SLC12A3 gene mutation. Using the CRISPR-Cas9 system, we corrected CMC-GIT-001 mutations and hence generated CMC-GIT-001corr. Both hiPSCs were differentiated into kidney organoids, and we analyzed the GIT phenotype. The number of matured kidney organoids from the CMC-GIT-001corr group was significantly higher, 3.3-fold, than that of the CMC-GIT-001 group . In qRT-PCR, performed using harvested kidney organoids, relative sodium chloride cotransporter (NCCT) mRNA levels  were increased in the CMC-GIT-001corr group compared with the CMC-GIT-001 group . Consistently, immunoblot analysis revealed increased levels of NCCT protein, in addition to other tubular proteins markers, such as LTL and ECAD, in the CMC-GIT-001corr group compared to the CMC-GIT-001 group. Furthermore, we found that increased immunoreactivity of NCCT in the CMC-GIT-001corr group was colocalized with ECAD  using confocal microscopy. Kidney organoids from GIT patient-derived iPSC recapitulated the Gitelman’s disease phenotype, and correction of SLC12A3 mutation utilizing CRISPR-Cas9 technology provided therapeutic insight.The aim of this study is to explore the possibility of modeling Gitelman’s disease (GIT) with human-induced pluripotent stem cell (hiPSC)-derived kidney organoids and to test whether gene correction using CRISPR/Cas9 can rescue the disease phenotype of GIT. To model GIT, we used the hiPSC line CMCi002 (CMC-GIT-001), generated using PBMCs from GIT patients with  SLC12A3) gene, which encodes the sodium chloride cotransporter (NCCT) . To. To8]. TSLC12A3 correction, CMC-GIT-001corr hiPSC. The AccIII restriction fragment length polymorphism was specific to the corrected allele, and DNA sequencing also confirmed that SLC12A3+/− was correctly targeted without any footprint. As shown in AccIII enzyme into 294 bp and 231 bp fragments for exon 1, respectively. In addition, we also checked the DNA sequence of the generated hiPSC lines following targeted DNA sequencing . However, we found that the number of matured kidney organoids was recovered in the CMC-GIT-001corr hiPSC-derived kidney organoids compared to that of CMC-GIT-001 hiPSCs   C. Upon qcorr hiPSCs differentiated into kidney organoids with normal nephron marker expression and patterns, as shown in Immunofluorescence staining and examination under confocal microscopy revealed that WTC-11 as wild-type (WT) was successfully differentiated into kidney organoids without major structural differences, expressing markers of nephron structure, including PODXL in glomerular epithelial cells, LTL in the proximal tubule, and E-cadherin (ECAD) in the distal tubule, in appropriately patterned and contiguous segments as shown in high magnification in SLC12A3, we evaluated the expression of NCCT in kidney organoids derived from CMC-GIT-001corr hiPSCs. Using qRT-PCR, NCCT mRNA levels were significantly decreased in CMC-GIT-001 compared to WTC-11-derived organoids . However, this level was recovered in CMC-GIT-001corr, as shown in p < 0.05) also significantly increased in the CMC-GIT-001corr compared to the CMC-GIT-001  (To confirm the targeted gene correction of 1 group) B,C. The corr 1 restored the expression of NCCT as well as ECAD compared to those of CMC-GIT-001, as shown in We next performed double immunolabeling with NCCT and ECAD to determine the localization and expression level of NCCT, which is mainly localized to the apical membrane of ECAD-positive tubular cells in WTC-11 hiPSC kidney organoids. However, the immature kidney organoids derived from CMC-GIT-001 hiPSC did not react with the antibodies against both NCCT and ECAD. On the other hand, the kidney organoids from CMC-GIT-00corr hiPSCs. Consistent with the immunofluorescence staining results, reduced LTL  and ECAD  protein levels in the CMC-GIT-001 compared with WTC-11 significantly recovered in CMC-GIT-001corr organoids , as shown in corr generated by CRISPR/Cas9-mediated gene correction significantly restored their expression as well as morphological maturation.We next compared the protein expression of the other nephron markers, LTL and ECAD, in the kidney organoids from WTC-11 hiPSC, CMC-GIT-001, and CMC-GIT-001In this study, we used CRISPR/Cas9 in GIT patient-derived hiPSCs to correct the mutation responsible for disease development and differentiated the corrected hiPSCs into kidney organoids. As a result, in the gene-corrected kidney organoids, the structure showed a more mature form and expression representative of renal tubular cells, demonstrating rescue of the GIT disease phenotype.First, we used the previously reported patient-derived hiPSCs as a source to model GIT with a kidney organoid and for the application of CRISPR/Cas9 . In prevSLC12A3 gene mutation using CRISPR/Cas9. Some previous studies already suggest that CRISPR/Cas9-mediated gene correction has potential as a therapeutic strategy for genetic diseases, especially when an appropriate treatment strategy is unavailable, such as GIT [Next, we tried to correct for h as GIT ,19. For h as GIT ,21. In ah as GIT . In this−/− mice) [SLC12A3 gene recovered not only NCCT but also the expression of PODXL, LTL and ECAD, and the maturity of kidney organoids. Nevertheless, further study is needed to determine the mechanisms for the morphological and functional defects of the nephron following downregulation of the SLC12A3 gene. For example, ion movement with a fluorescent indicator  or patch-clamp tool may be helpful in understanding of the NCCT channel function.Interestingly, the proportion of mature kidney organoids was significantly low, and tubular proteins, such as LTL and ECAD, showed decreased expression, and PODXL was not detected in CMC-GIT-001 kidney organoids in comparison with WT (WTC-11) kidney organoids. In contrast to the human kidney, in which nephrons comprise only a small portion of the whole kidney, most of a kidney organoid is comprised of tubular structures , and it /− mice) . TherefoSLC12A3 gene and showed compound heterozygous variants and single mutation, which can induce clinical manifestation, and is very rare [Interestingly, even though only one mutation (c. 47_48del in exon 1) out of two pathogenic mutations in this hiPSC was successfully corrected by CRISPR/Cas9, expression of NCCT and other tubule markers was successfully rescued. It can be accounted for by the previous knowledge that most GIT patients are confirmed to have biallelic mutation in the ery rare ,26. In tery rare . The cliery rare . Using oOur study has some limitations to be solved. First, most genetic disorders, including GIT, are not monogenic; hence, direct gene correction would be difficult to translate to all patients uniformly. Alternatively, targeted disruption or insertion of other specific genes associated with disease development or protection can be applied to all patients universally, irrespective of mutation type ,30. TherSLC12A3 gene mutation rescue the disease phenotype. Our results suggest that CRISPR/Cas9-mediated gene correction can be proposed as a new therapeutic strategy for the treatment of GIT.Taken together, kidney organoids from GIT patient-derived hiPSCs recapitulate the disease phenotype in terms of decreased expression of NCCT, and CRISPR/Cas9-mediated correction of SLC12A3 gene sequencing revealed heterozygous c. 47_48del and c.2963 T > C mutations. The PBMC obtained from patients was cultured for 4 days at 37 °C in an incubator with 5% CO2 in StemSpan medium , which includes StemSpan CC100  to expand CD34-positive cells. The expanded PBMCs were transfected using the CytoTune-iPS Sendai Reprogramming Kit , which includes the Yamanaka factors . PBMCs were induced to form hiPSCs via centrifugation, and the resulting attached cells were expanded and purified using colony picking.GIT patient hiPSCs were generated using peripheral blood samples as described in a previous study  . SLC12A3ACCIII enzyme site insert for exon 1.Single guide RNA (sgRNA) and ssODN for gene correction in the target regions were designed using the ToolGen CRISPR/Cas9 technology service. sgRNA are listed as follows: 5′-GTGAAGCGCCCGCTGCAAAGTGG-3′ (reverse frame) for exon 1. ssODN are listed as follows: 5′-TGGACACCCAGGCGACAATGGCAGAACTGCCCACAACAGAGACGCCTGGGGACGCCACTTTGTGCAGCGGGCGCTTCACCATCAGCACACTGCTGAGCAGTGATGAGCCCTCTCCACCAGCTGCCTATGAC-3′ for exon 1. We designed an ssODN donor with the To check sgRNA efficiency in GIT patient hiPSCs, we mixed the following components: 250 ng of sgRNA, 500 ng of Cas9 protein , 3 μL of genomic hiPSC DNA, and reaction buffer, then incubated the solution at 37 °C for 1.5 h. A total of 1μL of RNase was incubated for a further 20 min at 37 °C, followed by the addition of stop solution. The efficiency of Cas9-mediated cleavage was analyzed through the fragmented PCR products obtained using gel electrophoresis using primer sets .6 hiPSC with 100μL of Opti-MEM Medium . At least 72 h after transfection, genomic DNA (gDNA) was extracted using the Genomic DNA Extraction Kit  according to the manufacturer’s protocol. To identify the gene editing efficiency and exact sequence edited, we utilized the IN/DEL analysis service for CRISPR validation . In addition, correction of targeted genes was confirmed by AccIII restriction digestion of PCR products before and after gene correction.sgRNA and Cas9 protein were introduced by electroporation using NEPAGENE  as the ribonuleoprotein (RNP) complex. Briefly, the RNP complex was formed by mixing 30 μg Cas9 protein with 25 μg sgRNA and incubating the mixture at room temperature for 10 min. These were then electroporated to 1 × 10hiPSCs cells were dissociated using TE . The cells were washed twice with FACS buffer , permeabilized for 30 min using flow cytometry fixation and permeabilization solution , washed with wash buffer, and stained with stage-specific embryonic antigens SSEA-4  or TRA-1–81  surface antibodies for 30 min. Intracellular staining for NANOG  was performed by sequential incubations with fixation and permeabilization solutions . These cells were incubated with FITC-conjugated secondary antibody (BD Bioscience). IgG1 isotypes, control to FITC (NANOG) cells, were analyzed using a FACS Canto II flow cytometer (BD Biosciences). The data were analyzed using FlowJo software version 10.8.1. .hiPSCs or kidney organoids were washed once with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 10 min at 4 °C, and were blocked in 5% donkey serum in PBS-T (0.3% Triton X-100 in PBS) for 1 h at room temperature (RT). hiPSCs or kidney organoids were incubated with primary antibodies, anti-NANOG , anti-SSEA4 , anti–TRA-1-81 , anti-PAX6 , anti-SM22A , anti-FOXA2 , biotinylated Lotus Lectin , E-cadherin , podocalyxin , and anti-NCC  overnight at 4 °C. Then, the cells were stained with secondary antibodies; Alexa Fluor 488-donkey anti-mouse IgG , Alexa Fluor 647-donkey anti-goat IgG , Cyanine3(Cy3)-streptavidin , Alexa Fluor 488-donkey anti-rat IgG , and Cy3-conjugated donkey anti-rabbit . Nucleic acid staining was performed by incubation with 4′,6-diamidine-2-phenylindole  for 30 min at room temperature. Images were obtained using a Zeiss LSM700 confocal microscope .Regarding trilineage differentiation, a StemMACS™ Trilineage Differentiation Kit  was used. Putative hiPSCs were cultivated for seven days in three different chemically defined media, driving the differentiation into three germ layers. The three differentiated germ layers were fixed in 4% paraformaldehyde and stained with the following antibodies: anti-PAX6 antibody for the ectoderm, anti-SM22A antibody for the mesoderm, and anti-FOXA2 antibody for the endoderm.Pluripotent cells were cultured in culture plates coated with Matrigel in conditioned media for 3~5 days. The cells were transported to GenDix Research Center , where cell harvest and karyotype analysis of metaphase chromosomes was performed using G-banding.Mycoplasma analysis was performed using the e-Myco VALiD Mycoplasma PCR kit (iNtRON Biotechnology) and was absent in all cases.corr hiPSCs were differentiated into kidney organoids following the previously published protocol [® hESC Qualified Matrix. After 24 h, the medium was exchanged with 2.5% of Matrigel® in mTeSR1. On the fourth day, the medium was replaced with Advanced RPMI  supplemented with 12 µM CHIR-99021 (STEMCELL Technologies). Approximately 36 h later, the medium was changed to Advanced RPMI with B27 supplement . Organoids were cultured in this medium until collection on day 21.Healthy control WTC-11), GITpatient, and GITpatientprotocol ,11,12,14, GITpatiTotal RNA was extracted from cells using RNA-Bee  as per the manufacturer’s instructions. Around 1 μg of total RNA was employed for cDNA synthesis with a cDNA synthesis kit . RT-qPCR was performed using SYBR Green Master Mix  on a LightCycler 480 system  using a primer set for NCC or GAPDH . All data were normalized against GAPDH and were calculated using the ΔΔCq method.4. Equal amounts of protein were separated on 7% SDS-PAGE gels and transferred to a polyvinylidene difluoride membrane  using the iBlot2 Gel Transfer Device . Membranes were blocked with 5% skim milk in PBS with 0.1% Tween 20 detergent (T-PBS) for 1 h at room temperature and incubated with anti-NCC  and biotinylated Lotus Lectin , E-cadherin , and b-actin  at 4 °C overnight. Membranes were then washed 3 times with T-PBS and incubated for 1 h with a goat anti-rabbit IgG-HRP conjugate , a goat anti-mouse IgG-HRP conjugate , and a Strep Tactin-HRP conjugate . After 4 washes with T-PBS, the protein of interest was detected using an enhanced chemiluminescence system . Quantification of relative densities was performed with the control group set at 100%; densities were normalized to those of b-actin bands from the same gel .Kidney organoids were washed with PBS and lysed with 10mM Tris (pH 7.5) containing 1% sodium dodecyl sulfate (SDS) and 1 mM NaVOp < 0.05.Data have been expressed in terms of mean ± standard error (SE) from at least three independent experiments. Multiple comparisons between groups were performed by one-way analysis of variance with the Bonferroni post hoc test using Prism software . Statistical significance was set at


Transthyretin (TTR) is a homo-tetramer protein involved in the transport of thyroid hormone  in the plasma and cerebrospinal fluid. Many pollutants have been shown to bind to TTR, which could be alarming as disruption in the thyroid hormone system can lead to several physiological problems. It is also indicated that the monomerization of tetramer and destabilization of monomer can lead to amyloidogenesis. Many compounds are identified that can bind to tetramer and stabilize the tetramer leading to the inhibition of amyloid fibril formation. Other compounds are known to bind tetramer and induce amyloid fibril formation. Among the pollutants, per- and polyfluoroalkyl substances (PFAS) are known to disrupt the thyroid hormone system. The molecular mechanisms of thyroid hormone disruption could be diverse, as some are known to bind with thyroid hormone receptors, and others can bind to membrane transporters. Binding to TTR could also be one of the important pathways to alter thyroid signaling. However, the molecular interactions that drive thyroid-disrupting effects of long-chain and short-chain PFASs are not comprehensively understood at the molecular level. In this study, using a computational approach, we show that carbon chain length and functional group in PFASs are structural determinants, in which longer carbon chains of PFASs and sulfur-containing PFASs favor stronger interactions with TTR than their shorter-chained counterparts. Interestingly, short-chain PFAS also showed strong binding capacity, and the interaction energy for some was as close to the longer-chain PFAS. This suggests that short-chain PFASs are not completely safe, and their use and build-up in the environment should be carefully regulated. Of note, TTR homologs analysis suggests that thyroid-disrupting effects of PFASs could be most likely translated to TTR-like proteins and other species.The online version contains supplementary material available at 10.1007/s00204-022-03434-8. Per- and polyfluoroalkyl substances (PFASs) are a large family of environmentally persistent, man-made fluorinated compounds  that regulate metabolism, cellular growth, immune system, reproduction, development, heat production and circadian rhythm Brent . T3 and The increasing levels of environmental pollutants have been implicated in thyroid-related problems  was selected from literature survey    were taken from the X-ray crystal structure of human transthyretin  . A total of 12 homologs of TTR were found from 12 different vertebrate species, namely Danio rerio (Zebrafish), Xenopus tropicalis (Clawed frog), Gallus gallus (Junglefowl), Mus musculus (House mouse), Rattus norvegicus (Brown rat), Bos taurus (Cattle), Chlorocebus aethiops (Grivet), Macaca fascicularis (Macaque), Macaca mulatta (Rhesus macaque), Macaca fuscata (Japanese macaque), Pongo abelii (Sumatran orangutan), Pan troglodytes (Chimpanzee), and their sequences were retrieved from NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi)  . With respect to molecular interactions, T4 forms H-bonds with L110 and A109 of chain C (amino acid AA of chain A and C are referred to as AA(A) and AA(C), respectively, throughout the text, e.g., L110 of chain C is L110(C)). On the other hand, T3 forms H-bonds with T106(A), S52(C), and T123(A), respectively. Captivatingly, both T3 and T4 formed hydrophobic interactions with various amino acids, e.g., K15(A), K15(C), and L17(C) as shown in Fig. . We first aimed to identify which PFASs have stronger binding affinity with TTR than T4 and T3. To this end, binding energy analysis showed that all the 16 long-chain PFASs exerted stronger binding affinity towards TTR ranging between – 9.8 kcal/mol and – 6.7 kcal/mol  compared to T3 and T4 as shown in Table S1, whereas 10 out of 15 short-chain PFASs showed a stronger binding affinity with TTR compared to T4 and T3 (Tables S1 and S3). We thus next investigated key amino acids K15, A108, A109, L110, S117, T118, and T119 from chain A and K15, A108, L110, S117, and T119 from chain C of TTR interact with long-chain PFASs. Similarly, short-chain PFASs interacted with amino acid L17, A108, A109, L110, S117, T118, and T119 from chain A and A108, A109, L110, S117, T118, and T119 from chain C of TTR (Tables S1–S3). It should the noted that K15 was frequently interacting with long-chain PFASs and not with short-chain PFASs. Besides, residues M13, E54, T106, and V121 interacted less frequently with TTR (Tables S2-S3).In respect of PFASs, we calculated binding energy, interacting amino acids, and type of molecular interactions with TTR as explained in Table S2 and Table S3, respectivelyRegarding the molecular interactions, we observed that H-bonds and hydrophobic interactions drive PFASs binding with TTR. Hydrophobic interactions were major contributors to the molecular interactions, followed by H-bonds Fig.  and 3. FIn the case of short-chain PFASs, when we analyzed binding energy, we found a similar stronger binding affinity  like long-chain PFASs for 10 out of 15 short-chain PFASs as elucidated in Fig. PFASs having a stronger binding affinity towards TTR than T4 and T3 have the potential to displace natural ligands  from the binding site of TTR. As apparent from Fig. To get a clearer picture of molecular interactions, we sought to detect key interacting residues with TTR. Comparative analysis of data revealed that K15, A108, A109, L110, and S117 are the key interacting amino acid residues of TTR that were involved in the majority of molecular interactions with PFAS (Table S4). Detailed analyses are shown in Figs. We next inquired whether the identified amino acid substitution with glycine alters the binding efficiency of PFASs with TTR. Critical amino acid residues from TTR involved in interacting with PFASs have been identified as mentioned before (Table S4). To understand the role of each key residue, we generated protein variants of TTR using in silico substitution of amino acid residue with glycine, including two single amino acid variants (K15G and L110G), one double amino acid variant (K15G/L110G), and one multiple amino acids variant (K15G/L110G/S117G). We then directly tested whether these substitutions in TTR could account for the resulting binding energy changes with identified six strongest and six weakest PFASs, respectively (Tables S5-S16). According to the binding energy results, the binding affinity of the selected PFASs was significantly reduced due to substitution compared to wild-type TTR as described in Table Furthermore, single variants, K15G and L110G, were less effective in reducing the binding affinity and reducing molecular interactions as compared to double amino acid substituted variants (K15G/L110G), whereas multiple amino acid-substituted variant (K15G/L110G/S117G) has been observed to be most effective in reducing binding affinity for both long-chain and short-chain PFASs. Of note, there was a slight increase in the binding affinity of three short-chain PFASs  and one long-chain PFAS (N-methyl perfluorooctane sulfonamidoethanol) due to K15G and L110G substitutions, respectively. Strikingly, there are three sulfur-containing PFASs, i.e., perfluoroethane sulfonic acid, trifluoromethane sulfonic acid, and N-methylperfluorooctanesulfonamidoethanol, which showed slight increase in the binding affinity due glycine substitution. Although its structural determinant remains to be investigated, from this observation, it can be suggested that sulfur containing PFASs are more resistant and hence more toxic , Xenopus tropicalis (Clawed frog), Gallus gallus (Junglefowl), Mus musculus (House mouse), Rattus norvegicus (Brown rat), Bos taurus (Cattle), Chlorocebus aethiops (Grivet), Macaca fascicularis (Macaque), Macaca mulatta (Rhesus macaque), Macaca fuscata (Japanese macaque), Pongo abelii (Sumatran orangutan), and Pan troglodytes (Chimpanzee). Besides, we also found that invertebrates, including Caenorhabditis elegans (roundworms), Daphnia pulex (Daphnia), and Drosophila navojoa (flies) are enriched with TTR orthologs. It can be noted that A108I, S117E, and T119R substitutions were observed in Caenorhabditis elegans  from binding sites of TTR and cause toxicity. Besides, to strengthen our data, we compared the binding affinity of 11 Long-chain PFASs and 4 Short-chain PFASs with TTR and PPARγ (peroxisome proliferator-activated receptor gamma) as previously described   Fig. . As conses) Fig. , the proes) Fig. . These rIn summary, the results of the present study establish a discrete but comprehensive toxicity pattern of long-chain and short-chain PFASs toward TTR. Obtained results disclosed that the binding potency of a PFAS relies on its carbon chain length and functional group, in which longer carbon chains of PFASs and sulfur-containing PFASs favor stronger interactions with TTR than their shorter-chained counterparts. Moreover, these results pointed out the thyroid-disrupting effects of short-chain PFASs and their in-depth toxicity profile needs to be studied to understand the underlying biochemical processes under physiological and pathophysiological conditions. Furthermore, we identified and highlighted the significance of residues K15, A108, L110, S117, and T119 in establishing H-bonding and hydrophobic interaction networks with PFASs. Interestingly, TTR homology analysis strengthens the hypothesis that the thyroid-disrupting effects of PFASs are not only limited to humans, but to other species as well. Taken together, the data presented here facilitate molecular feature-based toxicity prediction of chemicals depending on their binding affinity with biomolecules.Supplementary file1 (DOCX 64353 KB)Supplementary file2 (DOCX 73 KB)Below is the link to the electronic supplementary material.


At least one T2C was identified in 66%, 69%, and 46% of M/S asthma, M/S CRSwNP, and M/S AD cohorts, respectively, and 24%, 36% and 16% had at least two T2Cs; trends were similar in the US and EUR5. In patients with M/S asthma or M/S CRSwNP, T2Cs commonly presented as mild or moderate. The comorbidity burden suggests that an integrated treatment approach is warranted to address underlying type 2 inflammation in patients with M/S type 2 diseases.This observational study assessed the prevalence of co-existing type 2 inflammatory conditions  in patients with moderate-to-severe (M/S) type 2 asthma, M/S CRSwNP, or M/S AD, in the real-world setting. Data from 761 physicians in the US and EUR5 were sourced from Adelphi Disease-Specific Programmes covering patients with M/S asthma (The online version contains supplementary material available at 10.1007/s00408-023-00603-z. The overexpression of type 2 inflammatory pathways, involving activation of T helper type 2 cells and type 2 innate lymphoid cells , signifiQuantifying the overlap of co-existing type 2 inflammatory diseases (T2Cs) in asthma, CRSwNP, and AD will allow a better understanding of the associated prevalence and disease burden. Patients with comorbid diseases are more refractory to treatment for their primary disease, and such data will help provide insight for clinicians regarding disease overlap. Limited data reporting on prevalence and severity of T2Cs among patients with moderate-to-severe (M/S) asthma, M/S CRSwNP, and M/S AD currently exists. In this study, prevalence of common T2Cs was evaluated in patients with M/S asthma, M/S CRSwNP, or M/S AD. The severity of T2Cs was also reported in patients with M/S asthma and patients with M/S CRSwNP.This was an observational, real-world study based on country-specific, point-in-time survey data collected from January 2017 through February 2020 from physicians practicing in the United States (US) and in five European countries .Real-world data were sourced from three Adelphi Disease-Specific Programmes (DSPs) covering asthma, CRSwNP, and AD. DSPs are cross-sectional, country-specific surveys of physicians and patients in routine clinical practice . A sampland ≥ 1 exacerbation, with either blood eosinophil count ≥ 150 cells/μL or intermediate/high fractional exhaled nitric oxide (based on physician judgement); or alternatively were included if they had persistent (≥ 3 months) maintenance oral corticosteroid (OCS) dependence. Patients with M/S CRSwNP were required to have maintenance or acute OCS in their treatment history or ≥ 1 prior sinus surgery for CRSwNP and M/S “nasal blockage” in the past 2 weeks. Patients with M/S AD were required to have received topical therapy  and either had disease described by their physician as changeable, deteriorating slowly, deteriorating rapidly or were suitable candidates for a systemic therapy (immunosuppressants or injectable corticosteroids or biologics) according to their physician; or alternatively were currently receiving or had previously received a systemic therapy for AD.To be included in the analyses, patients with M/S type 2 asthma needed to have persistent use (≥ 3 months) of medium-to-high dose inhaled corticosteroid/long-acting beta-2 agonist Common T2Cs evaluated for patients with M/S asthma, M/S CRSwNP, or M/S AD were AR , asthma , AD , and CRSwNP .Descriptive analyses were conducted to determine patient demographics and prevalence of T2Cs among patients with M/S asthma, M/S CRSwNP, or M/S AD. Physician-assessed distribution of severity of T2Cs among patients with M/S asthma and M/S CRSwNP was also evaluated.Overall, 761 physicians from the US and EUR5 identified 899 patients with M/S asthma, 683 with M/S CRSwNP, and 1497 with M/S AD who met the study inclusion criteria. Patient demographics are summarized by disease cohort in Online Resource 1.Overall, at least one T2C was identified in 66%, 69%, and 46% of patients with M/S asthma, M/S CRSwNP, and M/S AD, respectively. Similar proportions were reported in the US  and EUR5 . Among patients with a primary diagnosis of M/S asthma, 3% had all three of the common T2Cs, 21% had two T2Cs, and 43% had one T2C. Similar proportions were reported in the US  and EUR5 . For patients with a primary diagnosis of M/S CRSwNP, 4%, 32%, and 34% had three, two, and one T2Cs, respectively, with similar proportions reported in the US  and EUR5 . Among patients with a primary diagnosis of M/S AD, the respective proportions were 1%, 15%, and 30%. Similar proportions were reported in the US  and EUR5 .Among patients in the M/S asthma cohort, there was a high prevalence (60%) of comorbid AR, while comorbid AD and comorbid CRSwNP occurred in 15% and 17% of cases , and economic burden , 10, 11.In this study, a high proportion (~ 50–70%) of patients with M/S asthma, M/S CRSwNP, or M/S AD presented with at least one T2C, and many patients had at least two T2Cs. In patients with M/S asthma or M/S CRSwNP, T2Cs commonly presented in mild or moderate forms and less frequently in severe forms; these trends were consistent within US and EUR5 cohorts. These findings are consistent with previous observations in patients with M/S type 2 diseases. Prevalence of comorbid asthma has reportedly been as high as 65% in a study assessing 40 consecutive patients with CRSwNP from a single Danish center . Our preThe high comorbidity burden highlighted in this study, in terms of number and severity of T2Cs, suggests the importance of identifying patients with type 2 inflammation. The high prevalence of co-existing diseases in these patients highlights the importance of assessing comorbidities as part of routine care and the need for an integrated, multidisciplinary treatment approach to address underlying type 2 inflammation, and to improve HRQoL and overall outcomes in patients with type 2 diseases.Supplementary file1 (DOCX 397 kb)Below is the link to the electronic supplementary material.


These functional copolymers are bearing different types of complexing units  which are well-known ligands for various metals. They have been prepared by reversible addition–fragmentation chain-transfer (RAFT) polymerization in order to obtain well-defined gradient copolymers. The copolymers have been characterized by proton nuclear magnetic resonance (1H-NMR) spectroscopy, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, thermal gravimetric analysis (TGA), dynamical scanning calorimetry (DSC) and cloud point measurements in dense CO2. All the investigated metal-complexing copolymers are soluble in dense CO2 under mild conditions (pressure lower than 30 MPa up to 65 °C), confirming their potential applications in processes such as metal-catalyzed reactions in dense CO2, metal impregnation,  or metal extraction from various substrates (solid or liquid effluents). Particularly, it opens the door to greener and less energy-demanding processes for the recovery of metals from spent catalysts compared to more conventional pyro- and hydro-metallurgical methods.The synthesis and characterization of a platform of novel functional fluorinated gradient copolymers soluble in liquid and supercritical CO Most industrial processes involve the usage of metals ,2,3. TheDifferent processes are already used at an industrial scale for metal recovery. These treatments are usually performed through pyrometallurgy, hydrometallurgy, and leaching methods ,16,17. T2 . Above its critical point , scCO2 has properties in between those of liquid and gas, such as high density and high diffusivity. Furthermore, dense CO2 is a non-polar solvent, unable to solubilize metals; thus, additives are required for metal extractions. So far, different reports have shown the use of low molecular weight compounds,  which can act as complexing additives malononitrile (DCTB) diluted at 20 mg/mL in dichloromethane and sodium trifluoroacetate (Naco-FDA) gradient copolymer, the matrix was dithranol diluted at 20 mg/mL in dichloromethane and the cationizing agent was NaI diluted at 5 mg/mL in acetone. The polymer concentration was 10 mg/mL in hexafluoro-isopropanol (HFIP). The polymer and matrix were mixed in a 1:5 volume ratio. The NaI layer was first deposited on the target and dried. After evaporation of solvent, the mixture, composed of polymer and matrix, was placed on the top of the NaI layer in the MALDI target.Regarding the P, leading to gradient copolymer structures. In addition, we observed by 1H-NMR of the crude reaction medium at a given time that the complexing monomers  polymerize faster than FDA, which confirmed the theoretical data (2-philic monomer units (gradient structure).The synthesis of the proposed (co)polymers  has beenA = 0.45 , Q4VP =  = 0.84, , QAAEM =M = 0.13 . The Q a = −0.58 . From th = −0.58 : r4VP = cal data . Thus, tn,targeted) (co-FDA). The associated results are gathered in The targeted molecular weight (Margeted)  was set n,targeted and Mn,NMR. The higher values obtained for Mn,NMR may be explained by (co)polymer chain fractionation during the precipitation step, causing the loss of lower molecular weight chains.P(FDA) with a molecular weight of around 5000 g/mol and four copolymers with a molecular weight of ca. 10,000 g/mol were successfully obtained. All the polymers were purified by precipitation in pentane. P(FDA) homopolymer and the respective copolymers with DPPS and 4VP were obtained as a fine red powder. Instead, the fluorinated copolymers containing AAEM and StySAc units were isolated as a pink sticky gum after precipitation. After drying under a vacuum, they become fragile foams that can be easily milled into powder. High monomer conversions were reached (from 80 to 100% conversion) and quite good correlations are observed between M1H-NMR analyses were performed to determine polymer chain lengths, as well as the ratio between complexing and CO2-philic monomer units  for the other (co)polymers. The selection of this unusual solvent was due to the different solubility of the polymers in common solvents: the fluorinated copolymers with AAEM and StySAc units were also soluble in acetone and ethyl acetate, while the copolymer with 4VP is soluble in chloroform; contrarily, the other FDA homo- and copolymers were soluble exclusively in fluorinated solvents such as TFT or CFC-113. To calculate the polymer composition, the aromatic signal of the RAFT agent has been used as a reference. 1H-NMR of the P(FDA) homopolymer permits to easily identify the typical signal of the fluorinated units once polymerized, as well as the CTA signals  for the P(FDA), n,precipitated,P(FDA) equal to 5850 g/mol and DPFDA of 10.8 were estimated by 1H-NMR.To calculate the mean number and average degree of polymerization DP1H-NMR spectrum of P(4VP-co-FDA) showed the appearance of two broad peaks at δ = 6.5 and 8.5 ppm, in addition to the typical FDA signal at δ = 4 ppm ( = 4 ppm . These sAs for the P(FDA) homopolymer, 1H-NMR spectrum obtained after FDA and AAEM copolymerization and recorded using acetone-d6 as a solvent, shows two different forms of the AAEM moiety, as ketone and as enol, with 4.5 mol% of the enolic conformation (The ormation .AAEM = 18.9 and DPFDA = 17.7 for the copolymer P(AAEM-co-FDA) according to 1H-NMR spectrum  copolymer is only soluble in TFT and CFC-113 solvents, allowing exclusively to perform the 1H-NMR analysis using C6D6 capillaries for locking (P(DPPS- locking . A broadco-FDA) were estimated using co-FDA)n,precipitated,P copolymer is slightly soluble in acetone, and soluble in chloroform. Its 1H-NMR spectrum shows a broad peak between δ = 6.5 and 7 ppm relative to the aromatic protons of the StySAc monomer (P(StySAc- monomer .co-FDA), it needs to take into account the overlapping at δ = 4 ppm of the typical methylene signal of the FDA units with the benzylic signal of StySAc units, using To calculate the degrees of polymerization and the molecular weight of the P(StySAc-co-FDA) copolymer were thus estimated.Through all these data, it was possible to estimate the weight fractions of complexing monomer units and FDA monomer units for all the copolymers according to general (Co)polymer structures were also confirmed by MALDI-TOF-MS analyses using reflectron positive ion mode see .co-FDA) was detected between 1800 and 5750 m/z CHCH3(AAEMn-co-FDAm)SC(S)C6H5 + Na+, i.e., one CTA end-group associated with some units of AAEM and some units of FDA monomer incremented with one sodium cation due to ionization.The main population corresponds to the species ionized with sodium with the formula CHm/z; FDA = 518.02 m/z), , or 214 (MAAEM = 214.08 m/z), . The number of repeating units of AAEM (n) and FDA (m) confirms the presence of copolymer. The comparison of the experimental mass/charge (m/z)expt values with the calculated monoisotopic mass/charge (m/z)calc values are presented in AAEM = 214.08 m/z, MFDA = 518.02 m/z, MCTA = 254.04 m/z and MNa = 22.99 m/zA more detailed analysis of the MALDI-TOF-MS data in the region corresponding to the strongest peaks expt values for these series of peaks were in good correlation with the calculated (m/z)calc values  copolymer exhibit a similar shape and are therefore in good agreement (co-FDA) copolymer.Lastly, note that the isotope patterns of theoretical and experimental sodium adducts of P homopolymer and four other P(FDA)-based copolymers constituted of complexing monomer units were therefore successfully synthesized by RAFT and well characterized.2-soluble metal-complexing (co)polymers. The aminolysis of the different fluorinated (co)polymers has been performed using three equivalents of piperidine. In order to avoid disulfide formation, triphenylphosphine was added as a reductant in a previously degassed solution of the polymer in TFT (We took advantage of the RAFT technique to introduce a thiol group in our (co)polymers by aminolysis of the (co)polymer chain end. Indeed, thiols are known to complex some metals,  and can therefore bring additional properties to our COr in TFT .Usually, end-group removal of RAFT polymers is performed by primary amines, such as hexylamine, butylamine, or cyclohexylamine ,81. The 1H-NMR spectra of the polymer before and after aminolysis by n-butylamine, it is possible to see some differences in the signal related to the acetoacetoxy protons  led to a secondary reaction for the fluorinated copolymer containing AAEM units. Indeed, comparing the  protons .1H-NMR spectra of the aminolyzed copolymers are given in the In The polymers before and after aminolysis were thermally characterized in order to investigate their thermal stability  as well as the presence of thermal transitions   Table 3Table 3. 2wt%loss (temperature where 2% of the weight is lost) higher than 200 °C for all the polymers, except the aminolyzed P(FDA)SH homopolymer (197 °C) and P(StySAc-co-FDA) copolymer (160 °C). In addition, higher T2wt%loss are obtained for the copolymers after aminolysis (except for P(FDA) homopolymer): this behavior can be explained by the thermal elimination of the terminal phenyldithio group. Indeed, the cleavage of the dithio group in the case of unreacted CTA (R-S-C(S)Z, in this work, R = EtOC(O)CH(CH3)- and Z = Ph) occurs close to 200 °C, depending on the R group of the CTA. The higher residual mass by TGA after removing the end-group coming from the RAFT agent is also consistent with some works showing the lower thermal stability of RAFT polymers, even sometimes opening the route to a free depolymerization approach [co-FDA) > P(AAEM-co-FDA) > P(DPPS-co-FDA) > P(StySAc-co-FDA).TGA analyses show Tapproach ,85. On tapproach ,86,87. Tm), respectively, at 63 and 56 °C, before and after aminolysis, while no glass transition (Tg) was detected for both homopolymers. On the other hand, all the gradient copolymers exhibit a glass transition temperature and a melting temperature before and after aminolysis, except P(DPPS-co-FDA) and P(DPPS-co-FDA)SH copolymers that are 100% amorphous with only a Tg visible at 67 and 68 °C, respectively. Note that Tg-values are higher after aminolysis of the gradient copolymers. A decrease in the Tg suggests lower intermolecular interactions between the polymer chains, which can be caused by the lower Lewis base character of AAEM and StySAc moieties compared to 4VP and DPPS.DSC analyses show that P(FDA) and P(FDA)SH homopolymers present a melting temperature  to the cloudy suspension (polymer insoluble) and vice versa. The obtained data, recorded in a cooling process (meaning screening the temperature from 65 down to 25 °C), are shown in The phase behavior in dense COis forms . To this2, which is a major industrial application of supercritical fluids, is routinely operated at a pressure of 22 MPa and a temperature of 90 °C [All the synthesized polymers are soluble at pressures lower than 27 MPa up to 65 °C, which can be considered mild conditions. For the sake of comparison, decaffeination by scCOof 90 °C . Mild opg . On the contrary, the copolymer containing DPPS and 4VP units have shown an easier precipitation behavior during the purification step, forming a powder and showing a higher glass transition temperature . These facts allow us to draw a correlation between the Tg of the copolymers and their solubility in dense CO2: the higher Tg indicates an increased interaction between the polymer chains, decreasing the solubility of the polymers in dense CO2 [2 once incorporated in a polymeric chain, bringing a reduced solubility in dense CO2 [2-philicity [co-FDA) ≅ P(AAEM-co-FDA) > P(DPPS-co-FDA) ≅ P(4VP-co-FDA).Notably, three different trends can be identified in ense CO2 . These bense CO2 . Insteadhilicity . In summ2. Interestingly, the aminolysis reaction, with the introduction of a polar thiol group as polymer chain end instead of dithiobenzoate moiety, did not influence significantly the solubility of these polymers: the cloud point pressure only increased by a few MPa  at 65 °C) at a given temperature. Thus, this limited difference in cloud point pressure does not preclude the good solubility in dense CO2 of such thiol-terminated polymers. The mild conditions necessary to solubilize this wide range of polymers in a green solvent such as CO2 demonstrate the remarkable relevance of this library of metal-complexing CO2-soluble polymers for their application as extractants for the removal of metals from industrial or urban waste.Furthermore, the solubility of the aminolyzed polymers has also been investigated in dense CO-co-FDA) copolymers showed their ability to be used as palladium ligands [-co-FDA)-PdII supramolecular complex. The resulting polymer-supported Pd catalyst was successfully used for various applications such as a catalyst for Heck reaction, generation of Pd0 nanoparticles in the polymer matrix, or PdII impregnation of mesoporous silica in scCO2 followed by reduction to Pd0 . In addition to Pd, P(AAEM-co-FDA) copolymer has also shown its ability to complex cobalt and more precisely cobalt acetate hydrate. Complexes Co-P(AAEM-co-FDA) exhibit cloud point pressure approximately 30 to 40 bar higher than the copolymer alone [-co-FDA) and aminolized P(DPPS-co-FDA)SH gradient copolymers were used to extract palladium from commercial Pd/Al2O3 supported catalysts [2 under mild conditions (40 °C and 25 MPa). Polymers were found to successfully extract up to ca. 40% of Pd from the Pd/Al2O3 catalysts. P(DPPS-co-FDA)SH was the most efficient system, requiring a much lower quantity than the two other polymers to achieve the same level of extraction in one hour. Lastly, P(4VP-co-FDA) and aminolyzed P(4VP-co-FDA)SH gradient copolymers showed their ability to extract Pd from the aluminosilica-supported catalyst in scCO2 under mild conditions (40 °C and 25 MPa). More precisely, ca. 70% of the Pd was removed from an aluminosilica support in the presence of P(4VP-co-FDA) copolymer [Preliminary experiments have already been performed by our team to verify the ability of such polymers to complex metals. Thus, P. These (co)polymers were successfully synthesized by the reversible-deactivation radical polymerization technique (RAFT). 1H-NMR characterization allowed us to determine the composition as well as the molecular weight of the synthesized (co)polymers. In addition, the thermal properties of the (co)polymers have been studied by TGA and DSC, which confirms the good thermal stability of the (co)polymers  = 263 °C > Tco-FDA)2wt%loss,P(4VP- = 249 °C > Tco-FDA)2wt%loss,P(AAEM- = 239 °C > Tco-FDA)2wt%loss,P(DPPS- = 215 °C > Tco-FDA)2wt%loss,P(StySAc- = 160 °C) and their difference in Tm (Tco-FDA)m,P(AAEM- = 41 °C < Tco-FDA)m,P = 63 °C < Tco-FDA)m,P(4VP- = 85 °C) and Tg transition temperatures (Tco-FDA)g,P(AAEM- = 8 °C < Tco-FDA)g,P(StySAc- = 24 °C < Tco-FDA)g,P(4VP- = 61 °C < Tco-FDA)g,P(DPPS- = 67 °C). Finally, their phase behavior in dense CO2 has been studied by cloud point measurements, showing that these metal-complexing (co)polymers are soluble at mild pressure conditions (lower than 27 MPa at 65 °C), which is a key factor for their eventual application as metal extraction agents. Through the synthesis of well-defined copolymers  and their promising CO2 solubility, this library of metal-complexing CO2-soluble polymers show good candidates for the recovery or the decontamination of end-of-life metals-containing liquid or solid wastes. The investigation of their performances in metal extractions from supported catalysts is in progress and will be reported in forthcoming articles.In this work, we have presented the synthesis of ten (co)polymers which are constituted by a CO


Meanwhile, increasing slaughter weight reduced sarcomere length, the proportion of protein-bound water, and most kinds of muscular total amino acid contents except for tryptophan and arginine, while increased contents of muscular free lysine, tryptophan, leucine, isoleucine, valine, alanine, and arginine in the M. longissimus thoracis (P < 0.05). Health lipid indices based on fatty acid composition of intramuscular lipid were improved as the slaughter weight increased (P < 0.05). Notably, pigs received N-R (V/I) diet improved carcass traits in terms of thinner backfat thickness and higher fat-free lean index, as well as increased meat flavor-contributing amino acids at the cost of reduced intramuscular fat content and increased shear force of cooked meat compared with the pigs fed L-R (V/I) and H-R(V/I) diets (P < 0.05). H-R (V/I) diet decreased ultimate pH value and sarcomere length of the skeletal muscle but increased the proportion of free water (T23), consequently, increased drip loss and cooking loss of fresh meat in pigs (P < 0.05). In conclusion, both slaughter weight and dietary ratio of valine to isoleucine exerted significant impacts on carcass characteristics, meat quality and nutrition values. In particular, carcass traits and meat color of lighter pigs were more susceptible to the influence of dietary R (V/I) relative to heavier pigs.The aim of this study was to investigate effects of dietary ratio of valine to isoleucine [R(V/I)] on carcass characteristics and meat quality of finishing pigs and whether slaughter weight influence the effect. We carried out a 2 × 3 factorial trial with two slaughter weight (105 vs. 130 kg) and three R(V/I) , respectively). Data show that increasing slaughter weight significantly increased meat color (a* Branched-chain amino acids (BCAAs), including leucine, isoleucine and valine, play a vital role in skeletal muscle protein synthesis and metabolism. The amount of dietary leucine usually exceeds the requirement of pigs in commercial diets, while isoleucine and valine are regarded as limiting amino acids following lysine, methionine, threonine and tryptophan for obtaining optimal growth performance of pigs. The considerable wide range of BCAA combinations  has been reported to increase lipid accumulation in skeletal muscle and promote protein metabolism and muscle growth in growing pigs . FurtherThe compositions of muscular free amino acids and fatty acids are of great interest in meat industry since they are important for meat edible quality , 6. We sBesides, slaughter weight is an important factor determining the economic profitability of pork production. Presently, pig slaughter weight especially in China trends to increase from 110 kg to more than 130 kg in order to reduce production costs and earn more margin profit . HoweverThe effect of dietary nutrient level on carcass characteristics and meat quality may be influenced by slaughter weight . HoweverTogether, given economic benefit in the swine industry, the nutrition and eating quality of meat, we aimed to explore the effects of dietary ratio of valine to isoleucine [R(V/I)] on carcass characteristics and meat quality of finishing pigs and whether slaughter weight influence the effect.All procedures conducted in the present study were approved by the Institutional Animal Care and Use Committee of China Agriculture University (ID: SKLAB-B-2010-003).2) with 50% of slatted floors in an environmentally controlled room in FengNing Swine Research Unit of China Agricultural University .Fifty-four crossbred castrated male pigs (Duroc × Landrace × Yorkshire) with an initial body weight of 74.1 ± 1.3 kg were allocated into three dietary treatments in a randomized complete block design based on the initial body weight (BW), with six replicates (pens) per treatment and three piglets per pen. All the pigs were housed in pens (1.8 × 2.1 mn = 6). The remaining 36 pigs shift to diets for 100–135 kg BW  after overnight starvation for 16 h. Serum was separated and stored at −20°C for further analysis. Pigs were transported to local abattoir. After rest at least 4 h, the pigs were electrically stunned, exsanguinated and eviscerated according to the standard commercial procedure. Moreover, at 30 min postmortem, chops of the M. longissimus thoracis from the 10th to 12th ribs of each left carcass were separated and trimmed off extra-muscular fat and connective tissues. After the determination of meat color and pH at 45 min (pH45 min), these chops were vacuum-packed at 4°C to measure subsequently meat quality, including muscle pH at 24 h (pH24 h), drip loss, cooking loss and shear force as well as NMR transverse relaxation (T2) measurement. About 100 g of chops were stored at –20°C for measurement of contents of moisture, crude protein and IMF, as well as amino acid and fatty acid composition. For myofibril ultrastructure examination, the M. longissimus thoracis between the 9th and 10th rib was cut to 1 mm3 and stored immediately in fixing solution .Before the slaughter, blood samples were collected from the precaval vein of pigs close to the average BW of each pen (2) = loin eye height (cm) × width (cm) × 0.7).Dressing percentage was calculated by dividing the hot carcass weight by slaughter weight. Backfat depth at the thickest shoulder, last rib, 6th to 7th rib, 10th rib, and the last lumbar vertebra were recorded. Fat-free lean index = 50.767 + [0.035 × hot carcass weight (Ib)] – [8.979 × the last rib fat thickness (in.)]  . Loin ey45 min value was measured with a SPK pH meter . Meanwhile, the pH24 h value was determined at 24 h postmortem in 4°C chilling room.At 45 min postmortem, meat color  was measured with a Minolta Chroma Meter , pH2 parallel to the muscle fiber orientation to measure the tenderness using a digital-display muscle tenderness meter . Twelve replicates of each sample were measured.Drip loss was determined by a suspension method and calculated as follows: drip loss (%) = [/initial weight] × 100. The pork cooking loss and shear force was measured. Briefly, the M. longissimus thoracis chops were cooked to an internal temperature of 70°C for 30 min in 75°C thermostatic water bath. Chops were allowed to cool to room temperature, wiped with absorbent paper to remove residual moisture and reweighed to calculate cooking loss. Then the cooked chops were cut to 1 × 1 × 3 cmSamples of the M. longissimus thoracis were cut into 2–3 mm thin slices and freeze-dried using vacuum frozen dryer  and ground into powder for analysis of crude protein, IMF, fatty acid and amino acid. The IMF content was analyzed by Soxhlet petroleum-ether extraction. The crude protein content was measured according to Association of Analytical Chemists methods .T21), immobilized water  and free water .Low field NMR transverse relaxation was measured as described previously . BrieflyAs previously described , 2.5% glg, 4°C for 10 min to collect the supernatant.The free amino acid concentration in the M. longissimus thoracis was analyzed using ultra high-performance liquid chromatograph  and high-resolution mass spectrometer  as previously described . About 3Total amino acid (mainly protein-bound amino acids) profile of the M. longissimus thoracis was determined based on the standard methods in AOAC .g for 3 min. The supernatant was analyzed by the Gas Chromatography .The composition of fatty acids of the M. longissimus thoracis was analyzed as described previously . About 1The health quality of the lipid components of muscle was assessed by calculating: (1) the index of atherogenicity (IA), (2) the index of thrombogenicity (IT), and (3) the ratio between the hypocholesterolemic and hypercholesterolemic fatty acids (HH).The index of atherogenicity (IA) indicates the relationship between the sum of the main saturated fatty acids and that of the main classes of unsaturated ones. Saturated fatty acids are considered pro-atherogenic (favoring the adhesion of lipids to cells of the immune and circulatory system). However, unsaturated fatty acids are qualified as anti-atherogenic .The index of thrombogenicity (IT), defined as the ratio of the pro-thrombogenetic (saturated) to the anti-thrombogenetic (unsaturated) fatty acids, shows the tendency to form clots in the blood vessels.The following equations were used to calculate these indexes:- Atherogenic Index - Thrombogenic Index - Ratio between hypocholesterolemic and hypercholesterolemic fatty acids where Σ = Summatory, MUFA = monounsaturated fatty acids, and PUFA = polyunsaturated fatty acids.P-value for interaction terms was above 0.05, the interaction was excluded from the statistical model. If the interaction was significant, Tukey’s test was used to compare treatment means within a slaughter weight. Differences were considered significant when P < 0.05.Data were analyzed using the mixed procedure SAS 9.2  following a completely randomized design according to a 3 (R(V/I)) × 2 (slaughter weights) factorial arrangement with individual pig as the experimental unit. The statistical model included the fixed effects of the R(V/I), slaughter weight and their interaction and the pig as random effect of the experiment. When P < 0.05). Pigs offered N-R (V/I) diet had the thinnest backfat thickness at the shoulder, last rib, and highest fat-free lean index among all pigs slaughtered at 105 kg BW (P < 0.05). In contrast, no effect of R (V/I) on the last rib fat thickness and fat-free lean index was observed in pigs slaughtered at 130 kg BW. Significant interactions existed between R (V/I) and slaughter weight on the last rib fat thickness and fat-free lean index.As shown in min45  (P = 0.03), b*45 min, pHh24  value, shear force, marbling score and drip loss as well as intramuscular fat content significantly increased (P < 0.01), while crude protein level decreased (P < 0.01). Dietary R (V/I) significantly affected most indices of meat quality, especially in N-R (V/L) treatment, shear force (P = 0.02) was the highest while intramuscular fat content (P = 0.05) was the lowest among dietary treatments. Additionally, significant interaction between slaughter weight and dietary R (V/I) was observed on pH values (P < 0.01), a*min45  (P = 0.03) and marbling score (P = 0.02).As shown in T21) and free water (T23) decreased with the slaughter weight increased, while the proportion of immobilized water (T22) increased (P < 0.01). Pigs fed with L-R (V/I) diets had the highest proportion of bound water and pigs fed with H-R (V/I) diets had the highest proportion of free water, while pigs fed N-R (V/L) had the lowest proportion of free water and highest proportion of immobilized water (P < 0.01). The significant interaction between slaughter weight and R (V/I) was found on the proportions of bound water (P = 0.02) and immobilized water distribution (P = 0.03).As shown in P < 0.01). The myofibrils of pigs fed with L-R(V/I) diet were tightly integrated with visible I and A band, and the Z lines, actin and myosin could be distinguished. However, the most decayed myofibrils structure could be found in the M. longissimus thoracis of pigs fed H-R(V/I) diet, in which myofibrils gradually fractured along the Z line. What’s more, along with dietary R (V/I) increased, the sarcomere length decreased in pigs slaughtered at 105 kg body weight, but no effect observed in pigs slaughtered at 130 kg body weight.The myofibril ultrastructure was shown in P = 0.03), leucine, isoleucine, valine, alanine, arginine, and bitter amino acid were significantly increased (P < 0.01), while methionine, asparagic acid and glycine were decreased (P < 0.01). Besides, the amounts of muscular free phenylalanine, leucine, valine, alanine, asparagic acid and glutamic acid, glycine were significantly altered by dietary R (V/I) treatments (P < 0.05). Highest amounts of umami and sweet amino acid were observed in pigs fed N-R (V/I) diet compared with those in pigs fed H-R (V/I) and L-R (V/I) diet (P < 0.05). The significant interactions were observed between slaughter weight and dietary R (V/I) on muscular free methionine, phenylalanine, tryptophan, leucine, isoleucine, asparagic acid, proline, tyrosine and bitter amino acid (P < 0.05). The effects of dietary R (V/I) on these muscular free amino acids depended on the slaughter weight.Muscular free amino acid profile was presented in P < 0.01) (P < 0.05). There was no interaction between diet R (V/I) and slaughter weight.We determined amounts of protein-bound amino acids of fresh meat. Interestingly, we found that muscular contents of most protein-bound amino acids except tryptophan and arginine significantly decreased with the slaughter weight increased ( < 0.01) . H-R (V/P < 0.01)  .P < 0.05). Meanwhile, L-R (V/I) and N-R (V/I) dietary treatments increased the composition of C10:0 and C17:0 compared with H-R (V/I) treatment. Interaction between R (V/I) and slaughter weight were observed for C20:3n3 and C22:6n3.Highest compositions of C14:1, C20:3n6, C20:4n6, C20:3n3, C24:0, C22:6n3 and C24:1 were observed in pigs fed N-R (V/I) diet compared with those in pigs fed H-R (V/I) and L-R (V/I) diet , drip loss and shear force of cooked meat increased as pig slaughter weight increased, while loin eye area of carcasses remained unchanged. The result of slaughter weight on meat color supported by previous studies, the M. longissimus thoracis of pigs slaughtered at heavy body weight was redder  than that of pigs slaughtered at slight body weight in crossbred Duroc × Landrace × Yorkshire pigs , immobilized water (T22) and free water (T23). Besides, the free water reflects extra- myofibrillar water, which is mainly contributing to potential drip loss (T22) was higher whereas the peak area ratio (%) of bound water (T21) was lower in pigs slaughtered at 130 kg BW than at 105 kg BW, as a consequence, the drip loss of fresh meat was higher in pigs slaughtered at 130 kg BW than pigs at 105 kg BW. And for the first time, we found that increasing slaughter weight reduces sarcomere length, which might also be partly responsible for the increased drip loss by the increased slaughter weight.According to previous studies , water drip loss . CompareIt is generally accepted that the fatty acid composition of intramuscular lipid in pork changed greatly with the increased slaughter weight . ConsistCompared with lighter pigs, heavier pigs exhibited higher IMF content and lower content of protein in the heavier pigs, which is in line with the previous study in crossbred Duroc × Landrace × Yorkshire pigs . HoweverT23), consequently, undermined fresh meat WHC. Compared with pigs fed N-R (V/I) diet, pigs fed L-R(V/I) or H-R(V/I) diet had increased backfat thickness and IMF content, indicating that unbalanced dietary supply of valine and isoleucine (L-R(V/I) and H-R(V/I) diets) could lead to the obese in finishing pigs.The proportion of branched-chain amino acids could alter porcine fat mass . ParticuAlthough dietary R (V/I) exerted a secondary impact on muscular fatty acid profile compared with slaughter weight, we observed that balanced dietary valine and isoleucine supply (N-R (V/I) increased several PUFA, such as C20:3n6, C20:4n6, C20:3n3 and C22:6n3. In addition, we found that pigs received N-R (V/I) treatment increased muscular free amino acids that contribute to meat flavor, including the umami taste amino acids (aspartic acid and glutamic acid), sweet taste amino acids , but did not affect bitter taste amino acids  compared with pigs received other two dietary treatments. Pigs fed the diet with N-R (V/I) had the highest nutritional value in terms of the muscular abundance of essential amino acids  and non-essential amino acids .We also observed significant interaction between dietary R(V/I) and slaughter weight on the last rib backfat thickness, fat-free lean index, and meat color. The effect of R(V/I) on these indicators weakened with the increase of slaughter weight, indicating that pigs with heavier slaughter weights are more resistant to the effected of dietary R(V/I) treatments.Increased slaughter weight markedly favored meat color and IMF content but undermined water holding capacity of fresh meat and tenderness of cooked meat in finishing crossbred Duroc × Landrace × Yorkshire pigs. Pigs received N-R (V/I) diet improved carcass traits and meat flavor-contributing amino acids at the cost of reduction in IMF content and increase in shear force of cooked meat compared with the pigs fed L-R (V/I) and H-R(V/I) diets. In particular, the effects of dietary R(V/I) on carcass traits and meat color were influenced by slaughter weight.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of China Agricultural University.DX performed the animal experiments, analyzed the data, and wrote the manuscript. JY, XZ, and DX contributed to the experimental design and manuscript modification. YW was mainly responsible for the sample collection and data curation. EY, LH, LW, CM, and PZ carried out meat quality test. All authors approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Despite several CAR-T products being approved and commercialized in the USA, Europe, and China, CAR-T products still require additional optimization to ensure reproducible and cost-effective manufacture. Here, we investigated the critical parameters in the CD3+ T cell isolation process using CTS™ Dynabeads™ CD3/CD28. Different isolation media, cell-bead co-incubation time, and cell density were examined in this study. Activated CD3+ T cells were transduced with a gamma retroviral vector carrying the CD19 or BCMA CAR sequence. The CAR-T cells proliferated in a culture medium supplemented with interleukin 2 (IL-2).CAR-T cells were prepared from cryopreserved peripheral blood mononuclear cells (PBMC). The thawed PBMC was rested overnight before the CD3+ monocytes hindered T-cell isolation when X-VIVO 15 basic medium was used as the selection buffer. The activation of T cells was blocked because monocytes actively engulfed CD3/28 beads. In contrast, when DPBS was the selection medium, the T-cell isolation and activation were no longer blocked, even in patients whose PBMC contained abnormally high CD14+ monocytes and a low level of CD3+ T cells.CD14+ T-cell isolation media is critical for improving T-cell activation, transduction, and CAR-T proliferation. Using DPBS as a CD3+ T cell isolation buffer significantly improved the success rate and shortened the duration of CAR-T production. The optimized process has been successfully applied in our ongoing clinical trials.In this study, we discovered that selecting CD3Trial registration NCT03798509: Human CD19 Targeted T Cells Injection Therapy for Relapsed and Refractory CD19-positive Leukemia. Date of registration: January 10, 2019. NCT03720457: Human CD19 Targeted T Cells Injection (CD19 CAR-T) Therapy for Relapsed and Refractory CD19-positive Lymphoma. Date of registration: October 25, 2018. NCT04003168: Human BCMA Targeted T Cells Injection Therapy for BCMA-positive Relapsed/Refractory Multiple Myeloma. Date of registration: July 1, 2019The online version contains supplementary material available at 10.1186/s12967-022-03833-6. Chimeric antigen receptor T (CAR-T) cell therapy is a profound breakthrough that significantly changed the medical practice for hematological malignancies in the twenty-first century. The long-lasting efficacy in hematological malignancies, including acute lymphoblastic leukemia (ALL) and B-cell non-Hodgkin’s lymphoma (NHL), offers the cure for many patients –3. NeverUnlike many traditional pharmaceutical products, autologous CAR-T products are living products manufactured from whole blood or leukapheresis collected from each patient as the starting material. Generally, the manufacture of CAR-T cells consists of five principal processes. The first step is the processing of starting material. The leukapheresis or whole blood goes through either peripheral blood mononuclear cells (PBMC) separation by density gradient centrifugation and wash procedure to remove red blood cells. The second step is the T-cell enrichment step, typically achieved using paramagnetic beads by either a positive or negative selection method. Beads conjugated with anti-CD3 with and without anti-CD28 antibodies could be used for positive selection and T-cell activation in one step. Alternatively, PBMCs were first depleted of non-T cells using paramagnetic beads conjugate with various antibodies targeting CD19/CD14/CD16/CD56 to deplete B cells, monocytes, and NK cells T-cell activation reagents were added later to promote T cells viral transduction and proliferation. Third, T cells are transduced with CAR genes targeting specific antigens, such as CD19 or B-cell maturation antigen (BCMA), via a viral vector or non-viral techniques. Fourth, transduced CAR-T cells proliferate and expand in media supplemented with recombinant interleukin 2 (IL-2) and other cytokines.Finally, expanded CAR-T cells are cryopreserved and kept in liquid nitrogen before infusion –10. Desp+ T cells. PBMC, isolated from leukapheresis and the expected starting material for CAR-T production, consists of normal lymphocytes, monocytes, leukemia, and lymphoma cells. Therefore, the T-cell purification step is necessary for successful CAR gene transduction and subsequent T-cell expansion, but also it is crucial for safety considerations. This process is usually achieved by incubating PBMC with anti-CD3/CD28 antibody-coated paramagnetic beads or degradable nano matrix in a suitable isolation buffer, followed by a capture step to positively select CD3+ T cells with a magnum [+ cell isolation step and the degree of CAR-T cell expansion. Subsequently, we found that buffer types are the key input influencing CAR-T cell expansion response via establishing the “Design of experiments” (DoE) to evaluate factors of the CD3 isolation process, including buffer types and other process parameters. The optimized process based on this DoE analysis was applied to our ongoing clinical trials and greatly improved CAR-T cell expansion in monocyte-rich PBMC samples. The modification presented in this study could be easily implemented in an already established and widely-used protocol to improve CAR-T products’ quality and success rate significantly.The essential step in CAR-T manufacture is the purification and activation of CD3a magnum . Previou+ T-cell isolation step. Our original process, which strictly followed the manufacturer’s procedure, used Dulbecco's phosphate-buffered saline (DPBS) or X-VIVO15 basic medium as the isolation buffer depending on the CD3+ cell percentage in PBMC [+% higher than or equal to 30%, whereas we used X-VIVO 15 basic medium if the CD3+% was below 30%. To simplify the process, we tested the possibility of using a CAR-T culturing medium as the isolation buffer regardless what the percentage of CD3+ T cells in the purified PBMC. Three batches of PBMC samples from patients’ apheresis were randomly chosen as starting material for this experiment. Patient CHZH-10-053 was diagnosed with NHL, while the other two were diagnosed with multiple myeloma (MM). Although the efficiency of CD3+ cell selection was similar when different donor PBMCs were used, we observed that T cells isolated from one donor PBMC was poorly activated  or registered clinical trials . We reasoned that more patients would be treated if we could improve the manufacturing process to reduce manufacturing failure. At the beginning of this study, we encountered a problem during the first step of CAR-T manufacture, e.g., the bead-based CD3 in PBMC . DPBS wa+ T cells could be detected, which was consistent with the failure of T-cell activation   were significantly higher than healthy donors (around 5%) [+% in PBMC were higher. By dividing patients into two groups using 40% CD14+% in PBMC as the cut-off  were purposely selected and used to examine how the success rate of CAR-T manufacture would be affected by the content of monocytes. As done previously, thawed PBMCs were incubated with CD3/28 Dynabeads in an X-VIVO15 basic medium. T cells were isolated by positive selection and put into T-75 tissue culture flasks for additional incubation. After carefully examining flasks under an inverted microscope, we noticed many adherent cells on the culture surface, and cells were actively engulfing Dynabeads  in the positively selected PBMC from two of the three donors whose CD14+ cells before the selection was relatively higher , but the T-cell expansion was still less than usual. We also found that positively selected T cells were still contaminated with 0.5% CD14+ cells. We suspected that these minor populations of CD14+ cells were still significant enough to consume Dynabeads and resulted in poor T-cell stimulation. However, we could not exclude the possibility that these CD14+ monocytes were immunosuppressive MDSC found in cancer patients that could actively suppress the T-cell activation [+% in the starting material.Both CD25 and CD69 expression were barely increased after activation, and T-cell blast formation was delayed until 5 or 7 days after retroviral transduction Fig. c–e. Althfer Fig. f, g. Agativation . These o6 cells/mL), and incubation duration . We focus on the output responses CAR-T expansion fold at the experiment endpoint and upregulation of T-cell activation markers after T-cell selection and activation, including CD25 expression and CD69 expression in T cells and cell diameter. Experiment groups in the DoE and the levels of factors investigated are listed in Table + cells  were used in this DoE to ensure the complete application of this optimized process in the future clinical trials where a wide range of CD3+% in PBMC will be encountered. The DoE was repeated three times, and nine PBMC samples were used.To systematically optimize parameters that determine the optimal condition for CAR-T manufacture, we used DoE further to define an optimal T-cell selection and activation parameter. In addition to the choice of selection buffer, other parameters such as T-cell density during Dynabeads incubation and incubation duration were also included. In DoE, the process parameters we wanted to optimize were “input factors,” while the results we obtained were “output responses.” To create a design space or determine a better option for process parameters, we set up different “levels” of an input factor at the beginning of DoE. Input factors of our DoE in the T-cell isolation step are choice of isolation media , T-cell density during Dynabeads incubation . This simple solution to use DPBS instead of X-VIVO culture medium in T-cell selection will significantly impact the CAR-T manufacturing process development and is currently adapted into several CAR-T product lines for our ongoing phase I/II clinical trials.Because of the apparent improvement with DPBS as the T-cell selection buffer over our original process, we implemented the change to our manufacturing process. We produced CAR-T cells to treat 13 additional MM patients in an ongoing phase I BCMA CAR-T clinical trial NCT04003168). A wide range of CD148. A wideAdopting T cell therapies for cancer using genetically modified T cells started in the 1980s, and only recently, CAR-T-cell immunotherapy has experienced rapid development and clinical success . From 19The first challenge we face in CAR-T manufacture is how to enrich and activate T cells from a patient’s PBMC since a proper activation of T cells is critical for efficient CAR gene transduction and proliferation of transduced T cells. The Dynabeads™ Human T-Activator CD3/CD28, superparamagnetic micron beads coated with anti-CD3 and anti-CD28 mAbs are widely used for research, clinical trial, or commercial CAR-T products. The first FDA-approved CAR-T product, Kymriah, used Dynabeads . T-Cell +% found in PBMC. We discovered that failed activation and low proliferation of T cells happened more often when PBMC contained a higher percentage of CD14+ monocytes. Initially, we suspected that the high CD14+% in PBMC was the potential culprit for the failure. Several publications in the literature showed that CD14+ monocytes in mobilized peripheral stem cells (PSC) and PBMC isolated from cancer patients inhibited T-cell activation. In the 1990s, scientists realized that CD14+ monocytes in granulocyte–macrophage colony-stimulating factor (GM-CSF) mobilized PSC products, and peripheral blood post-transplantation could induce apoptosis of activated T cells [First, when using a medium containing human plasma as the isolation buffer in the Dynabeads co-incubation step, T cells from some donors were poorly activated, and their CAR-T cells barely proliferated. To determine the underlining reason, we did a retrospective analysis of data from our clinical trial. The original process development strictly followed the recommended choice of T-cell selection buffer based on the CD3 T cells , 23. As  T cells , 25.+ cell depletion in the CAR-T manufacturing [+ monocyte content threshold for an additional plastic adhesion step needed to remove adherent monocytes for an overall successful manufacturing rate of 97%. Noaks et al. presented similar results: CAR-T cells made from CD14+ cells-depleted samples outperformed untouched leukapheresis in various aspects, including transduction efficiency, cytotoxicity, and T cell stemness [. a few years ago also suggested that plastic adhesion helps remove CD14+ cells and benefit the CAR-T cell expansion [In 2021, two groups presented data conveying the importance of CD14acturing , 27. Wanacturing . Their sxpansion , 28.+ monocyte and from whom we failed to generate enough CAR-T cells to treat patients. One major characteristic of DPBS is that it is free of calcium and magnesium ions. These two divalent cations are closely related to cell adhesion on solid surfaces and the cell–cell interaction [Next, we planned experiments with the DoE tool to optimize the critical parameters. Surprisingly, we found that the choice of isolation buffer between X-VIVO15 and DPBS was the critical feature that determined the success of T-cell activation and CAR-T cell expansion. By using DPBS as the isolation buffer, we significantly improved the T-cell activation and proliferation of CAR-T cells as compared with cell culture media used in our original process, even for patients whose PBMC contained more than 60% CD14eraction –32.Without calcium and magnesium ions, we suspected that fewer monocytes would be attached to the surface of the culture flask. Although most of the composition is not revealed in the datasheet provided, the X-VIVO15 medium we used for the study and the clinical trials mentioned in this article contains human native transferrin. One study published in 2019 has shown increased phagocytic capacity and expansion rate of microglial cells treated with transferrin . Microgl+ cells in PBMC nor by the types of T cell isolation buffer. However, Wang et al. demonstrated that depleting CD14+ cells by plastic adhesion could elevate CAR transduction efficiency and CAR-T cell expansion at the same time [+ cells were not depleted, but the T cell isolation buffer change mitigated their capability of inhibiting T cell activation and proliferation. While in the research done by Wang et al. apheresis composition was changed after plastic adhesion.In our study, unlike CAR-T cell expansion, CAR transduction efficiency was not affected by the proportion of CD14ame time . One of . addressed the problem of the CD14 + cells by enriching apheresis through CD14+ cells depletion or using a depletion kit with a combination of blood cell capturing beads other than CD3+ T cells [+ cells depletion apheresis and isolated CD3+ T cells. Stroncek et al. used plastic adherence to deplete CD14 + cells and measured the CAR transduction efficiency for GD2 CAR-T production but not for the CD19 CAR-T production [+ cell depletion. It might depend on the methods and CAR-T constructs used in CAR-T production.Noaks et al T cells . Their soduction , 28. To + monocytes, including Percoll density gradient centrifugation, plastic adhesion exploiting the characteristic of monocytes, and immunomagnetic beads selection pursuing higher purity with anti-CD14 beads. However, these techniques, used in an academic setting or clinical trials, pose a significant hurdle to developing a cGMP-compliant process suitable for the pharmaceutical manufacture of CAR-T cells. Some methods, such as plate adhesion, are easy to perform in open procedures with culture well-plates and centrifuge tubes; it could often take an unsurmountable effort to adapt this step to a closed system. Removing CD14+ monocyte remains an open process, despite all other actions being performed in a closed system [There are many methods to deplete CD14d system .+ monocyte retention in the T-cell isolation process results in poor T-cell activation and diminished CAR-T proliferation. We also demonstrated that a simple change of T cell isolation buffer from X-VIVO to DPBS in the CD3/28 Dynabeads co-incubation step dramatically improved the success rate of manufacturing CAR-T products from PBMC with a high level of CD14+ monocytes and a low percentage of CD3+ T cells. This simple amendment is currently adapted by our ongoing phase I/II clinical trials, and we are planning to use this modification in closed or automated CAR-T manufacturing systems. The simple solution described in this study is the most effective method to eliminate monocyte problems and is readily employed in a close process.A 9% failure rate has been reported in one of the Tecartus clinical trials . A univeFurthermore, we have successfully modified our manufacturing process. Our ongoing clinical trials showed that CAR-T products manufactured with this new process are highly effective for our MM clinical trial patients. We have improved the manufacturing process with a straightforward change in the T-cell selection buffer. Our study provides an example of how crucial continuous manufacturing process optimization is via discovering manufacturing obstacles in previous clinical trials and doing experiments with the DoE tool.PBMC was isolated from apheresis products obtained from consented patients enrolled in clinical trials conducted by Hrain Biotechnology Co., Ltd. The isolation of PBMC fraction was performed with Sepax C-Pro Cell Processing System  and its built-in NeatCell C-Pro Protocol developed by the manufacturer. In brief, the tubing system of the bags containing apheresis product, Ficoll , wash buffer, and freezing media was connected to CT-90.1 C-Pro Sepax Kit , which was then loaded onto C-Pro. By running the NeatCell program on the instrument, leukapheresis was fractioned. The PBMC layer was pushed into freezing bags after a few wash rounds and resuspended in freezing media. Freezing bags with PBMC were sealed and separated from the tubing system. PBMC were cryopreserved using a controlled-rate freezer and stored in a liquid nitrogen tank.l-cysteine  and 5% human plasma in X-VIVO15 . Then, PBMC was rested for 20–24 h at 37 ℃ with 5% CO2 in a cell culture flask. Finally, PBMC has been washed and resuspended in an isolation buffer for CD3+ T cell selection. This study tested three isolation media: DPBS , X-VIVO15  basic medium, and CAR-T culturing medium. X-VIVO15 basic medium consists of X-VIVO15  with 0.2% NAC , 1% HEPES , and 1% GlutaMAX . Positive selection of CD3+ T cells was made by co-incubating CTS™ Dynabeads™ CD3/CD28  with PBMC at a 1:1 ratio at room temperature followed by magnetic capture of bead-bound cells on DynaMag™-5 Magnet . Selected cells were resuspended in CAR-T culturing medium supplemented with 300 IU/mL IL-2  and incubated at 37 ℃ with 5% CO2 for 44–52 h to complete T cell activation.Cryopreserved PBMC was thawed at a 37 ℃-water bath and washed and resuspended in CAR-T culturing medium composed of 1% GlutaMAX , 1% HEPES , 0.2% N-Acetyl-2, cells were washed and transferred to a new PL70-2G  bag for CAR-T proliferation under the same media and culture condition as during transduction. Again, CAR-T cell numbers were counted, and the cell density was adjusted to 3 × 105 cells/mL every 1–3 days.Before retroviral transduction, the mixture of activated T cells and beads was first applied to a DynaMag™-5 Magnet for the bead removal and then washed with CAR-T culturing medium. Next, gamma retroviral vector carrying CD19 CAR sequence and activated cell suspension were transferred into RetroNectin-coated PermaLife cell culture bags PL70-2G  with the addition of 300 IU/mL IL-2 . After 18–24 h coculture at 37 ℃ with 5% COFlow cytometry was performed with MACS Quant with the following antibodies: human BD Fc Block , Viability Dye , Pacific Blue anti-human CD14 Antibody , FITC anti-human CD19 Antibody , PE-Cy7 anti-human CD56 Antibody , APC-Cy7 anti-human CD3 Antibody , APC anti-human CD16 Antibody , BV421 anti-human CD25 Antibody , FITC anti-human CD69 Antibody , PerCP anti-human CD8a Antibody , PE-Cy7 anti-human CD4 Antibody , Biotin Rabbit Anti-Mouse FMC63 scFv , and BV421-Streptavidin .GraphPad Prism and JMP were used for conducting statistical analysis.Additional file 1: Cell density in cell-bead co-incubation is not critical in the T cell selection process. Description: During the cell-bead co-incubation step, cell density (ranging from 4*106 to 10*106 cells/mL) did not affect T cell activation, CAR-T proliferation, and CAR transduction efficiency. (a) CD25 expression percentage in CD3+ cells, (b) CD69 expression percentage in CD3+ cells, and (c) live cell diameter before CD3+ T cell selection and after 48-hour T cell activation. In (a), (b), and (c), an unpaired t-test was used, and two-tailed P value was calculated between 4*106 cells/mL and 7*106 cells/mL, 4*106 cells/mL and 10*106 cells/mL, and 7*106 cells/mL and 10*106 cells/mL; ns, P>0.3000. (d) Changes of CD25 expression percentage in CD3+ cells, (e) CD69 expression percentage in CD3+ cells, and (f) live cell diameter in the CAR-T manufacturing process. (g) Expansion fold of CAR-T cells after retroviral transduction. (h) CAR expression percentage in CD3+ cells at days 6 and 9 after retroviral transduction.Additional file 2: Cell-bead co-incubation duration is not critical in the T-cell selection process. The cell-bead co-incubation time, ranging from 30 minutes to 60 minutes, during T cell selection did not affect T cell activation, CAR-T proliferation, and CAR transduction efficiency. (a) CD25 expression percentage in CD3+ cells, (b) CD69 expression percentage in CD3+ cells, and (c) live cell diameter before CD3+ T cell selection and after 48-hour T cell activation. In (a), (b), and (c), an unpaired t-test was used, and a two-tailed P value was calculated between 30 minutes and 45 minutes, 30 minutes and 60 minutes, and 45 minutes and 60 minutes; ns, P>0.3000. (d) Changes in CD25 expression percentage in CD3+ cells, (e) CD69 expression percentage in CD3+ cells, and (f) live cell diameter in the CAR-T manufacturing process. (g) Expansion fold of CAR-T cells after retroviral transduction. (h) CAR expression percentage in CD3+ cells at days 6 and 9 after retroviral transduction.