In alcoholic hepatitis (AH), dysfunctional T lymphocytes may contribute to the high mortality from infections. T lymphocyte activation is governed by the expression of co-stimulatory receptors such as 4-1BB balanced by inhibitory receptors such as Programmed Death receptor 1 (PD-1). 4-1BB expression is unaccounted for in AH, while PD-1 is elevated. We characterized expression of 4-1BB and PD-1 and the associated T lymphocyte functional status in AH and investigated whether these were associated with short-term mortality.in vitro stimulated receptor expression were quantified by flow cytometry, and plasma proteins by ELISA.Thirty-five patients with AH (at diagnosis and days 7 and 90) were compared with healthy controls (HC). Spontaneous and + T lymphocytes. Also, the frequencies of PD-1+ T lymphocytes both with and without co-expressed 4-1BB were increased. Further, interferon-gamma was predominantly produced in T lymphocytes co-expressing 4-1BB. A decrease in the frequency of spontaneous 4-1BB+ T lymphocytes and an increase in soluble 4-1BB during the first week after diagnosis were associated with higher mortality at day 90 in AH. PD-1 expression showed no systematic dynamics related to mortality.At diagnosis, the patients showed increased stimulated 4-1BB responses of CD4We found an increased stimulated 4-1BB response of T lymphocytes in AH and early loss of these lymphocytes was associated with a higher short-term mortality. This suggests a role of T lymphocyte 4-1BB expression in the progression of AH. Patients with alcoholic hepatitis have a high susceptibility to infections which carries a high mortality , 2. Ther4-1BB is a glycosylated, co-stimulatory receptor which is induced on activated T lymphocytes and vital for T-cell receptor (TCR) signal transduction , 6. 4-1BPatients with AH often have lymphopenia. Moreover, the inhibitory receptors Programmed Death receptor-1 (PD-1) and T-cell immunoglobulin Mucin-3 (TIM-3) are upregulated on T lymphocytes, and this is associated with reduced antimicrobial functions and a cytokine production dominated by the anti-inflammatory interleukin-10 (IL-10) . FurtherWe hypothesized that T lymphocyte 4-1BB expression is increased in AH and associated with better T lymphocyte antimicrobial cytokine production and reduced mortality. We, therefore, characterized the T lymphocyte expression of 4-1BB in relation to PD-1 and Gal-9 and short-term mortality in patients with AH.The study was approved by Danish Central Region Committee of health research ethics (1-10-72-40-13) and Datatilsynet (2007-58-0016). All patients provided written, informed consent prior to study inclusion. The study population consisted of 35 patients with AH consecutively recruited from the Department of Hepatology and Gastroenterology, Aarhus University Hospital between March 2013 and March 2018. Patients were included when diagnosed with AH based on the following criteria: a history of excessive alcohol consumption with a period of abstinence of less than 4 weeks prior to disease presentation; acute jaundice with presentation within the previous 2 weeks (serum bilirubin > 80 μmol); age between 18 and 75 years. Exclusion criteria were other underlying liver disease, hepatocellular carcinoma, biliary stones, uncontrolled infection, upper gastrointestinal bleeding, or immunomodulatory therapy within the past 8 weeks. According to local clinical guidelines at the time of study, patients were treated with either pentoxifylline (prior to 2015) or prednisolone when their Glasgow alcoholic hepatitis score (GAHS) was 9 or above. Blood samples were collected at the day of diagnosis prior to initiation of pentoxifylline or prednisolone and at days 7 and 90 after diagnosis and liver biopsies were obtained when possible at diagnosis n = 21). Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA-whole blood samples by Ficoll-Hypaque  gradient centrifugation and stored until analysis at -140°C. Plasma and serum were obtained from centrifugation of whole blood at 1800g for 10 minutes and stored at -80°C. Blood samples from age- and sex-matched healthy controls were obtained from the Department’s biobank. Healthy liver tissue was collected from the rim of resectates during operations for metastatic colon cancer at the Department of Abdominal Surgery, Aarhus University Hospital [. Periphe6 cells/mL) in culture media (RPMI + Penicillin and Streptomycin) with 0.1 ug/mL anti-CD28  in wells pre-coated with 0.1 ug/mL anti-CD3  and incubated for 48 hours at 37°C in a 5% CO2 atmosphere. Cells were harvested in PBS on ice.At the time of analysis, PBMCs were thawed  and either stained immediately or incubated  pr. mL cell suspension and incubated for 15 minutes at 4°C. Afterwards, surface staining was performed on unstimulated and stimulated cells using optimised volumes of the following fluorescent-conjugated monoclonal antibodies: a T lymphocytes panel with anti-CD4-PerCP-Vio700 , anti-CD8-Pe-Vio770 , anti-4-1BB-APC , anti-Gal9-BV421 (Biolegend cat.number 348920), anti-PD-1-PE , anti-TIM3-eFlour-780  and anti-OX40-FITC , and a monocyte panel with anti-CD14-FITC , anti-CD16-APC-Vio770 , anti-4-1BBL-APC , anti-Gal3-PE  and anti-Gal9-BV421 (Biolegend cat.number 348920). Both panels included a dump channel with anti-CD56-BV510  and viability-viogreen  . Followi-CD56-CD4+CD8-, cytotoxic T lymphocytes as viability-CD56-CD4-CD8+ and classical monocytes as viability-CD56-CD14+CD16-. The lymphocyte gate was set on a forward-side scatter plot. T lymphocytes were identified as viabilityD14+CD16- Figs. CDphocytes . The expTo assess cytokine production potentials, PBMCs were stimulated as described above and 50 ug/mL phorbol 12-myristate 13-acetate  and 10 ug/mL Golgi-blocking agent brefeldin A  was added for the last four hours of incubation. Additionally, half of the wells were also treated with 400 ng/mL recombinant humanised Galectin-9 . Cell permeabilization was performed as previously described  and staiThe levels of s4-1BB and Gal-9 in plasma were quantified using optimised, commercial s4-1BB and Gal-9 ELISA kits  according to manufacturer’s instructions using a Thermo Scientific Multiskan GO . Cut-offLiver expression of Gal-9 was measured using RNA sequencing on liver biopsies as previously described  and compData were tested for normality using QQ-plots and histograms. A T test was used for comparison between two groups when data was normal distributed. Otherwise, log-transformation was attempted and when normal distribution was not achieved by log-transformation, a Mann-Whitney rank sum test was used for comparison between two groups. Association analyses were performed using the Spearman’s rank correlation test. Statistical analyses were performed using STATA . The results are expressed as median and interquartile range. A two-tailed p-value < 0.05 was considered statistically significant.Clinical and biochemical characteristics of the patients with alcoholic hepatitis (n = 35) are presented in + CD4+ and CD8+ T lymphocytes were increased compared with HC following anti-CD3 and anti-CD28 stimulation  lymphocytes, and monocytes  , 30. ThuIn light of this, it may be desirable to cautiously boost 4-1BB signalling in AH. Gal-9 was an obvious candidate to attempt to balance co-stimulatory and inhibitory receptor expression, as Gal-9 can bind both 4-1BB and the inhibitory TIM-3 receptor and modulate receptor signaling , 18, 19.In conclusion, stimulated T lymphocyte 4-1BB response is increased in patients with AH and loss of 4-1BB is associated with higher short-term mortality. This suggests a role of 4-1BB on T lymphocyte activation in alcoholic hepatitis.S1 Table(DOCX)Click here for additional data file.S1 Fig-Viability-. CD4-CD8- cells were excluded. CD4+ and CD8+ T lymphocytes were identified and their receptor expressions were based on flourescence-minus-5 controls.Flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) stimulated for 48 hours with anti-CD3 and anti-CD28. Lymphocytes were identified on a forward scatter (FSC) versus side scatter (SSC) plot. Duplets were excluded. Alive lymphocytes were identified as CD56(TIF)Click here for additional data file.S2 Fig-Viability-. Classical monocytes were identified as CD14+CD16- and receptor expression of galectin-9  were based on fluorescence-minus-three controls.Flow cytometry analysis of peripheral blood mononuclear cells (PBMCs). Monocytes were identified on a forward scatter (FSC) versus side scatter (SSC) plot. Duplets were excluded. Alive monocytes were identified as CD56(TIF)Click here for additional data file.S3 FigFlow cytometry analysis of peripheral blood mononuclear cells comparing the gating strategy (A) with and (B) without anti-CD3.(TIF)Click here for additional data file.S4 Fig+ and (B) CD8+ T lymphocytes from peripheral blood mononuclear cells after 48 hours stimulation with anti-CD3 and anti-CD28 and 4 hours with phorbol 12-myristate 13-acetate (PMA) and Golgi-blocking agent brefeldin A (BFA).Flow cytometry identification of (A) CD4(TIF)Click here for additional data file.S5 Fig+ CD4+ and (B) CD8+ T lymphocytes were compared between patients with alcoholic hepatitis (AH) at diagnosis and healthy controls (HC). Median ± interquartile range shown. T test.Peripheral blood mononuclear cells were analyzed using flow cytometry and the frequency of (A) 4-1BB(TIF)Click here for additional data file.S6 FigFlow cytometry analysis of peripheral blood mononuclear cells (PBMCs) stimulated for 48 hours with anti-CD3 and anti-CD28. Frequencies of CD4+ T Lymphocytes expressing (A) OX40, (B) PD-1, and (C) TIM-3 in healthy controls (HC) compared with alcoholic hepatitis patients (AH). T test used for comparisons between groups. Graph shown as median with interquartile range.(TIF)Click here for additional data file.S7 Fig+ CD4+ T lymphocytes measured using flow cytometry (A) and plasma s4-1BB measured with enzyme-linked immunosorbent assay (B) from day 0 to day 7 in patients with alcoholic hepatitis. Patients infected at day 7 (n = 2) compared with patients without infection. Flow cytometry data compared using T test and ELISA data compared using Mann-Whitney rank sum test. Graphs shown as median ± interquartile range.Change in frequencies of 4-1BB(TIF)Click here for additional data file.S8 Fig+ and (B) OX40+ CD4+ T lymphocytes from day 0 to day 7 measured using flow cytometry. Patients dead by day 90  compared with those still alive using T test. Graph shown as median ± interquartile range.Change in frequencies of (A) PD-1(TIF)Click here for additional data file.


Background: microRNAs (miRNAs) have drawn more attention to the progression of atherosclerosis (AS), due to their noticeable inflammation function in cardiovascular disease. Macrophages play a crucial role in disrupting atherosclerotic plaque, thereby we explored the involvement of miR-223-3p in the inflammatory response in macrophages.Methods: RT-qPCR was used to analyze the miR-223-3p levels in carotid arteries and serum of AS patients. ROC curve was used to assess the diagnostic value of miR-223-3p. Movat staining was applied to evaluate the morphological differences. FISH was used to identify the expression of miR-223-3p in macrophages of atherosclerotic lesions. Bioinformatic analysis was performed. Double-immunofluorescence and western blot were performed to assess the inflammatory cytokine secretion and p-ERK1/2. C16-PAF was injected into the culture medium of the miR-223-3p mimic/NC-transfected macrophages with ox-LDL.in vivo and in vitro. C16-PAF reversed the effects of miR-223-3p mimics on inflammation and ERK1/2 signaling pathway.Results: MiR-223-3p was up-regulated in AS patients and was associated with a higher overall survival rate. MiR-223-3p was co-localized with CD68+ macrophages in vulnerable atherosclerotic lesions. MiR-223-3p mimics decreased atherosclerotic lesions, macrophages numbers whereas increased SMCs numbers in the lesions. The TNF-a immune-positive areas were reduced by miR-223-3p mimics. MAP2K1 was negatively associated with miR-223-3p. MiR-223-3p mimics reduced the inflammation and the MEK1/ERK1/2 signaling pathway Conclusions: MiR-223-3p negatively regulates inflammatory responses by the MEK1/ERK1/2 signaling pathway. Our study provides new insight into how miR-223-3p protects against atherosclerosis, representing a broader therapeutic prospect for treating atherosclerosis by miR-223-3p. Atherosclerosis (AS) is the cause of most cardiovascular disease types, which becomes the primary reason for morbidity and mortality worldwide , 2. AtheThe Raf/mitogen-activated protein kinase (MEK/MAPKK)/extracellular signal-regulated kinase 1/2 (ERK 1/2) signaling pathway regulates multiple cellular processes such as proliferation, differentiation, and cell growth, and ERK phosphorylation is activated by MEK , 9. On aPrevious studies have described an association between the modifications in microRNA (miRNA) and atherosclerosis –16. MiRNTherefore, the present study was conducted to explore the effects and regulatory mechanisms of miR-223-3p /MEK/ERK1/2 in arteriosclerosis. ApoE-/- mice were fed with a high-fat diet (HFD) to induce AS models, macrophages treated with ox-LDL were served as cell AS models.in situ Hybridization (FISH) assay to identify the miR-223-3p in macrophages of the intima and vulnerable atherosclerotic lesions. We found miR-223-3p co-localization with CD68+ macrophages in vulnerable atherosclerotic lesions of AS patients  mice by a high-fat diet. To further elucidate the in vivo effects of miR-223-3p in AS, ApoE-/- mice were assigned to high-fat diet (HFD) + miR-223-3p negative control (NC), and HFD + miR-223-3p mimics. Mice carotid roots and myometrial tissues were obtained and stained with Movat staining. Representative staining results were shown in in vitro experiments. . In brief, these in vivo experimental data implied that miR-223-3p mimics could prevent AS from developing.To functionally investigate the role of miR-223-3p in vulnerable atherosclerotic lesions, we applied the well-established mice atherosclerosis HFDs, in which atherosclerotic lesions were induced in apolipoprotein-E deficient  assay revealed miR-223-3p co-localization with CD68+ macrophages in vulnerable atherosclerotic lesions of AS patients, as showed in AS is an inflammatory diseases, the macrophages play a significant roles in the formation and progression of atherosclerosis, therefore the factors regulated the inflammatory factors and associated modulatory pathway proteins secreted by lesional macrophages, deserved of research , 30. Pre-/- mice were treated with miR-223-3p negative control and mimic and pathological analysis results showed that the increase of miR-223-3p decreased lesional areas following with macrophage decrease, TNF-α secretion and smooth muscle cells increase. Lesional macrophages is the major cellular resource responding for inflammatory factors secretion, TNF-α, IL-6, IFN-γ, et al., resulting in accumulating circulating monocytes and macrophages into AS and promoting outbreak of inflammatory response [The disease based on atherosclerosis, such as myocardial infarction, apoplexia, cerebral infarction, obliterans of the Lower Extremities and et al., attracted from youth to middle aged and old aged people , 35. Theresponse –39, thusresponse , 41. OurMiRNAs exert their function by reducing their downstream expression of target genes. The variety of functions of miR-223 has been related to the suppression of many different target genes in inflammatory pathologies . TherefoDouble-immunofluorescence exhibited significant co-localization of the macrophage marker CD68+ and inflammatory factor TNF-α, indicating the TNF-α secreted by macrophage in AS lesions. To further investigate the possible role of miR-223-3p in the inflammatory response of AS, protein expression changes in mice carotid tissues were determined by quantitative analysis and western blots. Respectively, we found significantly decreased expression levels of inflammatory cytokine (IL-6 and TNF-a) in carotid tissues from the miR-223-3p mimics group compared with the NC group. Evidence has indicated that the MAPK1 signaling pathway is involved in miR-223-3p inhibition by combining with 3’-UTR of MAPK1 for further degradation . As the in vitro experiment demonstrated that miR-223-3p could play an anti-atherosclerosis role by modulating ERK activity and inflammatory factor secretion.The macrophages might initiate and exacerbate inflammation with ox-LDL stimulation and prompt the destabilization of the atherosclerotic plaques . Using wIn conclusion, our study elucidates that miR-223-3p prevents the development of AS and reduced inflammatory response through down-regulation of MEK1/ERK1/2, which suggests that miR-223-3p /MEK1/ERK1/2 can be potentially used as an attractive therapeutic for AS.8 patients with stable plaques n=4, and vulnerable plaques n=4 underwent diagnostic carotid CT and ultrasonic testing to intent to revascularize the culprit arteries shown in −/− mice (C57Bl/6 background) were obtained from Beijing HFK Biotechnology Co., Ltd . The animal use protocol for this study has been reviewed and approved by the Laboratory Animal Ethical Committee, Hebei Medical University first Affiliated Hospital. ApoE−/− mice established adeno-associated virus serotype-9 (AAV9) mediated miR-223-3p overexpression via the tail vein. The groups were set as follows: high-fat diet (HFD) + miR-223-3p negative control (NC), and HFD + miR-223-3p mimics groups via tail vein injection. The mice were fed a high-fat diet (HFD) to induce early atherosclerotic lesions. After providing a high-fat diet for two months and transfection with AAV9, the mice were sacrificed.The ApoEAfter injection with pentobarbital sodium, blood samples were gathered from the carotid artery of AS patients and controls. The serum contents were treated with regional citrate anticoagulation (RCA) and centrifuged at 2500g for 20 min at four ° C. Blood cells were recovered by centrifugation at 700 × g for 20 min at 4° C. The ELISA kit  was conducted to identify the plasma and macrophages expressed according to the kit instructions. The serum levels of inflammatory cytokines interleukin-6 (IL-6) (eBioscience) and tumor necrosis factor-α (TNF-α) (eBioscience) were examined using ELISA kits .After thoroughgoing washing with water, the aortic tissues were rinsed by 60% isopropanol for 5 min and stained with the Movat-staining dyeing solution for 2 h, then differentiated and rinsed by 60% isopropanol for 6~7 times. Finally, the images were captured under an optical microscope .Mouse macrophage-like cell line RAW 264.7 was obtained from Peking Union Medical College. After adding Dulbecco's modified eagle's medium  with 10% fetal bovine serum (FBS) , the cells were cultured at 37° C with 5% CO2. Forty-eight hours later, the cells were treated with 0.25% trypsin (Biotime Biotechnology) and re-cultured in a 75 mL flask. The cells were seeded in 6-well plates at 2.5 × 105 cells per well and cultured in a medium before transfection. According to the manufacturer's instructions, cells were then transfected using Lipofectamine 2000 . The miRNA and Lipofectamine 2000 reagent were individually diluted to 20% in serum-free DMEM and incubated for 10 mins. Then cultured macrophages were transfected with miR-223-3P mimics, miR-223-3P mimics negative control (NC). The primary culture medium was detached three hours after transfection, and the cells were cultured for an additional 24 hours in the transfection complex. Twenty-four hours after transfection, the culture medium was removed, and the cells were re-cultured for an extra 48 hours. Consecutively diluted ox-LDL at 50, 100, and 200 mg/L were used to treat the cells for 12 h, 24 h, and 48 h. qPCR was conducted on a Step One Plus system and Chromo4 using specific primers . Values of miR-223-3P were normalized (cultured macrophages) and presented as 2−ΔΔCT equation through boxplot with smallest and largest whiskers.The FISH assay was applied to detect the location and expression of miR-223-3P in aortic tissues. The aortic tissues were fixed with 4% paraformaldehyde at room temperature and embedded in paraffin (4-μm thickness). Subsequently, the tissues were deparaffinized by mineral oil. They rehydrated in graded dilutions of ethanol , digested with 16 μg of proteinase K  at 37° C for 15 min, rinsed in 0.2% glycine in PBS (Sigma-Aldrich) for 10 min, fixed with 4% paraformaldehyde phosphate-buffered saline (PBS) at room temperature for 10 min. Then, paraffin slides were hybridized with fluorescent (Cy3-labeled) oligonucleotide miR-223-3P probes (5′-UGUCAGUUUGUCAA AUACCCC-3′) at 37° C overnight. After probe hybridized, tissues were incubated in extraction buffer , at 37° C for 20 min. Tissues were stained with CD68+ with purified ab81289  at 1:200. The conventional heat-mediated antigen retrieval was conducted with Tris-EDTA buffer . Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody at 1/5000 dilution. 4,6-diamidino-2-phenylindole (DAPI) staining was used for cell nuclear counterstain with laser scanning confocal microscopy . A light microscope carried out the qualitative analysis . Images of the sections were captured by fluorescence microscope  and analyzed using Image-Pro Plus 6.0 software .The protein extracts from aortic tissues and cells were denatured. The protein samples were separated by SDS-PAGE (10% gel) and transferred to PVDF membranes . After washing with Tris-buffered saline with Tween- 20 (TBST), the protein samples were incubated with primary and secondary antibodies (Abcam) for 1h. Band intensities were detected by an Odyssey infrared imaging system .Total RNA was isolated from macrophages and aortas using a Trizol reagent  and purified by the RNA Easy kit . qPCR fluorophore SYBR-Green was purchased from Beijing Solarbio Science and Technology Co., Ltd. Reverse transcription-quantitative PCR (qRT-PCR) of the target mRNA was carried out using primers, The mice primers: miR-223-3p’ F 5'-GTG CAG GG TCC GAG GT-3';R 5'-CGG GCT GTC AGT TTG TCA-3'; U6’ F 5'-CTC GCT TCG GCA GCA CA-3'; R 5'-AAC GCT TCA CGA ATT TGC GT-3'. human primers : miR-223-3p F 5’-ACA CTC CAG CTG GGT GTC AGT TTG TCA AAT-3’; R 5’-CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG TGG GGT AT-3’; U6 F 5’- CGC TTC GGC AGC ACA TAT AC -3’; R 5’- AAA TAT GGA ACG CTT CAC GA -3’. Changes in gene expression levels of >2.5-fold were considered significant.All data were expressed as mean ± standard deviation (SD). Statistically significant differences among groups were evaluated by one-way analysis of variance (ANOVA) or Student's t-test using SPSS 20.0 software. Statistical significance was signified when p values <0.05.The data used to support the findings of this study are included within the article.The animal use protocol for this study has been reviewed and approved by the Laboratory Animal Ethical Committee, Hebei Medical University Second Affiliated Hospital.The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.


Weight gain after the initiation of antiretroviral therapy (ART) is becoming a major clinical issue in treatment-naïve people living with human immunodeficiency virus (PLWH). However, limited data exist for the Asian populations. We aimed to investigate changes in weight after the initiation of ART therapy in treatment-naïve Asian patients.We evaluated adult, treatment-naïve Asian PLWH who started integrase strand transfer inhibitor (INSTI)-, protease inhibitor (PI)-, or nonnucleoside reverse transcriptase inhibitor (NNRTI)-based ART at AIDS Clinical Center, Tokyo, between January 2005 and February 2019. They were followed up until October 2019. Multivariate linear mixed-effects models were used to generate marginal predictions of weight over time. Predicted weight by ART class , each key drug , and each key drug with or without the use of tenofovir alafenamide (TAF)/emtricitabine (FTC) was reported at 3-month intervals until censoring or 5 years.Among the 1,579 study patients, 610 (38.6%), 929 (58.8%), and 40 (2.5%) started INSTI-, PI-, and NNRTI-based ART. After 5 years, PLWH who initiated DTG- (5.3 kg), DRV- (4.0 kg), and EVG-based treatment (4.6 kg) gained more weight than those who initiated RAL-based treatment (1.8 kg). PLWH who initiated DTG plus TAF/FTC (6.7 kg) gained the largest weight.In the Asian PLWH population, ART-associated weight gain continues to increase for 5 years after treatment initiation. DTG plus TAF/FTC was associated with the largest weight gain.All Authors: No reported disclosures


Biosci Rep (2019) 39(3), https://doi.org/10.1042/BSR20190249) would like to correct The authors of the original article “Down-regulation of miR-543 expression increases the sensitivity of colorectal cancer cells to 5-Fluorouracil through the PTEN/PI3K/AKT pathway” (


Journal of Materials Science: Materials in Medicine (2021) 32:20Correction to: 10.1007/s10856-021-06491-0 published online 27 February 2021The authors agree to withdraw the Fig. Supporting Information-2


On PDF page 2, under “Severe COVID-19 is associated with higher ADCD and lower ADCP compared to mild COVID-19,” the following sentences are incorrect: “S1-specific antibodies from hospitalized individuals induced significantly higher NK cell degranulation and intracellular cytokine production (a surrogate of ADCC) than did S1-specific antibodies from nonhospitalized individuals . In contrast, RBD-specific antibodies from nonhospitalized individuals induced significantly higher ADCC surrogates than did RBD antibodies from hospitalized individuals .” Instead, these sentences should read as follows: “S1-specific antibodies from hospitalized individuals induced significantly lower NK cell degranulation and intracellular cytokine production (a surrogate of ADCC) than did S1-specific antibodies from nonhospitalized individuals . In contrast, RBD-specific antibodies from nonhospitalized individuals induced significantly lower ADCC surrogates than did RBD antibodies from hospitalized individuals .” This change does not change the conclusions of the paper.Volume 12, no. 2, e00281-21, 2021,


Drosophila eye, the Hippo pathway has been implicated in blue- and green-sensitive PR subtype fate specification. Specifically, Hippo pathway activation promotes green-sensitive PR fate at the expense of blue-sensitive PRs. Here, using a sensitized triple heterozygote-based genetic screening approach, we report the identification of the single Drosophila zonula occludens-1 (ZO-1) protein Polychaetoid (Pyd) as a new regulator of the Hippo pathway during the blue- and green-sensitive PR subtype binary fate choice. We demonstrate that Pyd acts upstream of the core components and the upstream regulator Pez in the Hippo pathway. Furthermore, We found that Pyd represses the activity of Su(dx), a E3 ligase that negatively regulates Pez and can physically interact with Pyd, during PR subtype fate specification. Together, our results identify a new mechanism underlying the Hippo signaling pathway in post-mitotic neuronal fate specification.The generation of a diversity of photoreceptor (PR) subtypes with different spectral sensitivities is essential for color vision in animals. In the  Drosophila eye, the Hippo pathway promotes green- and represses blue-sensitive photoreceptor (PR) subtype fate specification. Taking advantage of this binary PR fate choice, we screened Drosophila chromosomal deficiency lines to seek new regulators of the Hippo signaling pathway. We identified the Drosophila membrane-associated ZO-1 protein Pyd as an upstream regulator of the Hippo pathway to specify PR subtypes. Our results have demonstrated that Pyd represses Su(dx)’s activity in the Hippo pathway to specify PR subtypes. Our results demonstrate a new mechanism underlying the Hippo signaling pathway in post-mitotic neuronal fate specification.The Hippo signaling pathway was originally discovered for its critical role in tissue growth and organ size control. Its evolutionarily conserved roles in various biological processes, including cell differentiation, stem cell regeneration and homeostasis, innate immune biology, as well as tumorigenesis, have been subsequently found in other species. During the development of the  Drosophila eye as a model to understand the role of the Hippo pathway in post-mitotic neuronal terminal differentiation.Generating neuronal diversity during the development of a sensory organ is a prerequisite for the organ to perceive and discriminate various external stimuli. For example, the perception of color relies on comparing the outputs of multiple light-sensing photoreceptor (PR) subtypes with different spectral sensitivities –3. DurinDrosophila eye is a powerful model to understand the principles of neuronal development NP4419, which insertion is 8bp upstream of the pyd gene, was able to drive the expression of the reporter gene UAS-nuclear GFP (UAS-nGFP) in PR cells. The expression was found in all PR cells and also in non-neuronal cells . We further tested its expression in melt GOF (otd>melt) or wts GOF (otd>wts) flies and found that its expression was not affected by melt or wts misexpression . All these data suggest that pyd is transcriptionally expressed in both pR8 and yR8 subtypes, and its expression is not regulated by wts or melt.As wts, hpo or sav in wild type retinas was sufficient to induce yR8 fate and repress pR8 fate in most or all R8 cells NP4419, p[GawB]NP7518, p[GawB]NP0961 and p[GawB]NP4414 were from the Kyoto Stock Center. lGMR-GAL4, pWIZ-wΔ13 and UAS-Dicer2 lines were recombined onto a single chromosome for use in RNAi-mediated knockdown experiments [UAS-pez and pydex180, both on the 3rd chromosome, were recombined and used for misexpression of pez in pyd LOF flies. All flies and crosses were raised on standard cornmeal-molasses media at 25°C with 12 hr:12hr light-dark cycles except that GMR-GAL4>UAS-su(dx)  ,17, melt [meltΔ1 , sdΔB [8Δ1 [sdΔB , lGMR-GAGMR-GAL4 , UAS-pydANi-#450 , sensR8-sR8-GAL4 , ykiB5 [4 [ykiB5 , UAS-hpo[UAS-hpo , GMR-sav[GMR-sav , pWIZ-wΔAi line) . UAS-pydGMR-GAL4 , UAS-LucydRANi KK5581, UAS(dx) for  were in Fly head cryosections, dissection for whole mount retinas, and antibody staining were performed as previously described with modifications ,86. AdulPolyclonal antiserum against Rh6 was generated against a KLH-conjugated peptide from the Rh6 deduced amino acid sequence (CLACGKDDLTSDSRTQAT corresponding to amino acids 344–361). Peptide synthesis, KLH-conjugation, rabbit immunizations and bleeds were performed by GenScript .S1 Text(DOC)Click here for additional data file.S1 Fig(A-I) Cryosections of adult eyes immunostained for Rh5 (red) and Rh6 (green) in control, heterozygous, double heterozygous and triple heterozygous flies with Hippo pathway mutations. Compared to control and heterozygous mutations (A-E), double heterozygous Hippo pathway mutations slightly increased Rh5-expressing R8s and reduced Rh6-expressing R8s (F-H). In flies with triple heterozygous Hippo pathway mutations, the number of the Rh5-expressing R8s were dramatically increased at the expense of the Rh6-expressing R8s (I). See (TIF)Click here for additional data file.S2 Fig(A) Diagram of deficiencies on the third chromosome  that are used in the R8 subtype determinant screening. (B) Diagram showing the fly crossing strategy in the screening.(TIF)Click here for additional data file.S3 Figpyd mutant or pyd knock-down eyes. (A) Control, (B) pydB12/pydB12, (C) pydB12/pydJ4, (D) lGMR>pydRNAi-HMS00263 and (E) lGMR>pydRNAi-#450. (F) Adult eye tangential cryosection immunostained for Rh5 (red) and Rh6 (green) in pyd LOF (pydex180/pydB12) flies. (G) Quantification of R8 subtypes in pyd mutants and pyd knock-down eyes. Error bars represent standard deviation. NS: not significant. ** p < 0.001. lGMR-GAL4: n = 10 retinas, n = 1822 R8s; pydB12/pydB12: n = 4 retinas, n = 820 R8s; pydB12/pydJ4: n = 5 retinas, n = 909 R8s; lGMR>pydRNAi-HMS00263: n = 5 retinas, n = 840 R8s; lGMR>pydRNAi-#450: n = 5 retinas, n = 806 R8s. (H-I) Retina whole mount staining for control (I) and pyd LOF (pydex180/pydB12) eyes. (J) Quantification of R8 subtypes in control and pyd LOF eyes (the retina whole mount staining). ** p < 0.001. Control: n = 3 retinas, n = 725 R8s; pydex180/pydB12: n = 3 retinas, n = 524 R8s.(A-E) Adult eye cryosections immunostained for Rh5 (red) and Rh6 (green) in control, (TIF)Click here for additional data file.S4 Fig(Related to  (A-B) Adult eye cryosections immunostained for Rh5 (red) and Rh6 (green) in knock-down of sd  and knock-down of sd in pyd LOF  flies.(TIF)Click here for additional data file.S5 Fig(Related to  (A-C) Adult eye cryosections immunostained for Rh5 (red) and Rh6 (green) in knock-down of melt , pyd LOF , and knock-down of melt in pyd LOF  flies. (D-F) Adult eye tangential cryosections immunostained for Rh5 (red) and Rh6 (green) in melt mutant , pyd knock-down , and knock-down of pyd in melt mutant  flies.(TIF)Click here for additional data file.S6 Figsu(dx) misexpression eyes. (A) Control (lGMR-GAL4), (B) su(dx) misexpression (lGMR-GAL4>su(dx)). (C) Quantification of R8 subtypes in su(dx) misexpression eyes. Error bars represent standard deviation. ** p < 0.001. lGMR-GAL4: n = 10 retinas, n = 1822 R8s; lGMR>su(dx): n = 6 retinas, n = 816 R8s.(A-B) Adult eye cryosections immunostained for Rh5 (red) and Rh6 (green) in control (A) and (TIF)Click here for additional data file.S7 Fig(Related to  (A-C) Adult eye cryosections immunostained for Rh5 (red) and Rh6 (green) in kib knock-down , pyd overexpression  and kib knock-down in pyd overexpression  flies. UAS-Luciferase (UAS-Luc) was used as a control to balance the number of UAS sites. (D) Quantification of the Rh5- and Rh6-expressing R8s in the eyes with the indicated genotypes. NS: not significant. Error bars represent standard deviation. GMR>kibRNAi-HMC03256+Luc: n = 4 retinas, n = 901 R8s; GMR>pyd+Luc: n = 6 retinas, n = 912 R8s. GMR>pyd+kibRNAi-HMC03256+Luc: n = 4 retinas, n = 921 R8s.(TIF)Click here for additional data file.


Mutations that disrupt S-palmitoylation are associated with severe immunologic and inflammatory diseases such as Crohn’s disease, whereas mutations associated with Blau syndrome demonstrate increased S-palmitoylation and membrane localization (Nucleotide-binding and oligomerization domain containing protein 2 (NOD2) is a cytosolic pattern-recognition receptor that detects intracellular peptidoglycan (muramyl dipeptide) from bacteria. Membrane association of NOD2 is essential for its ability to activate nuclear factor κB and mitogen-activated protein kinase signaling pathways via the kinase, receptor-interacting serine/threonine-protein kinase 2. The post-translational addition of palmitate to NOD2 results in an acylated protein with increased affinity for membrane bilayers . PalmitoD: 5IRL) . The modEQUIPMENT: The following equipment were used: A Zeiss Axiovert 200M microscope with a Hamamatsu ImageEM x2 camera, Yokogawa spinning disc, and a 63× 1.4 NA oil objective.REAGENTS: Alkynyl-palmitate (Click Chemistry Tools), azide-PEG3-FLAG (Jena Bioscience), hydroxylamine, and other copper-catalyzed azide–alkyne cycloaddition reagents  were obtained from MilliporeSigma.SOFTWARE: The following software were used: MetaMorph, ImageJ, Python, PyMOL, and AutoDock (AutoDock .


Glioblastoma multiform (GBM) is the most common malignant brain tumor. Recent immunotherapy has demonstrated potential to treat GBM. However, the immune suppressive tumor environment in the brain represents a significant barrier for the treatment of GBM. Overexpression of programmed death ligand-1 (PD-L1) in GBM tumor cells and macrophages plays a key role in GBM vitality, proliferation, and migration, while also suppressing the immune system. We developed a CRISPR/Cas9 gene-editing system to delete whole cell PD-L1. Human PD-L1 targeted sgRNA were cloned into CRISPR/Cas9 plasmids with or without an HDR templet. CRISPR/Cas9 were treated to human GBM U87 cells for 15, 30, 60, 120 and 240 minutes. The intracellular concentration of CRISPR/Cas9 exhibited a time-dependent increases. A GFP tagged CRISPR/Cas9 plasmid was developed to test the transfection efficacy. Higher levels of GFP+ U87 cells were observed at day 3. CRISPR/Cas9 showed a greater PD-L1 knockout at day 3. The PD-L1 reduction limited the proliferation of U87 cells. A scratch assay showed that PD-L1 deletion inhibited the migration of U87 cells. An in vitro GBM model was developed by co-cultivation of U87 cells and macrophages. CRISPR/Cas9 treated co-cultures changed the ratios of U87 cells and macrophages and polarized tumor associated macrophages (TAM) from M2 toward M1. CRISPR/Cas9 gene-editing effectively deleted PD-L1 in U87 cells. Successful deletion of PD-L1 prevented U87 cells growth and migration, and altered the TAMs plasticity and the tumor environment.


Heterologous primary immunization against SARS-CoV-2 is part of applied recommendations. However, little is known about duration of immune responses after heterologous vaccine regimens. To evaluate duration of immune responses after primary vaccination with homologous adeno-vectored ChAdOx1 nCoV-19 vaccine (ChAd) or heterologous ChAd/BNT162b2 mRNA vaccine (BNT), anti-spike-IgG and SARS-CoV-2 VOC-neutralizing antibody responses were measured in 354 healthcare workers (HCW) at 2 weeks, 3 months, 5 months and 6 months after the second vaccine dose. T-cell responses were investigated using a whole blood interferon gamma (IFN-γ) release assay 2 weeks and 3 months post second vaccine dose. Two hundred and ten HCW immunized with homologous BNT were enrolled for comparison of antibody responses. In study participants naïve to SARS-CoV-2 prior to vaccination, heterologous ChAd/BNT resulted in 6-fold higher peak anti-spike IgG antibody titers compared to homologous ChAd vaccination. The half-life of antibody titers was 3.1 months (95% CI 2.8–3.6) following homologous ChAd vaccination and 1.9 months (95% CI 1.7–2.1) after heterologous vaccination, reducing the GMT difference between the groups to 3-fold 6 months post vaccination. Peak T-cell responses were stronger in ChAd/BNT vaccinees, but no significant difference was observed 3 months post vaccination. SARS-CoV-2 infection prior to vaccination resulted in substantially higher peak GMTs and IFN-γ levels and enhanced SARS-CoV-2 specific antibody and T cell responses over time. Heterologous primary SARS-CoV-2 immunization with ChAd and BNT elicits a stronger initial immune response compared to homologous vaccination with ChAd. However, although the differences in humoral responses remain over 6 months, the difference in SARS-CoV-2 specific T cell responses are no longer significant three months after vaccination. In the global struggle to achieve and maintain efficient COVID-19 vaccine programs, heterologous vaccine regimens attract great interest ,4,5,6,7.n = 210) were enrolled for comparison of VOC-neutralizing responses.In this study, T-cell responses against SARS-CoV-2 wild type and antibody responses against SARS-CoV-2 wild type and four variants of concern , were analyzed over six months after two doses adeno-vectored ChAdOx1 nCoV-19 vaccine (ChAd) or one dose ChAd followed by one dose BNT162b2 mRNA vaccine (BNT) in 354 healthcare workers (HCW) with and without SARS-CoV-2 infection prior to vaccination. Healthcare workers vaccinated with two doses BNT . Samples were collected at 2 weeks (n = 190), 3 months (n = 337), 5 months (n = 102) and 6 months (n = 92) after second vaccine dose (ChAd or BNT), n = 6) were censored upon detection of breakthrough infection. Of the 354 study participants, 113 (median age 50 (range 22–68) years, 88% women, 40 SARS-CoV-2 recovered) received homologous ChAd vaccination with a median dose interval of 83 (range 70–98) days. Two hundred and forty-one study participants (median age 48 (range 20–69), 88% women, 67 SARS-CoV-2 recovered) received heterologous vaccination with ChAd followed by BNT, with a median dose interval of 91 (range 70–98) days, Participants that had received a first dose of ChAd were included in this sub study  against wild type and variants B.1.1.7 (Alpha), B.1.351 (Beta), P1 (Gamma) and 1.617.2 (Delta) were measured using the V-PLEX SARS-CoV-2 Panel 13 for IgG and ACE2 , USA. Cat no. K15463U and K15466U) using a similar approach as previously described . ReferenA subset of 17 samples collected 18–21 days (median 20) following homologous ChAd and 17 samples collected 18–20 days (median 19) following heterologous ChAd/BNT  were analysed using a live-virus microneutralization assay based on cytopathic effects (CPE) with SARS-CoV-2 wild type and Delta strains as previously described . Serum wn = 190) and 3 months (n = 337) post second vaccine dose. Whole blood was stimulated with an in-house generated peptide pool containing in total 16 peptides based on the spike, nucleocapsid, membrane and open reading frame 3 and 7 proteins [T-cell responses were assessed 2 weeks . Interactions were applied between sex and age, dose interval and infection status with a three-way interaction for vaccine combination, infection status and time since vaccination. Non-linearity was evaluated using ANOVA in nested models and modelled using natural splines where the number of knots was decided by optimal AIC-value.p-values from Fisher’s test.Risk of side effects were evaluated using a logistic regression model by vaccine type and infection status, interaction adjusted for age and sex with 2 function. Estimate comparisons are provided as multiplication factors and presented with 95% confidence intervals (CI). The effect on antibody titers by side effects was measured using a linear regression model for the first measured antibody titer similarly adjusted with the addition of time since vaccination. Analyses were performed in statistical program R version 4.1.2 with the nlme-package version 3.1.152.Antibody response was transformed using the logTo minimize interference of variation of time between second dose and sampling when comparing VOC-neutralizing antibody titers three months post vaccination, a sub-cohort of samples collected 3 months (75–105 days) post second vaccine dose was established.Estimates of correlation between assays were obtained with Spearman’s rank correlation using GraphPad PRISM version 9.2.0. For comparison of VOC neutralizing geometric mean titers (GMTs) Wilcoxson rank-sum test was used .The study complies with the declaration of Helsinki and was approved by the Swedish Ethical Review Authority (dnr 2020-01653). Informed written consent was obtained from all study participants. higher peak anti-spike (WT) IgG GMT compared to homologous ChAd vaccination in individuals naïve to SARS-CoV-2 prior to vaccination, A. As exp. ConsequA.SARS-CoV-2 recovered participants reached substantially higher peak anti-spike IgG GMTs following both vaccine regimens . Half-liThere were no significant dose interval dependent effects on peak serological responses. However, there was an association between shorter dose interval and lower antibody titers at 3 months post vaccination among participants who were SARS-CoV-2 naïve prior to vaccination, diminishing at approximately 80 days dose interval.p = 0.025), but not against the VOCs, as compared to heterologous ChAd/BNT.Surrogate virus neutralization assay was performed in 205 participants sampled three months post second dose. In participants naïve to SARS-CoV-2 prior to vaccination, GMTs of neutralizing antibodies against all four VOCs’ spike were significantly higher following heterologous ChAd/BNT vaccination as compared to homologous ChAd vaccination . Three mGMT of neutralizing antibodies among participants with SARS-CoV-2 infection prior to vaccination exceeded those in naïve participants, regardless of vaccine regimen .A live-virus microneutralization assay was performed on 34 post vaccination samples. There was a strong correlation between live virus neutralization titer (NT), titers of anti-WT-spike IgG and VOC specific surrogate neutralization antibody levels .p < 0.001). At 3 months post second vaccine dose, a comparatively larger decline of IFN-γ responses was noted among recipients of heterologous ChAd/BNT, resulting in a no longer significant difference between the regimens (p = 0.18).SARS-CoV-2 specific T-cell memory responses were assessed based on IFN-γ release after stimulation of fresh whole blood with a SARS-CoV-2-specific peptide pool . When adp = 0.028) and ChAd/ChAd (p = 0.058)). Differences were retained at three months post vaccine (2.5-fold higher after both ChAd/BNT (p < 0.001) and ChAd/ChAd (p = 0.003), although without a significant difference between regimens (p = 0.28).Importantly, participants that had been SARS-CoV-2 infected prior to vaccination showed higher peak IFN-γ responses 2 weeks post vaccination compared to naïve participants irrespective of vaccine regimen , n = 26) reported fever or pronounced illness (bed bound or sick leave) at a higher frequency (p < 0.001). There was a non-significant trend towards stronger humoral responses three months post vaccine among participants with pronounced illness after the second vaccine dose.There were no self-reported severe adverse events following either of the vaccine regimens, but mild systemic and local events were more frequent after heterologous ChAd/BNT vaccination (62%) than after homologous ChAd (29%)  which remained detectable 3 months after vaccination. There were, however, large inter-individual variations in neutralizing antibody titers against the four VOCs in SARS-CoV-2 naïve participants, particularly after homologous ChAd. In contrast, this variation was much less pronounced among SARS-CoV-2 recovered participants, supporting a more robust humoral response in vaccinated SARS-CoV-2 recovered individuals .As expected ,33, SARSThis study is limited by the observational study design and the female dominance of the study cohort. Furthermore, we expect the longer dose interval in the ChAd/BNT group to enhance, but not fully explain, the stronger humoral response seen in this group.Taken together, our findings provide further support for the use of heterologous primary SARS-CoV-2 immunization with ChAd and BNT. The regimen is tolerable and results in durable immune responses. Despite an initial rapid decline in titers, heterologous vaccination induced antibody titers and T-cell responses that remain high over time compared to those evoked by homologous ChAd vaccination. Robust and durable immune responses to heterologous vector/mRNA immunization is of great importance to mitigate consequences of variations in vaccine supply, to avoid concerns related to vector immunity and to meet updated mRNA-vaccines.


However, newly developed probes  image the fine molecular changes involved in the pathogenesis of TC and enable target-specific diagnosis and treatment of TC. Furthermore, novel molecular probes have high specificity and sensitivity, imparting a high level of objectivity to the research areas of TC.Molecular imaging utilizes radionuclides or artificially modified molecules to image particular targets or pathways which are important in the pathogenesis of a certain disease. Transporter-based probes like radioiodine and [18F]FDG has been frequently used in disease evaluation for several decades now. Recently, advances in molecular imaging have led to the development of novel tracers based on aptamer, peptide, antibody, nanobody, antibody fragment, and nanoparticle platforms. The emerging targets—including HER2, CD54, SHP2, CD33, and more—are promising targets for clinical translation soon. The significance of these tracers may be realized by outlining the way they support the management of TC. The provided examples focus on where preclinical investigations can be translated. Furthermore, advances in the molecular imaging of TC may inspire the development of novel therapeutic or theranostic tracers. In this review, we summarize TC-targeting probes which include transporter-based and immuno-based imaging moieties. We summarize the most recent evidence in this field and outline how these emerging strategies may potentially optimize clinical practice.An essential aspect of thyroid cancer (TC) management is personalized and precision medicine. Functional imaging of TC with radioiodine and [ Thyroid cancer (TC) is one of the most common cancer types and its occurrence has been rapidly increasing over the last several years . TC repr131I (a beta-emitting radioisotope of iodine) therapy TlCl, [99mTc]Tc-sestamibi ([99mTc]Tc-MIBI), [99mTc]Tc-tetrofosmin, [99mTc]Tc-depreotide, [111In]In-diethylenetriaminepentaacetic acid-octreotide ([111In]In-DTPA-octreotide), and [18F]fluoro-D-glucose ([18F]FDG) have been synthesized, tested, and validated as beneficial for diagnosing TC [18F]FDG has been widely applied in the management of TC NaI can track thyroid and TC cells with γ radiation on SPECT, and damage those cells by emitting β- radiation [131I]NaI allows ablation of thyroid remnant, adjuvant therapy of TC, and therapy of TC, which vastly improves the prognosis of patients with TC NaI-PET/CT has superior spatial resolution and quantification ability over [131I]NaI-SPECT hexafluorophosphate ([18F]HFP) have been discovered for imaging DTCs TFB is an analog to radioiodine, having similar NIS affinities, same charge, and similar ionic radius to iodide. Therefore, [18F]TFB can be transported by NIS [18F]TFB can be readily synthesized at medical cyclotrons, and it provides a satisfactory half-life, dosing, biodistribution, and PET imaging quality [18F]TFB-PET can exclusively reveal NIS expression in tumor cells, therefore reclassifying TENIS metastases into partial or complete dedifferentiation, and helping metastasis localization and prognosis evaluation [18F]FS and [18F]HFP are two other newly discovered NIS-targeting tracers having favorable targeting efficiency, image contrasts, and biodistribution features [18F]HFP and [18F]FS are superior to [18F]TFB. The clinical evaluation of the [18F]HFP and [18F]FS are still needed.FDG uptake [18F]FDG-PET/CT has shown great sensitivity in patients who otherwise do not benefit from 131I treatment. This is because metastases without radioiodine uptake tend to have high glycolytic rates, causing enhanced [18F]FDG uptake [18F]FDG can help detect TC recurrence or metastases and predict radioiodine uptake [18F]FDG maximum standard unit value (SUVmax) higher than 4.0 would predict poor radioiodine uptake FDG PET/CT imaging is useful in initial diagnosis, subsequent disease grading, treatment, and follow-up after treatment. Even though numerous glucose analog tracers other than [18F]FDG were created [18F]FDG are lacking.[ imaging . AggressG uptake . [18F]FDG uptake . Furthere uptake ,55. Moree uptake . Besides created , their h18F]FDG are a subject of interest. Occasionally, [18F]FDG may yield inexplicable images. The false-positive [18F]FDG uptake happens in Hashimoto’s disease and Graves’ disease. In addition, it is hard to find brain metastasis on [18F]FDG PET/CT images because of the intense background signals [18F]FDG. Amino acids probes include [18F]fluoro-α-methyl tyrosine ([18F]FAMT), [18F]fluoro-dihydroxyphenylalanine ([18F]FDOPA), L-[methyl-11C]-methionine ([11C]MET), [18F]fluoroethyl-tyrosine ([18F]FET), [18F]fluoroglutamine ([18F]FGln), and the newly discovered [18F]NKO-035. Of these, [18F]FDOPA, [11C]MET, and [18F]FGln have been investigated in TCs FDOPA is a large neutral amino acid that resembles natural L-dopa, which can be transported by L-type amino acid transporter 1  and L-type amino acid transporter 2  [18F]FDOPA is a satisfactory probe for detecting MTC metastasis, persistence, and residual disease [18F]FDOPA imaging is negative or unavailable, [18F]FDG should be considered, especially for aggressive MTCs displaying signs of dedifferentiation or rising carcinoma embryonic antigen (CEA) concentration in serum [[ SLC7A8) . [18F]FD disease . Howeverin serum ,65.11C]MET, transported mainly by SLC7A5, is often used to visualize parathyroid adenoma and enable focused parathyroidectomy [11C]MET in TC is limited. Only one case with hyperparathyroidism has been reported, which showed intense focal [11C]MET uptake in a cold nodule with highly increased sestamibi uptake. The nodule was finally diagnosed as FTC, indicating the incremental value of [11C]MET in imaging DTCs [11C]MET is currently being studied as a surrogate for [18F]FDG in other tumor types, such as brain tumors [11C]MET over [18F]FDG. Although complementary uptake of 11C-MET and [18F]FDG has been reported in recurrent or metastatic DTCs [11C]MET in clinic settings. The downside of [11C]MET is the short half-life of 11C (20.4 min), which limits its broad application MEn tumors ,70 and ln tumors . To datetic DTCs , furtherlication .18F]FGln, an analog of natural glutamine regulated by several glutamine (Gln) transporters , has been tested and subsequently considered as a promising probe for assessing glutamine metabolism in tumors [18F]FGln can further complement the diagnostic capacity of [18F]FDG by detecting Gln metabolic changes in PTCs [18F]FGln imaging, excellent contrast images can be made only 10 min after injection, while late-phase imaging (60 min) would cause a high background to some extent [[n tumors . Its usen tumors . [18F]FG in PTCs . In [18Fe extent   has been tested to date. [18F]FLT, which can be taken up by equilibrative nucleoside transporter 1 (ENT1), is a marker of cell proliferation [18F]FLT and [18F]FDG on PET/CT. While 69% of the metastatic lesions were identified by focal increases in [18F]-FLT uptake, a lower result than the 92% identified by [18F]FDG PET/CT. It is also demonstrated that [18F]FDG has the advantage in terms of specificity and accuracy over [18F]FLT in finding local lymph node malignancy and distant metastases [18F]FLT PET/CT has not progressed very far in diagnosing TCs.Radiolabeled or fluorescent nucleobase analogs are currently used to diagnose solid tumors, including cancers of the bladder, breast, lung, ovary, and pancreas. Regarding diagnosis of TC specifically, only Ga-dodecane tetraacetic acid labeled RGD2 , [68Ga]Ga-prostate specific membrane antigen ligand ([68Ga]Ga-PSMA) with conjugates of N,N’-bis[2 -hydroxy-5-(carboxyethyl)benzyl]ethylene diamine-N,N′-diacetic acid (HBED-CC) or DOTA, [68Ga]Ga-DOTA-DGlu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH2 ([68Ga]Ga-DOTA-MGS5), [111In]In-DTPA-octreotide, and other somatostatin analogs have been developed for imaging TCs, particularly MTCs and RR-DTCs Ga-DOTA--octreotide ([68Ga]Ga-DOTANOC), [68Ga]Ga-DOTA(0)-Phe(1)-Tyr(3)-octreotide ([68Ga]Ga-DOTATOC), and [68Ga]Ga-DOTA-(Tyr3)-octreotate ([68Ga]Ga-DOTATATE), are valuable diagnostic tools showing excellent performance in the majority of patients with NETs Ga-DOTA-RGD2 has been successfully applied for PET imaging of RR-DTCs in clinical settings [18F]FDG of 82.3%, 50%, and 75%, respectively. For RR-DTCs, the advantage provided by [68Ga]Ga-DOTA-RGD2 is the ability to detect lesions not detected by [18F]FDG [68Ga]Ga-DOTA-RGD2 is better accompanied by [177Lu]Lu-DOTA-RGD2, a potential treatment option for RR-DTCs [68Ga]Ga-DOTA-RGD2 and [177Lu]Lu-DOTA-RGD2 are a useful theranostic pair for RR-DTCs, the potential to improve the theranostic landscape of RR-DTCs by sequentially using these agents is high. Nuclear medicine approaches have revolutionized the theranostic arsenal for DTCs, and we are confident that there is room to optimize the management of RR-DTCs with these novel agents.The integrin αvβ3 expression on epithelial cells and mature endothelial cells is relatively low, however, it is commonly and highly expressed in solid tumors. RGD and RGDrin αvβ3 . Recentlsettings , showing[18F]FDG  , who underwent consecutive [68Ga]Ga-DOTA-MGS5 and [18F]FDOPA-PET/CT. [68Ga]Ga-DOTA-MGS5 found three obvious liver lesions with higher uptake than [18F]FDOPA-PET/CT (SUVmax = 6.4–8.3 vs. 3.7) and showed a good lesion-to-background contrast in the liver, which might yield complementary information to [18F]FDOPA-PET in patients with recurrent MTC [68Ga]Ga-DOTA-MGS5 seems higher than that of [18F]FDOPA in our opinion.CCK2R is highly expressed in 90% of MTC, 50% of small cell lung cancers, 60% of astrocytomas, insulinomas, stromal ovarian cancers, gastrointestinal stromal tumors, and more than 20% of gastroenteropancreatic tumors ,94,95. Arent MTC   or Gal-3 mAb with F(ab’)2 conjunction 2) has shown good binding to TC in vivo, allowing it to be potentially used for the detection of recurrence and metastases [89Zr]Zr-DFO-Gal3-F(ab’)2, a protein formed of two F(ab’) fragments, results in faster blood clearance and lower liver uptake than traditional mAb-based tracers [89Zr]Zr-DFO-Gal3-F(ab’)2 in kidneys is due to the urinary excretion [89Zr]Zr-DFO-Gal3-F(ab’)2 might be an excellent candidate for translation into the preoperative evaluation and postoperative follow-up.Gal-3 is a protein that is undetectable in normal and benign thyroid tissues but highly expressed in DTC cytosol, cell membranes, and intercellular substance . The exptastases ,125  probes have filled the vacancy of single target IgG probes in theranostics by providing higher antigen-binding capacity in tumor tissues than the monomeric counterparts . Additio111In or 131I labeled haptens-peptides Ga-IMP288, with a time lag (one or two days) between the two injections Zr-DFO-αGal3-Fab-PAS200, an imunoPET probe fused with 200 Pro, Ala, and Ser residues (PAS200) and conjugated with [89Zr]Zr-deferoxamine ([89Zr]Zr-DFO), is a recently reported Fab-based probe derived from the rat anti-Gal3 mAb. Similar to the full-size [89Zr]Zr-labeled Gal-3 mAb  with excellent contrast FDG PET/CT results in 21% of the high-risk DTCs [For the diagnosis of TC, the superiority of MI over conventional anatomical imaging is clear, with advantages of favorable spatial and temporal resolution and functional imaging . The emeisk DTCs . We supp177Lu, 225Ac 188Re, 67Cu, 47Sc, 166Ho, 90Y, 161Tb, 149Tb, 212Pb, and 213Bi emitting α-particles, β-particles, and Auger electrons can be feasibly chelated with DOTA, NOTA, etc., which is similar to the diagnostic isotopes mentioned in this review, 64Cu or 68Ga chelated with NOTA or DOTA [64Cu) can be used to map the target and assess the therapeutic potential of the same probe labelled with the same chelating agent and a therapeutic radionuclide  [149Tb is another theranostic radioisotope that has not been investigated in thyroid cancer research to date. 149Tb can simultaneously emit positrons (β+ particles), α-particles, and γ-radiation, allowing PET and α particle-based therapy to go on at the same time [For TC therapy, despite the rapid progress in molecular imaging, we firmly believe that the innovation of therapeutic agents should accompany the development of diagnostic agents. For example, radionuclides such as nd 67Cu) . 149Tb iIn the last two decades, there has been a general increase in the prevalence of TC. The phenomenon is partially due to environmental factors , but is also due to overdiagnosis by increased screening with more sensitive methods , especially in developed countries , leading to unnecessary treatment. As mentioned previously, most incident TCs are low-risk DTCs that tend to retain their stability over the years until death due to aging. Therefore, it is unnecessary to treat indolent TCs because of the low cost-effectiveness and the practically unchanged mortality. Something to note is that the ever-developing MI techniques might more sensitively detect TCs, potentially causing overdiagnosis for indolent TC and overtreatment via invasive methods  or noninvasive therapeutics . To avoid these pitfalls, future work could focus on discovering prognosis- or progress-related targets or probes, thereby classifying TCs into indolent and active disease. In other words, future MI research should not be limited to the field of finding latent TC lesions.


Human astroviruses are an important cause of viral gastroenteritis globally, yet few studies have investigated the serostatus of adults to establish rates of previous infection. Here, we applied biolayer interferometry immunosorbent assay (BLI-ISA), a recently developed serosurveillance technique, to measure the presence of blood plasma IgG antibodies directed towards the human astrovirus capsid spikes from serotypes 1–8 in a cross-sectional sample of a United States adult population. The seroprevalence rates of IgG antibodies were 73% for human astrovirus serotype 1, 62% for serotype 3, 52% for serotype 4, 29% for serotype 5, 27% for serotype 8, 22% for serotype 2, 8% for serotype 6, and 8% for serotype 7. Notably, seroprevalence rates for capsid spike antigens correlate with neutralizing antibody rates determined previously. This work is the first seroprevalence study evaluating all eight classical human astrovirus serotypes. Astroviruses are a diverse family of small, nonenveloped, positive-sense RNA viruses that infect mammalian and avian species . AstroviHuman astroviruses (HAstVs) are a leading worldwide cause of viral gastroenteritis but are among the most poorly characterized enteric viruses . Young cSeveral lines of evidence highlight the importance of anti-astrovirus antibodies developed in childhood in preventing reinfection in adulthood. Firstly, astrovirus infection is rare in adults . AlthougPrevious structural studies have defined the structural domains of the human astrovirus capsid A,B 27,2,29,30. TSerosurveillance investigations bring the frequency of previous human astrovirus infection in a particular cohort into focus, but only a few have been performed on specific astrovirus subsets , or turkey astrovirus 2) or in specific populations (turkey growers and abattoir workers)  10,23,3,35,36. MThe accession numbers of sequences used for sequence alignments of the human astrovirus capsid core are as follows for serotypes 1 to 8 (Core- accession number: residues (depository)): Core 1- AAC34717.1: 80-410 (Genbank), Core 2- AZB52195.1: 80-409 (Genbank), Core 3- Q9WFZ0: 82-412 (Uniprot), Core 4- Q3ZN05: 80-410 (Uniprot), Core 5- Q4TWH7: 79-409 (Uniprot), Core 6- AZB52207.1: 80-410 (Genbank), Core 7- Q96818: 81-411 (Uniprot), and Core 8- Q9IFX1: 80-410 (Uniprot). The accession numbers of sequences used for sequence alignments of the human astrovirus capsid spike are as follows for serotypes 1 to 8 (Spike-accession number: residues (depository)): Spike 1-AAC34717.1: 430-644 (Genbank), Spike 2-KY964327.1: 12-228 (Genbank), Spike 3-Q9WFZ0: 432-645 (Uniprot), Spike 4-Q3ZN05: 430-644 (Uniprot), Spike 5-Q4TWH7: 429-641 (Uniprot), Spike 6-AZB52207.1: 430-642 (Genbank), Spike 7-Q96818: 431-644 (Uniprot), and Spike 8- Q9IFX1: 431-644 (Uniprot).Anti-Penta-His (HIS1K) sensor tips , Benzonase , bovine serum albumin (BSA) , CD OptiCHO expression medium , CHO CD EfficientFeed A , ChonBlock , 4 nm Colloidal Gold-AffiniPure Goat Anti-Human IgG Fcγ fragment-specific  rehydrated in 1 mL deionized water per the manufacturer’s instructions, 4 nm Colloidal Gold-AffiniPure Goat Anti-Rabbit IgG (H + L)  rehydrated in 1 mL deionized water per the manufacturer’s instructions, glucose , 1 mL HiTrap Protein A HP column , HT supplement , L-glutamine , phosphate buffered saline (PBS) tablets , Pierce IgG Elution Buffer , Pierce Normal Rabbit Serum Control , Pierce Protein A IgG Binding Buffer , Pluronic F68 , protease inhibitor cocktail set V EDTA-free , sodium butyrate (Sigma-Aldrich B5887), Superdex 75 10/300 and 16/600 , TALON metal affinity resin , tilted-bottom (TW384) microplates , Tween-20 , yeastolate .E. coli strain BL21 (DE3). Expression was induced with 1 mM IPTG and carried out at 18 °C for 16 h. Cell pellets were resuspended in 20 mM Tris-HCl pH 8.0, 500 mM NaCl, 20 mM imidazole (Buffer A) containing 2 mM MgCl2, 0.0125 U/μL benzonase, and 1x protease inhibitor cocktail set V EDTA-free. Cells were lysed using ultrasonication, and spikes were batch purified using TALON metal affinity resin. Elutions were carried out using Buffer A with 500 mM imidazole. Spikes were dialyzed into 10 mM Tris-HCl pH 8.0, 150 mM NaCl (TBS) with 1 mM DTT and purified using size exclusion chromatography in TBS with a Superdex 75 16/600  or Superdex 75 10/300 column .Synthetic cDNA encoding Spike 1 ), Spike 2 , a partial HAstV2 capsid sequence), Spike 3 ), Spike 4 ), Spike 5 ), Spike 6 ), Spike 7 ), or Spike 8 ) were cloned separately into pET52b (EMD Millipore) in frame with a C-terminal linker, thrombin cleavage site, and 10-Histidine tag. The plasmids were verified by DNA sequencing. Spike plasmids were transformed into The SARS-CoV-2 receptor-binding domain (RBD) in frame with a 10-Histidine tag and AviTag at the C-terminus was expressed and purified, as described in .Mouse hybridoma cells producing mAb 3B4 were generated, as reported in . The amiRabbit polyclonal serum to human astrovirus 1 was generated as described in .Human plasma samples were obtained from Discovery Life Sciences. These 63 de-identified samples were collected from adults ages 19–78  between 2012–2016 (before the emergence of SARS-CoV-2). Plasma samples were heated at 56 °C for 1 h before use. BLI-ISA studies were performed on an Octet RED384 instrument at 22 °C with shaking at 1000 rpm. BLI-ISA assay buffer consists of PBS pH 7.4, 2% BSA, and 0.1% Tween-20, which was 0.22-μm filtered. Before use, Anti-Penta-His (HIS1K) biosensors were placed into the wells of a biosensor-holding plate and pre-hydrated in BLI-ISA buffer for at least 10 min. Tilted-bottom 384-well microplates were loaded with 48 μL per well. The assay plate was prepared as follows: column 1 (BLI-ISA buffer), column 2 (10 nM His-tagged human astrovirus spikes 1–8 or 70 nM His-tagged SARS-CoV-2 RBD in BLI-ISA buffer), column 3 (a 1:4 dilution of ChonBlock in BLI-ISA buffer), column 4 (a 1:8 dilution of plasma samples: 6 μL plasma + 42 μL ChonBlock/BLI-ISA buffer), column 5 (BLI-ISA buffer), and column 6 .The BLI-ISA method was set as follows, with minimal adaptation from . BaselinAnalysis of BLI-ISA data was performed and automated as previously described in .E. coli and purified them by affinity chromatography and size exclusion chromatography. Purity was verified by SDS-PAGE . BLI-ISA is a recently developed method for rapid and semi-quantitative measurement of antibodies in blood plasma directed towards a particular antigen . In a siTo confirm whether BLI-ISA can be applied to human astrovirus serosurveillance, we assessed Spike 1 antigen binding with commercially available normal rabbit serum and rabbit serum positive for human astrovirus serotype 1 . As a poNext, we conducted dilution-series experiments to assess the optimal human plasma dilution to use in BLI-ISA serological assays for human astrovirus. Dzimianski et al. established a 1:8 plasma dilution as optimal after recent SARS-CoV-2 exposure, providing an estimate of the dilution range for serosurveillance studies using other antigens . Sixty-tAfter validation of the control samples and confirmation of the optimal 1:8 plasma dilution, we used BLI-ISA to determine the seroprevalence of antibodies in the 63 human plasma samples to HAstV spikes from serotypes 1 to 8 . This inE. coli and for their variable sequence identity (41.4–75.7%) which should minimize antibody cross-reactivity between spikes from different serotypes. Indeed, most known neutralizing monoclonal antibodies that target the spike are serotype specific . Th. Th10]. Finally, we present


N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) complex, whose disassembly in turn ensures the recycle of individual SNARE components. The disassembly of post-fusion SNARE complexes is controlled by the AAA+ ATPase N-ethylmaleimide-sensitive factor (Sec18/NSF) and soluble NSF attachment protein (Sec17/α-SNAP) in yeast and metazoans. Although non-canonical α-SNAPs have been functionally characterized in soybeans, the biological function of canonical α-SNAPs has yet to be demonstrated in plants. We report here that the canonical α-SNAP in Arabidopsis is essential for male and female gametophytic development. Functional loss of the canonical α-SNAP in Arabidopsis results in gametophytic lethality by arresting the first mitosis during gametogenesis. We further show that Arabidopsis α-SNAP encodes two isoforms due to alternative splicing. Both isoforms interact with the Arabidopsis homolog of NSF whereas have distinct subcellular localizations. The presence of similar alternative splicing of human α-SNAP indicates that functional distinction of two α-SNAP isoforms is evolutionarily conserved.The development of male and female gametophytes is a pre-requisite for successful reproduction of angiosperms. Factors mediating vesicular trafficking are among the key regulators controlling gametophytic development. Fusion between vesicles and target membranes requires the assembly of a fusogenic soluble  α-SNAP arrest mitosis and causes gametophytic lethality. In addition, we demonstrated that Arabidopsis α-SNAP encodes two functional isoforms. Interesting, the single α-SNAP gene in human also encodes two similarly distinct isoforms. Thus, the study of Arabidopsis α-SNAP provides important hints to that of human α-SNAP.Vesicular trafficking among different endomembrane compartments ensures membrane homeostasis, protein targeting, and finally the survival of eukaryotic cells. Fusion between vesicle and target membranes is mediated by tetrameric SNARE complexes, whose disassembly ensures the recycle of individual SNARE components. The disassembly of post-fusion SNARE complexes is controlled by NSF/Sec18 and α-SNAP/Sec17 both in yeast and metazoans. Recent reports show that a naturally occurring, truncated α-SNAP allele suppresses parasitic nematode infection in soybeans. However, a role of the canonical α-SNAP has yet to be demonstrated in plants. We report here that the canonical α-SNAP in Arabidopsis is essential for fertility. Mutations in  The development of male and female gametophytes is a pre-requisite for successful reproduction of angiosperms. In angiosperms, megagametogenesis  and micrN-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are coiled-coil domain proteins regulating vesicular fusion [Regulators of mitosis –7, of rir fusion , 21 betwr fusion , 21. Mutr fusion –19, 22. r fusion , suggest+ ATPase N-ethylmaleimide-sensitive factor (NSF/Sec18) and soluble NSF attachment protein (α-SNAP/Sec17), which perform ATP-dependent disassembly of cis-SNARE complexes, liberating SNAREs for subsequent assembly of trans-complexes for fusion [Vesicle-target membrane fusion not only depends on the assembly of tetrameric SNARE complex but also its disassembly so that the components of post-fusion SNARE complexes can be recycled . Studiesr fusion , 26. In r fusion  or moonlr fusion , 28.Recent studies in soybean showed that a naturally occurring, truncated α-SNAP allele, i.e. non-canonical α-SNAP, suppresses parasitic nematode infection –31. The α-SNAP in the Arabidopsis genome, designated ASNAP, is essential for male and female gametophytic development. By CRISPR/Cas9-mediated genomic editing, we generated and characterized asnap mutants. Functional loss of ASNAP resulted in gametophytic lethality such that both male and female gametophytes could not be transmitted. Specifically, functional loss of ASNAP caused mitotic arrest of unicellular microspores and of functional megaspores (FM), suggesting that ASNAP is essential for mitotic cell cycle progression during gametophytic development. We show that Arabidopsis ASNAP encodes two isoforms due to alternative splicing, both of which interact with the Arabidopsis NSF. The presence of similar alternative splicing of human α-SNAP indicates that functional distinction of two α-SNAP isoforms is evolutionarily conserved.We report here that the canonical α-SNAP whose protein products contain N-terminal, central, and C-terminal domains similar to animal α-SNAP homologs are encoded in the Arabidopsis genome. To determine its expression pattern, we generated ASNAPg:GUS transgenic plants expressing genomic-GUS translational fusion of ASNAP. By histochemical GUS staining, we detected GUS signals in various tissues and developmental stages, including seedlings, leaves, roots, reproductive organs, trichomes, root hairs as well as pollen tubes  by confocal laser scanning microscopy (CLSM) and whole-mount ovule clearing. At 24 HAP, wild-type ovules contained elongating zygotes or early embryos and peripheral endosperms , we confirmed that the GPR1p:amiR-ASNAP transgene did reduce the expression levels of ASNAP  into asnap-1/+, in which the Cas9 target site was mutated without affecting the corresponding amino acids. We obtained wild-type-like plants with the ASNAPg;asnap-1 genotype from the transformants . Indeed, treatment of root epidermal cells with Brefeldin A (BFA), a fungal toxin that causes the accumulation of TGN/EE vesicles into so-called BFA compartments, resulted in the co-localization of GFP-ASNAP.1 and FM4-64 into BFA compartments . Root epidermal cells expressing GFP-ASNAP.1 or GFP-ASNAP.2 were pulse-labeled with the lipophilic dye FM4-64, which first indicates the plasma membrane (PM) and then is internalized to different endomembrane compartments . Examinaartments , confirmP13-mRFP , a markeP13-mRFP , indicat1 plants , we deteBy contrast, GFP-ASNAP.2 was mostly present in the cytoplasm . Partialin vitro pull-down assays, in which his-tagged NSF was able to pull-down both ASNAP isoforms  compromised vacuolar formation and fusion [asnap [AP-1μ/HAPLESS13 [2-metabolizing enzymes [Although it is still unclear whether and how vesicular dynamics affect the first mitosis during male or female gametogenesis, studies in recent years suggested a direct link between defective vacuolar dynamics and gametophytic mitosis , 49, 50. enzymes , 15, as  enzymes .ASNAP loss-of-function could not be transmitted either through the male or the female  was cloned by using the primer pair ZP10000/ZP10001 or ZP9284/ZP9285/ZP9286/ZP9287, respectively. The full-length CDS of ASNAP.2 and Cas9-resistant ASNAP.2 (crASNAP.2) was cloned by using the primer pair ZP333/ZP397 or ZP9284/ZP9285/ZP9286/ZP9287, respectively. Entry vectors were used in LR reactions with the destination vector UBQ10p:GFP-GW and 35Sp:GFP-GW [UBQ10p:GFP-ASNAP.1, UBQ10p:GFP-crASNAP.1, UBQ10p:GFP-ASNAP.2, UBQ10p:GFP-crASNAP.2, 35Sp:GFP-ASNAP.1, and 35Sp:GFP-ASNAP.2. The full-length CDS of NSF was cloned by using the primer pair ZP9294/ZP9295. Entry vector for NSF was used in LR reactions with the destination vector 35Sp:mCherry-GW to generate 35Sp:mCherry-NSF.All constructs were generated using the Gateway technology (Invitrogen) except for CRISPR/Cas9 constructs. pENTR/D/TOPO (Invitrogen) was used to generate all entry vectors. Full-length genomic sequence of r GW:GUS  to generp:GFP-GW , 56 to gasnap mutants, the target site on ASNAP was selected using an online bioinformatics tool (http://www.genome.arizona.edu/crispr/CRISPRsearch.html) and was incorporated into forward and reverse PCR primers. The ASNAP-CRISPR/Cas9 cassette was generated by PCR amplifications from pCBC-DT1T2 [BsaI and inserted into pHSE401, resulting in pHSE401-ASNAP. To verify that the CRISPR-Cas9 construct resulted in the genomic editing of ASNAP, the primer pair ZP5203/ZP5204 were used to amplify the genomic sequences of pHSE401-ASNAP-transformed plants. The primer ZP5203 was used to sequence the amplified genomic fragment. For the amiR-ASNAP construct, the target site and sequence-specific primers for ASNAP were determined using an online tool (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi). The amiR-ASNAP cassette was generated by PCR amplifications from pRS300 with the primers ZP9288/ZP9289/ZP9290/ZP9291. The resultant PCR products were cloned into pENTR/D/TOPO. The entry vector was used in LR reactions with the destination vector GPR1p:GW-GFP and UBQ10p:GW-GFP.For the CRISPR/Cas9 construct used to generate the BC-DT1T2  with thein vitro pull-down assays were generated by double digestions and ligations. Coding sequences were amplified with the following primer pairs: ZP10836/ZP10837 for NSF, ZP10961/ZP10962 for ASNAP.1, and P732/P733 for ASNAP.2. PCR products were digested either with BamHI/SalI (for NSF) or with BamHI/XhoI (for ASNAP.1 and ASNAP.2). Digested fragments were inserted into the destination vector pET-32a [BamHI/SalI or BamHI/XhoI using the pEASY-Uni Seamless Cloning and Assembly Kit (TRAN). Constructs were sequenced and analyzed using Vector NTI. All PCR amplifications were performed with Phusion hot-start high-fidelity DNA polymerase with the annealing temperature and extension times recommended by the manufacturer (Thermo Fisher Scientific). All primers are listed in Constructs used in BiFC assays were generated using the destination vectors pSITE-cEYFP-C1, pSITE-nEYFP-C1, pSITE-nEYFP-N1 . Express pET-32a  pre-digeASNAP at diverse tissues, total RNAs were isolated from seedlings and roots at 7 DAG, from leaves at 14 DAG, from stems at 25 DAG, or from reproductive tissues at 4–5 days after anthesis. For RT-qPCRs analyzing the expression of ASNAP in GPR1p:amiR-ASNAP, RNAs were extracted from inflorescences. RT-qPCRs were performed with the Bio-Rad CFX96 real-time system using SYBR Green real-time PCR master mix (Toyobo) as described [ASNAP, P56/P57 for ASNAP.1, P114/P115 for ASNAP.2, and ZP12/P53 for the exogenous ASNAP. Primers for GAPDH and ACTIN2 in RT-qPCRs were as described [Total RNAs were extracted by using a Qiagen RNeasy plant mini kit according to the manufacturer’s instructions. Oligo (dT)-primed cDNAs were synthesized by using SuperScript III reverse transcriptase with on-column DNase digestion (Invitrogen). For RT-qPCRs of escribed . Primersescribed . All priin vitro pull-down assays, GST-ASNAP.1, GST-ASNAP.2, or His-NSF were transformed into E. coli strain BL21 (Rosetta), cultured at 37°C in Lurani-Bertani medium at the presence of antibiotics (100 mg/mL ampicillin) to an OD600 of 0.6 to 0.8. Protein expression was induced by adding 0.8 mM isopropyl-b-D-1thiogalactopyranosid (IPTG). In vitro pull-down assays were performed as described [For the purification of recombinant proteins in escribed , 58. TheNicotiana tabacum) by transient transformations as described [35Sp:GFP-ASNAP.1, 35Sp:GFP-ASNAP.2, 35Sp:mCherry-NSF and 35Sp:mCherry were performed in Arabidopsis protoplasts by transient transformations as described [BiFCs were performed in tobacco . All statistical analyses, One-Way ANOVA (Tukey’s multiple comparisons test) and t-test, were performed with build-in analysis tools and parameters.Quantification data are analyzed by using GraphPad Prism 6.02  pollinated with wild-type pollen. (C) Quantification of seed sets. Results are means ± SD (n>10). Asterisk indicates significant difference . Bars = 1 mm. Supports (A-B) Representative seed set of a wild-type (A) or (PDF)Click here for additional data file.S2 Figasnap-1/+ anthers at stage 9, stage 10, stage 11, or stage 12. T stands for tapetum. Arrowheads point at defective microspores. Asterisks indicate degenerating pollen. (B-D) Quantitative analyses of pollen development by alexander staining for pollen viability (Viability) (B), by DAPI staining for the development of tricellular pollen (Nuclei) (C), and by SEM for the rugby-shaped morphology (Morphology) (D). Results are means ± SD (n>100). Different letters indicate significant different groups . Bars = 10 μm. Supports (A) CLSM of developing wild type or (PDF)Click here for additional data file.S3 FigASNAP (non-discriminative for splicing variants) in wild-type and two lines of the UBQ10p:ami-ASNAP seedlings (#1 and #2) at 1 week after germination (WAG). Results are means ± SE (n = 3). (B) Representative wild-type and two lines of the UBQ10p:ami-ASNAP seedlings at 1 WAG. Three seedlings of each genotype growing on the same plate are shown. (C) Primary root length at 1 WAG. Results are means ± SE (n = 9). For each biological replicate, 6 seedlings of each genotype from the same plates were examined. (D) Representative wild-type and two lines of the UBQ10p:ami-ASNAP plants at 3 WAG. (E) Representative siliques from wild type and two lines of the UBQ10p:ami-ASNAP plants. (F-K) Alexander staining of a maturing anther  or pollen grains  from wild type , line 1 , or line 2  of the UBQ10p:ami-ASNAP plants. (L-N) DAPI staining of pollen grains released from wild type (L), line 1 (M), or line 2 (N) of the UBQ10p:ami-ASNAP plants. DAPI channel and transmission channel images are shown from top to bottom. (O) Percentage of DAPI-stained tricellular pollen from wild type and two lines of the UBQ10p:ami-ASNAP plants. Results are means ± SE (n>10). (P-R) Representative scanning electron micrographs (SEMs) of pollen  or pollen coat structure (R) from wild type and two lines of the UBQ10p:ami-ASNAP plants. Different letters in  indicate significantly different groups . Bars = 1 mm for ; 100 μm for ; 50 μm for ; 20 μm for (L-N); 5 μm for (Q); 1 μm for (R). Supports Figs (A) Relative transcript abundance of (PDF)Click here for additional data file.S4 FigASNAP (non-discriminative for splicing variants) in wild-type and ASNAPg;asnap-1 seedlings at 1 WAG. Results are means ± SE (n = 3). P value (t-test) is shown on top of the columns. (B-D) Representative wild-type (left) or ASNAPg;asnap-1 plants (right) at 1 WAG (B), 3 WAG (C), or 5 WAG (D). Supports (A) Relative transcript abundance of (PDF)Click here for additional data file.S5 FigASNAP among different Arabidopsis tissues. Results shown are means ± SE (n = 3). Each biological replicate was repeated three times with similar results. (B) Sequence alignment of α-SNAPs and their splicing variants from yeast, human, and Arabidopsis. Yellow-highlighted amino acids are identical while green and blue highlighted amino acids are similar in side chains. Lilac boxes indicate predicted tetratricopeptide-repeat domain (TPR). The blue box indicates coil-coil domain (InterPro). Arabidopsis protein sequence were obtained from TAIR, whereas proteins from other species were obtained from the National Center for Biotechnology Information. Species prefixes are as follows: Sc, Saccharomyces cerevisiae (AAA35029.1); Hs, Homo sapiens (NP_003818.2 and XP_011525739.1); At, Arabidopsis thaliana (At3G56190.1 and At3G56190.2).(A) Quantitative real-time PCRs (RT-qPCRs) of (PDF)Click here for additional data file.S6 FigASNAP (non-discriminative for splicing variants) in wild type versus ASNAP.1 in the UBQ10p:GFP-crASNAP.1;asnap-1 line. Results are means ± SE (n = 3). There is no significant difference between wild type and the complementation line . (B-C) Representative wild type or UBQ10p:GFP-crASNAP.1;asnap-1 (Comp) at 1 WAG (B) or 6 WAG (C). (D-E) Representative alexander staining of a mature anther from wild type (D) or from the UBQ10p:GFP-crASNAP.1;asnap-1 line (E). (F-G) A representative silique from wild type (F) or from the UBQ10p:GFP-crASNAP.1;asnap-1 line (G). Out of 20 siliques examined, none from the UBQ10p:GFP-crASNAP.1;asnap-1 line set seeds. Bars = 2 mm for (B); 1 cm for (C); 100 μm for (D-E); 1 mm for . Supports Figs (A) Relative transcript abundance of (PDF)Click here for additional data file.S7 FigUBQ10p:GFP-ASNAP.1;WAVE22R or UBQ10p:GFP-ASNAP.1;HAP13g:mRFP transgenic seedlings. RFP channels indicate either a mRFP-labeled Golgi marker (WAVE22R) or TGN/EE marker (HAP13-mRFP). Bars = 10 μm. Supports CLSM of root epidermal cells from (PDF)Click here for additional data file.S8 FigRepresentative CLSM of tobacco leaf epidermal cells expressing corresponding proteins in bimolecular fluorescence complementation (BiFC) assays. For BiFC assays, agrobacterium transformed with 35S:CBL1-mRFP (as a marker for the PM) or with 35S:mRFP-INT1 (as a marker for the tonoplast) is co-infiltrated with agrobacterium transformed with YN- and YC-vectors in a 1:1 ratio and 90 RFP-positive cells for every BiFC combination from three replicates were examined for YFP signals. Numbers at the bottom indicate displayed/transformed epidermal cells. Bars = 10 μm. Supports (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally, and the number of worldwide cases continues to rise. The zoonotic origins of SARS-CoV-2 and its intermediate and potential spillback host reservoirs, besides humans, remain largely unknown. Because of ethical and experimental constraints and more important, to reduce and refine animal experimentation, we used our repository of well-differentiated airway epithelial cell (AEC) cultures from various domesticated and wildlife animal species to assess their susceptibility to SARS-CoV-2. We observed that SARS-CoV-2 replicated efficiently only in monkey and cat AEC culture models. Whole-genome sequencing of progeny viruses revealed no obvious signs of nucleotide transitions required for SARS-CoV-2 to productively infect monkey and cat AEC cultures. Our findings, together with previous reports of human-to-animal spillover events, warrant close surveillance to determine the potential role of cats, monkeys, and closely related species as spillback reservoirs for SARS-CoV-2. During the past 2 decades we have observed zoonotic outbreaks of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012  culture repository from the primary tracheobronchial airway tissue of 12 mammal species comprising companion animals, animal model candidates, livestock, and wild animals to assess their susceptibility to SARS-CoV-2 infection. To control for the quality of the AEC, we used influenza viruses that have known broad host tropism (https://www.thermofisher.com). We maintained cells at 37°C in a humidified incubator with 5% CO2.We cultured Vero E6 cells in Dulbecco Modified Eagle medium supplemented with 10% volume/volume percent (vol/vol) heat-inactivated fetal bovine serum, 1 mmol/L sodium pyruvate, 1x GlutaMAX, 100 μg/mL streptomycin, 100 IU/mL penicillin, 1% vol/vol nonessential amino acids, and 15 mmol/L HEPES buffering agent , and 15 mmol/L HEPES buffer. We determined viral titer by 50% tissue culture infective dose (TCID50) assay on MDCK-II cells as described elsewhere (https://www.atcc.org) for 96 h in the infection medium, with the adjustment of using 0.25 μg/mL of trypsin. We determined viral titer by TCID50 assay on HRT-18G cells as described elsewhere (We propagated SARS-CoV-2 (SARS-CoV-2/München-1.1/2020/929) virus stock in Vero E6 cells for 48 h then cleared virus-containing supernatant from cell debris by centrifuging for 5 min at 500 × g before aliquoting and storing it at –80°C. We determined viral titer by plaque forming unit (PFU) assay on Vero E6 cells as described elsewhere  200-401-A50 rabbit polyclonal antibody against SARS-CoV nucleocapsid protein, which has previously been shown to cross-react with SARS-CoV-2 (https://www.abcam.com) ab128193 mouse antibody against IAV clone C43 nucleoprotein to detect IAV-infected cells and a custom-made rabbit polyclonal antibody against the nucleoprotein of influenza D/bovine/Oklahoma/660/2013 strain  to detect IDV-infected cells. To visualize the distribution of ACE2 in the AEC cultures, we used Abcam ab15348 and Biorbyt (https://www.biorbyt.com) orb582208 rabbit polyclonal antibodies against ACE2. We used Alexa Fluor 488 conjugated donkey anti–rabbit or anti–mouse IgG (H + L) as secondary antibodies. We used Alexa Fluor 647-conjugated anti-β-tubulin (9F3) rabbit mAb (Thermo Fisher Scientific) to visualize cilia and Alexa Fluor 594 mouse anti-ZO1 1A12 monoclonal antibody to visualize tight junctions. We counterstained all samples using DAPI  to visualize the nuclei. We performed imaging using a Thermo Fisher EVOS FL Auto 2 imaging system equipped with a plan apochromat 40×/0.95 air objective; we processed images using Fiji software packages (https://fiji.sc) (https://github.com/mutterer/figurej)  for SARS-CoV-2 or 48 hpi for IAV- or IDV-infected AEC cultures and processed them as described elsewhere (https://www.mn-net.com), according to the manufacturer’s guidelines, on a Kingfisher Flex purification system (Thermo Fisher Scientific). We amplified 2 μL of extracted RNA using TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific) according to the manufacturer’s protocol. We used a forward primer, adapted from primers described elsewhere (https://www.microsynth.ch), targeting the envelope gene of SARS-CoV-2 (GenBank accession no. MN908947.3). As a positive control, we included a serial dilution of in vitro–transcribed RNA containing regions of the RNA-dependent RNA polymerase, envelope, and N genes derived from a SARS-CoV-2 synthetic construct (GenBank accession no. MT108784) to determine the genome copy number. We performed measurements and analysis using an Applied Biosystems ABI7500 instrument and associated software package (Thermo Fisher Scientific).We extracted viral RNA from 100 μL of 1:1 diluted apical wash using the NucleoMag VET , 10% heat-inactivated fetal bovine serum, 100 μg/mL streptomycin, and 100 IU/mL penicillin. We incubated cells at 37°C with 5% CO2 for 48 h and fixed them with 4% vol/vol neutral buffered formalin before staining with crystal violet  by reverse transcription PCR amplification of ACE2 mRNA, as described elsewhere  plugin in Geneious Prime (https://www.geneious.com) with the default settings. We selected ACE2 protein residues interacting with SARS-CoV-2 RBM based on previously described critical ACE2 residues interacting with SARS-CoV-2 receptor binding domains , rhesus macaques (accession no. NM_001135696.1), cats (accession no. XM_023248796.1), ferrets (accession no. NM_001310190.1), dogs (accession no. NM_001165260.1), rabbits (accession no. XM_002719845.3), pigs (accession no. NM_001123070.1), cattle (accession no. XM_005228428.4), goats (accession no. NM_001290107.1), and Bactrian camels (accession no. XM_010968001.1). We acquired the ACE2 sequences for https://nanoporetech.com) and sequenced on an Oxford Nanopore Technologies MinION R9.4.1 flow cell for 48 h, according to the manufacturer’s instructions. We used Oxford Nanopore MinION software version 20.06.4 to perform data acquisition and real-time high-accuracy basecalling. We performed demultiplexing and read filtering according to the ARTIC platform nCoV19 pipeline (https://artic.network/ncov-2019) and the experimental Medaka pipeline (https://community.artic.network/t/medaka-longshot-pipeline/107) to perform consensus calling. We aligned and further analyzed consensus sequences in Geneious 11.1.5 using SARS-CoV-2/Wuhan-Hu-1 (GenBank accession no. MN908947.3) as the reference sequence.We performed sequencing on viral RNA isolated from SARS-CoV-2 stock and the 96 hpi apical washes of SARS-CoV-2–infected monkey and cat AEC cultures according to the ARTIC platform nCoV19 protocols , but we To evaluate the susceptibility of a diverse set of animal species to SARS-CoV-2 infection, we infected a total of 12 different well-differentiated mammal AEC culture models and monitored the viral replication kinetics at both 33°C and 37°C. Quantification of the viral RNA load at both temperatures showed a progressive 4-log fold increase in viral RNA load at 72 and 96 hpi in rhesus macaque and cat AEC cultures. In contrast, for the remaining animal AEC cultures we detected either a continuous or declining level of viral RNA load throughout the entire time course . We showed that these 2 neotropical bats express ACE2 but are not susceptible to SARS-CoV-2, suggesting that they are not likely reservoir hosts for SARS-CoV-2 despite the detection of other coronaviruses and presumptive ACE2 receptor usage by SARS-CoV-2 in closely related bat species (Rousettus aegyptiacus) (Rhinolophus), which have previously been characterized as reservoir hosts for viruses with a close genetic relationship to the coronavirus associated with the 2003 SARS outbreak (It has been proposed that SARS-CoV-2 spillover into humans, as with SARS-CoV, originally occurred from bats, either directly or through an intermediate reservoir (Primer pool for study of use of animal epithelial cells to determine animal species’ susceptibility to severe acute respiratory syndrome coronavirus 2.Additional information about the use of animal epithelial cells to determine animal species’ susceptibility to severe acute respiratory syndrome coronavirus 2.


Dear Editor,1 These mutations include almost all the sites of existing VOCs .4 Importantly, the key mutations directly interact with the ACE2 receptor and constitute the main target of neutralizing antibodies (NAbs), which is believed to alter the sensitivity to a large number of NAbs.The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants threatens efforts to contain the coronavirus disease 2019 (COVID-19) pandemic. Omicron (B.1.1.529), the fifth novel SARS-CoV-2 variant of concern (VOC), harbors 15 mutations in the receptor-binding domain (RBD) of the spike (S) protein.5 and S309 (Class III) epitope, but do not overlap with the hACE2-binding region. To reduce the risk of a human-anti-mouse-antibody (HAMA) response in clinical trials, R33 was humanized through complementarity determining region (CDR) grafting onto human acceptor germline frameworks and named hu33.6 In comparison with the affinity constant (KD) values of ACE2/SARS-CoV-2 RBDs, the Surface Plasmon Resonance (SPR) results demonstrated that Beta and Delta variants partially affected the affinities of NAbs to RBD proteins. Importantly, Omicron fully decreased the binding ability of Class I/II antibodies  below the affinities of ACE2/Omicron RBD except for S309 (a Class III NAb) and hu33  currently being evaluated in late clinical trials, NAbs with Emergency Use Authorization (EUA), and mAb hu33 to SARS-CoV-2 RBDs. We previously isolated a large panel of SARS-CoV-2 RBD-binding mAbs from RBD mRNA vaccine immunized mice using a 10×Genomics-based antibody discovery platform. The most frequent germline presented by RBD-specific B cells was VH9-3 (15.6%). Among these mAbs, R33 exhibited ultra-potent neutralizing activity and targeted epitopes that located between P2B-2F6 (Class II)u33 Fig. . Using tu33 Fig. . Further8 To investigate the breadth of hu33 neutralizing activity, SARS-CoV-2 S proteins-bearing vesicular stomatitis virus (VSV delta G/luciferase) pseudoviruses infection of Huh7 cells model was employed. hu33 had shown high potency against prototype SARS-CoV-2 with a median neutralization dose (ND50) of 3.3 ng/mL, consistent with its efficacy of 12.5 and 4.7 ng/mL to Beta and Delta variants, respectively  were divided into three groups, intraperitoneally administered with 15 mg/kg hu33 or phosphate buffer (PBS) as a placebo, and challenged with 1 × 102 50% tissue culture infectious dose (TCID50) of the virus through intranasal route. The changes in mice bodyweight of the prophylactic and treatment groups were significantly lower than those of the placebo-treated group  regions of hu33 and a prefusion-stabilized Beta S trimer using single-particle cryo-electron microscopy (cryo-EM), and reconstructed a cryo-EM density map for the one RBD open state at an overall resolution of 3.1 Å (Supplementary Fig. In summary, this study showed that the Omicron variant escapes most EUA Class I/II NAbs, whereas the neutralization sensitivity of Class III mAbs, non-ACE2-blocking antibodies, was less affected by this variant. In the meantime, hu33 was a mutation-resistant and broadly neutralizing activity against Omicron-included SARS-CoV-2 variants. Structural and functional analyses support the idea that hu33 is a potential treatment option for treating the SARS-CoV-2 VOCs and combating the COVID-19 pandemic.Supplemental materialsValidation Report