EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization.

Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 µm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine spatially and molecularly defined subregions. EASI-FISH also facilitates iterative reanalysis of scRNA-seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.

Nanoparticle-assisted tubulin assembly is environment dependent.

Nanomaterials acquire a biomolecular corona upon introduction to biological media, leading to biological transformations such as changes in protein function, unmasking of epitopes, and protein fibrilization. Ex vivo studies to investigate the effect of nanoparticles on protein-protein interactions are typically performed in buffer and are rarely measured quantitatively in live cells. Here, we measure the differential effect of silica nanoparticles on protein association in vitro vs. in mammalian cells. BtubA and BtubB are a pair of bacterial tubulin proteins identified in Prosthecobacter strains that self-assemble like eukaryotic tubulin, first into dimers and then into microtubules in vitro or in vivo. Förster resonance energy transfer labeling of each of the Btub monomers with a donor (mEGFP) and acceptor (mRuby3) fluorescent protein provides a quantitative tool to measure their binding interactions in the presence of unfunctionalized silica nanoparticles in buffer and in cells using fluorescence spectroscopy and microscopy. We show that silica nanoparticles enhance BtubAB dimerization in buffer due to protein corona formation. However, these nanoparticles have little effect on bacterial tubulin self-assembly in the complex mammalian cellular environment. Thus, the effect of nanomaterials on protein-protein interactions may not be readily translated from the test tube to the cell in the absence of particle surface functionalization that can enable targeted protein-nanoparticle interactions to withstand competitive binding in the nanoparticle corona from other biomolecules.

SALT OVERLY SENSITIVE 1 is inhibited by clade D Protein phosphatase 2C D6 and D7 in Arabidopsis thaliana.

The salt overly sensitive (SOS) pathway is essential for maintaining sodium ion homeostasis in plants. This conserved pathway is activated by a calcium signaling-dependent phosphorylation cascade. However, the identity of the phosphatases and their regulatory mechanisms that would deactivate the SOS pathway remain unclear. In this study, we demonstrate that PP2C.D6 and PP2C.D7, which belong to clade D of the protein phosphatase 2C (PP2C) subfamily in Arabidopsis thaliana, directly interact with SOS1 and inhibit its Na+/H+ antiporter activity under non-salt-stress conditions. Upon salt stress, SOS3-LIKE CALCIUM-BINDING PROTEIN8 (SCaBP8), a member of the SOS pathway, interacts with the PP2Cs and suppresses their phosphatase activity; simultaneously, SCaBP8 regulates the subcellular localization of PP2C.D6 by releasing it from the plasma membrane. Thus, we identified two negative regulators of the SOS pathway that repress SOS1 activity under nonstress conditions. These processes set the stage for the activation of SOS1 by the kinase SOS2 to achieve plant salt tolerance. Our results suggest that reversible phosphorylation/dephosphorylation is crucial for the regulation of the SOS pathway, and that calcium sensors play dual roles in activating/deactivating SOS2 and PP2C phosphatases under salt stress.

CAR-Macrophage Therapy Alleviates Myocardial Ischemia-Reperfusion Injury.

BACKGROUND: Given the growing acknowledgment of the detrimental effects of excessive myocardial fibrosis on pathological remodeling after myocardial ischemia-reperfusion injury (I/R), targeting the modulation of myocardial fibrosis may offer protective and therapeutic advantages. However, effective clinical interventions and therapies that target myocardial fibrosis remain limited. As a promising chimeric antigen receptor (CAR) cell therapy, whether CAR macrophages (CAR-Ms) can be used to treat I/R remains unclear. METHODS: The expression of FAP (fibroblast activation protein) was studied in mouse hearts after I/R. FAP CAR-Ms were generated to target FAP-expressing cardiac fibroblasts in mouse hearts after I/R. The phagocytosis activity of FAP CAR-Ms was tested in vitro. The efficacy and safety of FAP CAR-Ms in treating I/R were evaluated in vivo. RESULTS: FAP was significantly upregulated in activated cardiac fibroblasts as early as 3 days after I/R. Upon demonstrating their ability to engulf FAP-overexpressing fibroblasts, we intravenously administered FAP CAR-Ms to mice at 3 days after I/R and found that FAP CAR-Ms significantly improved cardiac function and reduced myocardial fibrosis in mice after I/R. No toxicities associated with FAP CAR-Ms were detected in the heart or other organs at 2 weeks after I/R. Finally, we found that FAP CAR-Ms conferred long-term cardioprotection against I/R. CONCLUSIONS: Our proof-of-concept study demonstrates the therapeutic potential of FAP CAR-Ms in alleviating myocardial I/R and potentially opens new avenues for the treatment of a range of heart diseases that include a fibrotic phenotype.

Atomically Resolved Transition Pathways of Iron Redox.

The redox transition between iron and its oxides is of the utmost importance in heterogeneous catalysis, biological metabolism, and geological evolution. The structural characteristics of this reaction may vary based on surrounding environmental conditions, giving rise to diverse physical scenarios. In this study, we explore the atomic-scale transformation of nanosized Fe3O4 under ambient-pressure H2 gas using in-situ environmental transmission electron microscopy. Our results reveal that the internal solid-state reactions dominated by iron diffusion are coupled with the surface reactions involving gaseous O or H species. During reduction, we observe two competitive reduction pathways, namely Fe3O4 → FeO → Fe and Fe3O4 → Fe. An intermediate phase with vacancy ordering is observed during the disproportionation reaction of Fe2+ → Fe0 + Fe3+, which potentially alleviates stress and facilitates ion migration. As the temperature decreases, an oxidation process occurs in the presence of environmental H2O and trace amounts of O2. A direct oxidation of Fe to Fe3O4 occurs in the absence of the FeO phase, likely corresponding to a change in the water vapor content in the atmosphere. This work elucidates a full dynamical scenario of iron redox under realistic conditions, which is critical for unraveling the intricate mechanisms governing the solid-solid and solid-gas reactions.

Introducing Electron Buffers into Intermetallic Pt Alloys against Surface Polarization for High-Performing Fuel Cells.

Surface polarization under harsh electrochemical environments usually puts catalysts in a thermodynamically unstable state, which strictly hampers the thermodynamic stability of Pt-based catalysts in high-performance fuel cells. Here, we report a strategy by introducing electron buffers (variable-valence metals, M = Ti, V, Cr, and Nb) into intermetallic Pt alloy nanoparticle catalysts to suppress the surface polarization of Pt shells using the structurally ordered L10-M-PtFe as a proof of concept. Operando X-ray absorption spectra analysis suggests that with the potential increase, electron buffers, especially Cr, could facilitate an electron flow to form a electron-enriched Pt shell and thus weaken the surface polarization and tensile Pt strain. The best-performing L10-Cr-PtFe/C catalyst delivers superb oxygen reduction reaction (ORR) activity (mass activity = 1.41/1.02 A mgPt-1 at 0.9 V, rated power density = 14.0/9.2 W mgPt-1 in H2-air under a total Pt loading of 0.075/0.125 mgPt cm-2, respectively) and stability (20 mV voltage loss at 0.8 A cm-2 after 60,000 cycles of accelerated durability test) in a fuel cell cathode, representing one of the best reported ORR catalysts. Density functional theory calculations reveal that the optimized surface strain by introducing Cr on L10-PtFe/C accounts for the enhanced ORR activity, and the durability enhancement stems from the charge transfer contribution of Cr to the Pt shells and the increased kinetic energy barrier for Pt dissolution/Fe diffusion.

Ascorbate insufficiency disrupts glutamatergic signaling and alters electroencephalogram phenotypes in a mouse model of Alzheimer's disease.

Clinical studies have reported that increased epileptiform and subclinical epileptiform activity can be detected in many patients with an Alzheimer's disease (AD) diagnosis using electroencephalogram (EEG) and this may correlate with poorer cognition. Ascorbate may have a specific role as a neuromodulator in AD as it is released concomitantly with glutamate reuptake following excitatory neurotransmission. Insufficiency may therefore result in an exacerbated excitatory/inhibitory imbalance in neuronal signaling. Using a mouse model of AD that requires dietary ascorbate (Gulo-/-APPswe/PSEN1dE9), EEG was recorded at baseline and during 4 weeks of ascorbate depletion in young (5-month-old) and aged (20-month-old) animals. Data were scored for changes in quantity of spike trains, individual spikes, sleep-wake rhythms, sleep fragmentation, and brainwave power bands during light periods each week. We found an early increase in neuronal spike discharges with age and following ascorbate depletion in AD model mice and not controls, which did not correlate with brain amyloid load. Our data also show more sleep fragmentation with age and with ascorbate depletion. Additionally, changes in brain wave activity were observed within different vigilance states in both young and aged mice, where Gulo-/-APPswe/PSEN1dE9 mice had shifts towards higher frequency bands (alpha, beta, and gamma) and ascorbate depletion resulted in shifts towards lower frequency bands (delta and theta). Microarray data supported ascorbate insufficiency altering glutamatergic transmission through the decreased expression of glutamate related genes, however no changes in protein expression of glutamate reuptake transporters were observed. These data suggest that maintaining optimal brain ascorbate levels may support normal brain electrical activity and sleep patterns, particularly in AD patient populations where disruptions are observed.

Point-of-Care Testing of Whole Blood for Liver Injury Auxiliary Diagnosis with Biothiols Activable Chemiluminescent Probe.

Liver injury significantly affects a patient's health and quality of life. However, timely and convenient diagnosis of this disease via whole blood detection remains challenging due to the lack of user-friendly and fast readout blood test methods. Herein, we developed such a method for the swift auxiliary diagnosis of liver injury via whole blood detection using a customed point-of-care testing (POCT) system consisting of a biothiols-activatable chemiluminescent probe and a hand-held POCT device. Biothiols served as the target to build the activable chemiluminescence probe due to their abnormal level in liver injury. Compared with fluorescent and electrical POCTs, this method is more convenient and has strong universality. By incorporating cyclodextrin via host-guest chemistry, we intensified chemiluminescence while mitigating chemical hemolysis caused by the dissolution of organic molecules, making this system suitable for whole blood analysis. Preliminary assessments in aqueous solutions, living cells, and mouse models confirmed its sensitivity, reliability, and feasibility. Simply mixing blood with the probe for 30 min yielded a clear signal readout within 15 s on the POCT device. Utilizing this portable detector, the reduced biothiol level was tested in 18 liver injury patient blood samples, and the results were similar to those measured by a commercial kit and in vivo imaging system. Thus, this work provides a universal platform for the fast and convenient detection of other biomarkers in whole blood samples and opens up possibilities for the rapid clinical diagnosis of diseases, enabling patients to conduct home self-examinations with ease.

Biomineralization-Inspired Synthesis of Hybrid COF Nanosheets toward Efficient Desalination Membranes.

Synthesis of covalent organic framework nanosheets (CONs) with high aspect ratio is crucial to their assembly into advanced membranes. Nonetheless, the π-π stacking between covalent organic framework (COF) layers often leads to thick CONs. Herein, inspired by biomineralization process, a series of aspect ratio CONs >15 000 is synthesized by multifunctional polyelectrolytes which not only provide the nucleation sites for pre-assembly with COF monomer, but also suppress π-π interaction for anisotropic growth through protonation. The membrane assembled from CONs exhibited water permeance of 341 kg m-2 h-1 and salt rejection of 99.5% in desalination, outperforming ever-reported membranes. This method establishes a platform for the synthesis of crystalline nanosheets.

Covalent Organic Framework Membranes from Oppositely Charged Nanosheets toward Efficient Desalination.

Fabricating covalent organic framework (COF) membranes through the pre-assembly of nanosheets with different properties may open a novel avenue to the fabrication of advanced 2D membranes. Herein, COF membranes are fabricated using oppositely-charged COF nanosheets (CONs). Negatively-charged CONs and positively-charged CONs are pre-assembled through simple physical mixing, yielding the CONs with an aspect ratio of exceeding 10 000, which are assembled into three kinds of COF membranes. The optimal membranes exhibit the highest desalination performance with permeation flux of 132.66 kg m-2 h-1, salt rejection of 99.99%, and superior long-term operation stability.

Zwitterionic Nanochannels in Covalent Organic Framework Membranes for Improved Flux and Antifouling Property.

Zwitterionic membranes demonstrate excellent antifouling property in water purification. The covalent organic frameworks (COFs), due to the ordered channels and abundant organic functional groups, have distinct superiority in constructing zwitterionic surfaces.Here, the zwitterionic COF membrane is prepared with precise framework structures and uniform charge distribution. The negatively charged 4,4'-diaminobiphenyl-2,2'-sisulphonic acid sodium (SA) and positively charged ethidium bromide (EB) fragments are used to react with 1,3,5-triformylphloroglucinol (TP) at the gas-liquid interface to prepare zwitterionic COF membrane. The complementary charged fragments in the inter-layer and inner-layer facilitate the formation of continuous and tight hydration layer on the membrane surface and pore walls to resist the adsorption of pollutants. The zwitterionic COF membrane effectively resists both negatively charged bovine serum albumin and positively charged lysozyme pollutants with flux recovery ratio (FRR) of 97% and 85%, respectively. Furthermore, the regular nano-channels and balanced interactions between water and surface/pore walls of the zwitterionic membrane result in outstanding permeability of up to 146 L m-2 h-1 bar-1 and excellent dye/salt separation selectivity. The water permeation and antifouling mechanism of membranes are elucidated by experimental and molecular dynamics calculation. Zwitterionic COF membranes can find promising applications in preparing high-performance antifouling membranes.

Correction: Oncogenic Herpesvirus KSHV Hijacks BMP-Smad1-Id Signaling to Promote Tumorigenesis.

[This corrects the article DOI: 10.1371/journal.ppat.1004253.].

Serum Proteomics Identified TAFI as a Potential Molecule Facilitating the Migration of Peripheral Monocytes to Damaged White Matter During Chronic Cerebral Hypoperfusion.

Neuroinflammation is assumed as the critical pathophysiologic mechanism of white matter lesions (WMLs), and infiltrated peripheral monocyte-derived macrophages are implicated in the development of neuroinflammation. This study sought to explore the blood molecules that promote the migration of peripheral monocytes to the sites of WMLs. The serum protein expression profiles of patients and Sprague-Dawley rat models with WMLs were detected by data-independent acquisition (DIA) proteomics technique. Compared with corresponding control groups, we acquired 62 and 41 differentially expressed proteins (DEPs) in the serum of patients and model rats with WMLs respectively. Bioinformatics investigations demonstrated that these DEPs were linked to various Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms involved in neuroinflammation. Afterward, we identified thrombin-activatable fibrinolysis inhibitor (TAFI) as a shared and overexpressed protein in clinical and animal serum samples, which was further verified by enzyme-linked immunosorbent assay. Additionally, an upregulation of TAFI was also observed in the white matter of rat models, and the inhibition of TAFI impeded the migration of peripheral monocytes to the area of WMLs. In vitro experiments suggested that TAFI could enhance the migration ability of RAW264.7 cells and increase the expression of Ccr2. Our study demonstrates that neuroinflammatory signals can be detected in the peripheral blood of WMLs patients and model rats. TAFI may serve as a potential protein that promotes the migration of peripheral monocytes to WMLs regions, thereby providing a novel molecular target for further investigation into the interaction between the central and peripheral immune systems.

The Impact of Dipyridamole on Disease-Associated Microglia Phenotypic Transformation in White Matter Lesions Induced by Chronic Cerebral Hypoperfusion.

White matter lesions (WMLs) resulting from chronic cerebral hypoperfusion (CCH) are the leading cause of vascular dementia (VaD). This study aimed to investigate whether dipyridamole could alleviate WMLs by regulating the phenotype of disease-associated microglia (DAM) through equilibrative nucleoside transporter 2 (ENT2) and adenosine A2A receptor (Adora2a) and to clarify the underlying molecular mechanisms. CCH rat models were constructed to mimic VaD. Morris water maze and Luxol Fast Blue staining were employed to assess cognitive function and quantify the severity of WMLs, respectively. Immunofluorescent staining was performed to analyze the activation of glial cells and the phenotypic transformation of DAM. Additionally, levels of ENT2, proteins in the NF-κB and ERK1/2 pathways and inflammatory cytokines were detected. The results indicated that dipyridamole diminished the activation and proliferation of microglia and astrocytes, increased the expression of myelin basic protein and ameliorated WMLs and cognitive decline in CCH rats. Further study revealed that dipyridamole decreased the expression of ENT2 and inhibited the activation of ERK1/2 and NF-κB signaling pathways, which ultimately converted DAM to anti-inflammatory phenotype and suppressed the levels of TNF-α, IL-1ß, IL-6 in WMLs. However, Adora2a inhibitor (SCH58261) attenuated above effects. Our study demonstrates that dipyridamole facilitates the conversion of DAM to the anti-inflammatory phenotype through ENT2/Adora2a pathway and inhibits the activation of ERK1/2 and NF-κB signaling pathways, thereby alleviating neuroinflammation in WMLs. The current findings establish the basis for using dipyridamole to treat VaD.

Single-cell RNA Sequencing Identifies a Novel Subtype of Microglia with High Cd74 Expression that Facilitates White Matter Inflammation During Chronic Cerebral Hypoperfusion.

Vascular dementia (VaD) causes progressive cognitive decline in the elderly population, but there is short of available therapeutic measures. Microglia-mediated neuroinflammation is vigorously involved in the pathogenesis of VaD, but the traditional classification of microglial M1/M2 phenotypes remains restrictive and controversial. This study aims to investigate whether microglia transform into novel subtypes in VaD. Chronic cerebral hypoperfusion (CCH) rat model was constructed to mimic VaD. Microglia were isolated via magnetic-activated cell sorting and analyzed by single-cell RNA sequencing (scRNA-seq) and bioinformatics. The findings inferred from scRNA-seq and bioinformatics were further validated through in vivo experiments. In this study, microglia were divided into eight clusters. The proportion of MG5 cluster was significantly increased in the white matter of the CCH group compared with the Sham group and was named chronic ischemia-associated microglia (CIAM). Immunity- and inflammation-related genes, including RT1-Db1, RT1-Da, RT1-Ba, Cd74, Spp1, C3, and Cd68, were markedly upregulated in CIAM. Enrichment analysis illustrated that CIAM possessed the function of evoking neuroinflammation. Further studies unveiled that Cd74 is associated with the most abundant GO terms involved in inflammation as well as cell proliferation and differentiation. In addition, microglia-specific Cd74 knockdown mediated by adeno-associated virus decreased the abundance of CIAM in the white matter, thereby mitigating inflammatory cytokine levels, alleviating white matter lesions, and improving cognitive impairment for CCH rats. These findings indicate that Cd74 is the core molecule of CIAM to trigger neuroinflammation and induce microglial differentiation to CIAM, suggesting that Cd74 may be a potential therapeutic target for VaD.

Predictive Model Building for Aggregation Kinetics Based on Molecular Dynamics Simulations of an Antibody Fragment.

Computational methods including machine learning and molecular dynamics simulations have strong potential to characterize, understand, and ultimately predict the properties of proteins relevant to their stability and function as therapeutics. Such methods would streamline the development pathway by minimizing the current experimental testing required for many protein variants and formulations. The molecular understanding of thermostability and aggregation propensity has advanced significantly along with predictive algorithms based on the sequence-level or structural-level information on a protein. However, these approaches focus largely on a comparison of protein sequence variations to correlate the properties of proteins to their stability, solubility, and aggregation propensity. For therapeutic protein development, it is of equal importance to take into account the impact of the formulation conditions to elucidate and predict the stability of the antibody drugs. At the macroscopic level, changing temperature, pH, ionic strength, and the addition of excipients can significantly alter the kinetics of protein aggregation. The mechanisms controlling aggregation kinetics have been traced back to a combination of molecular features, including conformational stability, partial unfolding to aggregation-prone states, and the colloidal stability governed by surface charges and hydrophobicity. However, very little has been done to evaluate these features in the context of protein dynamics in different formulations. In this work, we have combined a range of molecular features calculated from the Fab A33 protein sequence and molecular dynamics simulations. Using the power of advanced, yet interpretable, statistical tools, it has been possible to uncover greater insights into the mechanisms behind protein stability, validating previous findings, and also develop models that can predict the aggregation kinetics within a range of 49 different solution conditions.

Occurrence and Flow Behavior for Oil Transport in Mixed Wetting Nanoscale Shale Bedding Fractures.

Shale reservoirs are characterized by an abundance of nanoscale porosities and microfractures. The states of fluid occurrence and flow behaviors within nanoconfined spaces necessitate novel research approaches, as traditional percolation mathematical models are inadequate for accurately depicting these phenomena. This study takes the Gulong shale reservoir in China as the subject of its research. Initially, the unique mixed wetting characteristics of the Gulong shale reservoir are examined and characterized using actual micropore images. Subsequently, the occurrence and flow behavior of oil within the nanoscale bedding fractures under various wettability scenarios are described through a combination of microscopic pore image and molecular dynamics simulations. Ultimately, a mathematical model is established that depicts the velocity distribution of oil and its apparent permeability. This study findings indicate that when the scale of the shale bedding fractures is less than 100 nm, the impact of the nanoconfinement effect is significant and cannot be overlooked. In this scenario, the state of oil occurrence and its flow behavior are influenced by the initial oil-wet surface area on the mixed wetting walls. The study quantifies the velocity and density distribution of oil in mixed wetting nanoscale shale bedding fractures through a mathematical model, providing a crucial theoretical basis for upscaling from the nanoscale to the macroscale.

In-Cell Association of a Bioorthogonal Tubulin.

Studies of proteins from one organism in another organism's cells have shown that such exogenous proteins stick more, pointing toward coevolution of the cytoplasm and protein surface to minimize stickiness. Here we flip this question around by asking whether exogenous proteins can assemble efficiently into their target complexes in a non-native cytoplasm. We use as our model system the assembly of BtubA and BtubB from Prosthecobacter hosted in human U-2 OS cells. BtubA and B evolved from eukaryotic tubulins after horizontal gene transfer, but they have low surface sequence identity with the homologous human tubulins and do not respond to tubulin drugs such as nocodazole. In U-2 OS cells, BtubA and B assemble efficiently into dimers compared to in vitro, and the wild-type BtubA and B proteins subsequently are able to form microtubules as well. We find that generic crowding effects (Ficoll 70 in vitro) contribute significantly to efficient dimer assembly when compared to sticking interactions (U-2 OS cell lysate in vitro), consistent with the notion that a generic mechanism such as crowding can be effective at driving assembly of exogenous proteins, even when protein-cytoplasm quinary structure and sticking have been modified in a non-native cytoplasm. A simple Monte Carlo model of in vitro and in-cell interactions, treating BtubA and B as sticky dipoles in a matrix of sticky or nonsticky crowders, rationalizes all the experimental trends with two adjustable parameters and reveals nucleation as the likely mechanism for the time-scale separation between dimer- and tubule formation in-cell and in vitro.

Palladium-Catalyzed Electrochemical Iodination of 1-Arylpyridine N-Oxides.

The palladium-catalyzed C-H iodination of 1-arylpyridine N-oxides proceeded under electrochemical oxidation conditions using I2 as an iodine source. The reaction of isoquinoline N-oxides possessing various para- or meta-substituted aryl groups at the 1-position proceeded to give the corresponding iodination products. Electron-donating groups on the aryl group facilitated the reaction to give relatively high yields of the product. The reaction was also found to be applicable to 2-aryl-3-picoline N-oxides.

Research progress in the mechanisms and functions of specialized pro-resolving mediators in neurological diseases.

The nervous system interacts with the immune system through a variety of cellular regulators, signaling pathways, and molecular mechanisms. Disruptions in these interactions lead to the development of multiple neurological diseases. Recent studies have identified that specialized pro-resolving mediators (SPMs) play a regulatory role in the neuroimmune system. This study reviews recent research on the function of SPMs in the inflammatory process and their association with the nervous system. The review aims to provide new perspectives for studying the pathogenesis of neurological diseases and identify novel targets for clinical therapy.