| 2021 | From spherical compartments to polymer films: exploiting vesicle fusion to generate solid supported thin polymer membranes. | Nanoscale | Solid supported polymer membranes as scaffold for the insertion of functional biomolecules provide the basis for mimicking natural membranes. They also provide the means for unraveling biomolecule-membrane interactions and engineering platforms for biosensing. Vesicle fusion is an established procedure to obtain solid supported lipid bilayers but the more robust polymer vesicles tend to resist fusion and planar membranes rarely form. Here, we build on vesicle fusion to develop a refined and efficient way to produce solid supported membranes based on poly(dimethylsiloxane)-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b-PMOXA) amphiphilic triblock copolymers. We first create thiol-bearing polymer vesicles (polymersomes) and anchor them on a gold substrate. An osmotic shock then provokes polymersome rupture and drives planar film formation. Prerequisite for a uniform amphiphilic planar membrane is the proper combination of immobilized polymersomes and osmotic shock conditions. Thus, we explored the impact of the hydrophobic PDMS block length of the polymersome on the formation and the characteristics of the resulting solid supported polymer assemblies by quarz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM) and spectroscopic ellipsometry (SE). When the PDMS block is short enough, attached polymersomes restructure in response to osmotic shock, resulting in a uniform planar membrane. Our approach to rapidly form planar polymer membranes by vesicle fusion brings many advantages to the development of synthetic planar membranes for bio-sensing and biotechnological applications. |
| 2020 | In-Situ Investigation on Nanoscopic Biomechanics of at Low pH Citric Acid Environments Using an AFM Fluid Cell. | Int J Mol Sci | () is widely regarded as the main cause of human dental caries via three main virulence factors: adhesion, acidogenicity, and aciduricity. Citric acid is one of the antibiotic agents that can inhibit the virulence capabilities of . A full understanding of the acidic resistance mechanisms (ARMs) causing bacteria to thrive in citrate transport is still elusive. We propose atomic force microscopy (AFM) equipped with a fluid cell to study the ARMs via surface nanomechanical properties at citric acid pH 3.3, 2.3, and 1.8. Among these treatments, at pH 1.8, the effect of the citric acid shock in cells is demonstrated through a significantly low number of high adhesion zones, and a noticeable reduction in adhesion forces. Consequently, this study paves the way to understand that ARMs are associated with the variation of the number of adhesion zones on the cell surface, which is influenced by citrate and proton transport. The results are expected to be useful in developing antibiotics or drugs involving citric acid for dental plaque treatment. |
| 2020 | Clinical characteristics and treatment effects of astragalus injection in non-pediatric patients with acute fulminant myocarditis. | Medicine (Baltimore) | To explore the clinical characteristics of non-pediatric patients with acute fulminant myocarditis (AFM) and evaluate the treatment effects of astragalus injection on this disease.A total of 54 AFM patients were screened out from 586 patients with acute myocarditis, admitted to the department of cardiology between January 2011 to June 2018. The demographic and clinical data, investigations, treatments, and short-term outcomes were collected and retrospectively analyzed.The mean age of the 54 AFM patients was 34 ± 16.5 years old (range: 13-70 years), including 24 (44.5%) men and 30 (55.5%) women, with a high incidence in 2 age groups: 13-19 and 40-49 years old, despite an inverse trend to the increase of age. All these cases were admitted in emergency conditions: 26 (48.1%) cardiogenic shock, 18 (33.4%) malignant arrhythmias, 8 (14.8%) severe heart failure, and 2 (3.7%) acute pericardial tamponade. Apart from first-aid measures, 37 (68.5%) patients received astragalus injection. During hospitalization, 11 (20.4%) patients died, and 4 (36.3%) of them were from astragalus group while 7 (63.7%) of them from without-astragalus group (P=0.03). Furthermore, the levels of cardiac injury biomarkers, renal function and left ventricular ejection fraction of astragalus group were significantly improved compared with those of without-astragalus group at discharge (all P < .05).Middle-aged people were also prone to AFM. And cardiac shock was the most common, while acute pericardial tamponade was a rare presentation in non-pediatric AFM patients. Astragalus was a potential adjuvant medicine for the treatment of AFM. |
| 2021 | ATP Impedes the Inhibitory Effect of Hsp90 on Aβ Fibrillation. | J Mol Biol | Heat shock protein 90 (Hsp90) is a molecular chaperone that assists protein folding in an Adenosine triphosphate (ATP)-dependent way. Hsp90 has been reported to interact with Alzheimeŕs disease associated amyloid-β (Aβ) peptides and to suppress toxic oligomer- and fibril formation. However, the mechanism remains largely unclear. Here we use a combination of atomic force microscopy (AFM) imaging, circular dichroism (CD) spectroscopy and biochemical analysis to quantify this interaction and put forward a microscopic picture including rate constants for the different transitions towards fibrillation. We show that Hsp90 binds to Aβ monomers weakly but inhibits Aβ from growing into fibrils at substoichiometric concentrations. ATP impedes this interaction, presumably by modulating Hsp90's conformational dynamics and reducing its hydrophobic surface. Altogether, these results might indicate alternative ways to prevent Aβ fibrillation by manipulating chaperones that are already abundant in the brain. |
| 2020 | Achatina fulica mucous improves cell viability and increases collagen deposition in UVB-irradiated human fibroblast culture. | J Stem Cells Regen Med | Ultraviolet radiation induces skin photoaging by increasing matrix metalloproteinase-1 (MMP-1). MMP-1 degrades type I and III collagen that comprise the dermal connective tissue. mucous (AFM) is a natural remedy that has protective effects on fibroblasts and collagen. To investigate the effects of AFM on cell viability and collagen deposition in UVB-irradiated human fibroblast culture. The mucous was extracted from 50 snails that were stimulated by a 5-10 Volt electricity shock for 30-60 seconds and converted into powder by the freeze-drying process. The human dermal fibroblast culture was divided into six groups: group 1 were normal fibroblasts without UVB irradiation as normal control, groups 2-5 consisted of 100 mJ/cm UVB-irradiated fibroblasts. Group 2 had no treatment as negative control, group 3 was treated by PRP 10% as positive control group and groups 4-6 were treated by various concentrations of AFM (3.9; 15.625 and 62.5 μg/mL). At the end of the experiment, the proliferation was assessed with MTT assay, furthermore collagen deposition was measured by Sirius red assay. Real Time-PCR (RT-PCR) was performed to quantify Coll I, Coll III and MMP-1 mRNA expression, then to measured COL 1/COL III ratio. UVB induced significant lower viability, upregulated MMP-1 and downregulated COL I and COL III mRNA expressions. Meanwhile AFM treated groups demonstrated higher cell viability with downregulation of MMP-1 and upregulation of COL I and COL III mRNA expressions. The ratio of COL I/ III expression was significantly (<0.05) lower in the AFM treated groups compared to the UVB group. Among AFM treated groups, administration of 62.5 μg/mL AFM represented the best result. AFM may ameliorate viability of UVB-irradiated human fibroblast culture which associates with downregulating MMP-1, upregulating COL I and Col III, and reducing COL I/III ratio. |
| 2020 | Novel synthesis of ZnO by Ice-cube method for photo-inactivation of . | Saudi J Biol Sci | The ZnO particle with varieties of morphology was prepared from ice-cube of zinc ammonium complex at boiling water surface in 1 min induction of thermal shock. The zinc ammonium complex in ice cube was developed using zinc acetate and biologically activated ammonia in 1 hr and kept in the freezer. Temperature gradient behaviour of the water medium during thermal shock was captured by the thermal camera and thermometer. Morphology study revealed a variety of flower-like ZnO particles with variable size from 1.0 to 2.5 μm. Further, ZnO particle morphologies were tuned by adding trisodium citrate and hexamine to obtain uniform spherical (2-3 μm) and flower (3-4 μm) shapes, respectively. XRD patterns revealed that all ZnO samples are of a hexagonal structure. Photocatalytic inactivation of has been investigated using various particle morphologies of ZnO in an aqueous solution/overcoated glass slide under sunlight. The photo-inactivation of by ZnO particles in suspension condition was better when compared to a coated glass slide method. AFM study confirmed the destruction of bacterial cell wall membrane by the photocatalytic effect. The particles morphology of photocatalyst is well dependent on antibacterial activity under sunlight. |
| 2020 | Studying heat shock proteins through single-molecule mechanical manipulation. | Cell Stress Chaperones | Imbalances of cellular proteostasis are linked to ageing and human diseases, including neurodegenerative and neuromuscular diseases. Heat shock proteins (HSPs) and small heat shock proteins (sHSPs) together form a crucial core of the molecular chaperone family that plays a vital role in maintaining cellular proteostasis by shielding client proteins against aggregation and misfolding. sHSPs are thought to act as the first line of defence against protein unfolding/misfolding and have been suggested to act as "sponges" that rapidly sequester these aberrant species for further processing, refolding, or degradation, with the assistance of the HSP70 chaperone system. Understanding how these chaperones work at the molecular level will offer unprecedented insights for their manipulation as therapeutic avenues for the treatment of ageing and human disease. The evolution in single-molecule force spectroscopy techniques, such as optical tweezers (OT) and atomic force microscopy (AFM), over the last few decades have made it possible to explore at the single-molecule level the structural dynamics of HSPs and sHSPs and to examine the key molecular mechanisms underlying their chaperone activities. In this paper, we describe the working principles of OT and AFM and the experimental strategies used to employ these techniques to study molecular chaperones. We then describe the results of some of the most relevant single-molecule manipulation studies on HSPs and sHSPs and discuss how these findings suggest a more complex physiological role for these chaperones than previously assumed. |
| 2020 | Prognostic factors for heart recovery in adult patients with acute fulminant myocarditis and cardiogenic shock supported with extracorporeal membrane oxygenation. | J Crit Care | Extracorporeal membrane oxygenation (ECMO) is an effective support method for acute fulminant myocarditis (AFM) with cardiogenic shock. However, deciding whether to bridge to a left ventricular assist device (LVAD) or to maintain ECMO support until heart recovery is still controversial.This was a retrospective observational study from a single center. Eighty-eight adults with AFM and ECMO support between 2006 and 2018 were included. The primary endpoint was heart recovery without heart transplantation or long-term LVAD support.The heart recovery group contained 43 patients, of whom 41 were discharged after being weaned off ECMO and the other two after LVAD. Five patients with heart transplants and one with long-term LVAD support were discharged, accounting for an overall survival of 55.7%. Multivariate logistic regression revealed that peak CK-MB level, severe intraventricular conduction disturbance (asystole) and malignant arrhythmia (VT or VF) were prognostic factors for nonrecovery (P = .027 and 0.017, respectively), while early intravenous immunoglobulin (IVIG) use before ECMO was highly likely to have a protective effect with a trend toward statistical significance (P = .079). A risk score was developed: 4 points for VT/VF/asystole, 1 point for every 100 μg/L increase in the peak CK-MB level, up to a maximum of 5 points, and -3 points for early IVIG use. The area under the receiver operating characteristic (ROC) curve (AUC) was 0.818.High CK-MB levels and VT/VF/asystole in patients with AFM are associated with poor heart recovery. Early IVIG use shows a potentially protective effect. |
| 2019 | Distinct mechanical properties in homologous spectrin-like repeats of utrophin. | Sci Rep | Patients with Duchenne muscular dystrophy (DMD) lack the protein dystrophin, which is a critical molecular component of the dystrophin-glycoprotein complex (DGC). Dystrophin is hypothesized to function as a molecular shock absorber that mechanically stabilizes the sarcolemma of striated muscle through interaction with the cortical actin cytoskeleton via its N-terminal half and with the transmembrane protein β-dystroglycan via its C-terminal region. Utrophin is a fetal homologue of dystrophin that can subserve many dystrophin functions and is therefore under active investigation as a dystrophin replacement therapy for DMD. Here, we report the first mechanical characterization of utrophin using atomic force microscopy (AFM). Our data indicate that the mechanical properties of spectrin-like repeats in utrophin are more in line with the PEVK and Ig-like repeats of titin rather than those reported for repeats in spectrin or dystrophin. Moreover, we measured markedly different unfolding characteristics for spectrin repeats within the N-terminal actin-binding half of utrophin compared to those in the C-terminal dystroglycan-binding half, even though they exhibit identical thermal denaturation profiles. Our results demonstrate dramatic differences in the mechanical properties of structurally homologous utrophin constructs and suggest that utrophin may function as a stiff elastic element in series with titin at the myotendinous junction. |
| 2018 | Microwave Spectroscopic Detection of Human Hsp70 Protein on Annealed Gold Nanostructures on ITO Glass Strips. | Biosensors (Basel) | Conductive indium-tin oxide (ITO) and non-conductive glass substrates were successfully modified with embedded gold nanoparticles (AuNPs) formed by controlled thermal annealing at 550 °C for 8 h in a preselected oven. The authors characterized the formation of AuNPs using two microscopic techniques: scanning electron microscopy (SEM) and atomic force microscopy (AFM). The analytical performances of the nanostructured-glasses were compared regarding biosensing of Hsp70, an ATP-driven molecular chaperone. In this work, the human heat-shock protein (Hsp70), was chosen as a model biomarker of body stress disorders for microwave spectroscopic investigations. It was found that microwave screening at 4 GHz allowed for the first time the detection of 12 ng/µL/cm² of Hsp70. |
| 2018 | Associations with the In-Hospital Survival Following Extracorporeal Membrane Oxygenation in Adult Acute Fulminant Myocarditis. | J Clin Med | Acute fulminant myocarditis (AFM) is a serious disease that progresses rapidly, and leads to failing respiratory and circulatory systems. When medications fail to reverse the patient's clinical course, extracorporeal membrane oxygenation (ECMO) is considered the most effective, supportive and adjunct strategy. In this paper we analyzed our experience in managing AFM with ECMO support.During October 2003 and February 2017, a total of 35 patients (≥18 years) were enrolled in the study. Twenty patients survived, and another 15 patients expired. General demographics, the hemodynamic condition, timing of ECMO intervention, and laboratory data were compared for the survival and non-survival groups. Univariate and multivariate Cox regression analyses were performed to identify the associations with in-hospital mortality following ECMO use in this situation.The survival rate was 57.1% during the in-hospital period. The average age, gender, severity of the hemodynamic condition, and cardiac rhythm were similar between the survival and non-survival groups. Higher serum lactic acid (initial and 24 h later), higher peak cardiac biomarkers, higher incidence of acute kidney injury and the need for hemodialysis were noted in the non-survival group. Higher 24-h lactic acid levels and higher peak troponin-I levels were associated with in-hospital mortality.When ECMO was used for AFM, related cardiogenic shock and decompensated heart failure, higher peak serum troponin-I levels and 24-h serum lactic acid levels following ECMO use were independently associated with in-hospital mortality. |
| 2018 | The Microbial Composition of Bacteroidetes Species in Ulcerative Colitis Is Effectively Improved by Combination Therapy With Fecal Microbiota Transplantation and Antibiotics. | Inflamm Bowel Dis | We previously reported that fresh fecal microbiota transplantation (FMT) after triple-antibiotic therapy (amoxicillin, fosfomycin, and metronidazole [AFM]; A-FMT) synergistically contributed to the recovery of phylum Bacteroidetes composition associated with the endoscopic severity and treatment efficacy of ulcerative colitis (UC). Here, we performed further microbial analyses using a higher-resolution method to identify the key bacterial species in UC and determine whether viable Bacteroidetes species from donor feces were successfully colonized by A-FMT.The taxonomic composition of Bacteroidetes in 25 healthy donors and 27 UC patients at baseline was compared at the species level using a heat-shock protein (hsp) 60-based microbiome method. Microbiota alterations before and after treatment of UC patients were also analyzed in 24 cases (n = 17 A-FMT; n = 3 mono-AFM; n = 4 mono-FMT).We found species-level dysbiosis within the phylum Bacteroidetes in UC samples, which was associated with reduced species diversity, resulting from hyperproliferation and hypoproliferation of particular species. Moreover, in responders treated with A-FMT, diversity was significantly recovered at 4 weeks after a fresh round of FMT, after which high degrees of similarity in Bacteroidetes species composition among recipients and donors were observed.A-FMT alleviated intestinal dysbiosis, which is caused by the loss of Bacteroidetes species diversity in patients with UC. Eradication of dysbiotic indigenous Bacteroidetes species by AFM pretreatment might promote the colonization of viable Bacteroidetes cells, thereby improving the intestinal microbiota dysbiosis induced by UC. Our findings serve as a basis for further investigations into the mechanisms of FMT. |
| 2018 | DNA Binding Induces a Nanomechanical Switch in the RRM1 Domain of TDP-43. | J Phys Chem Lett | Understanding the molecular mechanisms governing protein-nucleic acid interactions is fundamental to many nuclear processes. However, how nucleic acid binding affects the conformation and dynamics of the substrate protein remains poorly understood. Here we use a combination of single molecule force spectroscopy AFM and biochemical assays to show that the binding of TG-rich ssDNA triggers a mechanical switch in the RRM1 domain of TDP-43, toggling between an entropic spring devoid of mechanical stability and a shock absorber bound-form that resists unfolding forces of ∼40 pN. The fraction of mechanically resistant proteins correlates with an increasing length of the TG oligonucleotide, demonstrating that protein mechanical stability is a direct reporter of nucleic acid binding. Steered molecular dynamics simulations on related RNA oligonucleotides reveal that the increased mechanical stability fingerprinting the holo-form is likely to stem from a unique scenario whereby the nucleic acid acts as a "mechanical staple" that protects RRM1 from mechanical unfolding. Our approach highlights nucleic acid binding as an effective strategy to control protein nanomechanics. |
| 2018 | Molecular AFM imaging of Hsp70-1A association with dipalmitoyl phosphatidylserine reveals membrane blebbing in the presence of cholesterol. | Cell Stress Chaperones | Hsp70-1A-the major stress-inducible member of the HSP70 chaperone family-is being implicated in cancer diseases with the development of resistances to standard therapies. In normal cells, the protein is purely cytosolic, but in a growing number of tumor cells, a significant fraction can be identified on to the cell surface. The anchoring mechanism is still under debate, as Hsp70-1A lacks conventional signaling sequences for translocation from the cytosol to exoplasmic leaflet of the plasma membrane and common membrane binding domains. Recent reports propose a lipid-mediated anchoring mechanism based on a specific interaction with charged, saturated lipids such as dipalmitoyl phosphatidylserine (DPPS). Here, we prepared planar supported lipid bilayers (SLBs) to visualize the association of Hsp70-1A directly and on the single molecule level by atomic force microscopy (AFM). The single molecule sensitivity of our approach allowed us to explore the low concentration range of 0.05 to 1.0 μg/ml of Hsp70-1A which was not studied before. We compared the binding of the protein to bilayers with 20% DPPS lipid content both in the absence and presence of cholesterol. Hsp70-1A inserted exclusively into DPPS domains and assembled in clusters with increasing protein density. A critical density was reached for incubation with 0.5 μg/ml (7 nM); at higher concentrations, membrane defects were observed that originated from cluster centers. In the presence of cholesterol, this critical concentration leads to the formation of membrane blebs, which burst at higher concentrations supporting a previously proposed non-classical pathway for the export of Hsp70-1A by tumor cells. In the discussion of our data, we attempt to link the lipid-mediated plasma membrane localization of Hsp70-1A to its potential involvement in the development of resistances to radiation and chemotherapy based on our own findings and the current literature. |
| 2017 | BL23 Produces Microvesicles Carrying Proteins That Have Been Associated with Its Probiotic Effect. | Front Microbiol | Archaea, bacteria, and eukarya secrete membrane microvesicles (MVs) as a mechanism for intercellular communication. We report the isolation and characterization of MVs from the probiotic strain BL23. MVs were characterized using analytical high performance techniques, DLS, AFM and TEM. Similar to what has been described for other Gram-positive bacteria, MVs were on the nanometric size range (30-50 nm). MVs carried cytoplasmic components such as DNA, RNA and proteins. Using a proteomic approach (LC-MS), we identified a total of 103 proteins; 13 exclusively present in the MVs. The MVs content included cell envelope associated and secretory proteins, heat and cold shock proteins, several metabolic enzymes, proteases, structural components of the ribosome, membrane transporters, cell wall-associated hydrolases and phage related proteins. In particular, we identified proteins described as mediators of probiotic effects such as p40, p75 and the product of LCABL_31160, annotated as an adhesion protein. The presence of these proteins suggests a role for the MVs in the bacteria-gastrointestinal cells interface. The expression and further encapsulation of proteins into MVs of GRAS (Generally Recognized as Safe) bacteria could represent a scientific novelty, with applications in food, nutraceuticals and clinical therapies. |
| 2017 | Rapid dynamics of cell-shape recovery in response to local deformations. | Soft Matter | It is vital that cells respond rapidly to mechanical cues within their microenvironment through changes in cell shape and volume, which rely upon the mechanical properties of cells' highly interconnected cytoskeletal networks and intracellular fluid redistributions. While previous research has largely investigated deformation mechanics, we now focus on the immediate cell-shape recovery response following mechanical perturbation by inducing large, local, and reproducible cellular deformations using AFM. By continuous imaging within the plane of deformation, we characterize the membrane and cortical response of HeLa cells to unloading, and model the recovery via overdamped viscoelastic dynamics. Importantly, the majority (90%) of HeLa cells recover their cell shape in <1 s. Despite actin remodelling on this time scale, we show that cell-shape recovery time is not affected by load duration, nor magnitude for untreated cells. To further explore this rapid recovery response, we expose cells to cytoskeletal destabilizers and osmotic shock conditions, which uncovers the interplay between actin and osmotic pressure. We show that the rapid dynamics of recovery depend crucially on intracellular pressure, and provide strong evidence that cortical actin is the key regulator in the cell-shape recovery processes, in both cancerous and non-cancerous epithelial cells. |
| 2016 | [Extracorporeal membrane oxygenation for rescuing 12 children with acute fulminant myocarditis]. | Zhonghua Er Ke Za Zhi | To summarize clinical experience of using extracorporeal membrane oxygenation (ECMO) in rescuing children with acute fulminant myocarditis (AFM). Data of 12 children with acute fulminate myocarditis (6 boys and 6 girls, median age 8.3 (0.6, 13.0) years, median weight 33.1 (6, 61) kg) who were rescued with ECMO in Children's Hospital, Zhejiang University from September 2009 to August 2015 were analyzed retrospectively. The analysis focused on the intervene timing of ECMO for the cardiogenic shock and hypoperfusion caused by heart failure and(or) lethal arrhythmia and the essentials of ECMO cardiopulmonary resuscitation(ECPR) for cardiac arrest in pediatric AFM were summarized. The median ECMO duration was 110(22, 240) h. Ten cases survived and 2 were dead of the total of 12 patients. Six ECPR patients survived and 2 were dead in the total of 8 ECPR patients. The complication of 10 survivors were cannula site bleeding (3 cases), hypernatremia and intracranial hemorrhage (1 case), limping (1 case), hoarse voice (1 case), and cerebral injury (1 case). The key points of improving ECMO rescuing outcome for the AFM children are grasping the ECMO intervene timing and training skilled ECMO team. For ECPR patients, keeping effective chest compressions resuscitation is the key to achieve survival and improve the quality of life. |
| 2016 | Impact of Extracorporeal Membrane Oxygenation on Acute Fulminant Myocarditis-related Hemodynamic Compromise Arrhythmia in Children. | Pediatr Neonatol | Acute fulminant myocarditis (AFM) commonly presents as abrupt cardiogenic shock with or without dysrhythmia. This study evaluated the impact of extracorporeal membrane oxygenation (ECMO) on AFM-related hemodynamic compromise dysrhythmias. We also reported the clinical experience of AFM at our hospital.Eighteen children diagnosed with AFM were enrolled. Demographic variables, laboratory data, and clinical courses were reviewed. Thirteen surviving patients with hemodynamic compromise arrhythmia [complete atrioventricular block (CAVB) or ventricular tachycardia (VT)] during hospitalization were divided into Group A (ECMO group; n = 7) and Group B (non-ECMO group; n = 6).The overall survival rate was 78% (14/18). There were no cases of mortality after ECMO was introduced at our hospital. Common symptoms at diagnosis included general malaise (94%), gastrointestinal symptoms (89%), chest pain (56%), shortness of breath (56%), and seizure/syncope (56%). In addition to abnormal cardiac enzyme levels, all patients displayed elevated alanine aminotransferase levels during early disease stages. Electrocardiography at diagnosis revealed dysrhythmia in 15 patients, namely, CAVB in 11 patients (61%) and VT in four patients (22%). During hospitalization, the dysrhythmia shifted from CAVB to VT in 10 patients and from sinus tachycardia to VT in one patient. New episodes of VT were common (overall occurrence rate, 83%). Although myocardial damage and dysfunction were more severe in Group A, the time to rhythm recovery in this group was shorter than that in Group B (median time, 1.7 days vs. 7.35 days, p = 0.045). All surviving patients had normal ventricular function at 6-month follow-up.Hemodynamic compromise arrhythmia is common in AFM patients and may cause rapid deterioration. Simply correcting sinus rhythm is not always sufficient because of myocardium instability. Timely use of ECMO can improve the survival rate and shorten the time to recapture sinus rhythm in AFM patients with CAVB or VT. |
| 2016 | Investigation of antibacterial activity of aspidin BB against Propionibacterium acnes. | Arch Dermatol Res | In the present study, antibacterial activity of four kinds of phloroglucinol derivatives extracted from Dryopteris fragrans (L.) Schott against S. aureus, S. epidermidis and P. acnes has been tested. Aspidin BB exerted the strongest antibacterial activity with minimal inhibition concentration (MIC) values ranging from 7.81 to 15.63 μg/mL. The time-kill assay indicated that aspidin BB could kill P. acnes completely at 2 MIC (MBC) within 4 h. By using AFM, we demonstrated extensive cell surface alterations of aspidin BB-treated P. acnes. SDS-PAGE of supernatant proteins and lipid peroxidation results showed that aspidin BB dose-dependently affected membrane permeability of P. acnes. DNA damage and protein degradation of P. acnes were also verified. SDS-PAGE of precipitated proteins revealed possible targets of aspidin BB, i.e., heat shock proteins (26 kDa) and lipase (33 kDa) which could all cause inflammation. Aspidin BB also seriously increased the inhibition rate of lipase activity from 10.20 to 65.20 % to possibly inhibit the inflammation. In conclusions, the effective constituents of D. fragrans (L.) Schott to treat acne might be phloroglucinol derivatives including aspidin BB, aspidin PB, aspidinol and dryofragin. Among this, aspidin BB inhibited the growth of P. acnes by disrupting their membrane, DNA and proteins and finally leaded to the cell death. The obtained data highlighted the potential of using aspidin BB as an alternative treatment for acne vulgaris. |
| 2016 | Venoarterial Extracorporeal Membrane Oxygenation for Acute Fulminant Myocarditis in Adult Patients: A 5-Year Multi-Institutional Experience. | Ann Thorac Surg | Acute fulminant myocarditis (AFM) may represent a life-threatening event, characterized by rapidly progressive cardiac compromise that ultimately leads to refractory cardiogenic shock or cardiac arrest. Venoarterial extracorporeal membrane oxygenation (VA-ECMO) provides effective cardiocirculatory support in this circumstance, but few clinical series are available about early and long-term results. Data from a multicenter study group are reported which analyzed subjects affected by AFM and treated with VA-ECMO during a 5-year period.From hospital databases, 57 patients with diagnoses of AFM treated with VA-ECMO in the past 5 years were found and analyzed. Mean age was 37.6 ± 11.8 years; 37 patients were women. At VA-ECMO implantation, cardiogenic shock was present in 38 patients, cardiac arrest in 12, and severe hemodynamic instability in 7. A peripheral approach was used with 47 patients, whereas 10 patients had a central implantation or other access.Mean VA-ECMO support was 9.9 ± 19 days (range, 2 to 24 days). Cardiac recovery with ECMO weaning was achieved in 43 patients (75.5%), major complications were observed in 40 patients (70.1%), and survival to hospital discharge occurred in 41 patients (71.9%). After hospital discharge (median follow-up, 15 months) there were 2 late deaths. The 5-year actual survival was 65.2% ± 7.9%, with recurrent self-recovering myocarditis observed in 2 patients (at 6 and 12 months from the first AFM event), and 1 heart transplantation.Cardiopulmonary support with VA-ECMO provides an invaluable tool in the treatment of AFM, although major complications may characterize the hospital course. Long-term outcome appears favorable with rare episodes of recurrent myocarditis or cardiac-related events. |
| 2015 | Water flattens graphene wrinkles: laser shock wrapping of graphene onto substrate-supported crystalline plasmonic nanoparticle arrays. | Nanoscale | Hot electron injection into an exceptionally high mobility material can be realized in graphene-plasmonic nanoantenna hybrid nanosystems, which can be exploited for several front-edge applications including photovoltaics, plasmonic waveguiding and molecular sensing at trace levels. Wrinkling instabilities of graphene on these plasmonic nanostructures, however, would cause reactive oxygen or sulfur species to diffuse and react with the materials, decrease charge transfer rates and block intense hot-spots. No ex situ graphene wrapping technique has been explored so far to control these wrinkles. Here, we present a method to generate seamless integration by using water as a flyer to transfer the laser shock pressure to wrap graphene onto plasmonic nanocrystals. This technique decreases the interfacial gap between graphene and the covered substrate-supported plasmonic nanoparticle arrays by exploiting a shock pressure generated by the laser ablation of graphite and the water impermeable nature of graphene. Graphene wrapping of chemically synthesized crystalline gold nanospheres, nanorods and bipyramids with different field confinement capabilities is investigated. A combined experimental and computational method, including SEM and AFM morphological investigation, molecular dynamics simulation, and Raman spectroscopy characterization, is used to demonstrate the effectiveness of this technique. Graphene covered gold bipyramid exhibits the best result among the hybrid nanosystems studied. We have shown that the hybrid system fabricated by laser shock can be used for enhanced molecular sensing. The technique developed has the characteristics of tight integration, and chemical/thermal stability, is instantaneous in nature, possesses a large scale and room temperature processing capability, and can be further extended to integrate other 2D materials with various 0-3D nanomaterials. |
| 2016 | AuNPs modified, disposable, ITO based biosensor: Early diagnosis of heat shock protein 70. | Biosens Bioelectron | This paper describes a novel, simple, and disposable immunosensor based on indium-tin oxide (ITO) sheets modified with gold nanoparticles to sensitively analyze heat shock protein 70 (HSP70), a potential biomarker that could be evaluated in diagnosis of some carcinomas. Disposable ITO coated Polyethylene terephthalate (PET) electrodes were used and modified with gold nanoparticles in order to construct the biosensors. Optimization and characterization steps were analyzed by electrochemical techniques such as electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Surface morphology of the biosensor was also identified by electrochemical methods, scanning electron microscopy (SEM), and atomic force microscopy (AFM). To interpret binding characterization of HSP70 to anti-HSP70 single frequency impedance method was successfully operated. Moreover, the proposed HSP70 immunosensor acquired good stability, repeatability, and reproducibility. Ultimately, proposed biosensor was introduced to real human serum samples to determine HSP70 sensitively and accurately. |
| 2015 | Rhamnolipids functionalized AgNPs-induced oxidative stress and modulation of toxicity pathway genes in cultured MCF-7 cells. | Colloids Surf B Biointerfaces | Rhamnolipids extracted from Pseudomonas aeruginosa strain JS-11 were utilized for synthesis of stable silver nanoparticles (Rh-AgNPs). The Rh-AgNPs (23 nm) were characterized by Fourier transform infra-red (FTIR) spectroscopy, atomic force microscopy (AFM) and transmission electron microscopy (TEM). The cytotoxicity assays suggested significant decrease in viability of Rh-AgNPs treated human breast adenocarcinoma (MCF-7) cells, compared with normal human peripheral blood mononuclear (PBMN) cells. Flow cytometry data revealed 1.25-fold (p<0.05) increase in the fluorescence of 2',7'-dichlorofluorescein (DCF) at 0.25 μg/mL. However, at Rh-AgNPs concentrations of 0.5 and 1.0 μg/mL, much lesser fluorescence was noticed, which is attributed to cell death. Results with the fluorescent probe Rh123 demonstrated change in inner mitochondrial membrane and dissipation of membrane potential. The cell cycle analysis suggested 19.9% (p<0.05) increase in sub-G1 peak with concomitant reduction in G1 phase at 1 μg/mL of Rh-AgNPs, compared to 2.7% in untreated control. The real-time RT(2) Profiler™ PCR array data elucidated the overexpression of seven oxidative stress and DNA damage pathways genes viz. BAX, BCl2, Cyclin D1, DNAJA1, E2F transcription factor 1, GPX1 and HSPA4, associated with apoptosis signaling, proliferation and carcinogenesis, pro inflammatory and heat shock responses in Rh-AgNPs treated cells. Thus, the increased ROS production, mitochondrial damage and appearance of sub-G1 (apoptotic) population suggested the anti-proliferative activity, and role of oxidative stress pathway genes in Rh-AgNPs induced death of MCF-7 cancer cells. |
| 2015 | New, non-quinone fluorogeldanamycin derivatives strongly inhibit Hsp90. | Chembiochem | Streptomyces hygroscopicus is a natural producer of geldanamycin. Mutasynthetic supplementation of an AHBA-blocked mutant with all possible monofluoro 3-aminobenzoic acids provided new fluorogeldanamycins. These showed strong antiproliferative activity and inhibitory effects on human heat shock protein Hsp90. Binding to Hsp90 in the low nanomolar range was determined from molecular modelling, AFM analysis and by calorimetric studies. |
| 2014 | Ultrahigh interlayer friction in multiwalled boron nitride nanotubes. | Nat Mater | Friction at the nanoscale has revealed a wealth of behaviours that depart strongly from the long-standing macroscopic laws of Amontons-Coulomb. Here, by using a 'Christmas cracker'-type of system in which a multiwalled nanotube is torn apart between a quartz-tuning-fork-based atomic force microscope (TF-AFM) and a nanomanipulator, we compare the mechanical response of multiwalled carbon nanotubes (CNTs) and multiwalled boron nitride nanotubes (BNNTs) during the fracture and telescopic sliding of the layers. We found that the interlayer friction for insulating BNNTs results in ultrahigh viscous-like dissipation that is proportional to the contact area, whereas for the semimetallic CNTs the sliding friction vanishes within experimental uncertainty. We ascribe this difference to the ionic character of the BN, which allows charge localization. The interlayer viscous friction of BNNTs suggests that BNNT membranes could serve as extremely efficient shock-absorbing surfaces. |
| 2014 | Drinking water biofilm cohesiveness changes under chlorination or hydrodynamic stress. | Water Res | Attempts at removal of drinking water biofilms rely on various preventive and curative strategies such as nutrient reduction in drinking water, disinfection or water flushing, which have demonstrated limited efficiency. The main reason for these failures is the cohesiveness of the biofilm driven by the physico-chemical properties of its exopolymeric matrix (EPS). Effective cleaning procedures should break up the matrix and/or change the elastic properties of bacterial biofilms. The aim of this study was to evaluate the change in the cohesive strength of two-month-old drinking water biofilms under increasing hydrodynamic shear stress τw (from ∼0.2 to ∼10 Pa) and shock chlorination (applied concentration at T0: 10 mg Cl2/L; 60 min contact time). Biofilm erosion (cell loss per unit surface area) and cohesiveness (changes in the detachment shear stress and cluster volumes measured by atomic force microscopy (AFM)) were studied. When rapidly increasing the hydrodynamic constraint, biofilm removal was found to be dependent on a dual process of erosion and coalescence of the biofilm clusters. Indeed, 56% of the biofilm cells were removed with, concomitantly, a decrease in the number of the 50-300 μm(3) clusters and an increase in the number of the smaller (i.e., <50 μm(3)) and larger (i.e., >600 μm(3)) ones. Moreover, AFM evidenced the strengthening of the biofilm structure along with the doubling of the number of contact points, NC, per cluster volume unit following the hydrodynamic disturbance. This suggests that the compactness of the biofilm exopolymers increases with hydrodynamic stress. Shock chlorination removed cells (-75%) from the biofilm while reducing the volume of biofilm clusters. Oxidation stress resulted in a decrease in the cohesive strength profile of the remaining drinking water biofilms linked to a reduction in the number of contact points within the biofilm network structure in particular for the largest biofilm cluster volumes (>200 μm(3)). Changes in the cohesive strength of drinking water biofilms subsequent to cleaning/disinfection operations call into question the effectiveness of cleaning-in-place procedures. The combined alternating use of oxidation and shear stress sequences needs to be investigated as it could be an important adjunct to improving biofilm removal/reduction procedures. |
| 2014 | Uncovering by atomic force microscopy of an original circular structure at the yeast cell surface in response to heat shock. | BMC Biol | Atomic Force Microscopy (AFM) is a polyvalent tool that allows biological and mechanical studies of full living microorganisms, and therefore the comprehension of molecular mechanisms at the nanoscale level. By combining AFM with genetical and biochemical methods, we explored the biophysical response of the yeast Saccharomyces cerevisiae to a temperature stress from 30°C to 42°C during 1 h.We report for the first time the formation of an unprecedented circular structure at the cell surface that takes its origin at a single punctuate source and propagates in a concentric manner to reach a diameter of 2-3 μm at least, thus significantly greater than a bud scar. Concomitantly, the cell wall stiffness determined by the Young's Modulus of heat stressed cells increased two fold with a concurrent increase of chitin. This heat-induced circular structure was not found either in wsc1Δ or bck1Δ mutants that are defective in the CWI signaling pathway, nor in chs1Δ, chs3Δ and bni1Δ mutant cells, reported to be deficient in the proper budding process. It was also abolished in the presence of latrunculin A, a toxin known to destabilize actin cytoskeleton.Our results suggest that this singular morphological event occurring at the cell surface is due to a dysfunction in the budding machinery caused by the heat shock and that this phenomenon is under the control of the CWI pathway. |
| Impact of extracorporeal membrane oxygenation support on clinical outcome of pediatric patients with acute cardiopulmonary failure: a single-center experience. | Biomed J | Conventional therapy against acute pediatric cardiopulmonary failure (APCPF) caused by a variety of disease entities remains unsatisfactory with extremely high morbidity and mortality. For refractory APCPF, extracorporeal membrane oxygenation (ECMO) is one of the last resorts.In this study, the in-hospital outcomes of pediatric patients with refractory APCPF receiving ECMO support were reviewed.Between August 2006 and May 2011, a single-center cohort study was performed in pediatric patients who required ECMO support due to cardiogenic shock or severe hypoxemia. A total of 22 patients with mean age of 7.0 ± 6.3 years received ECMO (male = 11; female = 11). The indications included acute fulminant myocarditis (AFM) (n = 6), congenital diaphragmatic hernia (CDH) (n = 3), acute respiratory distress syndrome (ARDS) (n = 6), enterovirus 71 (n = 3), viral sepsis (n = 2), refractory ventricular fibrillation due to long QT syndrome (n = 1), and pulmonary edema with brain herniation (n = 1). Eighteen patients received veno-arterial (VA) mode ECMO, while another four patients undertook the veno-venous (VV) mode. The duration of ECMO use and hospitalization were 6.1 ± 3.1 and 24.4 ± 19.4 days, respectively. The survival rate in patients with AFM was 100% (n = 6). Successful ECMO weaning with uneventful discharge from hospital was noted in 14 (63.6%) patients, whereas in-hospital mortality despite successful ECMO weaning occurred in 5 patients (22.7%). Failure in ECMO weaning and in-hospital death was noted in 3 patients (13.6%).ECMO resuscitation is an effective strategy in the clinical setting of APCPF. |
| 2012 | Clinical features and prognosis in Chinese patients with acute fulminant myocarditis. | Acta Cardiol | There is still a lack of large-scale studies focusing on acute fulminant myocarditis (AFM) and knowledge of this disease is unimpressive. To better understand the clinical features of AFM and improve the diagnosis and treatment of this disease, we analysed the data of consecutive Chinese AFM patients admitted to our department.This retrospective observational study analysed the data of 40 patients with a diagnosis of AFM admitted to our hospital between January 2003 and March 2010.The mean age of patients was 28.5 +/- 18.4 years and 70% of patients were females. 90.0% of patients had a short viral prodrome, 77.5% had cardiogenic shock and 35.0% had multiple organ failure, especially hepatic dysfunction. The most common manifestations in ECG were low voltage in the limb leads (87.5%) and sinus tachycardia (75%). Myocardial infarctions like ECG changes were not uncommon. Left ventricular ejection fraction (LVEF) was significantly reduced (25.3 +/- 7.5%), but the left cardiac sizes were normal. More than 90% of the patients were treated with glucocorticoids. 5.0% needed an intra-aortic balloon pump (IABP), and 35.0% were treated with bi-level positive airway pressure (BiPAP). In all, 39 (95.0%) patients were discharged alive and one man died from ventricular fibrillation. LVEF and left cardiac chambers at follow-up did not change as compared with discharge. No death, new onset heart failure or arrhythmias occurred during the follow-up.This study describes the clinical features of Chinese AFM patients. The short- and mid-term prognosis of AFM is good. AFM patients may benefit from a treatment with glucocorticoids. |
| 2011 | Investigation of bacteriophage T4 by atomic force microscopy. | Bacteriophage | Bacteriophage T4 was visualized using atomic force microscopy (AFM). The images were consistent with, and complementary to electron microscopy images. Head heights of dried particles containing DNA were about 75 nm in length and 60 nm in width, or about 100 nm and 85 nm respectively when scanned in fluid. The diameter of hydrated tail assemblies was 28 nm and their lengths about 130 nm. Seven to eight pronounced, right-handed helical turns with a pitch of 15 nm were evident on the tail assemblies. At the distal end of the tail was a knob shaped mass, presumably the baseplate. The opposite end, where the tail assembly joins the head, was tapered and connected to the portal complex, which was also visible. Phage that had ejected their DNA revealed the internal injection tube of the tail assembly. Heads disrupted by osmotic shock yielded boluses of closely packed DNA that unraveled slowly to expose threads composed of multiple twisted strands of nucleic acid. Assembly errors resulted in the appearance of several percent of the phage exhibiting two rather than one tail assemblies that were consistently oriented at about 72° to one another. No pattern of capsomeres was visible on native T4 heads. A mutant that is negative for the surface proteins hoc and soc, however, clearly revealed the icosahedral arrangement of ring shaped capsomeres on the surface. The hexameric rings have an outside diameter of about 14 nm, a pronounced central depression, and a center-to-center distance of 15 nm. Phage collapsed on cell surfaces appeared to be dissolving, possibly into the cell membrane. |
| 2011 | Management and outcomes in pediatric patients presenting with acute fulminant myocarditis. | J Pediatr | To investigate factors associated with mechanical circulatory support and survival in patients with acute fulminant myocarditis (AFM).Retrospective cohort of AFM patients admitted to the cardiac intensive care unit during 1996-2008. AFM was defined as distinct onset of symptoms ≤14 days before admission, rapid-onset cardiogenic shock, and normal left ventricular size. Demographic and physiological variables were compared between patients treated with extracorporeal membrane oxygenation (ECMO) and those who were not and between survivors and nonsurvivors.Twenty patients (median age 12.7 years) met inclusion criteria. Seventeen patients (85%) survived to hospital discharge. One underwent heart transplantation. Ten (50%) patients required ECMO, and 7 (70%) of these survived. On admission, patients requiring ECMO had elevated lactate (9 vs 1 mmol/L), creatinine (0.8 vs 0.6 mg/dL), and aspartate aminotransferase (256 vs 35 IU/L) (all P < .05) and a trend towards increased incidence of dysrhythmias (80% vs 30%, P = .07). During hospitalization, non-survivors had higher peak lactate (10 vs 3 mmol/L), creatinine (1.5 vs 0.8 mg/dL), and aspartate aminotransferase (3007 vs 156 IU/L) (all P < .05) compared with survivors.Patients with AFM with end organ dysfunction or arrhythmias on admission may require mechanical circulatory support. The transplant-free survival rate in this critically ill cohort was excellent (80%). |
| 2010 | Changes of cell-surface thiols and intracellular signaling in human monocytic cell line THP-1 treated with diphenylcyclopropenone. | J Toxicol Sci | Changes of cell-surface thiols induced by chemical treatment may affect the conformations of membrane proteins and intracellular signaling mechanisms. In our previous study, we found that a non-toxic dose of diphenylcyclopropene (DPCP), which is a potent skin sensitizer, induced an increase of cell-surface thiols in cells of a human monocytic cell line, THP-1. Here, we examined the influence of DPCP on intracellular signaling. First, we confirmed that DPCP induced an increase of cell-surface thiols not only in THP-1 cells, but also in primary monocytes. The intracellular reduced-form glutathione/oxidized-form glutathione ratio (GSH/GSSG ratio) was not affected by DPCP treatment. By means of labeling with a membrane-impermeable thiol-reactive compound, Alexa Fluor 488 C5 maleimide (AFM), followed by two-dimensional gel electrophoresis and analysis by liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS), we identified several proteins whose thiol contents were modified in response to DPCP. These proteins included cell membrane components, such as actin and β-tubulin, molecular chaperones, such as heat shock protein 27A and 70, and endoplasmic reticulum (ER) stress-inducible proteins. Next, we confirmed the expression in DPCP-treated cells of spliced XBP1, a known marker of ER stress. We also detected the phosphorylation of SAPK/JNK and p38 MAPK, which are downstream signaling molecules in the IRE1α-ASK1 pathway, which is activated by ER stress. These data suggested that increase of cell-surface thiols might be associated with activation of ER stress-mediated signaling. |
| 2010 | Successful treatment of acute fulminant myocarditis with double sets of extracorporeal membrane oxygenation: report of a case and review of the literature. | Thorac Cardiovasc Surg | We report a case of acute fulminant myocarditis (AFM) with cardiogenic shock and hemodynamic collapse. We performed emergency extracorporeal life support (ECLS) with right femoral venoarterial cannulation by Seldinger procedure. Because of poor systemic perfusion over the upper trunk with low O₂ saturation due to inadequate venous return, a second ECLS was applied 16 hours after the first ECLS with right subclavian venoarterial cannulation by cut-down procedure. Total flow of both ECLS sets can achieve a flow of up to 6-8 l/min without massive destruction of blood cells. Combined with supportive intravenous immunoglobulin (IVIG) treatment, cardiac function recovered well and the ECLS sets were removed one-by-one. Follow-up studies one year after admission showed good cardiac systolic function and no sequelae of AFM. |
| 2010 | Imaging articular cartilage tissue using atomic force microscopy (AFM). | Cold Spring Harb Protoc | Cartilage is a complex avascular tissue composed of cells ("chondrocytes") embedded in an extracellular matrix (ECM) consisting of 70%-80% water. The primary components of the ECM are negatively charged aggrecans and collagen II fibrils, which possess a characteristic, ordered three-dimensional structure. The components interact to ensure that the cartilage is able to absorb shock and can function to protect the bone ends. Atomic force microscopy (AFM) can be used to examine structure-function relationships of cartilage at both micrometer and nanometer scales. When imaged at the micrometer scale with microspheres, only the ECM and chondrocytes can be distinguished. Correspondingly, mechanical testing of cartilage at the micrometer scale results in unimodal distribution of the stiffness because the bulk elastic property of the ECM is probed. In contrast, bare AFM tips are able to reveal the molecular components of the ECM at the nanometer scale. Mechanical testing at the nanometer scale reveals a bimodal distribution of the stiffness and reflects the distinct stiffness of the collagen network and the proteoglycan moiety. In this protocol, the corresponding AFM image and force map are used to reveal the distinct morphology of the collagen fibers and proteoglycan gel. Although, in principle, these experiments can be performed using any AFM, an AFM with tube scanners that have manual screws for tilting the sample is preferable because cartilage has macroscopically rough surface features. By manually tilting the probe over the sample, an optimal angle for tip approach can be achieved. |
| 2010 | Designed biomaterials to mimic the mechanical properties of muscles. | Nature | The passive elasticity of muscle is largely governed by the I-band part of the giant muscle protein titin, a complex molecular spring composed of a series of individually folded immunoglobulin-like domains as well as largely unstructured unique sequences. These mechanical elements have distinct mechanical properties, and when combined, they provide the desired passive elastic properties of muscle, which are a unique combination of strength, extensibility and resilience. Single-molecule atomic force microscopy (AFM) studies demonstrated that the macroscopic behaviour of titin in intact myofibrils can be reconstituted by combining the mechanical properties of these mechanical elements measured at the single-molecule level. Here we report artificial elastomeric proteins that mimic the molecular architecture of titin through the combination of well-characterized protein domains GB1 and resilin. We show that these artificial elastomeric proteins can be photochemically crosslinked and cast into solid biomaterials. These biomaterials behave as rubber-like materials showing high resilience at low strain and as shock-absorber-like materials at high strain by effectively dissipating energy. These properties are comparable to the passive elastic properties of muscles within the physiological range of sarcomere length and so these materials represent a new muscle-mimetic biomaterial. The mechanical properties of these biomaterials can be fine-tuned by adjusting the composition of the elastomeric proteins, providing the opportunity to develop biomaterials that are mimetic of different types of muscles. We anticipate that these biomaterials will find applications in tissue engineering as scaffold and matrix for artificial muscles. |
| 2008 | Persisting atypical and cystic forms of Borrelia burgdorferi and local inflammation in Lyme neuroborreliosis. | J Neuroinflammation | The long latent stage seen in syphilis, followed by chronic central nervous system infection and inflammation, can be explained by the persistence of atypical cystic and granular forms of Treponema pallidum. We investigated whether a similar situation may occur in Lyme neuroborreliosis.Atypical forms of Borrelia burgdorferi spirochetes were induced exposing cultures of Borrelia burgdorferi (strains B31 and ADB1) to such unfavorable conditions as osmotic and heat shock, and exposure to the binding agents Thioflavin S and Congo red. We also analyzed whether these forms may be induced in vitro, following infection of primary chicken and rat neurons, as well as rat and human astrocytes. We further analyzed whether atypical forms similar to those induced in vitro may also occur in vivo, in brains of three patients with Lyme neuroborreliosis. We used immunohistochemical methods to detect evidence of neuroinflammation in the form of reactive microglia and astrocytes.Under these conditions we observed atypical cystic, rolled and granular forms of these spirochetes. We characterized these abnormal forms by histochemical, immunohistochemical, dark field and atomic force microscopy (AFM) methods. The atypical and cystic forms found in the brains of three patients with neuropathologically confirmed Lyme neuroborreliosis were identical to those induced in vitro. We also observed nuclear fragmentation of the infected astrocytes using the TUNEL method. Abundant HLA-DR positive microglia and GFAP positive reactive astrocytes were present in the cerebral cortex.The results indicate that atypical extra- and intracellular pleomorphic and cystic forms of Borrelia burgdorferi and local neuroinflammation occur in the brain in chronic Lyme neuroborreliosis. The persistence of these more resistant spirochete forms, and their intracellular location in neurons and glial cells, may explain the long latent stage and persistence of Borrelia infection. The results also suggest that Borrelia burgdorferi may induce cellular dysfunction and apoptosis. The detection and recognition of atypical, cystic and granular forms in infected tissues is essential for the diagnosis and the treatment as they can occur in the absence of the typical spiral Borrelia form. |
| 2005 | Electrical discharge in a nanometer-sized air/water gap observed by atomic force microscopy. | J Am Chem Soc | We report a method to initiate and investigate electrical discharges of ambient air/water molecules in a nanometer-sized gap. Our methodology is based on a typical atomic force microscopy (AFM) setup, in which a cylinder discharge gap of < or =5 nm could be configured between the AFM probe and substrate. We observed highly localized stochastic nanoexplosions in which the discharge probability is dominated by the electric field, material-specific surface reactions, and humidity. AFM results, coupled with the boundary element method (BEM), finite element method (FEM), and method of characteristics (MOC) simulations, further revealed the generation of transient shock waves in the nanoscale discharge. The propagation of shock fronts significantly facilitates the radial expansion of the ionized particles, leading to the formation of microscale patterns on selected substrates. Our findings provide an initial understanding of nanoscale discharge and could be relevant to a few applications including nano/microstructuring, microelectronics, and plasma-assisted depositions. |
| 2005 | Exploring the mechanical behavior of single intermediate filaments. | J Mol Biol | Intermediate filaments (IFs) are structural elements of eukaryotic cells with distinct mechanical properties. Tissue integrity is severely impaired, in particular in skin and muscle, when IFs are either absent or malfunctioning due to mutations. Our knowledge on the mechanical properties of IFs is mainly based on tensile testing of macroscopic fibers and on the rheology of IF networks. At the single filament level, the only piece of data available is a measure of the persistence length of vimentin IFs. Here, we have employed an atomic force microscopy (AFM) based protocol to directly probe the mechanical properties of single cytoplasmic IFs when adsorbed to a solid support in physiological buffer environment. Three IF types were studied in vitro: recombinant murine desmin, recombinant human keratin K5/K14 and neurofilaments isolated from rat brains, which are composed of the neurofilament triplet proteins NF-L, NF-M and NF-H. Depending on the experimental conditions, the AFM tip was used to laterally displace or to stretch single IFs on the support they had been adsorbed to. Upon applying force, IFs were stretched on average 2.6-fold. The maximum stretching that we encountered was 3.6-fold. A large reduction of the apparent filament diameter was observed concomitantly. The observed mechanical properties therefore suggest that IFs may indeed function as mechanical shock absorbers in vivo. |
| 2005 | Myomesin is a molecular spring with adaptable elasticity. | J Mol Biol | The M-band is a transverse structure in the center of the sarcomere, which is thought to stabilize the thick filament lattice. It was shown recently that the constitutive vertebrate M-band component myomesin can form antiparallel dimers, which might cross-link the neighboring thick filaments. Myomesin consists mainly of immunoglobulin-like (Ig) and fibronectin type III (Fn) domains, while several muscle types express the EH-myomesin splice isoform, generated by the inclusion of the unique EH-segment of about 100 amino acid residues (aa) in the center of the molecule. Here we use atomic force microscopy (AFM), transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy for the biophysical characterization of myomesin. The AFM identifies the "mechanical fingerprints" of the modules constituting the myomesin molecule. Stretching of homomeric polyproteins, constructed of Ig and Fn domains of human myomesin, produces a typical saw-tooth pattern in the force-extension curve. The domains readily refold after relaxation. In contrast, stretching of a heterogeneous polyprotein, containing several repeats of the My6-EH fragment reveals a long initial plateau corresponding to the sum of EH-segment contour lengths, followed by several My6 unfolding peaks. According to this, the EH-segment is characterized as an entropic chain with a persistence length of about 0.3nm. In TEM pictures, the EH-domain appears as a gap in the molecule, indicating a random coil conformation similar to the PEVK region of titin. CD spectroscopy measurements support this result, demonstrating a mostly non-folded conformation for the EH-segment. We suggest that similarly to titin, myomesin is a molecular spring, whose elasticity is modulated by alternative splicing. The Ig and Fn domains might function as reversible "shock absorbers" by sequential unfolding in the case of extremely high or long sustained stretching forces. These complex visco-elastic properties of myomesin might be crucial for the stability of the sarcomere. |
| 2005 | Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy. | J Cell Sci | The highly conserved and ubiquitous heat shock proteins (HSP) are essential for the cellular homeostasis and efficiently trigger cellular responses to stress conditions. Both microbial and human HSP act as dominant antigens in numerous infectious and autoimmune diseases such as atherosclerosis, inducing a strong immune-inflammatory response. In the present study, the surface localization of HSP60 on stressed and unstressed human umbilical venous endothelial cells (HUVECs) was investigated using sensitive high resolution microscopy methods and flow cytometry. Confocal laser scanning microscopy (CLSM) revealed an increase of HSP60 in the mitochondria and on the surface of heat-stressed living and fixed HUVECs compared to unstressed cells. Atomic force microscopy (AFM), which has developed as sensitive surface-probe technique in biology, confirmed the presence of HSP60 on the membrane of stressed cells at an even higher lateral resolution by detecting specific single molecule binding events between the monoclonal antibody AbII-13 tethered to AFM tips and HSP60 molecules on cells. The interaction force (force required to break a single AbII-13/HSP60 bond) was 59+/-2 pN, which correlated nicely to the 51+/-1 pN measured with isolated HSP60 attached to mica surfaces. Overall, we found clear evidence for the occurrence of HSP60 on the surface of stressed HUVECs in a very similar patchy distribution pattern in living and fixed cells. The relevance of our findings with respect to the role of HSP60 in atherogenesis is discussed. |
| 2004 | Calibration of AFM cantilever stiffness: a microfabricated array of reflective springs. | Ultramicroscopy | Calibration of the spring constant of atomic force microscope (AFM) cantilevers is necessary for the measurement of nanonewton and piconewton forces, which are critical to analytical applications of AFM in the analysis of polymer surfaces, biological structures and organic molecules. We have developed a compact and easy-to-use reference standard for this calibration. The new artifact consists of an array of 12 dual spiral-cantilever springs, each supporting a mirrored polycrystalline silicon disc of 160 microm in diameter. These devices were fabricated by a three-layer polysilicon surface micromachining method, including a reflective layer of gold on chromium. We call such an array a Microfabricated Array of Reference Springs (MARS). These devices have a number of advantages. Cantilever calibration using this device is straightforward and rapid. The devices have very small inertia, and are therefore resistant to shock and vibration. This means they need no careful treatment except reasonably clean laboratory conditions. The array spans the range of spring constant from around 0.16 to 11 N/m important in AFM, allowing almost all contact-mode AFM cantilevers to be calibrated easily and rapidly. Each device incorporates its own discrete gold mirror to improve reflectivity. The incorporation of a gold mirror both simplifies calibration of the devices themselves (via Doppler velocimetry) and allows interferometric calibration of the AFM z-axis using the apparent periodicity in the force-distance curve before contact. Therefore, from a single force-distance curve, taking about one second to acquire, one can calibrate the cantilever spring constant and, optionally, the z-axis scale. These are all the data one needs to make accurate and reliable force measurements. |
| 1996 | Atomic force microscopy proposes a novel model for stem-loop structure that binds a heat shock protein in the Staphylococcus aureus HSP70 operon. | Biochem Biophys Res Commun | The Staphylococcus aureus HSP70 operon produces a polycistronic RNA in response to heat shock, and ORF37 is the first protein to be translated. The promoter of this operon contains a palindromic nucleotide sequence that may form a stem-loop structure. Structural analysis of the promoter regions by atomic force microscopy (AFM) revealed a quadruplet that consists of a pair of stem-loops. A novel "SL2S' (Stem-Loop-Loop-Stem) model was proposed for this structure. AFM also revealed the binding of ORF37 to the quadruplet, establishing a molecular mechanism for this heat shock gene expression; ORF37 acts as a regulator by binding to the SL2S structure in the promoter. |
| 1991 | Doppler and echocardiographic characteristics of patients having an Austin Flint murmur. | Circulation | The purpose of this study was to investigate the genesis of the Austin Flint murmur using Doppler and echocardiographic imaging.A total of 51 patients having significant aortic insufficiency and an anatomically normal mitral valve were evaluated. They were divided into two groups; 30 patients had an audible Austin Flint murmur (AFM+) and 21 did not (AFM-). All patients had a complete M-mode, two-dimensional, and Doppler echocardiographic examination to characterize left ventricular size and function, motion of the mitral valve, transmitral flow velocities, direction of the aortic insufficiency jet, and severity of aortic insufficiency. There was no significant difference in severity of aortic insufficiency between groups. There was, however, a significant difference in direction of the insufficiency jet. In the AFM+ group compared with the AFM- group, for the parasternal long-axis view 24 (80%) versus eight (38%) had their insufficiency jet directed at the mitral valve, for the apical five-chamber view the values were 25 (83%) versus five (24%), and for the apical long-axis view the values were 27 (90%) versus five (24%); for all comparisons p less than 0.01. There was also a greater frequency of localized anterior mitral leaflet distortion by two-dimensional echocardiography (AFM+:23 [77%] versus AFM-:five [24%]; p less than 0.001) and a greater frequency of Doppler striations overlying the aortic insufficiency jet (AFM+:25 [83%] versus AFM-:seven [33%]; p less than 0.001). Regarding transmitral flow velocities, there was no significant difference in filling patterns or absolute velocities during early or late diastole between groups. There was no gradient by Doppler analysis or by hemodynamics (n = 26) across the mitral valve in either group. There also was no difference in the frequency of preclosure of the mitral valve (AFM+:two versus AFM-:three). Systolic function was similar in both groups, but the left ventricular end-diastolic dimension was significantly greater in the AFM+ group (6.8 +/- 0.8 cm) than in the AFM- group (6.2 +/- 0.7 cm, p = 0.008).The results of this study suggest that the primary factor responsible for the Austin Flint murmur is the presence of an aortic insufficiency jet directed at the anterior mitral leaflet. This, combined with the biphasic pattern of transmitral flow, distorts the shape of the anterior mitral leaflet as it opens and closes during diastole, making it shudder. The leaflet's shuddering sets up vibrations and shock waves that distort the aortic insufficiency jet, causing the observed Doppler striations and probably the sound of the murmur. There is no evidence from this study to support prior theories that have proposed functional mitral stenosis or diastolic mitral regurgitation as the source of the murmur. |