Clinical Consultation Guide: Nerve-sparing Techniques for Retroperitoneal Lymph Node Dissection in Testicular Cancer.

Reptroperitoneal lymph node dissection (RPLND) is associated with a risk of morbidity and ejaculatory dysfunction. Nerve-sparing RPLND shows promise in preserving ejaculation alongside oncological efficacy. Laparoscopic and robot-assisted modalities are feasible with good outcomes, highlighting the need for ongoing scientific research and refinement of surgical skills.

α-Methylacyl-CoA Racemase from Mycobacterium tuberculosis-Detailed Kinetic and Structural Characterization of the Active Site.

α-Methylacyl-CoA racemase in M. tuberculosis (MCR) has an essential role in fatty acid metabolism and cholesterol utilization, contributing to the bacterium's survival and persistence. Understanding the enzymatic activity and structural features of MCR provides insights into its physiological and pathological significance and potential as a therapeutic target. Here, we report high-resolution crystal structures for wild-type MCR in a new crystal form (at 1.65 Å resolution) and for three active-site mutants, H126A, D156A and E241A, at 2.45, 1.64 and 1.85 Å resolutions, respectively. Our analysis of the new wild-type structure revealed a similar dimeric arrangement of MCR molecules to that previously reported and details of the catalytic site. The determination of the structures of these H126A, D156A and E241A mutants, along with their detailed kinetic analysis, has now allowed for a rigorous assessment of their catalytic properties. No significant change outside the enzymatic active site was observed in the three mutants, establishing that the diminution of catalytic activity is mainly attributable to disruption of the catalytic apparatus involving key hydrogen bonding and water-mediated interactions. The wild-type structure, together with detailed mutational and biochemical data, provide a basis for understanding the catalytic properties of this enzyme, which is important for the design of future anti-tuberculosis drug molecules.

Recombinant protein production for structural and kinetic studies: A case study using M. tuberculosis α-methylacyl-CoA racemase (MCR).

Modern drug discovery is a target-driven approach in which a particular protein such as an enzyme is implicated in the disease process. Commonly, small-molecule drugs are identified using screening, rational design, and structural biology approaches. Drug screening, testing and optimization is typically conducted in vitro, and copious amounts of protein are required. The advent of recombinant DNA technologies has resulted in a rise in proteins purified by affinity techniques, typically by incorporating an "affinity tag" at the N- or C-terminus. Use of these tagged proteins and affinity techniques comes with a host of issues. This chapter describes the production of an untagged enzyme, α-methylacyl-CoA racemase (MCR) from Mycobacterium tuberculosis, using a recombinant E. coli system. Purification of the enzyme on a 100 mg scale using tandem anion-exchange chromatographies (DEAE-sepharose and RESOURCE-Q columns), and size-exclusion chromatographies is described. A modified protocol allowing the purification of cationic proteins is also described, based on tandem cation-exchange chromatographies (using CM-sepharose and RESOURCE-S columns) and size-exclusion chromatographies. The resulting MCR protein is suitable for biochemical and structural biology applications. The described protocols have wide applicability to the purification of other recombinant proteins and enzymes without using affinity chromatography.

Analysis of enzyme reactions using NMR techniques: A case study with α-methylacyl-CoA racemase (AMACR).

α-Methylacyl-CoA racemase (AMACR; P504S) catalyzes the conversion of R-2-methylacyl-CoA esters into their corresponding S-2-methylacyl-CoA epimers enabling their degradation by ß-oxidation. The enzyme also catalyzes the key epimerization reaction in the pharmacological activation pathway of ibuprofen and related drugs. AMACR protein levels and enzymatic activity are increased in prostate cancer, and the enzyme is a recognized drug target. Key to the development of novel treatments based on AMACR inhibition is the development of functional assays. Synthesis of substrates and purification of recombinant human AMACR are described. Incubation of R- or S-2-methylacyl-CoA esters with AMACR in vitro resulted in formation of epimers (at a near 1-1 ratio at equilibrium) via removal of their α-protons to form an enolate intermediate followed by reprotonation. Conversion can be conveniently followed by incubation in buffer containing 2H2O followed by 1H NMR analysis to monitor conversion of the α-methyl doublet to a single peak upon deuterium incorporation. Incubation of 2-methylacyl-CoA esters containing leaving groups results in an elimination reaction, which was also characterized by 1H NMR. The synthesis of substrates, including a double labeled substrate for mechanistic studies, and subsequent analysis is also described.

Steady-state kinetic analysis of reversible enzyme inhibitors: A case study on calf intestine alkaline phosphatase.

Enzymes are important drug targets and inhibition of enzymatic activity is an important therapeutic strategy. Enzyme assays measuring catalytic activity are utilized in both the discovery and development of new drugs. Colorimetric assays based on the release of 4-nitrophenol from substrates are commonly used. 4-Nitrophenol is only partly ionized to 4-nitrophenolate under typical assay conditions (pH 7-9) leading to under-estimation of product formation rates due to the much lower extinction coefficient of 4-nitrophenol compared to 4-nitrophenolate. Determination of 4-nitrophenol pKa values based on absorbance at 405 nm as a function of experimental pH values is reported, allowing for calculation of a corrected extinction coefficient at the assay pH. Characterization of inhibitor properties using steady-state enzyme kinetics is demonstrated using calf intestine alkaline phosphatase and 4-nitrophenyl phosphate as substrate at pH ∼8.2. The following kinetic parameters were determined: Km= 40±3 µM; Vmax= 72.8±1.2 µmolmin-1mg protein-1; kcat= 9.70±0.16 s-1; kcat/Km= 2.44±0.16 × 105 M-1s-1 (mean± SEM, N = 4). Sodium orthovanadate and EDTA were used as model inhibitors and the following pIC50 values were measured using dose-response curves: 6.61±0.08 and 3.07±0.03 (mean±SEM, N = 4). Rapid dilution experiments determined that inhibition was reversible for sodium orthovanadate and irreversible for EDTA. A Ki value for orthovanadate of 51±8 nM (mean±SEM, N = 3) was determined. Finally, data analysis and statistical design of experiments are discussed.

Limits of Solid Solution and Evolution of Crystal Morphology in (La1-xREx)FeO3 Perovskites by Low Temperature Hydrothermal Crystallization.

The crystallization of a new series of A-site substituted lanthanum ferrite materials (La1-xREx)FeO3 was explored by the hydrothermal method at 240 °C, for rare earth (RE) = Nd, Sm, Gd, Ho, Er, Yb, and Y, with 0 ≤ x ≤ 1. The effect of elemental substitution on the morphological, structural, and magnetic properties of the materials was studied using high-resolution powder X-ray diffraction, energy dispersive spectroscopy (EDS) on the scanning electron microscope, Raman spectroscopy, and SQUID magnetometry. If the radius of the La3+ and the substituent ions is similar, such as for Nd3+, Sm3+, and Gd3+, homogeneous solid solutions are formed, with the orthorhombic GdFeO3-type structure, and a continuous evolution of Raman spectra with composition and distinct magnetic behavior from the end members. When the radius difference between substituents and La3+ is large, such as for Ho3+, Er3+, Yb3+, and Y3+, then instead of forming solid solutions, crystallization in separate phases is found. However, low levels of element mixing are found and intergrowths of segregated regions give composite particles. In this case, the Raman spectra and magnetic behavior are characteristic of mixtures of phases, while EDS shows distinctive elemental segregation. A-site replacement induces an evolution in the crystallite shape with an increasing amount of substituent ions and this is most evident for RE = Y from cube-shaped crystals seen for LaFeO3 to multipodal crystals for (La1-xYx)FeO3, providing evidence for a phase-separation-driven evolution of morphology.

Objective Hemodynamic Cardiovascular Autonomic Abnormalities in Post-Acute Sequelae of COVID-19.

BACKGROUND: Many COVID-19 patients are left with symptoms several months after resolution of the acute illness; this syndrome is known as post-acute sequalae of COVID-19 (PASC). We aimed to determine the prevalence of objective hemodynamic cardiovascular autonomic abnormalities (CAA), explore sex differences, and assess the prevalence of CAA among hospitalized vs nonhospitalized patients with PASC. METHODS: Patients with PASC (n = 70; female [F] = 56; 42 years of age; 95% confidence interval [CI], 40-48) completed standard autonomic tests, including an active stand test 399 days (338, 455) after their COVID-19 infection. Clinical autonomic abnormalities were evaluated. RESULTS: Most patients with PASC met the criteria for at least 1 CAA (51; 73%; F = 43). The postural orthostatic tachycardia syndrome hemodynamic (POTSHR) criterion of a heart rate increase of > 30 beats per minute within 5 to 10 minutes of standing was seen in 21 patients (30%; F = 20; P = 0.037 [by sex]). The initial orthostatic hypotension hemodynamic (IOH40) criterion of a transient systolic blood pressure change of > 40 mm Hg in the first 15 seconds of standing was seen in 43 (61%) patients and equally among female and male patients (63% vs 57%; P = 0.7). Only 9 (13%) patients were hospitalized; hospitalized vs nonhospitalized patients had similar frequencies of abnormalities (67% vs 74%; P = 0.7). CONCLUSIONS: Patients with PASC have evidence of CAA, most commonly IOH40, which will be missed unless an active stand test is used. Female patients have increased frequency of POTSHR, but IOH40 is equally prevalent between sexes. Finally, even nonhospitalized "mild" infections can result in long-term CAAs.

Glucokinase activity in diabetes: too much of a good thing?

Type 2 diabetes (T2D) is a global health problem characterised by chronic hyperglycaemia due to inadequate insulin secretion. Because glucose must be metabolised to stimulate insulin release it was initially argued that drugs that stimulate glucokinase (the first enzyme in glucose metabolism) would enhance insulin secretion in diabetes. However, in the long term, glucokinase activators have been largely disappointing. Recent studies show it is hyperactivation of glucose metabolism, not glucose itself, that underlies the progressive decline in beta-cell function in diabetes. This perspective discusses if glucokinase activators exacerbate this decline (by promoting glucose metabolism) and, counterintuitively, if glucokinase inhibitors might be a better therapeutic strategy for preserving beta-cell function in T2D.

Discovering cell-active BCL6 inhibitors: effectively combining biochemical HTS with multiple biophysical techniques, X-ray crystallography and cell-based assays.

By suppressing gene transcription through the recruitment of corepressor proteins, B-cell lymphoma 6 (BCL6) protein controls a transcriptional network required for the formation and maintenance of B-cell germinal centres. As BCL6 deregulation is implicated in the development of Diffuse Large B-Cell Lymphoma, we sought to discover novel small molecule inhibitors that disrupt the BCL6-corepressor protein-protein interaction (PPI). Here we report our hit finding and compound optimisation strategies, which provide insight into the multi-faceted orthogonal approaches that are needed to tackle this challenging PPI with small molecule inhibitors. Using a 1536-well plate fluorescence polarisation high throughput screen we identified multiple hit series, which were followed up by hit confirmation using a thermal shift assay, surface plasmon resonance and ligand-observed NMR. We determined X-ray structures of BCL6 bound to compounds from nine different series, enabling a structure-based drug design approach to improve their weak biochemical potency. We developed a time-resolved fluorescence energy transfer biochemical assay and a nano bioluminescence resonance energy transfer cellular assay to monitor cellular activity during compound optimisation. This workflow led to the discovery of novel inhibitors with respective biochemical and cellular potencies (IC50s) in the sub-micromolar and low micromolar range.

Altered glycolysis triggers impaired mitochondrial metabolism and mTORC1 activation in diabetic ß-cells.

Chronic hyperglycaemia causes a dramatic decrease in mitochondrial metabolism and insulin content in pancreatic ß-cells. This underlies the progressive decline in ß-cell function in diabetes. However, the molecular mechanisms by which hyperglycaemia produces these effects remain unresolved. Using isolated islets and INS-1 cells, we show here that one or more glycolytic metabolites downstream of phosphofructokinase and upstream of GAPDH mediates the effects of chronic hyperglycemia. This metabolite stimulates marked upregulation of mTORC1 and concomitant downregulation of AMPK. Increased mTORC1 activity causes inhibition of pyruvate dehydrogenase which reduces pyruvate entry into the tricarboxylic acid cycle and partially accounts for the hyperglycaemia-induced reduction in oxidative phosphorylation and insulin secretion. In addition, hyperglycaemia (or diabetes) dramatically inhibits GAPDH activity, thereby impairing glucose metabolism. Our data also reveal that restricting glucose metabolism during hyperglycaemia prevents these changes and thus may be of therapeutic benefit. In summary, we have identified a pathway by which chronic hyperglycaemia reduces ß-cell function.

Plasmonic Fluor-Enhanced Antigen Arrays for High-Throughput, Serological Studies of SARS-CoV-2.

Serological testing for acute infection or prior exposure is critical for patient management and coordination of public health decisions during outbreaks. Current methods have several limitations, including variable performance, relatively low analytical and clinical sensitivity, and poor detection due to antigenic drift. Serological methods for SARS-CoV-2 detection for the ongoing COVID-19 pandemic suffer from several of these limitations and serves as a reminder of the critical need for new technologies. Here, we describe the use of ultrabright fluorescent reagents, Plasmonic Fluors, coupled with antigen arrays that address a subset of these limitations. We demonstrate its application using patient samples in SARS-CoV-2 serological assays. In our multiplexed assay, SARS-CoV-2 antigens were spotted into 48-plex arrays within a single well of a 96-well plate and used to evaluate remnant laboratory samples of SARS-CoV-2 positive patients. Signal-readout was performed with Auragent Bioscience's Empower microplate reader, and microarray analysis software. Sample volumes of 1 µL were used. High sensitivity of the Plasmonic Fluors combined with the array format enabled us to profile patient serological response to eight distinct SARS-CoV-2 antigens and evaluate responses to IgG, IgM, and IgA. Sensitivities for SARS-CoV-2 antigens during the symptomatic state ranged between 72.5 and 95.0%, specificity between 62.5 and 100%, and the resulting area under the curve values between 0.76 and 0.97. Together, these results highlight the increased sensitivity for low sample volumes and multiplex capability. These characteristics make Plasmonic Fluor-enhanced antigen arrays an attractive technology for serological studies for the COVID-19 pandemic and beyond.

Optimizing Shape Complementarity Enables the Discovery of Potent Tricyclic BCL6 Inhibitors.

To identify new chemical series with enhanced binding affinity to the BTB domain of B-cell lymphoma 6 protein, we targeted a subpocket adjacent to Val18. With no opportunities for strong polar interactions, we focused on attaining close shape complementarity by ring fusion onto our quinolinone lead series. Following exploration of different sized rings, we identified a conformationally restricted core which optimally filled the available space, leading to potent BCL6 inhibitors. Through X-ray structure-guided design, combined with efficient synthetic chemistry to make the resulting novel core structures, a >300-fold improvement in activity was obtained by the addition of seven heavy atoms.

Lower body muscle preactivation and tensing mitigate symptoms of initial orthostatic hypotension in young females.

BACKGROUND: Initial orthostatic hypotension (IOH) is a form of orthostatic intolerance defined by a transient decrease in blood pressure upon standing. Current clinical recommendations for managing IOH includes standing up slowly or lower body muscle tensing (TENSE) after standing. Considering that IOH is likely due to a large muscle activation response resulting in excessive vasodilation with a refractory period (<2 minutes), we hypothesized that preactivating lower body muscles (PREACT) before standing would reduce the drop in mean arterial pressure (MAP) upon standing and improve presyncope symptoms. OBJECTIVE: The purpose of this study was to provide IOH patients with effective symptom management techniques. METHODS: Study participants completed 3 sit-to-stand maneuvers, including a stand with no intervention (Control), PREACT, and TENSE. Continuous heart rate and beat-to-beat blood pressure were measured. Stroke volume and cardiac output were then estimated from these waveforms. RESULTS: A total of 24 female IOH participants (mean ± SD: 32 ± 8 years) completed the study. The drops in MAP following PREACT (-21 ± 8 mm Hg; P <.001) and TENSE (-18 ± 10 mm Hg; P <.001) were significantly reduced compared to Control (-28 ± 10 mm Hg). The increase in cardiac output was significantly larger following PREACT (2.6 ± 1 L/min; P <.001) but not TENSE (1.9 ± 1 L/min; P = .2) compared to Control (1.4 ± 1 L/min). The Vanderbilt Orthostatic Symptom Score following PREACT (9 ± 8 au; P = .033) and TENSE (8 ± 8 au; P = .046) both were significantly reduced compared to Control (14 ± 9 au). CONCLUSION: Both the drop in MAP and symptoms upon standing improved with either PREACT or TENSE. These maneuvers provide novel symptom management techniques for patients with IOH.

Clinical Performance of a Lateral Flow SARS-CoV-2 Total Antibody Assay.

BACKGROUND: Serological assays for SARS-CoV-2 are important tools for diagnosis in patients with negative RT-PCR testing, pediatric patients with multisystem inflammatory syndrome, and serosurveillance studies. However, lateral flow-based serological assays have previously demonstrated poor analytical and clinical performance, limiting their utility. METHODS: We assessed the ADEXUSDx COVID-19 lateral flow assay for agreement with diagnostic RT-PCR testing using 120 specimens from RT-PCR-positive patients, 77 specimens from symptomatic RT-PCR-negative patients, and 47 specimens obtained prepandemic. Specimens collected <14 days from symptom onset in RT-PCR-positive patients were compared relative to the Abbott SARS-CoV-2 IgG assay. RESULTS: The ADEXUSDx COVID-19 Test yielded an overall positive percent agreement (PPA) of 92.5% (95%CI 85.8 to 96.3) and negative percent agreement of 99.2% (95% CI 94.9-100.0) relative to RT-PCR and in prepandemic specimens. Relative to days from symptom onset, the PPA after 13 days was 100% (95% CI 94.2-100); from 7 to 13 days, 89.7 (95% CI 71.5-97.2); and from 0 to 7 days, 53.8 (95% CI 26.1-79.6). The overall agreement between the Abbott and ADEXUSDx assays was 80.9%. Twenty-five specimens were positive by both assays, 9 specimens were negative by both assays, and 8 specimens were positive by only the ADEXUSDx assay. CONCLUSIONS: We demonstrate high PPA and negative percent agreement of the ADEXUSDx COVID-19 assay and diagnostic testing by RT-PCR, with PPA approximately 90% by 7 days following symptom onset. The use of waived testing for antibodies to SARS-CoV-2 with high sensitivity and specificity provide a further tool for combatting the COVID-19 pandemic.

Mitigating Initial Orthostatic Hypotension: Mechanistic Roles of Muscle Contraction Versus Sympathetic Activation.

BACKGROUND: Initial orthostatic hypotension (IOH) is defined by a large drop in blood pressure (BP) within 15 s of standing. IOH often presents during an active stand, but not with a passive tilt, suggesting that a muscle activation reflex involving lower body muscles plays an important role. To our knowledge, there is no literature exploring how sympathetic activation affects IOH. We hypothesized involuntary muscle contractions before standing would significantly reduce the drop in BP seen in IOH while increasing sympathetic activity would not. METHODS: Study participants performed 4 sit-to-stand maneuvers including a mental stress test (serial 7 mental arithmetic stress test), cold pressor test, electrical stimulation, and no intervention. Continuous heart rate and beat-to-beat BP were measured. Cardiac output and systemic vascular resistance were estimated from these waveforms. Data are presented as mean±SD. RESULTS: A total of 23 female IOH participants (31±8 years) completed the study. The drops in systolic BP following the serial 7 mental arithmetic stress test (-26±12 mm Hg; P=0.004), cold pressor test (-20±15 mm Hg; P<0.001), and electrical stimulation (-28±12 mm Hg; P=0.01) were significantly reduced compared with no intervention (-34±11 mm Hg). The drops in systemic vascular resistance following the serial 7 mental arithmetic stress test (-391±206 dyne×s/cm5; P=0.006) and cold pressor test (-386±179 dyne×s/cm5; P=0.011) were significantly reduced compared with no intervention (-488±173 dyne×s/cm5). Cardiac output was significantly increased upon standing (7±2 L/min) compared with during the sit (6±1 L/min; P<0.001) for electrical stimulation. CONCLUSION: Sympathetic activation mitigates the BP response in IOH, while involuntary muscle contraction mitigates the BP response and reduces symptoms. Active muscle contractions may induce both of these mechanisms of action in their pretreatment of IOH. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03970551.

Into Deep Water: Optimizing BCL6 Inhibitors by Growing into a Solvated Pocket.

We describe the optimization of modestly active starting points to potent inhibitors of BCL6 by growing into a subpocket, which was occupied by a network of five stably bound water molecules. Identifying potent inhibitors required not only forming new interactions in the subpocket but also perturbing the water network in a productive, potency-increasing fashion while controlling the physicochemical properties. We achieved this goal in a sequential manner by systematically probing the pocket and the water network, ultimately achieving a 100-fold improvement of activity. The most potent compounds displaced three of the five initial water molecules and formed hydrogen bonds with the remaining two. Compound 25 showed a promising profile for a lead compound with submicromolar inhibition of BCL6 in cells and satisfactory pharmacokinetic (PK) properties. Our work highlights the importance of finding productive ways to perturb existing water networks when growing into solvent-filled protein pockets.

Performance Evaluation of an Automated Fentanyl Immunoassay.

BACKGROUND: High-throughput fentanyl immunoassays have recently emerged for clinical use, but early reports have demonstrated relatively high false-positive rates. The purpose of this study was to compare 2 immunoassays, the ARK and ARK II fentanyl immunoassays, and to demonstrate the clinical impact of implementing the ARK II assay. METHODS: The ARK and ARK II fentanyl assays were performed on a Roche c 502 chemistry analyzer. Positive and negative percentage agreement was assessed for each assay with 112 residual patient specimens relative to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cross-reactivity was assessed with the primary metabolite, norfentanyl, and analogs acetylfentanyl, acrylfentanyl, and furanylfentanyl. The proportion of specimens that did not confirm was assessed retrospectively from the laboratory information system. RESULTS: The concordance of the ARK assay was 75% (kappa 0.46, 95%CI 0.28-0.63) and the ARK II was 93% (kappa 0.86, 95%CI 0.76-0.95) with LC-MS/MS. 30 ng/mL of norfentanyl was required for a positive result by ARK and 15 ng/mL by ARK II. Similar cross-reactivity was observed when fentanyl and norfentanyl were both present in the specimen and with fentanyl analogs. After implementing the ARK II assay, the proportion of specimens that did not confirm by LC-MS/MS decreased from 11.7% per month to 2.0% per month. CONCLUSIONS: The ARK II fentanyl immunoassay has improved concordance relative to the original ARK fentanyl immunoassay using LC-MS/MS as the comparator method. Improved analyte specificity resulted in a reduced proportion of clinical samples that do not confirm.

Racemases and epimerases operating through a 1,1-proton transfer mechanism: reactivity, mechanism and inhibition.

Racemases and epimerases catalyse changes in the stereochemical configurations of chiral centres and are of interest as model enzymes and as biotechnological tools. They also occupy pivotal positions within metabolic pathways and, hence, many of them are important drug targets. This review summarises the catalytic mechanisms of PLP-dependent, enolase family and cofactor-independent racemases and epimerases operating by a deprotonation/reprotonation (1,1-proton transfer) mechanism and methods for measuring their catalytic activity. Strategies for inhibiting these enzymes are reviewed, as are specific examples of inhibitors. Rational design of inhibitors based on substrates has been extensively explored but there is considerable scope for development of transition-state mimics and covalent inhibitors and for the identification of inhibitors by high-throughput, fragment and virtual screening approaches. The increasing availability of enzyme structures obtained using X-ray crystallography will facilitate development of inhibitors by rational design and fragment screening, whilst protein models will facilitate development of transition-state mimics.

Forearm vascular resistance responses to the Valsalva maneuver in healthy young and older adults.

PURPOSE: Effective end-organ peripheral vascular resistance responses are critical to blood pressure control while upright, and prevention of syncope (fainting). The Valsalva maneuver (VM) induces blood pressure decreases that evoke baroreflex-mediated vasoconstriction. We characterized beat-to-beat forearm vascular resistance (FVR) responses to the VM in healthy adults, evaluated the impact of age and sex on these responses, and investigated their association with orthostatic tolerance (OT; susceptibility to syncope). We hypothesized that individuals with smaller FVR responses would be more susceptible to syncope. METHODS: Healthy young (N = 36; 19 women; age 25.4 ± 4.6 years) and older (N = 21; 12 women; age 62.4 ± 9.6 years) adults performed a supine 40 mmHg, 20 s VM. Graded 60° head-up-tilt with combined lower body negative pressure continued to presyncope was used to determine OT. Non-invasive beat-to-beat blood pressure and heart rate (finger plethysmography) were recorded continuously. FVR was calculated as mean arterial pressure (MAP) divided by brachial blood flow velocity (Doppler ultrasound) relative to baseline. RESULTS: The VM produces a distinctive FVR pattern that peaks (+137.1 ± 11.6%) in phase 2B (17.5 ± 0.3 s) as the baroreflex responds to low-pressure perturbations. This response increased with age overall (p < 0.001) and within male (p = 0.030) and female subgroups (p < 0.001). Maximum FVR during the VM was significantly correlated with maximal tilt FVR (r = 0.364; p = 0.0153) and with OT when expressed relative to the MAP decrease in phase 2A (Max FVR (%)/MAP2A-1; r = 0.337; p = 0.0206). CONCLUSION: This is the first characterization of FVR responses to the VM. The VM elicits large baroreflex-mediated increases in FVR; small FVR responses to the VM may indicate susceptibility to syncope.