Magnetic tweezers characterization of the entropic elasticity of intrinsically disordered proteins and peptoids.

Understanding the conformational behavior of biopolymers is essential to unlocking knowledge of their biophysical mechanisms and functional roles. Single-molecule force spectroscopy can provide a unique perspective on this by exploiting entropic elasticity to uncover key biopolymer structural parameters. A particularly powerful approach involves the use of magnetic tweezers, which can easily generate lower stretching forces (0.1-20 pN). For forces at the low end of this range, the elastic response of biopolymers is sensitive to excluded volume effects, and they can be described by Pincus blob elasticity model that allow robust extraction of the Flory polymer scaling exponent. Here, we detail protocols for the use of magnetic tweezers for force-extension measurements of intrinsically disordered proteins and peptoids. We also discuss procedures for fitting low-force elastic curves to the predictions of polymer physics models to extract key conformational parameters.

Pincus blob elasticity in an intrinsically disordered protein.

Understanding the dynamic structure of intrinsically disordered proteins (IDPs) is important to deciphering their biological functions. Here, we exploit precision entropic elasticity measurements to infer the conformational behavior of a model IDP construct formed from the disordered tail of the neurofilament low molecular weight protein. The IDP construct notably displays a low-force power-law elastic regime, consistent with the Pincus blob model, which allows direct extraction of the Flory exponent, [Formula: see text], from the force-extension relationship. We find [Formula: see text] increases with added denaturant, transitioning from a nearly ideal chain to a swollen chain in a manner quantitatively consistent with measurements of IDP dimensions from other experimental techniques. We suggest that measurements of entropic elasticity could be broadly useful in the study of IDP structure.

Association between Hypertension and Stroke Recurrence as Modified by Pro-oxidant-Antioxidant Balance: A Multi-Center Study.

BACKGROUND: Hypertension and oxidative stress are involved in the pathophysiological mechanism of stroke. We aimed to investigate the modification impact of the pro-oxidant-anti-oxidant balance (PAB) on the association between hypertension and stroke recurrence (SR). METHODS: A cross-sectional design was conducted from December 2019 to December 2020 in 951 stroke patients in six hospitals across Vietnam. Hypertension was defined using antihypertensive medication or systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg. PAB was estimated using weighting methods based on smoking, drinking, and overweight/obesity with pro-oxidant capacity, diet quality, fruit intake, vegetable intake, and physical activity with antioxidant capacity. The higher PAB scores indicated a beneficial balance shifting toward antioxidant dominance. SR was diagnosed by neurologists. Moreover, sociodemographic and health conditions were included as covariates. Multiple logistic regression analyses were used to explore the associations and interactions. RESULTS: The hypertension and SR proportions were 72.8% and 17.5%, respectively. hypertension was associated with an increased SR likelihood (odds ratio (OR) = 1.93; p = 0.004), whereas a higher PAB score was associated with a lowered SR likelihood (OR = 0.87; p = 0.003). Moreover, hypertension interacting with every one-point increment of PAB was associated with a lowered SR likelihood (OR = 0.83; p = 0.022). CONCLUSIONS: The harmful impact of hypertension on SR could be alleviated by PAB. The interplay of health behaviors should be highlighted in the intervention strategies for stroke prevention.

Structures of the nitrogenase complex prepared under catalytic turnover conditions.

The enzyme nitrogenase couples adenosine triphosphate (ATP) hydrolysis to the multielectron reduction of atmospheric dinitrogen into ammonia. Despite extensive research, the mechanistic details of ATP-dependent energy transduction and dinitrogen reduction by nitrogenase are not well understood, requiring new strategies to monitor its structural dynamics during catalytic action. Here, we report cryo-electron microscopy structures of the nitrogenase complex prepared under enzymatic turnover conditions. We observe that asymmetry governs all aspects of the nitrogenase mechanism, including ATP hydrolysis, protein-protein interactions, and catalysis. Conformational changes near the catalytic iron-molybdenum cofactor are correlated with the nucleotide-hydrolysis state of the enzyme.

Negative Impact of Comorbidity on Health-Related Quality of Life Among Patients With Stroke as Modified by Good Diet Quality.

Background: Comorbidity, along with aging, affects stroke-induced health-related quality of life (HRQoL). We examined the potential role of diet quality in modifying the association between comorbidity and HRQoL in patients with stroke. Methods: A cross-sectional study was conducted on 951 patients with stroke from December 2019 to December 2020 across Vietnam. Comorbidity was assessed using the Charlson Comorbidity Index (CCI) items and classified into two groups (none vs. one or more). Diet quality was evaluated using the Dietary Approaches to Stop Hypertension Quality (DASH-Q) questionnaire, and HRQoL was measured using the RAND-36, with a higher score indicating better diet quality or HRQoL, respectively. Besides, socio-demographics, health-related behaviors (e.g., physical activity, smoking, and drinking), disability (using WHODAS 2.0), and health literacy were also assessed. Linear regression analysis was utilized to explore the associations and interactions. Results: The proportion of patients with stroke aged ≥65 years and having comorbidity were 53.7 and 49.9%, respectively. The HRQoL scores were 44.4 ± 17.4. The diet quality was associated with higher HRQoL score (regression coefficient, B, 0.14; (95% confidence interval, 95% CI, 0.04, 0.23; p = 0.004), whereas comorbidity was associated with lower HRQoL score (B, -7.36; 95% CI, -9.50, -5.23; p < 0.001). In interaction analysis, compared to patients without comorbidity and having the lowest DASH-Q score, those with comorbidity and higher DASH-Q score had a higher HRQoL score (B, 0.21; 95% CI, 0.03, 0.39; p = 0.021). Conclusion: The findings showed that good diet quality could modify the adverse impact of comorbidity on HRQoL in patients with stroke. Diet quality should be considered as a strategic intervention to improve the HRQoL of patients with stroke, especially those with comorbidity, and to promote healthier aging.

Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy.

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires a thin layer of vitreous ice containing the biomolecules of interest. Despite numerous technological advances in recent years that have propelled single-particle cryoEM to the forefront of structural biology, the methods by which specimens are vitrified for high-resolution imaging often remain the rate-limiting step. Although numerous recent efforts have provided means to overcome hurdles frequently encountered during specimen vitrification, including the development of novel sample supports and innovative vitrification instrumentation, the traditional manually operated plunger remains a staple in the cryoEM community due to the low cost to purchase and ease of operation. Here, we provide detailed methods for using a standard, guillotine-style manually operated blot-and-plunge device for the vitrification of biological specimens for high-resolution imaging by single-particle cryoEM. Additionally, commonly encountered issues and troubleshooting recommendations for when a standard preparation fails to yield a suitable specimen are also described.

Tuberculous otitis media in Vietnam: Clinical features and diagnostic difficulties.

OBJECTIVES: The purpose of this article is to draw the attention of otorhinolaryngologists to tuberculous otitis media in the light of two cases illustrating the diagnostic difficulties of this rare form of extrapulmonary tuberculosis, which often presents as subacute otomastoiditis or apparently benign chronic otitis media. It is characterized by symptoms such as atonic tympanic membrane perforation with granulation tissue, absence of any history of otitis media, facial paralysis or severe otosclerosis, and failure to respond to the usual treatments. CASE REPORTS: The first case was a 24-year-old man referred for right chronic otitis media present for 18 months despite topical treatment and tympanoplasty. The second case was a 21-year-old woman referred for right facial paralysis present for 8 days in a context of chronic otitis media, also treated medically for two years and by two tympanoplasties without success. CONCLUSION: The diagnosis of tuberculous otitis media is not based on histopathological examination, but bacteriological identification of Mycobacterium tuberculosis on an otorrhoea fluid sample collected according to the technique described here. A rigorous clinical approach should ensure early diagnosis and initiation of treatment to prevent complications and severe sequelae.

Physical Activity and Diet Quality Modify the Association between Comorbidity and Disability among Stroke Patients.

BACKGROUND: Comorbidity is common and causes poor stroke outcomes. We aimed to examine the modifying impacts of physical activity (PA) and diet quality on the association between comorbidity and disability in stroke patients. METHODS: A cross-sectional study was conducted on 951 stable stroke patients in Vietnam from December 2019 to December 2020. The survey questionnaires were administered to assess patients' characteristics, clinical parameters (e.g., Charlson Comorbidity Index items), health-related behaviors (e.g., PA using the International Physical Activity Questionnaire- short version), health literacy, diet quality (using the Dietary Approaches to Stop Hypertension Quality (DASH-Q) questionnaire), and disability (using the World Health Organization Disability Assessment Schedule II (WHODAS II)). Linear regression models were used to analyze the associations and interactions. RESULTS: The proportion of comorbidity was 49.9% (475/951). The scores of DASH-Q and WHODAS II were 29.2 ± 11.8, 32.3 ± 13.5, respectively. Patients with comorbidity had a higher score of disability (regression coefficient, B, 8.24; 95% confidence interval, 95%CI, 6.66, 9.83; p < 0.001) as compared with those without comorbidity. Patients with comorbidity and higher tertiles of PA (B, -4.65 to -5.48; p < 0.05), and a higher DASH-Q score (B, -0.32; p < 0.001) had a lower disability score, as compared with those without comorbidity and the lowest tertile of PA, and the lowest score of DASH-Q, respectively. CONCLUSIONS: Physical activity and diet quality significantly modified the negative impact of comorbidity on disability in stroke patients. Strategic approaches are required to promote physical activity and healthy diet which further improve stroke rehabilitation outcomes.

Suppression of Deflecting Forces in Planar-Symmetric Dielectric Wakefield Accelerating Structures with Elliptical Bunches.

Wakefield based accelerators capable of accelerating gradients 2 orders of magnitude higher than present accelerators offer a path to compact high energy physics instruments and light sources. However, for high gradient accelerators, beam instabilities driven by commensurately high transverse wakefields limit beam quality. Previously, it has been theoretically shown that transverse wakefields can be reduced by elliptically shaping the transverse sizes of beams in dielectric structures with planar symmetry. Here, we report experimental measurements that demonstrate reduced transverse wakefields for elliptical beams in planar symmetric structures which are consistent with theoretical models. These results may enable the design of gigavolt-per-meter gradient wakefield based accelerators that produce and stably accelerate high quality beams.

Conductivity Induced by High-Field Terahertz Waves in Dielectric Material.

An intense, subpicosecond, relativistic electron beam traversing a dielectric-lined waveguide generates very large amplitude electric fields at terahertz (THz) frequencies through the wakefield mechanism. In recent work employing this technique to accelerate charged particles, the generation of high-power, narrow-band THz radiation was demonstrated. The radiated waves contain fields with measured amplitude exceeding 2 GV/m, orders of magnitude greater than those available by other THz generation techniques at a narrow bandwidth. For fields approaching the GV/m level, a strong damping has been observed in SiO_{2}. This wave attenuation with an onset near 850 MV/m is consistent with changes to the conductivity of the dielectric lining and is characterized by a distinctive latching mechanism that is reversible on longer timescales. We describe the detailed measurements that serve to clarify the underlying physical mechanisms leading to strong field-induced damping of THz radiation (hω=1.59 meV, f=0.38 THz) in SiO_{2}, a bulk, wide band-gap (8.9 eV) dielectric.

Spatial Regulation of Mitochondrial Heterogeneity by Stromal Confinement in Micropatterned Tumor Models.

Heterogeneity of mitochondrial activities in cancer cells exists across different disease stages and even in the same patient, with increased mitochondrial activities associated with invasive cancer phenotypes and circulating tumor cells. Here, we use a micropatterned tumor-stromal assay (µTSA) comprised of MCF-7 breast cancer cells and bone marrow stromal cells (BMSCs) as a model to investigate the role of stromal constraints in altering the mitochondrial activities of cancer cells within the tumor microenvironment (TME). Using microdissection and RNA sequencing, we revealed a differentially regulated pattern of gene expression related to mitochondrial activities and metastatic potential at the tumor-stromal interface. Gene expression was confirmed by immunostaining of mitochondrial mass, and live microscopic imaging of mitochondrial membrane potential (ΔΨm) and optical redox ratio. We demonstrated that physical constraints by the stromal cells play a major role in ΔΨm heterogeneity, which was positively associated with nuclear translocation of the YAP/TAZ transcriptional co-activators. Importantly, inhibiting actin polymerization and Rho-associated protein kinase disrupted the differential ΔΨm pattern. In addition, we showed a positive correlation between ΔΨm level and metastatic burden in vivo in mice injected with MDA-MB-231 breast cancer cells. This study supports a new regulatory role for the TME in mitochondrial heterogeneity and metastatic potential.

Evaluating CAR-T Cell Therapy in a Hypoxic 3D Tumor Model.

Despite its revolutionary success in hematological malignancies, chimeric antigen receptor T (CAR-T) cell therapy faces disappointing clinical results in solid tumors. The poor efficacy has been partially attributed to the lack of understanding in how CAR-T cells function in a solid tumor microenvironment. Hypoxia plays a critical role in cancer progression and immune editing, which potentially results in solid tumors escaping immunosurveillance and CAR-T cell-mediated cytotoxicity. Mechanistic studies of CAR-T cell biology in a physiological environment has been limited by the complexity of tumor-immune interactions in clinical and animal models, as well as by a lack of reliable in vitro models. A microdevice platform that recapitulates a 3D tumor section with a gradient of oxygen and integrates fluidic channels surrounding the tumor for CAR-T cell delivery is engineered. The design allows for the evaluation of CAR-T cell cytotoxicity and infiltration in the heterogeneous oxygen landscape of in vivo solid tumors at a previously unachievable scale in vitro.

An inverse free electron laser acceleration-driven Compton scattering X-ray source.

The generation of X-rays and γ-rays based on synchrotron radiation from free electrons, emitted in magnet arrays such as undulators, forms the basis of much of modern X-ray science. This approach has the drawback of requiring very high energy, up to the multi-GeV-scale, electron beams, to obtain the required photon energy. Due to the limit in accelerating gradients in conventional particle accelerators, reaching high energy typically demands use of instruments exceeding 100's of meters in length. Compact, less costly, monochromatic X-ray sources based on very high field acceleration and very short period undulators, however, may enable diverse, paradigm-changing X-ray applications ranging from novel X-ray therapy techniques to active interrogation of sensitive materials, by making them accessible in energy reach, cost and size. Such compactness and enhanced energy reach may be obtained by an all-optical approach, which employs a laser-driven high gradient accelerator based on inverse free electron laser (IFEL), followed by a collision point for inverse Compton scattering (ICS), a scheme where a laser is used to provide undulator fields. We present an experimental proof-of-principle of this approach, where a TW-class CO2 laser pulse is split in two, with half used to accelerate a high quality electron beam up to 84 MeV through the IFEL interaction, and the other half acts as an electromagnetic undulator to generate up to 13 keV X-rays via ICS. These results demonstrate the feasibility of this scheme, which can be joined with other techniques such as laser recirculation to yield very compact photon sources, with both high peak and average brilliance, and with energies extending from the keV to MeV scale. Further, use of the IFEL acceleration with the ICS interaction produces a train of high intensity X-ray pulses, thus enabling a unique tool synchronized with a laser pulse for ultra-fast strobe, pump-probe experimental scenarios.

[Bilateral uveitis associated with nivolumab therapy]. / Uvéite bilatérale associée à un traitement par nivolumab.

Immune-related adverse events (IRAEs) are rare but serious adverse events that may be associated with inhibitors of few immune control points. The purpose here is to report the case of an inflammatory ocular disease, potentially linked to the immunity and use of nivolumab, a new immunological agent used for the treatment of a solid tumor. In spite of the involvement of this treatment in the onset of inflammation, we must always seek another cause. It is possible to continue this treatment by considering the benefit/risk balance for each patient. Close collaboration between oncologists and ophthalmologists is necessary in the diagnosis and rapid management of these IRAE ocular related to these new emerging therapies.

Experimental Characterization of Electron-Beam-Driven Wakefield Modes in a Dielectric-Woodpile Cartesian Symmetric Structure.

Photonic structures operating in the terahertz (THz) spectral region enable the essential characteristics of confinement, modal control, and electric field shielding for very high gradient accelerators based on wakefields in dielectrics. We report here an experimental investigation of THz wakefield modes in a three-dimensional photonic woodpile structure. Selective control in exciting or suppressing of wakefield modes with a nonzero transverse wave vector is demonstrated by using drive beams of varying transverse ellipticity. Additionally, we show that the wakefield spectrum is insensitive to the offset position of strongly elliptical beams. These results are consistent with analytic theory and three-dimensional simulations and illustrate a key advantage of wakefield systems with Cartesian symmetry: the suppression of transverse wakes by elliptical beams.

Bilateral uveitis associated with nivolumab therapy.

New anticancer therapies, immune pathway inhibitors, may cause immune-related adverse events (IRAE). Immune-related ocular toxicities are rare but are potentially serious adverse events. The purpose of this article is to report a case of ocular inflammatory involvement potentially related to the immune response and the use of nivolumab, a new immunologic agent used for the treatment of a solid tumor. Despite the implication of this therapy in the occurrence of inflammation, other causes must always be ruled out. It is possible to continue this therapy in consideration of the risk/benefit ratio for each patient. Close collaboration between oncologists and ophthalmologists is necessary in the diagnosis and timely management of IRAE related to these new emerging therapies.

A Microdevice Platform Recapitulating Hypoxic Tumor Microenvironments.

Hypoxia plays a central role in cancer progression and resistance to therapy. We have engineered a microdevice platform to recapitulate the intratumor oxygen gradients that drive the heterogeneous hypoxic landscapes in solid tumors. Our design features a "tumor section"-like culture by incorporating a cell layer between two diffusion barriers, where an oxygen gradient is established by cellular metabolism and physical constraints. We confirmed the oxygen gradient by numerical simulation and imaging-based oxygen sensor measurement. We also demonstrated spatially-resolved hypoxic signaling in cancer cells through immunostaining, gene expression assay, and hypoxia-targeted drug treatment. Our platform can accurately generate and control oxygen gradients, eliminates complex microfluidic handling, allows for incorporation of additional tumor components, and is compatible with high-content imaging and high-throughput applications. It is well suited for understanding hypoxia-mediated mechanisms in cancer disease and other biological processes, and discovery of new therapeutics.

Observation of acceleration and deceleration in gigaelectron-volt-per-metre gradient dielectric wakefield accelerators.

There is urgent need to develop new acceleration techniques capable of exceeding gigaelectron-volt-per-metre (GeV m(-1)) gradients in order to enable future generations of both light sources and high-energy physics experiments. To address this need, short wavelength accelerators based on wakefields, where an intense relativistic electron beam radiates the demanded fields directly into the accelerator structure or medium, are currently under intense investigation. One such wakefield based accelerator, the dielectric wakefield accelerator, uses a dielectric lined-waveguide to support a wakefield used for acceleration. Here we show gradients of 1.347±0.020 GeV m(-1) using a dielectric wakefield accelerator of 15 cm length, with sub-millimetre transverse aperture, by measuring changes of the kinetic state of relativistic electron beams. We follow this measurement by demonstrating accelerating gradients of 320±17 MeV m(-1). Both measurements improve on previous measurements by and order of magnitude and show promise for dielectric wakefield accelerators as sources of high-energy electrons.