Temperature-Controllable Liquid Crystalline Medium for Stereochemical Elucidation of Organic Compounds via Residual Chemical Shift Anisotropies.

The configuration elucidation of organic molecules continues to pose significant challenges in studies involving stereochemistry. Nuclear magnetic resonance (NMR) techniques are powerful for obtaining such structural information. Anisotropic NMR techniques, such as measurement of residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs), complementing isotropic NMR parameters, provide relative configuration information. RCSAs provide valuable structural information, especially for nonprotonated carbons, yet have been severely underutilized due to the lack of an easily operational alignment medium capable of rapid transition from anisotropic to isotropic environments, especially in aqueous conditions. In this study, an oligopeptide-based alignment media (FK)4 is presented for RCSA measurements. Temperature variation manipulates the assembly of (FK)4, yielding tunable anisotropic and isotropic phases without the requirement of any special devices or time-consuming correction procedures during data analysis. Decent observed ΔΔRCSA values from sp3 carbons benefit the utilization of RCSA measurements in the structural elucidation of organic molecules highly composed with sp3 carbons. Moreover, the (FK)4 alignment medium is applicable for both RDC and RCSA measurements in one sample, further advancing the configuration analysis of molecules of interest.

Real-Time NMR-Based Drug Discovery to Identify Inhibitors against Fatty Acid Synthesis in Living Cancer Cells.

Abnormal fatty acid metabolism is recognized as a key driver of tumor development and progression. Although numerous inhibitors have been developed to target this pathway, finding drugs with high specificity that do not disrupt normal cellular metabolism remains a formidable challenge. In this paper, we introduced a novel real-time NMR-based drug screening technique that operates within living cells. This technique provides a direct way to putatively identify molecular targets involved in specific metabolic processes, making it a powerful tool for cell-based drug screening. Using 2-13C acetate as a tracer, combined with 3D cell clusters and a bioreactor system, our approach enables real-time detection of inhibitors that target fatty acid metabolism within living cells. As a result, we successfully demonstrated the initial application of this method in the discovery of traditional Chinese medicines that specifically target fatty acid metabolism. Elucidating the mechanisms behind herbal medicines remains challenging due to the complex nature of their compounds and the presence of multiple targets. Remarkably, our findings demonstrate the significant inhibitory effect of P. cocos on fatty acid synthesis within cells, illustrating the potential of this approach in analyzing fatty acid metabolism events and identifying drug candidates that selectively inhibit fatty acid synthesis at the cellular level. Moreover, this systematic approach represents a valuable strategy for discovering the intricate effects of herbal medicine.

A method for rapid nanobody screening with no bias of the library diversity.

Nanobody, referred to the variable domain of heavy-chain-only antibodies, has several advantages such as small size and feasible Escherichia coli expression, making them promising for scientific research and therapies. Conventional nanobody screening and expression methods often suffer from the need for subcloning into expression vectors and amplification-induced diversity loss. Here, we developed an integrated method for simultaneous screening and expression. Nanobody libraries were cloned and secretly expressed in the culture medium. Target-specific nanobodies were isolated through 1-3 rounds of dilution and regrowth following the Poisson distribution. This ensured no dismissal of positive clones, with populations of positive clones increasing over 10-fold in each dilution round. Ultimately, we isolated 5 nanobodies against death domain receptor 5 and 5 against Pyrococcus furiosus DNA polymerase directly from their immunized libraries. Notably, our approach enables nanobody screening without specialized instruments, demonstrating broad applicability in routine monoclonal nanobody production for diverse biomedical applications.

Real-Time Observation of Conformational Changes and Translocation of Endogenous Cytochrome c within Intact Mitochondria.

Cytochrome c (cyt c) is a multifunctional protein with varying conformations. However, the conformation of cyt c in its native environment, mitochondria, is still unclear. Here, we applied NMR spectroscopy to investigate the conformation and location of endogenous cyt c within intact mitochondria at natural isotopic abundance, mainly using widespread methyl groups as probes. By monitoring time-dependent chemical shift perturbations, we observed that most cyt c is located in the inner mitochondrial membrane and partially unfolded, which is distinct from its native conformation in solution. When suffering oxidative stress, cyt c underwent oxidative modifications due to increasing reactive oxygen species (ROS), weakening electrostatic interactions with the membrane, and gradually translocating into the inner membrane spaces of mitochondria. Meanwhile, the lethality of oxidatively modified cyt c to cells was reduced compared with normal cyt c. Our findings significantly improve the understanding of the molecular mechanisms underlying the regulation of ROS by cyt c in mitochondria. Moreover, it highlights the potential of NMR to monitor high-concentration molecules at a natural isotopic abundance within intact cells or organelles.

NMR spectroscopy for metabolomics in the living system: recent progress and future challenges.

Metabolism is a fundamental process that underlies human health and diseases. Nuclear magnetic resonance (NMR) techniques offer a powerful approach to identify metabolic processes and track the flux of metabolites at the molecular level in living systems. An in vitro study through in-cell NMR tracks metabolites in real time and investigates protein structures and dynamics in a state close to their most natural environment. This technique characterizes metabolites and proteins involved in metabolic pathways in prokaryotic and eukaryotic cells. In vivo magnetic resonance spectroscopy (MRS) enables whole-organism metabolic monitoring by visualizing the spatial distribution of metabolites and targeted proteins. One limitation of these NMR techniques is the sensitivity, for which a possible improved approach is through isotopic enrichment or hyperpolarization methods, including dynamic nuclear polarization (DNP) and parahydrogen-induced polarization (PHIP). DNP involves the transfer of high polarization from electronic spins of radicals to surrounding nuclear spins for signal enhancements, allowing the detection of low-abundance metabolites and real-time monitoring of metabolic activities. PHIP enables the transfer of nuclear spin polarization from parahydrogen to other nuclei for signal enhancements, particularly in proton NMR, and has been applied in studies of enzymatic reactions and cell signaling. This review provides an overview of in-cell NMR, in vivo MRS, and hyperpolarization techniques, highlighting their applications in metabolic studies and discussing challenges and future perspectives.

A tale of dual functions of SERF family proteins in regulating amyloid formation.

The abnormal aggregation of proteins is a significant pathological hallmark of diseases, such as the amyloid formation associated with fused in sarcoma protein (FUS) in frontotemporal lobar degeneration and amyotrophic lateral sclerosis diseases. Understanding which cellular components and how these components regulate the process of abnormal protein aggregation in living organisms is crucial for the prevention and treatment of neurodegenerative diseases. MOAG-4/SERF is a conserved family of proteins with rich positive charged residues, which was initially identified as an enhancer for the formation of amyloids in C. elegans. Knocking out SERF impedes the amyloid formation of various proteins, including α-synuclein and ß-amyloid, which are linked to Parkinson's and Alzheimer's diseases, respectively. However, recent studies revealed SERF exhibited dual functions, as it could both promote and inhibit the fibril formation of the neurodegenerative disease-related amyloidogenic proteins. The connection between functions and structure basis of SERF in regulating the amyloid formation is still unclear. This review will outline the hallmark proteins in neurodegenerative diseases, summarize the contradictory role of the SERF protein family in promoting and inhibiting the aggregation of neurodegenerative proteins, and finally explore the potential structural basis and functional selectivity of the SERF protein.

Breast cancer risk factors and mammographic density among 12518 average-risk women in rural China.

BACKGROUND: Mammographic density (MD) is a strong risk factor for breast cancer. We aimed to evaluate the association between MD and breast cancer related risk factors among average-risk women in rural China. METHODS: This is a population-based screening study. 12518 women aged 45-64 years with complete MD data from three maternal and childcare hospitals in China were included in the final analysis. ORs and 95%CIs were estimated using generalized logit model by comparing each higher MD (BI-RADS b, c, d) to the lowest group (BI-RADS a). The cumulative logistic regression model was used to estimate the ORtrend (95%CI) and Ptrend by treating MD as an ordinal variable. RESULTS: Older age (ORtrend = 0.81, 95%CI: 0.79-0.81, per 2-year increase), higher BMI (ORtrend = 0.73, 95%CI: 0.71-0.75, per 2 kg/m2), more births (ORtrend = 0.47, 95%CI: 0.41-0.54, 3 + vs. 0-1), postmenopausal status (ORtrend = 0.42, 95%CI: 0.38-0.46) were associated with lower MD. For parous women, longer duration of breastfeeding was found to be associated with higher MD when adjusting for study site, age, BMI, and age of first full-term birth (ORtrend = 1.53, 95%CI: 1.27-1.85, 25 + months vs. no breastfeeding; ORtrend = 1.45, 95%CI: 1.20-1.75, 19-24 months vs. no breastfeeding), however, the association became non-significant when adjusting all covariates. Associations between examined risk factors and MD were similar in premenopausal and postmenopausal women except for level of education and oral hormone drug usage. Higher education was only found to be associated with an increased proportion of dense breasts in postmenopausal women (ORtrend = 1.08, 95%CI: 1.02-1.15). Premenopausal women who ever used oral hormone drug were less likely to have dense breasts, though the difference was marginally significant (OR = 0.54, P = 0.045). In postmenopausal women, we also found the proportion of dense breasts increased with age at menopause (ORtrend = 1.31, 95%CI: 1.21-1.43). CONCLUSIONS: In Chinese women with average risk for breast cancer, we found MD was associated with age, BMI, menopausal status, lactation, and age at menopausal. This finding may help to understand the etiology of breast cancer and have implications for breast cancer prevention in China.

Enrichment and Delivery of Target Proteins into the Cell Cytosol via Outer Membrane Vesicles.

Advanced intracellular delivery of proteins has profound applications in both scientific investigations and therapies. However, existing strategies relying on various chemical and physical methods have drawbacks such as the requirement of high concentrations of in vitro prepared target proteins and difficulty in labeling target proteins. Developing new delivery systems integrating the enveloping and labeling of target proteins would bring great advantages for efficient protein transfections. Here, we enriched a high concentration (62 mg/mL) of several target proteins into the outer membrane vesicles (OMVs) of Escherichia coli to employ the native property of OMVs to deliver proteins into the cytosol of eukaryotic cells. The results revealed a high protein transfection efficiency from 90 to 97% for different cell lines. Moreover, the free penetration of molecules less than 600 Da across the membrane of OMVs allows direct labeling of target proteins within OMVs, facilitating the visualization of target proteins. Importantly, the nanobody delivered intracellularly by OMVs retains the biological activity of binding with its target, highlighting the advantages of OMVs as an emerging tool for efficient intracellular delivery of proteins.

Integration of Handheld Ultrasound or Automated Breast Ultrasound among Women with Negative Mammographic Screening Findings: A Multi-center Population-based Study in China.

RATIONALE AND OBJECTIVES: This study assessed the role of second-look automated breast ultrasound (ABUS) adjunct to mammography (MAM) versus MAM alone in asymptomatic women and compared it with supplementing handheld ultrasound (HHUS). MATERIALS AND METHODS: Women aged 45 to 64 underwent HHUS, ABUS, and MAM among six hospitals in China from 2018 to 2022. We compared the screening performance of three strategies (MAM alone, MAM plus HHUS, and MAM plus ABUS) stratified by age groups and breast density. McNemar's test was used to assess differences in the cancer detection rate (CDR), the false-positive biopsy rate, sensitivity, and specificity of different strategies. RESULTS: Of 19,171 women analyzed (mean [SD] age, 51.54 [4.61] years), 72 cases of breast cancer (3.76 per 1000) were detected. The detection rates for both HHUS and ABUS combined with MAM were statistically higher than those for MAM alone (all p < 0.001). There was no significant difference in cancer yields between the two integration strategies. The increase in CRD of the integrated strategies was higher in women aged 45-54 years with denser breasts compared with MAM alone (all p < 0.0167). In addition, the false-positive biopsy rate of MAM plus ABUS was lower than that of MAM plus HHUS (p = 0.025). Moreover, the retraction in ABUS was more frequent in cases detected among MAM-negative results. CONCLUSION: Integrated ABUS or HHUS into MAM provided similar CDRs that were significantly higher than those for MAM alone in younger women (45-54 years) with denser breasts. ABUS has the potential to avoid unnecessary biopsies and provides specific image features to distinguish malignant tumors from HHUS.

Diverse Roles of ScSERF in Modifying the Fibril Growth of Amyloidogenic Proteins.

The aggregation of amyloidogenic proteins is often related to the occurrence of neurodegenerative diseases, including fused in sarcoma protein (FUS) in frontotemporal lobar degeneration and amyotrophic lateral sclerosis diseases. Recently, the SERF protein family has been reported to have a significant regulatory effect on amyloid formation, but it is still unclear about the detailed mechanisms of SERF acting on different amyloidogenic proteins. Herein, nuclear magnetic resonance (NMR) spectroscopy and fluorescence spectroscopy were used to explore interactions of ScSERF with three amyloidogenic proteins FUS-LC, FUS-Core, and α-Synuclein. NMR chemical shift perturbations reveal them sharing similar interaction sites on the N-terminal region of ScSERF. However, the amyloid formation of α-Synuclein protein is accelerated by ScSERF, while ScSERF inhibits fibrosis of FUS-Core and FUS-LC proteins. Both the primary nucleation and the total amount of fibrils produced are detained. Our results suggest a diverse role of ScSERF in regulating the fibril growth of amyloidogenic proteins.

Protein Conformational Exchanges Modulated by the Environment of Outer Membrane Vesicles.

Protein function, in many cases, is strongly coupled to the dynamics and conformational equilibria of the protein. The environment surrounding proteins is critical for their dynamics and can dramatically affect the conformational equilibria and subsequently the activities of proteins. However, it is unclear how protein conformational equilibria are modulated by their crowded native environments. Here we reveal that outer membrane vesicle (OMV) environments modulate the conformational exchanges of Im7 protein at its local frustrated sites and shift the conformation toward its ground state. Further experiments show both macromolecular crowding and quinary interactions with the periplasmic components stabilize the ground state of Im7. Our study highlights the key role that the OMV environment plays in the protein conformational equilibria and subsequently the conformation-related protein functions. Furthermore, the long-lasting nuclear magnetic resonance measurement time of proteins within OMVs indicates that they could serve as a promising system for investigating protein structures and dynamics in situ via nuclear magnetic spectroscopy.

The Effect of Chronotype on Risk-Taking Behavior: The Chain Mediation Role of Self-Control and Emotional Stability.

BACKGROUND: Although previous studies indicate that chronotype might be associated with risk-taking behavior, the specific mechanism has not been thoroughly discussed. This study aimed to fill this gap by exploring the mediating role of self-control and the chain mediating role of self-control and emotional stability between chronotype and risk-taking behavior. METHODS: A total of 547 Chinese college students between 18 and 24 years old were selected to complete the Morningness-Eveningness Questionnaire (MEQ), Self-Control Scale (SCS), Eysenck's Personality Questionnaire-neuroticism (EPQ-N), and Adolescent Risk-Taking Questionnaire: Risk Behavior Scale (ARQ-RB) to assess chronotype, risk-taking behavior, self-control, and emotional stability, respectively. Hayes' PROCESS macro for SPSS was used to test the relationships among these variables. RESULTS: Our result showed significant positive correlations among chronotype, self-control, emotional stability, and significant negative correlations between self-control, emotional stability, and risk-taking behavior. We also found that chronotype had a significant predictive effect on risk-taking behavior in the chain mediation model. Specifically, chronotype affected risk-taking behavior through two pathways: the separate mediating role of self-control and the serial mediation pathway of self-control → emotional stability. CONCLUSIONS: Our study provides direct evidence that chronotype is associated with risk-taking behavior. The results showed that the predictive function of chronotype was mediated by self-control and emotional stability. This study provides a new perspective on preventing and reducing risk-taking behavior.

The Relationship between Self-Concept and Negative Emotion: A Moderated Mediation Model.

Background: Emotional problems such as depression and anxiety are very serious among college students, especially during the COVID-2019 pandemic. The present study aimed to explore the mediating role of resilience in the relationship between self-concept and negative emotion, and the moderating role of exercise intensity in the direct and indirect effect of self-concept on negative emotion among college students. Methods: A total of 739 Chinese college students aged between 18 and 25 years (M = 20.13; SD = 1.67) were selected to complete the Tennessee Self-Concept Scale (TSCS), the Depression Anxiety Stress Self Rating Scale, the Adolescent Psychological Resilience Scale, and the Physical Exercise Scale (PARS-3) to assess self-concept, negative emotions, psychological resilience, and exercise intensity, respectively. Hayes' PROCESS macro for SPSS was used to test the relationships among these variables. Results: Self-concept was negatively correlated with negative emotions; psychological resilience partially mediated the association between self-concept and negative emotions; exercise intensity moderated the effect of self-concept on negative emotions, and college students with low intensity physical activity would strengthening the association between self-concept and psychological resilience, psychological resilience, and negative emotions. Conclusions: Psychological resilience is a critical mediating mechanism through which self-concept is associated with negative emotions among college students, and exercise intensity plays a role as a moderating variable in the direct and indirect influence of self-concept on negative emotions. Implications for preventing or reducing negative emotions are discussed.

Self-Assembled Oligopeptide (FK)4 as a Chiral Alignment Medium for the Anisotropic NMR Analysis of Organic Compounds.

Anisotropic NMR parameters have been proven to be powerful for the structural elucidation of organic molecules. Herein, we present an alignment medium based on the self-assembled (FK)4 oligopeptide, showing excellent properties in measurements of anisotropic NMR parameters in both D2O and CD3OD. The preparation of the (FK)4-based alignment medium is simple and rapid. The low viscosity of the anisotropic phase makes it easy to be transferred to the NMR tube. The alignment of the oligopeptide is fast, stable, and homogeneous, with weak background signals, permitting the acquirement of high-quality NMR spectra. The performance of this alignment medium in residual dipolar coupling measurements and diastereomer discriminations is demonstrated by analyzing several different analytes. The enantiodiscrimination property of the (FK)4 oligopeptide is revealed by the difference of residual chemical shift anisotropy of the two enantiomers in the 1D 13C spectrum, granting its potential use for the quantification and identification of enantiomers of small molecules.

Backbone resonance assignments and dynamics of S. cerevisiae SERF.

Abnormal protein aggregation and precipitation are associated with the perturbation of cellular function and underlie a variety of neurodegenerative diseases. S. cerevisiae SERF (ScSERF), a homolog of modifier of aggregation-4 (MOAG-4) and small EDRK-rich factor protein (SERF1a) is highly conserved and discovered as an enhancer of amyloid formation of Aß40 and α-synuclein both in vitro and in vivo. However, the detailed molecular mechanism whereby ScSERF and its homologs accelerate amyloid formation is not well known yet. Herein, we present the 1 H, 15 N and 13 C NMR assignments of the 68 amino acids long ScSERF. Although ScSERF displays a very high degree of disorder, secondary chemical shifts of Cα, Cß, 15 N{1 H}-NOE values and the residue-specific secondary structure propensity (SSP) scores indicate the segment spanning residues 36E-65 K has a strong helical propensity. This work sets the stage for further detailed structural and dynamic investigations of ScSERF and the molecular mechanism it utilizes in accelerating amyloid formation.

Molecular Insight into the Extracellular Chaperone Serum Albumin in Modifying the Folding Free Energy Landscape of Client Proteins.

Serum albumin (SA) is the most abundant extracellular chaperone protein presenting in various bodily fluids. Recently, several studies have revealed molecular mechanisms of SA in preventing the amyloid formation of amyloidogenic proteins. However, our insight into the mechanism SA employed to sense and regulate the folding states of full-length native proteins is still limited. Addressing this question is technically challenging due to the intrinsic dynamic nature of both chaperones and clients. Here using nuclear magnetic resonance spectroscopy, we show SA modifies the folding free energy landscape of clients and subsequently alters the equilibria between different compact conformations of its clients, resulting in the increased populations of excited states of client proteins. This modulation of client protein conformation by SA can change the client protein activity in a way that cannot be interpreted on the basis of its ground state structure; therefore, our work provides an alternative insight of SA in retaining a balanced functional proteome.

Research on the prediction of longevity from both individual and family perspectives.

Increasing human longevity is of global interest. The present study explored the prediction of longevity from both individual perspective and family perspective based on demographic and psychosocial factors. A total of 186 longevous family members and 237 ordinary elderly family members participated in a cross-sectional study, and a sample of 62 longevous elderly and 57 ordinary elderly were selected for comparative research. The results showed that it was three times more female than male in longevous elderly group. Up to 71.2% of longevous elderly had no experience in education, which was significantly lower than that of ordinary elderly. Due to such extreme age, more widowed (81.4%) elderly than those in married (18.6%). Less than one-seventh of the longevous elderly maintained the habit of smoking, and about one-third of them liked drinking, both were significantly lower than that of ordinary elderly. In terms of psychosocial factors, longevous elderly showed lower neuroticism and social support, while higher extraversion, compared with the ordinary elderly. However, there were no significant differences between the two family groups in demographic and psychosocial variables, except longevous families showing lower scores in neuroticism. Regression analysis found that neuroticism, social support and smoking habit had significant impact on individuals' life span, then, neuroticism and psychoticism were the key factor to predict families' longevity. We conclude that good emotional management, benign interpersonal support, and moderation of habits are important factors for individual longevity, and the intergenerational influence of personality is closely related to family longevity.

Chaperone Spy Protects Outer Membrane Proteins from Folding Stress via Dynamic Complex Formation.

Gram-negative bacteria have a multicomponent and constitutively active periplasmic chaperone system to ensure the quality control of their outer membrane proteins (OMPs). Recently, OMPs have been identified as a new class of vulnerable targets for antibiotic development, and therefore a comprehensive understanding of OMP quality control network components will be critical for discovering antimicrobials. Here, we demonstrate that the periplasmic chaperone Spy protects certain OMPs against protein-unfolding stress and can functionally compensate for other periplasmic chaperones, namely Skp and FkpA, in the Escherichia coli K-12 MG1655 strain. After extensive in vivo genetic experiments for functional characterization of Spy, we use nuclear magnetic resonance and circular dichroism spectroscopy to elucidate the mechanism by which Spy binds and folds two different OMPs. Along with holding OMP substrates in a dynamic conformational ensemble, Spy binding enables OmpX to form a partially folded ß-strand secondary structure. The bound OMP experiences temperature-dependent conformational exchange within the chaperone, pointing to a multitude of local dynamics. Our findings thus deepen the understanding of functional compensation among periplasmic chaperones during OMP biogenesis and will promote the development of innovative antimicrobials against pathogenic Gram-negative bacteria. IMPORTANCE Outer membrane proteins (OMPs) play critical roles in bacterial pathogenicity and provide a new niche for antibiotic development. A comprehensive understanding of the OMP quality control network will strongly impact antimicrobial discovery. Here, we systematically demonstrate that the periplasmic chaperone Spy has a role in maintaining the homeostasis of certain OMPs. Remarkably, Spy utilizes a unique chaperone mechanism to bind OmpX and allows it to form a partially folded ß-strand secondary structure in a dynamic exchange of conformations. This mechanism differs from that of other E. coli periplasmic chaperones such as Skp and SurA, both of which maintain OMPs in disordered conformations. Our study thus deepens the understanding of the complex OMP quality control system and highlights the differences in the mechanisms of ATP-independent chaperones.

The influence of music tempo on mental load and hazard perception of novice drivers.

INTRODUCTION: Many novice drivers listen to music while driving, but the effect of music-listening on novice driver's safety is uncertain. We explored how music tempo affects two key aspects of novice drivers' cognitive functioning, mental load and hazard perception. METHOD: In a within-subjects experimental design study, 37 novice drivers completed a hazard perception test in simulated traffic situations under four randomly-ordered conditions: while listening to fast, medium, slow tempo music, and without listening to any music. Mental load was recorded both subjectively and through psychophysiological measures during all conditions. RESULTS: When listening to fast-tempo music, novice drivers' subjective load value, heart rate and respiratory rate were significantly higher than that during the other two music tempo conditions and the no music condition. Skin temperature was significantly higher during fast-tempo music than during the slow music tempo and no music conditions. When listening to slow-tempo music, drivers' R-R interval (variability in heart rate, with longer intervals reflecting lower mental load) was significantly longer than while listening to fast and mid-tempo music, and their hazard perception scores were significantly higher than the other three conditions. CONCLUSIONS: Listening to fast tempo music was associated with increased mental load and reduced hazard perception ability in traffic among novice drivers. Listening to slow tempo music did not increase novice drivers' mental load and offered some benefit to their hazard perception.