Low-cost, bottom-up fabrication of large-scale single-molecule nanoarrays by DNA origami placement

Large-scale nanoarrays of single biomolecules enable high-throughput assays while unmasking the underlying heterogeneity within ensemble populations. Until recently, creating such grids which combine the unique advantages of microarrays and single-molecule experiments (SMEs) has been particularly challenging due to the mismatch between the size of these molecules and the resolution of top-down fabrication techniques. DNA Origami Placement (DOP) combines two powerful techniques to address this issue: (i) DNA origami, which provides a 100-nm self-assembled template for single-molecule organization with 5 nm resolution, and (ii) top-down lithography, which patterns these DNA nanostructures, transforming them into functional nanodevices via large-scale integration with arbitrary substrates. Presently, this technique relies on state-of-the-art infrastructure and highly-trained personnel, making it prohibitively expensive for researchers. Here, we introduce a bench-top technique to create meso-to-macro-scale DNA origami nanoarrays using self-assembled colloidal nanoparticles, thereby circumventing the need for top-down fabrication. We report a maximum yield of 74%, two-fold higher than the statistical limit of 37% imposed on non-specific molecular loading alternatives. Furthermore, we provide a proof-of-principle for the ability of this nanoarray platform to transform traditionally low-throughput, stochastic, single-molecule assays into high-throughput, deterministic ones, without compromising data quality. Our approach has the potential to democratize single-molecule nanoarrays and demonstrates their utility as a tool for biophysical assays and diagnostics

Development of an automated detection algorithm for patient motion blur in digital mammograms

The purpose is to develop and validate an automated method for detecting image unsharpness caused by patient motion blur in digital mammograms. The goal is that such a tool would facilitate immediate re-taking of blurred images, which has the potential to reduce the number of recalled examinations, and to ensure that sharp, high-quality mammograms are presented for reading. To meet this goal, an automated method was developed based on interpretation of the normalized image Wiener Spectrum. A preliminary algorithm was developed using 25 cases acquired using a single vendor system, read by two expert readers identifying the presence of blur, location, and severity. A predictive blur severity score was established using multivariate modeling, which had an adjusted coefficient of determination, R2 =0.63±0.02, for linear regression against the average reader-scored blur severity. A heatmap of the relative blur magnitude showed good correspondence with reader sketches of blur location, with a Spearman rank correlation of 0.70 between the algorithmestimated area fraction with blur and the maximum of the blur area fraction categories of the two readers. Given these promising results, the algorithm-estimated blur severity score and heatmap are proposed to be used to aid observer interpretation. The use of this automated blur analysis approach, ideally with feedback during an exam, could lead to a reduction in repeat appointments for technical reasons, saving time, cost, potential anxiety, and improving image quality for accurate diagnosis.</p

The NF2 tumor suppressor regulates microtubule-based vesicle trafficking via a novel Rac, MLK and p38SAPK pathway

© Macmillan Publishers, 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Oncogene 32 (2013): 1135–1143, doi:10.1038/onc.2012.135.Neurofibromatosis type 2 patients develop schwannomas, meningiomas and ependymomas resulting from mutations in the tumor suppressor gene, NF2, encoding a membrane-cytoskeleton adapter protein called merlin. Merlin regulates contact inhibition of growth and controls the availability of growth factor receptors at the cell surface. We tested if microtubule-based vesicular trafficking might be a mechanism by which merlin acts. We found that schwannoma cells, containing merlin mutations and constitutive activation of the Rho/Rac family of GTPases, had decreased intracellular vesicular trafficking relative to normal human Schwann cells. In Nf2−/− mouse Schwann (SC4) cells, re-expression of merlin as well as inhibition of Rac or its effector kinases, MLK and p38SAPK, each increased the velocity of Rab6 positive exocytic vesicles. Conversely, an activated Rac mutant decreased Rab6 vesicle velocity. Vesicle motility assays in isolated squid axoplasm further demonstrated that both mutant merlin and active Rac specifically reduce anterograde microtubule-based transport of vesicles dependent upon the activity of p38SAPK kinase. Taken together, our data suggest loss of merlin results in the Rac-dependent decrease of anterograde trafficking of exocytic vesicles, representing a possible mechanism controlling the concentration of growth factor receptors at the cell surface.This work was supported by NIH R01 CA118032 (to NR), and MBL research fellowships (to NR and GM), NIH R01 NS23868 (to STB)

Directly Comparing Handoff Protocols for Pediatric Hospitalists

BACKGROUND AND OBJECTIVES: Handoff protocols are often developed by brainstorming and consensus, and few are directly compared. We hypothesized that a handoff protocol (Flex 11) developed using a rigorous methodology would be more favorable in terms of clinicians’ attitudes, behaviors, cognitions, or time-on-task when performing handoffs compared with a prevalent protocol (Situation Background Assessment Recommendation [SBAR]). METHODS: Using a between-groups, randomized control trial design (Flex 11 versus SBAR) during a pilot study in a simulated environment, 20 clinicians (13 attending physicians and 7 residents) received 3 patient handoffs from a standardized physician, managed the patients, and handed off the patients to the same standardized physician. Participants completed surveys assessing their attitudes and cognitions, and behaviors and handoff duration were assessed through observations. RESULTS: All data were analyzed using independent samples t tests. For attitudes, “ease of use” ratings were lower for SBAR participants than Flex 11 participants (P , .01), and “being helpful” ratings were lower for SBAR participants than Flex 11 participants (P 5 .02). For behaviors, results indicate no significant difference in the information acquired between the SBAR and Flex 11 protocols. However, SBAR participants gave significantly less information than Flex 11 participants (P , .01). For cognitions, SBAR and Flex 11 participants reported similar workload except for frustration. For handoff duration, there were no significant differences between the protocols (P 5 .36). CONCLUSIONS: The results suggest that Flex 11 is an efficient, beneficial tool in a simulated environment with pediatric clinicians. Future studies should evaluate this protocol in the inpatient setting

Heat Shock Protein 70 Prevents both Tau Aggregation and the Inhibitory Effects of Preexisting Tau Aggregates on Fast Axonal Transport

Aggregation and accumulation of the microtubule-associated protein tau are associated with cognitive decline and neuronal degeneration in Alzheimer's disease and other tauopathies. Thus, preventing the transition of tau from a soluble state to insoluble aggregates and/or reversing the toxicity of existing aggregates would represent a reasonable therapeutic strategy for treating these neurodegenerative diseases. Here we demonstrate that molecular chaperones of the heat shock protein 70 (Hsp70) family are potent inhibitors of tau aggregation in vitro, preventing the formation of both mature fibrils and oligomeric intermediates. Remarkably, addition of Hsp70 to a mixture of oligomeric and fibrillar tau aggregates prevents the toxic effect of these tau species on fast axonal transport, a critical process for neuronal function. When incubated with preformed tau aggregates, Hsp70 preferentially associated with oligomeric over fibrillar tau, suggesting that prefibrillar oligomeric tau aggregates play a prominent role in tau toxicity. Taken together, our data provide a novel molecular basis for the protective effect of Hsp70 in tauopathies

Frontotemporal lobar dementia mutant tau impairs axonal transport through a protein phosphatase 1γ-dependent mechanism

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Combs, B., Christensen, K. R., Richards, C., Kneynsberg, A., Mueller, R. L., Morris, S. L., Morfini, G., Brady, S. T., & Kanaan, N. M. Frontotemporal lobar dementia mutant tau impairs axonal transport through a protein phosphatase 1γ-dependent mechanism. Journal of Neuroscience, 41(45), (2021): 9431-9451, https://doi.org/10.1523/JNEUROSCI.1914-20.2021.Pathologic tau modifications are characteristic of Alzheimer's disease and related dementias, but mechanisms of tau toxicity continue to be debated. Inherited mutations in tau cause early onset frontotemporal lobar dementias (FTLD-tau) and are commonly used to model mechanisms of tau toxicity in tauopathies. Previous work in the isolated squid axoplasm model demonstrated that several pathogenic forms of tau inhibit axonal transport through a mechanism involving activation of protein phosphatase 1 (PP1). Here, we determined that P301L and R5L FTLD mutant tau proteins elicit a toxic effect on axonal transport as monomeric proteins. We evaluated interactions of wild-type or mutant tau with specific PP1 isoforms (α, β, and γ) to examine how the interaction contributes to this toxic effect using primary rat hippocampal neurons from both sexes. Pull-down and bioluminescence resonance energy transfer experiments revealed selective interactions of wild-type tau with PP1α and PP1γ isoforms, but not PP1β, which were significantly increased by the P301L tau mutation. The results from proximity ligation assays confirmed the interaction in primary hippocampal neurons. Moreover, expression of FTLD-linked mutant tau in these neurons enhanced levels of active PP1, also increasing the pausing frequency of fluorescently labeled vesicles in both anterograde and retrograde directions. Knockdown of PP1γ, but not PP1α, rescued the cargo-pausing effects of P301L and R5L tau, a result replicated by deleting a phosphatase-activating domain in the amino terminus of P301L tau. These findings support a model of tau toxicity involving aberrant activation of a specific PP1γ-dependent pathway that disrupts axonal transport in neurons.This work was supported by National Institutes of Health (NIH) Grants R01 NS082730 (N.M.K. and S.T.B.), R01 AG044372 (N.M.K.), and R01 AG067762 (N.M.K.); NIH/National Institute on Aging, Michigan Alzheimer's Disease Research Center Grant 5P30AG053760 (B.C.); Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Alzheimer's Research Program Award W81XWH-20-1-0174 (B.C.); Alzheimer's Association Research Grants 20-682085 (B.C.), R01 NS118177 (G.A.M.), and R21NS120126 (G.A.M.); Zenith Award from the Alzheimer's Association (S.T.B.); Tau Consortium/Rainwater Foundation (S.T.B.); Neurodegenerative Research (G.A.M.); and the Secchia Family Foundation (N.M.K.)

Low-cost, bottom-up fabrication of large-scale single-molecule nanoarrays by DNA origami placement

Large-scale nanoarrays of single biomolecules enable high-throughput assays while unmasking the underlying heterogeneity within ensemble populations. Until recently, creating such grids which combine the unique advantages of microarrays and single-molecule experiments (SMEs) has been particularly challenging due to the mismatch between the size of these molecules and the resolution of top-down fabrication techniques. DNA Origami Placement (DOP) combines two powerful techniques to address this issue: (i) DNA origami, which provides a 100-nm self-assembled template for single-molecule organization with 5 nm resolution, and (ii) top-down lithography, which patterns these DNA nanostructures, transforming them into functional nanodevices via large-scale integration with arbitrary substrates. Presently, this technique relies on state-of-the-art infrastructure and highly-trained personnel, making it prohibitively expensive for researchers. Here, we introduce a bench-top technique to create meso-to-macro-scale DNA origami nanoarrays using self-assembled colloidal nanoparticles, thereby circumventing the need for top-down fabrication. We report a maximum yield of 74%, two-fold higher than the statistical limit of 37% imposed on non-specific molecular loading alternatives. Furthermore, we provide a proof-of-principle for the ability of this nanoarray platform to transform traditionally low-throughput, stochastic, single-molecule assays into high-throughput, deterministic ones, without compromising data quality. Our approach has the potential to democratize single-molecule nanoarrays and demonstrates their utility as a tool for biophysical assays and diagnostics

Prevention of Lower Urinary Tract Symptoms Research Consortium focus group Study of Habits, Attitudes, Realities, and Experiences of Bladder health

AimThe study purpose is to explore adolescent and adult women’s experiences, perceptions, beliefs, knowledge and behaviours related to bladder health across the life course using a socioecological perspective. Lower urinary tract symptoms affect between 20-40% of young adult to middle-aged women, with symptoms increasing in incidence and severity with aging. There is limited evidence to address bladder health promotion and prevention of dysfunction. This first study of the Prevention of Lower Urinary Tract Symptoms (PLUS) Research Consortium is designed to address gaps in existing qualitative research in this area.DesignThis focus group study will be implemented across seven geographically diverse United States research centres using a semi-structured focus group guide informed by a conceptual framework based on the socioecological model.MethodsThe study was approved in July 2017. A total of 44 focus groups composed of 6-8 participants representing six different age categories (ranging from 11 to over 65 years) will be completed. We aim to recruit participants with diverse demographic and personal characteristics including race, ethnicity, education, socioeconomic status, urban/rural residence, physical/health conditions, and urinary symptom experience. Six of the focus groups will be conducted in Spanish and translated into English. Focus group transcripts will undergo content analysis and data interpretation to identify and classify themes and articulate emerging themes.DiscussionThis foundational qualitative study seeks to develop an evidence base to inform future research on bladder health promotion in adolescent and adult women.ImpactThis study has the potential to provide new insights and understanding into adolescent and adult women’s lived experience of bladder health, the experience of lower urinary symptoms and knowledge and beliefs across the life course.ç ®ç æ ¬ç  ç©¶ç ç ®ç æ ¯ä» ç¤¾ä¼ ç æ å­¦ç è§ åº¦,æ ¢è®¨é å° å¹´å æ å¹´å¥³æ §å ¨äººç è¿ ç¨ ä¸­ä¸ è è ±å ¥åº·ç ¸å ³ç ç» éª ã è§ å¿µã 信念ã ç ¥è¯ å è¡ ä¸ºã ä¸ å°¿è·¯ç ç ¶å½±å 20-40%ç 中é å¹´å¥³æ §,é ç å¹´é¾ ç å¢ é ¿,ç ç ¶ç å ç ç å 严é ç¨ åº¦é ½å ¨å¢ é ¿ã å ³äº ä¿ è¿ è è ±å ¥åº·å é¢ é ²å è ½é ç¢ ç è¯ æ ®æ é ã æ ¬æ¬¡é¢ é ²ä¸ å°¿è·¯ç ç ¶(PLUS)ç  ç©¶è ç ç ç  ç©¶æ ¯é¦ ä¸ªå ³äº æ­¤æ ¹é ¢ç ç  ç©¶,æ ¨å ¨è§£å ³ç °æ ç å® æ §ç  ç©¶å ¨è¿ æ ¹é ¢ç å·®è· ã è®¾è®¡è¯¥é¡¹ç ¦ç ¹å° ç» ç  ç©¶å° å ¨ä¸ ä¸ªä¸ å ä½ ç½®ç ç¾ å ½ç  ç©¶ä¸­å¿ è¿ è¡ ,ä»¥å ºäº ç¤¾ä¼ ç æ 模å æ¦ å¿µæ¡ æ ¶ç å ç» æ å ç ç ¦ç ¹å° ç» æ å 为æ 导ã æ ¹æ³ è¯¥ç  ç©¶äº 2017å¹´7æ è ·å¾ æ ¹å ã ç ±6-8å 代表6ä¸ªä¸ å å¹´é¾ ç±»å «(ä» 11å² å °65å² ä»¥ä¸ )ç å ä¸ è ç» æ å ±44ä¸ªç ¦ç ¹å° ç» ã æ 们计å æ å ä¸ å äººå £å ä¸ªäººç ¹å¾ ç å ä¸ è ,ä¾ å¦ ç§ æ ã ç§ æ æ¸ æº ã æ è ²ç» å ã ç¤¾ä¼ ç» æµ å °ä½ ã å ä¹¡å± æ° ã èº«ä½ /å ¥åº·ç ¶å µå æ³ å°¿ç³»ç» ç ç ¶ç» å ã å ­ä¸ªç ¦ç ¹å° ç» ç ç  ç©¶å° ä»¥è¥¿ç ­ç è¯­è¿ è¡ ,å¹¶ç¿»è¯ æ è ±è¯­ã ç ¦ç ¹å° ç» ç èª æ ¬å° è¢«ç ¨äº å 容å æ å æ °æ ®è§£é ,ä»¥ç¡®å® å å ç±»ä¸»é¢ ,并é æ æ °å ºç °ç ä¸»é¢ ã è®¨è®ºè¿ é¡¹å ºç¡ æ §ç å® æ §ç  ç©¶æ ¨å ¨ä¸ºæ é« æ ªæ ¥é å° å¹´å æ å¹´å¦ å¥³ç è è ±å ¥åº·ç ç  ç©¶æ ä¾ è¯ æ ®å ºç¡ ã å½±å è¿ é¡¹ç  ç©¶æ å ¯è ½æ ä¾ å ³äº é å° å¹´å æ å¹´å¦ å¥³ç è è ±å ¥åº·ç ç æ´»ç» éª ,ç» éª ç ä¸ å°¿è·¯ç ç ¶å ç ¥è¯ å ç æ³ ç 人ç è¿ ç¨ ä¸­æ °ç è§ è§£å ç 解ãPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151981/1/jan14148_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151981/2/jan14148.pd

Prostate cancer treated with brachytherapy; an exploratory study of dose-dependent biomarkers and quality of life

BACKGROUND: Low-dose-rate permanent prostate brachytherapy (PPB) is an attractive treatment option for patients with localised prostate cancer with excellent outcomes. As standard CT-based post-implant dosimetry often correlates poorly with late treatment-related toxicity, this exploratory (proof of concept) study was conducted to investigate correlations between radiation − induced DNA damage biomarker levels, and acute and late bowel, urinary, and sexual toxicity. METHODS: Twelve patients treated with (125)I PPB monotherapy (145Gy) for prostate cancer were included in this prospective study. Post-implant CT based dosimetry assessed the minimum dose encompassing 90% (D(90%)) of the whole prostate volume (global), sub-regions of the prostate (12 sectors) and the near maximum doses (D(0.1cc), D(2cc)) for the rectum and bladder. Six blood samples were collected from each patient; pre-treatment, 1 h (h), 4 h, 24 h post-implant, at 4 weeks (w) and at 3 months (m). DNA double strand breaks were investigated by staining the blood samples with immunofluorescence antibodies to γH2AX and 53BP1 proteins (γH2AX/53BP1). Patient self-scored quality of life from the Expanded Prostate Cancer Index Composite (EPIC) were obtained at baseline, 1 m, 3 m, 6 m, 9 m, 1 year (y), 2y and 3y post-treatment. Spearman’s correlation coefficients were used to evaluate correlations between temporal changes in γH2AX/53BP1, dose and toxicity. RESULTS: The minimum follow up was 2 years. Population mean prostate D(90%) was 144.6 ± 12.1 Gy and rectal near maximum dose D(0.1cc) = 153.0 ± 30.8 Gy and D(2cc) = 62.7 ± 12.1 Gy and for the bladder D(0.1cc) = 123.1 ± 27.0 Gy and D(2cc) = 70.9 ± 11.9 Gy. Changes in EPIC scores from baseline showed high positive correlation between acute toxicity and late toxicity for both urinary and bowel symptoms. Increased production of γH2AX/53BP1 at 24 h relative to baseline positively correlated with late bowel symptoms. Overall, no correlations were observed between dose metrics (prostate global or sector doses) and γH2AX/53BP1 foci counts. CONCLUSIONS: Our results show that a prompt increase in γH2AX/53BP1foci at 24 h post-implant relative to baseline may be a useful measure to assess elevated risk of late RT − related toxicities for PPB patients. A subsequent investigation recruiting a larger cohort of patients is warranted to verify our findings. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13014-017-0792-1) contains supplementary material, which is available to authorized users

TOI-1685 b Is a Hot Rocky Super-Earth: Updates to the Stellar and Planet Parameters of a Popular JWST Cycle 2 Target

We present an updated characterization of the TOI-1685 planetary system, which consists of a P b = 0.69 day ultra-short-period super-Earth planet orbiting a nearby (d = 37.6 pc) M2.5V star (TIC 28900646, 2MASS J04342248+4302148). This planet was previously featured in two contemporaneous discovery papers, but the best-fit planet mass, radius, and bulk density values were discrepant, allowing it to be interpreted either as a hot, bare rock or a 50% H2O/50% MgSiO3 water world. TOI-1685 b will be observed in three independent JWST Cycle 2 programs, two of which assume the planet is a water world, while the third assumes that it is a hot rocky planet. Here we include a refined stellar classification with a focus on addressing the host star’s metallicity, an updated planet radius measurement that includes two sectors of TESS data and multicolor photometry from a variety of ground-based facilities, and a more accurate dynamical mass measurement from a combined CARMENES, InfraRed Doppler, and MAROON-X radial velocity data set. We find that the star is very metal-rich ([Fe/H] ≃ +0.3) and that the planet is systematically smaller, lower mass, and higher density than initially reported, with new best-fit parameters of R pl = 1.468 −0.051+0.050 R ⊕ and M pl = 3.03−0.32+0.33 M ⊕. These results fall in between the previously derived values and suggest that TOI-1685 b is a hot rocky planet with an Earth-like density (ρ pl = 5.3 ± 0.8 g cm−3, or 0.96 ρ ⊕), high equilibrium temperature (T eq = 1062 ± 27 K), and negligible volatiles, rather than a water world