Elasticity of Biomolecules: probing, pushing and pulling using atomic force microscopy

Wuite, G.J.L. [Promotor]Roos, W.H. [Copromotor

In Vitro-Reconstituted Nucleoids Can Block Mitochondrial DNA Replication and Transcription

SummaryThe mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000–10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A (TFAM). Here, we reconstitute nucleoid-like particles in vitro and demonstrate that small changes in TFAM levels dramatically impact the fraction of DNA molecules available for transcription and DNA replication. Compaction by TFAM is highly cooperative, and at physiological ratios of TFAM to DNA, there are large variations in compaction, from fully compacted nucleoids to naked DNA. In compacted nucleoids, TFAM forms stable protein filaments on DNA that block melting and prevent progression of the replication and transcription machineries. Based on our observations, we suggest that small variations in the TFAM-to-mtDNA ratio may be used to regulate mitochondrial gene transcription and DNA replication

Single-shot two-dimensional full-range optical coherence tomography achieved by dispersion control

We present a full-range Fourier-domain optical coherence tomography (OCT) system that is capable of acquiring two-dimensional images of living tissue in a single shot. By using line illumination of the sample in combination with a two-dimensional imaging spectrometer, 1040 depth scans are performed simultaneously on a sub-millisecond timescale. Furthermore, we demonstrate an easy and flexible real-time single-shot technique for full-range (complex-conjugate cancelled) OCT imaging that is compatible with both two-dimensional as well as ultrahighresolution OCT. By implementing a dispersion imbalance between reference and sample arms of the interferometer, we eliminate the complex-conjugate signal through numerical dispersion compensation, effectively increasing the useful depth range by a factor of two. The system allows us to record 6.7 × 3.2 mm images at 5 μm depth resolution in 0.2 ms. Data postprocessing requires only 4 s. We demonstrate the capability of our system by imaging the anterior chamber of a mouse eye in vitro, as well as human skin in vivo. © 2009 Optical Society of America

Nanocolloidal albumin-IRDye 800CW: a near-infrared fluorescent tracer with optimal retention in the sentinel lymph node

Purpose: At present, the only approved fluorescent tracer for clinical near-infrared fluorescence-guided sentinel node (SN) detection is indocyanine green (ICG), but the use of this tracer is limited due to its poor retention in the SN resulting in the detection of higher tier nodes. We describe the development and characterization of a next-generation fluorescent tracer, nanocolloidal albumin-IRDye 800CW that has optimal properties for clinical SN detection Methods: The fluorescent dye IRDye 800CW was covalently coupled to colloidal human serum albumin (HSA) particles present in the labelling kit Nanocoll in a manner compliant with current Good Manufacturing Practice. Characterization of nanocolloidal albumin-IRDye 800CW included determination of conjugation efficiency, purity, stability and particle size. Quantum yield was determined in serum and compared to that of ICG. For in vivo evaluation a lymphogenic metastatic tumour model in rabbits was used. Fluorescence imaging was performed directly after peritumoral injection of nanocolloidal albumin-IRDye 800CW or the reference ICG/HSA (i.e. ICG mixed with HSA), and was repeated after 24 h, after which fluorescent lymph nodes were excised. Results: Conjugation of IRDye 800CW to nanocolloidal albumin was always about 50% efficient and resulted in a stable and pure product without affecting the particle size of the nanocolloidal albumin. The quantum yield of nanocolloidal albumin-IRDye 800CW was similar to that of ICG. In vivo evaluation revealed noninvasive detection of the SN within 5 min of injection of either nanocolloidal albumin-IRDye 800CW or ICG/HSA. No decrease in the fluorescence signal from SN was observed 24 h after injection of the nanocolloidal albumin-IRDye 800CW, while a strong decrease or complete disappearance of the fluorescence signal was seen 24 h after injection of ICG/HSA. Fluorescence-guided SN biopsy was very easy. Conclusion: Nanocolloidal albumin-IRDye 800CW is a promising fluorescent tracer with optimal kinetic features for SN detection. © The Author(s) 2012

School Rehabilitation Program’s (SRP) Contribution to School Management and Students’ Academic Performance to Beneficiary Schools in Cebu Province, Philippines

In an educational setting, the management and environment are bound to affect the whole learning environment. This study determines the impact of the Ramon Aboitiz Foundation Incorporated School Rehabilitation Program (RAFI-SRP) to beneficiary schools. The Descriptive-correlational design is utilized to establish the level of assessments for school buildings regarding its milieu, environment, management, and students’ academic performance during their occupancy of the RAFI classrooms. In the exploratory data analyses, multivariate principal component analysis (PCA) and regression are used. Results show that RAFI-SRP affects school management. The more new school buildings are built, the better is the performance of the school administrators. Moreover, RAFI-SRP has a modest influence on the academic performance of students among the community beneficiaries as a whole. This result may be owed to the fact that academic performance is multifactorial, such that the interplay of factors may be intrinsic and extrinsic. RAFI-SRP is just one amongst the extrinsic factors to the learners' academic performance to contend with

Imaging and manipulation of single viruses by atomic force microscopy

The recent developments in virus research and the application of functional viral particles in nanotechnology and medicine rely on sophisticated imaging and manipulation techniques at nanometre resolution in liquid, air and vacuum. Atomic force microscopy (AFM) is a tool that combines these requirements and imaging of viruses dates back to the early days of AFM. AFM has a comparable resolution to electron microscopy, but has the advantage that it can be used to image in fluid. This implies that dynamic processes in physiologically relevant environments can be studied and one of the first examples of AFM imaging of viruses was the real-time observation of viral infection of living cells. Improvement of the imaging techniques led the way to image fragile viruses in non-destructive ways, to visualise viral capsomeres in liquid and to image genome uncoating. In addition to imaging, AFM is also used as a tool to manipulate viral nanoparticles. Translation, rotation and nano-dissection of viruses are possible and by performing nanoindentation experiments the viral material properties can be examined. Next to providing the Young's modulus and breaking force of viral shells, such experiments have also elucidated the impact of the genome on the overall viral mechanical properties and the effect of capsid structural modifications. The combination of modelling and AFM experiments finally, yields a deeper insight into their structure, function and behaviour. Here we review the early AFM experiments on viruses, the achievements made since then and the recent advances in imaging and manipulation. © 2010 The Royal Society of Chemistry

Time-of-Flight Measurement of a 355-nm Nd:YAG Laser-Produced Aluminum Plasma

An aluminum target in air was irradiated by a 355-nm Nd:YAG laser with a pulse width of 10 ns and arepetition rate of 10 Hz. The emission spectra of the laser-produced aluminum plasma were investigatedwith varying distances from the target surface. The results show the presence of a strong continuum veryclose to the target surface, but as the plasma evolve in space, the continuum gradually disappears and theemitted spectra are dominated by stronger line emissions. The observed plasma species are the neutraland singly ionized aluminum and their speeds were investigated using an optical time-of-flight measurementtechnique. Results show that the speeds of the plasma species decreases gradually with distance from thetarget surface. Comparison of the computed speeds of the plasma species shows that the singly ionizedspecies have relatively greater kinetic energy than the neutral species

Probing the impact of loading rate on the mechanical properties of viral nanoparticles

The effects of changes in the loading rate during the forced dissociation of single bonds have been studied for a wide variety of interactions. Less is known on the loading rate dependent behaviour of more complex systems that consist of multiple bonds. Here we focus on viral nanoparticles, in particular the protein shell (capsid) that protects the viral genome. As model systems we use the well-studied capsids of the plant virus Cowpea Chlorotic Mottle Virus (CCMV) and of the bacteriophages φ29 and HK97. By applying an atomic force microscopy (AFM) nanoindentation approach we study the loading rate dependency of their mechanical properties. Our AFM results show very diverse behaviour for the different systems. In particular, we find that not only the breaking force, but also the spring constant of some capsids depend on the loading rate. We describe and compare the measured data with simulation results from the literature. The unexpected complex loading rate dependencies that we report present a challenge for the current theoretical considerations aimed at understanding the molecular level interactions of highly ordered protein assemblies. © 2012 Elsevier Ltd

In Vitro-Reconstituted Nucleoids Can Block Mitochondrial DNA Replication and Transcription

The mechanisms regulating the number of active copies of mtDNA are still unclear. A mammalian cell typically contains 1,000-10,000 copies of mtDNA, which are packaged into nucleoprotein complexes termed nucleoids. The main protein component of these structures is mitochondrial transcription factor A (TFAM). Here, we reconstitute nucleoid-like particles in vitro and demonstrate that small changes in TFAM levels dramatically impact the fraction of DNA molecules available for transcription and DNA replication. Compaction by TFAM is highly cooperative, andat physiological ratios of TFAM to DNA, there are large variations in compaction, from fully compacted nucleoids to naked DNA. In compacted nucleoids, TFAM forms stable protein filaments on DNA that block melting and prevent progression of the replication and transcription machineries. Based on our observations, we suggest that small variations in the TFAM-to-mtDNA ratio may be used to regulate mitochondrial gene transcription and DNA replication. © 2014 The Authors