Institution-Specific Perinatal Emergency Checklists: Multicenter Report on Development, Implementation, and Sustainability.

OBJECTIVE: The American College of Obstetricians and Gynecologists and the Society for Maternal-Fetal Medicine endorse checklist use to improve obstetric care. However, there is limited research into development, implementation, and sustained use of perinatal emergency checklists to inform individual institutions. This study aimed to investigate the development and implementation of perinatal emergency checklists in diverse hospital settings in the United States. STUDY DESIGN: A qualitative study was conducted individually with clinicians from three health care systems. The participants developed and implemented institution-tailored perinatal emergency checklists. Interview transcriptions were coded using the Consolidated Framework for Implementation Research. RESULTS: The study sites included two health care systems and one individual hospital. Delivery volumes ranged from 3,500 to 48,000 deliveries a year. Interviews were conducted with all 10 participants approached. Checklists for 19 perinatal emergencies were developed at the three health care systems. Ten of the checklist topics were the same at all three institutions. Participants described the checklists as improving patient care during crises. The tools were viewed as opportunities to promote a shared mental model across clinical roles, to reduce redundancy and coordinate obstetric crisis management. Checklist were developed in small groups. Implementation was facilitated by those who developed the checklists. Participants agreed that simulation was essential for checklist refinement and effective use by response teams. Barriers to implementation included limited clinician availability. There was also an opportunity to strengthen integration of checklists workflow early in perinatal emergencies. Participants articulated that culture change took time, active practice, persistence, reinforcement, and process measurement. CONCLUSION: This study outlines processes to develop, implement, and sustain perinatal emergency checklists at three institutions. Participants agreed that multiple, parallel implementation tactics created the culture shift for integration. The overview and specific Consolidated Framework for Implementation Research components may be used to inform adaptation and sustainability for others considering implementing perinatal emergency checklists. KEY POINTS: · Perinatal emergency checklists reduce redundancy and coordinate obstetric crisis management.. · Perinatal emergency simulation is essential for checklist refinement and effective team use.. · Integrations of perinatal emergency checklists requires culture change and process measurement..

Bioorthogonal Protein Conjugation: Application to the Development of a Highly Sensitive Bioluminescent Immunoassay for the Detection of Interferon-γ.

Bioorthogonal conjugation eliminates the shortcomings of classical conjugation methods. The conjugation of antibodies to reporter proteins, such as bioluminescent protein, can be controlled with orthogonal conjugation methods. Here we report a bioluminescent immunoassay for the sensitive detection of interferon-γ (IFN-γ) that utilizes orthogonal conjugation of bioluminescent protein, Gaussia luciferase to anti-IFN-γ antibody. The IFN-γ is produced by the immune system and the detection of the IFN-γ is pivotal for the detection of persistent viral and bacterial infections. A bioorthogonal conjugation approach is used to conjugate an anti-IFN-γ antibody with a GLuc mutant containing the N-terminal tyrosine using formylbenzene diazonium hexafluorophosphate reagent (FBDP) in hydrophilic mild pH environment yielding high conjugation efficiency (60%). This reagent is shown to be specific for tyrosine (Tyr) residues. Therefore, conjugation through Tyr was orthogonal and not detrimental to the bioluminescence activity of GLuc. The immunoassay described in this paper is a sandwich type assay and involves a capture and a detection antibody. The assay was validated for its robustness, precision, accuracy, limit of detection, and recovery.

Expression of a soluble truncated Vargula luciferase in Escherichia coli.

Marine luciferases are regularly employed as useful reporter molecules across a range of various applications. However, attempts to transition expression from their native eukaryotic environment into a more economical prokaryotic, i.e. bacterial, expression system often presents several challenges. Specifically, bacterial protein expression inherently lacks chaperone proteins to aid in the folding process, while Escherichia coli presents a reducing cytoplasmic environment in. These conditions contribute to the inhibition of proper folding of cysteine-rich proteins, leading to incorrect tertiary structure and ultimately inactive and potentially insoluble protein. Vargula luciferase (Vluc) is a cysteine-rich marine luciferase that exhibits glow-type bioluminescence through a reaction between its unique native substrate and molecular oxygen. Because most other commonly used bioluminescent proteins exhibit flash-type emission kinetics, this emission characteristic of Vluc is desirable for high-throughput applications where stability of emission is required for the duration of data collection. A truncated form of Vluc that retains considerable bioluminescence activity (55%) compared to the native full-length protein has been reported in the literature. However, expression and purification of this luciferase from bacterial systems has proven difficult. Herein, we demonstrate the expression and purification of a truncated form of Vluc from E. coli. This truncated Vluc (tVluc) was subsequently characterized in terms of both its biophysical and bioluminescence properties.

A paper-based platform for detection of viral RNA.

Viral detection presents a host of challenges for even the most sensitive analytical techniques, and the complexity of common detection platforms typically preclude portability. With these considerations in mind, we designed a paper microzone plate-based virus detection system for the detection of viral genetic material that can be performed with simple instruments. The sensing system can detect viral cDNA reverse-transcribed from total RNA extraction by utilizing a biotinylated capture probe and an Alexa Fluor® 647-labeled reporter probe. The biotinylated capture probe was linked to the paper surface via NeutrAvidin® that was physically adsorbed on the paper. After addition of reverse-transcribed sample and reporter probe in sequence, the reverse-transcribed target captured the reporter probe and tethered it to the capture probe in a bridged format. Fluorescence intensity was imaged using a Western blot imaging system, and higher target concentration was visible by the increased emission intensity from Alexa Fluor® 647. By utilizing paper, this detection setup could also serve as a sample concentration method via evaporation, which could remarkably lower the detection limit if needed. This detection platform used Epstein-Barr virus (EBV) RNA as a proof-of-concept by sensing cDNA resulting from reverse transcription and can be further expanded as a general method for other pathogens. EBV is a well-known human tumor virus, which has also recently been linked to the development of cervical cancer. The assay was accomplished within two hours including the room-temperature RNA extraction and reverse transcription steps. Also, this paper microzone plate-based platform can potentially be applicable for the development of point-of-care (POC) detection kits or devices due to its robust design, convenient interface, and easy portability. The experiment could be stopped after each step, and continued at a later time. The shelf-life of the modified paper plate setup was at least 3 months without a discernible change in signal, and the result from day 1 could be read at 3 months - both of which are important criteria for POC analytical testing tools, especially in resource-poor settings. All of the required assay steps could potentially be performed without any significant equipment using inexpensive paper microzone plates, which will be ideal for further development of POC testing devices. Although, this platform is not at the stage where it can be directly used in a point-of-care setting, it does have fundamental characteristics such as a stable platform, a simple detection method, and relatively common reagents that align closely with a POC system.

Design and development of high bioluminescent resonance energy transfer efficiency hybrid-imaging constructs.

Here we describe the design and construction of an imaging construct with high bioluminescent resonance energy transfer (BRET) efficiency that is composed of multiple quantum dots (QDs; λem = 655 nm) self-assembled onto a bioluminescent protein, Renilla luciferase (Rluc). This is facilitated by the streptavidin-biotin interaction, allowing the facile formation of a hybrid-imaging construct (HIC) comprising up to six QDs (acceptor) grafted onto a light-emitting Rluc (donor) core. The resulting assembly of multiple acceptors surrounding a donor permits this construct to exhibit high resonance energy transfer efficiency (∼64.8%). The HIC was characterized using fluorescence excitation anisotropy measurements and high-resolution transmission electron microscopy. To demonstrate the application of our construct, a generation-5 (G5) polyamidoamine dendrimer (PAMAM) nanocarrier was loaded with our HIC for in vitro and in vivo imaging. We envision that this design of multiple acceptors and bioluminescent donor will lead to the development of new BRET-based systems useful in sensing, imaging, and other bioanalytical applications.

Improved visual detection of DNA amplification using pyridylazophenol metal sensing dyes.

Detection of nucleic acid amplification has typically required sophisticated laboratory instrumentation, but as the amplification techniques have moved away from the lab, complementary detection techniques have been implemented to facilitate point-of-care, field, and even at-home applications. Simple visual detection approaches have been widely used for isothermal amplification methods, but have generally displayed weak color changes or been highly sensitive to sample and atmospheric effects. Here we describe the use of pyridylazophenol dyes and binding to manganese ion to produce a strong visible color that changes in response to nucleic acid amplification. This detection approach is easily quantitated with absorbance, rapidly and clearly visible by eye, robust to sample effects, and notably compatible with both isothermal and PCR amplification. Nucleic acid amplification and molecular diagnostic methods are being used in an increasing number of novel applications and settings, and the ability to reliably and sensitively detect them without the need for additional instrumentation will enable even more access to these powerful techniques.

Profiling Thermus thermophilus Argonaute Guide DNA Sequence Preferences by Functional Screening.

Prokaryotic Argonautes (pAgo) are an increasingly well-studied class of guided endonucleases, and the underlying mechanisms by which pAgo generate nucleic acid guides in vivo remains an important topic of investigation. Recent insights into these mechanisms for the Argonaute protein from Thermus thermophilus has drawn attention to global sequence and structural feature preferences involved in oligonucleotide guide selection. In this work, we approach the study of guide sequence preferences in T. thermophilus Argonaute from a functional perspective. Screening a library of 1,968 guides against randomized single- and double-stranded DNA substrates, endonuclease activity associated with each guide was quantified using high-throughput capillary electrophoresis, and localized sequence preferences were identified which can be used to improve guide design for molecular applications. The most notable preferences include: a strong cleavage enhancement from a first position dT independent of target sequence; a significant decrease in activity with dA at position 12; and an impact of GC dinucleotides at positions 10 and 11. While this method has been useful in characterizing unique preferences of T. thermophilus Argonaute and criteria for creating efficient guides, it could be expanded further to rapidly characterize more recent mesophilic variants reported in the literature and drive their utility toward molecular tools in biology and genome editing applications.

Global distribution, trends, and drivers of flash drought occurrence.

Flash drought is characterized by a period of rapid drought intensification with impacts on agriculture, water resources, ecosystems, and the human environment. Addressing these challenges requires a fundamental understanding of flash drought occurrence. This study identifies global hotspots for flash drought from 1980-2015 via anomalies in evaporative stress and the standardized evaporative stress ratio. Flash drought hotspots exist over Brazil, the Sahel, the Great Rift Valley, and India, with notable local hotspots over the central United States, southwestern Russia, and northeastern China. Six of the fifteen study regions experienced a statistically significant increase in flash drought during 1980-2015. In contrast, three study regions witnessed a significant decline in flash drought frequency. Finally, the results illustrate that multiple pathways of research are needed to further our understanding of the regional drivers of flash drought and the complex interactions between flash drought and socioeconomic impacts.

6-Alkylquinolone-3-carboxylic acid tethered to macrolides synthesis and antimicrobial profile.

Two series of clarithromycin and azithromycin derivatives with terminal 6-alkylquinolone-3-carboxylic unit with central ether bond in the linker were prepared and tested for antimicrobial activity. Quinolone-linker intermediates were prepared by Sonogashira-type C(6)-alkynylation of 6-iodo-quinolone precursors. In the last step, 4'' site-selective acylation of 2'-protected macrolides was completed with the EDC reagent, which selectively activated a terminal, aliphatic carboxylic group in dicarboxylic intermediates. Antimicrobial activity of the new series of macrolones is discussed. The most potent compound, 4''-O-{6-[3-(3-carboxy-1-ethyl-4-oxo-1,4-dihydroquinolin-6-yl)-propoxy]-hexanoyl}-azithromycin (10), is highly active against bacterial respiratory pathogens resistant to macrolide antibiotics and represents a promising lead for further investigation.

Resonance energy transfer methods of RNA detection.

Quantitation of RNA is important in diagnostics, environmental science, and basic biomedical research. RNA is considered a signature for pathogen identification, and its expression profile is linked with disease pathogenesis, allowing for biomarker identification. RNA-based diagnostics is an emerging field of research. This expansion of interest in studying RNA has generated demand for its accurate and sensitive detection. Several methods have therefore been developed to detect RNA. Resonance energy transfer methods of RNA detection are highly promising in terms of simplicity and high sensitivity. In this review, we have focused on the latest developments in resonance energy transfer methods of RNA detection that utilize various probe designs. The probe designs discussed here are molecular beacons, quenched autoligation probes, and linear oligonucleotide probes. Resonance energy transfer methods based on both fluorescence and bioluminescence detection are discussed.

Enhanced Recovery After Surgery to Change Process Measures and Reduce Opioid Use After Cesarean Delivery: A Quality Improvement Initiative.

OBJECTIVE: To evaluate implementation of an enhanced recovery after surgery (ERAS) program for patients undergoing elective cesarean delivery by comparing opioid exposure, multimodal analgesia use, and other process and outcome measures before and after implementation. METHODS: An ERAS program was implemented among patients undergoing elective cesarean delivery in a large integrated health care delivery system. We conducted a pre-post study of ERAS implementation to compare changes in process and outcome measures during the 12 months before and 12 months after implementation. RESULTS: The study included 4,689 patients who underwent an elective cesarean delivery in the 12 months before (pilot sites: March 1, 2015-February 29, 2016, all other sites: October 1, 2015-September 30, 2016), and 4,624 patients in the 12 months after (pilot sites: April 1, 2016-March 31, 2017, all other sites: November 1, 2016-October 31, 2017) ERAS program implementation. After ERAS implementation mean inpatient opioid exposure (average daily morphine equivalents) decreased from 10.7 equivalents (95% CI 10.2-11.3) to 5.4 equivalents (95% CI 4.8-5.9) controlling for age, race-ethnicity, prepregnancy body mass index, patient reported pain score, and medical center. The use of multimodal analgesia (ie, acetaminophen and neuraxial anesthesia) increased from 9.7% to 88.8%, the adjusted risk ratio (RR) for meeting multimodal analgesic goals was 9.13 (RR comparing post-ERAS with pre-ERAS; 95% CI 8.35-10.0) and the proportion of time patients reported acceptable pain scores increased from 82.1% to 86.4% (P<.001). Outpatient opioids dispensed at hospital discharge decreased from 85.9% to 82.2% post-ERAS (P<.001) and the average number of dispensed pills decreased from 38 to 26 (P<.001). The hours to first postsurgical ambulation decreased by 2.7 hours (95% CI -3.1 to -2.4) and the hours to first postsurgical solid intake decreased by 11.1 hours (95% CI -11.5 to -10.7). There were no significant changes in hospital length of stay, surgical site infections, hospital readmissions, or breastfeeding rates. CONCLUSIONS: Implementation of an ERAS program in patients undergoing elective cesarean delivery was associated with a reduction in opioid inpatient and outpatient exposure and with changes in surgical process measures of care without worsened surgical outcomes.

Red fluorescent protein variants with incorporated non-natural amino acid analogues.

Fluorescent proteins are important tools in biotechnology applications and biosensing. DsRed, a red fluorescent protein, has expanded the colors of fluorescent proteins beyond the more commonly used green fluorescent protein. Many genetic modifications have been performed on DsRed to overcome some of its drawbacks. These primarily focused on overcoming the oligomerization detrimental to DsRed activity, and the parasitic green fluorescence caused by the immature chromophore. One such variant, DsRed-monomer, has minimal green fluorescence and no oligomerization. A few traditional mutagenesis studies have been done with DsRed and its mutants to shift the fluorescence wavelengths creating additions to the pallet of fluorescent protein colors. We have explored incorporation of non-natural amino acid analogues into DsRed-Monomer, obtaining variants with differing emission properties. In this work, two such analogues of tyrosine have been incorporated into DsRed-Monomer: 3-amino-l-tyrosine and 3-fluoro-l-tyrosine. Tyrosine analogues were chosen due to the role of tyrosine in the formation and structure of the protein's chromophore. The variants obtained in our study showed altered emission wavelengths and spectral characteristics. Our study demonstrates that incorporation of non-natural analogues into DsRed-Monomer is a viable approach to alter the spectral characteristics of the protein. We envision that this study will open up the door to non-natural mutagenesis studies with red fluorescent proteins and its mutants.

Multidisciplinary management of peripartum cardiomyopathy during repeat cesarean delivery: a case report.

Peripartum cardiomyopathy is a potentially fatal form of heart failure associated with pregnancy. A 29-year-old African American woman, gravida 3, para 2, at 36 weeks' gestation had a history of cardiomyopathy, morbid obesity (body mass index > 70 kg/m2), uncontrolled hypertension, obstructive sleep apnea, and required a repeat cesarean delivery. The patient was admitted to the hospital several times throughout her pregnancy for congestive heart failure, pulmonary edema, and headaches. Two years previously the patient received a diagnosis of peripartum cardiomyopathy 3 weeks after the delivery of her second child. This case report illustrates the recognition of peripartum cardiomyopathy and the risks early in pregnancy. It also describes the appropriate medical management, including transesophageal echocardiography and the need for collaboration of multiple medical specialists before and during delivery to provide the best possible outcome for both mother and infant.

Single-stranded binding proteins and helicase enhance the activity of prokaryotic argonautes in vitro.

Prokaryotic argonautes are a unique class of nucleic acid-guided endonucleases putatively involved in cellular defense against foreign genetic elements. While their eukaryotic homologs and Cas protein counterparts require single-stranded RNAs as guides, some prokaryotic argonautes are able to utilize short single-stranded DNAs as guides for sequence-specific endonuclease activity. Many complications currently prevent the use of prokaryotic argonautes for in vivo gene-editing applications; however, they do exhibit potential as a new class of in vitro molecular tools if certain challenges can be overcome, specifically the limitations on substrate accessibility which leads to unequal levels of activity across a broad palate of substrates and the inability to act on double-stranded DNA substrates. Here we demonstrate the use of accessory factors, including thermostable single-stranded DNA binding proteins and UvrD-like helicase, in conjunction with prokaryotic argonautes to significantly improve enzymatic activity and enable functionality with a broader range of substrates, including linear double-stranded DNA substrates. We also demonstrate the use of Thermus thermophilus argonaute with accessory factors as a programmable restriction enzyme to generate long, unique single-stranded overhangs from linear double-stranded substrates compatible with downstream ligation.

Sources of Black Carbon Deposition to the Himalayan Glaciers in Current and Future Climates.

WRF-Chem and a modified version of the ECLIPSE 5a emission inventory were used to investigate the sources impacting black carbon (BC) deposition to the Himalaya, Karakoram, and Hindu Kush (HKHK) region. This work extends previous studies by simulating deposition to the HKHK region not only under current conditions, but also in the 2040-2050 period under two realistic emission scenarios and in three different phases of the El Niño-Southern Oscillation (ENSO). Under current conditions, sources from outside our South Asian modelling domain have a similar impact on total BC deposition to the HKHK region (35-87%, varying with month) as South Asian anthropogenic sources (13-62%). Industry (primarily brick kilns) and residential solid fuel burning combined account for 45-66% of the in-domain anthropogenic BC deposition to the HKHK region. Under a no further control emission scenario for 2040-2050, the relative contributions to BC deposition in the HKHK region are more skewed toward in-domain anthropogenic sources (45-65%) relative to sources outside the domain (26-52%). The in-domain anthropogenic BC deposition has significant contributions from industry (32-42%), solid fuel burning (17-28%), and diesel fuel burning (17-27%). Under a scenario in which emissions in South Asia are mitigated, the relative cotribution from South Asian anthropogenic sources is significantly reduced to 11-34%. The changes due to phase of ENSO do not seem to follow consistent patterns with ENSO. Future work will use the high-resolution deposition maps developed here to determine the impact of different sources of BC on glacier melt and water availability in the region.

The lattice stiffening transition in UO2 single crystals.

The effective Debye temperatures ([Formula: see text]) of the surface region of UO2 single crystals, prepared by the hydrothermal synthesis technique, were obtained from temperature-dependent x-ray photoemission in the temperature range of 300 K-623 K. A lattice stiffening transition, characterized by different regions of different effective Debye temperature, 500 ± 59 K below 475 K and 165 ± 21 K above 475 K is identified. A comparison of the temperature dependence of the effective UO2 Debye temperature, with the changes in the lattice expansion coefficient for UO2, support strong lattice-phonon interaction arising from the Jahn-Teller distortion.

Truncated Variants of Gaussia Luciferase with Tyrosine Linker for Site-Specific Bioconjugate Applications.

Gaussia luciferase (Gluc)-with its many favorable traits such as small size, bright emission, and exceptional stability-has become a prominent reporter protein for a wide range of bioluminescence-based detection applications. The ten internal cysteine residues crucial to functional structure formation, however, make expression of high quantities of soluble protein in bacterial systems difficult. In addition to this challenge, the current lack of structural data further complicates the use of Gluc for in vitro applications, such as biosensors, or cellular delivery, both of which rely heavily on robust and reproducible bioconjugation techniques. While Gluc is already appreciably small for a luciferase, a reduction in size that still retains significant bioluminescent activity, in conjunction with a more reproducible bioorthogonal method of chemical modification and facile expression in bacteria, would be very beneficial in biosensor design and cellular transport studies. We have developed truncated variants of Gluc, which maintain attractive bioluminescent features, and have characterized their spectral and kinetic properties. These variants were purified in high quantities from a bacterial system. Additionally, a C-terminal linker has been incorporated into these variants that can be used for reliable, specific modification through tyrosine-based bioconjugation techniques, which leave the sensitive network of cysteine residues undisturbed.

MicroRNA Detection: Current Technology and Research Strategies.

The relatively new field of microRNA (miR) has experienced rapid growth in methodology associated with its detection and bioanalysis as well as with its role in -omics research, clinical diagnostics, and new therapeutic strategies. The breadth of this area of research and the seemingly exponential increase in number of publications on the subject can present scientists new to the field with a daunting amount of information to evaluate. This review aims to provide a collective overview of miR detection methods by relating conventional, established techniques [such as quantitative reverse transcription polymerase chain reaction (RT-qPCR), microarray, and Northern blotting (NB)] and relatively recent advancements [such as next-generation sequencing (NGS), highly sensitive biosensors, and computational prediction of microRNA/targets] to common miR research strategies. This should guide interested readers toward a more focused study of miR research and the surrounding technology.

Bioluminescent stem-loop probes for highly sensitive nucleic acid detection.

Here, we report the first bioluminescent stem-loop probe, which is 50 times more sensitive and able to achieve a LOD 25 times lower than fluorescent stem-loop probes. Chemical generation of a signal from Renilla luciferase reduces background noise for improved quantitative utility in nucleic acid biomarker detection.