α-Adrenergic regulation of skeletal muscle blood flow during exercise in patients with heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) has been characterized by lower blood flow to exercising limbs and lower peak oxygen utilization ( V ̇ O 2 ${{\dot{V}}_{{{{\mathrm{O}}}_{\mathrm{2}}}}}$ ), possibly associated with disease-related changes in sympathetic (α-adrenergic) signaling. Thus, in seven patients with HFpEF (70 ± 6 years, 3 female/4 male) and seven controls (CON) (66 ± 3 years, 3 female/4 male), we examined changes (%Δ) in leg blood flow (LBF, Doppler ultrasound) and leg V ̇ O 2 ${{\dot{V}}_{{{{\mathrm{O}}}_{\mathrm{2}}}}}$ to intra-arterial infusion of phentolamine (PHEN, α-adrenergic antagonist) or phenylephrine (PE, α1-adrenergic agonist) at rest and during single-leg knee-extension exercise (0, 5 and 10 W). At rest, the PHEN-induced increase in LBF was not different between groups, but PE-induced reductions in LBF were lower in HFpEF (-16% ± 4% vs. -26% ± 5%, HFpEF vs. CON; P < 0.05). During exercise, the PHEN-induced increase in LBF was greater in HFpEF at 10 W (16% ± 8% vs. 8% ± 5%; P < 0.05). PHEN increased leg V ̇ O 2 ${{\dot{V}}_{{{{\mathrm{O}}}_{\mathrm{2}}}}}$ in HFpEF (10% ± 3%, 11% ± 6%, 15% ± 7% at 0, 5 and 10 W; P < 0.05) but not in controls (-1% ± 9%, -4% ± 2%, -1% ± 5%; P = 0.24). The 'magnitude of sympatholysis' (PE-induced %Δ LBF at rest - PE-induced %Δ LBF during exercise) was lower in patients with HFpEF (-6% ± 4%, -6% ± 6%, -7% ± 5% vs. -13% ± 6%, -17% ± 5%, -20% ± 5% at 0, 5 and 10 W; P < 0.05) and was positively related to LBF, leg oxygen delivery, leg V ̇ O 2 ${{\dot{V}}_{{{{\mathrm{O}}}_{\mathrm{2}}}}}$ , and the PHEN-induced increase in LBF (P < 0.05). Together, these data indicate that excessive α-adrenergic vasoconstriction restrains blood flow and limits V ̇ O 2 ${{\dot{V}}_{{{{\mathrm{O}}}_{\mathrm{2}}}}}$ of the exercising leg in patients with HFpEF, and is related to impaired functional sympatholysis in this patient group. KEY POINTS: Sympathetic (α-adrenergic)-mediated vasoconstriction is exaggerated during exercise in patients with heart failure with preserved ejection fraction (HFpEF), which may contribute to limitations of blood flow, oxygen delivery and oxygen utilization in the exercising muscle. The ability to adequately attenuate α1-adrenergic vasoconstriction (i.e. functional sympatholysis) within the vasculature of the exercising muscle is impaired in patients with HFpEF. These observations extend our current understanding of HFpEF pathophysiology by implicating excessive α-adrenergic restraint and impaired functional sympatholysis as important contributors to disease-related impairments in exercising muscle blood flow and oxygen utilization in these patients.

Evidence for a HURP/EB free mixed-nucleotide zone in kinetochore-microtubules.

Current models infer that the microtubule-based mitotic spindle is built from GDP-tubulin with small GTP caps at microtubule plus-ends, including those that attach to kinetochores, forming the kinetochore-fibres. Here we reveal that kinetochore-fibres additionally contain a dynamic mixed-nucleotide zone that reaches several microns in length. This zone becomes visible in cells expressing fluorescently labelled end-binding proteins, a known marker for GTP-tubulin, and endogenously-labelled HURP - a protein which we show to preferentially bind the GDP microtubule lattice in vitro and in vivo. We find that in mitotic cells HURP accumulates on the kinetochore-proximal region of depolymerising kinetochore-fibres, whilst avoiding recruitment to nascent polymerising K-fibres, giving rise to a growing "HURP-gap". The absence of end-binding proteins in the HURP-gaps leads us to postulate that they reflect a mixed-nucleotide zone. We generate a minimal quantitative model based on the preferential binding of HURP to GDP-tubulin to show that such a mixed-nucleotide zone is sufficient to recapitulate the observed in vivo dynamics of HURP-gaps.

Molecular mechanics underlying flat-to-round membrane budding in live secretory cells.

Membrane budding entails forces to transform flat membrane into vesicles essential for cell survival. Accumulated studies have identified coat-proteins (e.g., clathrin) as potential budding factors. However, forces mediating many non-coated membrane buddings remain unclear. By visualizing proteins in mediating endocytic budding in live neuroendocrine cells, performing in vitro protein reconstitution and physical modeling, we discovered how non-coated-membrane budding is mediated: actin filaments and dynamin generate a pulling force transforming flat membrane into Λ-shape; subsequently, dynamin helices surround and constrict Λ-profile's base, transforming Λ- to Ω-profile, and then constrict Ω-profile's pore, converting Ω-profiles to vesicles. These mechanisms control budding speed, vesicle size and number, generating diverse endocytic modes differing in these parameters. Their impact is widespread beyond secretory cells, as the unexpectedly powerful functions of dynamin and actin, previously thought to mediate fission and overcome tension, respectively, may contribute to many dynamin/actin-dependent non-coated-membrane buddings, coated-membrane buddings, and other membrane remodeling processes.

Kinetochore tracking in 3D from lattice light-sheet imaging data with KiT.

MOTIVATION: Lattice light-sheet microscopy (LLSM) is revolutionizing cell biology since it enables fast, high-resolution extended imaging in three dimensions combined with a drastic reduction in photo-toxicity and bleaching. However, analysis of such datasets still remains a major challenge. RESULTS: Automated tracking of kinetochores, the protein complex facilitating and controlling microtubule attachment of the chromosomes within the mitotic spindle, provides quantitative assessment of chromosome dynamics in mitosis. Here, we extend existing open-source kinetochore tracking software (KiT) to track (and pair) kinetochores throughout prometaphase to anaphase in LLSM data. One of the key improvements is a regularization term in the objective function to enforce biological information about the number of kinetochores in a human mitotic cell, as well as improved diagnostic tools. This software provides quantitative insights into how kinetochores robustly ensure congression and segregation of chromosomes during mitosis. AVAILABILITY AND IMPLEMENTATION: KiT is free, open-source software implemented in MATLAB and can be downloaded as a package from https://github.com/cmcb-warwick/KiT. The source repository is available at https://bitbucket.org/jarmond/kit (tag v2.4.0) and under continuing development. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Epidural Steroid Injections Acutely Suppress Bone Formation Markers in Postmenopausal Women.

CONTEXT: Over 9 million epidural steroid injections (ESIs) are performed annually in the United States. Although these injections effectively treat lumbar radicular pain, they may have adverse consequences, including bone loss. OBJECTIVE: To investigate acute changes in bone turnover following ESI. We focused on postmenopausal women, who may be at greatest risk for adverse skeletal consequences due to the combined effects of ESIs with aging and estrogen deficiency. METHODS: Single-center prospective observational study. Postmenopausal women undergoing lumbar ESIs and controls with no steroid exposure were included. Outcomes were serum cortisol, markers of bone formation, osteocalcin, and procollagen type-1 N-terminal propeptide (P1NP), and bone resorption by C-telopeptide (CTX) measured at baseline, 1, 4, 12, 26, and 52 weeks after ESIs. RESULTS: Among ESI-treated women, serum cortisol declined by ~50% 1 week after injection. Bone formation markers significantly decreased 1 week following ESIs: osteocalcin by 21% and P1NP by 22%. Both markers remained suppressed at 4 and 12 weeks, but returned to baseline levels by 26 weeks. There was no significant change in bone resorption measured by CTX. Among controls, there were no significant changes in cortisol or bone turnover markers. CONCLUSION: These results provide evidence of an early and substantial reduction in bone formation markers following ESIs. This effect persisted for over 12 weeks, suggesting that ESIs may have lasting skeletal consequences. Given the large population of older adults who receive ESIs, further investigation into the long-term skeletal sequelae of these injections is warranted.

Accuracy of Ultrasound Jugular Venous Pressure Height in Predicting Central Venous Congestion.

BACKGROUND: Assessment of volume status through the estimation of central venous pressure (CVP) is integral in the care of heart failure (HF). Bedside assessment is limited by obesity, variation in physical examination skills, and expertise in ultrasonography. OBJECTIVE: To validate the accuracy of quantitative and qualitative point-of-care ultrasonography assessment of jugular venous pressure (JVP) in predicting elevated CVP. DESIGN: Prospective observational study using convenience sampling. SETTING: 2 U.S. academic hospitals. PATIENTS: Adult patients undergoing right heart catheterization between 5 February 2019 and 1 March 2021. MEASUREMENTS: Estimation of the JVP height by handheld ultrasound device (uJVP), JVP by traditional physical examination, and qualitative presence of a distended uJVP in the upright position (upright-uJVP) was done before invasive measurements. Receiver-operating characteristic analysis of the uJVP was compared with invasive hemodynamics. RESULTS: In 100 participants undergoing right heart catheterization for HF indications (mean age, 59.6 years; 44% with preserved ejection fraction), the uJVP in a reclined position accurately predicted elevated right atrial pressure (RAP) (>10 mm Hg), with an area under the curve of 0.84. A positive uJVP in the upright position was 94.6% specific for predicting elevated RAP. LIMITATION: Limited examiners, only 2 centers, and convenience sampling. CONCLUSION: Point-of-care ultrasonography assessment of the uJVP is feasible, reproducible, and accurately predictive of elevated CVPs in patients undergoing right heart catheterization. Further investigation of clinical application of ultrasound-measured JVP seems warranted. PRIMARY FUNDING SOURCE: None.

Kinetochore life histories reveal an Aurora-B-dependent error correction mechanism in anaphase.

Chromosome mis-segregation during mitosis leads to aneuploidy, which is a hallmark of cancer and linked to cancer genome evolution. Errors can manifest as "lagging chromosomes" in anaphase, although their mechanistic origins and likelihood of correction are incompletely understood. Here, we combine lattice light-sheet microscopy, endogenous protein labeling, and computational analysis to define the life history of >104 kinetochores. By defining the "laziness" of kinetochores in anaphase, we reveal that chromosomes are at a considerable risk of mis-segregation. We show that the majority of lazy kinetochores are corrected rapidly in anaphase by Aurora B; if uncorrected, they result in a higher rate of micronuclei formation. Quantitative analyses of the kinetochore life histories reveal a dynamic signature of metaphase kinetochore oscillations that forecasts their anaphase fate. We propose that in diploid human cells chromosome segregation is fundamentally error prone, with an additional layer of anaphase error correction required for stable karyotype propagation.

Subcellular Euclidean distance measurements with multicolor fluorescence localization imaging in cultured cells.

This protocol measures the 3D Euclidean distance (Δ3D) between two/three fluorescently labeled kinetochore components in fixed samples using Kinetochore Delta software (KiDv1.0.1, MATLAB based). Overestimation of mean Δ3D is corrected through a Bayesian algorithm, with ΔEC distances reflecting the ensemble average positions of fluorophores within a kinetochore population. This package also enables kinetochore categorization, which can be used to sub-sample kinetochores and measure ΔEC. Together, this allows the dynamic architecture of human kinetochores to be investigated (tested in hTERT-RPE1 cells). For complete details on the use and execution of this protocol, please refer to Roscioli et al. (2020).

MRI-based Texture Analysis of Trabecular Bone for Opportunistic Screening of Skeletal Fragility.

CONTEXT: Many individuals at high risk for osteoporosis and fragility fracture are never screened by traditional methods. Opportunistic use of imaging obtained for other clinical purposes is required to foster identification of these patients. OBJECTIVE: The aim of this pilot study was to evaluate texture features as a measure of bone fragility, by comparing clinically acquired magnetic resonance imaging (MRI) scans from individuals with and without a history of fragility fracture. METHODS: This study retrospectively investigated 100 subjects who had lumbar spine MRI performed at our institution. Cases (n = 50) were postmenopausal women with osteoporosis and a confirmed history of fragility fracture. Controls (n = 50) were age- and race-matched postmenopausal women with no known fracture history. Trabecular bone from the lumbar vertebrae was segmented to create regions of interest within which a gray level co-occurrence matrix was used to quantify the distribution and spatial organization of voxel intensity. Heterogeneity in the trabecular bone texture was assessed by several features, including contrast (variability), entropy (disorder), and angular second moment (homogeneity). RESULTS: Texture analysis revealed that trabecular bone was more heterogeneous in fracture patients. Specifically, fracture patients had greater texture variability (+76% contrast; P = 0.005), greater disorder (+10% entropy; P = 0.005), and less homogeneity (-50% angular second moment; P = 0.005) compared with controls. CONCLUSIONS: MRI-based textural analysis of trabecular bone discriminated between patients with known osteoporotic fractures and controls. Further investigation is required to validate this promising methodology, which could greatly expand the number of patients screened for skeletal fragility.

Comparison of irrigant activation devices and conventional needle irrigation on smear layer and debris removal in curved canals. (Smear layer removal from irrigant activation using SEM).

AIM: To compare the effectiveness of smear layer and debris removal in the final rinse of curved canals of permanent molars using different commercially available irrigant activation devices. METHODS: The mesial roots of 74 extracted maxillary and mandibular molars were instrumented using the Mtwo nickel-titanium rotary system (VDW GmbH, Munich, Germany). They were then randomly assigned to one of three groups, varying in their final rinse protocol. Group 1 (n = 15) - conventional needle irrigation with 4% NaOCl; Group 2 (n = 19) - EndoActivator® (Dentsply Tulsa Dental Specialties, Tulsa, OK, USA) with 4% NaOCl; Group 3 (n = 17) - XP-endo® Finisher (FKG Dentaire SA, La Chaux-de-Fonds, Switzerland) with 4% NaOCl. After the final rinse, all canals were flushed with 1 mL 15% EDTA for 60 s and then flushed with saline. The roots were split longitudinally and prepared for scanning electron microscope imaging. ImageJ for Windows was utilised to assess the images for smear layer removal, while two blinded investigators assessed debris presence in the middle and apical thirds using a 5-point scale. RESULTS: There was no significant difference in smear layer and debris removal between treatment and control groups in the same canal zones. A significant difference was noted across different canal zones both within and across the groups. CONCLUSION: There is no statistically significant difference in effectiveness between activated irrigation techniques and manual activation. Further investigations are required to evaluate all methods available and determine the most efficient technique to irrigate successfully.

Patients with abnormal microarchitecture have an increased risk of early complications after spinal fusion surgery.

Spine fusion is one of the most common orthopedic surgeries, with more than 400,000 cases performed annually. While these procedures correct debilitating pain and deformities, complications occur in up to 45%. As successful fusion rests upon early stability of hardware in bone, patients with structural skeletal deficits may be at particular risk for complications. Few studies have investigated this relationship, and none have used higher order imaging to evaluate microstructural mechanisms for complications. Standard DXA measurements are subject to artifact in patients with spinal disease and therefore provide limited information. The goal of this prospective study was to investigate pre-operative bone quality as a risk factor for early post-operative complications using high resolution peripheral QCT (HR-pQCT) measurements of volumetric BMD (vBMD) and microarchitecture. We hypothesized that patients with low vBMD and abnormal microarchitecture at baseline would have more skeletal complications post-operatively. Conversely, we hypothesized that pre-operative DXA measurements would not be predictive of complications. Fifty-four subjects (mean age 63 years, BMI 27 kg/m2) were enrolled pre-operatively and followed for 6 months after multi-level lumbar spine fusion. Skeletal complications occurred in 14 patients. Patients who developed complications were of similar age and BMI to those who did not. Baseline areal BMD and Trabecular Bone Score by DXA did not differ. In contrast, HR-pQCT revealed that patients who developed complications had lower trabecular vBMD, fewer and thinner trabeculae at both the radius and tibia, and thinner tibial cortices. In summary, abnormalities of both trabecular and cortical microarchitecture were associated the development of complications within the first six months following spine fusion surgery. Our results suggest a mechanism for early skeletal complications after fusion. Given the burgeoning number of fusion surgeries, further studies are necessary to investigate strategies that may improve bone quality and lower the risk of post-operative complications.

An automatic adaptive method to combine summary statistics in approximate Bayesian computation.

To infer the parameters of mechanistic models with intractable likelihoods, techniques such as approximate Bayesian computation (ABC) are increasingly being adopted. One of the main disadvantages of ABC in practical situations, however, is that parameter inference must generally rely on summary statistics of the data. This is particularly the case for problems involving high-dimensional data, such as biological imaging experiments. However, some summary statistics contain more information about parameters of interest than others, and it is not always clear how to weight their contributions within the ABC framework. We address this problem by developing an automatic, adaptive algorithm that chooses weights for each summary statistic. Our algorithm aims to maximize the distance between the prior and the approximate posterior by automatically adapting the weights within the ABC distance function. Computationally, we use a nearest neighbour estimator of the distance between distributions. We justify the algorithm theoretically based on properties of the nearest neighbour distance estimator. To demonstrate the effectiveness of our algorithm, we apply it to a variety of test problems, including several stochastic models of biochemical reaction networks, and a spatial model of diffusion, and compare our results with existing algorithms.

Primary central nervous system marginal zone B-cell lymphoma arising from the dural meninges: A case report and review of literature.

Primary central nervous system (CNS) marginal zone B-cell lymphoma (MZBCL) arising from the dural meninges is a rare but indolent disease. This malignancy can present in various ways, hence making it difficult to diagnose. Biopsy results dictate an appropriate treatment plan, which commonly consists of a combination of surgical resection, whole brain radiotherapy and systemic therapy.

Differentiation agents increase the potential AraC therapy of AML by reactivating cell death pathways without enhancing ROS generation.

Acute myeloid leukemia (AML) has a poor prognosis and requires new approaches for treatment. We have reported that a combination of vitamin D-based cell differentiation agents (doxercalciferol/carnosic acid [D2/CA]) added following the cytotoxic drug arabinocytosine (AraC) increases AML cell death (CD), a model for improved therapy of this disease. Because AraC-induced CD is known to involve reactive oxygen species (ROS) generation, here we investigated if the modulation of cellular REDOX status plays a role in the enhancement of cell death (ECD) by D2/CA. Using thiol antioxidants, such as N-acetyl cysteine (NAC), we found a significant inhibition of ECD, yet this occurred in the absence of any detectable change in cellular ROS levels. In contrast, NAC reduced the vitamin D receptor (VDR) abundance and its signaling of ECD. Importantly, VDR knockdown and NAC similarly inhibited ECD without producing an additive effect. Thus, the proposed post-AraC therapy may be compromised by agents that reduce VDR levels in AML blasts.

Testing Models of mRNA Localization Reveals Robustness Regulated by Reducing Transport between Cells.

Robust control of gene expression in both space and time is of central importance in the regulation of cellular processes and for multicellular development. However, the mechanisms by which robustness is achieved are generally not identified or well understood. For example, messenger RNA (mRNA) localization by molecular motor-driven transport is crucial for cell polarization in numerous contexts, but the regulatory mechanisms that enable this process to take place in the face of noise or significant perturbations are not fully understood. Here, we use a combined experimental-theoretical approach to characterize the robustness of gurken/transforming growth factor-α mRNA localization in Drosophila egg chambers, where the oocyte and 15 surrounding nurse cells are connected in a stereotypic network via intracellular bridges known as ring canals. We construct a mathematical model that encodes simplified descriptions of the range of steps involved in mRNA localization, including production and transport between and within cells until the final destination in the oocyte. Using Bayesian inference, we calibrate this model using quantitative single molecule fluorescence in situ hybridization data. By analyzing both the steady state and dynamic behaviors of the model, we provide estimates for the rates of different steps of the localization process as well as the extent of directional bias in transport through the ring canals. The model predicts that mRNA synthesis and transport must be tightly balanced to maintain robustness, a prediction that we tested experimentally using an overexpression mutant. Surprisingly, the overexpression mutant fails to display the anticipated degree of overaccumulation of mRNA in the oocyte predicted by the model. Through careful model-based analysis of quantitative data from the overexpression mutant, we show evidence of saturation of the transport of mRNA through ring canals. We conclude that this saturation engenders robustness of the localization process in the face of significant variation in the levels of mRNA synthesis.

Autoimmune Diseases and Rosai-Dorfman Disease Coexist More Commonly than Expected: Two Case Reports.

BACKGROUND The educational objective of this study was to describe 2 case reports in which patients were found to have an autoimmune disease concomitantly with a rare, benign histiocytic disorder known as Rosai-Dorfman disease (RDD). It is unclear if there is an underlying association between autoimmune disease and RDD. Lymphadenopathy, although most frequently seen bilaterally in the cervical region in RDD, may be present anywhere. A biopsy with histologic confirmation is required to not only evaluate for malignancy in these cases, but also necessary to diagnose RDD. CASE REPORT We describe 2 cases in which RDD was found incidentally in 2 patients who concomitantly had known autoimmune diseases. The first patient's history included Factor II deficiency, antiphospholipid syndrome, and autoimmune hemolytic anemia; whereas the second patient had a positive antinuclear antibody test, elevated rheumatoid factor, positive lupus anticoagulant, and positive beta-2 glycoprotein 1 antibodies, as well as positive anticardiolipin antibody panel, immune mediated thrombocytopenia, and pernicious anemia. Lymphadenopathy and an enlarged mass were seen in these cases respectively, which were histologically proven to be RDD. Steroid therapy was the mainstay of treatment. CONCLUSIONS Autoimmune diseases are relatively common in the general population and it appears that RDD coexists more often than suspected. When lymphadenopathy or a mass is seen, especially in those with other autoimmune diseases, RDD should remain within the differential diagnosis. Further research is required to determine characteristics and optimal management of RDD. We have observed in the cases presented, that if the autoimmune disease is well controlled, RDD can be an indolent disease.