Biology subjects

Suhling, K.

Publications and source records attributed to Suhling, K..

5 recordsLinked to original sources

bioRxiv2024

Fluorescence Anisotropy Analysis of the Interaction between Doxorubicin and DNA Origami Nanostructures

Owing to doxorubicins high DNA binding affinity, doxorubicin-loaded DNA origami nanostructures (DOX-DONs) are promising nanocarriers against cancer. However, understanding the interactions between doxorubicin (DOX) and DNA origami nanostructures (DONs) is important to ensure the quality of DOX-DONs. This interaction is often taken for granted and the influence of DOX loading conditions is poorly characterized. Exploiting the inherent fluorescence of DOX, steady-state and time-resolved fluorescence anisotropy spectroscopy techniques are used for characterizing non-destructively the binding between DOX and DONs, and the purity of formed complexes. The difference in fluorescence anisotropy between free DOX and DOX-DONs confirms the DOX-DON complex formation. Further, at loading ratios of DOX to DNA base pairs > 0.5, homo-Forster resonance energy transfer (homo-FRET) between closely packed DOX molecules is observed. Moreover, time-resolved anisotropy reveals DOX aggregation on DONs at high loading ratios > 1. For loading ratios > 0.1, spin-filtration to remove excess free DOX is efficient and necessary, though at loading ratios > 1 some DOX aggregates remain attached to the DONs. In summary, fluorescence anisotropy analysis provides more detailed information and insight into DOX-DONs compared to regularly used fluorescence intensity-based characterization methods, and these results can help designing more efficient and safer DNA intercalator-based nanocarriers.

biochemistry↗

bioRxiv2024

A novel dual-excitation pulse-amplitude-modulation fluorometer for investigating photosynthesis of plants

A modular instrument was developed to measure the fluorescence yield in plants subject to a combination of two harmonically-oscillating blue lights with independently controlled frequencies and phases.It uses the pulse-amplitude-modulation (PAM) method to measure the fluorescence yield independently of the plant irradiance. Compared to existing commercial instruments, it uses a higher measuring frequency ({approx} 60 kHz) and higher measuring flash irradiance. This enables averaging over a number of subsequent measurement data points to achieve a higher signal-to-noise ratio. The manuscript describes the design, testing, and characterization of the operational limits of the instrument. It identifies its current weaknesses and makes recommendations for improvements. It is accompanied by supplementary materials containing the electronic schematics and the source code. The instrument was used to study a plant response to a mixture of two oscillating lights. It resulted in an excellent signal-to-noise ratio of the measured fluorescent yield. The measurements clearly demonstrated that the fluorescence yield of a plant subject to a combination of two harmonically-oscillating lights is not the same as the sum of the responses to the two oscillating lights applied independently. The observed non-linearity leads to the important conclusion that the time- and frequency-domain cannot be connected by a Fourier transform. Therefore, the frequency-domain approach will yield novel information that is not redundant to the well-established time-domain measurements.

plant biology↗

bioRxiv2023

Age-specific and compartment-dependent changes in mitochondrial homeostasis and cytoplasmic viscosity in mouse peripheral neurons

Mitochondria are dynamic bioenergetic hubs that become compromised with age. In neurons, declining mitochondrial axonal transport has been associated with reduced cellular health. However, it is still unclear to what extent the decline of mitochondrial transport and function observed during ageing are coupled, and if somal and axonal mitochondria display compartment-specific features that make them more susceptible to the ageing process. It is also not known whether the biophysical state of the cytoplasm, thought to affect many cellular functions, changes with age to impact mitochondrial trafficking and homeostasis. Focusing on the mouse peripheral nervous system, we show that age-dependent decline in mitochondrial trafficking is accompanied by reduction of mitochondrial membrane potential and intra-mitochondrial viscosity, but not calcium buffering, in both somal and axonal mitochondria. Intriguingly, we observe a specific increase in cytoplasmic viscosity in the neuronal cell body, where mitochondria are most polarised, which correlates with decreased cytoplasmic diffusiveness. Increasing cytoplasmic crowding in the somatic compartment of DRG neurons grown in microfluidic chambers reduces mitochondrial axonal trafficking, suggesting a mechanistic link between the regulation of cytoplasmic viscosity and mitochondrial dynamics. Our work provides a reference for studying the relationship between neuronal mitochondrial homeostasis and the viscoelasticity of the cytoplasm in a compartment-dependent manner during ageing.

neuroscience↗

bioRxiv2020

Cytoplasm Biophysical Properties Madolute Microtubule Dynamics

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions in vivo remain poorly understood. Here, we probed the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that both the rates of microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, while a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules in vitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of cytoplasm modulates the reactions underlying microtubule dynamic behaviors.

cell biology↗

bioRxiv2020

Bottom-Illuminated Orbital Shaker for Microalgae Cultivation

A bottom-illuminated orbital shaker designed for the cultivation of microalgae suspensions is described in this open-source hardware report. The instrument agitates and illuminates microalgae suspensions grown inside flasks. It was optimized for low production cost, simplicity, low power consumption, design flexibility, consistent, and controllable growth light intensity. The illuminated orbital shaker is especially well suited for low-resource research laboratories and education. It is an alternative to commercial instruments for microalgae cultivation. It improves on typical do-it-yourself microalgae growth systems by offering consistent and well characterized illumination light intensity. The illuminated growth area is 20 cm x 15 cm, which is suitable for three T75 tissue culture flasks or six 100 ml Erlenmeyer flasks. The photosynthetic photon flux density, is variable in eight steps (26 - 800 mol {middle dot} m-2 {middle dot} s-1) and programmable in a 24-hour light/dark cycle. The agitation speed is variable (0 - 210 RPM). The overall material cost is around {pound}300, including an entry-level orbital shaker. The build takes two days, requiring electronics and mechanical assembly capabilities. The instrument build is documented in a set of open-source protocols, design files, and source code. The design can be readily modified, scaled, and adapted for other orbital shakers and specific experimental requirements. The instrument function was validated by growing fresh-water microalgae Desmodesmus quadricauda and Chlorella vulgaris. The cultivation protocols, microalgae growth curves, and doubling times are included in this report. Specifications table O_TBL View this table: org.highwire.dtl.DTLVardef@23c347org.highwire.dtl.DTLVardef@1e96f84org.highwire.dtl.DTLVardef@985cb5org.highwire.dtl.DTLVardef@ee9a76org.highwire.dtl.DTLVardef@dee9ab_HPS_FORMAT_FIGEXP M_TBL C_TBL

cell biology↗