Emergency upscaling of video consultation during the COVID-19 pandemic: Contrasting user experience with data insights from the electronic health record in a large academic hospital.

BACKGROUND: Video consultation (VC) has been scaled up at our academic centre attempting to facilitate and accommodate patient-provider interaction in times of social distancing during the recent and ongoing COVID-19 pandemic. OBJECTIVES: This study evaluates qualitative outcomes with data insights from the electronic health record, to contrast satisfaction outcomes with the actual use of VC. METHODS: Healthcare providers and patients using VC during the COVID-19 pandemic at a large academic centre in the Netherlands were surveyed for user satisfaction and experiences with VC. In addition, quantitative technical assessment was performed using data related to VC from the EHR record. RESULTS: In total, 1,027/4,443 patients and 87/166 healthcare providers completed their online questionnaire. Users rated the use of VC during a pandemic with an average score of 8.3/10 and 7.6/10 respectively. Both groups believed the use of VC was a good solution to continue the provision of healthcare during this pandemic. The use of VC increased from 92 in March 2020 to 837 in April 2020. CONCLUSION: This study strongly signals that VC is an important modality in futureproofing outpatient care during and beyond pandemic times. Further development in end-user technology is needed for EHR integrated VC solutions. Guidelines needs to be developed advising both patients and healthcare providers. Such guidelines should not solely focus on technical implementation and troubleshooting, but must also consider important aspects such as digital health literacy, patient and provider authentication, privacy and ethics.

A new high-quality set of singly ((2) H) and doubly ((2) H and (18) O) stable isotope labeled reference waters for biomedical and other isotope-labeled research.

RATIONALE: Research using water with enriched levels of the rare stable isotopes of hydrogen and/or oxygen requires well-characterized enriched reference waters. The International Atomic Energy Agency (IAEA) did have such reference waters available, but these are now exhausted. New reference waters thus had to be produced in sufficient quantity, and higher characterization quality was desired. METHODS: The reference waters have been prepared gravimetrically from three parent waters: natural water, pure (2) H water and highly (18) O-enriched water. These parent waters have been thoroughly assessed for their full isotopic compositions. To ensure the integrity and correctness of the gravimetric procedure, validation measurements have been carried out on the isotopic composition of the produced reference waters by two of our laboratories. These measurements corroborate the values obtained on the basis of gravimetric data. RESULTS: Two new sets of three reference waters enriched in the stable isotopes have been produced and certified: one set of singly labeled waters, only enriched in (2) H, and another set of Doubly Labeled Waters, enriched in both (2) H and (18) O. They cover δ(2) H and δ(18) O values in the range of 800-16000 ‰ and 100-2000 ‰, respectively. The process has led to highly accurate isotopic values for these waters. CONCLUSIONS: These reference waters are now available (called IAEA-604 to IAEA-609). They will be valuable as reference materials for all fields using isotope labeling of water, most prominently, but not exclusively, biomedical research (body composition analyses, metabolic rate measurements). The two waters with the lowest enrichments will also be useful as anchor values for isotope measurements around the natural range.

A thoroughly validated spreadsheet for calculating isotopic abundances (2H, 17O, 18O) for mixtures of waters with different isotopic compositions.

RATIONALE: Oxygen and hydrogen stable isotopes are widely used tracers for studies on naturally occurring and laboratory mixtures of isotopically different waters. Although the mixing calculations are straightforward to perform, there are ample possibilities to make mistakes, especially when dealing with a large number of mixed fluids. To facilitate isotope mixing calculations and to avoid computational mistakes, a flexible tool to carry out these calculations is in demand. METHODS: We developed, in three independent efforts, spreadsheets to carry out the mixing calculations for a combination of waters with different isotopic compositions using the isotope mass balance equation. We validated our calculations by comparison of the results of the three spreadsheets for a large number of test calculations. For all the cases, we obtained identical results down to the 12(th) to 14(th) significant digit. RESULTS: We present a user-friendly, thoroughly validated spreadsheet for calculating (2) H, (17) O and (18) O stable isotopic abundances and respective isotope delta values for mixtures of waters with arbitrary isotopic compositions. The spreadsheet allows the mixing of up to 10 different waters, of which up to five can be specified using their isotopic abundances and up to five others using their isotope delta values. The spreadsheet is implemented in Microsoft Excel and is freely available from our research groups' websites. CONCLUSIONS: The present tool will be applicable in the production and characterization of singly and doubly labeled water (DLW) mother solutions, the analysis of isotope dilution measurements, the deduction of unknown isotope values of constituents for mixtures of natural waters, and many other applications.

Translational repression and eIF4A2 activity are critical for microRNA-mediated gene regulation.

MicroRNAs (miRNAs) control gene expression through both translational repression and degradation of target messenger RNAs (mRNAs). However, the interplay between these processes and the precise molecular mechanisms involved remain unclear. Here, we show that translational inhibition is the primary event required for mRNA degradation. Translational inhibition depends on miRNAs impairing the function of the eIF4F initiation complex. We define the RNA helicase eIF4A2 as the key factor of eIF4F through which miRNAs function. We uncover a correlation between the presence of miRNA target sites in the 3' untranslated region (3'UTR) of mRNAs and secondary structure in the 5'UTR and show that mRNAs with unstructured 5'UTRs are refractory to miRNA repression. These data support a linear model for miRNA-mediated gene regulation in which translational repression via eIF4A2 is required first, followed by mRNA destabilization.

Doubly Labelled Water analysis: preparation, memory correction, calibration and quality assurance for δ2H and δ18O measurements over four orders of magnitudes.

RATIONALE: The Doubly Labelled Water (DLW) method is an established way of determining the metabolic rate in humans and animals, with the advantage that the subjects need not be confined. The method, however, needs accurate determination of both the δ(2)H and the δ(18)O isotope values over a wide range of enrichments. METHODS: In this paper we describe a number of crucial steps in the process of isotope determination in body fluids. These steps include micro-distillation, correction of the measurements for sample-to-sample memory and calibration of the isotope scales over many orders of magnitudes. In contrast to several published protocols and guidelines, we also take highly enriched samples into account, as they are required for studying the metabolic rate of birds and small mammals. For our isotope scale calibration, we made a set of gravimetrically prepared, double labelled waters with known isotope values. Our quality assurance includes a scheme for easy calculation of the error propagation, leading to a reliable estimate of the analytical error in the metabolic rate. RESULTS: Our memory correction algorithm assumes the existence of three water "pools" that have different sizes and exchange rates with the injected samples. We show that the method can correct even huge memory signals, without the need for "true" values. CONCLUSIONS: With the presented building blocks, we show how to assure a reliable and accurate isotope analysis for the DLW method, both for human and for animal applications. Although our measurements have been performed using isotope ratio mass spectrometry, most of the procedures are also useful for laser spectrometry.

Carbon-14 based determination of the biogenic fraction of industrial CO(2) emissions - application and validation.

The (14)C method is a very reliable and sensitive method for industrial plants, emission authorities and emission inventories to verify data estimations of biogenic fractions of CO(2) emissions. The applicability of the method is shown for flue gas CO(2) samples that have been sampled in 1-h intervals at a coal- and wood-fired power plant and a waste incineration plant. Biogenic flue gas CO(2) fractions of 5-10% and 48-50% have been measured at the power plant and the waste incineration plant, respectively. The reliability of the method has been proven by comparison of the power plant results with those based on carbon mass input and output data of the power plant. At industrial plants with relatively low biogenic CO(2) fraction (<10%) the results need to be corrected for sampled (14)CO(2) from atmospheric air.

Melanoma of the rectum: a rare entity.

A 41-year-old man presented with a 6-mo history of changed defecation and rectal bleeding. A 3-cm polypoid tumor of the lower rectum was found at rectosigmoidoscopy, which proved to be a leiomyosarcoma upon biopsy. Dissemination studies did not show any metastases. He was underwent to an abdomino-perineal resection (APR). Histopathology of the specimen showed a melanoma (S-100 stain positive). Two years after the resection, metastases in the abdomen and right lung were found. He died one and half years later. Primary anorectal melanoma is a rare and very aggressive disorder. According to current data, one should always perform a S-100 stain when anorectal sarcoma is suspected. A positive S-100 stain suggests the tumour to be most likely a melanoma. Subsequently, thorough dissemination studies need to be performed. Depending on the outcome of the dissemination studies, a surgical resection has to be performed. Nowadays, a sphincter-saving local excision combined with adjuvant loco-regional radiotherapy should be preferred in case of small tumors. The same loco-regional control is achieved with less "loss of function" compared to non-sphincter saving surgery. Only in the case of large and obstructing tumors an abdomino-perineal resection is the treatment of choice.

Changes in isotope ratios during domestic wastewater production.

This paper presents considerations for the application of the natural water isotope method on catchment areas. For the estimation of the amount of infiltration and inflow in sewer systems the paper shows two applications in the Netherlands: one successful application on a relatively small catchment area with a simple geo-hydrological groundwater system and one unsuccessful application in an area that shows a large heterogeneity of delta 18O values in groundwater. Also, the paper focuses on the validity of the assumption that the isotopic ratios of drinking water are equal to those of strict domestic wastewater. In the transition from drinking water to strict wastewater it is shown that changes in isotopic composition of the water due to evaporation in common household appliances and effects inside the human body are insignificant. However, the presence of high-efficiency condensing boilers in an area can significantly influence the delta 18O value of strict wastewater, especially in winter months. This effect should be taken into account when applying the isotope method in such areas.

Validation of the DLW method in Japanese quail at different water fluxes using laser and IRMS.

In Japanese quail (Coturnix c. japonica; n = 9), the doubly labeled water (DLW) method ((2)H, (18)O) for estimation of CO(2) production (l/day) was validated. To evaluate its sensitivity to water efflux levels (r(H(2))O(e); g/day) and to assumptions of fractional evaporative water loss (x; dimensionless), animals were repeatedly fed a dry pellet diet (average r(H(2))O(e) of 34.8 g/day) or a wet mash diet (95.8 g/day). We simultaneously compared the novel infrared laser spectrometry (LS) with isotope ratio mass spectrometry. At low r(H(2))O(e), calculated CO(2) production rate exhibited little sensitivity to assumptions concerning x, with the best fit being found at 0.51, and only little error was made employing an x value of 0.25. In contrast, at high r(H(2))O(e), sensitivities were much higher with the best fit at x = 0.32. Conclusions derived from isotope ratio mass spectrometry and LS were similar, proving the usefulness of LS. Within a threefold range of r(H(2))O(e), little error in the DLW method is made when assuming one single x value of 0.25 (recommended by Speakman JR, Doubly Labelled Water. Theory and Practice. London: Chapman & Hall, 1997), indicating its robustness in comparative studies.

Determination of the 2H/1H, 17O/16O, and 18O/16O isotope ratios in water by means of tunable diode laser spectroscopy at 1.39 microm.

We demonstrate the feasibility of the accurate and simultaneous measurement of the 2H/1H, 17O/16O, and 18O/16O isotope ratios in water vapor by means of tunable diode laser spectroscopy. The absorptions are due to the v1 + v3 combination band, observed using a room temperature, distributed feedback (DFB) diode laser at 1.39 microm. The precision of the instrument is approximately 3, 1, and 0.5/1000 for the 2H, 17O, and 18O isotope ratios, respectively, and is at present limited by residual optical feedback to the laser. The signal-to-noise, however, is superior to that obtained in a similar experiment using a color center laser at 2.7 microm. Replacing the current laser with a better unit, we are confident that a precision well below 1/1000 is attainable for all three isotope ratios. The diode laser apparatus is ideally suited for applications demanding a reliable, cheap, and/or portable instrument, such as the biomedical doubly labeled water method and atmospheric sensing.

Stable isotope ratio measurements on highly enriched water samples by means of laser spectrometry.

We demonstrate the feasibility of using laser spectrometry (LS) to analyze isotopically highly enriched water samples (i.e., delta2H < or = 15,000/1,000, 18O < or = 1,200/1,000), as often used in the biomedical doubly labeled water (DLW) method to quantify energy metabolism. The method is an important extension of a recently developed infrared laser direct absorption spectrometer. The measurements on highly enriched, small-size (10 microL liquid water) samples show a clearly better accuracy for the 2H/1H ratio. In the case of 18O/16O, the same level of accuracy is obtained as with conventional isotope ratio mass spectrometer (IRMS) analysis. With LS, the precision is better for both 18O/ 16O and 2H/1H. New is the ability to measure 17O/16O with the same accuracy as 18O/16O. A major advantage of the present technique is the absence of chemical sample preparation. The method is proven to be reliable and accurate and is ready to be used in many biomedical applications.

Cloning and analysis of the untranslated regions of the Xenopus laevis Connexin30 mRNA.

The full-length 5' and 3' untranslated regions (UTRs) of Xenopus laevis Connexin30 (Cx30) mRNA were cloned and sequenced. The Cx30 messenger contains a 148 nt 5' UTR and a 480 nt 3' UTR. Four different constructs were made to enable the analysis of the role of the Cx30 UTRs in translation efficiency and in protein localization in the early Xenopus embryo. Transcripts encoded the Green Fluorescent Protein (GFP) reporter and contained the 5' and 3' UTR of either Cx30 or globin. In vivo analyses after injection of the transcripts into one cell-stage Xenopus embryos showed that the Cx30 3' UTR enables very efficient translation. The 5' UTR was slightly inhibitory compared with the globin 5' UTR. The localization of the produced GFP was analyzed. GFP was ubiquitously expressed in all parts of the embryo. Based on this observation we conclude that neither the 5' UTR nor the 3' UTR confers specific localization of the translation of the Cx30 mRNA in the embryo.

Validation of the doubly labeled water method in Japanese Quail Coturnix c. japonica chicks: is there an effect of growth rate?

The Doubly Labeled Water (DLW) method was validated against respiration gas analysis in growing Japanese Quail chicks (between 1 week and 3 weeks of age) as well as in birds after having achieved sexual maturity (7 weeks of age). A comparison was made between a strain selected for high growth rates (P-strain, n = 18), and a non-selected strain (C-strain, n = 18). Relative growth rates of individual chicks during the measurement ranged from -13.8% day(-1) to 23.1% day(-1). When employing a single-pool model (eq. 34, Lifson and McClintock 1966), it was found that the relative error of the DLW method was sensitive to assumptions concerning fractional evaporative water loss. The best fit was obtained after taking a fractional evaporative water loss value of 0.33. When applying this value for all chicks, it was found that neither strain, relative growth rate of the chick during measurement, nor age significantly contributed to the explained variance. When employing two-pool models, it was found that the DLW method significantly underestimated the true rates of CO2 production at all assumed levels of fractional evaporative water loss. Based on an evaluation of DLW validation studies in growing shorebirds, terns, and quail we recommend Speakman's Eq. 7.17 (Speakman 1997) for general use in young birds.

Translational control of the Xenopus laevis connexin-41 5'-untranslated region by three upstream open reading frames.

The Xenopus laevis Connexin-41 (Cx41) mRNA contains three upstream open reading frames (uORFs) in the 5'-untranslated region (UTR). We analyzed the translation efficiency of constructs containing the Cx41 5'-UTR linked to the green fluorescent protein reporter after injection of transcripts into one-cell stage Xenopus embryos. The translational efficiency of the wild-type Cx41 5'-UTR was only 2% compared with that of the beta-globin 5'-UTR. Mutation of each of the three uAUGs into AAG codons enhanced translation 82-, 9-, and 4-fold compared with the wild-type Cx41 5'-UTR. Based on these increased translation efficiencies, the percentages of ribosomes that recognized the uAUGs were calculated. Only 0.03% of the ribosomes that entered at the cap structure scanned the entire 5'-UTR and translated the main ORF. The results indicate that all uAUGs are recognized by the majority of the scanning ribosomes and that the three uAUGs strongly modulate translation efficiency in Xenopus laevis embryos. Based on these data, a model of ribosomal flow along the mRNA is postulated. We conclude that the three uORFs may play an important role in the regulation of Cx41 expression.

Expression of the organizer specific homeobox gene goosecoid (gsc) in porcine embryos.

The homeobox gene goosecoid is one of the first genes expressed in the organizer region of vertebrates and specifies future dorsal regions along the anterior/posterior axis of the embryo. Goosecoid (gsc) expression marks the posterior end of the anterior/posterior axis and might be a good marker to visualise early events in embryonic axis formation and differentiation processes in the epiblast at the onset of gastrulation. The aim of the present study was to evaluate gsc expression in porcine embryos. For this the homeobox containing region of the porcine gsc was isolated using RT-PCR. The sequence of the PCR product appeared to be highly homologous to the sequence in the mouse, human, and chicken. We concluded that the isolated region represents part of the porcine gsc messenger. Relative levels of gsc expression were estimated in porcine embryos from day 9 to day 12 of pregnancy. Gsc was expressed in embryos of all ages and localisation on one side of the embryoblast was demonstrated with in situ hybridisation on whole- mount embryos at day 10 of pregnancy. In embryos collected at day 13 of pregnancy gsc expression was localised anterior to the primitive streak. The correlation between embryo size and level of gsc expression was low. Levels and pattern of expression varied within and between litters collected at similar days of pregnancy. It is concluded that gsc expression can be used as an early marker of differentiation and to describe embryo diversity in the pig.

Simultaneous determination of the (2)h/(1)h, (17)o/(16)o, and (18)o/(16)o isotope abundance ratios in water by means of laser spectrometry.

We demonstrate the first successful application of infrared laser spectrometry to the accurate, simultaneous determination of the relative (2)H/(1)H, (17)O/(16)O, and (18)O/(16)O isotope abundance ratios in water. The method uses a narrow line width color center laser to record the direct absorption spectrum of low-pressure gas-phase water samples (presently 10 µL of liquid) in the 3-µm spectral region. It thus avoids the laborious chemical preparations of the sample that are required in the case of the conventional isotope ratio mass spectrometer measurement. The precision of the spectroscopic technique is shown to be 0.7‰ for δ(2)H and 0.5‰ for δ(17)O and δ(18)O (δ represents the relative deviation of a sample's isotope abundance ratio with respect to that of a calibration material), while the calibrated accuracy amounts to about 3 and 1‰, respectively, for water with an isotopic composition in the range of the Standard Light Antarctic Precipitation and Vienna Standard Mean Ocean Water international standards.