The gut microbiota modifies antibody durability and booster responses after SARS-CoV-2 vaccination.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are pivotal in combating coronavirus disease 2019 (COVID-19); however, the declining antibody titers postvaccination pose challenges for sustained protection and herd immunity. Although gut microbiome is reported to affect the early antibody response after vaccination, its impact on the longevity of vaccine-induced antibodies remains unexplored. METHODS: A prospective cohort study was conducted involving 44 healthy adults who received two doses of either the BNT162b2 or ChAdOx1 vaccine, followed by a BNT162b2 booster at six months. The gut microbiome was serially analyzed using 16S rRNA and shotgun sequencing, while humoral immune response was assessed using a SARS-CoV-2 spike protein immunoassay. RESULTS: Faecalibacterium prausnitzii was associated with robust and persistent antibody responses post-BNT162b2 vaccination. In comparison, Escherichia coli was associated with a slower antibody decay following ChAdOx1 vaccination. The booster immune response was correlated with metabolic pathways involving cellular functions and aromatic amino acid synthesis. CONCLUSIONS: The findings of this study underscored the potential interaction between the gut microbiome and the longevity/boosting effect of antibodies following vaccination against SARS-CoV-2. The identification of specific microbial associations suggests the prospect of microbiome-based strategies for enhancing vaccine efficacy.

Respiratory microbiome and clinical course of carbapenem-resistant Acinetobacter baumannii pneumonia in critically Ill patients.

Carbapenem-resistant Acinetobacter baumannii (CRAB) pneumonia has been a serious problem in the intensive care unit (ICU). However, defined characteristics of respiratory microbiome in CRAB pneumonia are lacking nowadays. This study aimed to analyze respiratory microbiome of CRAB pneumonia compared to non-CRAB pneumonia and reveal the clinical significance of respiratory microbiome data in these patients. Patients diagnosed with severe pneumonia with mechanical ventilation were enrolled in the ICU of a tertiary care hospital. Respiratory specimens were collected on days 1, 4, 7, and 14 in each participant via tracheal aspiration. Clinical data and outcomes of each enrolled patient were collected via electronic medical records. Microbiome analysis was conducted with collected respiratory specimens undergone by next-generation sequencing of microbial 16S ribosomal DNA. Six CRAB pneumonia, 4 non-CRAB pneumonia and 5 healthy controls were enrolled. In CRAB pneumonia, CRAB was detected in 3 patients by sputum culture at day 1, while it was negative at day 1 and detected later in the others by follow-up sputum culture. Beta diversity plot analysis showed differences between each group. Shannon index was decreased markedly at day 4 in CRAB pneumonia compared to the others. Among CRAB pneumonia cases, 3 respiratory specimens were culture-negative, but positive by microbiome analysis. Lower respiratory microbiome in CRAB pneumonia had distinct characteristics and early loss of diversity compared to non-CRAB pneumonia, which might be related to poor clinical course. Moreover, CRAB acquisition and colonization would be predicted by preemptive microbiome analysis, which will contribute to effective infection control in the ICU.

Superficial Anaplastic Lymphoma Kinase-Rearranged Myxoid Spindle Cell Neoplasm in the Buttock: A Case Report.

Anaplastic lymphoma kinase (ALK) is detected in both normal and oncological developmental tissues. Among ALK-related tumors, superficial ALK-rearranged myxoid spindle cell neoplasm (SAMS) is a rare, soft tissue tumor characterized by the immunophenotypical co-expression of CD34 and S100. Here, we describe a patient with this rare tumor and outline its clinical and radiological characteristics. A 28-year-old woman with diabetes, hypertension, and panic disorder presented with discomfort caused by a rubbery mass on the left buttock that had persisted for 10 years. Computed tomography revealed a multilobulated hypodense mass with small internal enhancing foci, posing challenges for the exact diagnosis of the lesion. The entire lesion was excised with clear resection margins. An 8.0 × 6.0 cm, well-circumscribed tumor with a lobular growth pattern was observed in the deep subcutaneous tissue. Light microscopy revealed epithelioid, ovoid, and spindle-shaped cells with a reticular cordlike pattern. Immunohistochemistry results were positive for S100, CD34, and vimentin. Break-apart fluorescence in situ hybridization assay results for ALK were also positive. These findings were consistent with those of SAMS. This case suggests that SAMS should be considered when identifying large nonspecific masses during clinical and imaging evaluation.

Precise base editing without unintended indels in human cells and mouse primary myoblasts.

Base editors are powerful tools for making precise single-nucleotide changes in the genome. However, they can lead to unintended insertions and deletions at the target sites, which is a significant limitation for clinical applications. In this study, we aimed to eliminate unwanted indels at the target sites caused by various evolved base editors. Accordingly, we applied dead Cas9 instead of nickase Cas9 in the base editors to induce accurate substitutions without indels. Additionally, we tested the use of chromatin-modulating peptides in the base editors to improve nucleotide conversion efficiency. We found that using both dead Cas9 and chromatin-modulating peptides in base editing improved the nucleotide substitution efficiency without unintended indel mutations at the desired target sites in human cell lines and mouse primary myoblasts. Furthermore, the proposed scheme had fewer off-target effects than conventional base editors at the DNA level. These results indicate that the suggested approach is promising for the development of more accurate and safer base editing techniques for use in clinical applications.

Clinical implications of gut microbiota and cytokine responses in coronavirus disease prognosis.

Objectives: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects gut luminal cells through the angiotensin-converting enzyme-2 receptor and disrupts the gut microbiome. We investigated whether the gut microbiome in the early stage of SARS-CoV-2 infection was associated with the prognosis of coronavirus disease (COVID-19). Methods: Thirty COVID-19 patients and 16 healthy controls were prospectively enrolled. Blood and stool samples and clinical details were collected on days 0 (enrollment), 7, 14, and 28. Participants were categorized into four groups by their clinical course. Results: Gut microbiota composition varied during the clinical course of COVID-19 and was closely associated with cytokine levels (p=0.003). A high abundance of the genus Dialister (linear discriminant analysis [LDA] effect size: 3.97856, p=0.004), species Peptoniphilus lacrimalis (LDA effect size: 4.00551, p=0.020), and Anaerococcus prevotii (LDA effect size: 4.00885, p=0.007) was associated with a good prognosis. Starch, sucrose, and galactose metabolism was highly activated in the gut microbiota of the poor prognosis group. Glucose-lowering diets, including whole grains, were positively correlated with a good prognosis. Conclusion: Gut microbiota may mediate the prognosis of COVID-19 by regulating cytokine responses and controlling glucose metabolism, which is implicated in the host immune response to SARS-CoV-2.

Establishment of multicenter COVID-19 therapeutics preclinical test system in Republic of Korea.

Throughout the recent COVID-19 pandemic, South Korea led national efforts to develop vaccines and therapeutics for SARS-CoV-2. The project proceeded as follows: 1) evaluation system setup (including Animal Biosafety Level 3 (ABSL3) facility alliance, standardized nonclinical evaluation protocol, and laboratory information management system), 2) application (including committee review and selection), and 3) evaluation (including expert judgment and reporting). After receiving 101 applications, the selection committee reviewed pharmacokinetics, toxicity, and efficacy data and selected 32 final candidates. In the nonclinical efficacy test, we used golden Syrian hamsters and human angiotensin-converting enzyme 2 transgenic mice under a cytokeratin 18 promoter to evaluate mortality, clinical signs, body weight, viral titer, neutralizing antibody presence, and histopathology. These data indicated eight new drugs and one repositioned drug having significant efficacy for COVID-19. Three vaccine and four antiviral drugs exerted significant protective activities against SARS-CoV-2 pathogenesis. Additionally, two anti-inflammatory drugs showed therapeutic effects on lung lesions and weight loss through their mechanism of action but did not affect viral replication. Along with systematic verification of COVID-19 animal models through large-scale studies, our findings suggest that ABSL3 multicenter alliance and nonclinical evaluation protocol standardization can promote reliable efficacy testing against COVID-19, thus expediting medical product development.

Targeted Mutagenesis in Mice Using a Base Editor.

Base editors, such as cytosine and adenine base editors, are composed of nickase Cas9 (nCas9) and deaminase and serve as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based enzymatic tools for specific nucleotide substitutions. They are mainly the most effective genome editing tools for introducing point mutations, such as C-to-T and A-to-G conversions. The enhanced base editor, a C-to-G base editor (CGBE), can perform other nucleotide substitutions, such as C-to-G conversions. Here, we introduce a method for generating mouse models with point mutations using a base editing system.

Cucurbitacin and volatile compound profiling reveals independent domestication of cucumber (Cucumis sativus L.) fruit.

Profiling of metabolites that confer bitter taste and flavor to cucumber products is necessary to produce preferred products. In this study, cucurbitacins A, B, C, D, E, and I and untargeted volatile compounds were analyzed using the fruit of 69 inbred cucumber of diverse cultivars. Only cucurbitacin C was detected in six inbreds. They were classified into four clusters based on the profiles of cucurbitacins and volatile compounds. Clusters 2 and 3 showed the largest difference with the highest and lowest volatile contents, respectively. Clusters 1 and 4 showed different fruit phenotypes of length and color. Fifteen F1 hybrids from selected inbreds were analyzed. Total volatile compound (TVC) content, especially for the alcohol content, was lower in the F1 hybrids than the mid-parent values, and the ratio of aldehyde to TVC was increased. This profiling will contribute to produce cucumber products with no bitter taste and improved flavor.

Mouse models of lung-specific SARS-CoV-2 infection with moderate pathological traits.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) has been a global health concern since 2019. The viral spike protein infects the host by binding to angiotensin-converting enzyme 2 (ACE2) expressed on the cell surface, which is then processed by type II transmembrane serine protease. However, ACE2 does not react to SARS-CoV-2 in inbred wild-type mice, which poses a challenge for preclinical research with animal models, necessitating a human ACE2 (hACE2)-expressing transgenic mouse model. Cytokeratin 18 (K18) promoter-derived hACE2 transgenic mice [B6.Cg-Tg(K18-ACE2)2Prlmn/J] are widely used for research on SARS-CoV-1, MERS-CoV, and SARS-CoV-2. However, SARS-CoV-2 infection is lethal at ≥105 PFU and SARS-CoV-2 target cells are limited to type-1 alveolar pneumocytes in K18-hACE2 mice, making this model incompatible with infections in the human lung. Hence, we developed lung-specific SARS-CoV-2 infection mouse models with surfactant protein B (SFTPB) and secretoglobin family 1a member 1 (Scgb1a1) promoters. After inoculation of 105 PFU of SARS-CoV-2 to the K18-hACE2, SFTPB-hACE2, and SCGB1A1-hACE2 models, the peak viral titer was detected at 2 days post-infection and then gradually decreased. In K18-hACE2 mice, the body temperature decreased by approximately 10°C, body weight decreased by over 20%, and the survival rate was reduced. However, SFTPB-hACE2 and SCGB1A1-hACE2 mice showed minimal clinical signs after infection. The virus targeted type I pneumocytes in K18-hACE2 mice; type II pneumocytes in SFTPB-hACE2 mice; and club, goblet, and ciliated cells in SCGB1A1-hACE2 mice. A time-dependent increase in severe lung lesions was detected in K18-hACE2 mice, whereas mild lesions developed in SFTPB-hACE2 and SCGB1A1-hACE2 mice. Spleen, small intestine, and brain lesions developed in K18-hACE2 mice but not in SFTPB-hACE2 and SCGB1A1-hACE2 mice. These newly developed SFTPB-hACE2 and SCGB1A1-hACE2 mice should prove useful to expand research on hACE2-mediated respiratory viruses.

Corrigendum: Mouse models of lung-specific SARS-CoV-2 infection with moderate pathological traits.

[This corrects the article DOI: 10.3389/fimmu.2022.1055811.].

Comprehensive Comparison of Chemical Composition and Antioxidant Activity of Panax ginseng Sprouts by Different Cultivation Systems in a Plant Factory.

In this study, the primary (such as amino acids, fatty acids, and minerals) and secondary (including ginsenosides, phenolic acids, and flavonols) metabolites and antioxidant effects of Panax ginseng sprouts (PGSs) by different cultivation systems, such as soil-substrate cultivation (SSC) and deep-water cultivation (DWC), in a plant factory has been observed. There was no significant difference in the total fatty acid (FA) contents. Particularly, the major FAs of PGSs were palmitic acid (207.4 mg/100 g) of saturated FAs and linoleic acid (397.6 mg/100 g) and α-linolenic acid (222.6 mg/100 g) of unsaturated FAs in the SSC system. The values of total amino acids were all higher in SSC than in DWC. In the case of ginsenosides, the total protopanaxtriol product was 30.88 mg/g in SSC, while the total protopanaxdiol product was 34.83 mg/g in DWC. In particular, the values of total phenolic acids and total flavonols were 133.36 and 388.19 ug/g, respectively, and SSC had a higher content than DWC. In conclusion, the SSC system was shown to be higher in nutritional constituents and antioxidant activities in soil cultivation, suggesting that PGS with SSC has a positive effect on the quality of PGS in a plant factory.

Metabolic Analyses and Evaluation of Antioxidant Activity in Purple Kohlrabi Sprouts after Exposed to UVB Radiation.

Various metabolites act as plant defense molecules due to their antioxidant abilities. This study aimed to investigate the influence of UVB irradiation on the accumulation of metabolites, including primary metabolites (sugar, sugar alcohols, amino acids, organic acids, and an amine) and secondary metabolites (anthocyanins, fatty acids, and phenolic acids), and its synergistic antioxidant ability, in purple kohlrabi sprouts. Metabolite analyses revealed a total of 92 metabolites in the sprouts. Specifically, the levels of most amino acids increased after 24 h of UVB treatment, and then slightly decreased in the kohlrabi sprouts. The levels of most sugars and sugar alcohols increased after 24 h of UVB treatment and then decreased. The levels of TCA cycle intermediates and phenolic acids gradually increased during the UVB treatment. Furthermore, the levels of some fatty acids gradually increased during the UVB treatment, and the levels of the other fatty acids increased after 6 h of UVB treatment and then decreased. In particular, the levels of most anthocyanins, known to be strong antioxidants, gradually increased after 24 h of UVB treatment. In the in vitro ABTS scavenging assay, UVB-treated purple kohlrabi sprouts showed increased scavenging ability. This may be attributed to the increased accumulation of metabolites acting as antioxidants, in response to UVB treatment. This study confirmed that UVB irradiation induced the alteration of primary and secondary metabolism in the kohlrabi sprouts.

Laboratory information management system for COVID-19 non-clinical efficacy trial data.

BACKGROUND: As the number of large-scale studies involving multiple organizations producing data has steadily increased, an integrated system for a common interoperable format is needed. In response to the coronavirus disease 2019 (COVID-19) pandemic, a number of global efforts are underway to develop vaccines and therapeutics. We are therefore observing an explosion in the proliferation of COVID-19 data, and interoperability is highly requested in multiple institutions participating simultaneously in COVID-19 pandemic research. RESULTS: In this study, a laboratory information management system (LIMS) approach has been adopted to systemically manage various COVID-19 non-clinical trial data, including mortality, clinical signs, body weight, body temperature, organ weights, viral titer (viral replication and viral RNA), and multiorgan histopathology, from multiple institutions based on a web interface. The main aim of the implemented system is to integrate, standardize, and organize data collected from laboratories in multiple institutes for COVID-19 non-clinical efficacy testings. Six animal biosafety level 3 institutions proved the feasibility of our system. Substantial benefits were shown by maximizing collaborative high-quality non-clinical research. CONCLUSIONS: This LIMS platform can be used for future outbreaks, leading to accelerated medical product development through the systematic management of extensive data from non-clinical animal studies.

Targeted mutagenesis in mouse cells and embryos using an enhanced prime editor.

Prime editors, novel genome-editing tools consisting of a CRISPR-Cas9 nickase and an engineered reverse transcriptase, can induce targeted mutagenesis. Nevertheless, much effort is required to optimize and improve the efficiency of prime-editing. Herein, we introduce two strategies to improve the editing efficiency using proximal dead sgRNA and chromatin-modulating peptides. We used enhanced prime-editing to generate Igf2 mutant mice with editing frequencies of up to 47% and observed germline transmission, no off-target effects, and a dwarf phenotype. This improved prime-editing method can be efficiently applied to cell research and to generate mouse models.

Transcriptome Analysis of the Fruit of Two Strawberry Cultivars "Sunnyberry" and "Kingsberry" That Show Different Susceptibility to Botrytis cinerea after Harvest.

Gray mold (Botrytis cinerea) is a fungal plant pathogen causing postharvest decay in strawberry fruit. Here, we conducted a comparative transcriptome analysis to identify differences in gene expression between the immature-green (IG) and mature-red (MR) stages of the "Sunnyberry" (gray mold-resistant) and "Kingsberry" (gray mold susceptible) strawberry cultivars. Most of the genes involved in lignin and alkane-type wax biosynthesis were relatively upregulated in "Sunnyberry". However, pathogenesis-related proteins encoding R- and antioxidant-related genes were comparatively upregulated in "Kingsberry". Analysis of gene expression and physiological traits in the presence and absence of B. cinerea inoculation revealed that the defense response patterns significantly differed between IG and MR rather than the cultivars. "Kingsberry" showed higher antioxidant induction at IG and upregulated hemicellulose-strengthening and R genes at MR. Hence, "Sunnyberry" and "Kingsberry" differed mainly in terms of the expression levels of the genes forming cuticle, wax, and lignin and controlling the defense responses. These discrepancies might explain the relative difference between these strawberry cultivars in terms of their postharvest responses to B. cinerea.

Comparative transcriptome and metabolome analyses of two strawberry cultivars with different storability.

Postharvest storability is an important trait for breeding strawberry (Fragaria × ananassa Duch.). We evaluated the postharvest fruit quality of five strawberry cultivars ('Durihyang', 'Kingsberry', 'Maehyang', 'Seolhyang', and 'Sunnyberry') and identified differences in their fruit ripening during the transition from the big-green to fully-red stage between two cultivars with the highest ('Sunnyberry') and lowest ('Kingsberry') storability, using comparative transcriptome and -metabolome analysis. The differentially expressed genes revealed transcriptome changes related to anthocyanin biosynthesis and cell walls. Consistently, the metabolites of both cultivars showed general changes during ripening along with cultivar-specific characteristics in sugar and amino acid profiles. To identify the genes responsible for storability differences, we surveyed the expression of transcription factors, and found that the expression levels of WRKY31, WRKY70, and NAC83 correlated with delayed senescence and increased storability. Among them, the expression levels of NAC83, and its downstream target genes, in the five cultivars suggested that NAC83 expression can be used to predict postharvest strawberry fruit storability.

Revealing biomass heterosis in the allodiploid xBrassicoraphanus, a hybrid between Brassica rapa and Raphanus sativus, through integrated transcriptome and metabolites analysis.

BACKGROUND: Heterosis is biologically important but the molecular basis of the phenomenon is poorly understood. We characterized intergeneric hybrids between B. rapa cv. Chiifu and R. sativus cv. WK10039 as an extreme example of heterosis. Taking advantage of clear heterosis phenotypes and the genetic distance between parents, we performed transcriptome and metabolite analysis to decipher the molecular basis of heterosis. RESULTS: The heterosis was expressed as fresh weight in the field and as inflorescence stem length in the glass house. Flowering time, distributed as a normal segregating population, ranged from the early flowering of one parent to the late flowering of the other, in contrast to the homogeneous flowering time in a typical F1 population, indicating unstable allelic interactions. The transcriptome and metabolome both indicated that sugar metabolism was altered, suggesting that the change in metabolism was linked to the heterosis. Because alleles were not shared between the hybridized genomes, classic models only partly explain this heterosis, indicating that other mechanisms are involved. CONCLUSION: The differential expression of genes for primary and secondary metabolism, along with the altered metabolite profiles, suggests that heterosis could involve a change in balance between primary and secondary metabolism.

Determination of radon leakage from sample container for gamma spectrometry measurement of 226Ra.

Gamma spectrometry is widely used for radionuclide activity measurement of environmental sample including 226Ra in soil because it is versatile, easy to implement, non-destructive, and relatively cheap compared to other methods. By gamma spectrometry, 226Ra can be determined directly using its energy peak of 186.2 keV or indirectly by its decay products which are in secular equilibrium with 226Ra, i.e. 214Bi and 214Pb. However, both methods have challenges where for direct measurement, interfering from 235U energy peak of 185.7 keV can add significant contribution to the 186.2 keV of 226Ra gamma peak while for indirect measurement using energy peaks of 214Bi and 214Pb, decay products of 222Rn gas, longer time (at least 21 days) for their equilibrium ingrowth is needed and radon tightness of sample container must be ensured. In some measurements, the equilibrium could not be reached due to radon leakage from sample container. Therefore, in this paper, the radon leakage fraction from three different sealing methods of Marinelli beaker used in soil activity measurement were studied using HPGe detector and radon accumulation chamber with RAD7. The three sealing methods were only lid without sealing (MB1), sealed with paraffin film (MB2), sealed with silicone glue and vacuumed plastic bag (MB3) and their leakage fractions were 53%, 30%, and negligible leakage, respectively. Significant radon leakage from MB1 and MB2 have caused underestimation of 226Ra activity concentration measured by indirect gamma spectrometry. The study result recommends using MB3 sealing method for gamma spectrometry measurement because it is simple and cheap and has negligible leakage, moreover, its vacuumed plastic bag facilitates visual inspection to possible radon leakage.

Deep Neck Inflammation: Probable Kawasaki Disease in Korean Children.

OBJECTIVES: Deep neck infections (DNIs) can cause life-threatening complications, and prompt diagnosis and management are necessary. Kawasaki disease (KD) may be accompanied by deep neck inflammation; making it difficult to distinguish from DNIs. This study was performed to evaluate clinical features and outcomes of children with parapharyngeal and retropharyngeal inflammation. METHODS: Medical records of the children diagnosed with parapharyngeal and retropharyngeal cellulitis or abscess using cervical computed tomography (CT) between 2013 and 2017 were retrospectively reviewed. RESULTS: A total of 47 children were diagnosed with parapharyngeal and retropharyngeal inflammation. Eleven (23.4%) of them were eventually diagnosed with KD, and 36 (76.6%) were diagnosed with DNIs. There were no significantly different clinical and laboratory characteristics on admission between children diagnosed with KD and DNIs; however, significantly more children with KD were febrile for ≥3 days after admission compared to those with DNIs (P=0.009). Deep neck abscesses on CT were observed in 16 children with DNIs (44.4%) and in no child with KD (P=0.009). Among the 36 children with DNIs, 30 (83.3%) were cured with antibiotic therapy only. CONCLUSION: A quarter of children presenting with deep neck inflammation were diagnosed with KD. KD should be considered in children showing deep neck inflammation unresponsive to empirical antibiotic therapy after 3 days, especially in those presenting with deep neck cellulitis rather than deep neck abscess.