Counterion Exchange Enhances the Brightness and Photostability of a Fluorous Cyanine Dye.

Fluorofluorophores are a unique class of fluorophores that can be solubilized in perfluorocarbons (PFCs) and used to study biological systems. However, because of the low dielectric constant and high oxygen solubility in the fluorous phase, the brightness and photostability of the fluorofluorophores are significantly diminished. Here, we leveraged the tight ion pairing in the fluorous phase to improve the photophysical properties of a fluorous soluble pentamethine dye (FCy5) via counterion exchange. We found that larger, softer, fluorinated, aryl borate counterions promote the ideal polymethine state where charge delocalization across the polymethine chain increases the brightness (6-fold) and photostability (55-fold) of FCy5.

A missense variant in SLC39A8 confers risk for Crohn's disease by disrupting manganese homeostasis and intestinal barrier integrity.

Common genetic variants interact with environmental factors to impact risk of heritable diseases. A notable example of this is a single-nucleotide variant in the Solute Carrier Family 39 Member 8 (SLC39A8) gene encoding the missense variant A391T, which is associated with a variety of traits ranging from Parkinson's disease and neuropsychiatric disease to cardiovascular and metabolic diseases and Crohn's disease. The remarkable extent of pleiotropy exhibited by SLC39A8 A391T raises key questions regarding how a single coding variant can contribute to this diversity of clinical outcomes and what is the mechanistic basis for this pleiotropy. Here, we generate a murine model for the Slc39a8 A391T allele and demonstrate that these mice exhibit Mn deficiency in the colon associated with impaired intestinal barrier function and epithelial glycocalyx disruption. Consequently, Slc39a8 A391T mice exhibit increased sensitivity to epithelial injury and pathological inflammation in the colon. Taken together, our results link a genetic variant with a dietary trace element to shed light on a tissue-specific mechanism of disease risk based on impaired intestinal barrier integrity.

Macromolecular Crowding as an Intracellular Stimulus for Responsive Nanomaterials.

Stimuli-responsive materials are exploited in biological, materials, and sensing applications. We introduce a new endogenous stimulus, biomacromolecule crowding, which we achieve by leveraging changes in thermoresponsive properties of polymers upon high concentrations of crowding agents. We prepare poly(2-oxazoline) amphiphiles that exhibit lower critical solution temperatures (LCST) in serum above physiological temperature. These amphiphiles stabilize oil-in-water nanoemulsions at temperatures below the LCST but are ineffective surfactants above the LCST, resulting in emulsion fusion. We find that the transformations observed upon heating nanoemulsions above their surfactant's LCST can instead be induced at physiological temperatures through the addition of polymers and protein, rendering thermoresponsive materials "crowding responsive." We demonstrate that the cytosol is a stimulus for nanoemulsions, with droplet fusion occurring upon injection into cells of living zebrafish embryos. This report sets the stage for classes of thermoresponsive materials to respond to macromolecule concentration rather than temperature changes.

Stimuli Induced Uptake of Protein-Like Peptide Brush Polymers.

Recently, we presented a strategy for packaging peptides as side-chains in high-density brush polymers. For this globular protein-like polymer (PLP) formulation, therapeutic peptides were shown to resist proteolytic degradation, enter cells efficiently and maintain biological function. In this paper, we establish the role charge plays in dictating the cellular uptake of these peptide formulations, finding that peptides with a net positive charge will enter cells when polymerized, while those formed from anionic or neutral peptides remain outside of cells. Given these findings, we explored whether cellular uptake could be selectively induced by a stimulus. In our design, a cationic peptide is appended to a sequence of charge-neutralizing anionic amino acids through stimuli-responsive cleavable linkers. As a proof-of-concept study, we tested this strategy with two different classes of stimuli, exogenous UV light and an enzyme (a matrix metalloproteinase) associated with the inflammatory response. The key finding is that these materials enter cells only when acted upon by the stimulus. This approach makes it possible to achieve delivery of the polymers, therapeutic peptides or an appended cargo into cells in response to an appropriate stimulus.

Post-Traumatic Stress Disorder is Associated with further Increased Parkinson's Disease Risk in Veterans with Traumatic Brain Injury.

OBJECTIVE: Determining if traumatic brain injury (TBI) and post-traumatic stress disorder (PTSD) are risk factors for Parkinson's disease (PD). This constitutes a research priority for the Veterans Administration (VA) with implications for screening policy and prevention. METHODS: Population-based, matched case-control study among veterans using VA health care facilities from October 1, 1999, to September 30, 2013. We identified 176,871 PD cases and 707,484 randomly selected PD-free matched controls. PD, TBI, and PTSD were ascertained by validated International Classification of Disease 9th revision (ICD)-9 code-based algorithms. We examined the association between both risk factors and PD using race-adjusted conditional logistic regression. RESULTS: The overall study cohort prevalence for TBImild , TBInon-mild , and PTSD was 0.65%, 0.69%, and 5.5%, respectively. Both TBI and PTSD were significantly associated with PD in single-risk factor race-adjusted analyses (conditional odds ratio [cOR] = 2.99; 95% confidence interval [CI]: 2.69-3.32), 3.82 (95% CI: 3.67-3.97), and 2.71 (95% CI: 2.66-2.77) for TBImild , TBInon-mild , and PTSD, respectively). There was suggestive positive interaction observed with comorbid PTSD/TBI in dual-risk factor analyses, with significant 2.69-fold and 3.70-fold excess relative PD risk in veterans with TBImild and TBInon-mild versus those without TBI when PTSD was present versus 2.17-fold and 2.80-fold excess risk when PTSD was absent. INTERPRETATION: Our study was the first to demonstrate that both TBI and PTSD are independently associated with increased relative PD risk in a diverse nationwide cohort of military service veterans, and the first to suggest a potential modest synergistic excess risk in those with comorbid TBI/PTSD. Longitudinal research is needed to confirm these suggestive findings. ANN NEUROL 2020 ANN NEUROL 2020;88:33-41.

Oligomerization of the Sec7 domain Arf guanine nucleotide exchange factor GBF1 is dispensable for Golgi localization and function but regulates degradation.

Members of the large Sec7 domain-containing Arf guanine nucleotide exchange factor (GEF) family have been shown to dimerize through their NH2-terminal dimerization and cyclophilin binding (DCB) and homology upstream of Sec7 (HUS) domains. However, the importance of dimerization in GEF localization and function has not been assessed. We generated a GBF1 mutant (91/130) in which two residues required for oligomerization (K91 and E130 within the DCB domain) were replaced with A and assessed the effects of these mutations on GBF1 localization and cellular functions. We show that 91/130 is compromised in oligomerization but that it targets to the Golgi in a manner indistinguishable from wild-type GBF1 and that it rapidly exchanges between the cytosolic and membrane-bound pools. The 91/130 mutant appears active as it integrates within the functional network at the Golgi, supports Arf activation and COPI recruitment, and sustains Golgi homeostasis and cargo secretion when provided as a sole copy of functional GBF1 in cells. In addition, like wild-type GBF1, the 91/130 mutant supports poliovirus RNA replication, a process requiring GBF1 but believed to be independent of GBF1 catalytic activity. However, oligomerization appears to stabilize GBF1 in cells, and the 91/130 mutant is degraded faster than the wild-type GBF1. Our data support a model in which oligomerization is not a key regulator of GBF1 activity but impacts its function by regulating the cellular levels of GBF1.

Dissecting components of the Campylobacter jejuni fetMP-fetABCDEF gene cluster under iron limitation.

IMPORTANCE: Campylobacter jejuni is a bacterium that is prevalent in the ceca of farmed poultry such as chickens. Consumption of ill-prepared poultry is thus the most common route by which C. jejuni infects the human gut to cause a typically self-limiting but severe gastrointestinal illness that can be fatal to very young, old, or immunocompromised people. The lack of a vaccine and an increasing resistance to current antibiotics highlight a need to better understand the mechanisms that make C. jejuni a successful human pathogen. This study focused on the functional components of one such mechanism-a molecular system that helps C. jejuni thrive despite the restriction on growth-available iron by the human body, which typically defends against pathogens. In providing a deeper understanding of how this system functions, this study contributes toward the goal of reducing the enormous global socioeconomic burden caused by C. jejuni.

Development steps of multimodal exercise interventions for older adults with multimorbidity: A systematic review.

Background and Aims: Multicomponent exercise interventions are recommended for older adults and for those with chronic diseases. While multiple programs have been tested, no one has yet explored how these programs were developed. This review set out to determine what development steps multicomponent exercise intervention studies that include older adults with multimorbidity have taken. Methods: Systematic review and narrative synthesis. Results: One hundred and thirty-eight studies meeting review criteria (Population: adults ≥60 years with multimorbidity; Intervention: exercise interventions with ≥2 components; Comparator: any considered; Outcome: any considered) were retrieved. Most studies (70%) do not report intervention development actions as suggested by available guidance. Notable deviations from recommendations include limited performance of systematic review of previously published evidence, lack of engagement with theory, and few examples of design then refine. Conclusions: Exercise interventions for older adults with multimorbidity do not appear to follow best practice in terms of their developing. Disregard of development recommendations risks contributing to research redundancy and/or avoidable waste, as important steps that make sure the intervention is warranted, suitable for the population in question, and tested using optimal methods and outcome measures are overlooked.

Promoting diversity in clinical trials: insights from planning the ALUMNI AD study in historically underrepresented US populations with early symptomatic Alzheimer's disease.

Clinical trial participation across disease areas, including Alzheimer's disease (AD), has been biased towards White participants of European ancestry. To support clinical decision-making across diverse populations, we must recognize and address barriers to trial participation. To inform the design of ALUMNI AD, a trial focused on historically underrepresented AD populations, we held advice-seeking fora with key stakeholders to understand barriers and identify potential solutions to maximize trial participation of underrepresented racial and ethnic groups in the US. Strategies identified from this process include: obtaining and implementing recommendations from community stakeholders; establishing a simple and inclusive prescreening and screening process; supporting participants and care partners; identifying and activating community-centric clinical sites; and demonstrating community commitment. While ALUMNI AD did not commence, we hope that our insights could be incorporated into future studies to improve diversity, equity, and inclusion in AD clinical research. Funding: This study was sponsored by Genentech, Inc.

Activation of picornaviral IRESs by PTB shows differential dependence on each PTB RNA-binding domain.

Polypyrimidine tract binding protein (PTB) is an RNA-binding protein with four RNA-binding domains (RBDs). It is a major regulator of alternative splicing and also stimulates translation initiation at picornavirus IRESs (internal ribosome entry sites). The sites of interaction of each RBD with two picornaviral IRESs have previously been mapped. To establish which RBD-IRES interactions are essential for IRES activation, point mutations were introduced into the RNA-binding surface of each RBD. Three such mutations were sufficient to inactivate RNA-binding by any one RBD, but the sites of the other three RBD-IRES interactions remained unperturbed. Poliovirus IRES activation was abrogated by inactivation of RBD1, 2, or 4, but the RBD3-IRES interaction was superfluous. Stimulation of the encephalomyocarditis virus IRES was reduced by inactivation of RBD1, 3, or 4, and abrogated by mutation of RBD2, or both RBDs 3 and 4. Surprisingly, therefore, the binding of PTB in its normal orientation does not guarantee IRES activation; three native RBDs are sufficient for correct binding but not for activation if the missing RBD-IRES interaction is critical.

Dissecting components of the Campylobacter jejuni fetMP-fetABCDEF gene cluster in iron scavenging.

Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. Acute infection can be antecedent to highly debilitating long-term sequelae. Expression of iron acquisition systems is vital for C. jejuni to survive the low iron availability within the human gut. The C. jejuni fetMP-fetABCDEF gene cluster is known to be upregulated during human infection and under iron limitation. While FetM and FetP have been functionally linked to iron transport in prior work, here we assess the contribution by each of the downstream genes ( fetABCDEF ) to C. jejuni growth during both iron-depleted and iron-replete conditions. Significant growth impairment was observed upon disruption of fetA , fetB, fetC , and fetD , suggesting a role in iron acquisition for each encoded protein. FetA expression was modulated by iron-availability but not dependent on the presence of FetB, FetC, FetD, FetE or FetF. Functions of the putative thioredoxins FetE and FetF were redundant in iron scavenging, requiring a double deletion (Δ fetEF ) to exhibit a growth defect. C. jejuni FetE was expressed and the structure solved to 1.50 Å, revealing structural similarity to thiol-disulfide oxidases. Functional characterization in biochemical assays showed that FetE reduced insulin at a slower rate than E. coli Trx and that together, FetEF promoted substrate oxidation in cell extracts, suggesting that FetE (and presumably FetF) are oxidoreductases that can mediate oxidation in vivo . This study advances our understanding of the contributions by the fetMP-fetABCDEF gene cluster to virulence at a genetic and functional level, providing foundational knowledge towards mitigating C. jejuni -related morbidity and mortality.

Spectrally Selective Time-Resolved Emission through Fourier-Filtering (STEF).

We demonstrate a method for separating and resolving the dynamics of multiple emitters without the use of conventional filters. By directing the photon emission through a fixed path-length imbalanced Mach-Zehnder interferometer, we interferometrically cancel (or enhance) certain spectral signatures corresponding to one emissive species. Our approach, Spectrally selective Time-resolved Emission through Fourier-filtering (STEF), leverages the detection and subtraction of both outputs of a tuned Mach-Zehnder interferometer, which can be combined with time-correlated single photon counting (TCSPC) or confocal imaging to demix multiple emitter signatures. We develop a procedure to calibrate out imperfections in Mach-Zehnder interferometry schemes. Additionally, we demonstrate the range and utility of STEF by performing the following procedures with one measurement: (1) filtering out laser scatter from a sample, (2) separating and measuring a fluorescence lifetime from a binary chromophore mixture with overlapped emission spectra, (3) confocally imaging and separately resolving the standard fluorescent stains in bovine pulmonary endothelial cells and nearly overlapping fluorescent stains on RAW 264.7 cells. This form of spectral balancing can allow for robust and tunable signal sorting.

What dietary interventions have been tested in heart failure with preserved ejection fraction? A systematic scoping review.

AIMS: To determine what dietary interventions have been tested in heart failure with preserved ejection fraction (HFpEF), the modulation method, and outcomes employed and to summarize any evidence for benefit. METHODS AND RESULTS: We performed key word searches in five bibliographic databases from 2001 to 2021, to identify randomized or experimental dietary interventions tested in HFpEF or mixed heart failure (HF) samples. Study characteristics were summarized according to population, intervention, comparator, outcome categories and intervention complexity was assessed. Twenty-five clinical investigations were retrieved; only 10 (40%) were conducted exclusively in HFpEF; the remainder enrolled mixed HF samples. Most studies employed either highly tailored prescribed diets (n = 12, 48%) or dietary supplementation (n = 10, 40%) modalities. Dietary pattern interventions (n = 3, 12%) are less well represented in the literature. CONCLUSION: Heterogeneity made pooling studies challenging. Better reporting of baseline characteristics and the use of standardized HF lexicon would ensure greater confidence in interpretation of studies involving mixed HF populations. The field would benefit greatly from explicit reporting of the biological mechanism of action (e.g. the causal pathway) that an intervention is designed to modulate so that studies can be synthesized via their underlying mechanism of action by which diet may affect HF. An extension of the current set of core outcomes proposed by the European Society of Cardiology Heart Failure Association would ensure dietary clinical endpoints are more consistently defined and measured. REGISTRATION: PROSPERO: CRD42019145388.

What are the outcomes of dietary interventions in Heart Failure with preserved Ejection Fraction? A systematic review and meta-analysis.

AIMS: To determine the efficacy of dietary interventions in Heart Failure with preserved Ejection Fraction (HFpEF). METHOD AND RESULTS: Keyword searches were performed in five bibliographic databases to identify randomized or controlled studies of dietary interventions conducted in HFpEF or mixed heart failure (HF) samples published in the English language. Studies were appraised for bias and synthesized into seven categories based on the similarity of the intervention or targeted population. The quality of the body of evidence was assessed via the GRADE (Grading of Recommendations, Assessment, Development, and Evaluations) framework. Twenty-five unique interventions were identified; 17 were considered for meta-analysis. Most studies were judged to be at high risk of bias. There was moderate-quality evidence that caloric restriction led to clinically meaningful improvements in blood pressure and body weight. There was moderate-quality evidence that carbohydrate restriction resulted in meaningful reductions in blood pressure. There was very low-quality evidence that protein supplementation improved blood pressure and body weight and moderate-quality evidence for clinically meaningful improvements in function. CONCLUSIONS: While some types of dietary interventions appeared to deliver clinically meaningful change in critical outcomes; the study heterogeneity and overall quality of the evidence make it difficult to make firm recommendations. Greater transparency when reporting the nutritional composition of interventions would enhance the ability to pool studies. REGISTRATION: PROSPERO CRD42019145388.

Genetic vulnerability to Crohn's disease reveals a spatially resolved epithelial restitution program.

Effective tissue repair requires coordinated intercellular communication to sense damage, remodel the tissue, and restore function. Here, we dissected the healing response in the intestinal mucosa by mapping intercellular communication at single-cell resolution and integrating with spatial transcriptomics. We demonstrated that a risk variant for Crohn's disease, hepatocyte growth factor activator (HGFAC) Arg509His (R509H), disrupted a damage-sensing pathway connecting the coagulation cascade to growth factors that drive the differentiation of wound-associated epithelial (WAE) cells and production of a localized retinoic acid (RA) gradient to promote fibroblast-mediated tissue remodeling. Specifically, we showed that HGFAC R509H was activated by thrombin protease activity but exhibited impaired proteolytic activation of the growth factor macrophage-stimulating protein (MSP). In Hgfac R509H mice, reduced MSP activation in response to wounding of the colon resulted in impaired WAE cell induction and delayed healing. Through integration of single-cell transcriptomics and spatial transcriptomics, we demonstrated that WAE cells generated RA in a spatially restricted region of the wound site and that mucosal fibroblasts responded to this signal by producing extracellular matrix and growth factors. We further dissected this WAE cell-fibroblast signaling circuit in vitro using a genetically tractable organoid coculture model. Collectively, these studies exploited a genetic perturbation associated with human disease to disrupt a fundamental biological process and then reconstructed a spatially resolved mechanistic model of tissue healing.

Novel C-terminal motif within Sec7 domain of guanine nucleotide exchange factors regulates ADP-ribosylation factor (ARF) binding and activation.

ADP-ribosylation factors (ARFs) and their activating guanine nucleotide exchange factors (GEFs) play key roles in membrane traffic and signaling. All ARF GEFs share a ∼200-residue Sec7 domain (Sec7d) that alone catalyzes the GDP to GTP exchange that activates ARF. We determined the crystal structure of human BIG2 Sec7d. A C-terminal loop immediately following helix J (loop>J) was predicted to form contacts with helix H and the switch I region of the cognate ARF, suggesting that loop>J may participate in the catalytic reaction. Indeed, we identified multiple alanine substitutions within loop>J of the full length and/or Sec7d of two large brefeldin A-sensitive GEFs (GBF1 and BIG2) and one small brefeldin A-resistant GEF (ARNO) that abrogated binding of ARF and a single alanine substitution that allowed ARF binding but inhibited GDP to GTP exchange. Loop>J sequences are highly conserved, suggesting that loop>J plays a crucial role in the catalytic activity of all ARF GEFs. Using GEF mutants unable to bind ARF, we showed that GEFs associate with membranes independently of ARF and catalyze ARF activation in vivo only when membrane-associated. Our structural, cell biological, and biochemical findings identify loop>J as a key regulatory motif essential for ARF binding and GDP to GTP exchange by GEFs and provide evidence for the requirement of membrane association during GEF activity.

Recent Advances in Spectroscopic Techniques for the Analysis of Microplastics in Food.

Microplastic pollution has become a worldwide concern in aquatic and terrestrial environments. Microplastics could also enter the food chain, causing potential harm to human health. To facilitate the risk assessment of microplastics to humans, it is critically important to have a reliable analytical technique to detect, quantify, and identify microplastics of various materials, sizes, and shapes from environmental, agricultural, and food matrices. Spectroscopic techniques, mainly vibrational spectroscopy (Raman and infrared), are commonly used techniques for microplastic analysis. This review focuses on recent advances of these spectroscopic techniques for the analysis of microplastics in food. The fundamental, recent technical advances of the spectroscopic techniques and their advantages and limitations were summarized. The food sample pretreatment methods and recent applications for detecting and quantifying microplastics in different types of food were reviewed. In addition, the current technical challenges and future research directions were discussed. It is anticipated that the advances in instrument development and methodology innovation will enable spectroscopic techniques to solve critical analytical challenges in microplastic analysis in food, which will facilitate the reliable risk assessment.

Toward a greener approach to detect inorganic arsenic using the Gutzeit method and X-ray fluorescence spectroscopy.

Inorganic arsenic is a carcinogen repeatedly found in water and foods threatening global human health. Prior work applied the Gutzeit method and X-ray fluorescence spectroscopy to quantify inorganic arsenic based on a harmful chemical, i.e., mercury bromide, to capture the arsine gas. In this project, we explored silver nitrate as an alternative to mercury bromide for the capture and detection of inorganic arsenic. To compare the performance of mercury bromide and silver nitrate, two standard curves were established in the range from 0 to 33.3 µg/L after optimization of reaction conditions such as the quantity of reagents and reaction time. Our result shows silver nitrate-based standard curve had a lower limit of detection and limit of quantification at 1.02 µg/L and 3.40 µg/L, respectively, as compared to the one built upon mercury bromide that has limit of detection of 4.86 µg/L and limit of quantification of 16.2 µg/L. The relative higher sensitivity when using silver nitrate was contributed by the less interfering elements for X-ray fluorescence analysis and thus lower background signals. A commercial apple juice was studied for matrix inference, and the results show 85%-99% recoveries and 7.4%-24.5% relative standard deviation. In conclusion, we demonstrated silver nitrate is a better choice in terms of safety restrictions and detection capability at lower inorganic arsenic concentrations.

Clinical and demographic correlates of accelerometer-measured physical activity in participants enrolled in the OPTIMISE HFpEF study.

AIMS: This study aimed to measure physical activity (PA) in participants with suspected heart failure with preserved ejection fraction (HFpEF) and assess associations between PA and participant characteristics. METHODS AND RESULTS: Adults with presumed HFpEF were recruited and received diagnostic evaluation and clinical assessment. Physical activity was objectively measured using accelerometers over 7 days. To examine predictors of PA, a best subset analysis was used, with the optimal model defined as that with the lowest Bayesian information criterion. One hundred and twenty-four participants with presumed HFpEF who had valid accelerometer data were included in this study. Seventy-six were confirmed by a cardiologist as meeting the European Society of Cardiology diagnosis criteria for HFpEF. The median age of all participants was 80.1 years, and 47.4% were female. Patients spent most of each 24-h period at low-intensity PA and few or no durations at high-intensity PA, with lower activity for those with HFpEF. Gait speed was the best univariate correlate of activity levels (adjusted R2 0.29). The optimal model using best subsets regression included six variables and improved adjusted R2 to 0.47. In the model, lower levels of PA were associated with slower gait speed, lower levels of anxiety, higher levels of depression, past smoking history, a confirmed HFpEF diagnosis, and higher body mass index. CONCLUSION: Participants demonstrated very low PA levels. The study has identified important patient characteristics associated with PA, which may help to identify those most in need of interventions. Notably, participants with confirmed HFpEF were more inactive than participants with other heart failure phenotypes.

210-Day Kinetics of Total, IgG, and Neutralizing Spike Antibodies across a Course of 3 Doses of BNT162b2 mRNA Vaccine.

Introduction: We tested the total spike antibody (S-Ab), IgG/IgM S-Ab, and neutralizing antibody (N-Ab) responses of COVID-19-naïve subjects from before their first BNT162b2 vaccination up to 210 days after boosting. Methods: We studied 136 COVID-19-naïve subjects who received three doses of the Pfizer mRNA vaccine (39 males, 97 females, mean age 43.8 ± 13.5 years) from January 2021 to May 2022. Serum was assessed for total S-Ab (Roche), IgG/M (Abbott), and N-Ab (Snibe). Results: Peak antibody levels were measured 20-30 days after each dose, with booster dosing eliciting significantly higher peak antibodies than the second dose: total S-Ab 2219 vs. 19,551 BAU/mL (difference 16,667 BAU/mL, p < 0.0001); IgG 2270 vs. 2932 BAU/mL (difference 660 BAU/mL, p = 0.04); and N-Ab 3.52 vs. 26.4 µg/mL (difference 21.4 µg/mL, p < 0.0001). Only IgM showed a lower peak post-booster antibody titer (COI 2.11 vs. 0.23, difference 1.63, 95% CI 1.05 to 2.38, p < 0.0001). By 180−210 days after the second or third vaccination, total S-Ab/IgG/N-Ab had decreased by 68.7/93.8/73.6% vs. 82.8/86.3/79.5%. The half-lives of IgG and N-Ab antibodies were longer after the third vaccination (IgG: 65 vs. 34 days, N-Ab: 99 vs. 78 days). Conclusion: Total S-Ab/IgG/N-Ab showed a greater increase post-booster, with IgG/N-Ab having a longer half-life.