Through-bond and through-space radiofrequency amplification by stimulated emission of radiation.

Radio Amplification by Stimulated Emission of Radiation (RASER) is a phenomenon observed during nuclear magnetic resonance (NMR) experiments with strongly negatively polarized systems. This phenomenon may be utilized for the production of very narrow NMR lines, background-free NMR spectroscopy, and excitation-free sensing of chemical transformations. Recently, novel methods of producing RASER by ParaHydrogen-Induced Polarization (PHIP) were introduced. Here, we show that pairwise addition of parahydrogen to various propargylic compounds induces RASER activity of other protons beyond those chemically introduced in the reaction. In high-field PHIP, negative polarization initiating RASER is transferred via intramolecular cross-relaxation. When parahydrogen is added in Earth's field followed by adiabatic transfer to a high field, RASER activity of other protons is induced via both J-couplings and cross-relaxation. This through-bond and through-space induction of RASER holds potential for the ongoing development and expansion of RASER applications and can potentially enhance spectral resolution in two-dimensional NMR spectroscopy techniques.

A pilot study to evaluate the combination of surgery and brachytherapy for local tumor control in young children with perianal rhabdomyosarcoma.

Background: Perianal rhabdomyosarcoma ((P)RMS) are rare and have a poor prognosis. Data in young children are limited and local therapy is not well defined. Combined brachytherapy and surgery is routinely being used for RMS at other sites in children as it provides good oncologic outcomes and allows for organ-sparing surgery. The objective of this study was to evaluate this combination treatment for local tumor control and organ-sparing surgery in young children with (P)RMS. Methods: A retrospective review of the medical records of all children who underwent surgery and brachytherapy for (P)RMS at our institution since 2009 was conducted. Results: Surgery for (P)RMS was performed in 6 patients at a median age of 19 months (range 8-50). Embryonal RMS was diagnosed in 4 patients and alveolar RMS in 2 patients, of which 1 patient had FOXO1 fusion-positive RMS. All patients underwent postoperative high-dose rate (HDR) brachytherapy. Organ-preserving surgery was achieved in 5 of 6 patients (83 %). In 1 patient, the entire sphincter was infiltrated, making organ-preserving resection impossible. 5 of 6 patients (83 %) exhibited an event-free and overall survival at a median follow-up of 26 months (range 8-107). One patient died due to locoregional recurrence. Complications were urethral leakage in 1 patient followed by urethral stenosis and delayed wound healing and vaginal stenosis in another patient. No patient reported fecal incontinence. Conclusions: Combined treatment with surgery and HDR brachytherapy is feasible in very young children with (P)RMS and leads to a favorable oncologic outcome. Preliminary data show a good functional preservation.

Affecting Charges and Structures of {Bi6} Architectures by 12-Electron Transition Metal Fragments.

Molecules based on polyatomic bismuth substructures are currently attracting a lot of attention owing to this heavy and essentially non-toxic element's uncommon chemical and physical properties, which include unprecedented bonding properties. Hexaatomic {Bi6} substructures that underly more complex cluster structures were recently reported to adopt different structures or exhibit different structural details as a consequence of the charge of the {Bi6} unit. This leads to either crown-shaped cycles for a nominal Bi66- or differently distorted trigonal prisms for compositions close to Bi62-. It was predicted by quantum chemistry that Bi64- should adopt a distinctly distorted boat-like shape, yet a corresponding compound has remained elusive. Here, we report a proof of this assumption by the synthesis and isolation of [K(crypt-222)]2[Bi6{Zn(hmds)}2]∙1.5THF (1), comprising a bimetallic [Bi6{Zn(hmds)}2]2- cluster that fulfils the prediction for the geometric and electronic structure of the missing link (crypt-222 = 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo-[8.8.8]hexa-cosane, hmds = hexamethyldisilazanid). A detailed quantum chemical study shows how the nature of Lewis-acidic transition metal complexes - in particular, 12-electron fragments - control and fine-tune the resulting {Bi6} architectures in accordance with the degree of electron-withdrawal from the polybismuthide core.

Rapid in situ carbon-13 hyperpolarization and imaging of acetate and pyruvate esters without external polarizer.

Hyperpolarized 13C MRI visualizes real-time metabolic processes in vivo. In this study, we achieved high 13C polarization in situ in the bore of an MRI system for precursor molecules of most widely employed hyperpolarized agents: [1-13C]acetate and [1-13C]pyruvate ethyl esters in their perdeuterated forms, enhancing hyperpolarization lifetimes, hyperpolarized to P13C ≈ 28% at 80 mM concentration and P13C ≈ 19% at 10 mM concentration, respectively. Using vinyl esters as unsaturated Parahydrogen-Induced Polarization via Side-Arm Hydrogenation (PHIP-SAH) precursors and our novel polarization setup, we achieved these hyperpolarization levels by fast side-arm hydrogenation in acetone-d6 at elevated temperatures (up to 90°C) and hydrogenation pressures (up to 32 bar). We optimized the hyperpolarization process, reducing it to under 10 s, and employed advanced pulse sequences to enhance the polarization transfer efficiency. The hyperpolarization system has a small footprint, allowing it to be positioned in the same magnet, where 13C MRI is performed. We exemplified the utility of the design with sub-second in situ 13C MRI of ethyl [1-13C]pyruvate-d6. However, challenges remain in side-arm cleavage and purification in the MRI system to extract highly polarized aqueous agent solutions. Our results showcase efficient and rapid 13C hyperpolarization of these metabolite precursors in an MRI system with minimal additional hardware, promising to enhance future throughput and access to hyperpolarized 13C MRI.

Anionic N-Heterocyclic Carbenes from Mesoionic Imidazolium-4-pyrrolides: The Influence of Substituents, Solvents, and Charge on their 77Se NMR Chemical Shifts.

Mesoionic compounds are the starting material for the synthesis of unique anionic N-heterocyclic carbenes. Herein, mesoionic imidazolium pyrrolides synthesized from pyrrole-2-carbaldehyde via various N-alkyl-4-pyrroyl-imidazoles are described. These were converted into nine new 4-(pyrrol-2-yl)-substituted imidazolium salts and transformed into the mesoionic title compounds using an anion exchange resin. The DFT-calculated (B3LYP/6-311++G**) CREF values indicate a great potential for the formation of anionic N-heterocyclic carbenes by deprotonation, which were generated and reacted with selenium to obtain selenoureas. The 77Se NMR shifts investigated under systematic variation of conditions are dependent on the substitution pattern (ΔδSe = 133 ppm) and the steric demand of the substituents. Solvent dependencies of the 77Se NMR shifts were investigated applying toluene-d8, THF-d8, CDCl3, CD2Cl2, pyridine-d5, acetone-d6, DMSO-d6, CD3CN, AcOD, and MeOD. The influences of the referencing method on the 77Se shifts using external or internal Me2Se or Ph2Se2 and solvent can add up to ΔδSe = ca. 80 ppm. In addition, we observed a temperature dependence of both the selenoureas and the reference reagent Ph2Se2 as well as a 77Se shift difference of the analyte caused by interaction with internally added Ph2Se2. The negative charge of deprotonated selenoureas shifts the values by an additional -20 ppm.

Induction of oxidative- and endoplasmic-reticulum-stress dependent apoptosis in pancreatic cancer cell lines by DDOST knockdown.

The dolichyl-diphosphooligosaccharide-protein glycosyltransferase non-catalytic subunit (DDOST) is a key component of the oligosaccharyltransferase complex catalyzing N-linked glycosylation in the endoplasmic reticulum lumen. DDOST is associated with several cancers and congenital disorders of glycosylation. However, its role in pancreatic cancer remains elusive, despite its enriched pancreatic expression. Using quantitative mass spectrometry, we identify 30 differentially expressed proteins and phosphopeptides (DEPs) after DDOST knockdown in the pancreatic ductal adenocarcinoma (PDAC) cell line PA-TU-8988T. We evaluated DDOST / DEP protein-protein interaction networks using STRING database, correlation of mRNA levels in pancreatic cancer TCGA data, and biological processes annotated to DEPs in Gene Ontology database. The inferred DDOST regulated phenotypes were experimentally verified in two PDAC cell lines, PA-TU-8988T and BXPC-3. We found decreased proliferation and cell viability after DDOST knockdown, whereas ER-stress, ROS-formation and apoptosis were increased. In conclusion, our results support an oncogenic role of DDOST in PDAC by intercepting cell stress events and thereby reducing apoptosis. As such, DDOST might be a potential biomarker and therapeutic target for PDAC.

Three-dimensional radial echo-planar spectroscopic imaging for hyperpolarized 13C MRSI in vivo.

PURPOSE: To demonstrate the feasibility of 3D echo-planar spectroscopic imaging (EPSI) technique with rapid volumetric radial k-space sampling for hyperpolarized (HP) 13C magnetic resonance spectroscopic imaging (MRSI) in vivo. METHODS: A radial EPSI (rEPSI) was implemented on a 3 T clinical PET/MR system. To enable volumetric coverage, the sinusoidal shaped readout gradients per k-t-spoke were rotated along the three spatial dimensions in a golden-angle like manner. A distance-weighted, density-compensated gridding reconstruction was used, also in cases with undersampling of spokes in k-space. Measurements without and with HP 13C-labeled substances were performed in phantoms and rats using a double-resonant 13C/1H volume resonator with 72 mm inner diameter. RESULTS: Phantom measurements demonstrated the feasibility of the implemented rEPSI sequence, as well as the robustness to undersampling in k-space up to a factor of 5 without advanced reconstruction techniques. Applied to measurements with HP [1-13C]pyruvate in a tumor-bearing rat, we obtained well-resolved MRSI datasets with a large matrix size of 123 voxels covering the whole imaging FOV of (180 mm)3 within 6.3 s, enabling to observe metabolism in dynamic acquisitions. CONCLUSION: After further optimization, the proposed rEPSI method may be useful in applications of HP 13C-tracers where unknown or varying metabolite resonances are expected, and the acquisition of dynamic, volumetric MRSI datasets with an adequate temporal resolution is a challenge.

Sustainability initiatives in inpatient psychiatry: tackling food waste.

Background: Food plays a dual role in promoting human health and environmental sustainability. Yet, current food systems jeopardize both. Food waste poses a major global challenge due to its significant economic, social, and environmental impacts. Healthcare facilities generate the largest amounts of food waste compared to other forms of catering provision. Food waste correlates with environmental unsustainability and diminished patient satisfaction, compounding the prevalent challenge of hospital malnutrition and contributing to suboptimal patient outcomes. Materials and methods: In a three-year interventional study (2020-2022) at a psychiatric tertiary care center, we assessed and mitigated food waste using evidence-based measures. We conducted systematic food wastage audits over three years (2020-2022) in May and June, each lasting four weeks. Costs were analyzed comprehensively, covering food, staff, infrastructure, and disposal. Environmental impact was assessed using Umweltbelastungspunkte (UBP) and CO2e/kg emissions, alongside water usage (H2O - l/kg). Results: Economic losses due to food wastage were substantial, primarily from untouched plates and partially consumed dinners, prompting meal planning adjustments. Despite a >3% increase in meals served, both food waste mass and costs decreased by nearly 6%. Environmental impact indicators showed a reduction >20%. Vegetables, salad, and fruits constituted a significant portion of waste. Overproduction minimally contributed to waste, validating portion control efficacy. Conclusion: Our study highlights significant economic and environmental losses due to hospital food waste, emphasizing the importance of resource efficiency. The strategies outlined offer promising avenues for enhanced efficiency. The decrease in food waste observed over the three-year period underscores the potential for improvement.

Spatial proteomics of skeletal muscle using thin cryosections reveals metabolic adaptation at the muscle-tendon transition zone.

Morphological studies of skeletal muscle tissue provide insights into the architecture of muscle fibers, the surrounding cells, and the extracellular matrix (ECM). However, a spatial proteomics analysis of the skeletal muscle including the muscle-tendon transition zone is lacking. Here, we prepare cryotome muscle sections of the mouse soleus muscle and measure each slice using short liquid chromatography-mass spectrometry (LC-MS) gradients. We generate 3,000 high-resolution protein profiles that serve as the basis for a network analysis to reveal the complex architecture of the muscle-tendon junction. Among the protein profiles that increase from muscle to tendon, we find proteins related to neuronal activity, fatty acid biosynthesis, and the renin-angiotensin system (RAS). Blocking the RAS in cultured mouse tenocytes using losartan reduces the ECM synthesis. Overall, our analysis of thin cryotome sections provides a spatial proteome of skeletal muscle and reveals that the RAS acts as an additional regulator of the matrix within muscle-tendon junctions.

Toward Ultra-High-Quality-Factor Wireless Masing Magnetic Resonance Sensing.

It has recently been shown that a bolus of hyperpolarized nuclear spins can yield stimulated emission signals similar in nature to maser signals, potentially enabling new ways of sensing hyperpolarized contrast media, including most notably [1-13C]pyruvate that is under evaluation in over 50 clinical trials for metabolic imaging of cancer. The stimulated NMR signal emissions lasting for minutes do not require radio-frequency excitation, offering unprecedented advantages compared to conventional MR sensing. However, creating nuclear spin maser emission is challenging in practice due to stringent fundamental requirements, making practical in vivo applications hardly possible using conventional passive MR detectors. Here, we demonstrate the utility of a wireless NMR maser detector, the quality factor of which was enhanced 22-fold (to 1,670) via parametric pumping. This active-feedback technique breaks the intrinsic fundamental limit of NMR detector circuit quality factor. We show the use of parametric pumping to reduce the threshold requirement for inducing nuclear spin masing at 300 MHz resonance frequency in a preclinical MRI scanner. Indeed, stimulated emission from hyperpolarized protons was obtained under highly unfavorable conditions of low magnetic field homogeneity (T2* of 3 ms). Greater gains of the quality factor of the MR detector (up to 1 million) were also demonstrated.

X-ray beam diagnostics at the MID instrument of the European X-ray Free-Electron Laser Facility.

The Materials Imaging and Dynamics (MID) instrument at the European X-ray Free-Electron Laser Facility (EuXFEL) is equipped with a multipurpose diagnostic end-station (DES) at the end of the instrument. The imager unit in DES is a key tool for aligning the beam to a standard trajectory and for adjusting optical elements such as focusing lenses or the split-and-delay line. Furthermore, the DES features a bent-diamond-crystal spectrometer to disperse the spectrum of the direct beam to a line detector. This enables pulse-resolved characterization of the EuXFEL spectrum to provide X-ray energy calibration, and the spectrometer is particularly useful in commissioning special modes of the accelerator. Together with diamond-based intensity monitors, the imager and spectrometer form the DES unit which also contains a heavy-duty beamstop at the end of the MID instrument. Here, we describe the setup in detail and provide exemplary beam diagnostic results.

Photo-switchable Collectors for the Flotation of Lithium Aluminate for the Recycling of the Critical Raw Material Lithium.

Flotation of the mineral lithium aluminate by application of the natural product punicine from Punica granatum and some derivatives as collectors is examined. Punicines, 1-(2',5'-dihydroxyphenyl)-pyridinium compounds, are switchable molecules whose properties can be changed reversibly. They exist as cations, neutral mesomeric betaines, anions, and dianions depending on the pH. In light, they form radicals. Five punicine derivatives were prepared which possess ß-methyl, ß-chlorine, γ-tert.-butyl, and γ-acetyl groups attached to the pyridinium ring, and a pyrogallol derivative. On the other hand, LiAlO2 reacts with water to give species such as LiAl2(OH)7 on its surface. Flotations were performed applying the punicines in daylight (3000 lux), in darkness (<40 lux) and under UV-irradiation (4500 lux, 390-400 nm). The pH of the suspension, the collector's concentration, the conditioning time as well as the flotation time were varied. The recovery rates strongly depend on these parameters. For example, the recovery rate of lithium aluminate was increased by 116 % on changing the lighting condition from daylight to darkness, when the pyrogallol derivative of punicine was applied. UV, FTIR, TGA and zeta potential measurements as well as DFT calculations were performed in order to gain insight into the chemistry of punicines on the surface of LiAlO2 and LiAl2(OH)7 in water which influence the flotation's results.

Lithium aluminate flotation by pH- and light-switchable collectors based on the natural product punicine.

Derivatives of the natural product punicine [1-(2',5'-dihydroxyphenyl)pyridinium chloride] were developed as switchable collectors for the flotation of lithium-containing engineered artifical minerals (EnAMs). These EnAMs are e.g. formed by pyrometallurgical processing of end-of-life lithium-ion batteries. Depending on the pH value and the lighting conditions, punicines exist in water as cations, two different electrostatically neutral mesomeric betaines, anionic tripoles, radical cations or radical anions. The radical species form by photochemically induced disproportionation reactions. We prepared punicine derivatives introducing alkyl chains in the pyridinium moiety (4-methyl, 4-ethyl, 4-octyl and 4-undecanyl) to install hydrophobic groups and examined the recovery rates of the flotation of lithium aluminate (LiAlO2). We varied the lighting conditions (darkness, daylight, LED irradiation at λ = 390-400 nm) and the pH value, the collector's and frother's concentration, and the flotation time. With our collectors, recovery rates of lithium aluminate up to 90% were accomplished when the flotation was conducted in Hallimond tubes exposed to daylight at pH 11 in water.

MATRESHCA: Microtesla Apparatus for Transfer of Resonance Enhancement of Spin Hyperpolarization via Chemical Exchange and Addition.

We present an integrated, open-source device for parahydrogen-based hyperpolarization processes in the microtesla field regime with a cost of components of less than $7000. The device is designed to produce a batch of 13C and 15N hyperpolarized (HP) compounds via hydrogenative or non-hydrogenative parahydrogen-induced polarization methods that employ microtesla magnetic fields for efficient polarization transfer of parahydrogen-derived spin order to X-nuclei (e.g., 13C and 15N). The apparatus employs a layered structure (reminiscent of a Russian doll "Matryoshka") that includes a nonmagnetic variable-temperature sample chamber, a microtesla magnetic field coil (operating in the range of 0.02-75 microtesla), a three-layered mu-metal shield (to attenuate the ambient magnetic field), and a magnetic shield degaussing coil placed in the overall device enclosure. The gas-handling manifold allows for parahydrogen-gas flow and pressure control (up to 9.2 bar of total parahydrogen pressure). The sample temperature can be varied either using a water bath or a PID-controlled heat exchanger in the range from -12 to 80 °C. This benchtop device measures 62 cm (length) × 47 cm (width) × 47 cm (height), weighs 30 kg, and requires only connections to a high-pressure parahydrogen gas supply and a single 110/220 VAC power source. The utility of the device has been demonstrated using an example of parahydrogen pairwise addition to form HP ethyl [1-13C]acetate (P13C = 7%, [c] = 1 M). Moreover, the Signal Amplification By Reversible Exchange in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) technique was employed to demonstrate efficient hyperpolarization of 13C and 15N spins in a wide range of biologically relevant molecules, including [1-13C]pyruvate (P13C = 14%, [c] = 27 mM), [1-13C]-α-ketoglutarate (P13C = 17%), [1-13C]ketoisocaproate (P13C = 18%), [15N3]metronidazole (P15N = 13%, [c] = 20 mM), and others. While the vast majority of the utility studies have been performed in standard 5 mm NMR tubes, the sample chamber of the device can accommodate a wide range of sample container sizes and geometries of up to 1 L sample volume. The device establishes an integrated, simple, inexpensive, and versatile equipment gateway needed to facilitate parahydrogen-based hyperpolarization experiments ranging from basic science to preclinical applications; indeed, detailed technical drawings and a bill of materials are provided to support the ready translation of this design to other laboratories.

Carbon-13 Radiofrequency Amplification by Stimulated Emission of Radiation of the Hyperpolarized Ketone and Hemiketal Forms of Allyl [1-13C]Pyruvate.

13C hyperpolarized pyruvate is an emerging MRI contrast agent for sensing molecular events in cancer and other diseases with aberrant metabolic pathways. This metabolic contrast agent can be produced via several hyperpolarization techniques. Despite remarkable success in research settings, widespread clinical adoption faces substantial roadblocks because the current sensing technology utilized to sense this contrast agent requires the excitation of 13C nuclear spins that also need to be synchronized with MRI field gradient pulses. Here, we demonstrate sensing of hyperpolarized allyl [1-13C]pyruvate via the stimulated emission of radiation that mitigates the requirements currently blocking broader adoption. Specifically, 13C Radiofrequency Amplification by Stimulated Emission of Radiation (13C RASER) was obtained after pairwise addition of parahydrogen to a pyruvate precursor, detected in a commercial inductive detector with a quality factor (Q) of 32 for sample concentrations as low as 0.125 M with 13C polarization of 4%. Moreover, parahydrogen-induced polarization allowed for the preparation of a mixture of ketone and hemiketal forms of hyperpolarized allyl [1-13C]pyruvate, which are separated by 10 ppm in 13C NMR spectra. This is a good model system to study the simultaneous 13C RASER signals of multiple 13C species. This system models the metabolic production of hyperpolarized [1-13C]lactate from hyperpolarized [1-13C]pyruvate, which has a similar chemical shift difference. Our results show that 13C RASER signals can be obtained from both species simultaneously when the emission threshold is exceeded for both species. On the other hand, when the emission threshold is exceeded only for one of the hyperpolarized species, 13C stimulated emission is confined to this species only, therefore enabling the background-free detection of individual hyperpolarized 13C signals. The reported results pave the way to novel sensing approaches of 13C hyperpolarized pyruvate, potentially unlocking hyperpolarized 13C MRI on virtually any MRI system─an attractive vision for the future molecular imaging and diagnostics.

Highly Sensitive Switchable Sensors for Hydroxide on Glass Surfaces Based on Isoquinolinium-Quinolinium-substituted Acetylenes.

Bi-substituted acetylenes with a quinolinium and an isoquinolinium substituent are described, which reversibly form intensely colored adducts with O-nucleophiles and thus enable the detection of >0,5 ppm hydroxide on the surfaces of various glasses. Acids reconstitute the colorless bi-substituted acetylenes. The quinolinium and isoquinolinium rings are bound via their 2-, 3-, 4- and 1-, 3-, 4-positions to the triple bond, respectively. The choice of substitution sites of the hetarenium rings enables the design of mixed conjugated/cross-conjugated π-electron systems. Depending on the combination of binding sites, the frontier orbital profile, the triple bond polarization, the fluorescence behaviour, and the sensitivity to hydroxide differs.

New Horizons in Hyperpolarized 13C MRI.

Hyperpolarization techniques significantly enhance the sensitivity of magnetic resonance (MR) and thus present fascinating new directions for research and applications with in vivo MR imaging and spectroscopy (MRI/S). Hyperpolarized 13C MRI/S, in particular, enables real-time non-invasive assessment of metabolic processes and holds great promise for a diverse range of clinical applications spanning fields like oncology, neurology, and cardiology, with a potential for improving early diagnosis of disease, patient stratification, and therapy response assessment. Despite its potential, technical challenges remain for achieving clinical translation. This paper provides an overview of the discussions that took place at the international workshop "New Horizons in Hyperpolarized 13C MRI," in March 2023 at the Bavarian Academy of Sciences and Humanities, Munich, Germany. The workshop covered new developments, as well as future directions, in topics including polarization techniques (particularly focusing on parahydrogen-based methods), novel probes, considerations related to data acquisition and analysis, and emerging clinical applications in oncology and other fields.

Genetic and functional analysis of chymotrypsin-like protease (CTRL) in chronic pancreatitis.

BACKGROUND: Genetic predisposition is crucial in the pathogenesis of early-onset chronic pancreatitis (CP). So far, several genetic alterations have been identified as risk factors, predominantly in genes encoding digestive enzymes. However, many early-onset CP cases have no identified underlying cause. Chymotrypsins are a family of serine proteases that can cleave trypsinogen and lead to its degradation. Because genetic alterations in the chymotrypsins CTRC, CTRB1, and CTRB2 are associated with CP, we genetically and functionally investigated chymotrypsin-like protease (CTRL) as a potential risk factor. METHODS: We screened 1005 non-alcoholic CP patients and 1594 controls for CTRL variants by exome sequencing. We performed Western blots and activity assays to analyse secretion and proteolytic activity. We measured BiP mRNA expression to investigate the potential impact of identified alterations on endoplasmic reticulum (ER) stress. RESULTS: We identified 13 heterozygous non-synonymous CTRL variants: five exclusively in patients and three only in controls. Functionality was unchanged in 6/13 variants. Four alterations showed normal secretion but reduced (p.G20S, p.G56S, p.G61S) or abolished (p.S208F) activity. Another three variants (p.C201Y, p.G215R and p.C220G) were not secreted and already showed reduced or no activity intracellularly. However, intracellular retention did not lead to ER stress. CONCLUSION: We identified several CTRL variants, some showing potent effects on protease function and secretion. We observed these effects in variants found in patients and controls, and CTRL loss-of-function variants were not significantly more common in patients than controls. Therefore, CTRL is unlikely to play a relevant role in the development of CP.

Super-resolution Reflection Microscopy via Absorbance Modulation.

In recent years, fluorescence microscopy has been revolutionized. Reversible switching of fluorophores has enabled circumventing the limits imposed by diffraction. Thus, resolution down to the molecular scale became possible. However, to the best of our knowledge, the application of the principles underlying super-resolution fluorescence microscopy to reflection microscopy has not been experimentally demonstrated. Here, we present the first evidence that this is indeed possible. A layer of photochromic molecules referred to as the absorbance modulation layer (AML) is applied to a sample under investigation. The AML-coated sample is then sequentially illuminated with a one-dimensional (1D) focal intensity distribution (similar to the transverse laser mode TEM01) at wavelength λ1 = 325 nm to create a subwavelength aperture within the AML, followed by illumination with a Gaussian focal spot at λ2 = 633 nm for high-resolution imaging. Using this method, called absorbance modulation imaging (AMI) in reflection, we demonstrate a 2.4-fold resolution enhancement over the diffraction limit for a numerical aperture (NA) of 0.65 and wavelength (λ) of 633 nm.

Spontaneous Confinement of mRNA Molecules at Biomolecular Condensate Boundaries.

Cellular biomolecular condensates, termed ribonucleoprotein (RNP) granules, are often enriched in messenger RNA (mRNA) molecules relative to the surrounding cytoplasm. Yet, the spatial localization and diffusion of mRNAs in close proximity to phase separated RNP granules are not well understood. In this study, we performed single-molecule fluorescence imaging experiments of mRNAs in live cells in the presence of two types of RNP granules, stress granules (SGs) and processing bodies (PBs), which are distinct in their molecular composition and function. We developed a photobleaching- and noise-corrected colocalization imaging algorithm that was employed to determine the accurate positions of individual mRNAs relative to the granule's boundaries. We found that mRNAs are often localized at granule boundaries, an observation consistent with recently published data. We suggest that mRNA molecules become spontaneously confined at the RNP granule boundary similar to the adsorption of polymer molecules at liquid-liquid interfaces, which is observed in various technological and biological processes. We also suggest that this confinement could be due to a combination of intermolecular interactions associated with, first, the screening of a portion of the RNP granule interface by the polymer and, second, electrostatic interactions due to a strong electric field induced by a Donnan potential generated across the thin interface.