Best practices for the execution, analysis, and data storage of plant single-cell/nucleus transcriptomics.

Single-cell and single-nucleus RNA-sequencing technologies capture the expression of plant genes at an unprecedented resolution. Therefore, these technologies are gaining traction in plant molecular and developmental biology for elucidating the transcriptional changes across cell types in a specific tissue or organ, upon treatments, in response to biotic and abiotic stresses, or between genotypes. Despite the rapidly accelerating use of these technologies, collective and standardized experimental and analytical procedures to support the acquisition of high-quality data sets are still missing. In this commentary, we discuss common challenges associated with the use of single-cell transcriptomics in plants and propose general guidelines to improve reproducibility, quality, comparability, and interpretation and to make the data readily available to the community in this fast-developing field of research.

Factors Shaping the Implementation of Strategies to Prevent Acute Kidney Injury: A Qualitative Study.

Reducing the prevalence of acute kidney injury (AKI) is an important patient safety objective set forth by the National Quality Forum. Despite international guidelines to prevent AKI, there continues to be an inconsistent uptake of these interventions by cardiac teams across practice settings. The IMPROVE-AKI study was designed to test the effectiveness and implementation of AKI preventive strategies delivered through team-based coaching activities. Qualitative methods were used to identify factors that shaped sites' implementation of AKI prevention strategies. Semi-structured interviews were conducted with staff in a range of roles within the cardiac catheterization laboratories, including nurses, laboratory managers, and interventional cardiologists (N = 50) at multiple time points over the course of the study. Interview transcripts were qualitatively coded, and aggregated code reports were reviewed to construct main themes through memoing. In this paper, we report insights from semi-structured interviews regarding workflow, organizational culture, and leadership factors that impacted implementation of AKI prevention strategies.

Organizing your space: The potential for integrating spatial transcriptomics and 3D imaging data in plants.

Plant cells communicate information for the regulation of development and responses to external stresses. A key form of this communication is transcriptional regulation, accomplished via complex gene networks operating both locally and systemically. To fully understand how genes are regulated across plant tissues and organs, high resolution, multi-dimensional spatial transcriptional data must be acquired and placed within a cellular and organismal context. Spatial transcriptomics (ST) typically provides a two-dimensional spatial analysis of gene expression of tissue sections that can be stacked to render three-dimensional data. For example, X-ray and light-sheet microscopy provide sub-micron scale volumetric imaging of cellular morphology of tissues, organs, or potentially entire organisms. Linking these technologies could substantially advance transcriptomics in plant biology and other fields. Here, we review advances in ST and 3D microscopy approaches and describe how these technologies could be combined to provide high resolution, spatially organized plant tissue transcript mapping.

Application of the threshold of toxicological concern (TTC) in the evaluation of drinking water contact chemicals.

The Threshold of Toxicological Concern (TTC) is an approach for assessing the safety of chemicals with low levels of exposure for which limited toxicology data are available. The original TTC criteria were derived for oral exposures from a distributional analysis of a dataset of 613 chemicals that identified 5th percentile no observed effect level (NOEL) values grouped within three tiers of compounds having specific structural functional groups and/or toxic potencies known as Cramer I, II and III classifications. Subsequent assessments of the TTC approach have established current thresholds to be scientifically robust. While the TTC has gained acknowledgment and acceptance by many regulatory agencies and organizations, use of the TTC approach in evaluating drinking water chemicals has been limited. To apply the TTC concept to drinking water chemicals, an exposure-based approach that incorporates the current weight of evidence for the target chemical is presented. Such an approach provides a comparative point of departure to the 5th percentile TTC NOEL using existing data, while conserving the allocation of toxicological resources for quantitative risk assessment to chemicals with greater exposure or toxicity. This approach will be considered for incorporation into NSF/ANSI/CAN 600, a health effects standard used in the safety evaluation of chemicals present in drinking water from drinking water contact additives and materials certified to NSF/ANSI/CAN 60 and 61, respectively.

Intra-cavity frequency-doubled VECSEL system for narrow linewidth Rydberg EIT spectroscopy.

High-power, narrow-linewidth light sources in the visible and UV spectra are in growing demand, particularly as quantum information and sensing research proliferates. Vertical external-cavity surface-emitting lasers (VECSELs) with intra-cavity frequency conversion are emerging as an attractive platform to fill these needs. Using such a device, we demonstrate 3.5 MHz full-width half-maximum Rydberg-state spectroscopy via electromagnetically induced transparency (EIT). The laser's 690 mW of output power at a wavelength of 475 nm enables large Rabi frequencies and strong signal-to-noise ratio in shorter measurement times. In addition, we characterize the frequency stability of the VECSEL using the delayed self-heterodyne technique and direct comparison with a commercial external-cavity diode laser (ECDL). We measure the pre-doubled light's Lorentzian linewidth to be 2π × 5.3(2) kHz, and the total linewidth to be 2π × 23(2) kHz. These measurements provide evidence that intra-cavity frequency-doubled VECSELs can perform precision spectroscopy at and below the MHz level, and are a promising tool for contemporary, and future, quantum technologies.

Spin-Wave Multiplexed Atom-Cavity Electrodynamics.

We introduce multiplexed atom-cavity quantum electrodynamics with an atomic ensemble coupled to a single optical cavity mode. Multiple Raman dressing beams establish cavity-coupled spin-wave excitations with distinctive spatial profiles. Experimentally, we demonstrate the concept by observing spin-wave vacuum Rabi splittings, selective superradiance, and interference in the cavity-mediated interactions of two spin waves. We highlight that the current experimental configuration allows rapid, interchangeable cavity coupling to 4 profiles with an overlap parameter of less than 10%, enough to demonstrate, for example, a quantum repeater network simulation in the cavity. With further improvements to the optical multiplexing setup, we infer the ability to access more than 10^{3} independent spin-wave profiles.

Quantum-Limited Atomic Receiver in the Electrically Small Regime.

We use a quantum sensor based on thermal Rydberg atoms to receive data encoded in electromagnetic fields in the extreme electrically small regime, with a sensing volume over 10^{7} times smaller than the cube of the electric field wavelength. We introduce the standard quantum limit for data capacity, and experimentally observe quantum-limited data reception for bandwidths from 10 kHz up to 30 MHz. In doing this, we provide a useful alternative to classical communication antennas, which become increasingly ineffective when the size of the antenna is significantly smaller than the wavelength of the electromagnetic field.

Derivation of an oral reference dose (RfD) for di 2-ethylhexyl cyclohexan-1,4-dicarboxylate (DEHCH), an alternative to phthalate plasticizers.

Di 2-ethylhexyl cyclohexane-1,4-dicarboxylate (DEHCH, CAS 84731-70-4) is an ester of polycarboxylic acid assessed in a variety of mammalian toxicity assays as a substitute for phthalate ester-type plasticizers. An OECD 422 combined systemic toxicity study with a reproductive/developmental toxicity screening test in SD rats found minimal effects on the liver, spleen, and thyroid and no indication that DEHCH is a developmental or reproductive toxicant. In a 90-day feeding study in SD rats, no toxicologically relevant effects were noted. Low genotoxic potential of DEHCH is indicated by the lack of mutagenicity or clastogenicity in vitro. No studies assessing mode of action were identified. Where data gaps exist for DEHCH, a read-across approach was used to assess other toxicological endpoints of interest. Di-ethylhexyl terephthalate (DEHT, CAS 6422-86-2) and 1,2-cyclohexane dicarboxylic acid, diisononyl ester (DINCH, CAS 474919-59-0) have higher tiered studies to supplant the data lacking for health-based standard setting. DEHT and DINCH were chosen as the source substances due to similar physical/chemical properties and thus anticipated metabolism and toxicological characteristics. An oral reference dose (RfD) for DEHCH was calculated using the human equivalent NOAEL from the OECD 422 study. A total uncertainty factor of 100 was comprised of interspecies (3x), intraspecies (10x), subchronic to chronic (1x), LOAEL to NOAEL uncertainty (1x) and database uncertainty (3x) factors, resulting in an RfD of 0.3 mg/kg-day.

Regulation of cotton (Gossypium hirsutum) drought responses by mitogen-activated protein (MAP) kinase cascade-mediated phosphorylation of GhWRKY59.

Drought is a key limiting factor for cotton (Gossypium spp.) production, as more than half of the global cotton supply is grown in regions with high water shortage. However, the underlying mechanism of the response of cotton to drought stress remains elusive. By combining genome-wide transcriptome profiling and a loss-of-function screen using virus-induced gene silencing, we identified Gossypium hirsutum GhWRKY59 as an important transcription factor that regulates the drought stress response in cotton. Biochemical and genetic analyses revealed a drought stress-activated mitogen-activated protein (MAP) kinase cascade consisting of GhMAP3K15-Mitogen-activated Protein Kinase Kinase 4 (GhMKK4)-Mitogen-activated Protein Kinase 6 (GhMPK6) that directly phosphorylates GhWRKY59 at residue serine 221. Interestingly, GhWRKY59 is required for dehydration-induced expression of GhMAPK3K15, constituting a positive feedback loop of GhWRKY59-regulated MAP kinase activation in response to drought stress. Moreover, GhWRKY59 directly binds to the W-boxes of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 2 (GhDREB2), which encodes a dehydration-inducible transcription factor regulating the plant hormone abscisic acid (ABA)-independent drought response. Our study identified a complete MAP kinase cascade that phosphorylates and activates a key WRKY transcription factor, and elucidated a regulatory module, consisting of GhMAP3K15-GhMKK4-GhMPK6-GhWRKY59-GhDREB2, that is involved in controlling the cotton drought response.

Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric miRNA precursors.

Artificial microRNAs (amiRNAs) are used for selective gene silencing in plants. However, current methods to produce amiRNA constructs for silencing transcripts in monocot species are not suitable for simple, cost-effective and large-scale synthesis. Here, a series of expression vectors based on Oryza sativa MIR390 (OsMIR390) precursor was developed for high-throughput cloning and high expression of amiRNAs in monocots. Four different amiRNA sequences designed to target specifically endogenous genes and expressed from OsMIR390-based vectors were validated in transgenic Brachypodium distachyon plants. Surprisingly, amiRNAs accumulated to higher levels and were processed more accurately when expressed from chimeric OsMIR390-based precursors that include distal stem-loop sequences from Arabidopsis thaliana MIR390a (AtMIR390a). In all cases, transgenic plants displayed the predicted phenotypes induced by target gene repression, and accumulated high levels of amiRNAs and low levels of the corresponding target transcripts. Genome-wide transcriptome profiling combined with 5'-RLM-RACE analysis in transgenic plants confirmed that amiRNAs were highly specific.

Deterministic Squeezed States with Collective Measurements and Feedback.

We demonstrate the creation of entangled, spin-squeezed states using a collective, or joint, measurement and real-time feedback. The pseudospin state of an ensemble of N=5×10^{4} laser-cooled ^{87}Rb atoms is deterministically driven to a specified population state with angular resolution that is a factor of 5.5(8) [7.4(6) dB] in variance below the standard quantum limit for unentangled atoms-comparable to the best enhancements using only unitary evolution. Without feedback, conditioning on the outcome of the joint premeasurement, we directly observe up to 59(8) times [17.7(6) dB] improvement in quantum phase variance relative to the standard quantum limit for N=4×10^{5} atoms. This is one of the largest reported entanglement enhancements to date in any system.

Reduced Mortality Associated with Acute Kidney Injury Requiring Dialysis in the United States.

BACKGROUND: Dialysis-requiring acute kidney injury (AKI-D) is a documented complication of hospitalization and procedures. Temporal incidence of AKI-D and related hospital mortality in the US population has not been recently characterized. We describe the epidemiology of AKI-D as well as associated in-hospital mortality in the US. METHODS: Retrospective cohort of a national discharge data (n = 86,949,550) from the Healthcare Cost and Utilization Project's National Inpatient Sample, 2001-2011 of patients' hospitalization with AKI-D. Primary outcomes were AKI-D and in-hospital mortality. We determined the annual incidence rate of AKI-D in the US from 2001 to 2011. We estimated ORs for AKI-D and in-hospital mortality for each successive year compared to 2001 using multiple logistic regression models, adjusted for patient and hospital characteristics, and stratified the analyses by sex and age. We also calculated population-attributable risk of in-hospital mortality associated with AKI-D. RESULTS: The adjusted odds of AKI-D increased by a factor of 1.03 (95% CI 1.02-1.04) each year. The number of AKI-D-related (19,886-34,195) in-hospital deaths increased almost 2-fold, although in-hospital mortality associated with AKI-D (28.0-19.7%) declined significantly from 2001 to 2011. Over the same period, the adjusted odds of mortality for AKI-D patients were 0.60 (95% CI 0.56-0.67). Population-attributable risk of mortality associated with AKI-D increased (2.1-4.2%) over the study period. CONCLUSIONS: The incidence rate of AKI-D has increased considerably in the US since 2001. However, in-hospital mortality associated with AKI-D hospital admissions has decreased significantly.