presence of allosteric modulators. Activation of the homodimeric CaS receptor requires a break in the transmembrane 6 (TM6) helix of each subunit, which facilitates the formation of a TM6-mediated homodimer interface and expansion of homodimer interactions. This transformation in TM6 occurs without a positive allosteric modulator. Two modulators with opposite functional roles bind to overlapping sites within the transmembrane domain through common interactions, acting to stabilize distinct rotamer conformations of key residues on the TM6 helix. The positive modulator reinforces TM6 distortion and maximizes subunit contact to enhance receptor activity, while the negative modulator strengthens an intact TM6 to dampen receptor function. In both active and inactive states, the receptor displays symmetrical transmembrane conformations that are consistent with its homodimeric assembly.

Critical to the maintenance of Ca²⁺ homeostasis, the extracellular calcium-sensing (CaS) receptor was the first G protein–coupled receptor (GPCR) discovered to sense ions (1–3). The CaS receptor detects fluctuations in plasma Ca²⁺ at the parathyroid. In response to increases in Ca²⁺, it transmits signals to inhibit the release of parathyroid hormone, in turn preventing further rises in Ca²⁺ concentration (2, 3). In the cortical thick ascending limb of the renal nephron, the CaS receptor is also activated by surges in plasma Ca²⁺ and responds by inhibiting Ca²⁺ reabsorption. The excess urinary calcium excretion arising from CaS receptor activation lowers the plasma Ca²⁺ level. The CaS receptor is implicated in various pathologies associated with hypercalcemia and hypocalcemia (4). It has also been linked to the progression of diseases such as breast and colon cancer, in which the receptor modulates tumor growth (3, 5–7).The CaS receptor senses a diverse array of extracellular stimuli. During normal function, it activates multiple intracellular signaling pathways involving G_q/11, G_i/o, or G_12/13; in tumor cells, it is coupled to G_s (2, 3, 8, 9). In addition to the principal agonist Ca²⁺, the receptor is directly activated by aromatic l-amino acids (10, 11). Other CaS agonists include various divalent and trivalent cations (12), referred to as type I calcimimetics for mimicking the action of Ca²⁺ (13).The activity of the CaS receptor is also subject to allosteric modulation. Positive allosteric modulators (PAMs) are classified as type II calcimimetics for increasing the receptor sensitivity for Ca²⁺ (12–16). The prototypical PAM molecules share a phenylalkylamine structure, including cinacalcet and NPS R-568 (abbreviated as R-568). Cinacalcet was the first drug described to target a GPCR allosterically, and it is used clinically to treat hyperparathyroidism in patients with chronic kidney diseases (15). Negative allosteric modulators (NAMs) of the CaS receptor are referred to as calcilytics for suppressing the receptor response to Ca²⁺ (12–16). Synthetic calcilytics such as NPS-2143 and ronacaleret are also structurally related to phenylalkylamines. Recently, inorganic phosphate has been identified as an inhibitor of the receptor (11, 17).The CaS receptor rests within the class C family of GPCRs and functions as an obligate homodimer. Like other class C GPCRs, each CaS subunit contains a large extracellular domain (ECD) involved in orthosteric ligand binding, a seven-helix transmembrane (TM) domain responsible for G protein coupling, followed by an extended cytoplasmic tail (18–23). The conformations of the CaS ECDs in both the inactive and active states have been determined by X-ray crystallography (11, 24). The ECD structures also revealed how the receptor recognizes various extracellular ligands, including Ca²⁺, the amino acid l-Trp, and inorganic phosphate. Although the role of amino acids is still under debate (25), recent structural studies of full-length CaS receptor further confirmed that Ca²⁺ and amino acids cooperate to activate the receptor (26–28).The TM domain of the CaS receptor harbors the binding sites for PAM and NAM molecules according to previous mutagenesis studies (29–32). Recently reported modulator-bound CaS receptor structures revealed asymmetric TM configurations that are stabilized by PAM molecules binding in different poses within the separate subunits of the homodimer (33). We have determined PAM- and NAM-bound, as well as PAM-free, structures of a near–full-length CaS receptor using cryogenic-electron microscopy (cryo-EM) that display symmetric TM dimers and modulator poses, instead. This finding presents the possibility of receptor activation without requiring asymmetric conformational transition. Our structures also illustrate how distortion of TM6 provides the driving force for receptor activation. Furthermore, the presence of a PAM or NAM stabilizes distinct TM6 helix conformations to promote specific dimer arrangements and differentially modulate receptor function.     

Coulomb interaction,phonons, and superconductivity in twisted bilayer graphene

The polarizability of twisted bilayer graphene, due to the combined effect of electron–hole pairs, plasmons, and acoustic phonons, is analyzed. The screened Coulomb interaction allows for the formation of Cooper pairs and superconductivity in a significant range of twist angles and fillings. The tendency toward superconductivity is enhanced by the coupling between longitudinal phonons and electron–hole pairs. Scattering processes involving large momentum transfers, Umklapp processes, play a crucial role in the formation of Cooper pairs. The magnitude of the superconducting gap changes among the different pockets of the Fermi surface.

Twisted bilayer graphene (TBG) shows a complex phase diagram which combines superconducting and insulating phases (1, 2) and resembles strongly correlated materials previously encountered in condensed matter physics (3–6). On the other hand, superconductivity seems more prevalent in TBG (7–11), while in other strongly correlated materials magnetic phases are dominant.The pairing interaction responsible for superconductivity in TBG has been intensively studied. Among other possible pairing mechanisms, the effect of phonons (12–19) (see also ref. 20), the proximity of the chemical potential to a van Hove singularity in the density of states (DOS) (21–25) and excitations of insulating phases (26–28) (see also refs. 29–31), and the role of electronic screening (32–35) have been considered.In the following, we analyze how the screened Coulomb interaction induces pairing in TBG. The calculation is based on the Kohn–Luttinger formalism (36) for the study of anisotropic superconductivity via repulsive interactions. The screening includes electron–hole pairs (37), plasmons (38), and phonons (note that acoustic phonons overlap with the electron–hole continuum in TBG). Our results show that the repulsive Coulomb interaction, screened by plasmons and electron–hole pairs only, leads to anisotropic superconductivity, although with critical temperatures of order T_c ∼ 10⁻³ to 10⁻² K. The inclusion of phonons in the screening function substantially enhances the critical temperature, to T_c ∼ 1 to 10 K.     

Single-molecule optofluidic microsensor with interface whispering gallery modes

Label-free sensors are highly desirable for biological analysis and early-stage disease diagnosis. Optical evanescent sensors have shown extraordinary ability in label-free detection, but their potentials have not been fully exploited because of the weak evanescent field tails at the sensing surfaces. Here, we report an ultrasensitive optofluidic biosensor with interface whispering gallery modes in a microbubble cavity. The interface modes feature both the peak of electromagnetic-field intensity at the sensing surface and high-Q factors even in a small-sized cavity, enabling a detection limit as low as 0.3 pg/cm². The sample consumption can be pushed down to 10 pL due to the intrinsically integrated microfluidic channel. Furthermore, detection of single DNA with 8 kDa molecular weight is realized by the plasmonic-enhanced interface mode.

Detecting biological molecules and monitoring their dynamics are of crucial importance in biomedical analysis and disease diagnosis (1, 2). Practical applications generally involve complex biological environments, in which an engineered interface is highly desirable to enable the enrichment, detection, and analysis of specific biomolecules (3). Over the past decades, many techniques on interfacial molecular analysis have been developed, such as lateral flow immunoassay, electrochemical analytical techniques, and optical biosensors (4–7). Among them, optical evanescent microsensors, such as microspheres (8, 9), microtoroids (10–14), and nanowaveguides (15–18), have attracted considerable research interest since they can detect unlabeled molecules and monitor their interactions in real time and in situ with ultrahigh sensitivity, fast response, and miniature footprint.Despite these advantages, potentials of optical evanescent microsensors have not been fully explored. With the peak field intensity confined inside the cavity, these sensors can utilize the weak tail of the evanescent field only on the sensing surface (8, 10–12, 15, 16), thus limiting their sensitivities. Moreover, ultrasmall sample consumption is desired for high-efficiency sensing yet challenging in evanescent microsensors, since they require delicate sample delivery designs such as an additional chamber (9, 12) or a precisely aligned fluidic channel (19, 20). Therefore, an integrated microfluidic platform with ultimate sensitivity is highly demanded.In this work, we demonstrate an ultrasensitive optofluidic microbubble biosensor by exploiting the whispering gallery modes (WGMs) peaked at the interface between the optical resonator and the analyte solution, which are termed as the interface modes. Previously, the microbubble resonator has been widely used for measuring refractive index, biomolecule concentration, and single nanoparticle generally by the mode localized in the liquid core (21–29). Here, we find that the profile of the WGM field can be tuned by varying the wall thickness, and the interface mode emerges when the maximum of the field intensity is drawn onto the interface. Compared with conventional evanescent sensors, the present scheme utilizing interface modes promises maximum sensitivity for interfacial molecular analysis, pushing the detection limit down to 0.3 pg/cm². The scheme is also compatible with the widely adopted techniques to enhance signal- to-noise ratio (SNR) such as plasmonic hybridization (30–33) and frequency tracking (12, 15, 18). As a proof of concept, single-molecule detection is demonstrated with a plasmonic-enhanced interface mode. Naturally integrated into a microfluidic system, the sensor with single-molecule sensitivity exhibits ultrasmall sample consumption down to 10 pL, providing an automatic platform for biomedical analysis.     

Two-dimensional electronic–vibrational sum frequency spectroscopy for interactions of electronic and nuclear motions at interfaces

Interactions of electronic and vibrational degrees of freedom are essential for understanding excited-states relaxation pathways of molecular systems at interfaces and surfaces. Here, we present the development of interface-specific two-dimensional electronic–vibrational sum frequency generation (2D-EVSFG) spectroscopy for electronic–vibrational couplings for excited states at interfaces and surfaces. We demonstrate this 2D-EVSFG technique by investigating photoexcited interface-active (E)-4-((4-(dihexylamino) phenyl)diazinyl)-1-methylpyridin-1- lum (AP3) molecules at the air–water interface as an example. Our 2D-EVSFG experiments show strong vibronic couplings of interfacial AP3 molecules upon photoexcitation and subsequent relaxation of a locally excited (LE) state. Time-dependent 2D-EVSFG experiments indicate that the relaxation of the LE state, S₂, is strongly coupled with two high-frequency modes of 1,529.1 and 1,568.1 cm⁻¹. Quantum chemistry calculations further verify that the strong vibronic couplings of the two vibrations promote the transition from the S₂ state to the lower excited state S₁. We believe that this development of 2D-EVSFG opens up an avenue of understanding excited-state dynamics related to interfaces and surfaces.

Electronic and vibrational degrees of freedom are the most important physical quantities in molecular systems at interfaces and surfaces. Knowledge of interactions between electronic and vibrational motions, namely electronic–vibrational couplings, is essential to understanding excited-states relaxation pathways of molecular systems at interfaces and surfaces. Many excited-states relaxation processes occur at interfaces and surfaces, including charge transfer, energy transfer, proton transfer, proton-coupled electron transfer, configurational dynamics, and so on (1–11). These relaxation processes are intimately related to the electronic–vibrational couplings at interfaces and surfaces. Strong electronic–vibrational couplings could promote nonadiabatic evolution of excited potential energy and thus, facilitate chemical reactions or intramolecular structural changes of interfacial molecules (10, 12, 13). Furthermore, these interactions of electronic and vibrational degrees of freedom are subject to solvent environments (e.g., interfaces/surfaces with a restricted environment of unique physical and chemical properties) (9, 14, 15). Despite the importance of interactions of electronic and vibrational motions, little is known about excited-state electronic–vibrational couplings at interfaces and surfaces.Interface-specific electronic and vibrational spectroscopies enable us to characterize the electronic and vibrational structures separately. As interface-specific tools, second-order electronic sum frequency generation (ESFG) and vibrational sum frequency generation (VSFG) spectroscopies have been utilized for investigating molecular structure, orientational configurations, chemical reactions, chirality, static potential, environmental issues, and biological systems at interfaces and surfaces (16–52). Recently, structural dynamics at interfaces and surfaces have been explored using time-resolved ESFG and time-resolved VSFG with a visible pump or an infrared (IR) pump thanks to the development of ultrafast lasers (6–9, 13–15, 49, 53–61). Doubly resonant sum frequency generation (SFG) has been demonstrated to probe both electronic and vibration transitions of interfacial molecular monolayer (15, 62–71). This frequency-domain two-dimensional (2D) interface/surface spectroscopy could provide information regarding electronic–vibrational coupling of interfacial molecules. However, contributions from excited states are too weak to be probed due to large damping rates of vibrational states in excited states (62, 63). As such, the frequency-domain doubly resonant SFG is used only for electronic–vibrational coupling of electronic ground states. Ultrafast interface-specific electronic–vibrational spectroscopy could allow us to gain insights into how specific nuclear motions drive the relaxation of electronic excited states. Therefore, development of interface-specific electronic–vibrational spectroscopy for excited states is needed.In this work, we integrate the specificity of interfaces and surfaces into the capabilities of ultrafast 2D spectroscopy for dynamical electronic–vibrational couplings in excited states of molecules; 2D interface-specific spectroscopies are analogous to those 2D spectra in bulk that spread the information contained in a pump−probe spectrum over two frequency axes. Thus, one can better interpret congested one-dimensional signals. Two-dimensional vibrational sum frequency generation (2D-VSFG) spectroscopy was demonstrated a few year ago (72–74). Furthermore, heterodyne 2D-VSFG spectroscopy using middle infrared (mid-IR) pulse shaping and noncollinear geometry 2D-VSFG experiments have also been developed to study vibrational structures and dynamics at interfaces (31, 75–78). Recently, two-dimensional electronic sum frequency generation (2D-ESFG) spectroscopy has also been demonstrated for surfaces and interfaces (79). On the other hand, bulk two-dimensional electronic–vibrational (2D-EV) spectroscopy has been extensively used to investigate the electronic relaxation and energy transfer dynamics of molecules, biological systems, and nanomaterials (80–90). The 2D-EV technique not only provides electronic and vibrational interactions between excitons or different excited electronic states of systems but also, identifies fast nonradiative transitions through nuclear motions in molecules, aggregations, and nanomaterials. However, an interface-specific technique for two-dimensional electronic–vibrational sum frequency generation (2D-EVSFG) spectroscopy has yet to be developed.Here, we present the development of 2D-EVSFG spectroscopy for the couplings of electronic and nucleic motions at interfaces and surfaces. The purpose of developing 2D-EVSFG spectroscopy is to bridge the gap between the visible and IR regions to reveal how structural dynamics for photoexcited electronic states are coupled with vibrations at interfaces and surfaces. As an example, we applied this 2D-EVSFG experimental method to time evolution of electronic–vibrational couplings at excited states of interface-active molecules at the air–water interface.     

Childhood bullying involvement predicts low-grade systemic inflammation into adulthood

Bullying is a common childhood experience that involves repeated mistreatment to improve or maintain one’s status. Victims display long-term social, psychological, and health consequences, whereas bullies display minimal ill effects. The aim of this study is to test how this adverse social experience is biologically embedded to affect short- or long-term levels of C-reactive protein (CRP), a marker of low-grade systemic inflammation. The prospective population-based Great Smoky Mountains Study (n = 1,420), with up to nine waves of data per subject, was used, covering childhood/adolescence (ages 9–16) and young adulthood (ages 19 and 21). Structured interviews were used to assess bullying involvement and relevant covariates at all childhood/adolescent observations. Blood spots were collected at each observation and assayed for CRP levels. During childhood and adolescence, the number of waves at which the child was bullied predicted increasing levels of CRP. Although CRP levels rose for all participants from childhood into adulthood, being bullied predicted greater increases in CRP levels, whereas bullying others predicted lower increases in CRP compared with those uninvolved in bullying. This pattern was robust, controlling for body mass index, substance use, physical and mental health status, and exposures to other childhood psychosocial adversities. A child’s role in bullying may serve as either a risk or a protective factor for adult low-grade inflammation, independent of other factors. Inflammation is a physiological response that mediates the effects of both social adversity and dominance on decreases in health.The social and psychological effects of bullying involvement are independent of other childhood experiences, pleiotropic, and long lasting, with the worst effects for those who are both victims and bullies (e.g., refs. 1–4). To date, the primary focus of bullying research has been on such psychosocial outcomes. Bullied children, however, also have adverse physical health functioning (1, 5–7), including a broad range of somatic issues, such as sleep problems, abdominal pain, appetite suppression, headaches, and frequency of illnesses. In contrast, there is evidence to suggest that those who perpetrate only, pure bullies, may be healthier than their peers, emotionally and physically (6, 8). Little is known about how this social adversity becomes biologically embedded to influence health status.One potential mechanism is chronic systemic low-grade inflammation (9). Inflammation is activated similarly by a diverse range of health risk behaviors (poor diet, lack of exercise, and sleep disturbance) and environmental challenges [low socioeconomic status (SES), psychosocial stress] (10–14). Elevated inflammation markers are part of the phenomenology of common psychological disorders (particularly depression) across the lifespan (for reviews, refs. 15, 16). One marker of inflammation, C-reactive protein (CRP), has been the focus of extensive epidemiologic investigation because of the association of elevated plasma CRP levels (>3 mg/L) with cardiovascular risk (17, 18) and aspects of metabolic syndrome (19–21). Subclinical levels of inflammation may be a nonspecific marker for a broad range of organismic challenges, but they have not been studied as a mechanism for the social adversity of bullying involvement on health.The aim of this study was to use a prospective, longitudinal study that has followed a sample of 1,420 children up to nine times to test whether involvement in childhood bullying affects low-grade inflammation as measured by CRP levels short term within childhood/adolescence (ages 9–16) and long term into adulthood (ages 19 and 21). Chronic victims and bully/victims display the worst health and psychosocial outcomes (1, 2, 4). It is hypothesized that both these groups will have more systemic inflammation because of the social strain of victimization. Almost no attention has been paid to the biological consequences to bullying itself in the absence of being a victim. Children may use bullying techniques in efforts to elevate their social status (22). In adults, such elevated social status, measured by income or education level, is associated with lower levels of inflammatory markers (23–25). The role of elevated social status inflammatory markers has not yet been tested, but we expected that pure bullies would display lower levels of CRP than those uninvolved in bullying.     

Synaptotagmin-1 is a bidirectional Ca2+ sensor for neuronal endocytosis

Exocytosis and endocytosis are tightly coupled. In addition to initiating exocytosis, Ca²⁺ plays critical roles in exocytosis–endocytosis coupling in neurons and nonneuronal cells. Both positive and negative roles of Ca²⁺ in endocytosis have been reported; however, Ca²⁺ inhibition in endocytosis remains debatable with unknown mechanisms. Here, we show that synaptotagmin-1 (Syt1), the primary Ca²⁺ sensor initiating exocytosis, plays bidirectional and opposite roles in exocytosis–endocytosis coupling by promoting slow, small-sized clathrin-mediated endocytosis but inhibiting fast, large-sized bulk endocytosis. Ca²⁺-binding ability is required for Syt1 to regulate both types of endocytic pathways, the disruption of which leads to inefficient vesicle recycling under mild stimulation and excessive membrane retrieval following intense stimulation. Ca²⁺-dependent membrane tubulation may explain the opposite endocytic roles of Syt1 and provides a general membrane-remodeling working model for endocytosis determination. Thus, Syt1 is a primary bidirectional Ca²⁺ sensor facilitating clathrin-mediated endocytosis but clamping bulk endocytosis, probably by manipulating membrane curvature to ensure both efficient and precise coupling of endocytosis to exocytosis.

Endocytosis and subsequent vesicle recycling are spatiotemporally coupled to exocytosis, which is critical for neurons and endocrinal cells to maintain the integrity of plasma membrane architecture, intracellular homeostasis, and sustained neurotransmission (1–3). In addition to triggering vesicular exocytosis, neural activity/Ca²⁺ also play an executive role in the coupling of endocytosis to exocytosis (1, 2, 4–6). Following a pioneering study 40 y ago (7), extensive studies have been conducted and showed that Ca²⁺ triggers and facilitates vesicle endocytosis in neurons and nonneuronal secretory cells (1, 8–11). Accumulating evidence also shows that intracellular Ca²⁺ may inhibit endocytosis (12–15), which has been challenged greatly due to the apparently lower occurrences in few preparations and the missing underlining mechanisms, making the endocytic role of Ca²⁺ a four-decades–long dispute (1, 2, 4, 6).Machineries and regulators involved in exocytosis–endocytosis coupling have been extensively studied for over 30 y. The soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) and synaptophysin play critical dual roles in exocytosis and endocytosis during neurotransmission (2, 3, 16, 17). Calmodulin and synaptotagmin-1 (Syt1) are currently known primary Ca²⁺ sensors facilitating endocytosis (1, 9, 16, 18, 19). Ca²⁺/calmodulin activate calcineurin, which dephosphorylates endocytic proteins (e.g., dynamin, synaptojanin, and amphiphysin) to facilitate clathrin-mediated endocytosis (CME) and clathrin-independent fast endocytosis (1, 2). Syt1 is a dual Ca²⁺ sensor for both exocytosis and endocytosis (5, 16, 18–20). It promotes CME through binding with the endocytic adaptors adaptor protein-2 (AP-2) and stonin-2 (21–24). In contrast to the well-established Ca²⁺ sensors that promote endocytosis, the mechanism of Ca²⁺-dependent inhibition in endocytosis remains unknown.CME is the classical but slow endocytosis pathway for vesicle retrieval under resting conditions or in response to mild stimulation, while the accumulated Ca²⁺ also triggers calmodulin/calcineurin-dependent bulk endocytosis, which takes up a large area of plasma membrane to fulfill the urgent requirement for high-speed vesicle exocytosis (1–3). They cooperate with kiss-and-run and ultrafast endocytosis to ensure both sufficient and precise membrane retrieval following exocytosis (3, 25–27). These endocytic pathways are all initiated from membrane invagination and are critically controlled by neural activity. However, how the switch between different endocytic modes is precisely determined remains largely unknown.Here, by combining electrophysiological recordings, confocal live imaging, superresolution stimulated emission depletion (STED) imaging, in vitro liposome manipulation, and electron microscope imaging of individual endocytic vesicles, we define Syt1 as a primary and bidirectional Ca²⁺ sensor for endocytosis, which promotes CME but inhibits bulk endocytosis, probably by mediating membrane remodeling. The balance between the facilitatory and inhibitory effects of Syt1 on endocytosis offers a fine-tuning mechanism to ensure both efficient and precise coupling of endocytosis to exocytosis. By including a non-Ca²⁺–binding Syt as the constitutive brake, this work also explains the four-decades–long puzzle about the positive and negative Ca²⁺ effects on endocytosis.