Resolving Structure and Mechanical Properties at the Nanoscale of Viruses with Frequency Modulation Atomic Force Microscopy

Structural Biology (SB) techniques are particularly successful in solving virus structures. Taking advantage of the symmetries, a heavy averaging on the data of a large number of specimens, results in an accurate determination of the structure of the sample. However, these techniques do not provide true single molecule information of viruses in physiological conditions. To answer many fundamental questions about the quickly expanding physical virology it is important to develop techniques with the capability to reach nanometer scale resolution on both structure and physical properties of individual molecules in physiological conditions. Atomic force microscopy (AFM) fulfills these requirements providing images of individual virus particles under physiological conditions, along with the characterization of a variety of properties including local adhesion and elasticity. Using conventional AFM modes is easy to obtain molecular resolved images on flat samples, such as the purple membrane, or large viruses as the Giant Mimivirus. On the contrary, small virus particles (25–50 nm) cannot be easily imaged. In this work we present Frequency Modulation atomic force microscopy (FM-AFM) working in physiological conditions as an accurate and powerful technique to study virus particles. Our interpretation of the so called “dissipation channel” in terms of mechanical properties allows us to provide maps where the local stiffness of the virus particles are resolved with nanometer resolution. FM-AFM can be considered as a non invasive technique since, as we demonstrate in our experiments, we are able to sense forces down to 20 pN. The methodology reported here is of general interest since it can be applied to a large number of biological samples. In particular, the importance of mechanical interactions is a hot topic in different aspects of biotechnology ranging from protein folding to stem cells differentiation where conventional AFM modes are already being used

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3

ObjectivesCARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain.MethodsIn total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis.ResultsIn total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were blaOXA–48 (263/377), blaKPC–3 (62/377), blaVIM–1 (28/377), and blaNDM–1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5).ConclusionThis study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3

Spatiotemporal Characteristics of the Largest HIV-1 CRF02_AG Outbreak in Spain: Evidence for Onward Transmissions

Background and Aim: The circulating recombinant form 02_AG (CRF02_AG) is the predominant clade among the human immunodeficiency virus type-1 (HIV-1) non-Bs with a prevalence of 5.97% (95% Confidence Interval-CI: 5.41–6.57%) across Spain. Our aim was to estimate the levels of regional clustering for CRF02_AG and the spatiotemporal characteristics of the largest CRF02_AG subepidemic in Spain.Methods: We studied 396 CRF02_AG sequences obtained from HIV-1 diagnosed patients during 2000–2014 from 10 autonomous communities of Spain. Phylogenetic analysis was performed on the 391 CRF02_AG sequences along with all globally sampled CRF02_AG sequences (N = 3,302) as references. Phylodynamic and phylogeographic analysis was performed to the largest CRF02_AG monophyletic cluster by a Bayesian method in BEAST v1.8.0 and by reconstructing ancestral states using the criterion of parsimony in Mesquite v3.4, respectively.Results: The HIV-1 CRF02_AG prevalence differed across Spanish autonomous communities we sampled from (p < 0.001). Phylogenetic analysis revealed that 52.7% of the CRF02_AG sequences formed 56 monophyletic clusters, with a range of 2–79 sequences. The CRF02_AG regional dispersal differed across Spain (p = 0.003), as suggested by monophyletic clustering. For the largest monophyletic cluster (subepidemic) (N = 79), 49.4% of the clustered sequences originated from Madrid, while most sequences (51.9%) had been obtained from men having sex with men (MSM). Molecular clock analysis suggested that the origin (tMRCA) of the CRF02_AG subepidemic was in 2002 (median estimate; 95% Highest Posterior Density-HPD interval: 1999–2004). Additionally, we found significant clustering within the CRF02_AG subepidemic according to the ethnic origin.Conclusion: CRF02_AG has been introduced as a result of multiple introductions in Spain, following regional dispersal in several cases. We showed that CRF02_AG transmissions were mostly due to regional dispersal in Spain. The hot-spot for the largest CRF02_AG regional subepidemic in Spain was in Madrid associated with MSM transmission risk group. The existence of subepidemics suggest that several spillovers occurred from Madrid to other areas. CRF02_AG sequences from Hispanics were clustered in a separate subclade suggesting no linkage between the local and Hispanic subepidemics

Mapping in vitro local material properties of intact and disrupted virions at high resolution using multi-harmonic atomic force microscopy

Understanding the relationships between viral material properties (stiffness, strength, charge density, adhesion, hydration, viscosity, etc.), structure (protein sub-units, genome, surface receptors, appendages), and functions (self-assembly, stability, disassembly, infection) is of significant importance in physical virology and nanomedicine. Conventional Atomic Force Microscopy (AFM) methods have measured a single physical property such as the stiffness of the entire virus from nano-indentation at a few points which severely limits the study of structure-property-function relationships. We present an in vitro dynamic AFM technique operating in the intermittent contact regime which synthesizes anharmonic Lorentz-force excited AFM cantilevers to map quantitatively at nanometer resolution the local electro-mechanical force gradient, adhesion, and hydration layer viscosity within individual phi 29 virions. Furthermore, the changes in material properties over the entire phi 29 virion provoked by the local disruption of its shell are studied, providing evidence of bacteriophage depressurization. The technique significantly generalizes recent multi-harmonic theory (A. Raman, et al., Nat. Nanotechnol., 2011, 6, 809-814) and enables high-resolution in vitro quantitative mapping of multiple material properties within weakly bonded viruses and nanoparticles with complex structure that otherwise cannot be observed using standard AFM techniques

Stiffness as a source for dissipation.

<p>(a) and (d) FM-AFM topographies showing, each of them, two virus MVM particles containing genetic material. In both images the particles have different spring constant since they are sitting on different fold-symmetries <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030204#pone.0030204-Carrasco2" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030204#pone.0030204-Carrasco3" target="_blank">[11]</a>. (b) and (e) Drive amplitude maps taken simultaneously with a and d. Since the adhesion is negligible, the compositional map is only plotting the elastic modulus. As expected for the different fold-symmetries, the virus showing the 5 fold-symmetry has lower stiffness than the other one which is showing the 3 fold-symmetry. Furthermore the viruses are softer than the substrate. (c) Simulation of the drive signal versus elastic modulus and viscosity. If the adhesion is close to zero, the drive signal is basically the elastic modulus of the sample, with the exception of samples that could have an extremely high viscosity orders of magnitude higher that the expected for virus particles. Fig c despites the corresponding profiles across the two virus particles observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030204#pone-0030204-g006" target="_blank">fig. 6a</a>. Cantilever oscillation amplitude 1.0 nm and frequency shift 32 Hz, scan speed 350 nm/s, Resonance frequency 21 KHz, calibrated spring constant of 0.6 N/m.</p

High resolution FM-AFM. Seeing individual features.

<p>(a) Image of a muscovite mica surface (001) showing true atomic resolution. The mica was immersed into a physiological buffer to ensure that under these conditions we are able to obtain true atomically resolved images. Parameters of the image: cantilever oscillation amplitude 0.5 nm and frequency shift 36 Hz, scan speed 800 nm/s, Resonance frequency 130 KHz, nominal spring constant 40 N/m. (b) View of a MVM particle oriented with a 5-fold symmetry axis on top as obtained from a molecular surface model derived from crystallographic data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030204#pone.0030204-AgbandjeMcKenna1" target="_blank">[36]</a> as rendered in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030204#pone.0030204-Carrasco3" target="_blank">[11]</a>. The capsid diameter is 25 nm. (c) FM-AFM topographic image of an individual MVM particle in the same orientation shown in (b).Virus particles were adsorbed on a glass surface immersed into a physiological buffer (PBS, 137 mM NaCl, 2.7 mM KCl, 1.5 mM NaH₂PO₄, 8.1 mM KH2PO₄). Parameters of the image: cantilever oscillation amplitude 0.9 nm and frequency shift 30 Hz, speed 350 nm/s, Resonance frequency 19 KHz. Calibrated spring constant of 0.6 N/m. (d) Cryo-EM model of the φ29 bacteriophage <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030204#pone.0030204-Tang1" target="_blank">[24]</a> (equatorial diameter ∼45 nm) after a <i>dilation</i> process using a size tip of 0.2 nm. Reconstruction techniques are extremely precise but require the merging and averaging of a large set of images of individual viruses and, consequently, they tend to neglect individual particle features. (e) FM-AFM topographic image of an individual φ29 particle in TMS buffer (50 mM Tris pH7.8, 10 mM Mg Cl₂, 100 mM Na Cl). This virus presents a damaged tail (see white arrow). Parameters of the image: cantilever oscillation amplitude 1.0 nm and frequency shift 31 Hz, scan speed 350 nm/s, Resonance frequency 35 KHz, calibrated spring constant of 1.8 N/m. (f) Another φ29 virus particle with a clear and well resolved tail but with some material bound to its head, forming extra protrusions. Parameters of the image: cantilever oscillation amplitude 1.0 nm and frequency shift 25 Hz, scan speed 350 nm/s, Resonance frequency 19 KHz calibrated spring constant of 0.6 N/m.</p

Adhesion force as a source of dissipation.

<p>(a) Bare HOPG topography taken in FM-AFM in the same buffer solution used for the MVM viruses. (b) Dissipation map simultaneously taken with a. (d) and (e) are 2 force distance curves taken in the bright and dark regions of the dissipation respectively. As can be seen from the curves there is a clear correspondence between adhesion and dissipation. Cantilever oscillation amplitude 1.0 nm and frequency shift 35 Hz, scan speed 350 nm/s, Resonance frequency 21 KHz, calibrated spring constant of 0.6 N/m. (c) Simulation of the drive signal versus elastic modulus and adhesion. For a given sample with a homogeneous elastic modulus, the drive signal increases as the adhesion gets higher.</p

<i>In liquid</i> FM-AFM basis.

<p>(a) Experimental (solid line) and simulated (dashed line) average deflection (mean force) of the cantilever as the tip approaches the sample surface. The horizontal axis is the piezo elongation. (b) Experimental (solid lines) and simulated (dashed lines) frequency shift and drive amplitude as the tip approaches de sample surface. The horizontal axis is the piezo elongation. All the magnitudes in (a) and (b) were acquired simultaneously. Oscillation amplitude 1.2 nm., calibrated spring constant of 0.6 N/m.</p

<i>In liquid</i> FM-AFM basis.

<p>Artistic draw of a virus particle immersed in a buffer solution and adsorbed on a surface. The colored environment represents the total gradient of short and long-range interactions between the cantilever tip and the sample. FM-AFM follows the constant gradient lines to get the sample topography.</p