Phenotyping Protocol Research Articles

Prediction accuracy and repeatability of UAV based biomass estimation in wheat variety trials as affected by variable type, modelling strategy and sampling location

BackgroundThis study explores the use of Unmanned Aerial Vehicles (UAVs) for estimating wheat biomass, focusing on the impact of phenotyping and analytical protocols in the context of late-stage variety selection programs. It emphasizes the importance of variable selection, model specificity, and sampling location within the experimental plot in predicting biomass, aiming to refine UAV-based estimation techniques for enhanced selection accuracy and throughput in variety testing programs.ResultsThe research uncovered that integrating geometric and spectral traits led to an increase in prediction accuracy, whilst a recursive feature elimination (RFE) based variable selection workflowled to slight reductions in accuracy with the benefit of increased interpretability. Models, tailored to specific experiments were more accurate than those modelling all experiments together, while models trained for broad-growth stages did not significantly increase accuracy. The comparison between a permanent and a precise region of interest (ROI) within the plot showed negligible differences in biomass prediction accuracy, indicating the robustness of the approach across different sampling locations within the plot. Significant differences in the within-season repeatability (w2) of biomass predictions across different experiments highlighted the need for further investigation into the optimal timing of measurement for prediction.ConclusionsThe study highlights the promising potential of UAV technology in biomass prediction for wheat at a small plot scale. It suggests that the accuracy of biomass predictions can be significantly improved through optimizing analytical and modelling protocols (i.e., variable selection, algorithm selection, stage-specific model development). Future work should focus on exploring the applicability of these findings under a wider variety of conditions and from a more diverse set of genotypes.

Abstract Mo025: Noninvasive cardiac phenotyping in patients treated with chemo- and immunotherapy for breast cancer

Current breast cancer diagnosis and treatment protocols achieve survival rates of>80% at 10 years.However, long-term cancer survivors have>4-fold higher incidence of cardiovascular complications, including heart failure.Early potentially reversible changes that may underlie downstream irreversible loss of mechanical function are unknown.Therefore, we aimed to test the feasibility of a noninvasive cardiac phenotyping protocol in patients undergoing treatment for breast cancer with doxorubicin, without or with immune checkpoint inhibitors, including multiparametric proton cardiac magnetic resonance(CMR) imaging and novel metabolic carbon13MR. A comprehensive set of studies included conventional 1HCMR,as well as metabolic 13CCMR using hyperpolarized(HP)[1-13C] pyruvate, following an oral glucose load. 13CCMR with HP[1-13C] pyruvate allows the detection of downstream metabolites of injected pyruvate within the myocardium: bicarbonate from mitochondrial oxidation and lactate from anaerobic cytosolic metabolism.HP[1-13C]pyruvate was prepared using dynamic nuclear polarization in a commercial polarizer.Imaging and spectroscopy(MRS) of hyperpolarized pyruvate was conducted using a 13C transmit/receive Helmholtz loop-pair coil on a 3Tscanner.13C data were acquired from a short-axis slice, ECG-triggered in end-systole. ProtonCMR images were analyzed using Circle, and 13CCMR images were analyzed using MATLAB. The left ventricular mid-myocardium and blood pool were segmented for metabolite quantitation. Baseline cardiac studies were performed in a consecutive series of 25 women treated for breast cancer. Of these 14(56%) were Caucasian, 3(12%) were Hispanic, 5(20%) Black, and 3(12%) Asian, with ages ranging 46±11 years.Patients received standard-of-care treatment plans based on the tumor type: 11 patients were treated with dose-dense anthracycline and cyclophosphamide, and 14 also received pembrolizumab. The comprehensive baseline phenotyping protocol is planned to be repeated after standard of care therapy in all patients. Baseline data are presented in Table1, with values reported as mean±standard deviation. Noninvasive cardiac phenotyping, including dynamic detection of metabolic substrate utilization is feasible,without showing significant adverse effects,and is well tolerated in a diverse group of patients.Completion of this protocol post-oncological therapy may allow to detect early changes in cardiac function due to chemo-and immunotherapy in susceptible patients.

High Throughput Image-Based Phenotyping for Determining Morphological and Physiological Responses to Single and Combined Stresses in Potato.

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions over time. By using multiple imaging sensors, many traits of interest can be assessed, including plant biomass, photosynthetic efficiency, canopy temperature, and leaf reflectance indices. Plants are frequently exposed to multiple stresses under field conditions where severe heat waves, flooding, and drought events seriously threaten crop productivity. When stresses coincide, resulting effects on plants can be distinct due to synergistic or antagonistic interactions. To elucidate how potato plants respond to single and combined stresses that resemble naturally occurring stress scenarios, five different treatments were imposed on a selected potato cultivar (Solanum tuberosum L., cv. Lady Rosetta) at the onset of tuberization, i.e. control, drought, heat, waterlogging, and combinations of heat, drought, and waterlogging stresses. Our analysis shows that waterlogging stress had the most detrimental effect on plant performance, leading to fast and drastic physiological responses related to stomatal closure, including a reduction in the quantum yield and efficiency of photosystem II and an increase in canopy temperature and water index. Under heat and combined stress treatments, the relative growth rate was reduced in the early phase of stress. Under drought and combined stresses, plant volume and photosynthetic performance dropped with an increased temperature and stomata closure in the late phase of stress. The combination of optimized stress treatment under defined environmental conditions together with selected phenotyping protocols allowed to revealthe dynamics of morphological and physiological responses to single and combined stresses. Here, a useful tool is presented for plant researchers looking to identify plant traits indicative of resilience to several climate change-related stresses.

Phenotypic Evaluation of a Lemon Hybrid Population to Identify Sources of Resistance to Plenodomus tracheiphilus

Mal secco, caused by the fungus Plenodomus tracheiphilus, is a xylem disease that is a limiting factor for lemon production in the Mediterranean. Resistance or field tolerance are major goals for lemon breeders; however, there is scant information regarding the heritability of mal secco resistance in breeding populations. As with other vascular diseases, phenotyping is the bottleneck for ascertaining resistance and susceptibility, and a validated protocol for greenhouse phenotyping would be valuable to accelerate the selection of tolerant trees before field evaluation. We report phenotyping of 148 hybrids of Khasi papeda (Citrus latipes; tolerant to mal secco) × lemon (susceptible to the disease) in field and greenhouse conditions. Field evaluation was performed on all hybrids for 2 to 3 consecutive years on trees subjected to high natural-pathogen pressure. Detection of the fungal infection was performed by visual observation and real-time polymerase chain reaction (PCR). The first infections occurred ≈6 months after planting, but 2 years of observations were needed for a reliable estimation of susceptibility. The spread of the disease did not occur uniformly throughout the plot, with patterns of spread within rows, probably resulting from infections from plant to plant. The possible errors in the estimation of susceptibility as a result of the uneven distribution of infections in the plot were reduced by using more than one replicate tree per hybrid. The correlation between phenotyping scores and cycle threshold values was weak (r = –0.48, P < 0.001). Three years after planting, hybrids clustered into three groups—susceptible, tolerant, and intermediate—based on symptom progression. A subset of 65 self-rooted hybrids was also subjected to stem inoculation in an unheated greenhouse, with two to seven biological replicates per hybrid. Three months after inoculation, the samples were monitored for symptoms appearance and subjected to real-time PCR pathogen quantification. We observed a weak (r = 0.41) but significant (P < 0.001) correlation between phenotypes in the field and the greenhouse, indicating that, in our conditions, field evaluation remains the best method for phenotyping. However, artificial inoculations might help to discard the highly susceptible hybrids before field evaluation.

Identification of potential morpho-biochemical determinants conferring salt tolerance in wheat (Triticum aestivum L.) through seedling- and -reproductive stage phenotyping

To optimize a reproductive-stage-specific phenotyping protocol and isolate potential determinants conferring salinity tolerance in wheat, two consecutive experiments were conducted using salt-tolerant varieties (Binagom-1 and BARI Gom 25) and a sensitive variety (BARI Gom 20). In the first experiment, seedlings were grown hydroponically, and 14-day-old seedlings were subjected to two different levels of salt stress (EC=12 dS/m and 16 dS/m) for 7 days. Based on the results of tolerant and susceptible varieties, parameters such as root and shoot weight, shoot Na+/K+ ratio, chlorophyll content, proline content, methylglyoxal content, H2O2 content and lipid peroxidation content and the activities of enzymes such as ascorbate peroxidase, peroxidase, and glyoxalase I were considered as potential determinants of salt tolerance. The second experiment employed four leaf cutting treatments under both control and salinity stress conditions. Seedlings were grown in perforated pots filled with field soil, and at the heading stage, plants were subjected to salt stress (12 dS/m) after trimming as indicated. The combined analysis of control and salt stress data obtained from setup B reflected a significant decrease in yield and yield-attributing traits; however, a lesser decrease was observed in tolerant varieties. Correlation studies revealed that grain yield per spike exhibited a significant positive correlation with the number of seeds per spike, spike weight, plant height, and days to first flowering under both stress and control conditions. Additionally, different stress tolerance indices also supported the results of reproductive stage phenotyping. However, further studies will be required to tag the genes/QTLs controlling salinity tolerance in wheat at various growth phases.

WirPheno: an affordable medium throughput root phenotyping protocol for assessment of waterlogging induced roots in cowpea

WirPheno: an affordable medium throughput root phenotyping protocol for assessment of waterlogging induced roots in cowpea

Leveraging the potential of big genomic and phenotypic data for genome-wide association mapping in wheat

Genome-wide association mapping studies (GWAS) based on Big Data are a potential approach to improve marker-assisted selection in plant breeding. The number of available phenotypic and genomic data sets in which medium-sized populations of several hundred individuals have been studied is rapidly increasing. Combining these data and using them in GWAS could increase both the power of QTL discovery and the accuracy of estimation of underlying genetic effects, but is hindered by data heterogeneity and lack of interoperability. In this study, we used genomic and phenotypic data sets, focusing on Central European winter wheat populations evaluated for heading date. We explored strategies for integrating these data and subsequently the resulting potential for GWAS. Establishing interoperability between data sets was greatly aided by some overlapping genotypes and a linear relationship between the different phenotyping protocols, resulting in high quality integrated phenotypic data. In this context, genomic prediction proved to be a suitable tool to study relevance of interactions between genotypes and experimental series, which was low in our case. Contrary to expectations, fewer associations between markers and traits were found in the larger combined data than in the individual experimental series. However, the predictive power based on the marker-trait associations of the integrated data set was higher across data sets. Therefore, the results show that the integration of medium-sized to Big Data is an approach to increase the power to detect QTL in GWAS. The results encourage further efforts to standardize and share data in the plant breeding community.

Genetic background and sex influence somatosensory sensitivity and oxycodone analgesia in the Hybrid Rat Diversity Panel.

Opioid use disorder (OUD) is an ongoing public health concern in the United States, and relatively little work has addressed how genetic background contributes to OUD. Understanding the genetic contributions to oxycodone-induced analgesia could provide insight into the early stages of OUD development. Here, we present findings from a behavioral phenotyping protocol using several inbred strains from the Hybrid Rat Diversity Panel. Our behavioral protocol included a modified "up-down" von Frey procedure to measure inherent strain differences in the sensitivity to a mechanical stimulus on the hindpaw. We also performed the tail immersion assay, which measures the latency to display tail withdrawal in response to a hot water bath. Initial withdrawal thresholds were taken in drug-naïve animals to record baseline thermal sensitivity across the strains. Oxycodone-induced analgesia was measured after administration of oxycodone over the course of 2 h. Both mechanical and thermal sensitivity are shaped by genetic factors and display moderate heritability (h2 = 0.23-0.40). All strains displayed oxycodone-induced analgesia that peaked at 15-30 min and returned to baseline by 2 h. There were significant differences between the strains in the magnitude and duration of their analgesic response to oxycodone, although the heritability estimates were quite modest (h2 = 0.10-0.15). These data demonstrate that genetic background confers differences in mechanical sensitivity, thermal sensitivity, and oxycodone-induced analgesia.

Digital Phenotyping for Real-Time Monitoring of Nonsuicidal Self-Injury: Protocol for a Prospective Observational Study.

Nonsuicidal self-injury (NSSI) is a major global health concern. The limitations of traditional clinical and laboratory-based methodologies are recognized, and there is a pressing need to use novel approaches for the early detection and prevention of NSSI. Unfortunately, there is still a lack of basic knowledge of a descriptive nature on NSSI, including when, how, and why self-injury occurs in everyday life. Digital phenotyping offers the potential to predict and prevent NSSI by assessing objective and ecological measurements at multiple points in time. This study aims to identify real-time predictors and explain an individual's dynamic course of NSSI. This study will use a hybrid approach, combining elements of prospective observational research with non-face-to-face study methods. This study aims to recruit a cohort of 150 adults aged 20 to 29 years who have self-reported engaging in NSSI on 5 or more days within the past year. Participants will be enrolled in a longitudinal study conducted at 3-month intervals, spanning 3 long-term follow-up phases. The ecological momentary assessment (EMA) technique will be used via a smartphone app. Participants will be prompted to complete a self-injury and suicidality questionnaire and a mood appraisal questionnaire 3 times a day for a duration of 14 days. A wrist-worn wearable device will be used to collect heart rate, step count, and sleep patterns from participants. Dynamic structural equation modeling and machine learning approaches will be used. Participant recruitment and data collection started in October 2023. Data collection and analysis are expected to be completed by December 2024. The results will be published in a peer-reviewed journal and presented at scientific conferences. The insights gained from this study will not only shed light on the underlying mechanisms of NSSI but also pave the way for the development of tailored and culturally sensitive treatment options that can effectively address this major mental health concern. DERR1-10.2196/53597.

Protocol for the combined cardiometabolic deep phenotyping and registry-based 20-year follow-up study of the Inter99 cohort

IntroductionThe population-based Inter99 cohort has contributed extensively to our understanding of effects of a systematic screening and lifestyle intervention, as well as the multifactorial aetiology of type 2 diabetes (T2D)...

Effective protocol for rice phenotyping based on grain quality: Ranking of rice samples from a segregating BC2 population

The physical properties of rice backcross samples were studied in connection with the development of an effective breeding methodology for creating rice genotypes based on an accelerated breeding process. The purpose of the research was to study the traits of size, vitreousity, and fracturing of samples of the segregating population of BC2 reseedings from the 2022 and 2023 yield. As part of the program for developing varieties based on the use of modern post-genomic and cellular technologies, BC2 samples were used; as parent forms – samples of the USI “Collection of Federal Scientific Rice Centre” of various groups according to the shape of the grain. The crop was grown in artificial climate chambers under controlled conditions of light and air temperature and on the growing site of Federal Scientific Rice Centre. In the study, white grain samples from the segregating BC2 population were isolated for large-scale genotyping. The cultivation of parental forms for hybridization and the crossing process were carried out in artificial climate chambers. Hybridizations were performed using pneumocastration, which included cutting off the flower scales by 1/3 and then removing the anthers using a vacuum compressor. White grain BC2 samples were isolated for research. Seeds were sown on the growing area in containers with 6 kg of soil and Pryanishnikov’s mixture as the main fertilizer. After ripening, the seeds were manually harvested. Plant phenotyping of experimental populations was carried out basing on grain quality traits using certified equipment in accordance with the instructions for the devices and GOST standards. For 15 combinations, samples with the best characteristics of grain quality were selected. In connection with the indicators of physical grain quality traits, BC2 samples were classified into three groups with indicators for each trait: high, medium, low.

Antibacterial activity of two actinomycetes species isolated from black sand in North Egypt

Increasingly high levels of multidrug-resistant (MDR) pathogens have necessitated the discovery of novel bioactive compounds. For this reason, two actinomycetes strains, Streptomyces griseorubens and Streptomyces rochei, were isolated for the first time from the black sand shores of Kafr El Sheikh in Egypt, which is home to several large fish farms. Isolates were identified via phenotypic, biochemical and 16S rRNA sequence protocols. Both strains exhibited powerful antimicrobial activity against three serious MDR pathogens: Bacillus subtilis, Salmonella enteritidis and Pseudomonas aeruginosa. The bioactive compounds of isolates’ filtrates were identified using gas chromatography–mass spectroscopy (GC-MS). For S. griseorubens, the detectable antibacterial compounds were hexanoic acid, 2-ethyl-, 2-ethylhexyl ester, n-Decane, hexadecanoic acid methyl ester, benzene acetic acid, ricinolic acid, and ethylparaben, while S. rochei secretes heptadecane, 2,6-dimethyl-, benzene acetic acid, dibutyl phthalate, octacosane, hexacosane, and vitamin A aldehyde. These results strongly encourage the use of these eco-friendly isolates as a biocontrol against MDR pathogens that attack fish farms. Significance: Streptomyces spp. act as strong weapons for fighting multidrug resistance in pathogenic bacteria – one of the most important current threats to public health. They are additionally regarded as eco-friendly organisms that can be used as a biocontrol agent against infections that endanger fish farms.

3 Smartphone Digital Phenotyping for Unobtrusive and Continuous Assessment of Everyday Cognition and Movement Trajectories in Older Adults

Objective:To evaluate the feasibility, usability, and preliminary validity of a digital phenotyping protocol to capture everyday cognition and activities in vivo among older adults.Participants and Methods:Eight participants (M age=69.1 + 2.6; M education=18.0 + 1.4; 50% female; 88% non-Hispanic White) with normal cognition or mild cognitive impairment used an open-source smartphone application (mindLAMP) to passively and continuously capture sensor data including global positioning system (GPS) trajectories for a 4-week study period. Baseline neuropsychological tests and measures of depression, self-reported cognitive decline and mobility patterns were collected as external validators for digital data. Participants downloaded mindLAMP onto their smartphones and resumed their daily routines for 4 weeks before removing mindLAMP and completing a debriefing questionnaire. A cognitive composite was derived by averaging T-scores across domains of attention, executive functioning, processing speed, memory, and language. GPS raw data were processed to generate monthly average and standard deviation mobility metrics for each participant, including time spent at home, distance travelled, radius of gyration, flight length, and circadian routine. Feasibility and usability findings are presented along with correlation coefficients >.4 between GPS metrics and external validators.Results:100% of enrolled participants completed the 4-week study without requesting to withdraw. Usability ratings ranged from poor to excellent. 75% of participants agreed that mindLAMP was easy to use, whereas only 1 participant enjoyed using mindLAMP. 100% of participants were satisfied with the study team’s explanation of procedures, privacy safeguards, data encryption methods and risks/benefits, reflected in an average score of 98.8% on the comprehension of consent quiz. No participants reported feeling uncomfortable, suspicious, or paranoid due to the study application running on their smartphone. No participants endorsed new problems using their smartphone, though 75% reported charging it more frequently during the study period. On average each day, participants spent 1121 + 227 minutes at home, travelled 38727 + 36210 geodesic units, and had 201 + 149 minutes of missing GPS data. Overall, greater amounts of activity (monthly average) and higher variability (monthly standard deviation) in GPS metrics were associated with better outcomes. Specifically, less time spent at home, greater distance travelled, larger radius of gyration, greater flight length, and greater variability in home time, distance travelled, radius of gyration and flight length were associated with less depression, less self-reported cognitive decline, better cognition, and greater self-reported mobility (.40< |r| <.69). On the other hand, greater circadian routine was associated with more self-reported cognitive decline (r=.66) and less self-reported mobility (r=-.43).Conclusions:Smartphone digital phenotyping is a feasible and acceptable method to capture everyday activities in older adults. Continuous collection of data from personal devices warrants caution; however, participants denied privacy concerns and expressed an overall positive experience. High frequency GPS data collection impacts battery life and should be considered among relative risks and confounds to naturalistic assessment. Patterns of behavior from passive smartphone data show promise as an unobtrusive method to identify cognitive risk and resilience in older adults. Subsequent analyses will evaluate additional sensor metrics across a larger and more heterogeneous cohort.

Unveiling the phenotypic landscape of stalk lodging resistance in diverse maize hybrids

ContextStalk lodging causes up to 43 % of yield losses in maize (Zea mays L.) worldwide, significantly worsening food and feed shortages. Stalk lodging resistance is a complex trait specified by several structural, material, and geometric phenotypes. However, the identity, relative contribution, and genetic tractability of these intermediate phenotypes remain unknown. ObjectiveThe study is designed to identify and evaluate plant-, organ-, and tissue-level intermediate phenotypes associated with stalk lodging resistance following standardized phenotyping protocols and to understand the variation and genetic tractability of these intermediate phenotypes. MethodsWe examined 16 diverse maize hybrids in two environments to identify and evaluate intermediate phenotypes associated with stalk flexural stiffness, a reliable indicator of stalk lodging resistance, at physiological maturity. Engineering-informed and machine learning models were employed to understand relationships among intermediate phenotypes and stalk flexural stiffness. ResultsStalk flexural stiffness showed significant genetic variation and high heritability (0.64) in the evaluated hybrids. Significant genetic variation and comparable heritability for the cross-sectional moment of inertia and Young's modulus indicated that geometric and material properties are under tight genetic control and play a combinatorial role in determining stalk lodging resistance. Among the twelve internode-level traits measured on the bottom and the ear internode, most traits exhibited significant genetic variation among hybrids, moderate to high heritability, and considerable effect of genotype × environment interaction. The marginal statistical model based on structural engineering beam theory revealed that 74–80 % of the phenotypic variation for flexural stiffness was explained by accounting for the major diameter, minor diameter, and rind thickness of the stalks. The machine learning model explained a relatively modest proportion (58–62 %) of the variation for flexural stiffness. ConclusionsCharacterization of stalk and internode properties using standard phenotyping methods revealed tremendous variation for intermediate phenotypes underlying stalk lodging resistance. The intermediate phenotypes showed moderate to high heritability, indicating their genetic tractability for improving stalk lodging resistance. Stalk geometric and material properties showed complementarity in determining stalk flexural stiffness. Engineering-informed models outperformed machine learning approaches in explaining variation for flexural stiffness. ImplicationsIdentification of genetically tractable intermediate phenotypes will boost efforts toward genetic improvement of stalk lodging resistance in maize. Discovering the genetic architecture of the intermediate traits will enhance our understanding of the biological underpinning of stalk lodging resistance.

Standardized high-dimensional spectral cytometry protocol and panels for whole blood immune phenotyping in clinical and translational studies.

Flow cytometry is the method of choice for immunophenotyping in the context of clinical, translational, and systems immunology studies. Among the latter, the Milieu Intérieur (MI) project aims at defining the boundaries of a healthy immune response to identify determinants of immune response variation. MI used immunophenotyping of a 1000 healthy donor cohort by flow cytometry as a principal outcome for immune variance at steady state. New generation spectral cytometers now enable high-dimensional immune cell characterization from small sample volumes. Therefore, for the MI 10-year follow up study, we have developed two high-dimensional spectral flow cytometry panels for deep characterization of innate and adaptive whole blood immune cells (35 and 34 fluorescent markers, respectively). We have standardized the protocol for sample handling, staining, acquisition, and data analysis. This approach enables the reproducible quantification of over 182 immune cell phenotypes at a single site. We have applied the protocol to discern minor differences between healthy and patient samples and validated its value for application in immunomonitoring studies. Our protocol is currently used for characterization of the impact of age and environmental factors on peripheral blood immune phenotypes of >400 donors from the initial MI cohort.

FRI142 The Pathophysiologic Spectrum Of Primary Aldosteronism Determined Via Saline Suppression Testing In Overweight Hypertensive And Pre-hypertensive People

Abstract Disclosure: W.W. Parksook: None. J.M. Brown: Consulting Fee; Self; Bayer, Inc. J. Milks: None. A. Moore: None. Y. Niebuhr: None. B. Honzel: None. A. Vaidya: Consulting Fee; Self; Corcept, HRA Pharmaceuticals, Mineralys. Background: Emerging evidence shows that primary aldosteronism is not a categorical condition, but rather a pathophysiologic continuum that manifests across a broad range of severity. Saline suppression testing has long been used to “confirm” primary aldosteronism while employing relatively arbitrary and categorical thresholds. Methods: Overweight to obese participants with metabolic risk factors and pre-hypertension or stage 1 hypertension with suppressed renin activity (<1.0 ng/mL/h) (n=42) underwent deep phenotyping protocols to assess the pathophysiologic continuum of the primary aldosteronism phenotype. Participants completed a seated saline suppression test (SSST), to maximally suppress Angiotensin II-mediated aldosterone production, and dexamethasone suppression and cosyntropin stimulation testing, to maximally suppress and stimulate ACTH-mediated aldosterone production. The association of each parameter with the continuum of aldosterone levels was assessed using linear regression. Results: Following SSST, there was a continuum of non-suppressible and renin-independent aldosterone production. Categorically, 29% of participants had post-SSST plasma aldosterone levels ≥10 ng/dL, and another 45% had levels 6-10 ng/dL; however, there was a continuous spectrum of aldosterone values above and below these arbitrary thresholds. Across the continuum of post-SSST aldosterone levels, greater non-suppressibility of aldosterone was associated with greater non-suppressibility of aldosterone post-dexamethasone (β= 1.05, P<0.001), greater stimulation of aldosterone post-cosyntropin administration (β= 1.42, P<0.001), and greater aldosterone-to-renin ratio (β= 3.44, P=0.01). Conclusion: Despite existing categorical thresholds for SSST, dynamic phenotyping studies in overweight/obese stage I hypertensive and pre-hypertensive individuals demonstrate a continuum of non-suppressible and renin-independent aldosterone production following SSST that parallels higher levels of ACTH-dependent aldosterone production. These findings underscore the fallibility of currently recommended diagnostic limits for SSST and suggest that primary aldosteronism pathophysiology transcends these relatively arbitrary thresholds. Presentation: Friday, June 16, 2023

OMIP-097: High-parameter phenotyping of human platelets by spectral flow cytometry.

Using spectral flow cytometry, we developed a 16-color panel for analysis of platelet phenotype and function in human whole blood. The panel contains markers of clinical relevance and follows an optimized protocol for the high-parameter phenotyping of (phosphatidylserine positive) procoagulant platelets. Inclusion of established markers, such as CD62P and PAC-1, allows the subsetting of classic (proinflammatory and proaggregatory) phenotypes, while addition of novel markers, such as TLR9, allows the resolution of platelets with nonclassic functions. Multiple inducible (C3b, CD63, CD107a, CD154, and TLT-1) and constitutive (CD29, CD31, CD32, CD36, CD42a, CD61, and GPVI) markers are also measurable, and we demonstrate the use of automatic gating for platelet analysis. The panel is widely applicable to research and clinical settings and can be readily modified, should users wish to tailor the panel to more specific needs.

Digital phenotyping: An equal opportunity approach to reducing disparities in Alzheimer's disease and related dementia research.

A rapidly aging world population is fueling a concomitant increase in Alzheimer's disease (AD) and related dementias (ADRD). Scientific inquiry, however, has largely focused on White populations in Australia, the European Union, and North America. As such, there is an incomplete understanding of AD in other populations. In this perspective, we describe research efforts and challenges of cohort studies from three regions of the world: Central America, East Africa, and East Asia. These cohorts are engaging with the Davos Alzheimer's Collaborative (DAC), a global partnership that brings together cohorts from around the world to advance understanding of AD. Each cohort is poised to leverage the widespread use of mobile devices to integrate digital phenotyping into current methodologies and mitigate the lack of representativeness in AD research of racial and ethnic minorities across the globe. In addition to methods that these three cohorts are already using, DAC has developed a digital phenotyping protocol that can collect ADRD-related data remotely via smartphone and/or in clinic via a tablet to generate a common data elements digital dataset that can be harmonized with additional clinical and molecular data being collected at each cohort site and when combined across cohorts and made accessible can provide a global data resource that is more racially/ethnically represented of the world population.

Emergence of New Delhi metallo-β-lactamase-1 (NDM-1) producing Enterobacterales from water sources: An impending public health challenge in Adamawa-north senatorial zone, Nigeria

Background: The emergence of New Delhi metallo-beta-lactamase-1 (NDM-1) among Enterobacterales in water sources has raised a major public health concern and constitute critical threat to human health as these organisms exhibits high level of resistance to available potent antibiotics. The aim of this study is to detect the presence of NDM-1 gene among carbapenem resistant Enterobacterales (CRE) isolates from water sources.
 Methodology: A total of 256 water samples were collected from randomly selected hand-dug wells (128 samples) and river/stream (128 samples) for each of dry and rainy seasons in four out of the five local government areas (LGAs) of Adamawa-north senatorial zone, Nigeria. The water samples were filtered using membrane filtration technique and the filters introduced into appropriate bacteriologic media for bacterial growth. The bacterial isolates recovered were identified by both phenotypic and molecular protocols. Phenotypic carbapenem (imipenem) resistance was determined by disc diffusion test, blaNDM-1 gene was detected by specific polymerase chain reaction (PCR) test, and plasmid DNA was extracted and electrophoresed by standard procedure.
 Results: Of the 256 water samples analyzed for bacteria growth, 300 bacterial isolates of the order Enterobacterales were recovered. Of these, only 45 (12.6%) isolates were phenotypically resistant to carbapenem (imipenem) antibiotic and blaNDM-1 gene was detected in 30 (66.7%) of these. While blaNDM-1 gene was detected in all the isolates of Klebsiella oxytoca, Klebsiella variicola, Enterobacter aerogenes, Enterobacter hormaechei, Enterobacter asburiae, Citrobacter freundii, and Morganella morganii that were resistant to imipenem, other isolates harbored blaNDM-1 gene in varying proportion. Most of the isolates positive for blaNDM-1 also harbored R-plasmids.
 Conclusion: Emergence of carbapenem resistance mediated by NDM-1 gene in Enterobacterales isolated from water sources constitutes an emerging public health challenge with potential transmission to humans, thereby complicating the treatment of infections caused by these resistant pathogens in man. As such, the urgent need for antimicrobial surveillance and stewardship is of utmost importance.

Development of a phenotyping protocol for combined drought and salinity stress at seedling stage in rice.

The case of combined drought and salinity stress is increasingly becoming a constraint to rice production, especially in coastal areas and river deltas where low rainfall not only reduces soil moisture levels but also reduces the flow of river water, resulting in intrusion of saline sea-water. A standardized screening method is needed in order to systematically evaluate rice cultivars under combined drought+salinity at the same time because sequential stress of salinity followed by drought or vice-versa is not similar to simultaneous stress effects. Therefore, we aimed to develop a screening protocol for combined drought+salinity stress applied to soil-grown plants at seedling stage. The study system used 30-L soil-filled boxes, which allowed a comparison of plant growth under control conditions, individual drought and salinity stress, as well as combined drought+salinity. A set of salinity tolerant and drought tolerant cultivars were tested, together with several popular but salinity and drought-susceptible varieties that are grown in regions prone to combined drought+salinity. A range of treatments were tested including different timings of the drought and salinity application, and different severities of stress, in order to determine the most effective that resulted in visible distinction among cultivars. The challenges related to determining a protocol with repeatable seedling stage stress treatment effects while achieving a uniform plant stand are described here. The optimized protocol simultaneously applied both stresses by planting into saline soil at 75% of field capacity which was then allowed to undergo progressive drydown. Meanwhile, physiological characterization revealed that chlorophyll fluorescence at seedling stage correlated well with grain yield when drought stress was applied to vegetative stage only. The drought+salinity protocol developed here can be used for screening rice breeding populations as part of a pipeline to develop new rice varieties with improved adaptation to combined stresses.