Large conformational changes of the epsilon subunit in the bacterial F1F0 ATP synthase provide a ratchet action to regulate this rotary motor enzyme

The F(1)F(0) ATP synthase is the smallest motor enzyme known. Previous studies had established that the central stalk, made of the gamma and epsilon subunits in the F(1) part and c subunit ring in the F(0) part, rotates relative to a stator composed of alpha(3)beta(3)deltaab(2) during ATP hydrolysis and synthesis. How this rotation is regulated has been less clear. Here, we show that the epsilon subunit plays a key role by acting as a switch of this motor. Two different arrangements of the epsilon subunit have been visualized recently. The first has been observed in beef heart mitochondrial F(1)-ATPase where the C-terminal portion is arranged as a two-alpha-helix hairpin structure that extends away from the alpha(3)beta(3) region, and toward the position of the c subunit ring in the intact F(1)F(0). The second arrangement was observed in a structure determination of a complex of the gamma and epsilon subunits of the Escherichia coli F(1)-ATPase. In this, the two C-terminal helices are apart and extend along the gamma to interact with the alpha and beta subunits in the intact complex. We have been able to trap these two arrangements by cross-linking after introducing appropriate Cys residues in E. coli F(1)F(0), confirming that both conformations of the epsilon subunit exist in the enzyme complex. With the C-terminal domain of epsilon toward the F(0), ATP hydrolysis is activated, but the enzyme is fully coupled in both ATP hydrolysis and synthesis. With the C-terminal domain toward the F(1) part, ATP hydrolysis is inhibited and yet the enzyme is fully functional in ATP synthesis; i.e., it works in one direction only. These results help explain the inhibitory action of the epsilon subunit in the F(1)F(0) complex and argue for a ratchet function of this subunit.     

Na+/H+ exchange mediates postprandial ileal water and electrolyte transport

Feeding stimulates fluid and electrolyte absorption in the small intestine. Previous studies have suggested that Na⁺/glucose cotransport is important in initiating this response in the jejunum. The purpose of this study was to determine whether Na⁺/H⁺ exchange plays a role in meal-induced absorption. Exteriorized, neurovascularly intact jejunal and ileal loops (25 cm) were constructed in dogs. Following a two-week period of postoperative recovery, the loops of awake dogs were perfused with standard buffer alone or with increasing concentrations of amiloride, a Na⁺/H⁺ exchange inhibitor. Water, sodium, and chloride fluxes were calculated following a meal using [¹⁴C]PEG as a volume marker. The meal significantly increased absorption in both the jejunum (P<0.001) and ileum (P<0.01) in those animals perfused with buffer alone. More significantly, amiloride suppressed the increased absorption seen following a meal in the ileum (P<0.001) but not the jejunum. The response in the ileum was dose dependent. These findings suggest that a major mediator of postprandial sodium and water absorption in the ileum is the Na⁺/H⁺ exchanger.Supported by NIH R29-DK-47326 (S.W.A.), R01-DK-39879 (M.J.Z.), and a VA Merit Review (D.W.M.).Portions of this work were presented at the Annual Meeting of the American Gastroenterological Association, May 1993, Boston, Massachusetts.     

Biocompatible surface functionalization architecture for a diamond quantum sensor

Quantum metrology enables some of the most precise measurements. In the life sciences, diamond-based quantum sensing has led to a new class of biophysical sensors and diagnostic devices that are being investigated as a platform for cancer screening and ultrasensitive immunoassays. However, a broader application in the life sciences based on nanoscale NMR spectroscopy has been hampered by the need to interface highly sensitive quantum bit (qubit) sensors with their biological targets. Here, we demonstrate an approach that combines quantum engineering with single-molecule biophysics to immobilize individual proteins and DNA molecules on the surface of a bulk diamond crystal that hosts coherent nitrogen vacancy qubit sensors. Our thin (sub–5 nm) functionalization architecture provides precise control over the biomolecule adsorption density and results in near-surface qubit coherence approaching 100 μs. The developed architecture remains chemically stable under physiological conditions for over 5 d, making our technique compatible with most biophysical and biomedical applications.

Recent developments in quantum engineering and diamond processing have brought us considerably closer to performing nanoscale NMR and electron paramagnetic resonance (EPR) spectroscopy of small ensembles and even individual biomolecules. Notably, these advances have enabled the detection of the nuclear spin noise from a single ubiquitin protein (1) and the probing of the EPR spectrum of an individual paramagnetic spin label conjugated to a protein (2) or DNA molecule (3). More recently, lock-in detection and signal reconstruction techniques (4, 5) have enabled one- and multidimensional NMR spectroscopy with 0.5-Hz spectral resolution (6–8). More advanced control sequences at cryogenic temperatures have further enabled mapping the precise location of up to 27 ¹³C nuclear spins inside of diamond (9). Yet biologically meaningful spectroscopy on intact biomolecules remains elusive. One of the main outstanding challenges, which is required to perform nanoscale magnetic resonance spectroscopy of biomolecules, is the need to immobilize the target molecules within the 10- to 30-nm sensing range (2, 3, 7) of a highly coherent nitrogen vacancy (NV) qubit sensor. Immobilization is necessary because an untethered molecule would otherwise diffuse out of the detection volume within a few tens of microseconds.Various avenues to the functionalization of high-quality, single-crystalline diamond chips have been pursued over the last decade (10–12). However, none of the currently known approaches has led to the desired results of interfacing a coherent quantum sensor with target biomolecules. For example, hydrogen-terminated diamond surfaces can be chemically modified and form biologically stable surfaces (10, 13); but near-surface NV centers are generally charge-unstable under hydrogen termination (14), posing open challenges for NV sensing. On the other hand, oxygen-terminated diamond surfaces have been used to create charge stable NV^– centers with exceptional coherence times within 10 nm from the diamond surface (15). However, perfectly arranged, ether-terminated diamond surfaces generally lack chemically functionalizable surface groups (such as carboxyl or hydroxyl groups), making it difficult to control immobilization density and surface passivation. Other platforms such as diamond nanocrystals can generally be functionalized (16, 17) because of their heterogeneous surface chemistry, but they do not possess the coherence times needed for nanoscale magnetic resonance spectroscopy. Our approach (Fig. 1A) overcomes these limitations by utilizing a 2-nm-thick Al₂O₃ layer deposited onto an oxygen-terminated diamond surface by atomic layer deposition (ALD). This Al₂O₃ “adhesion” layer is silanized by N-[3-(trimethoxysilyl)propyl]ethylenediamine to create an amine (–NH₂) -terminated surface, which in turn is then grafted with a monolayer of heterobifunctional polyethylene glycol (PEG) via an N-hydroxysuccinimide (NHS) reaction, a process also referred as PEGylation. The PEG layer serves two purposes. First, it passivates the diamond surface to prevent nonspecific adsorption of biomolecules. Second, by adjusting the density of PEG molecules with functional groups (e.g., biotin or azide), we can control the immobilization density of proteins or DNA target molecules on the diamond surface. Furthermore, the small persistence length of the PEG linker (∼0.35 nm) allows the immobilized biomolecules to undergo rotational diffusion (18). This tumbling motion is the basis for motional averaging of the NMR spectra and helps to prevent immobilization of molecules in biologically inactive orientations.Open in a separate windowFig. 1.Architecture and characterization of the diamond functionalization approach. (A) Schematic illustration of the functionalization process. A thin layer of Al₂O₃ (gray) was deposited to the pristine, oxygen-terminated diamond surfaces (blue), followed by silanization (purple) and PEGylation (green). Functional groups (biotin, yellow circle; azide, red triangle) allow for cross-linking with target biomolecules. AFM characterization of the surfaces (B) and XPS Al2p signal after each step of the functionalization (C). (D) Illustration of the overall chemical functionalization architecture (not to scale), with corresponding thicknesses. (E) Illustration of SPAAC reaction. (F) A lithographically fabricated Al₂O₃ pattern on the diamond surface by lift-off, with a thickness of ∼2.1 nm. The Al₂O₃ layer is uniform without the presence of pin holes. The elevated edges originate from lift-off combined with ALD deposition.     

Preeclampsia is associated with a serum factor cytotoxic to human endothelial cells

Preeclampsia occurs in 7% to 10% of pregnancies and is a leading cause of morbidity for mothers and their infants. Intensive investigation has failed to reveal the cause of the multiple organ dysfunction characteristic of this disorder, which abates completely with delivery. However, several observations suggest that endothelial cell dysfunction is a central pathophysiologic event. We report that serum from preeclamptic women is cytotoxic to endothelial cells in vitro. Consistent with the reversal of the clinical condition after delivery, cytotoxic activity in serum of preeclamptic women is reduced after 24 to 48 hours post partum. In contrast, cytotoxic activity of serum from normal pregnant women increases after delivery.     

Recent advances in the diagnostic evaluation of pancreatic cystic lesions

Pancreatic cystic lesions (PCLs) are becoming more prevalent due to more frequent abdominal imaging and the increasing age of the general population. It has become crucial to identify these PCLs and subsequently risk stratify them to guide management. Given the high morbidity associated with pancreatic surgery, only those PCLs at high risk for malignancy should undergo such treatment. However, current diagnostic testing is suboptimal at accurately diagnosing and risk stratifying PCLs. Therefore, research has focused on developing new techniques for differentiating mucinous from non-mucinous PCLs and identifying high risk lesions for malignancy. Cross sectional imaging radiomics can potentially improve the predictive accuracy of primary risk stratification of PCLs at the time of detection to guide invasive testing. While cyst fluid glucose has reemerged as a potential biomarker, cyst fluid molecular markers have improved accuracy for identifying specific types of PCLs. Endoscopic ultrasound guided approaches such as confocal laser endomicroscopy and through the needle microforceps biopsy have shown a good correlation with histopathological findings and are evolving techniques for identifying and risk stratifying PCLs. While most of these recent diagnostics are only practiced at selective tertiary care centers, they hold a promise that management of PCLs will only get better in the future.