The study of atmospheric ice-nucleating particles via microfluidically generated droplets

Ice-nucleating particles (INPs) play a significant role in the climate and hydrological cycle by triggering ice formation in supercooled clouds, thereby causing precipitation and affecting cloud lifetimes and their radiative properties. However, despite their importance, INP often comprise only 1 in 10³–10⁶ ambient particles, making it difficult to ascertain and predict their type, source, and concentration. The typical techniques for quantifying INP concentrations tend to be highly labour-intensive, suffer from poor time resolution, or are limited in sensitivity to low concentrations. Here, we present the application of microfluidic devices to the study of atmospheric INPs via the simple and rapid production of monodisperse droplets and their subsequent freezing on a cold stage. This device offers the potential for the testing of INP concentrations in aqueous samples with high sensitivity and high counting statistics. Various INPs were tested for validation of the platform, including mineral dust and biological species, with results compared to literature values. We also describe a methodology for sampling atmospheric aerosol in a manner that minimises sampling biases and which is compatible with the microfluidic device. We present results for INP concentrations in air sampled during two field campaigns: (1) from a rural location in the UK and (2) during the UK’s annual Bonfire Night festival. These initial results will provide a route for deployment of the microfluidic platform for the study and quantification of INPs in upcoming field campaigns around the globe, while providing a benchmark for future lab-on-a-chip-based INP studies

Immersion freezing of ice nucleation active protein complexes

Utilising the Leipzig Aerosol Cloud Interaction Simulator (LACIS), the immersion freezing behaviour of droplet ensembles containing monodisperse particles, generated from a Snomax™ solution/suspension, was investigated. Thereto ice fractions were measured in the temperature range between −5 °C to −38 °C. Snomax™ is an industrial product applied for artificial snow production and contains <i>Pseudomonas</i> syringae} bacteria which have long been used as model organism for atmospheric relevant ice nucleation active (INA) bacteria. The ice nucleation activity of such bacteria is controlled by INA protein complexes in their outer membrane. <br><br> In our experiments, ice fractions increased steeply in the temperature range from about −6 °C to about −10 °C and then levelled off at ice fractions smaller than one. The plateau implies that not all examined droplets contained an INA protein complex. Assuming the INA protein complexes to be Poisson distributed over the investigated droplet populations, we developed the CHESS model (stoCHastic modEl of similar and poiSSon distributed ice nuclei) which allows for the calculation of ice fractions as function of temperature and time for a given nucleation rate. Matching calculated and measured ice fractions, we determined and parameterised the nucleation rate of INA protein complexes exhibiting class III ice nucleation behaviour. Utilising the CHESS model, together with the determined nucleation rate, we compared predictions from the model to experimental data from the literature and found good agreement. <br><br> We found that (a) the heterogeneous ice nucleation rate expression quantifying the ice nucleation behaviour of the INA protein complex is capable of describing the ice nucleation behaviour observed in various experiments for both, Snomax™ and <i>P. syringae</i> bacteria, (b) the ice nucleation rate, and its temperature dependence, seem to be very similar regardless of whether the INA protein complexes inducing ice nucleation are attached to the outer membrane of intact bacteria or membrane fragments, (c) the temperature range in which heterogeneous droplet freezing occurs, and the fraction of droplets being able to freeze, both depend on the actual number of INA protein complexes present in the droplet ensemble, and (d) possible artifacts suspected to occur in connection with the drop freezing method, i.e., the method frequently used by biologist for quantifying ice nucleation behaviour, are of minor importance, at least for substances such as <i>P. syringae</i>, which induce freezing at comparably high temperatures. The last statement implies that for single ice nucleation entities such as INA protein complexes, it is the number of entities present in the droplet population, and the entities' nucleation rate, which control the freezing behaviour of the droplet population. Quantities such as ice active surface site density are not suitable in this context. <br><br> The results obtained in this study allow a different perspective on the quantification of the immersion freezing behaviour of bacterial ice nucleation

Immersion freezing of ice nucleation active protein complexes

Utilising the Leipzig Aerosol Cloud Interaction Simulator (LACIS), the immersion freezing behaviour of droplet ensembles containing monodisperse particles, generated from a Snomax™ solution/suspension, was investigated. Thereto ice fractions were measured in the temperature range between −5 °C to −38 °C. Snomax™ is an industrial product applied for artificial snow production and contains <i>Pseudomonas</i> syringae} bacteria which have long been used as model organism for atmospheric relevant ice nucleation active (INA) bacteria. The ice nucleation activity of such bacteria is controlled by INA protein complexes in their outer membrane. <br><br> In our experiments, ice fractions increased steeply in the temperature range from about −6 °C to about −10 °C and then levelled off at ice fractions smaller than one. The plateau implies that not all examined droplets contained an INA protein complex. Assuming the INA protein complexes to be Poisson distributed over the investigated droplet populations, we developed the CHESS model (stoCHastic modEl of similar and poiSSon distributed ice nuclei) which allows for the calculation of ice fractions as function of temperature and time for a given nucleation rate. Matching calculated and measured ice fractions, we determined and parameterised the nucleation rate of INA protein complexes exhibiting class III ice nucleation behaviour. Utilising the CHESS model, together with the determined nucleation rate, we compared predictions from the model to experimental data from the literature and found good agreement. <br><br> We found that (a) the heterogeneous ice nucleation rate expression quantifying the ice nucleation behaviour of the INA protein complex is capable of describing the ice nucleation behaviour observed in various experiments for both, Snomax™ and <i>P. syringae</i> bacteria, (b) the ice nucleation rate, and its temperature dependence, seem to be very similar regardless of whether the INA protein complexes inducing ice nucleation are attached to the outer membrane of intact bacteria or membrane fragments, (c) the temperature range in which heterogeneous droplet freezing occurs, and the fraction of droplets being able to freeze, both depend on the actual number of INA protein complexes present in the droplet ensemble, and (d) possible artifacts suspected to occur in connection with the drop freezing method, i.e., the method frequently used by biologist for quantifying ice nucleation behaviour, are of minor importance, at least for substances such as <i>P. syringae</i>, which induce freezing at comparably high temperatures. The last statement implies that for single ice nucleation entities such as INA protein complexes, it is the number of entities present in the droplet population, and the entities' nucleation rate, which control the freezing behaviour of the droplet population. Quantities such as ice active surface site density are not suitable in this context. <br><br> The results obtained in this study allow a different perspective on the quantification of the immersion freezing behaviour of bacterial ice nucleation

Effects of Ice Nucleation Protein Repeat Number and Oligomerization Level on Ice Nucleation Activity

Ice nucleation active bacteria have attracted particular attention due to their unique ability to produce specific ice nucleation proteins (INpros), which are the most efficient ice nuclei known as they induce nucleation at temperatures close to 0°C. Our model bacterium Pseudomonas syringae strain R10.79 produced INpros containing 67 tandem repeats, forming the proposed ice-binding surface. To understand the role of the INpro repeats as well as the role of intermolecular interactions between INpros for their ice nucleation behavior, we produced a truncated version of the protein with only 16 tandem repeats (INpro16R). The purified INpro16R produced oligomers of varying sizes. Immersion freezing ice nucleation behavior of purified INpro16R was characterized by droplet-freezing assays and in the Leipzig Aerosol Cloud Interaction Simulator. Predominant INpro16R oligomers introduced into Leipzig Aerosol Cloud Interaction Simulator as single particles with diameters of 50 nm or 70 nm were ice nucleation active at temperatures of -26°C and -24°C, respectively. These are much lower temperatures compared to that of intact INpros (-12°C). The data clearly indicated that the number of repeats determines the ice nucleation temperature. In addition, ice nucleation between -9°C and -10°C, comparable to the activity of intact INpro, was caused by higher-order INpro16R oligomers. This supported previous observations that INpro oligomerization increases the ice-binding surface, thereby affecting ice nucleation activity. In conclusion, both repeat number and oligomerization contribute in a seemingly independent manner to the nucleation mechanism of INpros

Association of the ACE rs4646994 and rs4341 polymorphisms with the progression of carotid atherosclerosis in slovenian patients with type 2 diabetes mellitus

The current study was designed to reveal possible associations between the angiotensin-converting-enzyme (ACE) gene polymorphisms (rs4646994 and rs4341) with markers of carotid atherosclerosis in patients with type 2 diabetes mellitus (T2DM) in a 4-year-long follow-up study. Five hundred and ninety-five T2DM subjects and 200 control subjects were enrolled. Genotyping of ACE polymorphisms was performed using KASPar assays, and ultrasound examinations were performed twice (at the enrollment and at follow-up). With regard to the progression of atherosclerosis in subjects with T2DM, statistically significant differences were demonstrated in the change of the sum of carotid plaques thickness for the rs4646994 polymorphism. We did not demonstrate an association between the tested polymorphisms (rs4646994 and rs4341) and either carotid intima media thickness (CIMT) or CIMT progression in a 3.8-year period. In our study, we demonstrated that subjects with T2DM with the DD genotype of the rs4646994 [ACE insertion/deletion (I/D)] polymorphism had faster progression of atherosclerosis in comparison to subjects with other genotypes

Temperature-controlled airflow ventilation in operating rooms compared with laminar airflow and turbulent mixed airflow

Aim: To evaluate three types of ventilation systems for operating rooms with respect to air cleanliness [in colony-forming units (cfu/m3)], energy consumption and comfort of working environment (noise and draught) as reported by surgical team members. Methods: Two commonly used ventilation systems, vertical laminar airflow (LAF) and turbulent mixed airflow (TMA), were compared with a newly developed ventilation technique, temperature-controlled airflow (TcAF). The cfu concentrations were measured at three locations in an operating room during 45 orthopaedic procedures: close to the wound (<40cm), at the instrument table and peripherally in the room. The operating team evaluated the comfort of the working environment by answering a questionnaire. Findings: LAF and TcAF, but not TMA, resulted in less than 10cfu/m3 at all measurement locations in the room during surgery. Median values of cfu/m3 close to the wound (250 samples) were 0 for LAF, 1 for TcAF and 10 for TMA. Peripherally in the room, the cfu concentrations were lowest for TcAF. The cfu concentrations did not scale proportionally with airflow rates. Compared with LAF, the power consumption of TcAF was 28% lower and there was significantly less disturbance from noise and draught. Conclusion: TcAF and LAF remove bacteria more efficiently from the air than TMA, especially close to the wound and at the instrument table. Like LAF, the new TcAF ventilation system maintained very low levels of cfu in the air, but TcAF used substantially less energy and provided a more comfortable working environment than LAF. This enables energy savings with preserved air quality