Exploration of Materials for Three-Dimensional NMR Microcoil Production via CNC Micromilling and Laser Etching.

The excellent versatility of 5-axis computer numerical control (CNC) micromilling has led to its application for prototyping NMR microcoils tailored to mass-limited samples (reducing development time and cost). However, vibrations during 5-axis milling can hinder the creation of complex 3D volume microcoils (i.e., solenoids and saddle coils). To address these limitations, a high-resolution NSCNC ELARA 4-axis milling machine was developed with the extra precision required for making complex 3D volume microcoils. Upon investigating the performance of resonators made with various copper-coated dielectrics, resonators with poly(methyl methacrylate) (PMMA) provided the best SNR/line shape. Thus, complex 1.7 mm microcoil designs were machined from Cu-coated PMMA. A milled 6.4 mm solenoid also provided 6.6× the total carbon signal for a 13C-labeled broccoli seed compared to a commercial inverse 5 mm NMR probe (demonstrating potential for larger coil designs). However, the manufacture of coils <1.7 mm with copper-coated PMMA rods was challenging as ∼0.5 mm of remaining PMMA was needed to retain their structural integrity. To manufacture smaller microcoils, both a solenoid and saddle coil (both with 1 mm O.D., 0.1 mm thick walls) were etched from Cu-coated glass capillaries using a UV picosecond laser that was mounted onto an NSCNC 5-axis MiRA7L. Both resonators showed excellent signal and identified a wide range of metabolites in a 13C-labeled algae extract, while the solenoid was further tested on two copepod egg sacs (∼4 µg of total sample). In summary, the flexibility to prototype complex microcoils in-house allows laboratories to tailor microcoils to specific mass-limited samples while avoiding the costs of cleanrooms.

Integrated Digital Microfluidics NMR Spectroscopy: A Key Step toward Automated In Vivo Metabolomics.

Toxicity testing is currently undergoing a paradigm shift from examining apical end points such as death, to monitoring sub-lethal toxicity in vivo. In vivo nuclear magnetic resonance (NMR) spectroscopy is a key platform in this endeavor. A proof-of-principle study is presented which directly interfaces NMR with digital microfluidics (DMF). DMF is a "lab on a chip" method allowing for the movement, mixing, splitting, and dispensing of µL-sized droplets. The goal is for DMF to supply oxygenated water to keep the organisms alive while NMR detects metabolomic changes. Here, both vertical and horizontal NMR coil configurations are compared. While a horizontal configuration is ideal for DMF, NMR performance was found to be sub-par and instead, a vertical-optimized single-sided stripline showed most promise. In this configuration, three organisms were monitored in vivo using 1H-13C 2D NMR. Without support from DMF droplet exchange, the organisms quickly showed signs of anoxic stress; however, with droplet exchange, this was completely suppressed. The results demonstrate that DMF can be used to maintain living organisms and holds potential for automated exposures in future. However, due to numerous limitations of vertically orientated DMF, along with space limitations in standard bore NMR spectrometers, we recommend future development be performed using a horizontal (MRI style) magnet which would eliminate practically all the drawbacks identified here.

Development of Low-Magnetic Susceptibility Microcoils via 5-Axis Machining for Analysis of Biological and Environmental Samples.

In environmental research, it is critical to understand how toxins impact invertebrate eggs and egg banks, which, due to their tiny size, are very challenging to study by conventional nuclear magnetic resonance (NMR) spectroscopy. Microcoil technology has been extensively utilized to enhance the mass-sensitivity of NMR. In a previous study, 5-axis computer numerical control (CNC) micromilling (shown to be a viable alternative to traditional microcoil production methods) was used to create a prototype copper slotted-tube resonator (STR). Despite the excellent limit of detection (LOD) of the resonator, the quality of the line shape was very poor due to the magnetic susceptibility of the copper resonator itself. This is best solved using magnetic susceptibility-matched materials. In this study, approaches are investigated that improve the susceptibility while retaining the versatility of coil milling. One method involves machining STRs from various copper/aluminum alloys, while the other involves machining ones from an aluminum 2011 alloy and electroplating them with copper. In all cases, combining copper and aluminum to produce resonators resulted in improved line shape and SNR compared to pure copper resonators due to their reduced magnetic susceptibility. However, the copper-plated aluminum resonators showed optimal performance from the devices tested. The enhanced LOD of these STRs allowed for the first 1H-13C heteronuclear multiple quantum coherence (HMQC) of a single intact 13C-labeled Daphnia magna egg (∼4 µg total biomass). This is a key step toward future screening programs that aim to elucidate the toxic processes in aquatic eggs.

Evaluation of double-tuned single-sided planar microcoils for the analysis of small 13 C enriched biological samples using 1 H-13 C 2D heteronuclear correlation NMR spectroscopy.

Microcoils provide a cost-effective approach to improve detection limits for mass-limited samples. Single-sided planar microcoils are advantageous in comparison to volume coils, in that the sample can simply be placed on top. However, the considerable drawback is that the RF field that is produced by the coil decreases with distance from the coil surface, which potentially limits more complex multi-pulse NMR pulse sequences. Unfortunately, 1 H NMR alone is not very informative for intact biological samples due to line broadening caused by magnetic susceptibility distortions, and 1 H-13 C 2D NMR correlations are required to provide the additional spectral dispersion for metabolic assignments in vivo or in situ. To our knowledge, double-tuned single-sided microcoils have not been applied for the 2D 1 H-13 C analysis of intact 13 C enriched biological samples. Questions include the following: Can 1 H-13 C 2D NMR be performed on single-sided planar microcoils? If so, do they still hold sensitivity advantages over conventional 5 mm NMR technology for mass limited samples? Here, 2D 1 H-13 C HSQC, HMQC, and HETCOR variants were compared and then applied to 13 C enriched broccoli seeds and Daphnia magna (water fleas). Compared to 5 mm NMR probes, the microcoils showed a sixfold improvement in mass sensitivity (albeit only for a small localized region) and allowed for the identification of metabolites in a single intact D. magna for the first time. Single-sided planar microcoils show practical benefit for 1 H-13 C NMR of intact biological samples, if localized information within ~0.7 mm of the 1 mm I.D. planar microcoil surface is of specific interest.

Comprehensive Multiphase NMR Probehead with Reduced Radiofrequency Heating Improves the Analysis of Living Organisms and Heat-Sensitive Samples.

Comprehensive multiphase (CMP) NMR, first described in 2012, combines all of the hardware components necessary to analyze all phases (solid, gel, and solution) in samples in their natural state. In combination with spectral editing experiments, it can fully differentiate phases and study the transfer of chemical species across and between phases, providing unprecedented molecular-level information in unaltered natural systems. However, many natural samples, such as swollen soils, plants, and small organisms, contain water, salts, and ionic compounds, making them electrically lossy and susceptible to RF heating, especially when using high-strength RF fields required to select the solid domains. While dedicated reduced-heating probes have been developed for solid-state NMR, to date, all CMP-NMR probes have been based on solenoid designs, which can lead to problematic sample heating. Here, a new prototype CMP probe was developed, incorporating a loop gap resonator (LGR) for decoupling. Temperature increases are monitored in salt solutions analogous to those in small aquatic organisms and then tested in vivo on Hyalella azteca (freshwater shrimp). In the standard CMP probe (solenoid), 80% of organisms died within 4 h under high-power decoupling, while in the LGR design, all organisms survived the entire test period of 12 h. The LGR design reduced heating by a factor of ∼3, which allowed 100 kHz decoupling to be applied to salty samples with generally ≤10 °C sample heating. In addition to expanding the potential for in vivo research, the ability to apply uncompromised high-power decoupling could be beneficial for multiphase samples containing true crystalline solids that require the strongest possible decoupling fields for optimal detection.

Expanding current applications and permitting the analysis of larger intact samples by means of a 7 mm CMP-NMR probe.

Comprehensive multiphase NMR combines the ability to study and differentiate all phases (solids, gels, and liquids) using a single NMR probe. The general goal of CMP-NMR is to study intact environmental and biological samples to better understand conformation, organization, association, and transfer between and across phases/interfaces that may be lost with conventional sample preparation such as drying or solubilization. To date, all CMP-NMR studies have used 4 mm probes and rotors. Here, a larger 7 mm probehead is introduced which provides ∼3 times the volume and ∼2.4 times the signal over a 4 mm version. This offers two main advantages: (1) the additional biomass reduces experiment time, making 13C detection at natural abundance more feasible; (2) it allows the analysis of larger samples that cannot fit within a 4 mm rotor. Chicken heart tissue and Hyalella azteca (freshwater shrimp) are used to demonstrate that phase-based spectral editing works with 7 mm rotors and that the additional biomass from the larger volumes allows detection with 13C at natural abundance. Additionally, a whole pomegranate seed berry (aril) and an intact softgel capsule of hydroxyzine hydrochloride are used to demonstrate the analysis of samples too large to fit inside a conventional 4 mm CMP probe. The 7 mm version introduced here extends the range of applications and sample types that can be studied and is recommended when 4 mm CMP probes cannot provide adequate signal-to-noise (S/N), or intact samples are simply too big for 4 mm rotors.

5-Axis CNC Micromilling for Rapid, Cheap, and Background-Free NMR Microcoils.

The superior mass sensitivity of microcoil technology in nuclear magnetic resonance (NMR) spectroscopy provides potential for the analysis of extremely small-mass-limited samples such as eggs, cells, and tiny organisms. For optimal performance and efficiency, the size of the microcoil should be tailored to the size of the mass-limited sample of interest, which can be costly as mass-limited samples come in many shapes and sizes. Therefore, rapid and economic microcoil production methods are needed. One method with great potential is 5-axis computer numerical control (CNC) micromilling, commonly used in the jewelry industry. Most CNC milling machines are designed to process larger objects and commonly have a precision of >25 µm (making the machining of common spiral microcoils, for example, impossible). Here, a 5-axis MiRA6 CNC milling machine, specifically designed for the jewelry industry, with a 0.3 µm precision was used to produce working planar microcoils, microstrips, and novel microsensor designs, with some tested on the NMR in less than 24 h after the start of the design process. Sample wells could be built into the microsensor and could be machined at the same time as the sensors themselves, in some cases leaving a sheet of Teflon as thin as 10 µm between the sample and the sensor. This provides the freedom to produce a wide array of designs and demonstrates 5-axis CNC micromilling as a versatile tool for the rapid prototyping of NMR microsensors. This approach allowed the experimental optimization of a prototype microstrip for the analysis of two intact adult Daphnia magna organisms. In addition, a 3D volume slotted-tube resonator was produced that allowed for 2D 1H-13C NMR of D. magna neonates and exhibited 1H sensitivity (nLODω600 = 1.49 nmol s1/2) close to that of double strip lines, which themselves offer the best compromise between concentration and mass sensitivity published to date.

Plants and their uses in dermatological recipes of the Receptarium of Burkhard III von Hallwyl from 16th century Switzerland - Data mining a historical text and preliminary in vitro screening.

ETHNOPHARMACOLOGICAL RELEVANCE: Historical texts on materia medica can be an attractive source of ethnopharmacological information. Various research groups have investigated corresponding resources from Europe and the Mediterranean region, pursuing different objectives. Regardless of the method used, the indexing of textual information and its conversion into data sets useful for further investigations represents a significant challenge. AIM OF THE STUDY: First, this study aims to systematically catalogue pharmaco-botanical information in the Receptarium of Burkhard von Hallwyl (RBH) in order to identify candidate plants in a targeted manner. Secondly, the potential of RBH as a resource for pharmacological investigations will be assessed by means of a preliminary in vitro screening. MATERIALS AND METHODS: We developed a relational database for the systematic recording of parameters composing the medical recipes contained in the historical text. Focusing on dermatological recipes, we explored the mentioned plants and their uses by drawing on specific literature. The botanical identities (candidate species) suggested in the literature for the historical plant names were rated based on their plausibility of being the correct attribution. The historical uses were interpreted by consulting medical-historical and modern clinical literature. For the subsequent in vitro screening, we selected candidate species used in recipes directed at the treatment of inflammatory or infectious skin disorders and wounds. Plants were collected in Switzerland and their hydroethanolic crude extracts tested for possible cytotoxic effects and for their potential to modulate the release of IL-6 and TNF in PS-stimulated whole blood and PBMCs. RESULTS: The historical text analysis points up the challenges associated with the assessment of historical plant names. Often two or more plant species are available as candidates for each of the 161 historical plant names counted in the 200 dermatological recipes in RBH. On the other hand, our method enabled to draw conclusions about the diseases underlying the 56 medical applications mentioned in the text. On this basis, 11 candidate species were selected for in vitro screening, four of which were used in RBH in herbal simple recipes and seven in a herbal compound formulation. None of the extracts tested showed a noteworthy effect on cell viability except for the sample of Sanicula europaea L. Extracts were tested at 50 µg/mL in the whole blood assay, where especially Vincetoxicum hirundinaria Medik. or Solanum nigrum L. showed inhibitory or stimulatory activities. In the PBMC assay, the root of Vincetoxicum hirundinaria revealed a distinct inhibitory effect on IL-6 release (IC50 of 3.6 µg/mL). CONCLUSIONS: Using the example of RBH, this study illustrates a possible ethnopharmacological path from unlocking the historical text and its subsequent analysis, through the selection and collection of plant candidates to their in vitro investigation. Fully documenting our approach to the analysis of historical texts, we hope to contribute to the discussion on solutions for the digital indexing of premodern information on the use of plants or other natural products.

Heat waves, incidence of infectious gastroenteritis, and relapse rates of inflammatory bowel disease: a retrospective controlled observational study.

OBJECTIVES: The objective of this study was to evaluate the effect of heat waves on flares of inflammatory bowel disease (IBD) and infectious gastroenteritis (IG). METHODS: In this retrospective controlled observational study, data from 738 IBD and 786 IG patients admitted to the University Hospital of Zurich in the years 2001-2005, as well as from 506 other noninfectious chronic intestinal inflammations, which were used as control, were collected. Climate data were obtained from the Swiss Federal Office for Meteorology and Climatology. RESULTS: The presence of a heat wave increased the risk of IBD flares by 4.6% (95% confidence interval (CI): 1.6-7.4%, P=0.0035) and of IG flares by 4.7% (95% CI: 1.8-7.4%, P=0.0020) for every additional day within a heat wave period. In the control group there was no significant effect (95% CI: -6.2-2.9%, P=0.53). Screening of alternative forms for the effect of heat waves suggested that for IG the effect is strongest when lagged by 7 days (risk increase per day: 7.2%, 95% CI: 4.6-9.7%, P<0.0001), whereas for IBD no such transformation was required. Other formulations with additive effects, interactions between heat waves and time of the year, and additional adjustments for daily average temperature did not show any improvement in model fit. CONCLUSIONS: In this retrospective controlled observational study, we found a substantial increase in hospital admissions because of flares of IBD and IG during heat wave periods. Whereas the effect on IG is strongest with a delay of 7 days, the effect on IBD flares is immediate, suggesting different mechanisms.

Climate change in Switzerland: Impact on hazel, birch, and grass pollen on the basis of half a century of pollen records (1969 - 2018).

As indicated by the Intergovernmental Panel on Climate Change (IPCC), human activities are estimated to have caused ~ 1.0 °C of global warming above pre-industrial levels. The impact of this global warming is diverse and pertains also plant biology. The start of the pollen season as well as the observed quantities of pollen have been considered indicators of the impact of climate change. Switzerland has one of the longest pollen time series now - more than half a century. It has been tested whether the impact of climate change is robust by checking with this long time series of different pollen like hazel, birch, and grass as prominent representatives of triggers of hay fever. The results indicate that based on a time series of 50 years, the pollen seasons of hazel, birch, and grass started earlier as the temperature increased. Comparing the annual amount of pollen, a relevant increase is only observed for hazel. However, it must be considered that in the observed 50 years there was a land use change from grass land to built-up land due to the increase of population, and yet certain pollen counts increased considerably.

Digital microfluidics and nuclear magnetic resonance spectroscopy for in situ diffusion measurements and reaction monitoring.

In recent years microcoils and related structures have been developed to increase the mass sensitivity of nuclear magnetic resonance spectroscopy, allowing this extremely powerful analytical technique to be extended to small sample volumes (<5 µl). In general, microchannels have been used to deliver the samples of interest to these microcoils; however, these systems tend to have large dead volumes and require more complex fluidic connections. Here, we introduce a two-plate digital microfluidic (DMF) strategy to interface small-volume samples with NMR microcoils. In this system, a planar microcoil is surrounded by a copper plane that serves as the counter-electrode for the digital microfluidic device, allowing for precise control of droplet position and shape. This feature allows for the user-determination of the orientation of droplets relative to the main axes of the shim stack, permitting improved shimming and a more homogeneous magnetic field inside the droplet below the microcoil, which leads to improved spectral lineshape. This, along with high-fidelity droplet actuation, allows for rapid shimming strategies (developed over decades for vertically oriented NMR tubes) to be employed, permitting the determination of reaction-product diffusion coefficients as well as quantitative monitoring of reactive intermediates. We propose that this system paves the way for new and exciting applications for in situ analysis of small samples by NMR spectroscopy.

The Impact of Cold Spells on the Incidence of Infectious Gastroenteritis and Relapse Rates of Inflammatory Bowel Disease: A Retrospective Controlled Observational Study.

GOALS: We aimed to assess the impact of very cold days on inflammatory bowel disease (IBD) flares and infectious gastroenteritis (IG). We defined a cold day using the World Meteorological definition of an ice day, which is a day with a maximum temperature below 0°C. BACKGROUND: Recently, we have shown that heat waves increase the risk for IG and IBD flares. STUDY: We retrospectively collected data from 738 IBD and 786 IG patients admitted to the University Hospital of Zurich between 2001 and 2005 and from 506 patients with other noninfectious chronic intestinal inflammations as controls. Climate data were received by the Swiss Federal Office for Meteorology and Climatology. RESULTS: There was no evidence for an increased risk of IBD flares (relative risk, RR = 0.99, 95% confidence interval, CI: 0.72-1.33, p = 0.94) or IG flares (RR = 1.16, 95% CI: 087-1.52, p = 0.30) on very cold days. This negative finding was confirmed in alternative formulations with lagged or cumulative (possibly lagged) effects. CONCLUSION: In this retrospective controlled observational study, no evidence for an increase in hospital admissions due to flares of IBD and IG during cold days was observed. This may be attributed to not relevantly altered bacterial growth conditions during cold days compared to heat waves.

T Cell Migration from Inflamed Skin to Draining Lymph Nodes Requires Intralymphatic Crawling Supported by ICAM-1/LFA-1 Interactions.

T cells are the most abundant cell type found in afferent lymph, but their migration through lymphatic vessels (LVs) remains poorly understood. Performing intravital microscopy in the murine skin, we imaged T cell migration through afferent LVs in vivo. T cells entered into and actively migrated within lymphatic capillaries but were passively transported in contractile collecting vessels. Intralymphatic T cell number and motility were increased during contact-hypersensitivity-induced inflammation and dependent on ICAM-1/LFA-1 interactions. In vitro, blockade of endothelial cell-expressed ICAM-1 reduced T cell adhesion, crawling, and transmigration across lymphatic endothelium and decreased T cell advancement from capillaries into lymphatic collectors in skin explants. In vivo, T cell migration to draining lymph nodes was significantly reduced upon ICAM-1 or LFA-1 blockade. Our findings indicate that T cell migration through LVs occurs in distinct steps and reveal a key role for ICAM-1/LFA-1 interactions in this process.

Climate change and its impact on birch pollen quantities and the start of the pollen season an example from Switzerland for the period 1969-2006.

As published by the Intergovernmental Panel on Climate Change (IPCC) global warming is a reality and its impact is huge like the increase of extreme weather events, glacier recession, sea level rise and also effects on human health. Among them allergies to airborne pollen might increase or change in pattern due to the invasion of new allergic plants or due to different behavior of plants like earlier flowering. In this study we used the longest Swiss airborne pollen data set to examine the influence of the temperature increase on the time of flowering. In the case of Basel, where pollen data for 38 years are available, it was shown that due to a temperature increase the start of flowering in the case of birch occurred about 15 days earlier. Apart from a shift of the start of the flowering there is also a trend towards higher annual birch pollen quantities and an increase of the highest daily mean pollen concentrations. Due to global warming and because symptoms may appear earlier in the year people suffering from a pollen allergy might face a new unaccustomed situation.

Trends in prevalence of allergic rhinitis and correlation with pollen counts in Switzerland.

In recent decades, a large number of epidemiological studies investigating the change of prevalence of hay fever showed an increase in the occurrence of this disease. However, other studies carried out in the 1990s yielded contradictory results. Many environmental factors have been hypothesized to contribute to the increasing hay fever rate, including both indoor and ambient air pollution, reduced exposure to microbial stimulation and changes in diets. However, the observed increase has not convincingly been explained by any of these factors and there is limited evidence of changes in exposure to these risk factors over time. Additionally, recent studies show that no further increase in asthma, hay fever and atopic sensitisation in adolescents and adults has been observed during the 1990s and the beginning of the new century. As the pattern of pollen counts has changed over the years, partly due to the global warming but also as a consequence of a change in the use of land, the changing prevalence of hay fever might partly be driven by this different pollen exposure. Epidemiological data for hay fever in Switzerland are available from 1926 until 2000 (with large gaps between 1926 and 1958 and 1958 to 1986) whereas pollen data are available from 1969 until the present. This allows an investigation as to whether these data are correlated provided the same time spans are compared. It would also be feasible to correlate the pollen data with meteorological data which, however, is not the subject of our investigation. Our study focuses on analyzing time series of pollen counts and of pollen season lengths in order to identify their trends, and to ascertain whether there is a relationship between these trends and the changes in the hay fever prevalence. It is shown in this paper that the pollen exposure has been decreasing in Basel since the beginning of the 1990s whereas the rate of the hay fever prevalence in Switzerland remained approximately unchanged in this period but with a slight tendency to decrease. In Locarno, most of the pollen species also show a decreasing trend, while in Zurich, the development is somewhat different as the pollen counts of most of the pollen types have been increasing. It is interesting, however, that some of the pollen counts of this station (grass, stinging nettle, mugwort and ragweed) have been decreasing in the period 1982--2007.