Acinetobacter spp. This new classification system may facilitate better clinical management of patients infected by TetX-producing strains.Recently, many TetX variants such as Tet(X3~14) were reported to confer resistance to tigecycline which is a last-resort antibiotic used to treat infections caused by multidrug-resistant bacteria. In this study, we identified essential residues including 329, 339, 340, 350, and 351 in TetX variants that mediated the evolution of the tigecycline-inactive Tet(X2) enzyme to the active forms of Tet(X3) and Tet(X4). Based on their amino acid sequences and functional features, we classified TetX variants into TetX-A class, TetX-B class and TetX-C class. We further found that TetX-A class variants originated from Bacteroidetes, with some variants further evolving to TetX-C class and acquired by Enterobacteriaceae. On the other hand, our data showed that some variants genes belonging to TetX-A class evolved directly to TetX-B class, which was further transmitted to  TetX variants such as Tet(X3~14) were reported to confer resistance to tigecycline which is a last-resort antibiotic used to treat infections caused by multidrug-resistant bacteria. Here, Cheng et al. propose a new classification system for TetX variants. Because of their broad-spectrum activity and the low cost, tetracyclines are extensively used in clinical treatment of human infections, as well as applications in the field of veterinary medicine and agriculture, since their discovery in the 1940s. Tetracyclines inhibit protein synthesis by binding reversibly to the bacterial 30 S ribosomal subunit and arresting translation, exerting steric hindrance effect on the docking of aminoacyl-transfer RNA (tRNA) during elongation2. Due to the widespread use of tetracyclines, resistance in clinical and food isolates is increasingly reported. A range of mechanisms of resistance are known to mediate tetracycline resistance, such as efflux pumps, ribosomal protection, rRNA mutations and enzymatic inactivation3. To counteract these resistance mechanisms, a semisynthetic glycylcycline known as tigecycline was approved for clinical use by FDA in 20054. Tigecycline has become increasingly important in treating bacterial infections, since it is one of the few antibiotics which is still effective against a range of newly emerged multidrug-resistant Gram-positive and Gram-negative bacterial strains5. Although tigecycline can overcome two main resistance mechanisms, namely ribosomal protection and efflux, resistance to tigecycline has been reported7. In particular, several plasmid-borne tet(X) variant genes that confer clinically significant level of tigecycline resistance have recently been detectable among clinical strains, compromising the effectiveness of this relatively new tetracycline drug in clinical treatment of bacterial infection12.Tetracyclines are a group of antibiotic compounds that have a common basic structure (a linear fused tetracyclic nucleus) which exhibit activity against a wide range of microorganisms, including Gram-positive and Gram-negative bacteria13. As a flavin-dependent monooxygenase, flavin adenine dinucleotide (FAD) as a cofactor bound to TetX and TetX strictly required exogenous nicotinamide adenine dinucleotide phosphate (NADPH) to catalyze the oxidation reaction which inactivates most of the tetracyclines in vitro, including tigecycline15. To date, several TetX variants, designated as, TetX, Tet(X1), Tet(X2), Tet(X3), Tet(X4), Tet(X5), Tet(X6), Tet(X7), Tet(X8), Tet(X9), Tet(X10), Tet(X11), Tet(X12), Tet(X13), and Tet(X14) have been identified in various bacterial species. Compared to TetX, Tet(X2) only has one mutation at residue 94 . Based on these findings, we hypothesize that an increasing number of TetX variants that can confer tigecycline-resistance will continue to emerge as a result of deep surveillance of clinical and food isolates. Designating the TetX variants an increment number is not an appropriate way to label a large number of functionally different enzyme variants. It is necessary to design a new system for functional classification for TetX variants to facilitate clinical management of infections caused by bacterial strains that produced different TetX variants, and therefore exhibit different levels of susceptibility to tigecycline. In this study, we propose that TetX variants should be classified into three major groups, namely TetX-A class, TetX-B class, and TetX-C class, depending on enzymatic activity and genetic features.TetX is one of the flagship tetracycline-inactivating enzymes that can catalyze the degradation of tetracyclines, which was first proposed as a resistance mechanism in 1984217, H279, T280, L282, E295, P297, V329, A339, D340, V350, K351 and I359 were found -producing strains . Strains that contain amino acid substitutions at three sites, such as those carrying the V329L/ A339T/D340N, V329L/ V350I/K351E, V329M/ A339T/D340N, and V329M/ V350I/K351E changes, exhibited tigecycline MIC of 8, 4, 4, 8 mg/L, which represent 8, 4, 4 and 8-fold increase, respectively. Multiple mutations, such as those which lead to as many as four amino acid changes (A339T/D340N/V350I/K351E), also caused the tigecycline MIC to increase to 8 mg/L. Furthermore, two mutants which contained five amino acid changes, namely V329L/A339T/D340N/V350I/K351E and V329M/A339T/D340N/V350I/K351E, both exhibited tigecycline MIC of 16 mg/L, which is similar to that of Tet(X3) and Tet(X4) (Table 329V/T339A/N340D/I350V/E351K and Tet(X4)-M329V/T339A/N340D/I350V/E351K exhibited decreasing MIC  against tigecycline comparing to the wild type, respectively (Table Tet(X3), Tet(X4), Tet(X5), Tet(X6), Tet(X7), TetX14) but not TetX and TetX2), were shown to confer resistance to tigecycline. We aligned the representative amino acid sequences of each of these enzymes in an attempt to identify common amino acid substitutions in Tet(X3) to Tet(X14) that might contribute to elevated tigecycline MIC. Several common changes at residues S4 but not, were shkcat/Km values of them are 8.09 × 105 M−1 S−1, 1.16 × 106 M−1 S−1 and 2.33 × 105 M−1 S−1, respectively -L329V/T339A/N340D/I350V/E351K and Tet(X4)-M329V/T339A/N340D/I350V/E351K was lower than that of Tet(X3) and Tet(X4) but was about 1.3–3.1 folds higher than that of Tet(X2) for hydrolysis of tigecycline  , Tet(X3), Tet(X4) and mutants were also detected by Western Blotting. All test proteins displayed small changes (0.8–1.5 folds) comparing to Tet(X2) under T7 promoter in , Tet(X3)196L, Q197H, S217A, H279R, T280S, T280V, T280L and L282S changes, which are commonly found in Tet(X3) to Tet(X14), occur within the second domain of the protein (cyan), which is implicated largely in tigecycline recognition. On the other hand, the other commonly found changes in the high activity variants of tetracycline, such as E295D, E295G, E295N, K296N, P297D, P297S, V329M, A339T, and D340N changes, were found to occur in the FAD-binding domain (deep olive). In addition, residues where the V350I, K351E, I359M and I359V occur were located in a C-terminal alpha-helix (red), which could stabilize the other two domains19. The T280 residue was closer to the putative O2 binding pockets and has previously been suggested to interfere with O2 diffusion. Therefore, the T280V or T280S change might also affect O2 transport within the enzyme. In previous directed evolution studies, a mutant carrying the T280A change could be selected in the presence of minocycline and tigecycline21. The structure of the Tet(X2) (T280A) complex with minocycline showed that position 280 was not directly involved in the catalytic mechanism of the enzyme but the T280A substitution Tet(X2) (T280A) changed the enzyme kinetics of Tet(X2) indirectly perhaps through altered the protein dynamics20. In the Tet(X2) (V329M/A339T/D340N/V350I/K351E) mutant model, the A339T substitution makes van der Waals contact with the side chain of N38  activity, we mapped the site of these substitutions against the complex structure of Tet(X2) with tigecycline Fig.  and found in TetX to Tet and Tet(X11) could be classified into new TetX-A class , Tet(X5), Tet(X6), Tet(X7), and Tet(X14)  and similar activity on tigecycline according to our mutational analysis data, which showed that the V329M, A339T/D340N, and V350I/K351E amino acid substitutions elevated tigecycline MICs to the same level as original Tet(X4), Tet(X5), Tet(X6) and Tet(X7) . We also propose that they should be classified as TetX-B class. Two independently reported TetX variants from Acinetobacter baumannii and Empedobacter brevis could be classified into this class22. This definition is based on the fact that it contains one different substitution at position 329 when compared to TetX-C defined above. Consistently, carriage of similar patterns of important amino acid substitutions including V329L, A339T/D340N, V350I/K351E, implied that the catalytic activity of the newly defined TetX-B enzyme is similar to TetX-C class and played a key role in elevating tigecycline MICs. Based on the effect of these amino acid changes in enzyme functions, there might be two branches of the evolution pathway for TetX variants. Apart from the one which involves evolution from TetX-A to the newly defined TetX-B and TetX-C, respectively  as the template sequence returned a total of 128 related homologs with the annotation of TetX. Based on the analysis of the multiple sequence alignment, we defined that sequences lacking the ABacteroides fragilis was used to root the phylogenetic tree, tree was divided into four substitution rate categories. Four major branches can be depicted by the phylogenetic tree and are aligned well to our newly defined TetX-A class, TetX-B class, and TetX-C class. TetX variants evolved originally from the TetX-A class to TetX-C class, and subsequently to TetX-B. It should be noted, however, that categorization of some TetX variants by functional classification is not consistent with their position in phylogenetic analysis. For example, three variants belonging the TetX-A class  were under a same phylogenetic branch with TetX-C class protein , Tet(X5), Tet(X6), Tet(X7), and Tet(X14). From the evolutionary viewpoint and the perspective of phylogenetic relationship, these variants exhibit the closest genetic relationship with TetX-A class and should therefore classified as TetX-C class. The newly TetX-B class comprise most of the original Tet(X3) enzymes; apart from the V329L/A339T/D340N/V350I/K351E changes found in Tet(X3), they also contained some conservative amino acid substitutions hence they should be regarded as a group derived from further evolution events that occur among the TetX-B class variants. Functional classification is more informative than phylogenetic classification alone. In this work we found newly TetX-C class were distributed in three parts basing on phylogenetic analysis  is known to confer resistance to tetracycline. Its significance was brought up recently due to the reports of new variants of Tet(X2), namely TetX3~14), which are responsible for causing resistance to tigecycline among members of Enterobacteriaceae, as tigecycline has become the last-resort antibiotic to treat clinical infections caused by Carbapenem-resistant Enterobacteriaceae (CRE). The increasing prevalence of clinical strains producing these TetX variants will undermine the choice of treatment for clinical CRE infections. However, due to presence of various TetX-related amino acid sequences available in GenBank and a lack of data regarding the functional characteristic of these protein sequences, the current definition of TetX variants is confusing. With the advent of sequencing technology in recent years, the number of sequences of TetX variants deposited into the Genbank will continue to increase, rendering current nomenclature of TetX variants insufficient to depict the functional types of these enzymes. It is urgent to develop a new classification system according to the functional and amino acid sequence characteristics of TetX variants. The key problem in developing a classification system for TetX variants lies in the large sequence variation between TetX variants. We believe that the best classification system for TetX variants should be a function-based system. In this study, we tested this logic and identified functionally amino acid substitutions that can help distinguish between Tet(X/X2) which exhibit no or very low catalytic activity on tigecycline, and TetX variants with high catalytic activity. Using sequence comparison and mutational analysis, we identified key residues that enable us to classify some TetX variants as a new TetX-C class. With the signature amino acid substitutions of V~14, whicBacteroides spp., Chryseobacterium spp. and Riemerella spp. being the dominant host species  and TetX-A variant (WP_150433355.1) were both from Empedobacter brevis strain SE1-3 but were located on different plasmid pSE1-3-9kb and pSE1-3-14kb, respectively  (WP_063856436.1), tet(X3) (WP024160783), and tet(X4) (WP094309310) genes were amplified from dairy cows’ clinical isolates25 by PCR with primers listed in Supplementary Table tet(X2), pBAD-18-tet(X3) and pBAD-18-tet(X4) were transformed into E. coli BW25113 and followed by antimicrobial susceptibility tests. In addition, the recombinant plasmids pET28-6×His-tet(X2), pET28-6×His-tet(X3) and pET28-6×His-tet(X4) were transformed into E. coli strain BL21(DE3) for protein purification. Point mutations were introduced into the tet(X2) gene, tet(X3) gene and tet(X4) by using the QuickChange (Stratagene) commercial kit, following the manufacturer’s instructions, and confirmed by sequencing. Primers used in mutagenesis are also listed in Supplementary Table The Bacteroides fragilis is same to that of the first identified TetX (Q01911)27. PSI-BLAST 28 was performed with the amino acid sequence of Tet(X2) (Accession number: WP_063856436.1)23 as the query sequence and searched on nr database with default value. The result yielded 128 TetX variants sequences with query cover >90% and percent identity >80%. These sequences were subjected to multiple sequence alignments by Clustal Omega29, the results were used to construct a phylogenetic tree using the online software PhyML 3.030. The tree was visualized using iTOL31.The sequence of a TetX variant (Accession number: WP_063856436.1) from 32. For tigecycline, the breakpoint was interpreted according to the FDA criteria 9. E. coli strain ATCC 25922 was used as a quality control.The MICs of three antibiotics  for strains were determined using the microbroth dilution method and the results were interpreted according to the CLSI guidelinesLuria Broth (LB) containing 50 mg/L kanamycin was inoculated with 1% overnight culture, followed by incubation with shaking at 37 °C until an optical density of 0.6 at 600 nm (OD600) was reached. Expression of enzymes was induced by 0.5 mM IPTG at 16 °C for 16 h. The cells were harvested by centrifugation at 7000 rpm for 15 min and resuspended in lysis buffer , and broken with sonication. The soluble fractions were passed through a Ni column, rinsed by 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 10–30 mM imidazole, and finally eluted with 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 250 mM imidazole. The 6xHis tag was removed by thrombin . The target proteins were further purified by gel filtration chromatography  in a buffer of 20 mM Tris (pH 7.5), 150 mM NaCl, and 1 mM DTT. The desired fractions were collected and concentrated. The purity of protein was determined by SDS-PAGE  for 3 minutes under a dim light condition at ambient temperature. Initial reaction velocities were determined for linear regression by the UVProbe 2.70 Software and fitted to the Michaelis–Menten equation using GraphPad Prism 8 .Each enzyme reaction contains 0.1 M TAPS buffer at pH 8.5 with 0–40 μM substrate, 5 mM MgClE. coli BL21(DE3) carrying pET28-6 × His-tet(X2), pET28-6 × His-tet(X3), pET28-6 × His-tet(X4) and the variants were diluted 1:100 into 5 mL LB broth containing 50 μg/mL kanamycin. Cells were grown to OD600 = 0.6 at 37 °C and induced by adding 1 mM IPTG for 4 h at 30 °C. Cell was harvested by centrifugation at 13,000 rpm. Cell lysates were solubilized by boiling with SDS running buffer for 10 minutes and were subsequently separated by SDS-PAGE. Proteins were transferred to a PVDF membrane followed by blocking by skimmed milk for 1 h and incubated with mouse anti-6 × His antibody  at 4 °C overnight. The goat anti-mouse antibody  was used as the secondary antibody. The signal was generated by HRP substrate and detected by ChemiDoc Touch System . Tet(X2) was used as a positive control on each protein gel and cells containing the empty vector was used as a negative control. The broad range anti-GADPH  was used as loading control. Band intensities were quantified using ImageJ software.Overnight cultures of http://www.rcsb.org/pdb/). Structures of TetX variants were generated according to their amino acid sequence, using the comparative protein-modeling SWISS-MODEL server33. The structures were analyzed and showed by the PyMOL software34.The structure of Tet(X2) (PDB accession number 4A6N) was obtained from the Protein Data Bank (Statistical analysis was conducted with GraphPad Prism 8. Statistical methods used in this work are described in methods part and the figure legends.Supplementary Information


Anas platyrhynchos)/South Korea/KNU 2019-48/2019(H6N6))—was isolated from Anas platyrhynchos in South Korea. Phylogenetic analysis results revealed that the hemagglutinin (HA) gene of this strain belongs to the Korean lineage, whereas the neuraminidase (NA) and polymerase basic protein 1 (PB1) genes belong to the Chinese lineage. Outstanding internal proteins such as PB2, polymerase acidic protein, nucleoprotein, matrix protein, and non-structural protein belong to the Vietnamese lineage. Additionally, a monobasic amino acid (PRIETR↓GLF) at the HA cleavage site; non-deletion of the stalk region (residue 59–69) in the NA gene; and E627 in the PB2 gene indicate that the KNU2019-48 isolate is a typical low-pathogenic avian influenza (LPAI) virus. The nucleotide sequence similarity analysis of HA revealed that the highest homology (97.18%) of this isolate is to that of A/duck/Jiangxi/01.14 NCJD125-P/2015(H6N6), and the amino acid sequence of NA (97.38%) is closely related to that of A/duck/Fujian/10.11_FZHX1045-C/2016 (H6N6). An in vitro analysis of the KNU2019-48 virus shows a virus titer of not more than 2.8 Log10 TCID 50/mL until 72 h post-infection, whereas in the lungs, the virus is detected at 3 dpi (days post-infection). The isolated KNU2019-48 (H6N6) strain is the first reported AIV in Korea, and the H6 subtype virus has co-circulated in China, Vietnam, and Korea for half a decade. Overall, our study demonstrates that Korean H6N6 strain PB1-S375N, PA-A404S, and S409N mutations are infectious in humans and might contribute to the enhanced pathogenicity of this strain. Therefore, we emphasize the importance of continuous and intensive surveillance of the H6N6 virus not only in Korea but also worldwide.The subtype H6N6 has been identified worldwide following the increasing frequency of avian influenza viruses (AIVs). These AIVs also have the ability to bind to human-like receptors, thereby increasing the risk of animal-human transmission. In September 2019, an H6N6 avian influenza virus—KNU2019-48 (A/Mallard ( Orthomyxoviridae family and are composed of eight single-stranded negative-sense RNA segments. The AIVs have been identified based on the antigenic properties of the hemagglutinin (HA) and neuraminidase (NA) glycoproteins. AIVs are categorized into 18 HA (H1–16 in wild birds and H17–18 in bats) and 11 NA (N1–9 in wild birds and N10–11 in bats) subtypes [Avian influenza viruses (AIVs) belong to the subtypes . Furthersubtypes ,4,5.Downie and Laver reported in 1973 that the H6 subtype AIVs were isolated in 1965 in Massachusetts in the United States . SimultaGlobally, H6 subtype AIVs were divided into the following two major clades: (i) The first group clade mainly consisted of H6N2 and H6N6 AIVs from Asian regions; (ii) The second group clade was mostly composed of H6N1 and H6N2 AIVs from Asian regions and other regions around the world . Huang eThe aim of this study is to perform a phylogenetic and mutational analysis of H6N6 AIVs isolated from mallard ducks in South Korea in 2019. Moreover, to the best of our knowledge, there are no reports related to the pathogenetic analysis of South Korean H6N6 AIVs; thus, for the first time, we elucidate the complete pathogenesis of South Korean H6N6 AIVs through in vitro and in vivo experimental infections in a mammalian model. During the surveillance period from January 2019 to December 2019, a total of approximately 4253 wild-bird fresh fecal samples were collected from the fields in Gyeonggi-do, South Korea (Latitude—37°23’46.8” N and Longitude—127°56’08.9” E) Fecal samples were processed according to our previously described protocol . The colHost bird species were determined by confirmation of a DNA barcode of a region consisting of 751 base pairs (bp) of the mitochondrial gene cytochrome c oxidase I (COXI) as previously described elsewhere . The ideA NucleoSpin RNA kit  was used to extract the viral RNA directly from the allantoic fluid of ECEs according to the manufacturer’s instructions. Finally, the extracted RNA was eluted in RNase-free water distributed with 20 units of RNase inhibitor and was stored at −80 °C for further analysis. Conventional real-time RT-PCR was performed to determine the presence of the influenza virus and its subtypes using total RNA following WHO  guidelines . NGS analysis was accompanied by GnCBIO  on the Illumina Hiseq X platform method as previously reported . Brieflyhttps://www.ncbi.nlm.nih.gov/ (accessed on 27 March 2022)) and GISAID ) databases. Data were merged, all duplicated sequences were deleted, and phylogenetic trees were finally constructed using MEGA6.0 software . Phylogenetic trees of all segments  of the KNU2019-48 (H6N6) viral isolate were generated by applying the maximum-likelihood with Tamura-Nei model and 1000 bootstrap replicates.Nucleotide blast analysis was used to identify pertinency of the viral genes. Entire reference sequences were downloaded from the NCBI  was used to measure TCIDreported , MDCK ceh method . To deteA assays , and EIDh method .50) of each supernatant was determined [The growth kinetics of virus isolates were evaluated in vitro. MDCK cells were infected with the viruses at an MOI (multiplicity of infection) of 0.001 in DMEM medium containing 1% antibiotic and 1 μg/mL TPCK-trypsin. Supernatants were collected every 12 hpi up to 72 hpi. ELISA was performed using anti-influenza nucleoproteins (PAN antibodies) to detect infected cells, and the virus titer (TCIDtermined .n = 11), which were intranasally infected with 105 EID50/mL of virus. Mice were anesthetized using 1% isoflurane following the manufacturer’s instructions . The survival rate and bodyweight of mice were observed for 15 days, following which the mice were euthanized, and their lungs (n = 3 mice) were collected at 3, 6, and 15 dpi. The lung tissue was homogenized, and the TCID50 was determined to analyze the viral titers of homogenate supernatants [The pathogenic potential of the new isolate was determined in mammals using 6-week-old female BALB/c mice purchased from Orient  . Results were presented as the mean ± SD. Statistical significance was set at p < 0.05.The calculation of mean and SD (standard deviation) and Student’s Anas platyrhynchos)/South Korea/KNU 2019-48/2019(H6N6), designated as KNU2019-48 (H6N6), samples were isolated from feces of Anas platyrhynchos in South Korea on September 18, 2019. The genome sequence information of the isolate was deposited in GenBank (accession numbers MW380639 to MW380646). The GenBank accession numbers of the eight gene segments and the highest nucleotide identities from the GenBank database are shown in A/Mallard  isolates in 2015. This was followed by the transmission of the HA gene from South Korea through the A/mallard/Korea/M219/2014 (H6N2) isolate in 2014. Similarly, based on the 2014–2015 report, the H6N6 strain NA gene co-circulated for a long time in China, which was later transmitted to South Korea. Next, the PB1 gene was transformed from Chinese isolates (A/duck/Guangdong/7.20_DGCP015-C/2017 (H6N6)).The other genes  were collectively reassorted from the Chinese isolate A/duck/China/330D17/2018 (H6N6) in 2018. The backbone of these isolates originated from the Vietnamese isolates of A/Muscovy duck/Vietnam/LBM755/2014 (H5N6) in 2014. Additionally, there might have been a major H6N6 transmission from China during the migration season of 2018–2019. The locations of the putative origins of genomic components of the KNU2019-48 (H6N6) strain are shown in Based on the evolutionary information, whole gene segments of our isolate KNU2019-48 (H6N6) originated from Chinese H6N6 isolates. It is possible that the Asian birds migrated through the East Asia–Australasian Flyway. Detailed information on the evolutionary re-assortment is presented in The HA cleavage site of KNU2019-48 (H6N6) contained the PRIETR↓GLF (↓denotes the cleavage site) sequence with a single basic amino acid in the HA cleavage site, which revealed a low pathogenic feature of the isolated virus. This HA receptor-binding site (RBS) suggests that H6 isolates would have a high preference toward α-2,6-linked sialic acid, which is abundant in the upper respiratory tract of avian species. We compared the HA protein of our isolate with those of the following four other isolated H6N6 strains that originated from birds and swine: (1) a low-pathogenic H6N6 isolate from domestic ducks in central China, labeled as A/duck/Hubei/10/2010 (H6N6); (2) A/swine/Guangdong/K6/2010 (H6N6) isolates, which originated from domestic ducks following whole gene reassortment; (3) isolates from poultry markets in Southern China that are a potential threat to mammals, labeled as A/duck/China/A729-2/2011(H6N6); (4) novel North American-origin avian influenza A (H6N5) virus isolated from bean goose in South Korea in 2018, which was published by our team in 2020, labeled as A/Bean goose/South Korea/KNU18-6/2018(H6N5).A reasonable analysis of HA RBS at positions 138, 186, 190, 226, and 228 (H3 numbering) shows no mutation in amino acid residues . The sin50 assay are shown in Since the molecular characterization shows reassortment via mutation, our viral isolate could increase replication efficiency; therefore, in vitro examination of the viral replication of our isolate along with other isolates was performed. To evaluate the growth kinetics of our isolate, human-origin viruses, A/California/07/2009 (H1N1) and H7N7, which are infectious to mammals, were used as controls. H1N1 replicated more efficiently in MDCK cells as compared to KNU2019-48 (H6N6) and H7N7 . Raw dat5 EID50/50 µL. The H1N1 and H7N7 strains were used as the controls for comparisons. The bodyweight of the mice infected with the positive control H1N1 strain decreased from 3 dpi and was gradually regained after 10 dpi, with the lowest weight (76.15 ± 1.96%) observed at 8 dpi. Similarly, our KNU2019-48 (H6N6) isolates and the H7N7 control strain demonstrated a stable body weight trend for 15 days  that were intranasally infected with 10 15 days A.50 assay. Based on the experimental results, control group H1N1 showed maximum virus titer in the lungs in comparison with the KNU2019-48 (H6N6) and H7N7 strains at 3 dpi , and these viral loads gradually decreased at 6 dpi   C. In conctively) .The H6N6 subtype AIV is widespread in poultry, and its host range extends to mammals such as pigs. Moreover, it has become an endemic disease in domestic animals . Our isoAccording to the molecular analysis of the H6N6 strain in our study, the HA cleavage site sequence PRIETR↓GLF was subtyped into a low-pathogenic virus. According to the results of phylogenetic analysis, the HA gene was closely related to A/mallard/Korea/M219/2014 (H6N2), while the genes of PB2, PA, NP, M, and NS were closer to the Chinese and Vietnamese isolates (A/duck/China/330D17/2018 (H6N6) and A/Muscovy duck/ Vietnam/LBM755/2014 (H5N6)). The PB1 gene was similar to that of the Chinese isolate, A/duck/Guangdong/7.20_DGCP015-C/2017 (H6N6), and the NA gene was similar to that of co-circulated Chinese isolates A/duck/Fujian/10.11_FZHX1045-C/2016 (H6N6) and A/chicken/Guangdong/8.30_DGCP022-O/2017(H6N6), respectively. These analyses strongly indicate that the H6 subtype virus has been frequently detected in migratory ducks in China ,64,65.Generally, H6 isolates receptor-binding sites were composed of HA protein residues (Q226 and G228), which preferentially bind to the α-2,3-linked sialic acid receptors in avian bird species . Similar10 TCID50/mL) was achieved at 24 hpi, and there were no changes until 72 h post-infection. Similarly, our isolate strongly exhibited low pathogenicity in the lungs of mice at 3 and 6 dpi compared to the positive control H1N1, which originated from humans. Additionally, we performed body weight and survival percentage analysis. Low pathogenicity was confirmed in intranasally-infected mice; mice lungs weighed the highest at 3 dpi, whereas at 6 and 15 dpi, no variance was observed, and the lung weight was normal  isolate using in vitro experiments. We monitored the growth kinetics of KNU2019-48 (H6N6) replication in MDCK cells as follows: the virus replicated promptly and exhibited no obvious signs of illness. A maximum virus titer (2.84 logs normal D. AlthouIn conclusion, this is the first study that broadens the knowledge of Korean H6N6 isolates from evolution to mammalian cell expressions and in vivo characterization. Continuous monitoring and molecular characterization of the H6N6 virus will be required for a better understanding of the evolutionary dynamics of the virus, which can further assist in improving control measures.


D− WT, D+ Cyp KO, and D− Cyp KO mice all exhibited significantly reduced survival compared to D+ WT mice. Importantly, survival was not the result of reduced viral replication, as influenza M gene expression in the lungs was similar for all animals. Based on these findings, additional experiments were performed using the mouse and hamster models of SARS-CoV-2 infection. In these studies, high dose vitamin D supplementation reduced lung inflammation in mice but not hamsters. A trend to faster weight recovery was observed in 1,25(OH)2D treated mice that survived SARS-CoV-2 infection. There was no effect of vitamin D on SARS-CoV-2 N gene expression in the lung of either mice or hamsters. Therefore, vitamin D deficiency enhanced disease severity, while vitamin D sufficiency/supplementation reduced inflammation following infections with H1N1 influenza and SARS-CoV-2.Vitamin D supplementation is linked to improved outcomes from respiratory virus infection, and the COVID-19 pandemic renewed interest in understanding the potential role of vitamin D in protecting the lung from viral infections. Therefore, we evaluated the role of vitamin D using animal models of pandemic H1N1 influenza and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. In mice, dietary-induced vitamin D deficiency resulted in lung inflammation that was present prior to infection. Vitamin D sufficient (D+) and deficient (D−) wildtype (WT) and D+ and D− Cyp27B1 (Cyp) knockout  Low vitamin D status is associated with poorer outcomes following acute respiratory diseases including influenza . Vitamin2D, 1,25D), has been implicated to play a role in the anti-viral response; however, this effect may be specific to different viruses. For example, 1,25D treatment of T cells from human immunodeficiency virus (HIV)-infected individuals in vitro resulted in a decrease in viral RNA transcription by a direct reduction in NF-κB, which reactivates proviral HIV [Vitamin D, and the active form of vitamin D iral HIV . Conversiral HIV . Productiral HIV ,11. Seveiral HIV . Catheliiral HIV ,15,16,17iral HIV ,19. Lung epithelial cells are vitamin D targets since they express the vitamin D receptor (VDR) and are regulated by 1,25D treatments. In mice, 1,25D treatments reduced inflammation following lipopolysaccharide-induced lung injury and regulated angiotensin converting enzyme (ACE) expression in the rat lung epithelium . In miceIfnb expression in the lung of mice following SARS-CoV-2 infection. Vitamin D treatments had no effect on the expression of viral RNA for either SARS-CoV-2 or H1N1 influenza in the lungs of hamsters or mice. Instead, the data support an important role for vitamin D and 1,25D in controlling the host inflammatory response to viruses in the lungs.Extensive association studies in humans led to the proposal that high dose vitamin D supplements could be beneficial for protection from severe influenza and SARS-CoV-2 infections. Since cause and effect are extremely difficult to determine in human studies, we sought to evaluate the effects of vitamin D on the lung anti-viral response in animal models. The data from mice and hamsters suggest that vitamin D supplementation reduces inflammation in the lung following pandemic H1N1 and SARS-CoV-2 infection. D− mice had lung inflammation even without infection. We showed previously that feeding D− diets to mice that cannot produce 1,25D (Cyp KO) resulted in severe vitamin D deficiency [C57BL/6 WT, K18hACE2 , and Cyp KO  mice were bred (mice) and housed (mice and hamsters) according to approved IACUC protocols at the Pennsylvania State University . For the experiments, age- and sex-matched mice were fed: chow diets (D+)  or purified diets with (D+) and without (D−) vitamin D . For some experiments, D+ mice were fed corn oil alone or corn oil with 1,25D. For some experiments, D− mice were fed corn oil alone or corn oil with one of two doses of vitamin D3 . Age- and sex-matched Golden Syrian Hamsters were purchased from Envigo  and maintained on the chow (D+) or D− diet  and orally fed corn oil or corn oil with vitamin D3. Serum was collected to monitor the vitamin D status of mice and hamsters. Serum 25D levels were measured using an ELISA kit and standards as per the manufacturer’s instructions . The limits of detection were 1.6 ng/mL 25D.n = 7–8/group) were anesthetized using isofluorane and infected with 100−1000 TCID50 units of SARS-CoV-2 in 50 μL phosphate buffered saline. Hamsters were sedated with ketamine (150 mg/kg), atropine (0.015 mg/kg), and xylazine (7.5 mg/kg) via intraperitoneal injection and intranasally inoculated with 10,000 TCID50 units of SARS-CoV-2 in 100 μL Dulbecco’s Modified Eagle Media. Hamsters were given atipamezole (1 mg/kg) subcutaneously to reverse the sedation. Equal numbers of males and females were used for all experiments, and body weights and symptoms were monitored daily until the endpoint criteria were reached or day 14 post-infection.SARS-CoV-2 USA-WA-1/2020  was used for infecting both mice and hamsters. hACE2 mice (n = 8–16/group). D− hACE2 mice were dosed orally with corn oil (D−), 0.125 μg vitamin D3/day (D+), or 2.5 μg vitamin D3/day (D++) beginning 8 weeks prior to infection and continuing throughout the experiment (n = 12–18/group). Hamsters were placed on D− diets with corn oil or 8 μg vitamin D3 (D+) in corn oil/day starting 11 days before infection and continuing throughout the experiment (n = 5–6/group).One series of experiments in hACE2 mice used standard D+ rodent chow  diets with oral dosing of corn oil or 10 ng/day of 1,25D diluted in 10 μL of corn oil beginning the day before infection and continuing until sacrifice. Additional experiments used mice or hamsters fed D− diets with or without oral vitamin D3 (n = 12–18/group) were fed identical diets with and without vitamin D. Mice were anesthetized with isofluorane to inoculate them intranasally with 10–30 TCID50 mouse-adapted A/H1N1/California/04/2009 influenza in 50 μL Gibco’s reduced-serum minimum essential media  [D+ and D− WT and D+ and D− Cyp KO littermates  . Body weAll studies with SARS-CoV-2 were conducted in a biosafety level 3 enhanced (BSL3+) laboratory. This facility is approved for BSL3+ respiratory pathogen studies by the U.S. Department of Agriculture and Centers for Disease Control. Studies with pandemic H1N1 influenza were conducted under biosafety level 2 enhanced conditions. All animal studies were conducted in compliance with the Animal Care and Use Committee under protocol numbers: 202001693, 202001516, 202001440, and 202001638. T method using GAPDH and uninfected control tissues. Gene expression was normalized to day 0 uninfected controls.Tissues were homogenized in TRIzol reagent . Total RNA was extracted from the tissues using chloroform-isopropanol precipitation and quantified using NanoDrop . A total of 1–2 µg RNA was reverse transcribed using AMV Reverse Transcriptase . Primers were purchased from Integrated DNA Technologies  and are listed in n = 2–5/group) [n = 4–6/group) [n = 4/group) [Lung tissues were collected from hamsters and mice and fixed in 10% formalin. These tissues were embedded in paraffin, sectioned, and H & E stained by the PSU Animal Diagnostic Lab Histology lab. Scoring of lung pathology associated with SARS-CoV-2 infection was conducted by veterinary pathologists certified by the American College of Veterinary Pathologists that were blinded to vitamin D3 treatment status. The criteria for the histopathologic evaluation of the tissue sections were conducted as described for H1N1 infected mouse lungs (5/group) ,28,29, S6/group) ,31, and 4/group) ,33,34. Tt-tests, one-way ANOVA, two-way ANOVA, mixed-effects analysis with Bonferroni multiple comparisons tests, the Kruskal–Wallis test with Dunn’s multiple comparison, the unpaired t-test, and the log-rank survival test as applicable. Data were checked for normal distribution and outliers. p < 0.05 was the cutoff to determine significance.Results are represented as the mean ± SEM. Statistical analysis was performed using Prism ver. 10  using multiple To evaluate the role of vitamin D deficiency during respiratory virus infection, WT and Cyp KO mice were fed a D+ or D− diet. Regardless of the genotype, serum 25D levels were significantly different between mice fed the D+ or D− diet A. As exp50 SARS-CoV-2, and the mice were evaluated for pre-determined euthanasia endpoints for up to 14 days post-infection. The dose of SARS-CoV-2 that resulted in the sacrifice of 50% of the mice was 200 TCID50 , the vitamin D receptor (Vdr), and the 24 hydroxlase that degrades vitamin D (Cyp24A1). There was no change in Vdr expression in the lung at day 2 or day 4 post-infection (Cyp27B1 and Cyp24A1 expression was higher at day 2 and day 4 post-infection than in the uninfected lung (Cyp27B1 expression was significantly lower in the day 4 than the day 2 post-infection lung (SARS-CoV-2 does not effectively infect WT mice. The transgenic expression of human (h)ACE-2 was shown to allow SARS-CoV-2 infection in mice . hACE-2 0 TCID50 A. The might loss A. The vinfection B. The N nfection B. Intereinoculum A,B. Lungnfection C. Converted lung C. Cyp27Bion lung C. Infect5 TCID50 SARS-CoV-2 lost weight shortly after infection, and none of the hamsters died following infection [Hamsters infected with SARS-CoV-2 are reflective of human SARS-CoV-2 infection in that the virus infects the lower respiratory tract, causing similar respiratory sequelae . We prevnfection . The weinfection .Vdr, Cyp27B1, Cyp24A1 was not different in D− and D++ mice at day 6 post-infection  , 0.125 μg/day (D+), or 2.5 μg/day (D++) for 8 weeks prior to infection. Serum 25D levels were higher in D+ mice and significantly higher in D++ mice before and at 14 days post-infection A. The D+nfection A. Ifnb anfection B. Mice snfection D,E. The nfection D,E. The ion (50) F. There ion (50) F. There Serum 25D levels in hamsters fed on the chow diet were not significantly different than serum 25D levels in hamsters fed D− diets for 4 weeks A. TherefCyp27B1 and Cyp24A1 in the lung of mice suggesting a role for vitamin D metabolites in the lung response to SARS-CoV-2 infection , or a transgenic mouse was needed to allow infection (hACE) with SARS-CoV-2. In addition, vitamin D metabolism is not identical in animals and humans. To our surprise, hamsters fed on standard chow diets replete in vitamin D were found to have low serum 25D levels. It is possible that vitamin D metabolism in hamsters is different than in the mouse and humans, which might impact our results. Mechanistic studies in humans are difficult, so it is important to use animals to determine pathways and processes that are regulated by vitamin D. The data in humans and animals do support a role of vitamin D to control the inflammatory responses in the lung providing protection following a viral infection with H1N1 or SARS-CoV-2.Together, the data support an important role for vitamin D and Cyp27B1 in the regulation of the host response to H1N1 and SARS-CoV-2 viruses. The role of vitamin D includes the restraining of the IFN response shortly after infection. Vitamin D-deficient hosts had pre-existing inflammation in the lungs that contributed to susceptibility to viral infection. Future experiments should continue to determine the mechanisms by which vitamin D regulates the anti-viral response in the lungs and whether there are differences in the effect of vitamin D on host resistance to H1N1 influenza and SARS-CoV-2.


There is limited information about diabetes and thyroid related autoantibodies in children with type 1 diabetes (T1D) or their siblings in Sri Lanka.To assess in T1D children and their unaffected siblings the prevalence of autoantibodies to (1) glutamic acid decarboxylase (GADA), insulinoma associated antigen-2 (IA-2A) and zinc transporter 8 (ZnT8A) using 3 Screen ICA™ (3-Screen) and individual ELISA assays; (2) insulin (IAA); and (3) thyroid peroxidase (TPOA), thyroglobulin (TgA) and the TSH receptor (TSHRA).We selected - (a) consecutive T1D children, and (b) their unaffected siblings of both sexes, from the T1D Registry at Lady Ridgeway Hospital, Colombo.(1) T1D children (a) 79.1% were 3-Screen positive; (b) all 3-Screen positives were individual antibody positive ; (c) and were younger (p=0.01 vs 3-Screen negatives); (d) multiple autoantibodies were present in 45.1%; (e) IA-2A (p=0.002) and ZnT8A (p=0.006) prevalence decreased with T1D duration. (f) TPOA and TgA prevalence was higher in T1D children compared to unaffected siblings . (2) Unaffected siblings (a) 6.3% were 3-Screen positive (p=0.001 vs T1D), and 2.4% were positive for IAA; (b) four subjects had two diabetes related autoantibodies, one of whom developed dysglycaemia during follow-up.The median age (IQR) of 235 T1D children and 252 unaffected siblings was 11  and 9  years respectively, and the duration of T1D was 23  months. The 3-Screen assay, used for the first time in Sri Lankan T1D children and their siblings as a screening tool, shows a high prevalence of T1D related Abs with a high correlation with individual assays, and is also a helpful tool in screening unaffected siblings for future T1D risk. The higher prevalence of thyroid autoantibodies in T1D children is consistent with polyglandular autoimmunity. Childhood type 1 diabetes mellitus (T1D) is an autoimmune disease with multiple factors affecting its pathophysiology and onset . HoweverAutoantibodies to islet cell antigens are a hallmark of T1D and are directed against the 65kDa isoform of glutamic acid decarboxylase (GADA), insulin (IAA), insulinoma associated antigen-2 (IA-2A) and zinc transporter 8 (ZnT8A) , 8. Thes(a) diagnosing T1D - there are only sparse data about T1D related autoantibody prevalence either at diagnosis or thereafter in Sri Lankan children . Sensiti(b) predicting T1D – this is possible in high-risk individuals and in the general population. First-degree relatives of patients with T1D have a high risk of developing T1D and are obvious targets for such predictive autoantibody testing . Data ab(c) understanding T1D aetiology – the variable prevalence of T1D autoantibodies in ethnically disparate groups may point to a variable role for autoimmunity in different population groups although other factors may be responsible. The coexistence of islet cell and thyroid autoimmunity in T1D has been shown previously in Sri Lankan subjects . HoweverTo assess in Sri Lankan T1D children and their unaffected siblings the prevalence of:(1) GADA, IA-2A and ZnT8A, using the combined three autoantibody screening assay [Elisa RSR™ 3 Screen ICA™ (3-Screen)], confirmed by individual specific autoantibody assays.(2) IAA, using a separate specific assay.(3) thyroid autoantibodies to – thyroid peroxidase (TPOA), thyroglobulin (TgA) and the TSH receptor (TSHRA).(1) Selection of T1D subjects and their siblings –Inclusion criteria – all children (a) with new and previously diagnosed T1D; (b) of both sexes; (c) and any age; and (d) their unaffected siblings.Exclusion criteria – children (a) with diabetes on immunosuppressive treatment; (b) with secondary diabetes ; and (c) with Maturity Onset Diabetes of the Young (MODY) if known or suspected(2) Study setting and subject recruitment – Subjects were identified from the T1D Registry of the Lady Ridgeway Hospital, Colombo 8, Sri Lanka. Informed consent was obtained from their parents or guardians. Consecutive T1D subjects and their siblings were tested during routine visits to the Diabetes Clinic. Approval was granted for this study by the Sri Lanka College of Paediatricians Ethics Committee (SLCP EC17/7/16). This study was significantly affected by the COVID pandemic and the civil unrest in the country and recruitment had to be curtailed as a result (see below)https://www.rsrltd.com) and assays for GADA, IA-2A, ZnT8A and IAA .(3) Blood sample collection and laboratory analysis - A venous blood sample was collected from subjects, centrifuged immediately and the serum frozen at -80°C. Samples were transported in dry ice to the FIRS Laboratories, RSR Ltd., Cardiff, for analysis. Samples were analyzed using 3-Screen  Assay Methodology –https://www.rsrltd.com).(a) Diabetes autoantibody ELISAs – All ELISAs used the bridging assay principle where the divalent autoantibody forms a bridge between the antigen coated onto the plates and a biotin labelled antigen added in a solution. In the assay, antigen coated plates were incubated with serum samples, calibrators and controls followed by a wash step and incubation with the antigen-biotin solution. After washing, the amount of antigen-biotin bound was determined by addition of streptavidin peroxidase and after a further wash the reaction was developed by addition of tetramethylbenzidine followed by a stop solution. The optical density (OD) of the plate wells was measured at 450 nm and 405 nm using an ELISA plate reader. Reading at 405nm allowed quantitation of high absorbances. All the ELISAs were performed following the manufacturer’s Instructions For Use  . The meaIA-2A - Samples were assayed in the Elisa RSR™ IA-2A Version 2 assay (IA-2A ELISA) . The meaZnT8A - Samples were assessed in the ELISA RSR™ ZnT8A™ assay (ZnT8A ELISA). This assay measures and detects ZnT8A specific to the 325R, 325W and residue 325 non-specific alleles , 15. The3 Screen – In the 3 Screen ELISA the plates coated with all three antigens GADA, IA-2A and ZnT8A  and a miRSR ELISAs’ reported sensitivity and specificity in International Autoantibody Standardization Program (IASP) workshop for 2020 were: 3 Screen , GADA , IA-2A  and ZnT8A , respectively.https://www.rsrltd.com). The measuring range was 0.4–50 units/mL (arbitrary RSR units). The cut off was ≥0.4 units/mL as indicated in the IFU.(b) IAA - Samples were assessed in the RIA RSR™ IAA (IAA RIA)  performe(c) Thyroid Autoantibodies - TPOA and TgA (cut off > 0.3 unit/mL) were measured using RIA assays (RSR Ltd.) , 18. TSH(5) Follow up of siblings positive for two or more T1D related antibodies -Unaffected siblings who were positive for two or more T1D related autoantibodies were monitored with an oral glucose tolerance test (OGTT) done at the time of autoantibody detection, and they were reviewed as follows – (a) in case of normal glucose tolerance – OGTT repeated every 6 months; (b) impaired glucose tolerance – OGTT repeated every 3 months with likely early insulin treatment; (c) diabetes mellitus - referred for routine diabetes care. Parents/guardians were informed, and educational and medical support offered.Statistical methodsSummary values are displayed as mean ± standard deviation (SD) or median  as appropriate. Comparisons of percentages of autoantibody prevalence between different subgroups of patients for categorical variables were made using the chi squared test. The comparison of means/medians between two groups was done using the unpaired Student’s t test and the Mann-Whitney test as appropriate . A statistically significant difference was defined as p< 0.05.Consent was obtained from parents/guardians of 300 T1D subjects and 261 siblings. However, 61 T1D subjects defaulted because of the current COVID19 pandemic, and political and civil unrest related disruption . Also, four T1D subjects and nine siblings, with inadequate information or aged below 6 months were excluded from analysis. Samples were therefore available from 235 T1D subjects and 252 siblings Table 1.Age and duration of diabetes(1) (a)T1D subjects - the median age  of T1D subjects at diagnosis and at sampling, and the median duration of T1D (IQR) at sampling are shown in Median age at sampling was higher in 3 Screen negative T1D children (p=0.01) but there was no difference in median age at diagnosis (p=0.55) and disease duration (p=0.83) between 3 Screen positive and negative subjects Table 1.All subjects classified as antibody negative T1D were on insulin at the time of recruitment, and none was obese or had acanthosis(b)Unaffected siblings – samples from unaffected siblings of 195 subjects with T1D were analysed (n=252). Their median age at sampling is shown in Autoantibody prevalence in T1D subjects and siblings –(2) (a)T1D subjects(i) 186/235 (79.1%) of T1D subjects were 3-Screen positive (66.8% of them had high concentrations of antibody i.e. were ≥4 times the cut-off ≥30 index). (ii) All 186 (100%) 3-Screen positive subjects were positive for at least one component autoantibody when tested with specific assays as follows: GADA 174/235 ; IA-2A 73/235 ; ZnT8A 91/235  Table 1.Of the 49 T1D subjects who were 3 Screen negative, three had one autoantibody  detected by individual ELISAs at low concentration (≥1.25 - 2.4 times cut off). Therefore there were 46 autoantibody negative T1D subjects in total. In addition, four T1D subjects who were autoantibody negative had siblings who were T1D autoantibody positive – GADA in 2, IA-2A in 1 and IAA in 1. These T1D subjects had disease durations of 42, 6, 140 and 141 months, respectively.(b)Unaffected siblings –(i) The prevalence of T1D autoantibodies amongst unaffected siblings was 16/252 (6.3%) in the 3-Screen assay. (ii) All 16 were positive for at least one single autoantibody in the specific ELISAs. For the individual autoantibody ELISAs the prevalences were – 15 GADA (6.0%), six IA-2A (2.4%) and four ZnT8A (1.6%). (iii) The prevalence of IAA was 2.4% (6/252) and these were likely true insulin autoantibodies. (iv) Multiple autoantibodies were present in 4/255 (1.6%) siblings (A single T1D related autoantibody at low concentration was found in four 3-Screen negative unaffected siblings using single specific autoantibody tests.Follow up of autoantibody positive unaffected siblings –(3) The four siblings who had two T1D autoantibodies were followed up as planned. One developed impaired glucose tolerance (2-hour post OGTT plasma glucose 9.3mmo/l) and was given insulin.Furthermore, one unaffected sibling who was only GADA positive, developed T1D four years after recruitment during a COVID19 related admission to hospital.Thyroid autoantibody prevalence(4) Samples were available for TPOA and TgA testing in 427 and TSHRA testing in 340 subjects Table 3.We have shown in this cross-sectional study that the prevalence of T1D related autoantibodies in Sri Lankan children using the relatively new screening tool, 3 Screen assay, is consistent with prevalence figures for established disease in Asian countries quoted in a recent review . All 3-SIn this study, 46 T1D subjects were autoantibody negative. The reasons for autoantibody negativity in T1D may include – unidentified MODY, long disease duration, mis-classified type 2 diabetes, coexisting diseases, autoantibody reversion, ethnic differences (in particular in Asian populations), defects in antibody production, immunodysregulation etc , 23. We Unaffected siblings had a significantly lower T1D autoantibody prevalence using 3 Screen. Furthermore, 4/252 (1.6%) unaffected siblings had two T1D related autoantibodies conferring a high risk of developing T1D of 70% in 10 years in one study . It shouThe minimum volume required to assay all autoantibodies in the study (including thyroid autoantibodies) was 410µL. However for future screening strategies a capillary blood sample of approximately 200µL should be sufficient for 3 Screen and IAA assays  the cross-sectional design with its inherent disadvantages, (b) the modest number of subjects in the two groups due to the COVID19 pandemic and local civil unrest related factors forcing the study to be discontinued prematurely, (c) the unavailability of previous studies with similar screening assay tools for comparison, (d) the lack of local age and sex derived reference ranges and (e) the absence of HLA DR and DQ allele analysis in this population – including these would add value to any future studies.However, it also has several advantages – (a) the use of a sensitive multiple T1D autoantibody screening assay which has a good correlation with individual specific autoantibody assays, used for the first time in this population, (b) the availability of patient data in a hospital based T1D Registry, and (c) establishing prevalence data for T1D related autoantibodies using a single screening assay (3-Screen) both in T1D children and their unaffected siblings for the first time in Sri Lanka.We have demonstrated the prevalence of T1D related autoantibodies in Sri Lanka in both T1D subjects and their unaffected siblings using a screening tool, 3-Screen. This assay is a valuable tool for screening T1D subjects and high-risk groups, showing a high correlation with individual specific autoantibody assays. Our study forms the basis for future investigations of autoantibody prevalence and T1D prediction models in the Sri Lankan population.The original contributions presented in the study are included in The studies involving human participants were reviewed and approved by Sri Lanka College of Paediatricians Colombo. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.NA, DdS, KdS, LDP conceived and developed the idea for the study and were involved in the planning of it. MP, JF, BRS also contributed to the study design. NA did the clinical investigations, and MP, JF, BRS contributed to analysing the data and writing the paper. MA performed the autoantibody assays and data analysis. All authors contributed to the article and approved the submitted version.


The crystalline products obtained from this synthetic approach include MPS3  and Cu3PS4. The successful reactions benefit from thermochemically favorable PCl3 elimination. This solvent-free route performed at moderate temperatures leads to mixed anion products with complex heteroatomic anions, such as P2S64−. The MPS3 phases are thermally metastable relative to the thermodynamically preferred separate MPx/ MSy and more metal-rich MPxSy phases. The micrometer-sized M-P-S products exhibit room-temperature optical and magnetic properties consistent with isolated metal ion structural arrangements and semiconducting band gaps. The MPS3 materials were examined as electrocatalysts in hydrogen evolution reactions (HER) under acidic conditions. In terms of HER activity at lower applied potentials, the MPS3 materials show the trend of Co > Ni >> Fe. Extended time constant potential HER experiments show reasonable HER stability of ionic and semiconducting MPS3  structures under acidic reducing conditions.The facile solvent-free synthesis of several known metal thiophosphates was accomplished by a chemical exchange reaction between anhydrous metal chlorides and elemental phosphorus with sulfur, or combinations of phosphorus with molecular P The the3 structures (3 (PDF #00-033-0672) and monoclinic NiPS3 (PDF #01-078-0499) with several small peaks for cubic NiS2 (PDF #04-003-4307). In the case of Co-P-S, two similar intensity crystalline phases are identified by XRD, monoclinic CoPS3 (PDF #01-078-0498), and cubic-phase CoP0.5S1.5 . This latter phase adopts the pyrite CoS2 structure with phosphorus substituting some sulfur in a solid-solution formula of CoPxS2-x where x = ~0.50 , such as CoPS3 [P2S5 + P].The XRD patterns of the products synthesized from the metal halides and P3 and Cu3PS4 products are shown in 3 materials with anion-rich layers nearest to the interlayer region. The MPS3 structures contain 2 M2+ cations with each P2S64− anion, which form PS3 pyramidal units that point into the interlayer spaces above/below the metal containing layer. The MPS3 form isostructural monoclinic structures with all three lattice parameters shrinking from Fe to Co to Ni in the MPS3 structure . The product yields for samples range from the ~60–95% range with higher yields for phase-pure products from P2S5 reactions. Energy dispersive spectroscopy (EDS) analysis performed on the M-P-S products shows low chlorine residues for FePS3 from the elemental P/S reaction to nearly undetectable chlorine (<2%) for other samples, indicating effective PCl3 elimination, consistent with our prior work on crystalline MP2/MP3 synthesis . Sp. Sp42]. x and MSy or MX2, where X is a combination of P/S, products are generally more thermodynamically stable, and therefore they are more easily produced compared to the mixed anion-layered MPS3 structures with P2S64− dumbbell anions. A comparison of standard heats of formation , where R is the diffuse reflectance of the sample. Extrapolation of the onset absorption events from a plot of the KM function versus energy yields estimated band gap (Eg) energies. The magnetic susceptibility of the M-P-S materials was analyzed at room temperature (298 K) using ground powders with a Johnson-Matthey MSB magnetic susceptibility balance. Molar magnetic susceptibility (χm) and spin only magnetic moment (μB) values were calculated from mass susceptibilities assuming paramagnetic behavior with correction for core diamagnetism.Powder X-ray diffraction (XRD) was performed on ground powders affixed to glass slides using a Bruker D8 Advance DaVinci diffractometer with nickel-filtered Cu Kα X-ray irradiation . Reference XRD patterns were generated from crystal structure data using the PowderCell program . The morwax electrode) similar to that previously reported by our group . Th. Thwax w2S5/P at 500 °C produces crystalline metal thiophosphates, with P2S5 reactions generally leading to the phase-pure products, MPS3  and Cu3PS4. The MPS3 materials contain M2+ cations and P2S64− anions in a non-metal rich layered structure that form as micrometer-sized and sometimes faceted particles. While these three thiophosphates have similar low energy band gaps (~1.5 eV), they show a great variation in electrocatalytic HER activity when embedded on a conducting wax electrode. Whereas CoPS3 shows the highest and most stable HER activity in acidic electrolyte, NiPS3 is less active and stable, and FePS3 appears highly resistant to performing HER. The effect of potential platinum counter electrode migration is identified for the lower activity NiPS3 and FePS3 samples. Post-electrocatalytic analysis of particles embedded on the wax electrode show strong evidence of bulk retention of the crystalline MPS3 structure.The direct solvent-free exchange reaction of anhydrous metal halides with either element P or S, P/S mixtures, or P


Eukaryotic translation elongation factors 1 δ (EEF1D), has garnered much attention with regards to their role in the drug resistance of cancers. In this paper, we investigated the effects and mechanisms of increasing the sensitivity of ovarian cancer cells to cisplatin or cis-dichlorodiammine platinum (DDP) by knockdown and knockout of EEF1D gene in cellular and animal models.The EEF1D gene was knocked-down or -out by siRNA or CRISPR/Cas9 respectively in human ovarian cancer cell SKOV3, DDP-resistant subline SKOV3/DDP, and EEF1D gene in human primary ovarian cancer cell from 5 ovarian cancer patients with progressive disease/stable disease (PD/SD) was transiently knocked down by siRNA interference. The mice model bearing xenografted tumor was established with subcutaneous inoculation of SKOV3/DDP.The results show that reducing or removing EEF1D gene expression significantly increased the sensitivity of human ovarian cancer cells to DDP in inhibiting viability and inducing apoptosis in vitro and in vivo, and also boosted DDP to inhibit xenografted tumor growth. Interfering with EEF1D gene expression in mice xenografted tumor significantly affected the levels of OPTN, p-Akt, Bcl-2, Bax, cleaved caspase-3 and ERCC1 compared to DDP treated mice alone, and had less effect on PI3K, Akt and caspase-3.The knocking down or out EEF1D gene expression could enhance the sensitivity of ovarian cancer cells to DDP partially, which may be achieved via inactivating the PI3K/AKT signaling pathway, thus inducing cell apoptosis and decreasing repairment of DNA damage. Our study provides a novel therapeutic strategy for the treatment of ovarian cancer.The online version contains supplementary material available at 10.1186/s12885-022-09699-7. Ovarian cancer is the primary cause of mortality in patients with gynecological malignant tumors . The inchttps://www.ncbi.nlm.nih.gov/gene/1936#gene-expression). EEF1D has also been found to be upregulated in numerous malignant tumors, such as liver cancer, esophageal cancer, small cell lung cancer and medulloblastoma  and electrotransfered onto PVDF membranes, which were performed using a standard protocol . ProteinP<0.05 was considered to indicate a statistically significant difference.All results are presented as the mean ± standard deviation. The differences among groups were analyzed using one-way ANOVA followed by Tukey’s post hoc test with SPSS software . POCC lines were successfully isolated and established from 5 patients with ovarian cancer with PD/SD who underwent debulking surgery at The First Affiliated Hospital of Anhui Medical University. These primary cells were cultured in the laboratory and morphologically represented typical cancer cells Fig.  A-D, wheP < 0.01; Fig. SP < 0.01; Fig. SBy constructing the pLJM1-shRNA EEF1D plasmid, lentivirus packaging, infecting the target cells and subculturing cells under puromycin screening, the four stable ovarian cancer cell lines  were successfully established, among which SKOV3/scrambled shRNA and SKOV3/DDP/scrambled shRNA cell lines were used as the controls. The expression of EEF1D in SKOV3 and SKOV3/DDP cells, in which EEF1D genes were knocked down, were determined via RT-PCR and Western blotting. The expression of EEF1D mRNA in SKOV3/EEF1D shRNA and SKOV3/DDP/EEF1D shRNA cells was significantly decreased compared with the control ; the effect in SKOV3/DDP cells was more significant than that in SKOV3 cells  or MK-2206 (10 μmol/l) respectively in the presence or absence of DDP (25 μmol/l). The results demonstrated that PI3K or Akt inhibitors decreased the cell viabilities of EEF1D KO/KD cells less than control cells, they particularly had a low inhibitory effect in KO cells  and Akt inhibitor (MK-2206) decreased the viabilities of EEF1D KO and KD cells less than control cells. Furthermore, EEF1D overexpression cell lines should be constructed in future experiments to determine whether the overexpression of EEF1D increases Akt activation, and whether the PI3K/Akt inhibitors reverse the effect of EEF1D on increasing the resistance of ovarian cancer cells to cisplatin. OPTN KD experiments showed that reducing OPTN gene expression could abate the effect of EEF1D. We reasoned that EEF1D might be achieved by increasing OPTN and reducing PI3K/Akt pathway function, or through the mutual influence of the two –36. The We successfully constructed human ovarian cancer cell model knocked down and out EEF1D gene with stable passage, and established an animal model of xenograft mice in human ovarian cancer cell SKOV3/DDP. The reducing or removing EEF1D gene expression could significantly increase the sensitivity of human ovarian cancer cells to DDP both in vitro and in vivo. This may be achieved through inactivating the PI3K/AKT signaling pathway, thus inducing cell apoptosis and decreasing repairment of DNA damage.Additional file 1: Figure S1. Knockdown of EEF1D gene was validated by reverse transcription polymerase chain reaction (RT-PCR). (A) PCR products of EEF1D gene were observed by agarose gel electrophoresis, and β-actin was used as the reference gene. (B) Relative intensity of DNA bands in A was determined using Quantity-One software and normalized using β-actin band intensity. Data shown in B are mean ± SD (n = 6).Additional file 2: Figure S2. Knockdown of EEF1D gene was validated by Western blotting. (A) EEF1D proteins in SKOV3 and SKOV3/DDP were detected with Western blotting, and β-actin was used to show the similar amount of protein loaded. (B) Relative intensity of protein band in A was determined using Quantity-One software and normalized using β-actin band intensity. (C) EEF1D protein in human primary ovarian cancer cell was detected with Western blotting, and β-actin was used to show the similar amount of protein loaded. (D) Relative intensity of protein band in C was determined using Quantity-One software and normalized using β-actin band intensity. Data shown in B and D are mean ± SD . POCC: primary ovarian cancer cell.Additional file 3: Figure S3. Knockout of EEF1D gene was validated by DNA sequencing and Western blotting. (A) Genomic sequence of EEF1D exon 2 region targeted by sgRNA editing. (B) Knockout of EEF1D gene in monoclonal cell mass was validated by sequencing. (C) EEF1D protein was determined by Western blotting.Additional file 4: Table S1. The DNA sequences of EEF1D shRNA and scrambled shRNA.Additional file 5: Table S2. The DNA sequences of OPTN shRNA and scrambled shRNA.


Although their absolute levels in the GIT are diet dependent, elevated IP7 metabolism still exists under dietary regimens devoid of exogenous IP7. Of the major GIT cells, enteric neurons selectively express the IP7-synthesizing enzyme IP6K2. We found that IP6K2-knockout mice exhibited significantly impaired IP7 metabolism in the various organs including the proximal GIT. In addition, our LC-MS analysis displayed that genetic ablation of IP6K2 significantly impaired IP7 metabolism in the gut and duodenal muscularis externa containing myenteric plexus. Whole transcriptome analysis of duodenal muscularis externa further suggested that IP6K2 inhibition significantly altered expression levels of the gene sets associated with mature neurons, neural progenitor/stem cells, and glial cells, as well as of certain genes modulating neuronal differentiation and functioning, implying critical roles of the IP6K2-IP7 axis in developmental and functional regulation of the enteric nervous system. These results collectively reveal an unexpected role of mammalian IP7—a highly active IP6K2-IP7 pathway is conducive to the enteric nervous system.Inositol pyrophosphates regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important. Here, we profiled inositol pyrophosphate levels in mammalian organs using an originally designed liquid chromatography–mass spectrometry (LC-MS) protocol and discovered that the gastrointestinal tract (GIT) contained the highest levels of diphosphoinositol pentakisphosphate (IP IP6K1 and IP6K2 are expressed in most mammalian tissues, with the highest expression in the brain and testis, whereas IP6K3 expression is mainly restricted to the muscles  are ubiquitously synthesized in all organisms and are involved in pleiotropic biological processes, most importantly in intracellular signaling . Among tositions , 3, 4, 5ositions  and bisdsphate IP are the ectively , 10, 11. muscles , 13, 14.al cells , 16, 17. obesity , 19, in  obesity  and in n obesity , amyotro obesity , and Alz obesity . Therefo obesity , 25. How3H-inositol labeling coupled with anion exchange chromatography  coupled with hydrophilic interaction liquid chromatography (HILIC) , where IP6Ks are highly expressed. Surprisingly, we also discovered that the highest IP7 production was observed in the gastrointestinal tract (GIT), even after depletion of dietary derived IP7. Of the major GIT cells, enteric neurons selectively expressed IP7-synthesizing enzyme IP6K2, which was revealed by assessment of single-cell RNA sequencing (scRNA-seq) data sets and confirmed by immunohistochemical detection. Our HILIC-MS/MS survey using IP6K2-knockout (IP6K2−/−) mice exhibited that IP6K2-dependent enhanced IP7 metabolism exists in the gut and duodenal muscularis externa where the myenteric plexus is located. We further performed whole transcriptome analysis of IP6K2-deficient and wildtype (WT) duodenal muscularis externa to define a physiological role of IP6K2-IP7 pathway in the enteric nervous system (ENS).We recently developed an analytical method that directly detects mammalian-derived IP (HILIC) , enablin6 and IP7 (A) and achieved clear detection of low abundance (10 pmol) of IP7 (B). We also determined the selected reaction monitoring (SRM) conditions for IP8 (D) to quantitate IP8 simultaneously with IP6 and IP7 (E). To benchmark this method for the detection of endogenous PP-IPs, we analyzed HCT116 cells treated with NaF, which is known to increase intracellular PP-IPs level (7 and IP8 SRM peaks in NaF-treated cells (F). We also observed a dose-dependent reduction in IP7 level and the IP7/IP6 ratio in HCT116 cells treated with the IP6K inhibitor TNP  of IP7 B. We als for IP8  by learn for IP8 D to quan and IP7 E. To benPs level . Our HILed cells F. We alsitor TNP . Thus, o6 and IP7, even after extensive rinsing of the organs with phosphate-buffered saline (PBS) to wash out the digested contents  but was not detected in other organs. While IP7 SRM peaks were clearly detected in CNS samples , IP7 levels in the CNS were modest compared with those in the GIT. Moreover, the IP7/IP6 ratio in the spinal cord appeared to be higher than that in the cerebrum .Using the newly developed HILIC-MS/MS protocol, we investigated the distribution of PP-IPs in experimental model rodents fed with a standard plant-based diet . Fifteen organs, including the CNS and GIT, were harvested from standard diet–fed C57BL/6J male mice. Surprisingly, HILIC-MS/MS analysis showed that the GIT had the highest levels of IP6, known as phytic acid) are present in a variety of crop seeds . We next investigated their concentrations in purified diets with minimal levels of plant-derived components . The two purified diets examined (iVid-neo and 70% casein) contained low amounts of IP6 and negligible amounts of IP7 and IP8. Quantitative analysis revealed that the levels of all PP-IPs in both purified diets were less than 2% of those in CE-2 . The concentrations of IP6 and IP7 and the relative level of IP8 in CE-2 and two purified diets were summarized in 6 and PP-IPs differ among different mouse diets, and standard mouse diet CE-2 substantially contains IP6 and PP-IPs, suggesting that dietary IP6 and IP7 affect the total levels of these molecules in the GIT of standard diet–fed mice.Several reports have shown that IPs : increased absorption of dietary IP7 or the presence of endogenous IP7 in these two GIT organs. To verify the former possibility , we analyzed the feces of mice fed on CE-2 and estimated the loss of IP6 and IP7 in the digestive system. Similar to those for CE-2 samples, IP6 and IP7 SRM peaks were clearly observed in mouse feces samples (C), suggesting that considerable amounts of IP6 and IP7 still remain in the feces. Quantitative analysis showed that approximately 50% of IP6 and IP7 in ingested food remained in the feces (D). Since the IP7/IP6 ratio remained unchanged between undigested CE-2 and feces (E), dietary IP6 and IP7 might not be disproportionately degraded and absorbed throughout the digestive system. Therefore, we could exclude the possibility that dietary IP7 is preferentially absorbed in the GIT, further highlighting that the high IP7/IP6 ratios of the stomach and duodenum are probably attributable to cellular IP7 metabolism.Two possibilities were considered to explain the high IPduodenum C: increa samples C, suggeshe feces D. Since nd feces E, dietar6 and PP-IPs present in the diet blur the direct detection of mammalian-derived IP6 and PP-IPs in the GIT of standard diet–fed mice. To more precisely validate the presence of endogenously synthesized IP6 and PP-IPs in the GIT, we prepared C57BL/6J mice maintained under two different conditions both of which remove dietary PP-IPs, namely, 2 months feeding of purified diet iVid-neo containing negligible amounts of PP-IPs (A and B and 6 and PP-IPs with those from standard CE-2 diet–fed counterparts (A). The GIT of both purified diet–fed and fasted mice showed a reduction in IP6 and IP7 levels compared with those of standard diet–fed mice; however, the levels were still close to (in the case of IP6) or far greater than (in the case of IP7) the CNS levels (B and C). IP8 was not detected in any of the tested organs of purified diet–fed and fasted mice. The SRM chromatograms of both purified diet–fed and fasted mice samples had explicit IP7 SRM peaks (D). Importantly, the stomach and duodenum of these mice showed prominently higher IP7/IP6 ratios than those of their standard diet–fed counterparts (E), implying further enhanced IP7 metabolism compensated for the overall reduced IP7 level. On the other hand, both purified diet–fed and fasted mice did not show any changes in the IP6 and IP7 levels as well as the IP7/IP6 ratio in the CNS and testis compared with those of mice fed a standard diet. However, as with standard diet–fed mice, both purified diet–fed and fasted mice showed higher IP7/IP6 ratios in the spinal cord than in the cerebrum, implying heterogeneous IP7 metabolic activity in the rostral and caudal CNS. We also investigated IP7 levels in the GIT of purified diet (70% casein)-fed Sprague–Dawley rats (A). Analogous to the results observed in the mouse model, both IP6 and IP7 levels in the GIT of these rats were drastically reduced compared with those in the standard diet–fed GIT and comparable with those in the CNS (B and C). In addition, the IP7/IP6 ratio was higher in the stomach and duodenum of purified diet–fed rats compared with that of standard diet–fed rats (D), further demonstrating very active IP7 metabolism in the mammalian proximal GIT.Dietary IPterparts A. The GIS levels , B and CRM peaks D. Importterparts E, implyiley rats A. Analog the CNS , B and Cfed rats D, furtheA, upper panel). In enteric neural cells, IP6K2 was selectively expressed across enteric neuron subsets, such as motor neurons, interneurons, and neuroendocrine cells but not in glial cells . This analysis was further supported by the IP6K quantitation using both E15.5 (A) and E18.5  mouse embryonic ENS scRNA-seq datasets . Other than enteric neurons, several cell types, including secretory progenitor cells, also expressed relatively high levels of IP6K2 (B). In addition, mouse enteric epithelial cell scRNA-seq data (C). Expression levels of IP6K1 and IP6K3 in entire embryonic intestinal cells were low and negligible, respectively (A and B). These results suggest that IP6K2 is highly expressed in mammalian enteric neurons.To investigate the expression levels of the three IP6Ks in each GIT cell type, we used scRNA-seq datasets and compared the expression levels of IP6Ks among GIT cell types. Quantitative analysis using a human embryonic intestinal cell scRNA-seq dataset  showed tFig. 4th E15.5 A and E18datasets . As in hof IP6K2 B. In addseq data  showed tseq data C. Expres7 synthesis in the mammalian organs including GIT, we employed a genetically modified mouse in which IP6K2 exon 6, encoding the kinase domain, was specifically deleted  (IP6K2-knockout (IP6K2−/−) or wildtype (WT) mice raised on the standard CE-2 diet were switched to a purified diet (iVid-neo) for 1 week and then fasted for 48 h before sacrifice . In WT mice, IP6K2 mRNA containing the exon 6 sequence was expressed in the proximal GIT but only marginally compared with the expression in the CNS (A). As expected, the IP6K2 transcript was absent in IP6K2−/− mouse organs. We confirmed the loss of IP6K2 expression in IP6K2−/− mice at the protein level using cerebrum lysate (B), because it has high IP6K2 protein expression and thus was useful for clearly validating the loss of IP6K2 in IP6K2−/− mice. HILIC-MS/MS analysis showed that IP6K2−/− mice had significantly lower levels of IP7 in various organs, including the stomach and duodenum, compared with those in their WT counterparts, while IP6 levels in each organ were almost the same between IP6K2−/− and WT mice (B and C). As previously observed (E and S3D), the IP7/IP6 ratios in the stomach and duodenum of WT mice were much higher than those in the other organs examined . These two organs of IP6K2−/− mice exhibited significant reduction in the IP7/IP6 ratios compared with those of WT mice, suggesting active IP6K2 pathway works in stomach and duodenum. Consistently, the IP7 SRM peaks for the IP6K2−/− mouse stomach and duodenum were smaller compared with those of WT mice, while IP6 levels were unchanged (C and D). Collectively, these data demonstrate that IP6K2 is required for enhanced IP7 metabolism in the mammalian proximal GIT.To estimate the importance of IP6K2 in endogenous IPt panel) . To avoi the CNS A. As expm lysate B, becausobserved E and S3Dnchanged , C and D7-synthesizing kinase IP6K2 is selectively expressed in enteric neurons  or fasted (dietary PP-IPs depleted) mice to more precisely survey the PP-IP profile in the proximal GIT. Some of these organs collected were subsequently used to isolate the muscularis externa where the myenteric plexus is located. These total GIT tissues and their muscularis externa were subjected to HILIC-MS/MS analysis to compare their IP7 metabolism (A). Similar to the results shown in 6 and IP7 levels with concomitant increase of the IP7/IP6 ratio in total GIT tissues . We found that IP6K2 deletion largely affected neither the morphological features nor the neuronal cell density in the duodenal myenteric plexus (B). We next prepared the muscularis externa from the stomach to the ileum of WT and IP6K2−/− mice, first depleting dietary IP7 in the GIT by 48 h fasting and performed HILIC-MS/MS analysis to evaluate IP7 metabolism in the ENS of IP6K2−/− proximal GITs (C). While IP6 levels in the muscularis externa were almost equivalent between WT and IP6K2−/− mice, IP7 levels and IP7/IP6 ratios were significantly reduced in the gut and duodenal muscularis externa of IP6K2−/− mice  and cholecystokinin B receptor (Cckbr), and investigated their mRNA levels in both the CNS and GIT by quantitative PCR (qPCR) (A). Compared with those in WT mice, these mRNA levels were explicitly increased from the stomach through the small intestine of IP6K2−/− mice, especially in the duodenum, but not the colon and CNS. To comprehensively appreciate the role of IP6K2-dependent IP7 metabolism in neuronal gene expression in the mammalian ENS, we isolated the duodenal muscularis externa from WT and IP6K2−/− mice and performed whole transcriptome analysis by RNA sequencing (RNA-Seq) (B). Gene set enrichment analysis showed that IP6K2 deletion suppressed certain gene sets associated with neural stem/progenitor cells, oligodendrocyte progenitor cells, and glial cells, concomitantly with the induction of those of mature neurons such as inhibitory, dopaminergic, or GABAergic neurons (C and S9A), implying that inhibition of the IP6K2-IP7 pathway triggers neurodevelopmental imbalance in the mammalian ENS. The RNA-Seq analysis also exhibited that 107 and 134 of 23,405 genes were more than 1.5-fold increased or decreased in IP6K2−/− with p-value less than 0.05, respectively (B). Pathway enrichment analysis of these genes showed that transcripts increased more than 1.5-fold in IP6K2−/− were significantly enriched for proteins involved in neuronal signaling (neuroactive ligand-receptor interaction of Kyoto Encyclopedia of Genes and Genomes Annotation) (C). In these transcripts, we observed that seven genes associated with neuronal function  or development  were prominently altered in their transcript levels between WT and IP6K2−/− (D). To validate our RNA-Seq results, differential expression of these seven neuronal genes were further assessed by qPCR and all of these candidate genes exhibited similar significant or prominent changes in transcript levels as observed in RNA-Seq results (D). qPCR analysis also showed that expression of other neuronal genes, including Drd5 and Cckbr, explicitly increased in IP6K2−/− duodenal muscularis externa (E). These changes were not observed in the RNA-Seq analysis possibly because they were below the lower detection limit and/or because of quantitation error  A. ComparRNA-Seq) B. Gene s neurons C and S9Aectively B. Pathwaotation) C. In theIP6K2−/− D. To val results D. qPCR a externa E. These on error , 45. Colin vivo PP-IP profiling of mammalian tissues is an important subject of research. However, this objective has been thwarted by various technical difficulties. Recently, we developed an HILIC-MS/MS analysis protocol for the sensitive and specific detection of IP7 and its precursor IP6 . Since standard diet CE-2 contains abundant IP6 and PP-IPs  still contained more abundant IP7 than that in the CNS (B and C and B and C). Furthermore, the IP7/IP6 ratio, an indicator of PP-IPs metabolism, was far higher in the proximal GIT than in the CNS irrespective of dietary condition (E and S3D), implying that endogenous IP7 synthesis is enhanced in the proximal GIT. In line with this result, IP7 levels in the stomach and duodenum were significantly diminished in IP6K2−/− mice under dietary PP-IP-depleted conditions (D). Therefore, our HILIC-MS/MS analysis unexpectedly revealed enhanced IP7 metabolism in the mammalian GIT.Surprisingly, we found that standard diet–fed rodents possess far more IPn organs B. Since ondition E and S3Dnditions D. Theref7/IP6 ratio than whole GIT tissues and the duodenal muscle layer has a much higher IP7/IP6 ratio than those of neighboring GIT segments (B). Considering the expression of the IP7-synthesizing enzyme IP6K2 in the myenteric plexus , these observations lead to the idea that IP6K2 actively synthesizes endogenous IP7 in the ENS of the proximal GIT. Our results also implied the presence of endogenous IP7 in other GIT layers because total GIT tissues of dietary IP7-depleted (fasted) mice contained a greater amount of IP7 than their corresponding muscle layers , the submucosal layer may contain endogenous IP7 to some extent. This PP-IP might also exist in mucosal epithelium because certain enteroendocrine cells, including tuft cells, express IP6Ks at the relatively high level (B and C). Tuft cell is one of rare cell types present in intestinal epithelium. Park et al. recently showed that tuft cell development is controlled by inositol polyphosphate multikinase, an enzyme responsible for driving IP metabolic pathway leading to IP7 synthesis (B and C) encourage to think that IP6K2 and IP7 might underlie tuft cell physiology as well. Future study is required for assessing cell type–specific IP7 metabolism to more precisely characterize IP7 metabolism in the GIT.The GIT consists of several histological layers including the muscularis externa that contains the myenteric plexus, a collection of large neuronal assemblies in the GIT. Our HILIC-MS/MS survey of the proximal GIT clarified that the muscularis externa has a higher IPsegments B. Considc plexus  and the e layers D, these e layers . Since agh level , B and Cynthesis . This faynthesis , B and CIP6K2 was initially cloned as a Pi uptake stimulator from a rabbit duodenum complementary DNA library . Given that the developmental lineage of enteric neurons comprises several differentiation points such as neural crest cell migration, neuron–glia bifurcation, and neural stem/progenitor cell differentiation into mature enteric neurons  . Notably, Nckipsd transcript, one of the transcripts most significantly induced in IP6K2−/− duodenal muscularis externa, contributes to the formation of neural dendrites  , 55, 56.tion (D) . Besidesunctions , D and Eendrites , 59 and endrites . These p the ENS . The molisorders , 62, IP6 WT mice . Thus, t7 metabolism links to various human diseases including neurodegenerative diseases, studying IP7 metabolism in human organs provides essential knowledge from the clinical point of view. The refined HILIC-MS/MS protocol we described in this study is capable of detecting IP6 and IP7 not only in rodent organs but also in human postmortem organs dissected after forensic intervention  were less abundant compared with those in human CNS. This is probably due to the high turnover rate of PP-IPs and the delay in dissecting human postmortem organs. Forensic intervention and subsequent organ dissection take hours after death. The presence of the intestinal flora may also facilitate the decomposition of these molecules in the GITs , sodium fluoride (Cat# S7920), and LC-MS grade medronic acid (Cat# 64255-1G-F) were purchased from Sigma-Aldrich. Hexadeutero-myo-inositol trispyrophosphate (ITPP-d6) was purchased from Toronto Research Chemicals (Cat# I666022). IP7 and IP8 were synthesized from myo-inositol using fluorenylmethyl phosphoramidite chemistry as described (LC-MS grade acetonitrile (Cat# 349672.5) and ammonium bicarbonate (Cat# 40867-50G) were purchased from Honeywell Burdick & Jackson. Ultrapure water (Cat# 210-01303) and formic acid (Cat# 067-04531) were obtained from Wako Pure Chemical Industries. Ultrapure grade ammonium hydroxide (28% w/v) was obtained from Kanto Chemical (Cat# 01266-3B). The InfinityLab deactivator additive was obtained from Agilent Technologies (Cat# 5191-3940). IPcid Cat# 255-1G-F 2. Cells prepared at 60% confluence in 10-cm dishes were incubated for 1 h with or without 50 mM sodium fluoride (Sigma-Aldrich). After washing twice with PBS, the cells were lysed in cell lysis buffer . A small aliquot was set aside for protein quantitation, and the rest was used for purification of IPs.HCT116 cells  were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum in 5% COIP6K2−/− (RRID: IMSR_JAX:036426) and WT mice maintained on a standard diet were switched to the purified diet for a week and subsequently fasted for 48 h before sacrifice. During fasting, mouse cages were changed to clean ones with new bedding every 24 h to reduce coprophagy. Mice and rats were anesthetized using isoflurane and then sacrificed by whole blood withdrawal from the left atrium. Before dissection of the organs, the animals were perfused transcardially with ice-cold PBS to wash out the residual blood and prevent the detection of IPs derived from blood cells. GIT organs  were cut open to remove feces and then extensively rinsed with PBS to wash out any dietary residuals. The duodenum, small intestine, and colon were harvested by cutting a 5-cm (mouse) or 10-cm (rat) segment from the distal end of stomach, between 10 and 15 cm (mouse) or 20 and 30 cm (rat) away from the duodenum and from the anus, respectively. The harvested organs were frozen until further use.All experiments involving animals were performed in accordance with protocols approved by institutional animal care guidelines (Tokai University School of Medicine). C57BL/6J mice and Sprague–Dawley rats obtained from Clea Japan  were maintained on a standard diet  or purified diet . Some mice fed a standard diet or purified diet were fasted for 48 h before sacrifice. The muscularis externa containing myenteric plexuses was prepared from mouse GITs as previously described with some modification , 66. ForThe human study was approved by the Ethics Committee of Tokai University , and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki . WrittenThe synthetic PP-IPs were validated using polyacrylamide gel electrophoresis as described . Briefly6 and IP7 concentrations with those in the diet. Crude lysate was mixed with an equal volume of 2 M perchloric acid, incubated on ice for 30 min, and centrifuged to remove tissue debris. After spiking with 3 nmol of ITPP-d6 as an internal control, 5 mg of titanium dioxide beads  were added to each sample. The beads were incubated at 4 °C for 30 min and washed twice with 1 M perchloric acid, and then 200 μl of 10% ammonium hydroxide was added for IP elution. The elution step was repeated to maximize recovery. The total eluate was dried using a SpeedVac concentrator (Thermo Fisher Scientific) and reconstituted in 125 μl of 100 mM ammonium carbonate/40% acetonitrile buffer, 50 μl of which was used for LC-MS.IPs in biological samples were purified as described , with so6, IP7, IP8, and the internal control ITPP-d6 was achieved using a modified version of a previously described procedure (Chromatographic experiments were performed using a Nexera UHPLC instrument (Shimadzu). HILIC-based chromatographic separation of IProcedure . The mobGIT segments and their muscularis externa were carefully collected and subjected to RNA extraction, as described . Total RIP6K2-deficient duodenal muscle externa with around 7.0 of RNA integrity number were subjected to RNA-Seq analysis. RNA sequencing libraries were prepared using TruSeq Stranded mRNA Kit  according to the manufacturer’s instructions. Each library was sequenced in 1 × 75 bp of single read mode using a NextSeq 500 platform (Illumina). Adapter sequences are removed from sequencing reads using Trim Galore . Sequence reads were aligned to mouse genome (mm10) by HISAT2 (version 2.1.1) (http://broadinstitute.github.io/picard/). The following genes were excluded before processing for the expression data analysis: highly expressing mucosal digestive enzyme genes  contaminated during the muscularis isolation, mitochondrial genes, and long noncoding RNAs. Expression levels of genes annotated in GENCODE (version M25) were quantitated by TPMCalculator (version 0.0.3) (http://www.gsea-msigdb.org/gsea/msigdb/index.jsp)   . Duplican 0.0.3) . The sofn 0.0.3) . Gene sedex.jsp) . Pathway_001881) .AB_10960426), anti-IP6K2 , and anti-β-actin  primary antibodies overnight at 4  °C. After rinsing 3 times in PBS containing 0.05% Tween-20, the membranes were incubated with the appropriate secondary antibodies conjugated with horseradish peroxidase (HRP) 2 fragment or sheep anti-mouse IgG, HRP-linked F(ab’)2 fragment; GE Healthcare). The immunoreactivities of the primary antibodies were visualized with Immobilon Western Chemiluminescent HRP Substrate  and recorded using an Ez-Capture Analyzer (ATTO).Western blot analysis was performed as described . MembranAB_221448) overnight at 4  °C. Rabbit immunoglobulin  and mouse IgG2b isotype control  were used to evaluate nonspecific binding. After rinsing thrice with 0.05% Tween-20 in TBS, the sections were incubated with secondary goat anti-rabbit IgG Alexa 488  and goat anti-mouse IgG Alexa 594  antibodies for 30 min at room temperature. The sections were then washed thrice with 0.05% Tween-20 in TBS and mounted using antifading medium . Confocal fluorescence images were obtained using an LSM 880 microscope (Carl Zeiss).Preparation of formalin-fixed, paraffin-embedded sections was performed as described . After dIP6K2−/− mice were blocked with 3% bovine serum albumin blocking solution containing the corresponding isotype control antibodies  for 2 days after fixation with 4% paraformaldehyde overnight. The muscle layers were then washed with PBS containing 0.05% Triton X-100 and incubated with diluted primary antibodies against HuC/D (Thermo Fisher Scientific) or βIII-Tubulin  for 3 days. After rinsing thrice with 0.05% Triton X-100 in PBS, the muscularis externa were then incubated with secondary goat anti-mouse IgG Alexa 594  for 3 h at room temperature. The samples were then washed thrice with 0.05% Triton X-100 in PBS and mounted using antifading medium . Fluorescence images were obtained using an LSM 880 confocal microscope (Carl Zeiss).Immunostaining of duodenal muscularis externa was performed as previously described with minor modifications , 66. Brihttps://simmonslab.shinyapps.io/FetalAtlasDataPortal/) and the GEO database (identifier: GSE149524), respectively. Mouse embryonic intestinal epithelial cell data (GSE92332). The above datasets were analyzed using the R package Seurat version 4.0.0 (RRID: SCR_007322) (Publicly available human embryonic intestine scRNA-seq processed data  and mousell data  were obt_007322)  to perfot test or one-way analysis of variance (ANOVA) followed by Bonferroni-type post hoc test, respectively. In RNA-Seq analyses, p values were determined using the corresponding analytical tools. Statistical significance was set at p < 0.05.Data are expressed as the mean ± SD. Differences between two or more groups were analyzed using two-tailed Student’s DRA014733. Other individual datasets and corresponding files generated in this study are available upon reasonable request from the corresponding authors.The raw RNA-Seq data have been deposited at the DNA Data Bank of Japan (DDBJ) and are publicly available with accession number This article contains The authors declare that they have no conflicts of interest with the contents of this article.