Link Brain-Wide Projectome to Neuronal Dynamics in the Mouse Brain.

Knowledge about the neuronal dynamics and the projectome are both essential for understanding how the neuronal network functions in concert. However, it remains challenging to obtain the neural activity and the brain-wide projectome for the same neurons, especially for neurons in subcortical brain regions. Here, by combining in vivo microscopy and high-definition fluorescence micro-optical sectioning tomography, we have developed strategies for mapping the brain-wide projectome of functionally relevant neurons in the somatosensory cortex, the dorsal hippocampus, and the substantia nigra pars compacta. More importantly, we also developed a strategy to achieve acquiring the neural dynamic and brain-wide projectome of the molecularly defined neuronal subtype. The strategies developed in this study solved the essential problem of linking brain-wide projectome to neuronal dynamics for neurons in subcortical structures and provided valuable approaches for understanding how the brain is functionally organized via intricate connectivity patterns.

Arg177 and Asp159 from dog prion protein slow liquid-liquid phase separation and inhibit amyloid formation of human prion protein.

Prion diseases are a group of transmissible neurodegenerative diseases primarily caused by the conformational conversion of prion protein (PrP) from α-helix-dominant cellular prion protein (PrPC) to ß-sheet-rich pathological aggregated form of PrPSc in many mammalian species. Dogs exhibit resistance to prion diseases, but the mechanism behind the phenomenon remains poorly understood. Compared with human PrP and mouse PrP, dog PrP has two unique amino acid residues, Arg177 and Asp159. Because PrPC contains a low-complexity and intrinsically disordered region in its N-terminal domain, it undergoes liquid-liquid phase separation (LLPS) in vitro and forms protein condensates. However, little is known about whether these two unique residues modulate the formation of PrPC condensates. Here, using confocal microscopy, fluorescence recovery after photobleaching assays, thioflavin T binding assays, and transmission electron microscopy, we report that Arg177 and Asp159 from the dog PrP slow the LLPS of full-length human PrPC, shifting the equilibrium phase boundary to higher protein concentrations and inhibit amyloid formation of the human protein. In sharp contrast, His177 and Asn159 from the human PrP enhance the LLPS of full-length dog PrPC, shifting the equilibrium phase boundary to lower protein concentrations, and promote fibril formation of the canid protein. Collectively, these results demonstrate how LLPS and amyloid formation of PrP are inhibited by a single residue Arg177 or Asp159 associated with prion disease resistance, and how LLPS and fibril formation of PrP are promoted by a single residue His177 or Asn159. Therefore, Arg177/His177 and Asp159/Asn159 are key residues in modulating PrPC liquid-phase condensation.

Prevalence and contributing factors of impaired awareness of hypoglycemia in patients with type 2 diabetes: a meta-analysis.

AIMS: To conduct a systematic review to summarize the definition, measurement tools, prevalence, and contributing factors of impaired awareness of hypoglycemia (IAH) in type 2 diabetes mellitus (T2DM). METHODS: A reproducible search strategy was used to identify factors affecting IAH in T2DM in PubMed, MEDLINE, EMBASE, Cochrane, PsycINFO, and CINAHL from inception until 2022. Literature screening, quality evaluation, and information extraction were performed independently by 2 investigators. A meta-analysis of prevalence was performed using Stata 17.0. RESULTS: The pooled prevalence of IAH in patients with T2DM was 22% (95%CI:14-29%). Measurement tools included the Gold score, Clarke's questionnaire, and the Pedersen-Bjergaard scale. IAH in T2DM was associated with sociodemographic factors (age, BMI, ethnicity, marital status, education level, and type of pharmacy patients visited), clinical disease factors (disease duration, HbAlc, complications, insulin therapy regimen, sulfonylureas use, and the frequency and severity of hypoglycemia), and behavior and lifestyle (smoking and medication adherence). CONCLUSION: The study found a high prevalence of IAH in T2DM, with an increased risk of severe hypoglycemia, suggesting that medical workers should take targeted measures to address sociodemographic factors, clinical disease, and behavior and lifestyle to reduce IAH in T2DM and thus reduce hypoglycemia in patients.

Happy 40th, NIH Shared Instrumentation Program! The NIH Shared Instrumentation Grant Program Embraces a Promising Future.

The National Institutes of Health (NIH) offers many types of funding programs and opportunities to support biomedical research. The best known of these programs, the NIH Research Project Grant Program, or R01, supports investigator-initiated research projects. Another well-known funding mechanism is the NIH Shared Instrumentation Grant Program, also known as SIG or S10. This year marks the S10's 40th anniversary. To commemorate this triumphant milestone and a successful 40 years, let's first review how this legendary and highly impactful program started.

Genetic prion disease-related mutation E196K displays a novel amyloid fibril structure revealed by cryo-EM.

Prion diseases are caused by the conformational conversion of prion protein (PrP). Forty-two different mutations were identified in human PrP, leading to genetic prion diseases with distinct clinical syndromes. Here, we report the cryo­electron microscopy structure of an amyloid fibril formed by full-length human PrP with E196K mutation, a genetic Creutzfeldt-Jakob disease­related mutation. This mutation disrupts key interactions in the wild-type PrP fibril, forming an amyloid fibril with a conformation distinct from the wild-type PrP fibril and hamster brain­derived prion fibril. The E196K fibril consists of two protofibrils. Each subunit forms five ß strands stabilized by a disulfide bond and an unusual hydrophilic cavity stabilized by a salt bridge. Four pairs of amino acids from opposing subunits form four salt bridges to stabilize the zigzag interface of the two protofibrils. Our results provide structural evidences of the diverse prion strains and highlight the importance of familial mutations in inducing different strains.

Cryo-EM structure of an amyloid fibril formed by full-length human prion protein.

Prion diseases are caused by the misfolding of prion protein (PrP). Misfolded PrP forms protease-resistant aggregates in vivo (PrPSc) that are able to template the conversion of the native form of the protein (PrPC), a property shared by in vitro-produced PrP fibrils. Here we produced amyloid fibrils in vitro from recombinant, full-length human PrPC (residues 23-231) and determined their structure using cryo-EM, building a model for the fibril core comprising residues 170-229. The PrP fibril consists of two protofibrils intertwined in a left-handed helix. Lys194 and Glu196 from opposing subunits form salt bridges, creating a hydrophilic cavity at the interface of the two protofibrils. By comparison with the structure of PrPC, we propose that two α-helices in the C-terminal domain of PrPC are converted into ß-strands stabilized by a disulfide bond in the PrP fibril. Our data suggest that different PrP mutations may play distinct roles in modulating the conformational conversion.

Neutralizing Mutations Significantly Inhibit Amyloid Formation by Human Prion Protein and Decrease Its Cytotoxicity.

Prion diseases, such as Creutzfeldt-Jakob disease and bovine spongiform encephalopathy, are fatal neurodegenerative diseases that affect many mammals including humans and are caused by the misfolding of prion protein (PrP). A naturally occurring protective polymorphism G127V in human PrP has recently been found to significantly attenuate prion diseases, but the mechanism has remained elusive. We herein report that the hydrophobic chain introduced in G127V significantly inhibits amyloid fibril formation by human PrP, highlighting the protective effect of the G127V polymorphism. We further introduce an amino acid with a different hydrophobic chain (Ile) at the same position and find that G127I has similar protective effects as G127V. Moreover, we show that these two neutralizing mutations, G127V and G127I, significantly decrease the human PrP cytotoxicity resulting from PrP fibril formation, mitochondrial damage, and elevated reactive oxygen species production enhanced by a strong prion-prone peptide PrP 106-126. These findings elucidate the molecular basis for a natural protective polymorphism in PrP and will enable the development of novel therapeutic strategies against prion diseases.

Special method to prepare quantum dot probes with reduced cytotoxicity and increased optical property.

Quantum dots (QDs) are widely used in the life sciences because of their novel physicochemical properties. However, the cytotoxity of these nonoparticles have attracted great attention recently because this has not been well resolved. Four probes were synthesized by chemical coupling and protein denaturation with CdSeZnS, CdTe QDs, and transferrin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and capillary electrophoresis were used to verify the conjugation of these luminescent probes. The cytotoxicity of these four luminescent probes and the original QDs were evaluated in HeLa cells. The results showed that over 92% of HeLa cells were still alive after being exposed to 3.2-microM CdSeZnS QDs capped with denatured transferrin for 72 h. Furthermore, while the probe preparation was very simple, the photoluminescence quantum yield of this probe was 7% higher than the original CdSeZnS QDs. This provides a new way for exploiting QD probes with low cytotoxicity, which will expand applications of nanocomposite assembly in biolabeling and imaging.

Quantification of instantaneous flow rate and dynamically changing effective orifice area using a geometry independent three-dimensional digital color Doppler method: An in vitro study mimicking mitral regurgitation.

OBJECTIVE: Our study was intended to test the accuracy of a 3-dimensional (3D) digital color Doppler flow convergence (FC) method for assessing the effective orifice area (EOA) in a new dynamic orifice model mimicking a variety of mitral regurgitation. BACKGROUND: FC surface area methods for detecting EOA have been reported to be useful for quantifying the severity of valvular regurgitation. With our new 3D digital direct FC method, all raw velocity data are available and variable Nyquist limits can be selected for computation of direct FC surface area for computing instantaneous flow rate and temporal change of EOA. METHODS: A 7.0-MHz multiplane transesophageal probe from an ultrasound system (ATL HDI 5000) was linked and controlled by a computer workstation to provide 3D images. Three differently shaped latex orifices (zigzag, arc, and straight slit, each with cutting-edge length of 1 cm) were used to mimic the dynamic orifice of mitral regurgitation. 3D FC surface computation was performed on parallel slices through the 3D data set at aliasing velocities (14-48 cm/s) selected to maximize the regularity and minimize lateral dropout of the visualized 3D FC at 5 points per cardiac cycle. Using continuous wave velocity for each, 3D-calculated EOA was compared with EOA determined by using continuous wave Doppler and the flow rate from a reference ultrasonic flow meter. Simultaneous digital video images were also recorded to define the actual orifice size for 9 stroke volumes (15-55 mL/beat with maximum flow rates 45-182 mL/s). RESULTS: Over the 9 pulsatile flow states and 3 orifices, 3D FC EOAs (0.05-0.63 cm(2)) from different phases of the cardiac cycle in each pump setting correlated well with reference EOA (r = 0.89-0.92, SEE = 0.027-0.055cm(2)) and they also correlated well with digital video images of the actual orifice peak (r = 0.97-0.98, SEE = 0.016-0.019 cm(2)), although they were consistently smaller, as expected by the contraction coefficient. CONCLUSION: The digital 3D FC method can accurately predict flow rate, and, thus, EOA (in conjunction with continuous wave Doppler), because it allows direct FC surface measurement despite temporal variability of FC shape.

A validation study of aortic stroke volume using dynamic 4-dimensional color Doppler: an in vivo study.

OBJECTIVE: To explore the feasibility of directly quantifying transaortic stroke volume with a newly developed dynamic 3-dimensional (3D) color Doppler flow measurement technique, an in vivo experimental study was performed. BACKGROUND: Traditional methods for flow quantification require geometric assumptions about flow area and flow profiles. Accurate quantification of flow across the aortic valve is clinically important as a means of estimating cardiac output. METHODS: Eight open-chest sheep were scanned with apical epicardial placement of a 7 to 4 MHz multiplane transesophageal probe scanning parallel to aortic flow and running on an ATL HDI 5000 system. An electromagnetic flow meter implanted on the ascending aorta was used as reference. Thirty different hemodynamic conditions were studied after steady states were obtained in the animals by administration of blood, angiotensin, and sodium nitroprusside. Electrocardiogram-gated digital color 3D velocity data were acquired for each of the 30 steady states. The aortic stroke volumes were computed by temporal and spatial integration of flow areas and actual velocities across a projected surface perpendicular to the direction of flow, at a level just below the aortic valve. RESULTS: There was close correlation between the 3D color Doppler calculated aortic stroke volumes and the electromagnetic data (r = 0.91, y = 0.96x + 1.01, standard error of the estimate = 2.6 mL/beat). CONCLUSION: Our results showed that dynamic 3D color Doppler measurements obtained in an open-chest animals provide the basis for accurate, geometry-independent quantitative evaluation of the aortic flow. Therefore, 3D digital color Doppler flow computation could potentially represent an important method for noninvasively determining cardiac output in patients.

A new dynamic three-dimensional digital color doppler method for quantification of pulmonary regurgitation: validation study in an animal model.

OBJECTIVES: The purpose of the present study was to validate a newly developed three-dimensional (3D) digital color Doppler method for quantifying pulmonary regurgitation (PR), using an animal model of chronic PR. BACKGROUND: Spectral Doppler methods cannot reliably be used to assess pulmonary regurgitation. METHODS: In eight sheep with surgically created PR, 27 different hemodynamic states were studied. Pulmonary and aortic electromagnetic (EM) probes and meters were used to provide reference right ventricular (RV) forward and pulmonary regurgitant stroke volumes. A multiplane transesophageal probe was placed directly on the RV and aimed at the RV outflow tract. Electrocardiogram-gated and rotational 3D scans were performed for acquiring dynamic 3D digital velocity data. After 3D digital Doppler data were transferred to a computer workstation, the RV forward and pulmonary regurgitant flow volumes were obtained by a program that computes the velocity vectors over a spherical surface perpendicular to the direction of scanning. RESULTS: Pulmonary regurgitant volumes and RV forward stroke volumes computed by the 3D method correlated well with those by the EM method (r = 0.95, mean difference = 0.51 +/- 1.89 ml/beat for the pulmonary regurgitant volume; and r = 0.91, mean difference = -0.22 +/- 3.44 ml/beat for the RV stroke volume). As a result of these measurements, the regurgitant fractions derived by the 3D method agreed well with the reference data (r = 0.94, mean difference = 2.06 +/- 6.11%). CONCLUSIONS: The 3D digital color Doppler technique is a promising method for determining pulmonary regurgitant volumes and regurgitant fractions. It should have an important application in clinical settings.

Direct quantification of transmitral flow volume with dynamic 3-dimensional digital color Doppler: a validation study in an animal model.

Accurately quantifying transmitral flow volume is clinically important not only as a measure of cardiac output, but also as a value from which to subtract aortic flow, for determining the severity of mitral regurgitation. However, controversy exists over the accuracy of pulsed Doppler for mitral flow quantification because of the complexity of mitral flow geometry and dynamic changes in flow profile and flow area. To explore the feasibility of directly quantifying transmitral flow volume with a newly developed dynamic 3-dimensional digital color Doppler technique, this in vivo experimental study was conducted to validate the method. Eight open chest sheep were imaged with a multiplane transesophageal (TEE) probe placed on the heart for digital 3-dimensional gated acquisition of mitral inflow over a 180-degree acquisition. The digital velocity data were contour detected for flow area after computing the velocity vectors and flow profile perpendicular to a spherical 3-dimensional surface across the mitral annulus. Flow areas and actual velocities were then integrated in time and space and the resulting flow volumes were compared with those obtained by a reference electromagnetic flowmeter on the aorta for 26 steady hemodynamic states. The flow volumes correlated closely to the electromagnetic references (y = 0.87x + 2.49, r = 0.92, SEE = 1.9 Ml per beat). Our study shows that transmitral flow volume can be accurately determined in vivo by this dynamic 3-dimensional digital color Doppler flow quantification method.