Celsr1 suppresses Wnt5a-mediated chemoattraction to prevent incorrect rostral migration of facial branchiomotor neurons.

In the developing hindbrain, facial branchiomotor (FBM) neurons migrate caudally from rhombomere 4 (r4) to r6 to establish the circuit that drives jaw movements. Although the mechanisms regulating initiation of FBM neuron migration are well defined, those regulating directionality are not. In mutants lacking the Wnt/planar cell polarity (PCP) component Celsr1, many FBM neurons inappropriately migrate rostrally into r3. We hypothesized that Celsr1 normally blocks inappropriate rostral migration of FBM neurons by suppressing chemoattraction towards Wnt5a in r3 and successfully tested this model. First, FBM neurons in Celsr1; Wnt5a double mutant embryos never migrated rostrally, indicating that inappropriate rostral migration in Celsr1 mutants results from Wnt5a-mediated chemoattraction, which is suppressed in wild-type embryos. Second, FBM neurons migrated rostrally toward Wnt5a-coated beads placed in r3 of wild-type hindbrain explants, suggesting that excess Wnt5a chemoattractant can overcome endogenous Celsr1-mediated suppression. Third, rostral migration of FBM neurons was greatly enhanced in Celsr1 mutants overexpressing Wnt5a in r3. These results reveal a novel role for a Wnt/PCP component in regulating neuronal migration through suppression of chemoattraction.

Versatile cell ablation tools and their applications to study loss of cell functions.

Targeted cell ablation is a powerful approach for studying the role of specific cell populations in a variety of organotypic functions, including cell differentiation, and organ generation and regeneration. Emerging tools for permanently or conditionally ablating targeted cell populations and transiently inhibiting neuronal activities exhibit a diversity of application and utility. Each tool has distinct features, and none can be universally applied to study different cell types in various tissue compartments. Although these tools have been developed for over 30 years, they require additional improvement. Currently, there is no consensus on how to select the tools to answer the specific scientific questions of interest. Selecting the appropriate cell ablation technique to study the function of a targeted cell population is less straightforward than selecting the method to study a gene's functions. In this review, we discuss the features of the various tools for targeted cell ablation and provide recommendations for optimal application of specific approaches.

A dynamic intracellular distribution of Vangl2 accompanies cell polarization during zebrafish gastrulation.

During vertebrate gastrulation, convergence and extension movements elongate embryonic tissues anteroposteriorly and narrow them mediolaterally. Planar cell polarity (PCP) signaling is essential for mediolateral cell elongation underlying these movements, but how this polarity arises is poorly understood. We analyzed the elongation, orientation and migration behaviors of lateral mesodermal cells undergoing convergence and extension movements in wild-type zebrafish embryos and mutants for the Wnt/PCP core component Vangl2 (Trilobite). We demonstrate that Vangl2 function is required at the time when cells transition to a highly elongated and mediolaterally aligned body. vangl2 mutant cells fail to undergo this transition and to migrate along a straight path with high net speed towards the dorsal midline. Instead, vangl2 mutant cells exhibit an anterior/animal pole bias in cell body alignment and movement direction, suggesting that PCP signaling promotes effective dorsal migration in part by suppressing anterior/animalward cell polarity and movement. Endogenous Vangl2 protein accumulates at the plasma membrane of mesenchymal converging cells at the time its function is required for mediolaterally polarized cell behavior. Heterochronic cell transplantations demonstrated that Vangl2 cell membrane accumulation is stage dependent and regulated by both intrinsic factors and an extracellular signal, which is distinct from PCP signaling or other gastrulation regulators, including BMP and Nodals. Moreover, mosaic expression of fusion proteins revealed enrichment of Vangl2 at the anterior cell edges of highly mediolaterally elongated cells. These results demonstrate that the dynamic Vangl2 intracellular distribution is coordinated with and necessary for the changes in convergence and extension cell behaviors during gastrulation.

The atypical cadherin Celsr1 functions non-cell autonomously to block rostral migration of facial branchiomotor neurons in mice.

The caudal migration of facial branchiomotor (FBM) neurons from rhombomere (r) 4 to r6 in the hindbrain is an excellent model to study neuronal migration mechanisms. Although several Wnt/Planar Cell Polarity (PCP) components are required for FBM neuron migration, only Celsr1, an atypical cadherin, regulates the direction of migration in mice. In Celsr1 mutants, a subset of FBM neurons migrates rostrally instead of caudally. Interestingly, Celsr1 is not expressed in the migrating FBM neurons, but rather in the adjacent floor plate and adjoining ventricular zone. To evaluate the contribution of different expression domains to neuronal migration, we conditionally inactivated Celsr1 in specific cell types. Intriguingly, inactivation of Celsr1 in the ventricular zone of r3-r5, but not in the floor plate, leads to rostral migration of FBM neurons, greatly resembling the migration defect of Celsr1 mutants. Dye fill experiments indicate that the rostrally-migrated FBM neurons in Celsr1 mutants originate from the anterior margin of r4. These data suggest strongly that Celsr1 ensures that FBM neurons migrate caudally by suppressing molecular cues in the rostral hindbrain that can attract FBM neurons.

Role of branchiomotor neurons in controlling food intake of zebrafish larvae.

The physical act of eating or feeding involves the coordinated action of several organs like eyes and jaws, and associated neural networks. Moreover, the activity of the neural networks controlling jaw movements (branchiomotor circuits) is regulated by the visual, olfactory, gustatory and hypothalamic systems, which are largely well characterized at the physiological level. By contrast, the behavioral output of the branchiomotor circuits and the functional consequences of disruption of these circuits by abnormal neural development are poorly understood. To begin to address these questions, we sought to evaluate the feeding ability of zebrafish larvae, a direct output of the branchiomotor circuits, and developed a qualitative assay for measuring food intake in zebrafish larvae at 7 days post-fertilization. We validated the assay by examining the effects of ablating the branchiomotor neurons. Metronidazole-mediated ablation of nitroreductase-expressing branchiomotor neurons resulted in a predictable reduction in food intake without significantly affecting swimming ability, indicating that the assay is robust. Laser-mediated ablation of trigeminal motor neurons resulted in a significant decrease in food intake, indicating that the assay is sensitive. Importantly, in larvae of a genetic mutant with severe loss of branchiomotor neurons, food intake was abolished. These studies establish a foundation for dissecting the neural circuits driving a motor behavior essential for survival.

The PCP protein Vangl2 regulates migration of hindbrain motor neurons by acting in floor plate cells, and independently of cilia function.

Vangl2, a core component of the Planar Cell Polarity pathway, is necessary for the caudal migration of Facial Branchiomotor (FBM) neurons in the vertebrate hindbrain. Studies in zebrafish suggest that vangl2 functions largely non-cell autonomously to regulate FBM neuron migration out of rhombomere 4 (r4), but the cell-type within which it acts is not known. Here, we demonstrate that vangl2 functions largely in floor plate cells to regulate caudal neuronal migration. Furthermore, FBM neurons fail to migrate caudally in the mouse Gli2 mutant that lacks the floor plate, suggesting an evolutionarily conserved role for this cell type in neuronal migration. Although hindbrain floor plate cilia are disorganized in vangl2 mutant embryos, cilia appear to be dispensable for neuronal migration. Notably, Vangl2 is enriched in the basolateral, but not apical, membranes of floor plate cells. Taken together, our data suggest strongly that Vangl2 regulates FBM neuron migration by acting in floor plate cells, independently of cilia function.

A smartphone application toward detection of systolic hypertension in underserved populations.

High systolic blood pressure (BP) is the most important modifiable risk factor for cardiovascular disease. Managing systolic hypertension is especially difficult in underserved populations wherein access to cuff BP devices is limited. We showed that ubiquitous smartphones without force sensing can be converted into absolute pulse pressure (PP) monitors. The concept is for the user to perform guided thumb and hand maneuvers with the phone to induce cuff-like actuation and allow built-in sensors to make cuff-like measurements for computing PP. We developed an Android smartphone PP application. The 'app' could be learned by volunteers and yielded PP with total error < 8 mmHg against cuff PP (N = 24). We also analyzed a large population-level database comprising adults less than 65 years old to show that PP plus other basic information can detect systolic hypertension with ROC AUC of 0.9. The smartphone PP app could ultimately help reduce the burden of systolic hypertension in underserved populations and thus health disparities.

The mouse Wnt/PCP protein Vangl2 is necessary for migration of facial branchiomotor neurons, and functions independently of Dishevelled.

During development, facial branchiomotor (FBM) neurons, which innervate muscles in the vertebrate head, migrate caudally and radially within the brainstem to form a motor nucleus at the pial surface. Several components of the Wnt/planar cell polarity (PCP) pathway, including the transmembrane protein Vangl2, regulate caudal migration of FBM neurons in zebrafish, but their roles in neuronal migration in mouse have not been investigated in detail. Therefore, we analyzed FBM neuron migration in mouse looptail (Lp) mutants, in which Vangl2 is inactivated. In Vangl2(Lp/+) and Vangl2(Lp/Lp) embryos, FBM neurons failed to migrate caudally from rhombomere (r) 4 into r6. Although caudal migration was largely blocked, many FBM neurons underwent normal radial migration to the pial surface of the neural tube. In addition, hindbrain patterning and FBM progenitor specification were intact, and FBM neurons did not transfate into other non-migratory neuron types, indicating a specific effect on caudal migration. Since loss-of-function in some zebrafish Wnt/PCP genes does not affect caudal migration of FBM neurons, we tested whether this was also the case in mouse. Embryos null for Ptk7, a regulator of PCP signaling, had severe defects in caudal migration of FBM neurons. However, FBM neurons migrated normally in Dishevelled (Dvl) 1/2 double mutants, and in zebrafish embryos with disrupted Dvl signaling, suggesting that Dvl function is essentially dispensable for FBM neuron caudal migration. Consistent with this, loss of Dvl2 function in Vangl2(Lp/+) embryos did not exacerbate the Vangl2(Lp/+) neuronal migration phenotype. These data indicate that caudal migration of FBM neurons is regulated by multiple components of the Wnt/PCP pathway, but, importantly, may not require Dishevelled function. Interestingly, genetic-interaction experiments suggest that rostral FBM neuron migration, which is normally suppressed, depends upon Dvl function.

Smartphone-Based Blood Pressure Monitoring via the Oscillometric Finger Pressing Method: Analysis of Oscillation Width Variations Can Improve Diastolic Pressure Computation.

OBJECTIVE: Oscillometric finger pressing is a potential method for absolute blood pressure (BP) monitoring via a smartphone. The user presses their fingertip against a photoplethysmography-force sensor unit on a smartphone to steadily increase the external pressure on the underlying artery. Meanwhile, the phone guides the finger pressing and computes systolic BP (SP) and diastolic BP (DP) from the measured blood volume oscillations and finger pressure. The objective was to develop and evaluate reliable finger oscillometric BP computation algorithms. METHODS: The collapsibility of thin finger arteries was exploited in an oscillometric model to develop simple algorithms for computing BP from the finger pressing measurements. These algorithms extract features from "width" oscillograms (oscillation width versus finger pressure functions) and the conventional "height" oscillogram for markers of DP and SP. Finger pressing measurements were obtained using a custom system along with reference arm cuff BP measurements from 22 subjects. Measurements were also obtained during BP interventions in some subjects for 34 total measurements. RESULTS: An algorithm employing the average of width and height oscillogram features predicted DP with correlation of 0.86 and precision error of 8.6 mmHg with respect to the reference measurements. Analysis of arm oscillometric cuff pressure waveforms from an existing patient database provided evidence that the width oscillogram features are better suited to finger oscillometry. CONCLUSION: Analysis of oscillation width variations during finger pressing can improve DP computation. SIGNIFICANCE: The study findings may help in converting widely available devices into truly cuffless BP monitors for improving hypertension awareness and control.

Tissue perfusion pressure enables continuous hemodynamic evaluation and risk prediction in the intensive care unit.

Treatment of circulatory shock in critically ill patients requires management of blood pressure using invasive monitoring, but uncertainty remains as to optimal individual blood pressure targets. Critical closing pressure, which refers to the arterial pressure when blood flow stops, can provide a fundamental measure of vascular tone in response to disease and therapy, but it has not previously been possible to measure this parameter routinely in clinical care. Here we describe a method to continuously measure critical closing pressure in the systemic circulation using readily available blood pressure monitors and then show that tissue perfusion pressure (TPP), defined as the difference between mean arterial pressure and critical closing pressure, provides unique information compared to other hemodynamic parameters. Using analyses of 5,988 admissions to a modern cardiac intensive care unit, and externally validated with 864 admissions to another institution, we show that TPP can predict the risk of mortality, length of hospital stay and peak blood lactate levels. These results indicate that TPP may provide an additional target for blood pressure optimization in patients with circulatory shock.

Mathematical Modeling of Oscillometric Blood Pressure Measurement: A Complete, Reduced Oscillogram Model.

OBJECTIVE: Oscillogram modeling is a powerful tool for understanding and advancing popular oscillometric blood pressure (BP) measurement. A reduced oscillogram model relating cuff pressure oscillation amplitude ( ∆O) to external cuff pressure of the artery ( Pe) is: [Formula: see text], where g(P) is the arterial compliance versus transmural pressure ( P) curve, Ps and Pd are systolic and diastolic BP, and k is the reciprocal of the cuff compliance. The objective was to determine an optimal functional form for the arterial compliance curve. METHODS: Eight prospective, three-parameter functions of the brachial artery compliance curve were compared. The study data included oscillometric arm cuff pressure waveforms and invasive brachial BP from 122 patients covering a 20-120 mmHg pulse pressure range. The oscillogram measurements were constructed from the cuff pressure waveforms. Reduced oscillogram models, inputted with measured systolic and diastolic BP and each parametric brachial artery compliance curve function, were optimally fitted to the oscillogram measurements in the least squares sense. RESULTS: An exponential-linear function yielded as good or better model fits compared to the other functions, with errors of 7.9±0.3 and 5.1±0.2% for tail-trimmed and lower half-trimmed oscillogram measurements. Importantly, this function was also the most tractable mathematically. CONCLUSION: A three-parameter exponential-linear function is an optimal form for the arterial compliance curve in the reduced oscillogram model and may thus serve as the standard function for this model henceforth. SIGNIFICANCE: The complete, reduced oscillogram model determined herein can potentially improve oscillometric BP measurement accuracy while advancing foundational knowledge.

Evolutionarily conserved function of Gbx2 in anterior hindbrain development.

The amino acid sequence across the DNA-binding homeodomain of Gbx2 is highly conserved across multiple species. In mice, Gbx2 is essential for establishment of the midbrain-hindbrain boundary (MHB), and in development of anterior hindbrain structures, rhombomeres (r) 1-r3, and the r2/r3-derived cranial nerve V. In contrast, studies in zebrafish have implicated gbx1 in establishment of the MHB. Therefore, we tested potential roles for gbx2 in anterior hindbrain development in zebrafish. gbx2 knockdown with antisense morpholino results in increased cell death in r2, r3, and r5 and a truncation of the anterior hindbrain, similar to the defect in Gbx2(-/-) mice. Moreover, there is abnormal clustering of cranial nerve V cell bodies in r2 and r3 indicative of defects in aspects of anterior hindbrain patterning. These phenotypes can be rescued by expression of the mouse GBX2 protein. These results suggest that gbx2/Gbx2 has an evolutionarily conserved role in anterior hindbrain development.

Photoplethysmography Fast Upstroke Time Intervals Can Be Useful Features for Cuff-Less Measurement of Blood Pressure Changes in Humans.

OBJECTIVE: Photoplethysmography (PPG) waveform analysis is being increasingly investigated for continuous, non-invasive, and cuff-less blood pressure (BP) measurement. However, the efficacy of this approach and the useful features and models remain largely unclear. The objectives were to develop easy-to-understand models relating PPG waveform features to BP changes (after a cuff calibration) and to determine their value in BP measurement accuracy. METHODS: The study data comprised finger, toe, and ear PPG waveforms, an ECG waveform, and reference manual cuff BP measurements from 32 human subjects (25% hypertensive) before and after slow breathing, mental arithmetic, cold pressor, and nitroglycerin administration. Stepwise linear regression was employed to create parsimonious models for predicting the intervention-induced BP changes from popular PPG waveform features, pulse arrival time (PAT, time delay between ECG R-wave and PPG foot), and subject demographics. Leave-one-subject-out cross validation was applied to compare the BP change prediction root-mean-squared-errors (RMSEs) of the resulting models to reference models in which PPG waveform features were excluded. RESULTS: Finger b-time (PPG foot to minimum second derivative time interval) and ear "STT" (PPG amplitude divided by maximum derivative), when combined with PAT, reduced the systolic BP change prediction RMSE of reference models by 6-7% (p 0.022). Ear STT together with pulse width reduced the diastolic BP change prediction RMSE of the reference model by 13% (p = 0.003). CONCLUSION: The two PPG fast upstroke time intervals can offer some added value in cuff-less BP trending. SIGNIFICANCE: This study offers important information towards achieving non-invasive and passive BP monitoring without a cuff.

Atypical cadherins Celsr1-3 differentially regulate migration of facial branchiomotor neurons in mice.

During hindbrain development, facial branchiomotor neurons (FBM neurons) migrate from medial rhombomere (r) 4 to lateral r6. In zebrafish, mutations in planar cell polarity genes celsr2 and frizzled3a block caudal migration of FBM neurons. Here, we investigated the role of cadherins Celsr1-3, and Fzd3 in FBM neuron migration in mice. In Celsr1 mutants (knock-out and Crash alleles), caudal migration was compromised and neurons often migrated rostrally into r2 and r3, as well as laterally. These phenotypes were not caused by defects in hindbrain patterning or neuronal specification. Celsr1 is expressed in FBM neuron precursors and the floor plate, but not in FBM neurons. Consistent with this, conditional inactivation showed that the function of Celsr1 in FBM neuron migration was non-cell autonomous. In Celsr2 mutants, FBM neurons initiated caudal migration but moved prematurely into lateral r4 and r5. This phenotype was enhanced by inactivation of Celsr3 in FBM neurons and mimicked by inactivation of Fzd3. Furthermore, Celsr2 was epistatic to Celsr1. These data indicate that Celsr1-3 differentially regulate FBM neuron migration. Celsr1 helps to specify the direction of FBM neuron migration, whereas Celsr2 and 3 control its ability to migrate.

Zebrafish trilobite identifies new roles for Strabismus in gastrulation and neuronal movements.

Embryonic morphogenesis is driven by a suite of cell behaviours, including coordinated shape changes, cellular rearrangements and individual cell migrations, whose molecular determinants are largely unknown. In the zebrafish, Dani rerio, trilobite mutant embryos have defects in gastrulation movements and posterior migration of hindbrain neurons. Here, we have used positional cloning to demonstrate that trilobite mutations disrupt the transmembrane protein Strabismus (Stbm)/Van Gogh (Vang), previously associated with planar cell polarity (PCP) in Drosophila melanogaster, and PCP and canonical Wnt/beta-catenin signalling in vertebrates. Our genetic and molecular analyses argue that during gastrulation, trilobite interacts with the PCP pathway without affecting canonical Wnt signalling. Furthermore, trilobite may regulate neuronal migration independently of PCP molecules. We show that trilobite mediates polarization of distinct movement behaviours. During gastrulation convergence and extension movements, trilobite regulates mediolateral cell polarity underlying effective intercalation and directed dorsal migration at increasing velocities. In the hindbrain, trilobite controls effective migration of branchiomotor neurons towards posterior rhombomeres. Mosaic analyses show trilobite functions cell-autonomously and non-autonomously in gastrulae and the hindbrain. We propose Trilobite/Stbm mediates cellular interactions that confer directionality on distinct movements during vertebrate embryogenesis.

Defective Neuronal Positioning Correlates With Aberrant Motor Circuit Function in Zebrafish.

Precise positioning of neurons resulting from cell division and migration during development is critical for normal brain function. Disruption of neuronal migration can cause a myriad of neurological disorders. To investigate the functional consequences of defective neuronal positioning on circuit function, we studied a zebrafish frizzled3a (fzd3a) loss-of-function mutant off-limits (olt) where the facial branchiomotor (FBM) neurons fail to migrate out of their birthplace. A jaw movement assay, which measures the opening of the zebrafish jaw (gape), showed that the frequency of gape events, but not their amplitude, was decreased in olt mutants. Consistent with this, a larval feeding assay revealed decreased food intake in olt mutants, indicating that the FBM circuit in mutants generates defective functional outputs. We tested various mechanisms that could generate defective functional outputs in mutants. While fzd3a is ubiquitously expressed in neural and non-neural tissues, jaw cartilage and muscle developed normally in olt mutants, and muscle function also appeared to be unaffected. Although FBM neurons were mispositioned in olt mutants, axon pathfinding to jaw muscles was unaffected. Moreover, neuromuscular junctions established by FBM neurons on jaw muscles were similar between wildtype siblings and olt mutants. Interestingly, motor axons innervating the interhyoideus jaw muscle were frequently defasciculated in olt mutants. Furthermore, GCaMP imaging revealed that mutant FBM neurons were less active than their wildtype counterparts. These data show that aberrant positioning of FBM neurons in olt mutants is correlated with subtle defects in fasciculation and neuronal activity, potentially generating defective functional outputs.

The cell adhesion molecule Tag1, transmembrane protein Stbm/Vangl2, and Lamininalpha1 exhibit genetic interactions during migration of facial branchiomotor neurons in zebrafish.

Interactions between a neuron and its environment play a major role in neuronal migration. We show here that the cell adhesion molecule Transient Axonal Glycoprotein (Tag1) is necessary for the migration of the facial branchiomotor neurons (FBMNs) in the zebrafish hindbrain. In tag1 morphant embryos, FBMN migration is specifically blocked, with no effect on organization or patterning of other hindbrain neurons. Furthermore, using suboptimal morpholino doses and genetic mutants, we found that tag1, lamininalpha1 (lama1) and stbm, which encodes a transmembrane protein Vangl2, exhibit pairwise genetic interactions for FBMN migration. Using time-lapse analyses, we found that FBMNs are affected similarly in all three single morphant embryos, with an inability to extend protrusions in a specific direction, and resulting in the failure of caudal migration. These data suggest that tag1, lama1 and vangl2 participate in a common mechanism that integrates signaling between the FBMN and its environment to regulate migration.

PPG Sensor Contact Pressure Should Be Taken Into Account for Cuff-Less Blood Pressure Measurement.

OBJECTIVE: Photo-plethysmography (PPG) sensors are often used to detect pulse transit time (PTT) for potential cuff-less blood pressure (BP) measurement. It is known that the contact pressure (CP) of the PPG sensor markedly alters the PPG waveform amplitude. The objective was to test the hypothesis that PTT detected via PPG sensors is likewise impacted by CP. METHODS: A device was built to measure the time delay between ECG and finger PPG waveforms (i.e., pulse arrival time (PAT) - a popular surrogate of PTT) and the PPG sensor CP at different CP levels. These measurements and finger cuff BP were recorded while the CP was slowly varied in 17 healthy subjects. RESULTS: Over a physiologic range of CP, the maximum deviations of PAT detected at the PPG foot and peak were 22±2 and 40±7 ms (p<0.05), which translate to ∼11 and ∼20 mmHg BP error based on the literature. The curve relating PAT detected at the PPG foot to CP was U-shaped with minimum near finger diastolic BP. A conceptual model accounting for finger artery viscoelasticity and nonlinearity explained this curve. CONCLUSION: Since the regulatory bias error for BP measurement is limited to 5 mmHg, PPG sensor CP should be taken into account for cuff-less BP measurement via PTT. SIGNIFICANCE: This study suggests that widely pursued PPG-based BP measurement devices including those that detect PTT should maintain the CP or include a CP measurement in the calibration equation for deriving BP.

Conventional pulse transit times as markers of blood pressure changes in humans.

Pulse transit time (PTT) represents a potential approach for cuff-less blood pressure (BP) monitoring. Conventionally, PTT is determined by (1) measuring (a) ECG and ear, finger, or toe PPG waveforms or (b) two of these PPG waveforms and (2) detecting the time delay between the waveforms. The conventional PTTs (cPTTs) were compared in terms of correlation with BP in humans. Thirty-two volunteers [50% female; 52 (17) (mean (SD)) years; 25% hypertensive] were studied. The four waveforms and manual cuff BP were recorded before and after slow breathing, mental arithmetic, cold pressor, and sublingual nitroglycerin. Six cPTTs were detected as the time delays between the ECG R-wave and ear PPG foot, R-wave and finger PPG foot [finger pulse arrival time (PAT)], R-wave and toe PPG foot (toe PAT), ear and finger PPG feet, ear and toe PPG feet, and finger and toe PPG feet. These time delays were also detected via PPG peaks. The best correlation by a substantial extent was between toe PAT via the PPG foot and systolic BP [- 0.63 ± 0.05 (mean ± SE); p < 0.001 via one-way ANOVA]. Toe PAT is superior to other cPTTs including the popular finger PAT as a marker of changes in BP and systolic BP in particular.