Human Studies Research Articles

Impact of DPP-4 inhibitors on interleukin levels in type 2 diabetes mellitus.

Accumulating evidence had implicated pathological involvement of interleukins (ILs) in progression and complications in patients with type 2 diabetes mellitus (T2DM). Dipeptidyl peptidase-4 inhibitors (DPP-4i) produced favorable effects on glucose homeostasis in T2DM. This study aimed to evaluate the impact of DPP-4i on interleukins (ILs) concentrations in T2DM. PubMed, Embase and the Cochrane library were systematically searched for relevant articles from inception to May 31, 2024. Related searching items were used including DPP-4i, T2DM and randomized controlled trials (RCTs). Placebo- or active agents-controlled human studies were screened. All the RCTs were identified if they provided detailed information on changes of ILs during DPP-4i treatment. A total of 14 RCTs involving 850 participants were identified. Pooled estimates revealed that DPP-4i significantly lower IL-6 concentrations (-0.54 pg/mL, 95% CI, -0.82 to -0.25, I2 = 10%, P = 0.0003) compared to placebo. Similar effects were demonstrated for IL-1β (-16.33 pg/mL, 95% CI, -19.56 to -13.11, I2 = 0%, P<0.00001), whereas the effect on IL-18 was not statistically significant (-13.55 pg/mL, 95% CI, -76.95 to 49.85, I2 = 0%, P = 0.68). Subgroup analysis on IL-6 demonstrated that marked effects were found in groups of basal IL-6 concentrations (< 5 pg/mL), BMI (≥ 28 kg/m2) and type of DPP-4i (linagliptin). DPP-4i favorably decreased concentrations of IL-6 in patients with T2DM. The impact of DPP-4i on IL-1β and IL-18 needed to be explored with more studies. Further trials should be performed to elucidate this anti-inflammatory effect of DPP-4i during treatment of T2DM.

Influence of sleep on physiological systems in atherosclerosis.

Sleep is a fundamental requirement of life and is integral to health. Deviation from optimal sleep associates with numerous diseases including those of the cardiovascular system. Studies, spanning animal models to humans, show that insufficient, disrupted or inconsistent sleep contribute to poor cardiovascular health by disrupting body systems. Fundamental experiments have begun to uncover the molecular and cellular links between sleep and heart health while large-scale human studies have associated sleep with cardiovascular outcomes in diverse populations. Here, we review preclinical and clinical findings that demonstrate how sleep influences the autonomic nervous, metabolic and immune systems to affect atherosclerotic cardiovascular disease.

The kinetics of SARS-CoV-2 infection based on a human challenge study

Studying the early events that occur after viral infection in humans is difficult unless one intentionally infects volunteers in a human challenge study. Here, we use data about severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in such a study in combination with mathematical modeling to gain insights into the relationship between the amount of virus in the upper respiratory tract and the immune response it generates. We propose a set of dynamic models of increasing complexity to dissect the roles of target cell limitation, innate immunity, and adaptive immunity in determining the observed viral kinetics. We introduce an approach for modeling the effect of humoral immunity that describes a decline in infectious virus after immune activation. We fit our models to viral load and infectious titer data from all the untreated infected participants in the study simultaneously. We found that a power-law with a power h < 1 describes the relationship between infectious virus and viral load. Viral replication at the early stage of infection is rapid, with a doubling time of ~2 h for viral RNA and ~3 h for infectious virus. We estimate that adaptive immunity is initiated ~7 to 10 d postinfection and appears to contribute to a multiphasic viral decline experienced by some participants; the viral rebound experienced by other participants is consistent with a decline in the interferon response. Altogether, we quantified the kinetics of SARS-CoV-2 infection, shedding light on the early dynamics of the virus and the potential role of innate and adaptive immunity in promoting viral decline during infection.

Sex- and site-specific associations of circulating lipocalin 2 and incident colorectal cancer: Results from the EPIC cohort.

Experimental research has uncovered lipocalin 2 (LCN2) as a novel biomarker implicated in the modulation of intestinal inflammation, metabolic homeostasis, and colon carcinogenesis. However, evidence from human research has been scant. We, therefore, explored the association of pre-diagnostic circulating LCN2 concentrations with incident colorectal cancer (CRC) in a nested case-control study within the in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. LCN2 was measured in 1267 incident CRC cases matched to 1267 controls using incidence density sampling. Conditional logistic regression was used to estimate incidence rate ratios (IRRs) and 95% confidence intervals (95% CIs) according to tumor subsite and sex. Weighted Cox proportional hazard regression was used to explore associations by adiposity status. In multivariable-adjusted analyses, the IRR [95% CI] per doubling in LCN2 concentration was 1.16 [0.98-1.37] for CRC overall, 1.26 [1.00-1.59] for colon cancer, and 1.08 [0.85-1.38] for rectal cancer. The association for colon cancer was more pronounced in women (IRR [95% CI], 1.66 [1.20-2.30]) and for proximal colon cancer (IRR [95% CI], 1.96 [1.15-3.34]), whereas no association was seen in men and distal colon cancer. The association for colon cancer was positive in individuals with high waist circumference (hazard ratio [95% CI], 1.69 [1.52-1.88]) and inverse in individuals with low waist circumference (hazard ratio [95% CI], 0.86 [0.76-0.98], P interaction<0.01). Overall, these data suggest that pre-diagnostic LCN2 concentrations were positively associated with colon cancer, particularly occurring in the proximal colon, in women and among individuals with abdominal adiposity.

Acute change in resting energy expenditure and vital signs in response to white tea consumption in females: a pilot study

BackgroundWhite tea, derived from the Camellia sinensis plant like other teas, uses tender buds and young leaves and undergoes minimal processing. This results in higher levels of antioxidants and bioactive substances, which may enhance thermogenesis more effectively than other teas. This first human study aimed to investigate the acute effects of white tea consumption on resting energy expenditure (REE) and some vital signs, including blood pressure (BP), heart rate (HR), and body temperature (BT).MethodsThirty-two healthy female volunteers with normal initial BP and whose caffeine intakes were < 300 mg/d were enrolled in the study. The caffeine and total phenolic content of white tea samples were determined by the high-performance liquid chromatography method and the Folin-Ciocalteu colorimetric method, respectively. After baseline measurements, participants consumed white tea containing 6 mg of caffeine per kilogram of lean body mass, and the white tea was prepared with bottled drinking water at 80 °C and brewed for 3 min. REE, BP, and BT were assessed at various intervals (baseline, 30 min, 120 min, and 180 min) post-consumption of the white tea.ResultsThe results revealed a significant increase in REE by 8.7% at 180 min after the consumption. In particular, there was a substantial difference in both values between the intervals of 30 min to 180 min and baseline to 180 min for REE (p < 0.05). Maximal oxygen consumption and BT also increased significantly over time (p < 0.05) and the observed increment in BT suggests a thermogenic effect associated with white tea consumption. However, systolic BP, diastolic BP, and heart rate showed no significant difference.ConclusionsThese findings suggest white tea consumption may acutely enhance REE and maximal oxygen consumption, so the results are promising for body weight management. This study is the first human study in the literature about the effects of white tea on energy expenditure and vital signs.

Leveraging Ion-Ion and Ion-Photon Activation to Improve the Sequencing of Proteins Carrying Multiple Disulfide Bonds: The Human Serum Albumin Case Study.

Gas-phase sequencing of large intact proteins (>30 kDa) via tandem mass spectrometry is an inherently challenging process that is further complicated by the extensive overlap of multiply charged product ion peaks, often characterized by a low signal-to-noise ratio. Disulfide bonds exacerbate this issue because of the need to cleave both the S-S and backbone bonds to liberate sequence informative fragments. Although electron-based ion activation techniques such as electron transfer dissociation (ETD) have been proven to rupture disulfide bonds in whole protein ions, they still struggle to produce extensive sequencing when multiple, concatenated S-S bonds are present on the same large polypeptide chain. Here, we evaluate the increase in sequence coverage obtained by combining activated-ion ETD (AI-ETD) and proton transfer charge reduction (PTCR) in the analysis of 66 kDa human serum albumin, which holds 17 disulfide bridges. We also describe the combination of AI-ETD with supplemental postactivation of the ETD reaction products via higher-energy collisional dissociation─a hybrid fragmentation method termed AI-EThcD. AI-EThcD leads to a further improvement compared to AI-ETD in both the global number of cleaved backbone bonds and the number of ruptured backbone bonds from disulfide-protected regions. Our results also demonstrate that the full potential of AI-ETD and AI-EThcD is unveiled only when combined with PTCR: reduction in overlap of ion signals leads to a sequence coverage as high as 39% in a single experiment, highlighting the relevance of spectral simplification in top-down mass spectrometry of large proteins.

Characterization of Chemical Exposome in A Paired Human Preconception Pilot Study.

Parental preconception exposure to synthetic chemicals may have critical influences on fertility and reproduction. Here, we present a robust LC-MS/MS method covering up to 95 diverse xenobiotics in human urine, serum, seminal and follicular fluids to support exposome-wide assessment in reproductive health outcomes. Extraction recoveries of validated analytes ranged from 62% to 137% and limits of quantification from 0.01 to 6.0 ng/mL in all biofluids. We applied the validated method to a preconception cohort of Australian couples (n = 30) receiving fertility treatment. In total, 36 and 38 xenobiotics were detected across the paired biofluids of males and females, respectively, including PFAS, parabens, organic UV-filters, plastic additives, antimicrobials, and other industrial chemicals. Results showed 39% of analytes in males and 37% in females were equally detected in paired serum, urine, and reproductive fluids. The first detection of the sunscreen ingredient avobenzone and the industrial chemical 4-nitrophenol in follicular and seminal fluids suggests it can cross both blood-follicle/testis barriers, indicating potential risks for fertility. Further, the blood-follicle transfer of perfluorobutanoic acid, PFOA, PFHxS, PFOS, and oxybenzone corroborate that serum concentrations can be reliable proxies for assessing exposure within the ovarian microenvironment. In conclusion, we observed significant preconception exposure to multiple endocrine disruptors in couples and identified potential xenobiotics relevant to male and female fertility impairments.

MS2Lipid: A Lipid Subclass Prediction Program Using Machine Learning and Curated Tandem Mass Spectral Data

Background: Untargeted lipidomics using collision-induced dissociation-based tandem mass spectrometry (CID-MS/MS) is essential for biological and clinical applications. However, annotation confidence still relies on manual curation by analytical chemists, despite the development of various software tools for automatic spectral processing based on rule-based fragment annotations. Methods: In this study, we present a novel machine learning model, MS2Lipid, for the prediction of known lipid subclasses from MS/MS queries, providing an orthogonal approach to existing lipidomics software programs in determining the lipid subclass of ion features. We designed a new descriptor, MCH (mode of carbon and hydrogen), to increase the specificity of lipid subclass prediction in nominal mass resolution MS data. Results: The model, trained with 6760 and 6862 manually curated MS/MS spectra for the positive and negative ion modes, respectively, classified queries into one or several of 97 lipid subclasses, achieving an accuracy of 97.4% in the test set. The program was further validated using various datasets from different instruments and curators, with the average accuracy exceeding 87.2%. Using an integrated approach with molecular spectral networking, we demonstrated the utility of MS2Lipid by annotating microbiota-derived esterified bile acids, whose abundance was significantly increased in fecal samples of obese patients in a human cohort study. This suggests that the machine learning model provides an independent criterion for lipid subclass classification, enhancing the annotation of lipid metabolites within known lipid classes. Conclusions: MS2Lipid is a highly accurate machine learning model that enhances lipid subclass annotation from MS/MS data and provides an independent criterion.

Efficacy of an mHealth intervention to support pain self-management and improve analgesia in patients with rib fractures: protocol for a randomised controlled trial

IntroductionIn light of the risks of over-reliance on opioid analgesia during recovery from rib fractures, there is increased interest in the efficacy of non-pharmacological approaches to pain management. This paper...

Cognitive bias modification for social anxiety: protocol for a living systematic review of human studies and meta-analysis

Background Social anxiety is a heightened fear and discomfort in social situations which can be experienced in varying degrees of severity. Cases of elevated distress and impaired functioning and quality of life can lead to a clinical diagnosis of social anxiety disorder. Altering cognitive biases associated with social anxiety has been suggested as potentially beneficial; however, little is known about the comparative effectiveness of such interventions. The aim of this living systematic review is to examine the efficacy of cognitive bias modification for reducing social anxiety, including in people who have not been diagnosed with the disorder. Methods We will search multiple electronic databases for randomised controlled trials evaluating the efficacy of cognitive bias modification for people diagnosed with social anxiety and people exposed to a simulated social stressor. The primary outcome will be change in social anxiety related symptoms; secondary outcomes will be changes in social functioning and quality of life and adverse events. Study selection, data extraction and risk of bias assessment will be done by at least two reviewers using pre-defined tools. We will synthesise data from people with social anxiety diagnosis and those subjected to a simulated social stressor separately using random effects meta-analyses. Heterogeneity will be evaluated by investigating characteristics of included studies. We will appraise the strength of the evidence for each outcome by reviewing the overall association, internal and external validity, and reporting biases. Where data allows, we will triangulate the evidence from both sources with a multidisciplinary group of experts. The review will begin in living mode and the database search will be rerun every three months to identify and integrate potential new evidence. We will co-produce this review with members of a global lived experience advisory board. This protocol was registered with PROSPERO (CRD42024601380) on 15.10.2024.

Human Dynamics Research in GIScience: challenges and opportunities

The Symposium on Human Dynamics Research, first organized at the 2015 AAG Meeting in Chicago, celebrated its 10th anniversary at the 2024 AAG Meeting in Honolulu, marking a decade of transformative advancements in the field. Over the past decade, the focus of human dynamics research has shifted from traditional spatial-temporal analyses to sophisticated modeling of human behavior in a hybrid physical-virtual world. This evolving field now examines the intricate interdependencies between physical and digital environments, addressing critical issues such as urban resilience, public health, social equity, and community sustainability. The symposium emphasized the growing importance of interdisciplinary collaboration, advanced data-driven analytical platforms, and innovative theoretical frameworks to better understand human interactions across these spaces. As human dynamics continue to shape global urban systems, these advancements are pivotal for future research and real-world problem-solving, offering novel insights into the interconnectedness of mobility, technology, and societal well-being in a rapidly changing world.

Synthesis, Analytical Characterization, and Human CB1 Receptor Binding Studies of the Chloroindole Analogues of the Synthetic Cannabinoid MDMB-CHMICA

Synthetic cannabinoids (SCs) are one of the largest groups of new psychoactive substances (NPSs). However, the relationship between their chemical structure and the affinity to human CB1 receptors (hCB1), which mediates their psychotropic activity, is not well understood. Herein, the synthesis of the 2-, 4-, 5-, 6- and 7-chloroindole analogues of the synthetic cannabimimetic MDMB-CHMICA, along with their analytical characterization via ultraviolet–visible (UV/VIS), infrared (IR), nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry, is described. Furthermore, all five derivatives of MDMB-CHMICA were analyzed for their hCB1 binding affinities. Chlorination at position 4 and 5 of the indole core reduced the binding affinity compared to MDMB-CHMICA, while the test compounds chlorinated in positions 2, 6, and 7 largely retained their binding affinities relative to the non-chlorinated parent compound.

Quercetin: A Promising Candidate for the Management of Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD)

Metabolic dysfunction-associated steatotic liver disease (MASLD) represents a chronic liver disease. The development of MASLD is influenced by a multitude of diseases associated with modern lifestyles, including but not limited to diabetes mellitus, hypertension, hyperlipidaemia and obesity. These conditions are often consequences of the adoption of unhealthy habits, namely a sedentary lifestyle, a lack of physical activity, poor dietary choices and excessive alcohol consumption. The treatment of MASLD is primarily based on modifying the patient’s lifestyle and pharmacological intervention. Despite the absence of FDA-approved pharmacological agents for the treatment of MASLD, several potential therapeutic modalities have demonstrated efficacy in reversing the histopathological features of the disease. Among the botanical ingredients belonging to the flavonoid group is quercetin (QE). QE has been demonstrated to possess a number of beneficial physiological effects, including anti-inflammatory, anticancer and antifungal properties. Additionally, it functions as a natural antioxidant. Preclinical evidence indicates that QE may play a beneficial role in reducing liver damage and improving metabolic health. Early human studies also suggest that QE may be an effective treatment for MASLD due to its antioxidant, anti-inflammatory, and lipid-regulating properties. This review aims to summarize the available information on the therapeutic effects of QE in MASLD.

Glucagon‐like Peptide-1 Agonists and Smoking Cessation: A Brief Review

The glucagon‐like peptide 1 (GLP-1) agonists such as semaglutide (Ozempic®, Wegovy®) and tirzepatide (Mounjaro®) have shown efficacy inducing weight loss in both diabetics and non-diabetics. According to the incentive sensitization theory of addiction, these drugs may prove useful in addictive disorders such as nicotine addiction. Animal data has been suggestive of a potential positive effect but early human studies have been mixed. This manuscript reviews the theory of addiction as well as the few animal and human studies available. Further human studies are needed to show GLP-1 agonist efficacy in smoking cessation.

High-quality multimodal MRI with simultaneous EEG using conductive ink and polymer-thick film nets

Objective. Combining magnetic resonance imaging (MRI) and electroencephalography (EEG) provides a powerful tool for investigating brain function at varying spatial and temporal scales. Simultaneous acquisition of both modalities can provide unique information that a single modality alone cannot reveal. However, current simultaneous EEG-fMRI studies are limited to a small set of MRI sequences due to the image quality and safety limitations of commercially available MR-conditional EEG nets. We tested whether the Inknet2, a high-resistance polymer thick film based EEG net that uses conductive ink, could enable the acquisition of a variety of MR image modalities with minimal artifacts by reducing the radiofrequency-shielding caused by traditional MR-conditional nets.Approach. We first performed simulations to model the effect of the EEG nets on the magnetic field and image quality. We then performed phantom scans to test image quality with a conventional copper EEG net, with the new Inknet2, and without any EEG net. Finally, we scanned five human subjects at 3 Tesla (3 T) and three human subjects at 7 Tesla (7 T) with and without the Inknet2 to assess structural and functional MRI image quality.Main results. Across these simulations, phantom scans, and human studies, the Inknet2 induced fewer artifacts than the conventional net and produced image quality similar to scans with no net present.Significance. Our results demonstrate that high-quality structural and functional multimodal imaging across a variety of MRI pulse sequences at both 3 T and 7 T is achievable with an EEG net made with conductive ink and polymer thick film technology.

Early subclinical stages of the inflammatory bowel diseases - insights from human and animal studies.

The inflammatory bowel diseases (IBD) occur in genetically susceptible individuals that mount inappropriate immune responses to their microbiota leading to chronic intestinal inflammation. The natural history of IBD progression begins with early subclinical stages of disease that occur before clinical diagnosis. Improved understanding of those early subclinical stages could lead to new or improved strategies for IBD diagnosis, prognostication or prevention. Here we review our current understanding of the early subclinical stages of IBD in humans including studies from first-degree relatives of IBD patients and members of the general population who go on to develop IBD. We also discuss representative mouse models of IBD that can be used to investigate disease dynamics and host-microbiota relationships during these early stages. In particular, we underscore how mouse models of IBD that develop disease later in life with variable penetrance may present valuable opportunities to discern early subclinical mechanisms of disease before histological inflammation and other severe symptoms become apparent.

Precision Cut Lung Slices: Emerging Tools for Preclinical and Translational Lung Research. An Official American Thoracic Society Workshop Report.

The urgent need for effective treatments for acute and chronic lung diseases underscores the significance of developing innovative preclinical human research tools. The 2023 ATS Workshop on Precision Cut Lung Slices (PCLS) brought together 35 experts to discuss and address the role of human tissue-derived PCLS as a unique tool for target and drug discovery and validation in pulmonary medicine. With increasing interest and usage, along with advancements in methods and technology, there is a growing need for consensus on PCLS methodology and readouts. The current document recommends standard reporting criteria and emphasizes the requirement for careful collection and integration of clinical metadata. We further discuss current clinically relevant readouts that can be applied to PCLS and highlight recent developments and future steps for implementing novel technologies for PCLS modeling and analysis. The collection and correlation of clinical metadata and multiomic analysis will further advent the integration of this preclinical platform into patient endotyping and the development of tailored therapies for lung disease patients.

Ocular biometric responses to simulated polychromatic defocus.

Evidence from human studies of ocular accommodation and studies of animals reared in monochromatic conditions suggest that chromatic signals can guide ocular growth. We hypothesized that ocular biometric response in humans can be manipulated by simulating the chromatic contrast differences associated with imposition of optical defocus. The red, green, and blue (RGB) channels of an RGB movie of the natural world were individually incorporated with computational defocus to create two different movie stimuli. The magnitude of defocus incorporated in the red and blue layers was chosen such that, in one case, it simulated +3 D defocus, referred to as color-signed myopic (CSM) defocus, and in another case it simulated -3 D defocus, referred to as color-signed hyperopic (CSH) defocus. Seventeen subjects viewed the reference stimulus (unaltered movie) and at least one of the two color-signed defocus stimuli for ∼1 hour. Axial length (AL) and choroidal thickness (ChT) were measured immediately before and after each session. AL and subfoveal ChT showed no significant change under any of the three conditions. A significant increase in vitreous chamber depth (VCD) was observed following viewing of the CSH stimulus compared with the reference stimulus (0.034 ± 0.03 mm and 0 ± 0.02 mm, respectively; p = 0.018). A significant thinning of the crystalline lens was observed following viewing of the CSH stimulus relative to the CSM stimulus (-0.033 ± 0.03 mm and 0.001 ± 0.03 mm, respectively; p = 0.015). Differences in the effects of CSM and CSH conditions on VCD and lens thickness suggest a directional, modulatory influence of chromatic defocus. On the other hand, ChT responses showed large variability, rendering it an unreliable biomarker for chromatic defocus-driven responses, at least for the conditions of this study.

Expression and distribution of rAAV9 intrathecally administered in juvenile to adolescent mice.

Intrathecal (IT) lumbar puncture delivery of recombinant adeno-associated virus serotype 9 (rAAV9) is a gene therapy approach being explored in preclinical studies and ongoing gene therapy clinical trials for neurological diseases. Few studies address IT rAAV9 vector distribution, tropism, and expression with respect to age of administration. Therefore, we IT delivered a rAAV9/GFP vector in mice at ages ranging from early postnatal development through adulthood (P10-P90). Tissues were assessed for transgene expression, cell tropism, and vector distribution. In the CNS, transduction was highest when delivered at post-natal day 10 (P10) and there was an age-dependent decline in transduction. We found higher transduction of astrocytes relative to neurons when rAAV9 was administered at younger ages and a switch to higher neuronal transduction with delivery at older timepoints. Biodistribution analysis of peripheral tissues showed that when delivered at P10, rAAV9 has the greatest distribution to the heart. Conversely, at P90 rAAV9 liver distribution was highest. As rAAV9 IT-delivered gene therapies continue to emerge for neurological diseases, careful consideration of the age of delivery should be taken in relation to the expected distribution and cell expression in animal models, and how this may translate to human studies.

Construction of Freezing Injury Grade Index for Nanfeng Tangerine Plants Based on Physiological and Biochemical Parameters.

Low-temperature freezing stress constitutes the most significant meteorological disaster during the overwintering period in the Nanfeng Tangerine (NT) production area, severely impacting the normal growth and development of the plants. Currently, the accuracy of meteorological disaster warnings and forecasts for NT orchards remains suboptimal, primarily due to the absence of quantitative meteorological indicators for low-temperature freezing stress. Therefore, this study employed NT plants as experimental subjects and conducted controlled treatment experiments under varying intensities of low-temperature freezing stress (0 °C, -2 °C, -5 °C, -7 °C, and -9 °C) and durations (1 h, 4 h, and 7 h). Subsequently, physiological and biochemical parameters were measured, including photosynthetic parameters, chlorophyll fluorescence parameters, reactive oxygen species, osmoregulatory substances, and antioxidant enzyme activities in NT plants. The results demonstrated that low-temperature freezing stress adversely affected the photosynthetic system of NT plants, disrupted the dynamic equilibrium of the antioxidant system, and compromised cellular stability. The severity of freezing damage increased with decreasing temperature and prolonged exposure. Chlorophyll (a/b) ratio (Chl (a/b)), maximum quantum yield of photosystem II (Fv/Fm), soluble sugar, and malondialdehyde (MDA) were identified as key indicators for assessing physiological and biochemical changes in NT plants. Utilizing these four parameters, a comprehensive score (CS) model of freezing damage was developed to quantitatively evaluate the growth status of NT plants across varying low-temperature freezing damage gradients and durations. Subsequently, the freezing damage grade index for NT plants during the overwintering period was established. Specifically, Level 1 for CS ≤ -0.50, Level 2 for -0.5 < CS ≤ 0, Level 3 for 0 < CS ≤ 0.5, and Level 4 for 0.5 < CS. The research results provide valuable data for agricultural meteorological departments to carry out disaster monitoring, early warning, and prevention and control.