High-throughput mechanobiology: Force modulation of ensemble biochemical and cell-based assays

Mechanobiology is focused on how the physical forces and mechanical properties of proteins, cells, and tissues contribute to physiology and disease. Although the response of proteins and cells to mechanical stimuli is critical for function, the tools to probe these activities are typically restricted to single-molecule manipulations. Here, we have developed a novel microplate reader assay to encompass mechanical measurements with ensemble biochemical and cellular assays, using a microplate lid modified with magnets. This configuration enables multiple static magnetic tweezers to function simultaneously across the microplate, thereby greatly increasing throughput. We demonstrate the broad applicability and versatility through in vitro and in cellulo approaches. Overall, our methodology allows, for the first time (to our knowledge), ensemble biochemical and cell-based assays to be performed under force in high-throughput format. This approach substantially increases the availability of mechanobiology measurements

Atomic Force Microscopy micro-rheology reveals large structural inhomogeneities in single cell-nuclei

During growth, differentiation and migration of cells, the nucleus changes size and shape, while encountering forces generated by the cell itself and its environment. Although there is increasing evidence that such mechanical signals are employed to control gene expression, it remains unclear how mechanical forces are transduced through the nucleus. To this end, we have measured the compliance of nuclei by applying oscillatory strains between 1 and 700 Hz to individual nuclei of multiple mammalian cell-lines that were compressed between two plates. The quantitative response varied with more than one order of magnitude and scaled with the size of the nucleus. Surprisingly, the qualitative behaviour was conserved among different cell-lines: all nuclei showed a softer and more viscous response towards the periphery, suggesting a reduced degree of crosslinking of the chromatin. This may be an important feature to regulate transcription via mechano-transduction in this most active and dynamic region of the nucleus

Establishment of cell-based transposon-mediated transgenesis in cattle

Transposon-mediated transgenesis is a well-established tool for genome modification in small animal models. However, translation of this active transgenic method to large animals warrants further investigations. Here, the piggyBac (PB) and sleeping beauty (SB) transposon systems were assessed for stable gene transfer into the cattle genome. Bovine fibroblasts were transfected either with a helper-independent PB system or a binary SB system. Both transposons were highly active in bovine cells increasing the efficiency of DNA integration up to 88 times over basal nonfacilitated integrations in a colony formation assay. SB transposase catalyzed multiplex transgene integrations in fibroblast cells transfected with the helper vector and two donor vectors carrying different transgenes (fluorophore and neomycin resistance). Stably transfected fibroblasts were used for SCNT and on in vitro embryo culture, morphologically normal blastocysts that expressed the fluorophore were obtained with both transposon systems. The data indicate that transpositionis a feasible approach for genetic engineering in the cattle genome.Fil: Alessio, Ana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Fili, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Garrels, Wiebke. Institut für Nutztiergenetik; Alemania. Gottfried Wilhelm Leibniz Universität Hannover; AlemaniaFil: Forcato, Diego Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Olmos Nicotra, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Liaudat, Ana Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Bevacqua, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Savy, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Hiriart, María Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Talluri, Thirumala R.. Institut für Nutztiergenetik; AlemaniaFil: Owens, Jesse B.. University of Hawaii at Manoa; Estados UnidosFil: Ivics, Zoltán. Paul-Ehrlich-Institute; AlemaniaFil: Salamone, Daniel Felipe. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Moisyadi, Stefan. University of Hawaii at Manoa; Estados UnidosFil: Kues, Wilfried A.. Institut für Nutztiergenetik; AlemaniaFil: Bosch, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentin

Acute ST-segment elevation myocardial infarction after amoxycillin-induced anaphylactic shock in a young adult with normal coronary arteries: a case report

BACKGROUND: Acute myocardial infarction (MI) following anaphylaxis is rare, especially in subjects with normal coronary arteries. The exact pathogenetic mechanism of MI in anaphylaxis remains unclear. CASE PRESENTATION: The case of a 32-year-old asthmatic male with systemic anaphylaxis, due to oral intake of 500 mg amoxycillin, complicated by acute ST-elevation MI is the subject of this report. Following admission to the local Health Center and almost simultaneously with the second dose of subcutaneous epinephrine (0.2 mg), the patient developed acute myocardial injury. Coronary arteriography, performed before discharge, showed no evidence of obstructive coronary artery disease. In vivo allergological evaluation disclosed strong sensitivity to amoxycillin and the minor (allergenic) determinants of penicillin. CONCLUSION: Acute ST-elevation MI is a rare but potential complication of anaphylactic reactions, even in young adults with normal coronary arteries. Coronary artery spasm appears to be the main causative mechanism of MI in the setting of "cardiac anaphylaxis". However, on top of the vasoactive reaction, a thrombotic occlusion, induced by mast cell-derived mediators and facilitated by prolonged hypotension, cannot be excluded as a possible contributory factor

Force spectroscopy in studying infection

Biophysical force spectroscopy tools - for example optical tweezers, magnetic tweezers, atomic force microscopy, - have been used to study elastic, mechanical, conformational and dynamic properties of single biological specimens from single proteins to whole cells to reveal information not accessible by ensemble average methods such as X-ray crystallography, mass spectroscopy, gel electrophoresis and so on. Here we review the application of these tools on a range of infection-related questions from antibody-inhibited protein processivity to virus-cell adhesion. In each case we focus on how the instrumental design tailored to the biological system in question translates into the functionality suitable for that particular study. The unique insights that force spectroscopy has gained to complement knowledge learned through population averaging techniques in interrogating biomolecular details prove to be instrumental in therapeutic innovations such as those in structure-based drug design

Fibrinogen beta variants confer protection against coronary artery disease in a Greek case-control study

<p>Abstract</p> <p>Background</p> <p>Although plasma fibrinogen levels are related to cardiovascular risk, data regarding the role of fibrinogen genetic variation in myocardial infarction (MI) or coronary artery disease (CAD) etiology remain inconsistent. The purpose of the present study was to investigate the effect of <it>fibrinogen A (FGA)</it>, <it>fibrinogen B (FGB) </it>and <it>fibrinogen G (FGG) </it>gene SNPs and haplotypes on susceptibility to CAD in a homogeneous Greek population.</p> <p>Methods</p> <p>We genotyped for rs2070022, rs2070016, rs2070006 in <it>FGA </it>gene, the rs7673587, rs1800789, rs1800790, rs1800788, rs1800787, rs4681 and rs4220 in <it>FGB </it>gene and for the rs1118823, rs1800792 and rs2066865 SNPs in <it>FGG </it>gene applying an arrayed primer extension-based genotyping method (APEX-2) in a sample of CAD patients (n = 305) and controls (n = 305). Logistic regression analysis was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs), before and after adjustment for potential confounders.</p> <p>Results</p> <p>None of the <it>FGA </it>and <it>FGG </it>SNPs and <it>FGA, FGB, FGG </it>and <it>FGA-FGG </it>haplotypes was associated with disease occurrence after adjustment. Nevertheless, rs1800787 and rs1800789 SNPs in <it>FGB </it>gene seem to decrease the risk of CAD, even after adjustment for potential confounders (OR = 0.42, 95%CI: 0.19-0.90, p = 0.026 and OR = 0.44, 95%CI:0.21-0.94, p = 0.039, respectively).</p> <p>Conclusions</p> <p><it>FGA </it>and <it>FGG </it>SNPs as well as <it>FGA, FGB, FGG </it>and <it>FGA-FGG </it>haplotypes do not seem to be important contributors to CAD occurrence in our sample. On the contrary, <it>FGB </it>rs1800787 and rs1800789 SNPs seem to confer protection to disease onset lowering the risk by about 50% in homozygotes for the minor alleles.</p

Onstage and off: The shifting relevance of gender in women’s prisons

uncorrected proofEven though international research on men’s prisons is no longer oblivious to gender, approaches to women’s prisons have tended to be more gender-bound as a whole. Besides having informed a specific reflexive agenda of representation, the angle of gender has presided to most research issues as an analytical overall parti pris: from the gendered nature of prison regimes to the gendered character of prison cultures, socialities and ‘pains of imprisonment’. This more ‘gendercentric’ agenda is however becoming more diversified for theoretical and empirical reasons alike. These involve a recognition of the diversity of women prisoners’ experiences and identities, and an attention to a wider variety of aspects of carceral life. Drawing on field approaches to the Portuguese carceral world spanning three decades, I propose to take this debate further by focusing on contextual shifts in the actual saliency of gender as a category of identity and social life in women’s prisons.(undefined)(undefined)info:eu-repo/semantics/publishedVersio

Phosphoinositide Regulation of Integrin Trafficking Required for Muscle Attachment and Maintenance

Muscles must maintain cell compartmentalization when remodeled during development and use. How spatially restricted adhesions are regulated with muscle remodeling is largely unexplored. We show that the myotubularin (mtm) phosphoinositide phosphatase is required for integrin-mediated myofiber attachments in Drosophila melanogaster, and that mtm-depleted myofibers exhibit hallmarks of human XLMTM myopathy. Depletion of mtm leads to increased integrin turnover at the sarcolemma and an accumulation of integrin with PI(3)P on endosomal-related membrane inclusions, indicating a role for Mtm phosphatase activity in endocytic trafficking. The depletion of Class II, but not Class III, PI3-kinase rescued mtm-dependent defects, identifying an important pathway that regulates integrin recycling. Importantly, similar integrin localization defects found in human XLMTM myofibers signify conserved MTM1 function in muscle membrane trafficking. Our results indicate that regulation of distinct phosphoinositide pools plays a central role in maintaining cell compartmentalization and attachments during muscle remodeling, and they suggest involvement of Class II PI3-kinase in MTM-related disease

Direct Interaction of Endogenous Kv Channels with Syntaxin Enhances Exocytosis by Neuroendocrine Cells

K+ efflux through voltage-gated K+ (Kv) channels can attenuate the release of neurotransmitters, neuropeptides and hormones by hyperpolarizing the membrane potential and attenuating Ca2+ influx. Notably, direct interaction between Kv2.1 channels overexpressed in PC12 cells and syntaxin has recently been shown to facilitate dense core vesicle (DCV)-mediated release. Here, we focus on endogenous Kv2.1 channels and show that disruption of their interaction with native syntaxin after ATP-dependent priming of the vesicles by Kv2.1 syntaxin–binding peptides inhibits Ca2+ -triggered exocytosis of DCVs from cracked PC12 cells in a specific and dose-dependent manner. The inhibition cannot simply be explained by the impairment of the interaction of syntaxin with its SNARE cognates. Thus, direct association between endogenous Kv2.1 and syntaxin enhances exocytosis and in combination with the Kv2.1 inhibitory effect to hyperpolarize the membrane potential, could contribute to the known activity dependence of DCV release in neuroendocrine cells and in dendrites where Kv2.1 commonly expresses and influences release

Urticaria and infections

Urticaria is a group of diseases that share a distinct skin reaction pattern. Triggering of urticaria by infections has been discussed for many years but the exact role and pathogenesis of mast cell activation by infectious processes is unclear. In spontaneous acute urticaria there is no doubt for a causal relationship to infections and all chronic urticaria must have started as acute. Whereas in physical or distinct urticaria subtypes the evidence for infections is sparse, remission of annoying spontaneous chronic urticaria has been reported after successful treatment of persistent infections. Current summarizing available studies that evaluated the course of the chronic urticaria after proven Helicobacter eradication demonstrate a statistically significant benefit compared to untreated patients or Helicobacter-negative controls without urticaria (p < 0.001). Since infections can be easily treated some diagnostic procedures should be included in the routine work-up, especially the search for Helicobacter pylori. This review will update the reader regarding the role of infections in different urticaria subtypes