Cellaca® PLX image cytometer as an alternative for immunophenotyping, GFP/RFP transfection efficiencies, and apoptosis analysis.

Cell and gene therapy is a fast-growing field for cancer therapeutics requiring reliable instrumentation and technologies. Key parameters essential for satisfying Chemistry Manufacturing and Controls criteria standards are routinely performed using flow cytometry. Recently, image cytometry was developed for cell characterization and cell-based assays but had not yet demonstrated sufficient sensitivity for surface marker detection. We developed the Cellaca® PLX image cytometry system and the respective methodologies required for immunophenotyping, GFP and RFP transfection/transduction efficiencies, and cell health analyses for routine cell characterization. All samples tested were compared directly to results from the CytoFLEX flow cytometer. PBMCs were stained with T-cell surface markers for immunophenotyping, and results show highly comparable CD3, CD4, and CD8 populations (within 5 %). GFP- or RFP-expressing cell lines were analyzed for transfection/transduction efficiencies, and the percentage positive cells and respective viabilities were equivalent on both systems. Staurosporine-treated Jurkat cells were stained for apoptotic markers, where annexin V and caspase-3 positive cells were within 5 % comparing both instruments. The proposed system may provide a complementary tool for performing routine cell-based experiments with improved efficiency and sensitivity compared to prior image cytometers, which may be significantly valuable to the cell and gene therapy field.

Automated Assessment of Cancer Drug Efficacy On Breast Tumor Spheroids in Aggrewell™400 Plates Using Image Cytometry.

Tumor spheroid models have proven useful in the study of cancer cell responses to chemotherapeutic compounds by more closely mimicking the 3-dimensional nature of tumors in situ. Their advantages are often offset, however, by protocols that are long, complicated, and expensive. Efforts continue for the development of high-throughput assays that combine the advantages of 3D models with the convenience and simplicity of traditional 2D monolayer methods. Herein, we describe the development of a breast cancer spheroid image cytometry assay using T47D cells in Aggrewell™400 spheroid plates. Using the Celigo® automated imaging system, we developed a method to image and individually track thousands of spheroids within the Aggrewell™400 microwell plate over time. We demonstrate the use of calcein AM and propidium iodide staining to study the effects of known anti-cancer drugs Doxorubicin, Everolimus, Gemcitabine, Metformin, Paclitaxel and Tamoxifen. We use the image cytometry results to quantify the fluorescence of calcein AM and PI as well as spheroid size in a dose dependent manner for each of the drugs. We observe a dose-dependent reduction in spheroid size and find that it correlates well with the viability obtained from the CellTiter96® endpoint assay. The image cytometry method we demonstrate is a convenient and high-throughput drug-response assay for breast cancer spheroids under 400 µm in diameter, and may lay a foundation for investigating other three-dimensional spheroids, organoids, and tissue samples.

Risk Classification for Progression to Subfoveal Geographic Atrophy in Dry Age-Related Macular Degeneration Using Machine Learning-Enabled Outer Retinal Feature Extraction.

BACKGROUND AND OBJECTIVE: To evaluate the utility of spectral-domain optical coherence tomography biomarkers to predict the development of subfoveal geographic atrophy (sfGA). PATIENTS AND METHODS: This was a retrospective cohort analysis including 137 individuals with dry age-related macular degeneration without sfGA with 5 years of follow-up. Multiple spectral-domain optical coherence tomography quantitative metrics were generated, including ellipsoid zone (EZ) integrity and subretinal pigment epithelium (sub-RPE) compartment features. RESULTS: Reduced mean EZ-RPE central subfield thickness and increased sub-RPE compartment thickness were significantly different between sfGA convertors and nonconvertors at baseline in both 2-year and 5-year sfGA risk assessment. Longitudinal change assessment showed a significantly higher degradation of EZ integrity in sfGA convertors. The predictive performance of a machine learning classification model based on 5-year and 2-year risk conversion to sfGA demonstrated an area under the receiver operating characteristic curve of 0.92 ± 0.06 and 0.96 ± 0.04, respectively. CONCLUSIONS: Quantitative outer retinal and sub-RPE feature assessment using a machine learning-enabled retinal segmentation platform provides multiple parameters that are associated with progression to sfGA. [Ophthalmic Surg Lasers Imaging. 2022;53:31-39.].

Understanding Leisure Centre-Based Physical Activity after Physical Activity Referral: Evidence from Scheme Participants and Completers in Northumberland UK.

Physical activity referral schemes (PARS) are a popular physical activity (PA) intervention in the UK. Little is known about the type, intensity and duration of PA undertaken during and post PARS. We calculated weekly leisure centre-based moderate/vigorous PA for PARS participants (n = 448) and PARS completers (n = 746) in Northumberland, UK, between March 2019-February 2020 using administrative data. We categorised activity levels (<30 min/week, 30-149 min/week and ≥150 min/week) and used ordinal regression to examine predictors for activity category achieved. PARS participants took part in a median of 57.0 min (IQR 26.0-90.0) and PARS completers a median of 68.0 min (IQR 42.0-100.0) moderate/vigorous leisure centre-based PA per week. Being a PARS completer (OR: 2.14, 95% CI: 1.61-2.82) was a positive predictor of achieving a higher level of physical activity category compared to PARS participants. Female PARS participants were less likely (OR: 0.65, 95% CI: 0.43-0.97) to achieve ≥30 min of moderate/vigorous LCPA per week compared to male PARS participants. PARS participants achieved 38.0% and PARS completers 45.3% of the World Health Organisation recommended ≥150 min of moderate/vigorous weekly PA through leisure centre use. Strategies integrated within PARS to promote PA outside of leisure centre-based activity may help participants achieve PA guidelines.

Multiple behavior-specific cancellation signals contribute to suppressing predictable sensory reafference in a cerebellum-like structure.

Movement induces sensory stimulation of an animal's own sensory receptors, termed reafference. With a few exceptions, notably vestibular and proprioception, this reafference is unwanted sensory noise and must be selectively filtered in order to detect relevant external sensory signals. In the cerebellum-like electrosensory nucleus of elasmobranch fish, an adaptive filter preserves novel signals by generating cancellation signals that suppress predictable reafference. A parallel fiber network supplies the principal Purkinje-like neurons (called ascending efferent neurons, AENs) with behavior-associated internal reference signals, including motor corollary discharge and sensory feedback, from which predictive cancellation signals are formed. How distinct behavior-specific cancellation signals interact within AENs when multiple behaviors co-occur and produce complex, changing patterns of reafference is unknown. Here, we show that when multiple streams of internal reference signals are available, cancellation signals form that are specific to parallel fiber inputs temporally correlated with, and therefore predictive of, sensory reafference. A single AEN has the capacity to form more than one cancellation signal, and AENs form multiple cancellation signals simultaneously and modify them independently during co-occurring behaviors. Cancellation signals update incrementally during continuous behaviors, as well as episodic bouts of behavior that last minutes to hours. Finally, individual AENs, independently of their neighbors, form unique AEN-specific cancellation signals that depend on the particular sensory reafferent input it receives. Together, these results demonstrate the capacity of the adaptive filter to utilize multiple cancellation signals to suppress dynamic patterns of reafference arising from complex co-occurring and intermittently performed behaviors.

The Contribution of Leisure Center Usage to Physical Activity in the United Kingdom: Evidence From a Large Population-Based Cohort.

BACKGROUND: Physical activity (PA) levels vary across specific population groups, contributing to health inequalities. Little is known about how local authority leisure centers contribute to population PA and whether this differs by age, sex, or socioeconomic group. METHODS: The authors calculated weekly leisure center-based moderate/vigorous PA for 20,904 registered adult users of local authority leisure facilities in Northumberland, United Kingdom, between July 2018 and June 2019, using administrative data. The authors categorized activity levels (<30, 30-149, and ≥150 min/wk) and used ordinal regression to examine predictors for activity category achieved. RESULTS: Registered users were mainly female (58.7%), younger (23.9% of users aged 18-29 y vs 10.1% of those aged 70+ y), and from the 2 most affluent socioeconomic quintiles (53.7%). Median weekly moderate/vigorous leisure center-based activity was 55 minutes per week (interquartile range: 30-99). Being female (odds ratio: 2.09; 95% confidence interval, 1.95-2.35), older (odds ratio: 1.14; 95% confidence interval, 1.11-1.16), and using a large facility (odds ratio: 1.21; 95% confidence interval, 1.03-1.42) were positive predictors of leisure center-based PA. CONCLUSION: Older adults and females were more likely to be active and achieve the recommended PA levels through usage of the centers. Widespread use of this novel measure of leisure center-based activity would improve the understanding of how local authority leisure centers can address physical inactivity and its associated inequalities.

An expanding bacterial colony forms a depletion zone with growing droplets.

Many species of bacteria have developed effective means to spread on solid surfaces. This study focuses on the expansion of Pseudomonas aeruginosa on an agar gel surface under conditions of minimal evaporation. We report the occurrence and spread of a depletion zone within an expanded colony, where the bacteria laden film becomes thinner. The depletion zone is colocalized with a higher concentration of rhamnolipids, the biosurfactants that are produced by the bacteria and accumulate in the older region of the colony. With continued growth in population, dense bacterial droplets occur and coalesce in the depletion zone, displaying remarkable fluid dynamic behavior. Whereas expansion of a central depletion zone requires activities of live bacteria, new zones can be seeded elsewhere by adding rhamnolipids. These depletion zones due to the added surfactants expand quickly, even on plates covered by bacteria that have been killed by ultraviolet light. We explain the observed properties based on considerations of bacterial growth and secretion, osmotic swelling, fluid volume expansion, interfacial fluid dynamics involving Marangoni and capillary flows, and cell-cell cohesion.

Incorporation and Use of Earth Remote Sensing Imagery within the NOAA/NWS Damage Assessment Toolkit.

Severe weather events including tornadoes, damaging winds, hail, and their combination produce changes in land surface vegetation and urban settings that are frequently observed through remote sensing. Capabilities continue to improve through a growing constellation of governmental and commercial assets, increasing the spatial resolution of visible, near to shortwave infrared, and thermal infrared remote sensing. Here, we highlight cases where visual interpretation of imagery benefitted severe weather damage assessments made within the NOAA/NWS Damage Assessment Toolkit. Examples demonstrate utility of imagery in assessing tracks and changes in remote areas where staffing limitations or access prevent a ground-based assessment.

Discovery of oscillations in rotational speed of body-tethered Caulobacter crescentus.

Swarmer cells of Caulobacter crescentus have been found to tether to glass at a point on the cell body. The rolling of the freely rotating flagellum near the glass surface causes the cell body to rotate. We describe the discovery of damped oscillations in the rotational speed of these cell bodies. We show that the damped oscillations are robust over multiple cells and that they depend more on the cell's accumulated rotation angle than on time. We also find that their phase is determined by the moment the flagellar motor changes the direction of its rotation. The oscillations occur only for one direction of cell rotation, when the flagellum is in pulling mode. We discuss possible explanations for these oscillations, including fluctuations in flagellar motor torque and periodic changes in flagellar orientation, and illustrate both of these cases using simplified computer models. Finally, we present the hypothesis that the oscillations are the result of fluctuations in the proton motive force, initiated by a sudden change in proton current that occurs when the motor switches rotation direction.

Spatial, Temporal, and Electrical Characteristics of Lightning in Reported Lightning-Initiated Wildfire Events.

Analysis was performed to determine if a lightning flash could be associated with every reported lightning-initiated wildfire that grew to at least 4 km2. In total, 905 lightning-initiated wildfires within CONUS between 2012 and 2015 were analyzed. Fixed and fire radius search methods showed that 81-88% of wildfires had a corresponding lightning flash within a 14 day period prior to the report date. The two methods showed that 52-60% of lightning-initiated wildfire were reported on the same day as the closest lightning flash. The fire radius method indicated the most promising spatial results, where the median distance between the closest lightning and the wildfire start location was 0.83 km, followed by a 75th percentile of 1.6 km, and a 95th percentile of 5.86 km. Ninety percent of the closest lightning flashes to wildfires were negative polarity. Maximum flash densities were less than 0.41 flashes km2 for the 24 hour period at the fire start location. The majority of lightning-initiated holdover events were observed in the Western CONUS, with a peak density in north-central Idaho. A twelve day holdover event from New Mexico was also discussed; outlining the opportunities and limitations of using lightning data to characterize wildfires.

Satellite-based assessment of electricity restoration efforts in Puerto Rico after Hurricane Maria.

A real-time understanding of the distribution and duration of power outages after a major disaster is a precursor to minimizing their harmful consequences. Here, we develop an approach for using daily satellite nighttime lights data to create spatially disaggregated power outage estimates, tracking electricity restoration efforts after disasters strike. In contrast to existing utility data, these estimates are independent, open, and publicly-available, consistently measured across regions that may be serviced by several different power companies, and inclusive of distributed power supply (off-grid systems). We apply the methodology in Puerto Rico following Hurricane Maria, which caused the longest blackout in US history. Within all of the island's settlements, we track outages and recovery times, and link these measures to census-based demographic characteristics of residents. Our results show an 80% decrease in lights, in total, immediately after Hurricane Maria. During the recovery, a disproportionate share of long-duration power failures (> 120 days) occurred in rural municipalities (41% of rural municipalities vs. 29% of urban municipalities), and in the northern and eastern districts. Unexpectedly, we also identify large disparities in electricity recovery between neighborhoods within the same urban area, based primarily on the density of housing. For many urban areas, poor residents, the most vulnerable to increased mortality and morbidity risks from power losses, shouldered the longest outages because they lived in less dense, detached housing where electricity restoration lagged. The approach developed in this study demonstrates the potential of satellite-based estimates of power recovery to improve the real-time monitoring of disaster impacts, globally, at a spatial resolution that is actionable for the disaster response community.

Flagellar Motor Switching in Caulobacter Crescentus Obeys First Passage Time Statistics.

A Caulobacter crescentus swarmer cell is propelled by a helical flagellum, which is rotated by a motor at its base. The motor alternates between rotating in clockwise and counterclockwise directions and spends variable intervals of time in each state. We measure the distributions of these intervals for cells either free swimming or tethered to a glass slide. A peak time of around one second is observed in the distributions for both motor directions with counterclockwise intervals more sharply peaked and clockwise intervals displaying a larger tail at long times. We show that distributions of rotation intervals fit first passage time statistics for a biased random walker and the dynamic binding of CheY-P to FliM motor subunits accounts for this behavior. Our results also suggest that the presence of multiple CheY proteins in C. crescentus may be responsible for differences between its switching behavior and that of the extensively studied E. coli.

Visualization of inflammation and demyelination in 2D2 transgenic mice with rodent MRI.

Research tools are urgently needed to elucidate the specificities of NMO and MS due to their clinical similarity at the early stage of the diseases. Herein, using high-field-strength MRI we characterized the optic nerve and spinal cord lesions in 2D2(tg) mice (MOG 35-55 specific TCR). Specifically, early Blood-brain Barrier breakdown was observed in 86% of the 2D2(tg) mice, while the majority of mice showed little to no brain lesions. Further, immunohistology showed inflammatory infiltrates and demyelination in the brain and spinal cord that mirrored sites of MRI lesions, along with a decrease in AQP4 protein at lesion sites. Collectively, 2D2(tg) mice develop optic and spinal cord lesions that can be visualized by high-field rodent MRI and verified by pathological assessment. The similarity of these lesions with those seen in NMO patients suggests that the 2D2(tg) mouse might serve as a model for NMO research.

Regulation of cytochrome P450 4F11 by nuclear transcription factor-κB.

Although the mechanisms that regulate CYP4F genes have been and are currently being studied in a number of laboratories, the specific mechanisms for the regulation of these genes are not yet fully understood. This study shows that nuclear factor κB of the light-chain-enhancer in activated B cells (NF-κB) can inhibit CYP4F11 expression in human liver carcinoma cell line (HepG2) as summarized below. Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, has been shown to activate NF-κB signaling while also activating the c-Jun NH(2)-terminal kinase (JNK) signaling pathway. Other studies have reported that JNK signaling can up-regulate CYP4F11 expression. The results of this study demonstrate that in the presence of TNF-α and the specific NF-κB translocation inhibitor N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (IMD-0354), there is a greater increase in CYP4F11 expression than that elicited by TNF-α alone, indicating that NF-κB plays an inhibitory role. Moreover, NF-κB stimulation by overexpression of mitogen-activated protein kinase kinase kinase inhibited CYP4F11 promoter expression. CYP4F11 promoter inhibition can also be rescued in the presence of TNF-α when p65, a NF-κB protein, is knocked down. Thus, NF-κB signaling pathways negatively regulate the CYP4F11 gene.

Gene regulation of CYP4F11 in human keratinocyte HaCaT cells.

Mechanisms regulating CYP4F genes remain under investigation, although characterization of CYP4F regulatory modalities would facilitate the discovery of new drug targets. This present study shows that all-trans- and 9-cis-retinoic acids can inhibit CYP4F11 expression in human keratinocyte-derived HaCaT cells. Transrepression of many genes by retinoic acids is mediated by interactions between retinoid receptors and the activator protein 1 (AP-1) complex. Proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta, which can activate the AP-1 complex, induce CYP4F11 transcription in HaCaT cells. The c-Jun N-terminal kinase (JNK)-specific inhibitor 1,9-pyrazoloanthrone (SP600125) blocked the induction of CYP4F11 by both cytokines, indicating involvement of the JNK pathway. Furthermore, TNF-alpha failed to induce CYP4F11 transcription when HaCaT cells were preincubated with retinoic acids. Retinoic acids are ligands for the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). The RXR agonist 6-(1(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl) nicotinic acid (LG268) greatly induced CYP4F11 transcription, whereas the RAR agonist 4-(2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid (TTNPB) markedly inhibited CYP4F11 transcription, indicating that down-regulation of CYP4F11 transcription by retinoic acid is mediated by RARs and may also be related to ligand competition for RXRs. Thus, the CYP4F11 gene is positively regulated by multiple signaling pathways in HaCaT keratinocytes, including RXR and JNK signaling pathways.

Attachment representation and suicidal ideation in Vietnam combat veterans with posttraumatic stress disorder.

Attachment representation has been linked to psychopathology and suicidality. Possible links between attachment representations and suicidal ideation in a PTSD sample were examined. Vietnam combat veterans in treatment for PTSD (N = 48) were assessed for PTSD severity, attachment representation and current and lifetime suicidality. Contrary to expectation individuals with secure attachment representations had higher levels of current suicidal ideation than those with insecure attachment representations, and unresolved/disorganized attachment was associated with lower levels of current suicidal ideation. Secure attachment may not provide protection against high levels of distress and suicidal ideation among combat veterans with PTSD.

Attachment organization in Vietnam combat veterans with posttraumatic stress disorder.

Attachment organization in a combat-related PTSD sample was investigated and compared with previously published clinical and non-clinical samples. The association between insecure attachment and unresolved mourning classification (U-loss) and between U-loss and PTSD symptoms was investigated. Vietnam combat veterans diagnosed with PTSD and in treatment (N = 48) were administered the Adult Attachment Interview, the SCID-IV, and CAPS. The PTSD sample was like non-clinical samples in the incidence of secure attachment (50%), but were more commonly unresolved. Veterans with insecure attachment organizations were more likely than those with secure attachment to be classified U-loss. U-loss classification was associated with greater likelihood of comorbid anxiety disorders and PTSD avoidance/numbing symptoms. The results suggest that while insecure attachment organization is associated with unresolved mourning in response to loss, it is not differentially associated with combat-related PTSD. The relationship between U-loss and PTSD is discussed in light of current literature.

Specific symptoms predict suicidal ideation in Vietnam combat veterans with chronic post-traumatic stress disorder.

Previous research documented the elevated risk of suicide and suicidal ideation among Vietnam veterans with post-traumatic stress disorder (PTSD). The aim of the current study was to examine which Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, PTSD symptom clusters are most associated with suicidal ideation in this population. Fifty Vietnam combat veterans enrolled in treatment for PTSD responded to the Beck Scale for Suicide Ideation and were interviewed with the Clinician-Administered PTSD Scale. In linear regression analysis, it was found that the reexperiencing symptom cluster was significantly associated with suicidal ideation but the other two symptom clusters (avoidance/numbing and increased arousal) were not. Furthermore, scores on a measure of severity of combat exposure were not found to be significantly related to PTSD symptoms or suicidal ideation. The results of this study suggest the importance of reexperiencing symptoms for predicting which individuals with combat-related PTSD are most at risk for suicidal ideation and behavior.

Impact of alcohol exposure after pregnancy recognition on ultrasonographic fetal growth measures.

BACKGROUND: More than 3 decades after Jones and Smith (1973) reported on the devastation caused by alcohol exposure on fetal development, the rates of heavy drinking during pregnancy remain relatively unchanged. Early identification of fetal alcohol exposure and maternal abstinence led to better infant outcomes. This study examined the utility of biometry for detecting alcohol-related fetal growth impairment. METHODS: We obtained fetal ultrasound measures from routine ultrasound examinations for 167 pregnant hazardous drinkers who were enrolled in a brief alcohol intervention study. The fetal measures for women who quit after learning of their pregnancies were compared with measures for women who continued some drinking throughout the course of their pregnancies. Because intensity of alcohol consumption is associated with poorer fetal outcomes, separate analyses were conducted for the heavy (average of >or=5 drinks per drinking day) alcohol consumers. Fetal measures from the heavy-exposed fetuses were also compared with measures from a nondrinking group that was representative of normal, uncomplicated pregnancies from our clinics. Analyses of covariance were used to determine whether there were differences between groups after controlling for influences of gestational age and drug abuse. RESULTS: Nearly half of the pregnant drinkers abstained after learning of their pregnancies. When women reportedly quit drinking early in their pregnancies, fetal growth measures were not significantly different from a non-alcohol-exposed group, regardless of prior drinking patterns. Any alcohol consumption postpregnancy recognition among the heavy drinkers resulted in reduced cerebellar growth as well as decreased cranial to body growth in comparison with women who either quit drinking or who were nondrinkers. Amphetamine abuse was predictive of larger cranial to body growth ratios. CONCLUSIONS: Alterations in fetal biometric measurements were observed among the heavy drinkers only when they continued drinking after becoming aware of their pregnancies. Although the reliance on self-reported drinking is a limitation in this study, these findings support the benefits of early abstinence and the potential for ultrasound examinations in the detection of fetal alcohol effects.