PROGRESSIVE SUPRANUCLEAR PALSY PRESENTING WITH PSYCHIATRIC FEATURES

SUMMARY A patient with progressive supranuclear palsy who presented with psychiatric features is reported. His case illustrates the difficulty of early diagnosis of this condition. Associated psychiatric symptoms are common and may precede the occurrence of gaze palsy. Our patient's behavioural problems responded to fluoxetine.     

Orthotopic liver transplantation in a patient with carbamyl phosphate synthetase deficiency and cystic fibrosis

A 15-year-old female with carbamyl phosphate synthetase deficiency, cystic fibrosis, and cystic fibrosis-related diabetes underwent orthotopic cadaveric liver transplantation. Metabolic control was maintained during the procedure with nutritional support and the use of intravenous sodium phenylacetate and benzoate. Her postoperative course was complicated by seizures and a transient decline in her pulmonary function tests, which returned to preoperative levels within one year of the transplant. Now, four years post-transplant, her quality of life has dramatically improved. There are only four Canadian centres with paediatric liver transplantation programs. However, expert medical care for adults with inborn error of metabolism is even more limited, suggesting that access to adult medical care is one of the many factors to be considered when liver transplantation is contemplated for patients with metabolically unstable conditions.     

Intravenous Lidocaine and Bupivacaine Dose-dependently Attenuate Bronchial Hyperreactivity in Awake Volunteers

Background: In standard textbooks, intravenous lidocaine is recommended for intubation of patients with bronchial hyperreactivity. However, whether and to what extent intravenous local anesthetics attenuate bronchial hyperreactivity in humans is unknown. Accordingly, nine awake volunteers with known bronchial hyperreactivity were subjected to an inhalational challenge with acetylcholine before and during intravenous infusion of lidocaine, bupivacaine, or placebo in a randomized, double-blinded fashion.

Methods: Baseline acetylcholine threshold concentrations were determined 3-5 days before initiation of the investigation. The response to the acetylcholine challenge was defined as hyperreactive, if forced expiratory volume in 1 s decreased by at least 20%. In addition, the acetylcholine threshold for a 100% increase in airway resistance was obtained by body plethysmography. On seven different days, the acetylcholine challenge was repeated at the end of a 30-min intravenous infusion period of three doses of lidocaine (1, 3, and 6 mg *symbol* min sup -1) or bupivacaine (0.25, 0.75, and 1.5 mg *symbol* min sup -1), during saline placebo infusion, respectively. Acetylcholine-threshold concentrations were presented with the respective plasma concentrations of the local anesthetic.

Results: The infusion of lidocaine and bupivacaine resulted in plasma concentrations (means+/-SD) of 0.29+/-0.11, 1.14 +/-0.39, and 2.02+/-0.5 micro gram *symbol* ml sup -1 for lidocaine and 0.11+/-0.04, 0.31+/-0.09, and 0.80 +/-0.18 micro gram *symbol* ml sup -1 for bupivacaine, respectively. Compared to baseline, the acetylcholine threshold for a 20% decrease of forced expiratory volume in 1 s as well as the threshold for a 100% increase in total airway resistance increased significantly with increasing plasma concentrations of both local anesthetics. Compared to placebo, acetylcholine threshold was almost quadrupled for lidocaine and tripled for bupivacaine with the highest plasma concentration of each local anesthetic.     

Prognostic significance of HIV-associated oral lesions and their relation to therapy

The oral manifestations of HIV infection have been considered to be of value in assessing disease progression in the developed world. However, the potential use of oral lesions as prognostic markers in resource-poor countries has yet to be fully investigated. There is reasonably compelling evidence in the developed world for an association between oral lesions and viral load. However, the true nature of this association is less clear and there are few data available from the developing world. With the introduction of HAART, a change in prevalence of the oral manifestations of HIV infection has been observed, including regression of oral candidiasis, Kaposi's sarcoma and oral hairy leukoplakia. However, oral condylomata and herpes simplex virus infection appear to persist with HMRT therapy. Further research in partnership with resource-poor countries is required to document disease progression and the associated oral lesions in both adults and children.     

A tethered ligand assay to probe SARS-CoV-2:ACE2 interactions

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections are initiated by attachment of the receptor-binding domain (RBD) on the viral Spike protein to angiotensin-converting enzyme-2 (ACE2) on human host cells. This critical first step occurs in dynamic environments, where external forces act on the binding partners and avidity effects play an important role, creating an urgent need for assays that can quantitate SARS-CoV-2 interactions with ACE2 under mechanical load. Here, we introduce a tethered ligand assay that comprises the RBD and the ACE2 ectodomain joined by a flexible peptide linker. Using magnetic tweezers and atomic force spectroscopy as highly complementary single-molecule force spectroscopy techniques, we investigate the RBD:ACE2 interaction over the whole physiologically relevant force range. We combine the experimental results with steered molecular dynamics simulations and observe and assign fully consistent unbinding and unfolding events across the three techniques, enabling us to establish ACE2 unfolding as a molecular fingerprint. Measuring at forces of 2 to 5 pN, we quantify the force dependence and kinetics of the RBD:ACE2 bond in equilibrium. We show that the SARS-CoV-2 RBD:ACE2 interaction has higher mechanical stability, larger binding free energy, and a lower dissociation rate compared to SARS-CoV-1, which helps to rationalize the different infection patterns of the two viruses. By studying how free ACE2 outcompetes tethered ACE2, we show that our assay is sensitive to prevention of bond formation by external binders. We expect our results to provide a way to investigate the roles of viral mutations and blocking agents for targeted pharmaceutical intervention.

A subset of coronaviruses (CoV) causes severe acute respiratory syndrome (SARS) in humans. We have seen three major recent outbreaks, including the first SARS pandemic from 2002 to 2004 (SARS-CoV-1), Middle East respiratory syndrome that emerged in 2012, and the ongoing COVID-19 pandemic (SARS-CoV-2). SARS-CoV-2 particles carry ∼100 copies of the trimeric viral glycoprotein Spike (S) on their surface (1), giving the appearance of an eponymous corona around the virus. Like SARS-CoV-1, SARS-CoV-2 attaches to human host cells by S binding to angiotensin-converting enzyme-2 (ACE2) (2–6) (Fig. 1A). Specifically, each of the three S1 subunits in an S trimer carries a receptor-binding domain (RBD) at its tip, which is presented in an up or down conformation and can bind ACE2 in the up conformation (Fig. 1B) (7). Binding of the virus to host cells occurs in dynamic environments (8, 9) where external forces act on the virus particle. In particular, in the respiratory tract, coughing, sneezing, and mucus clearance exert mechanical forces (10, 11) that the virus must withstand for productive infection. The magnitude and dynamics of these forces are not known precisely and are likely variable. A rough estimate from fluid dynamics suggests an upper limit of forces in the range of ∼2 pN to 2 nN (estimates are provided in SI Appendix).Open in a separate windowFig. 1.Single-molecule assays to probe the SARS-CoV-2 RBD:ACE2 interface under force. Motivation and overview of our tethered ligand assay for equilibrium and dynamic SMFS measurements in MT and AFM. (A) Schematic of a SARS-CoV-2 virus particle (green) presenting spike protein trimers (gray) that can bind to human ACE2 (red) on the cell surface via their RBDs (blue). The bond between RBD and ACE2 is formed in a dynamic environment, where it must withstand external mechanical forces (indicated by the red arrow), for example, caused by coughing or sneezing in the respiratory tract, in order to allow efficient infection of the human host cell (orange). (B) Crystal structure of the RBD:ACE2 complex (PDB ID: 6m0j). The N and C termini of the RBD (blue) and ACE2 (red) are indicated with yellow dots. (C) Schematic (not to scale) of the tethered ligand assay in MT. The tethered ligand construct consists of the ACE2 ectodomain (red square) and RBD (blue triangle) joined by a flexible polypeptide linker (black line) of 85 aa (31 nm contour length) or 115 aa (42 nm contour length). The tethered ligand construct is attached with one end covalently to the surface via an ELP linker (33) and with the other end to a superparamagnetic bead via a biotin–streptavidin bond. Permanent magnets above the flow cell enable the application of precisely calibrated stretching forces. (D) Tethered ligand construct in the absence of force, where the RBD remains bound to ACE2. (E) Stylized measurement of the tethered ligand construct in the MT. (Bottom) To probe RBD:ACE2 bond dynamics, time traces of the tether extension are recorded at different levels of applied force (indicated at the bottom). At low forces, reversible transitions between the bound configuration, with the RBD:ACE2 interface engaged, and a dissociated configuration, where the interface is broken and the peptide linker connecting the domains is stretched, are observed as jumps between two extension levels (red and blue dashed lines). At higher forces, further upward steps in the extension trace correspond to unfolding events of protein (sub)domains. (Top) From the MT time traces, both the fraction of time spent in the dissociated state and the dwell times in the bound and dissociated state can be determined as a function of applied force. (F) Schematic (not to scale) of the tethered ligand construct in the AFM. Here, covalent attachment to the surface uses a heterobifunctional PEG spacer, and the coupling to the AFM cantilever is accomplished via an Fgɣ tag on the protein that binds with very high force stability to the ClfA protein handle on the cantilever. (G) Stylized AFM measurement. (Bottom) The cantilever is retracted with constant velocity, and the force response to the applied extension is shown as a force-extension curve. With increasing extension, the RBD:ACE2 interface ruptures, protein subdomains unfold, and, finally, the ClfA:Fgɣ bond ruptures, giving rise to distinct peaks in the force-extension curve. Comparing two constructs with different linker lengths (31 nm black solid line and 42 nm gray/lilac alternative first peak) joining RBD and ACE2 allows assignment of the RBD:ACE2 interface rupture and unfolding of parts of the RBD to the first increment. Histograms of rupture forces (Top) are compiled from multiple measurements. The blue star refers to the RBD:ACE2 interface rupture.The SARS-CoV-2 S protein and its interaction with ACE2 have been the target of intense research activity, as they are critical in the first steps of SARS-CoV-2 infection, and S constitutes a major drug and the key vaccine target in the current fight against COVID-19. Further, differences in binding between ACE2 and the SARS-CoV-1 and SARS-CoV-2 RBDs have been linked to the different observed patterns in lower and upper respiratory tract infections by the two viruses (5). Despite their importance, many questions about RBD:ACE2 interactions, particularly about their stability under external forces, are unresolved. Consequently, there is an urgent need for assays that can probe the affinity and kinetics of the interaction under a wide range of external forces. In nature, receptor–ligand pairs are often held in spatial proximity by neighboring interactions, creating high effective concentrations. Engagement of multiple interactions has been suggested to be important in other viral infections, including influenza, rabies, and HIV (12–16). Since conventional affinity measurements do not take into account these effects, there is a need for novel in vitro assays mimicking these effects when measuring bond characteristics.Here, we present a tethered ligand assay to determine RBD interactions with ACE2 at the single-molecule level subject to defined levels of applied force. Our assay utilizes fusion protein constructs of SARS-CoV-1 or SARS-CoV-2 RBD and the human ACE2 ectodomain joined by flexible peptide linkers. To probe the linkage under a large range of mechanical forces and loading rates, we used two highly complementary single-molecule force spectroscopy (SMFS) approaches: an atomic force microscope (AFM) and magnetic tweezers (MT) (Fig. 1 C–G). We complemented the experiments with steered molecular dynamics (SMD) simulations to provide microscopic insights that are inaccessible experimentally.AFM force spectroscopy can probe molecular interactions and protein stability dynamically (15–29), typically measuring at constant loading rate, and can investigate even the most stable high-force host–pathogen interactions (at forces F > 2,000 pN) (18). In AFM experiments, the molecular construct of interest is stretched between a surface and the tip of an AFM cantilever. The cantilever is retracted at a constant velocity, and the force is monitored from the cantilever deflection. Molecular rupture or protein (sub)domain unfolding events give rise to a sawtooth-like pattern in the force vs. extension traces (Fig. 1G). In contrast, MT typically operate at constant force and can resolve very low forces (19, 20), down to F < 0.01 pN. In MT, molecules are tethered between a flow cell surface and magnetic beads. External magnets apply defined and constant stretching forces, and the tether extension is monitored by video microscopy. In MT, unbinding or unfolding events give rise to steps in the extension vs. time trace (Fig. 1E).Tethered ligand assays have provided insights into a range of critical molecular interactions under mechanical load (21–29). Under constant force, they allow observation of repeated interactions of the same binding partners, which are held in spatial proximity under mechanical control. Therefore, they can provide information on affinity, avidity, on and off rates, and mechanical stability (21, 23). Conversely, AFM force spectroscopy can perform dynamic measurements in a highly automated fashion and can reveal characteristic protein unfolding patterns, which can serve as molecular fingerprints (30) to select only properly folded and assembled molecular constructs for further analysis.Probing our tethered ligand construct by AFM force spectroscopy, we reveal the dynamic force stability of the assembly. In combination with SMD simulations, we assign the increments revealed by force spectroscopy and establish the ACE2 unfolding pattern as a molecular fingerprint to select properly assembled tethers. Using MT, we measure the on and off rates at different levels of mechanical load and extrapolate to the thermodynamic stability at zero load. We compare the stability of the SARS-CoV-1 and SARS-CoV-2 RBD:ACE2 interactions in all three assays and consistently find a lower force stability for SARS-CoV-1 across the different techniques.     

肾小球滤过率和蛋白尿预测心血管高危患者结局

<正>较低的肾小球滤过率(eGFR)和较高的尿白蛋白肌酐比值(ACR)与主要心血管复合终点相关,例如,对eGFR<30ml/min·1.73m2和非常高的尿ACR校正后风险比为2.53(95%CI:1.61～3.99)。然而,将有关eGFR和尿ACR的信息添加到危险再分类分析中,对分配至中危分类的患者比例并未带来有意义的