Heuristic genotype groupings and principal component analysis demonstrated concordance for Illumina sequencing, fragment analysis, and fluorescent PCR. However, Illumina sequencing and fragment analysis returned a range of fragment sizes, likely arising due to PCR “slippage”. Direct sequencing was accurate, but this method led to ambiguous electrophoregrams, hampering interpretation of heterozygotes. Gel sizing, pyrosequencing, and array-based technologies were less concordant. Pharmacoscan genotyping was concordant, but it does not ascertain 8(TA) genotypes that are common in African populations. Method-based genotyping differences were also observed in the publication record (p < 0.0046), although fragment analysis and direct sequencing were concordant (p = 0.11). Genotyping errors can have significant consequences in a clinical setting. At the present time, we recommend that all genotyping for this allele be conducted with fluorescent PCR (fPCR).To ensure accuracy of  UGT1A1) gene is involved in glucuronidation of a wide variety of substances, including drugs, endobiotics, dietary, and environmental compounds [UGT1A1 is associated with wide variability in UGT1A1 enzymatic activity that affects the pharmacokinetics and activity of over 50 therapeutics, including anticancer agents, antiretrovirals, NSAIDs, corticosteroids, anti-inflammatory agents, and many others [UGT1A1 genotyping in their package insert and several hundred published studies are devoted to UGT1A1-related pharmacogenomics associations [UGT1A1 genotype accuracy is an important issue to address [The uridine diphosphate glucuronosyltransferase 1A1  significantly contributes to enzyme activity by altering the expression of UGT1A1 and, thus, its enzyme activity. Individuals carrying (TA)5 or (TA)6 at this locus 7 or (TA)8  [UGT1A1*36/*36, UGT1A1*36/*1, and UGT1A1*1/*1 carriers; (2) intermediate metabolism (IM) status for UGT1A1*36/*28, UGT1A1*36/*37, UGT1A1*1/*28, and UGT1A1*1/*37; (3) and poor metabolism (PM) status for UGT1A1*28/*28, UGT1A1*28/*37, and UGT1A1*37/*37 [UGT1A1*60 , UGT1A1*93 (rs10929302), and UGT1A1*6 ) may also be involved in genetic determination of UGT1A1 glucuronidation capacity [Variation in TA-repeat length of a critical TATA box in the ctively) ,14. Cons1*37/*37 . Other sDuring assay development for two genotype-directed clinical trials involving a liposomal formulation of irinotecan  and an SN-38 conjugated HSP90 inhibitor , we noted several instances in which different genotyping technologies reported different results for rs3064744 in the same control samples. We therefore undertook a process to determine which genotyping technologies were concordant: direct sequencing, pyrosequencing, gel sizing, DMET Plus arrays, Pharmacoscan arrays, Illumina (MiSeq), fragment analysis, and fluorescent PCR (fPCR).UGT1A1 rs3064744  from eight different genotyping platforms: direct sequencing, Illumina (MiSeq), fragment analysis, fluorescent PCR , pyrosequencing, Agilent’s Bioanalyzer Lab-on-a-Chip Platform , DMET Plus , and Pharmacoscan . Hardy–Weinberg testing was not possible for DMET Plus because specific UGT1A1 genotypes are not reported 6) obtained by Illumina sequencing matched previously-reported data (p > 0.178) for Caucasians 6) = 0.76 vs. 0.70) [6) = 0.38 vs. 0.50) [6) = 0.70 vs. 0.62) [6) = 0.78 vs. 0.87) [Genotyping data were obtained on  samples . Genotyprted see  legend, s. 0.70) , Africanta p > 0.8 for Caus. 0.62) , and Asis. 0.87) . 6 homozygote revealed a range of fragment sizes that are attributed to PCR “slippage”: 2.5% (TA)4, 18.1% (TA)5, 57.5% (TA)6, 11.7% (TA)7, and 0.3% (TA)8. However, beause distributions of fragment size percentages had non-overlapping ranges, we developed an algorithm that utilizes raw percentages of fragment sizes (5/(TA)6 and (TA)5/(TA)7 genotypes in Illumina data due to the small number of carriers. . These results were also completely concordant with Illumina and fragment analysis data. We concluded that these methods were accurate on the basis of three lines of evidence: (1) the former three genotyping methods were completely concordant, (2) genotypes were concordant with controls, and (3) Illumina and fragment analysis demonstrated similar percentages of fragment size distributions that were unique enough to bin into different genotype categories.The consistency between percentages of fragment sizes between the two genotyping methods were remarkable and suggest that the polymerase used in both assays was similarly error prone. Such analysis led to complete concordance between the two genotyping methods . We sentUGT1A1*1/*28 carriers were only nominally different from those of UGT1A1*1/*37 carriers, and the same was true for UGT1A1*36/*1 and UGT1A1*36/*28 carriers  along with a larger band at 120-123 base pairs 8, whereas an extra T peak was observed in a (TA)7/(TA)8 carrier 8 fragment sizes.Five of six individuals harboring quencing B; howeveous call C. A lack5 or (TA)6 and (TA)7 or (TA)8, which are useful in the determination of metabolism status . When metabolism status is considered alone, the DMET Plus was 94.2% concordant with Illumina and fragment analysis (UGT1A1*1/*28 (n = 4) and UGT1A1*36/*28 (n = 5), for which DMET Plus determined to be normal metabolizers . Pharmacoscan was inconsistent with Illumina sequencing and fragment analysis in two patients carrying UGT1A1*1/*37 and UGT1A1*28/*37 genotypes as UGT1A1*1/*1 and UGT1A1*28/*28, respectively. This result was expected, as the latest version of the Affymetrix platform, Pharmacoscan, detects (TA)5, (TA)6, and (TA)7 individually, but it does not probe for (TA)8 repeats. We next engaged Nanostring  to design an assay detecting the UGT1A1 rs3064744 allele. At that point, Nanostring did not support customized SNV assay development without submission of several thousand samples. The DMET Plus array does not provide specific genotype calls for the platform reports (TA)analysis , and allbolizers . These rUGT1A1*1/*28 and UGT1A1*36/*28) as EMs, and (2) the Pharmacosan array misclassified an IM (UGT1A1*1/*37) as an EM. As the pyrosequencing assay often led to unclear results, UGT1A1 phenotype would be uncalled in seven cases using this technology. Direct sequencing and Pyromark gels would be likely to result in incorrect phenotypes in a multitude of cases. No platform for which we received raw data provided clear genotypes without significant post-processing, which could impede phenotype assessment in many cases.In spite of the non-concordance between genotype, all assays were phenotypically concordant with two exceptions: (1) the DMET Plus array misclassified nine IMs  and healthy individuals with African descent (n = 1707). We identified 136 publications appearing between 1996 and 2018 that genotyped this allele in populations who did not have disease or conditions directly related to UGT1A1 function . A total of 71 of these publications contained data comparable with the present study and prior publications utilizing direct sequencing (26 studies), fragment analysis (52 studies), gel sizing (17 studies), or pyrosequencing . This difference was primarily attributable to gel sizing, which has a lower frequency of (TA)6/(TA)6 genotypes (43.0%) and a higher frequency of (TA)6/(TA)7 (47.7%) than others (%(TA)6/(TA)6 = 44.3, 43.8, 46.1; and %(TA)6/(TA)7 = 44.3, 43.9, 45.9 for direct sequencing, fragment analysis, and pyrosequencing, respectively). When gel sizing was excluded, the three others also differed (P = 0.0046). This difference was attributable to pyrosequencing, which has a higher frequency of (TA)6/(TA)7 and a lower frequency of (TA)7/(TA)7 than others (%(TA)7/(TA)7 = 11.5, 12.3, 9.3, and 8.0 direct sequencing, fragment analysis, gel sizing, and pyrosequencing, respectively). Data obtained from direct sequencing and fragment analysis did not differ in either Caucasians (P = 0.11) or individuals with African descent (P = 0.92). We next searched the literature for publications ascertaining UGT1A1 genotyping for dosing or therapeutic choice may be underserved by many other current genotyping technologies, regardless of the CLIA certification of the Illumina sequencing, fragment analysis, DMET analysis, and pyrosequencing used herein. Genotyping errors have the potential to persist in the medical record, as the germline does not change throughout an individual’s lifetime. Such errors can expose patients to a variety of iatrogenic hazards [UGT1A1-related gene–drug interactions are identified and characterized, selection of the appropriate genotyping test for UGT1A1 rs3064744 is critical [The present study was undertaken to address concerns about genotype accuracy in two genotype-directed prospective clinical trials utilizing a liposomal irinotecan formulation (MM-398) and an HSP90 inhibitor conjugated to SN-38 , for which we chose to use fPCR by Quest Diagnostics. Although we demonstrated that fPCR  is an ac hazards ,21. ThusUGT1A1*37 alleles, and a patient who was UGT1A1*1/*37 (an IM) was called UGT1A1*1/*1 (an NM). Thus, even if one ignores specific genotypes and only classifies patients as NM, IM, or PM, then DMET Plus and Pharmacoscan could each introduce genotyping errors into the medical record. Because African Americans carry a higher frequency of (TA)5 and (TA)8 [We showed that only fPCR provides unambiguous results that require no post-processing of data; albeit, the Nichols Institute, which conducted the test, does not disclose methods for this assay . Althougnd (TA)8 , this pond (TA)8 , such geUGT1A1*28/*28 carriers receiving irinotecan, and further dosing is based on neutrophil count [UGT1A1*28/*28 carrier receiving 180–230 mg/m2 spaced by 2-3-week intervals and administering UGT1A1*1/*28 or UGT1A1*28/*28 carriers less than 240 mg/m2 [UGT1A1*36 and UGT1A1*37 carriers is sparse. As doses are increased on the basis of neutrophil count, risk is focused in patients who experience excessive toxicity due to a high starting dose. Thus, genotyping errors are of particular concern for UGT1A1*28/*28 carriers receiving 180–230 mg/m2 and UGT1A1*1/*28 or UGT1A1*28/*28 carriers receiving more than 240 mg/m2 of irinotecan. DMET genotyping would have incorrectly reported that nine intermediate metabolizers were extensive metabolizers; thus, these patients could have been subjected to irinotecan doses greater than 240 mg/m2 that could have been dangerous. Other genotyping platforms, except fPCR, could have led to non-calls and/or incorrect calls, as these platforms are often ambiguous or incorrect. Our results also extend to other clinical facets, including dosing and therapeutic selection of traditional irinotecan formulations. The Dutch Pharmacogenetics Working Group (DPWG) recommendations advise a 70% starting dose reduction for il count . The Fre40 mg/m2 . Recomme*1/*1 (NM) by Pharmacoscan, but was actually *1/*37 (IM) by other genotyping methods and ambiguous via pyrosequencing. Such a patient would not necessarily be at risk under the current dosing guidelines, but a patient carrying *37/*37 (PM) most likely would have been called *1/*1 and would have been treated improperly. Nevertheless, should a patient be assigned an incorrect genotype prior to atazanavir therapy, such an error would propagate in the medical record, potentially exposing such a patient to improper therapy in the future. Atazanavir prescribing is also based on UGT1A1 metabolism status: NM, IM, and PM. Patients with PM status are at risk of developing jaundice that will result in atazanavir discontinuation (approximately 20%–60% of carriers), and alternate agents should be considered . One patUGT1A1*36 and UGT1A1*37 carriers is also likely a function of genotyping errors in the literature. At the present time, we recommend that all genotyping for this allele be conducted with fPCR. Technological innovation to overcome genotype miscalls at this site is urgently needed for both clinical and scientific purposes.As the rs3064744 locus appears to have significant phenotypic consequences on a wide variety of agents, genotyping errors at this site are of particular concern as pharmacogenomics testing continues to discover new interactions with this variant. At best, ambiguity in genotype testing would lead to unnecessary delays in therapy when urgent treatment is required . At worsThe patient cohort was derived from a prospective pharmacogenomics trial . BrieflyPrimers were designed for the region of interest within the promoter of UGT1A1, specifically dbSNP ID: rs3064744. For the MiSeq amplicon design, the FW primer sequence was 5′- TTTATCTCTGAAAGTGAACTC-3′ and the RV was 5′- TGGGCGTCCGCCCTGGGACTC-3′. These primers were adapted with M13FW and M13RV tags: 5′-gtaaaacgacggccagt-3′ (FW strand) and 5′-ggaaacagctatgaccatg-3′ (RV strand). All primers were purchased from Thermo Scientific. For the PCR, Invitrogen’s High-Fidelity Taq System  and 10 mM dNTPs (Thermo Fisher Scientific) with 5% molecular grade dimethyl sulfoxide  were utilized. The M13-labeled primers were used to generate the target amplicons for the libraries from DNA with an Applied Biosystems Veriti 96-well thermal cycler (Thermo Fisher Scientific) using the following conditions: 95 °C for 5 min; followed by 20 cycles of 94 °C for 1 min, 58 °C for 1 min, 72 °C for 1 min; followed by 20 more cycles of 94 °C for 1 min, 65 °C for 1 min, 72 °C for 1 min, and completed with a final extension of 72 °C for 10 min, then holding at 4 °C. The resulting target specific amplicons were used directly in the adapter PCR that follows. www.Illumina.com) using Illumina P5 and P7 adapter-indexes, barcodes, and M13 adapters. These adapters were added to the amplicons to create the library samples with the Veriti thermal cycler and the following conditions: 95 °C for 2 min; followed by 15 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and completed with a final extension of 72 °C for 1 min, then holding at 4 °C. The PCR system was Invitrogen’s Platinum Taq along and dNTPs.For this step, barcoded primers for bi-directional coverage of each target amplicon were designed per Illumina indexing protocols  according to Agencourt/Beckman Coulter’s protocol. The DynaMag-96 Side Magnet was used (Thermo Fisher Scientific). Final libraries were eluted with Qiagen’s Elution Buffer (Buffer EB).The libraries were then analyzed for quality and quantity using Agilent’s 2100 Bioanalyzer  and Agilent’s DNA 1000 kit. Using Bioanalyzer software version 2100 Expert B.02.08 SI648, region tables were assigned to the libraries, giving approximate sizes and concentrations. Any libraries that failed to amplify well were repeated from the initial PCR step and not included in the final pool until optimal. Each library sample was also quality checked using Thermo Fisher Scientific’s nanodrop ND-8000 spectrophotometer. If required, library samples were normalized on the basis of the lowest concentration from the Bioanalyzer data, and then all libraries were pooled together to that equimolar concentration. The final library pool was then checked one last time for concentration with the ND-8000. Using the approximate base pair size and the final nanodrop concentration, the correct load concentration was determined for the library. The library was then processed according to Illumina’s MiSeq System Denature and Dilute Libraries Guide. The control used was PhiX Control kit v.3, . The library was sequenced using the Illumina MiSeq next generation sequencer (Illumina), MiSeq Controller software version 2.6.2.1., using an Illumina MiSeq Reagent Kit v.2 300 Cycle, (Illumina). This was in accordance with the Illumina MiSeq System Guide.Sample barcode demultiplexed FASTQ files from the MiSeq sequencing were evaluated for the presence of variants of the reference sequence TTTTTGCCATATATATATATATAGTAGGAGAGGGCGAACC. Variants were enumerated by collecting, for each sample, all sequence reads that were bounded by TTTTTGCCA and AGTAGGAGAGGGCGAACC. All variants for each sample were tabulated and sorted according to length and nucleotide sequence. Variant frequency was calculated as the ratio of the number of identical reads for each sample divided by the total number of qualifying reads in the sample.Primers were designed for the region of interest within the promoter of UGT1A1, specifically dbSNP ID: rs3064744. This design was a nested PCR, and the outer PCR primers were as follows: FW primer 5′-TTCTTCCTCTCTGGTAACACTT-3′, RV primer 5′-ACTCTTTCACATCCTCCCTT-3′. For the PCR assay, Invitrogen’s High Fidelity Taq System (Thermo Fisher Scientific) and Invitrogen 10 mM dNTPs (Thermo Fisher Scientific) with 5% molecular grade dimethyl sulfoxide (DMSO) (Sigma-Aldrich) were utilized. Samples were amplified with Applied Biosystems Veriti 96-well thermal cycler (Thermo Fisher Scientific) using the following conditions: 95 °C for 5 min; followed by 20 cycles of 94 °C for 1 min, 58 °C for 1 min, 72 °C for 1 min. This was followed by 20 more cycles of 94 °C for 1 min, 65 °C for 1 min, 72 °C for 1 min, with a final extension of 72 °C for 10 min, then holding at 4 °C.The generated PCR amplicons were then purified using exonuclease I  and shrimp alkaline phosphatase (Affymetrix), in accordance with the Exo-Sap protocol. The Exo-Sap-sample mixture was then incubated in the Veriti thermal cycler: 37 °C for 15 min, then 80 °C for 15 min, followed by a 4 °C hold. This purified amplicon was then used in the next, inner PCR as described below.For the inner PCR, the FW PCR primers were 5′-GCTCCACCTTCTTTATCTCTG-3′, 5′-FAM- GCTCCACCTTCTTTATCTCTG-3′, and 5′-GTTTCTGCTCCACCTTCTTTATCTCTG-3′ (pigtailed FW). The RV PCR primers were 5′-ATCAACAGTATCTTCCCAGC-3′, 5′-FAM- ATCAACAGTATCTTCCCAGC-3′, and 5′-GTTTCTATCAACAGTATCTTCCCAGC-3′ (pigtailed RV). Before amplification, primer mixes were created. For the FW FAM-labeled product, a 20 µM FAM-FW primer mix was created by adding 480 µL of molecular grade water, 18 µL of 500 µM RV, and 2 µL of 500 µM FAM-labeled FW primer. The amplification was then carried out with this FAM-FW/RV primer master mix and the pigtailed RV, each at 0.8 µM final concentrations. For the RV FAM-labeled product, a 20 µM FAM-RV primer mix was created by adding 480 µl of molecular grade water, 18 µL of 500 µM FW, and 2 µL of 500 µM FAM-labeled RV primer. The amplification was then carried out with this FAM-RV/FW primer master mix and the pigtailed FW, each at 0.8 µM final concentrations. All samples were tested with both the FAM FW primer master mix and the FAM RV primer master mix for comparison and confirmation. Samples from DNA were amplified for the inner PCR using Platinum Taq and dNTPs, along with the Veriti thermal cycler using the following conditions: 94 °C for 5 min, then 20 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s; followed by a final extension of 72 °C for 7 min, then holding at 4 °C. The resulting products were then checked for quality and concentration with Agilent’s 2100 Bioanalyzer (Agilent) and Agilent’s DNA 1000 kit, using Bioanalyzer software version 2100 Expert B.02.08 SI648. These samples were then diluted with molecular grade water at a 1:10 ratio to prepare them for running on fragment analysis. A master mix was created using Applied Biosystems Hi Di Formamide (Thermo Fisher Scientific) and Applied Biosystems Gene Scan LIZ 500 (Thermo Fisher Scientific). All incubations were carried out with the Veriti thermal cycler. Samples were then processed on Applied Biosystems 3730xl DNA Analyzer (Thermo Fisher Scientific), 96 capillary 50 cm array, using data generated with DS-33 Matrix Standard Kit (Dye Set 5) (Thermo Fisher Scientific). Fragment analysis data was reviewed with Thermo Fisher Scientific Peak Scanner 1.0 software.2, 0.2 mM deoxynucleotide triphosphates, 800nM of each primer , and 1.25 U Platinum Taq DNA polymerase (Invitrogen) using a GeneAmp PCR system 9700  as a thermocycler with the following thermal profile (primary PCR): 40 cycles of denaturation at 94 °C for 30 s, annealing at 66 °C for 30 s, and extension at 72 °C for 30 s. After amplification, the quality of the amplified PCR products was verified by agarose gel electrophoresis. The PCR products were then sequenced on an ABI Prism 3130xl Genetic Analyzer (Applied Biosystems) per the manufacturer’s instructions using the following sequencing primers: 5′-TCCTTCTTCCTCTCTGGTAAC-3′, 5′-ACATTATGCCCGAGACTAAC-3′.Primer pairs were designed on the basis of the gene sequence available at Genbank, and the sequences of each primer pair is listed as follows: 5′-AAGCGGGGGTACAGTTGTGTTC-3′, 5′-AAGAATACAGTGGGCAGAGACAG-3′. PCR reactions were carried out in a 50 μL reaction mixture containing 200 ng of genomic DNA, 1X PCR buffer , 1.5 mmol/L MgClPrimers were designed for the region of interest within the promoter of UGT1A1, specifically dbSNP ID: rs3064744, and ordered from Invitrogen. The FW Pyromark PCR primer was 5′Bio-CACGTGACACAGTCAAACATTAAC-3′, and the RV PCR primer was 5′- AGGTTCGCCCTCTCCTACTTATA-3′. The Pyromark sequence primer was 5′- CGCCCTCTCCTACTTATATAT-3′ . Assay dAll amplicons were checked for concentration and quality using Agilent’s 2100 Bioanalyzer  and Agilent’s DNA 1000 kit. Data analysis was performed using Bioanalyzer software version 2100 Expert B.02.08 SI648.Samples were then processed with Biotage’s MD Pyrosequencing System: Biotage Q96MD, Biotage RIA Vacuum Work Station, and Biotage HS Sample Prep Thermoplate D1200. Qiagen reagents, kits, and consumables were used as listed: Pyromark Q96 HS Plates, Pyromark Gold Q96 Reagents, Pyromark Control Oligo, Pyromark Annealing Buffer, Pyromark Denaturation Buffer, and Pyromark Binding Buffer. Additionally, GE Healthcare’s Streptavidin Sepharose High Performance was used for binding the DNA to the plate. N were determined manually and not called by the software.Samples were analyzed using Pyromark MD Software version 1.0, a custom dispensation of the sequence listed of was used—GATATATATCATATGCT, with the sequence to analyze being ATATATATGC/TGCAAAAACC/GAATCGATACACCAAGT. However, because this assay design was custom, the calls and analysis for the (TA)Samples were then processed using Applied Biosystems’ DMET Plus Starter kit in accordance with the DMET Plus assay protocol with supplementation of Qiagen’s Multiplex PCR kit for use in the mPCR step of the assay, Titanium Taq DNA Polymerase  for the amplification stage of the assay step, and Streptavidin R-Phycoerythrin Conjugate  for the preparation of the stain buffer solution used in the wash/stain protocol. All temperature holds and thermal cycling were performed on Applied Biosystems’ GeneAmp PCR System 9700 (Thermo Fisher Scientific). Following the cleanup and fragmentation steps of the assay protocol, sample amplification and fragmentation were assessed using Agilent’s 2100 Bioanalyzer (Agilent) and Agilent’s DNA 1000 kit, software version 2100 Expert B.02.08 SI648. Hybridization occurred in an Affymetrix GeneChip Hybridization Oven 645 (Thermo Fisher Scientific), arrays were registered using Affymetrix GeneChip Command Console (AGCC) Portal version 4.0.0.1567G, wash/stain protocol was performed on an Affymetrix GeneChip Fluidics Station 450 (Thermo Fisher Scientific) using AGCC Fluidics Control software version 4.0.0.1567, and arrays were scanned an Affymetrix GeneChip Scanner 3000 7G Plus (Thermo Fisher Scientific) with an autoloader (Thermo Fisher Scientific) using AGCC Scan Control software version 4.0.0.1567. Array controls were assessed using Affymetrix’s AGCC Viewer software version 4.0.0.1567 and assay results were analyzed on Affymetrix’s DMET Console software version 1.3.0.20. Pharmacoscan was conducted by RUDCR at Rutgers University on a fee-for-service basis.Hardy–Weinberg equilibrium and comparisons between genotypes and previously published studies were conducted with the chi-square test (or Fisher’s exact test where appropriate). Principal component analyses were conducted to confirm the heuristic groupings of genotypes between the different genotyping methods. Comparisons between previously published studies were conducted using the Fisher–Freeman–Halton test. All comparisons were conducted with Statistical Analysis Software (SAS).


Both Pcs 1 and 2 exhibited low melting points, i.e., 120 and 130 °C respectively, due to the reduction in intermolecular π-π interaction among the Pc rings caused by the steric hindrance of 2,6-dihexyloxybenzene units. The thermal behaviors were investigated with temperature-controlled polarizing optical microscopy, differential scanning calorimetry, powder X-ray diffraction, and absorption spectral analyses. Pc 1, having C4h molecular symmetry, organized into a lamellar structure containing lateral assemblies of Pc rings. In contrast, the other Pc 2 revealed the formation of metastable crystalline phases, including disordered stacks of Pcs due to rapid cooling from a melted liquid.The synthesis and characterization of two phthalocyanine (Pc) structural isomers,  After cyclic tetramerization of 3 or two phthalonitriles  by LiOC6H13 in n-hexanol, eight methoxy groups were cleaved by BBr3 to give 1,8,15,22- and 1,4,15,18-tetraphthalocyanines 7 and 8. Finally, two target compounds, 1 and 2, were obtained through the Williamson ether reaction between 7 or 8 and 1-bromohexane in the presence of K2CO3. All compounds were fully characterized by 1H-NMR, 13C-NMR, atmospheric pressure chemical ionization-time-of-flight (APCI-TOF) mass, and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectroscopies.Two Pcs, itrile 4  8,9]. T,9. T1 an2h, C2v, C4h, and Cs). The reaction mixture during the tetramerization of 3 at 157 °C showed a simple 1H-NMR spectrum, suggesting the formation of a single symmetrical isomer. Several attempts for the selective formation of C4h isomers from 3-substituted phthalonitriles have been demonstrated . Fur. Fur2 unskeleton .1 and 2 showed that the weight-losses started at around 300 °C under N2 atmosphere, indicating their thermal stability up to 300 °C  and differential scanning calorimetry (DSC) analysis. Compounds 1 and 2 melted at 120 and 130 °C, respectively, and the liquids showed no birefringence under POM observation. Liquid-crystalline Pcs with flexible alkyl chains at peripheral positions of the Pc core possessed two transitions, that is: a crystalline phase to a mesophase, and then a mesophase to an isotropic liquid [1 displayed only one reversible transition at 121 and 70 °C upon heating and cooling runs at 10 °C/min . The absorption spectral shape for the Q bands of Pcs is known to be sensitive to the local environment of individual molecules . Figure n theory , the redt 140 °C b. The XRssian 16 , the obsPc plain c. The otPc plain d. Howeve2 upon heating and cooling runs at 2 °C/min. During the first heating runs from 30 to 200 °C, 2 exhibited one endothermic peak at 130 °C. Subsequent cooling at 2 °C/min showed an exothermic peak at 95 °C, and the transition enthalpies for the heating and cooling processes were comparable. When 2 was cooling from an isotropic liquid at 2 °C/min, birefringent short needles appeared at 95 °C under TPOM (2 crystal contained two crystal structures possessing different monoclinic lattices of Pcs. The XRD peaks, as indicated by * in 2 along the a-axis agreed with the simulated molecular length with elongated hexyl chains. The difference between the a- and b-axes suggested the tilting of the Pc cores within the stacks. The Pc cores were probably tilted with respect to the c-axis, and the tilted angle of the cores in the stacks could be calculated to be 34° [2 was broadened, and the maximum of the Q band was blue-shifted to 687 nm, indicating the formation of a cofacial stack of Pc molecules (2 could form long-range ordered stacks while avoiding the steric hindrance of 2,6-dihexyloxybenzene substituents attached to the 1, 4, 15 and 18 positions of the Pc rings due to slow cooling from an isotropic liquid (der TPOM b, and shder TPOM c. Accordo be 34° . Two shaolecules e. Thus, c liquid f.2 from an isotropic liquid at 20 °C/min, an exothermic peak was observed at 43 °C, which was lower than that obtained at 2 °C/min (2 exhibited only a broad halo (2 was in a supercooled state due to rapid cooling from an isotropic liquid [2 film exhibited a red-shifted absorption band, observed at 726 nm (In the DSC cooling run of 2 °C/min a. The XRoad halo b, and noc liquid . The 2 ft 726 nm d. Upon h1 and 2, possessing different molecular symmetries, were investigated. The spatial overlapping of hexyloxy groups onto the π-surface of the Pc rings resulted in a significant decrease in the melting points, and large homogeneous domains were formed by cooling from the melting points. C4h isomer 1 organized into a layered structure by two-dimensional assembly without π-π stacking among Pcs. In contrast, D2h isomer 2 was able stack in the crystalline phase, and the ordering in the stacks depended on the cooling speeds from the isotropic liquid. Since Pc crystals have been used in several organic devices such as organic thin film transistors and organic photovoltaic cells due to their highly efficient electron or charge transportations [In summary, the crystalline structures of two structural isomers, rtations ,23, the


However, the level of restitution of circulatory function during long-lasting hypothermic cardiopulmonary resuscitation (CPR) remains largely unknown. We compared the effects of CPR in replacing spontaneous circulation during 3 h at 27°C vs. 45 min at normothermia by determining hemodynamics, global oxygen transport (DOn = 7) were immersion cooled to CA at 27°C. Predetermined variables were compared: (1) Before cooling, during cooling to 27°C with spontaneous circulation, after CA and subsequent continuous CPR (n = 7), vs. (2) before CA and during 45 min CPR in normothermic pigs (n = 4).Anesthetized pigs  After normothermic CPR for 15 min, CO was reduced by 71%, MAP by 54%, and DO2 by 63%. After 45 min, hemodynamic function had deteriorated significantly, organ blood flow was undetectable, serum lactate increased by a factor of 12, and mixed venous O2 content was reduced to 18%.When compared to corresponding values during spontaneous circulation at 38°C: (1) After 15 min of CPR at 27°C, cardiac output (CO) was reduced by 74%, mean arterial pressure (MAP) by 63%, DO2 delivery to maintain aerobic metabolism. This fundamental new knowledge is important in that it encourages early and continuous CPR in accidental hypothermia victims during evacuation and transport.The level to which CPR can replace CO and MAP during spontaneous circulation at normothermia was not affected by reduction in core temperature in our setting. Compared to spontaneous circulation at normothermia, 3 h of continuous resuscitation at 27°C provided limited but sufficient O Over the past years, patient case reports of accidental hypothermia with cardiac arrest (CA) indicate favorable neurologic outcome after in-hospital rewarming . These fNormothermic CPR can provide ∼30% of pre-arrest cardiac output (CO)  but is u2 transport (DO2) and O2 consumption (VO2)  . In addirmic CPR . In an iransport  and tissransport  are mainr 20 min . Subsequr 20 min  reportedr 20 min .New devices make it possible to sustain CPR during evacuation and transport for a longer period of time with a limited number of rescuers . UNN . Eleven castrated male pigs  from NOROC stock were used. On arrival, the animals were acclimated for 2–5 days before the terminal experiment. Animals were fed twice daily, had free access to water at all times, and received humane care in accordance to the Norwegian Animal Welfare Act.–1), midazolam (30 mg), and atropine (1 mg), and anesthesia was induced by a bolus infusion of fentanyl (10 μg kg–1) and pentobarbital-sodium (10 mg kg–1) in an ear vein. After tracheotomy and intubation, the animals were connected to a respirator , adjusted to maintain PaO2 > 10 kPa and PaCO2 at 4.5–6.0 kPa uncorrected for temperature (α-stat management). After instrumentation, animals were randomly assigned to two groups . During ventricular fibrillation (VF) and CPR, FiO2 was set to 1.0. Infusion of fentanyl (20 μg kg–1h–1), midazolam (0.3 μg kg–1h–1), and pentobarbital-sodium (4 mg kg–1h–1) was continued via a femoral vein catheter. In the hypothermia group, anesthesia was discontinued at 27°C. Microspheres were injected to the left ventricle through a 6F fluid filled pigtail catheter . Core temperature, CO, and venous and mixed venous blood gases were measured via a 7F Swan–Ganz thermodilution catheter  positioned in the pulmonary trunk. Thermodilution is described as the gold standard for measuring CO during CPR  were injected into the left ventricle . SimultaAnimals in the hypothermia group were immersion cooled in ice water to a blood temperature of 27°C, and VF was induced by stimulating the epicardial surface using an alternating current  conducted via a 15 cm-long needle electrode . The neeIn the normothermia group: during spontaneous circulation at 38°C before VF, and at 15 and 45 min during CPR. In the hypothermia group: during spontaneous circulation at 38°C, during cooling at 32° and 27°C before VF, and during CPR at 15 min and hourly thereafter. Simultaneously, we recorded electrocardiogram (standard leads), heart rate (HR), MAP, central venous pressure (CVP), and urine output, and microspheres were injected. Blood gases were analyzed by simultaneous sampling from arterial, central venous, and mixed venous blood.p < 0.05. Data are presented as means and SD.Statistical analyses were performed using SigmaPlot statistical software version 14 . Normal distribution was checked using the Shapiro–Wilk test. Comparisons between normothermia and hypothermia groups as well as intragroup comparisons at 38°C baseline, 15 min, and 45/60 min of CPR were performed by two-way repeated measures ANOVA. Where significant differences were found, all pairwise multiple comparison procedures were done using the Holm–Sidak test. To compare values within the hypothermia group, we used one-way repeated measures ANOVA for normal distributed variables, and Friedman repeated measures ANOVA on ranks for non-normal distributed variables. Where significant differences were found, Dunnett’s test was used to compare all values within the hypothermia group vs. 38°C baseline, as well as values obtained during CPR at 60, 120, and 180 min vs. CPR at 15 min. The level of significance was set at No statistically significant differences were found between groups in any of the variables recorded at the start of the experiments. Animals in both groups had spontaneous circulation before induction of VF. Stability of the actual pig model related to hemodynamic function and organ blood flow at normothermia has previously been documented in experiments lasting up to 7 h . Multipl–1 (-71%) and MAP was significantly reduced from 85 ± 5 to 38 ± 12 mmHg (-55%) (–1 and 18 ± 6 mmHg), respectively, but the reduction in MAP was statistically significant.Compared to spontaneous circulation at 38°C, after CPR for 15 min at this temperature, CO was significantly reduced from 3.0 ± 0.6 to 0.9 ± 0.2 L ming (-55%) . After 4–1 (-74%) and MAP from 89 ± 14 to 33 ± 9 mmHg (-63%). After 60 min of CPR at 27°C, CO and MAP were reduced by 74 and 65% (0.8 ± 0.2 L min–1 and 31 ± 8 mmHg), respectively.Compared to spontaneous circulation at 38°C, after CPR for 15 min at 27°C, CO was significantly reduced from 3.2 ± 0.6 to 0.8 ± 0.1 L minNo significant differences between groups in CO or MAP were found after 15 min of CPR. In the 38°C group, MAP after 45 min CPR was significantly lower than MAP after 60 min of CPR in the 27°C group. This is in essential contrast to the 27°C group where both MAP and CO remained unchanged during 60 min of CPR.2 decreased significantly from 13.6 ± 2.2 to 4.8 ± 1.9 ml min–1 kg–1 (-64%), and VO2 from 5.8 ± 0.5 to 4.1 ± 1.8 ml min–1kg–1 (-30%)  (crit) value (0.6–0.7) to provide aerobic metabolism (2 saturation (SvO2)  . AccordiDO2/VO2)  was signtabolism . After 4n (SvO2)  from 56 n (SvO2) , from 0.2 decreased significantly from 13.7 ± 1.7 to 3.6 ± 0.8 ml min–1 kg–1 (-73%), and VO2 from 5.7 ± 0.7 to 2.0 ± 0.7 ml min–1kg–1 (-64%) (crit (0.6–0.7)  . This ca0.6–0.7) . Simulta–0.7) , and MAP significantly reduced from 89 ± 14 to 60 ± 14 mmHg (-33%).Compared to corresponding values at 38°C, cooling with maintained spontaneous circulation gave an almost linear reduction in CO and MAP . At 27°C2 and VO2 (–1 kg–1 (-48%), and from 5.7 ± 0.7 to 1.8 ± 0.2 ml min–1 kg–1 (-68%), respectively, giving an ER , and a significant reduction in MAP from 89 ± 14 to 33 ± 9 mmHg (-63%) when compared to corresponding values during spontaneous circulation at 38°C , DO2 and VO2 were further significantly reduced first after 2 h of CPR . By 1 h of CPR, ERcrit   recommenSeveral studies using the pig as experimental model have explored the effects of different techniques for CPR on pressure generation and organ blood flow during normothermia, with and without the aid of compression devices . AlternaFrom clinical practice and preclinical experiments, we know that the effectiveness of normothermic CPR to maintain blood flow rapidly deteriorates. In the present experiment, hemodynamics and organ blood flow were largely maintained during the first 2 h of hypothermic CPR. After that, a modest reduction in these variables was accompanied with a small increase in lactate production, but far from ending in a circulatory collapse as demonstrated in normothermic animals after 45 min of CPR.In pig models of normothermic CPR, hemodynamic and organ blood flow data are well documented after 5–10 min of CPR . The pre2 will eventually become dependent on the DO2. The O2 extraction ratio (ER), the ratio between VO2 and DO2, can be used to demonstrate this physiologic mechanism. With limited O2 supply, ER approaches a critical value, ERcrit, where tissue O2 consumption is ruled by O2 supply (crit is unchanged (∼0.65)  , even wi (∼0.65) . During ewarming .2, and ER demonstrate that 3 h of CPR at 27°C provides marginal, but sufficient, O2 transport for aerobic metabolism in vital organs. Importantly, we also found that VO2 was maintained during 3 h of CPR, indicating a use-dependent O2 consumption. This is in essential contrast to normothermic CPR where DO2 after 15 min was unable to provide aerobic metabolism, reflected by the significant elevation of ER exceeding the ERcrit value, in parallel with a substantial elevation in serum lactate and lowering of SvO2.Taken together, the actual values for lactate production, SvOThe porcine model was chosen in our experiment because of its previous use in exploring the effects of CPR, especially when using a compression device . HoweverFrom a clinical perspective, to date, the only possible way to treat patients with continuous CPR during evacuation and transport in an ambulance car is by using an automated chest compression device. Therefore, to treat accidental hypothermia patients with cardiac arrest in our catchment area, we aim to equip all car ambulances with automated compression devices. Further, the beneficial physiologic effects of prolonged CPR for cardiac arrest at reduced core temperatures, emerges to build a new basis with unknown potential for patient survival after the ensuing in-hospital rewarming. From clinical reports already listed, extracorporal circulation has evolved to become the method of choice for rewarming these patients. Preferentially, extra corporal membrane oxygenation (ECMO) is increasingly being used. By applying ECMO for rewarming, cardio/respiratory support can be continued during an ensuing ICU stay for days, if needed. This is to treat rewarming injury, which often manifests itself as cardiac dysfunction caused by hypothermia/rewarming, in addition to the unknown consequences of prolonged CPR to cause physical damage to the heart, lungs, and the thoracic skeleton. However, although effects of extracorporal rewarming to increase survival seems promising, more experimental and clinical work is needed to customize this method for rewarming patients from accidental hypothermia.2 during a 3 h period at 27°C, combined with an apparent patent peripheral circulatory function, make continuous CPR the most valuable prehospital intervention as a bridge for hypothermic patients to receive in-hospital rewarming.The results of this study support our working hypothesis and show the favorable effects of CPR when given at 27°C in comparison to normothermic CPR. The beneficial effects of CPR in providing a limited, but adequate, DOThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.JN, TT, GS, and TK contributed to the conception and design. JN, SV, RM, TS, TN, and TK contributed to the completion of experiments and collection of data. TT, GS, TK, JN, SV, RM, and TS contributed to the data analysis and interpretation. JN, TT, and GS contributed to the drafting the manuscript for intellectual content. JN, SV, RM, TS, TK, TN, GS, and TT contributed to the revision of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Synechococcus-derived organic matter (SOM) by estuarine and coastal microbes during 180-day dark incubations. Variations in organic carbon, inorganic nutrients, fluorescent dissolved organic matter (FDOM), and total/active microbial communities were monitored. The entire incubations could be partitioned into three phases  based on the total organic carbon (TOC) consumption rates of 6.38–7.01, 0.53–0.64, and 0.10–0.13 μmol C L–1 day–1, respectively. This corresponded with accumulation processes of NH4+, NO2–, and NO3–, respectively. One tryptophan-like (C1) and three humic-like  FDOM components were identified. The intensity variation of C1 followed bacterial growth activities, and C2, C3, and C4 displayed labile, semi-labile, and refractory DOM characteristics, respectively. Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes, and Actinobacteria dominated the quickly consumed process of SOM (phase I) coupled with a substantial amount of NH4+ generation. Thaumarchaeota became an abundant population with the highest activities in phase II, especially in the free-living size-fraction, and these organisms could perform chemoautotroph processes through the ammonia oxidation. Microbial populations frequently found in the dark ocean, even the deep sea, became abundant during phase III, in which Nitrospinae/Nitrospirae obtained energy through nitrite oxidation. Our results shed light on the transformation of different biological availability of organic carbon by coastal microorganisms which coupled with the regeneration of different form of inorganic nitrogen.Phytoplankton are major contributors to labile organic matter in the upper ocean. Diverse heterotrophic bacteria successively metabolize these labile compounds and drive elemental biogeochemical cycling. We investigated the bioavailability of  Moreoveon of O2 .Phytoplankton are also an important source of sinking particles in the ocean and contribute to the biological pump (the process by which carbon is exported from the euphotic zone to the dark ocean) . A fractAlteromonas) display successive relationships in the decomposition of phytoplankton-derived organic matter  blooms are frequently observed in coastal waters and can drastically influence the cycling of carbon and other nutrients in eutrophic ecosystems (Synechococcus-derived organic matter (SOM) is labile for heterotrophic bacteria, and N-, P-, and S-containing organic matter exhibit a much shorter turnover time  was grown in the SN medium (Synechococcus-derived organic matter (SOM). Seawater was collected from a depth of 5 m in the coastal region near Xiamen Island (stations S03 and S05) on June 29, 2017  in our studied regions (7 cells mL–1) (6 cells mL–1)   and the ls mL–1)  suggestiMicrocystis aeruginosa bloom, it has been observed that bacteria attached to cyanobacterial cells or detritus during the decline phase of the bloom  and immediately stored at −20°C for further analysis. For dissolved organic carbon (DOC) and fluorescent dissolved organic matter (FDOM) samples, 20 mL samples were filtered through pre-combusted  0.7 μm pore-size GF/F filters  into 40 mL glass vials and stored at −20°C. To prevent carbon contamination, all glass materials used for sample collection and storage were acid-washed, Milli-Q (ion- and nuclease-free water) rinsed, and pre-combusted for 4 h at 450°C. For inorganic nutrients measurement, 50 mL samples were filtered through 0.45 μm polycarbonate membrane filters  and stored at −20°C for further analysis. For DNA and RNA samples, 300 mL water samples were immediately filtered through 0.2 μm polycarbonate membrane filters . For the control incubations, 300 mL of water samples were immediately filtered through 0.2 μm polycarbonate membrane filters  for DNA and RNA analyses. However, for the SOM-addition incubations 300 mL of water samples was filtered through 3 and 0.2 μm polycarbonate membrane filters to obtain two size fractions, the >3 μm (particle attached bacteria) and the 0.22–3 μm (free-living bacteria). This fractionation was performed because in the he bloom . This fr2–), nitrate (NO3–), and phosphate (PO43–), were measured via spectrophotometric methods (4+) concentration in samples  using a Shimadzu TOC-VCPH analyzer (Japan). Concentration of dissolved inorganic nutrients, including nitrite (NO methods  using a  samples . AlthougFluorescence measurements were performed using a 1 cm quartz cuvette and a Varian Cary Eclipse spectro-fluorometer (United States). Emission spectra were scanned every 2 nm at wavelengths from 280 to 600 nm, with excitation wavelengths ranging from 240 to 450 nm at 5 nm intervals . Slit wi@ 2.0 Fluorometer  and Agilent Bioanalyzer 2100 system (United States). Finally, the library was sequenced on an Illumina HiSeq 2500 platform, generating 450 bp paired-end reads that were then combined using FLASH software (V1.2.71). Raw data were first quality-filtered with QIIME to remove reads that did not meet the desired quality (2). The OTUs were taxonomically classified based on the SILVA database (Version 132). Sequence data were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive3 under BioProject PRJNA532855.DNA extraction was performed using the phenol-chloroform-isoamyl alcohol method . The qua quality . Chimera quality . OperatiNon-metric multidimensional scaling (NMDS) was used to determine the similarity of samples to each other based on Bray-Curtis similarities  with CANSynechococcus-derived organic matter was quickly consumed by bacteria during the 180-day incubations, and TOC concentration continually decreased from 148.9–153.5 to 75.2–85.9 μmol C L–1 in the SOM-addition groups and from 85.2–98.4 to 69.9–83.1 μmol C L–1 in the control groups (l groups . Accordi–1 (from 148.9 ± 5.8 to 99.8 ± 2.3 μmol C L–1) with a consumption rate of 7.01 ± 0.29 μmol C L–1 day–1 in phase I. The TOC concentration ranged from 99.8 ± 2.3 to 92.3 ± 0.8 μmol C L–1 in phase II with a consumption rate of 0.53 ± 0.10 μmol C L–1 day–1. The TOC concentration reduced to 75.2 ± 3.2 μmol C L–1 at the end of the incubations with a consumption rate of 0.10 ± 0.01 μmol C L–1 day–1. Compared to the SOM-addition groups, the TOC concentration had a relatively narrow range variation from 85.2 ± 1.6 to 69.9 ± 0.5 μmol C L–1 in the control groups during the incubations.In the station S05 incubation system , the TOC–1 with a consumption rate of 6.38 ± 0.54 μmol C L–1 day–1 in phase I. In phase II, the TOC concentration decreased to 105.5 μmol L–1 with a consumption rate of 0.64 μmol C L–1 day–1. The TOC concentration dropped to 85.9 ± 2.2 μmol C L–1 at day 180 with a consumption rate of 0.13 ± 0.03 μmol C L–1 day–1 in phase III. The TOC concentration decreased by 15.4 ± 0.3 μmol C L–1 from 98.4 ± 0.7 to 83.1 ± 0.3 μmol C L–1 in the control groups over the course of the incubations.In the S03 incubations , the TOCDOC concentrations were also monitored in our incubaitons . Althoug4+, NO2–, NO3–, and PO43–, were measured during the 180-day incubations  in phase III in the SOM-addition groups (2– was low (∼2.0 μmol L–1) in phase I, then increased to 17.7 ± 1.9 μmol L–1 at the 20th day and decreased back to a very low level (undetectable) from day 50. The NO3– concentration showed few variations before phase III ranging from 31.9 ± 0.4 to 28.4 ± 3.6 μmol L–1 (the 20th day), then gradually increased from 28.4 ± 3.6 to 52.7 ± 2.6 μmol L–1 during phase III , and then remained with relatively small variations in the following days. Clear transformation processes of nitrogen-containing nutrients from SOM (N-containing organic matter) to NH4+ in phase I, from NH4+ to NO2– in phase II, and from NO2– to NO3– in phase III were observed. In the control groups, the concentration of NH4+ (from 4.7 ± 0.5 to 0.8 ± 0.1 μmol L–1) and NO2– (from 2.0 ± 0.1 μmol L–1 to undetectable at day 180) gradually decreased over the course of the entire incubations. Meanwhile, the NO3– concentration gradually increased from 26.1 ± 0.09 to 33.2 ± 0.1 μmol L–1 at the end of the incubations. The PO43– concentration increased from 1.0 ± 0.04 to 1.5 ± 0.02 μmol L–1 in phase I  at the 20th day before being reduced to a low level (0.2 ± 0.04 μmol L–1) at day 50. Additionally, the NO3– concentration increased from 53.3 ± 0.9  to 67.6 ± 7.9 μmol L–1 (at the 50th day) and reached 81.5 ± 0.4 μmol L–1 by the end of the incubations. The concentration of PO43– increased from 1.3 ± 0.06 to 1.7 ± 0.07 μmol L–1 in phase I and reached 1.8 ± 0.1 μmol L–1 by the end of the incubations  were identified by PARAFAC analyses in our incubations. Component C1 exhibited fluorescence properties similar to those of the tryptophan-containing molecules , whereas components C2, C3, and C4 showed locations of maximum peak intensities typical of what are referred to as humic-like molecules . VariatiSynechococcus sp. CB0101  were obtained (after removing Cyanobacteria reads mainly from the SOM addition) on both size fractions from stations S05 and S03 incubations, respectively. Over the entire incubations, Gammaproteobacteria , Alphaproteobacteria (14.14 and 12.04%), Bacteroidetes (13.35% and 20.97%), Planctomycetes (9.29 and 7.80%), Thaumarchaeota (7.00 and 9.09%), and Actinobacteria (7.25 and 4.83%) were the dominant active microbial communities.In the station S05 incubations , the SOMIn the station S03 incubations, the active microbial responses and succession exhibited the similar pattern with that of in station S05 incubations . AlphaprThe variations of total microbial communities were also studied, and a total of 195,542 and 200,153 sequences (16S rDNA) were also obtained (after removing Cyanobacteria reads mainly from the SOM addition) from stations S05 and S03 incubations, respectively. Considering the similar microbial response pattern with active microbial populations, these results were put into The NMDS analyses showed that total  and acti–1 SOM, mainly in the form of POM, was added to the SOM-addition groups. Three phases could be clearly separated based on the organic carbon consumption rates in the SOM-addition groups. SOM was rapidly degraded by coastal heterotrophic microbes in phase I and II, suggesting its labile properties. Meanwhile, the concentration of NH4+ and PO43– rapidly accumulated with the consumption of SOM in phases I and II, and showed a significant negative correlation with TOC concentration, respectively  changes of DOC over the 180-day incubations. C1 presented a protein-like component, and its intensity variations displayed a close relationship with heterotrophic bacterial growth activities. It seems that component C1 was produced/released during high bacterial metabolic activities and was re-used after the environmental labile organic matter was depleted. Previous studies have also reported marine bacteria can mediate the production and consumption of peak T, and the fluorescence of peak T and the bacterial biomass had a significant positive linear correlation .Synechococcus sp. cells. The same fluorescent signal was also observed in a 15-day culture of Synechococcus sp. CB0101   . Here, TM range) . Thaumar strains .In the later period of our incubations, some species that were rare in the early phases became abundant, including Acidobacteria, Chloroflexi, Gemmatimonadetes, and Nitrospinae/Nitrospirae. These specific taxa have been commonly detected as dominant populations in the deep sea and in surface sediment . AcidobaSynechococcus sp. cells could be a significant source of high molecular weight dissolved organic nitrogen compounds, and abundant peptides from proteolysis of the light-harvesting protein phycoerythrin were detected , the generation and re-utilization of FDOM  and the production of recalcitrant FDOM  by microbial activity indicated that microbes could consume and transform SOM, affecting the bioavailability of DOM in the environment. Additionally, the variations in the microbial communities corresponded with the bioavailable TOC/SOM  in the long-term incubations, implying the possible functional diversity coupling with environmental nutrient conditions. The different dominant microbial communities were present in different phases during SOM degradation which suggested different bioavailable substrates provide distinct ecological habitats suitable for specialized populations. The three identified phases, based on organic carbon consumption rates, corresponded with the accumulation processes of NHThe coupling of carbon and nitrogen metabolic processes mediated by coastal microbes during the degradation of SOM provided greater insight into the biogeochemical cycles in the eutrophic environment. Additionally, the bacterial community succession and degradation of organic matter in the long-term shed light on the vertical transformation processes of POM from euphotic zone to the darker and deeper parts of the ocean. Future work should be focused on determining the necessity of applying advance mass spectrometry technologies , in combination with metagenome and metatranscriptome analyses to strengthen our understanding of the microbial behaviors and microbial metabolites.http://trace.ncbi.nlm.nih.gov/Traces/sra/) under BioProject PRJNA532855.The datasets generated for this study can be found in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (RX, QZ, and NJ conceived the study and designed the experiments. RX performed experiments and data analyses. YW undertook the 16S rDNA/rRNA analyses. QC assisted with the sampling. WG provided Varian Cary Eclipse spectro-fluorometer. RX and QZ wrote the manuscript. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Under the optimized conditions, sulfite ion is accurately and precisely measured under batch and flow injection modes of analysis. The sensor exhibits fast and linear response for 1.0 × 10−2–1.0 × 10−6 M (800–0.08 µg/mL) and 1.0 × 10−1–5.0 × 10−5 M (8000–4 µg/mL) sulfite with Nernstian slopes of −27.4 ± 0.3 and −23.7 ± 0.6 mV/concentration decade under static and hydrodynamic modes of operation, respectively. Results in good agreement with the standard iodometric method are obtained.Validation of the assay method is examined in details including precision, accuracy, bias, trueness, repeatability, reproducibility, and uncertainty and good performance characteristics of the method are obtained. The sensor response is stable over the pH range of 5 to 7 without any significant interference from most common anions. The advantages offered by the proposed sensor  suggest its use in the quality control/quality assurance routine tests in beverages industries, toxicological laboratories and by inspection authorities.A simple potentiometric sensor is described for accurate, precise, and rapid determination of sulfite additives in beverages. The sensor is based on the use of cobalt phthalocyanine as a recognition material, dispersed in a plasticized poly(vinyl chloride) matrix membrane.  Sulfite ions have been widely used for centuries as preservative for food, beverages, and pharmaceutical products ,2. It isPreviously described analytical methods for the determination of sulfite include spectrophotometry based on reactions with formaldehyde-pararosaniline , methyl 2O2 sensor or Clark-type oxygen electrode. An amperometric sulfite oxidase/cytochrome c-based sensor has been also used  sulfite PVC membrane sensor in 10−2 M acetate buffer and 10−3 M NaCl (pH 5) under batch and FIA modes of operation.−3 M −3 M NaCl background from pH 3 to 11 by adding aliquots of concentrated sodium hydroxide and/or acetic acid solutions. The corresponding mV readings at each pH value were recorded after each addition. − ion, while at low pH values of < 3, the sensor responded to the monovalent HSO3−(1pKa = 1.89 and 2pKa = 7.21 at 25 °C). A 10−2 M acetate buffer solution of pH 5, containing 10−3 M NaCl was used as a background solution and used for all subsequent measurements.The effect of pH on the potentiometric response of the sulfite sensor was evaluated by changing the pH of 1.0 × 10PotSulfite,J)(K of the sulfite sensor were measured according to the IUPAC guidelines using the fixed interfering method (FIM) [−2 M of the interfering ion solution was added to 9.0 mL of a 1:1 mix of 1.0 × 10−3 M NaCl and 1.0 × 10−3 M acetate buffer of pH 5 in presence of fixed concentration of sulfite ion (10−3 M). Equation (1) was used for calculation,ia and ja are the activities of the primary and interfering ions, respectively, and iz and jz are their respective charges.The potentiometric selectivity coefficients od (FIM) . In this+/o-NPOE-based sulfite sensor exhibited reasonable selectivity towards sulfite ion over most common ions, which was in a good agreement with data obtained by the spectrophotometric measurements. The selectivity coefficients for nitrate, nitrite, chloride, fluoride, azide, sulfate, phosphate, and acetate ranged from 10−2 to 10−3. Thiocyanate ion, however, significantly interferes but it is fortunate that thiocyanate ion rarely coexists with sulfite in real beverages.The obtained results revealed that the CoPC/TDMAt )value at 95% confidence level were made. No statistical difference was detected between the calculated and the theoretical values.Replicate measurements of 1.0 and 10.0 µg/mL internal quality control sulfite sample (IQS) under batch mode of operation and calculation of the student’s  of different sulfite calibration solutions using a CoPC/TDMAwCV), between-days-variability (bCV), ruggedness (robustness), and uncertainty.Validation of the proposed potentiometric method was performed, using the quality control/quality assurance standards and guidelines ,45, to vn = 6, each) showed relative standard deviations (RSD) in the range ± 0.9 ± 0.1–1.2 ± 0.2%. Theaverage of sulfite results (X) and the standard deviation (S) were calculated to evaluate the method precision using Equation (2),X is the mean of test results obtained for the sulfite reference standard sample and S is the standard deviation of X. Absolute uncertainty is expressed as: X sulfite value ± precision.Repeated measurements of an internal quality control sulfite samples containing 1.0, 10.0, 50.0, 100.0, 200.0, and 300.0 µg/mL sulfite  sulfite value was measured by spiking (adding) of a reference sulfite samples. The accuracy of the assay method across the dynamic range of the analytical procedure was calculated using Equation (3) and found to be 98.1 ± 0.7–97.3 ± 1.1%,X is the mean of test results obtained for the reference sample and µ is thetrue value of the reference sample. The mean trueness and bias percentages obtained, ranged between 98.0 ± 0.8–97.1 ± 1.2% and 2.0 ± 0.2–3.2 ± 0.8 %, respectively.Replicate analyses performed on reference sulfite samples  were also used to determine method trueness and bias according to Equations (4) and (5), respectively.R) is calculated from the standard deviation data of the results (Rs) (Equation (6)).= 2√2 and is derived from the normal or Gaussian distribution; ISO 5725). The data reproducibility within-day and between-days were found to be 0.7 ± 0.2–1.1 ± 0.4% and 0.9 ± 0.2–1.3 ± 0.5%, respectively.The spread of results when a sulfite sample (1.0–300.0 µg/mL) was measured in the same day and on different days to conform the agreement between results obtained with the sulfite reference sample under different conditions with different sensor assembly and pH meters at different times. The reproducibility (−6 M (0.08 µg/mL) and 1.0 × 10−5 M (0.8 µg/mL) under batch and flow injection analysis, respectively. The sensor displayed linear dynamic range between 1.0 × 10−2–1.0 × 10−6 M (800–0.08 µg/mL) and between 5.0 × 10−5–1.0 × 10−1 M (4–8000 µg/mL) with near-Nernstian slopes of −27.4 ± 0.3 and −23 ± 0.6 mV/decade with batch and flow injection analysis, respectively. All the results of the validation protocol which support the applicability and suitability of the proposed sensor for accurate routine analysis of sulfite ion in various beverages are shown in The lower limit of sulfite detection (LOD) was calculated according to the IUPAC guidelines . It is eX and R) were prepared for monitoring 0.5, 5.0, 100.0, 200.0, and 300.0 µg/mL sulfite standard solutions. The results were used to construct both X- and R-control chart. The distribution of measurements and range of the data under investigation indicated that they were in statistical control [Based on running duplicate measurements for 10 days, control charts  equipped with an Orion combination Ross glass pH electrode (Model 81-02) was used for pH adjustments. A cobalt phthalocyanine-PVC-based membrane sensor in conjunction with an Orion Ag/AgCl double junction reference electrode (Model 90–02) was used for potentiometric measurements of sulfite. Potentiometric signals were recorded using a home-made high-impedance data acquisition system connected to a PC through the interface ADC 16  and a PicoLog for windows (version 5.07) software  Potentiometric measurements were performed at 25 ± 1 °C.A laboratory-made potentiometric flow-through tubular cell was equipped with cobalt phthalocyanine-PVC membrane as previously described  and inco2SO3), potassium iodate, potassium iodide, sulfamic acid, sulfuric acid, acetic acid, sodium hydroxide, sodium bicarbonate, sodium chloride, dimethyl sulfoxide (DMSO), dioxane, and sodium chloride (NaCl) were obtained from Aldrich Chemical Co. . Tetrahydrofuran (THF), freshly distilled prior to use, o-nitrophenyloctyl ether (o-NPOE), and dioctylphthalate (DOP) were purchased from FlukaChemika-Biochemika .All reagents were prepared from analytical reagent grade chemicals unless otherwise specified and doubly distilled deionized water was used throughout. Cobalt phthalocyanine (CoPC) was obtained from Midcentury . Tri-dodecylmethyl- ammonium chloride (TDMAC), high molecular weight poly (vinyl chloride) (PVC), M.W. of 100,000, sodium sulfite  and covered with a piece of filter paper until complete evaporation of THF. A disk of about 6 mm in diameter was then cut, by the aid of a cork borer, and glued to a piece of Tygon tube  using THF. The Tygon tube was attached to the electrode glass body, and a 1:1 mixture of 10−3 M NaCl and 1.0 × 10−3 M acetate buffer of pH 5. Aliquots (1.0 mL of standard sulfite solutions (1.0 × 10−6 to 1.0 × 10−2 M) were sequentially added and the potential change after each addition was recorded. A calibration curve was constructed by plotting the potential change against the logarithm of sulfite concentration. The calibration graph was used for subsequent measurement of sulfite levels in real test samples under the same experimental conditions. The sensor was stored in the same solution between measurements.The sulfite sensor in conjunction with Ag/AgCl double-junction reference electrode was immersed into a 25 mL beaker containing 10 mL of a 1:1 mix of 1.0 × 10−3 M NaCl and 1.0 × 10−3 M acetate buffer of pH 5, add a 10.0 mL aliquot of the beverage sample and stir. The working and reference electrodes were immersed in the solution. The potential readings were recorded after stabilization and the concentrations of sulfite in the test samples were calculated using the calibration graph. A blank experiment was carried out under the same conditions without the beverage sample.Sulfite levels in some beverage samples were potentiometrically measured. The standard iodometric method for sulfide assessment was carried in parallel for comparison. The test samples were used without any further treatment. To a 50 mL beaker containing 10 mL of a 1:1 mix of 1.0 × 10−3 M NaCl and 1.0 × 10−3 M acetate buffer of pH 5) was propelled with a flow rate of 10 mL/min. The carrier solution was allowed to passes through the flow through-cell by means of a peristaltic pump and Tygon tubing. Successive injections of 20 µL aliquots of each sulfite standards (1.0 × 10−1–1.0 × 10−5 M) were made into the flowing stream. The corresponding signal heights and potential change was measured using a data acquisition system. The mV vs. logarithm sulfite concentration readings of the sulfite standards were recorded and used for construction of a calibration plot. Beverage solutions were similarly injected (in triplicate) in the flowing stream and the average potential readings of three runs was compared with those of the standard solutions. A blank experiment under the same conditions was carried out.A flow stream of the carrier solution (1:1 mix of 1.0 × 103 solution (0.4458 g KIO3 + 4.35 g KI + 310 mg NaHCO3 in 1 L bi-distilled water). The end point was signaled by the dark purple color due to interaction of the first excess of iodine with starch; 1.00 mL of the titrant ≡ 500 µg sulfite.A 100.0 mL aliquot of the beverage test solution was transferred to a 250 mL Erlenmeyer flask; add 1 mL of 1:1 sulfuric acid and 0.1 g sulfamic acid crystals. A 1 mL portion of a starch indicator solution was added and titration was conducted with a standard 0.002 M KI/KIO+/o-NPOE as an ion recognition sensing material. The method covers the range 1.0 × 10−6 to 2.2 × 10−3 M (0.08–176 µg/mL) with a low detection limit of 1.0 × 10−6 M (0.08 µg/mL), and good selectivity over many other common anions. The performance characteristics of the sensor are validated and proved to be suitable for spectrophotometric covers the linear range 16 to 80 µg/mL. Both methods are used for determination of sulfite in real beverages. The results show good correlation with the standard iodometric method. The proposed method offers many advantages over many of those previously described it needs no prior treatment of the sulfite sample, it has a fast response and is cost-effective, and uses simple equipment.Sulfite additives in real beverage samples are determined using a simple novel potentiometric sensor based on the use of cobalt phthalocyanine (CoPC) ionophore. The sensor consists of poly (vinyl chloride) membrane containing CoPC/TDMAC


Transglutaminase 2 (TG2), also known as tissue transglutaminase, is a calcium-dependent enzyme that has a variety of intracellular and extracellular substrates. TG2 not only increases in osteoarthritis (OA) tissue but also affects the progression of OA. However, it is still unclear how TG2 affects cartilage degradation in OA at the molecular level. Surgically induced OA lead to an increase of TG2 in the articular cartilage and growth plate, and it was dependent on TGFβ1 in primary chondrocytes. The inhibition of TG2 enzymatic activity with intra-articular injection of ZDON, the peptide-based specific TG2 inhibitor, ameliorated the severity of surgically induced OA as well as the expression of MMP-3 and MMP-13. ZDON attenuated MMP-3 and MMP-13 expression in TGFβ- and calcium ionophore-treated chondrocytes in a Runx2-independent manner. TG2 inhibition with ZDON suppressed canonical Wnt signaling through a reduction of β-catenin, which was mediated by ubiquitination-dependent proteasomal degradation. In addition, TG2 activation by a calcium ionophore enhanced the phosphorylation of AMPK and FoxO3a and the nuclear translocation of FoxO3a, which was responsible for the increase in MMP-13. In conclusion, TG2 plays an important role in the pathogenesis of OA as a major catabolic mediator that affects the stability of β-catenin and FoxO3a-mediated MMP-13 production. This change is accompanied by subchondral bone remodelling and osteophyte formation at the joint margin1. In this process, the proteases produced by chondrocytes, especially matrix metalloproteinase (MMP)-13 and A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS)-5, have a major role in the matrix degradation, and proteolytic generation of matrix fragments has been suggested to drive chondrocyte hypertrophy3. However, the regulatory mechanisms of MMPs and AMDATS production, as well as the post-transcriptional modification that stabilizes or activates these proteases, have not been fully elucidated4.Osteoarthritis (OA), which is the most common form of arthritis, is characterized by damage to the extracellular matrix of cartilage, resulting in the loss of hyaline articular cartilage6. In the presence of calcium, TG2 functions as a catalytic enzyme that modifies the protein-bound glutamine side chains, resulting in protein crosslinking through transamidation or deamidation7. When this protein binds to GTP intracellularly, it becomes closed conformationally and acts as a G-protein associated signaling molecule8. Through its enzymatic and signaling functions, TG2 has diverse biological roles, such as an enhancer of inflammation, apoptosis regulation, cellular differentiation and extracellular matrix interaction6. In chondrocyte biology, TG2 is known not only as a marker for hypertrophic chondrocyte but also as an inducer of chondrocyte maturation10. TG2 knockout mice showed less severe cartilage degradation and enhanced osteophyte formation compared to control wild-type mice in a surgically induced OA model11.Transglutaminase 2 (TG2) is a 78-kDa calcium-dependent enzyme of the protein-glutamine γ-glutamyl-transferase family12. This increased calcium level in the articular cartilage of early OA can affect the enzyme activity of TG2, which may affect the production and stability of MMPs, and consequently accelerate cartilage degradation. However, the molecular mechanisms of the catabolic role of TG2 in the progression of OA are still unclear. In the present study, we investigated the effect of inhibition of TG2 enzyme activity on the severity of surgically induced OA. At the molecular level, TG2 stabilized β-catenin to increase Wnt signaling and enhanced nuclear translocation of FoxO3a, which was responsible for the production of MMP-3 and MMP-13.Recently, we reported that enhanced subchondral bone remodelling in early OA can increase the calcium level in articular cartilage, and this change was related to MMP-3 and MMP-13 production12. Briefly, after 12-week-old C57BL/6 male mice were anaesthetized, the skin over the left knee was opened, and the meniscotibial ligament of the medial meniscus was sectioned in total 15 mice among which 5 mice were sacrificed at 4 weeks without treatment, and 5 mice were treated with ZDON and 5 with control DMSO for 8 weeks. The sham operation just visualized the meniscotibial ligament but not resected in total 13 mice . The treatment group mice were intra-articularly injected with 10 μl of ZDON (200 μM), a TG2 inhibitor  or DMSO alone as a control once a week for 7 weeks and sacrificed at 8 weeks after DMM surgery. After sacrifice, the knee joints were fixed in 4% paraformaldehyde  for 24 h and decalcified in 10% EDTA for 3 weeks. Embedded blocks were sectioned at a thickness of 5 μm and stained with safranin-O. OA severity was scored by two blinded observers using the Osteoarthritis Research Society International (OARSI) grading system13.Surgical OA was induced by destabilization of the medial meniscus (DMM) in mice as previously describedAll animal protocols were approved by the Institutional Animal Care and Use Committee of the Daegu Fatima Hospital (approved protocol number F-16–4). Mice were maintained in standard laboratory conditions and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health .14. Total cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes . The membranes were blocked with 5% bovine serum albumin (BSA) at room temperature (RT) for 1 h, and incubated with the primary antibodies listed next for 1 h with shaking. The primary antibodies against TG2, β-catenin, p-β-catenin, non-p-β-catenin, Ampka, p-Ampka, Foxo3a, p-Foxo3a, p65, p-p65, mTOR, p-mTOR, Axin2, Tcf1, Sirt1, and IκB were purchased from Cell Signaling Technology ; antibodies against Sox9, Runx2, Mmp3, Mmp13, and Adamts5 were from Abcam ; the antibodies against LaminB and haemagglutinin (HA) were from Santa Cruz Biotechnology , and β-actin antibody was from Sigma Aldrich . Membranes were washed, and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h. The membrane signal was visualized using Supersignal Chemiluminescent Substrates .The Western blot analysis was performed as reported previously14. For immunofluorescence staining, the rehydrated sections were retrieved in sodium citrate buffer . After the sections were blocked with 2% BSA in PBS for 1 h, they were incubated with the primary antibodies for TG2 (1:200 dilution), β-catenin (1:400), non-p-β-catenin (1:400), FoxO3a (1:200), and p-FoxO3a (1:200) or normal rabbit IgG (1:200) in 1% BSA in PBS at 4 ℃ overnight. For immunohistochemistry, the retrieved sections were immunostained for MMP3 (1:200) and MMP13 (1:400) in 1% BSA in PBS for 16 h at 4 ℃ and then with goat anti-rabbit IgG conjugated with HRP  for 1 h at RT.The immunofluorescence and immunohistochemical staining were performed on the tissue samples of surgical knee OA as reported previously4 cells/well, Thermo Fisher Scientific) and treated with TGF-β1 (10 ng/ml), with or without calcium ionophore (2 mM) or ZDON (100 μM). The cells were fixed with 4% formalin for 5 min, permeabilized with 0.25% Triton X-100 in PBS for 3 min, blocked with 5% BSA for 1 h, and incubated with rabbit anti-non-p-β-catenin (1:400) and mouse anti-TG2 (1:200) antibodies at RT for 1 h.For immunocyto-fluorescence, primary chondrocytes were plated on 8-chamber slides  supplemented with 0.2% BSA, 0.25 mM ascorbic acid, 1 mM β-glycerophosphate, 0.25% l-glutamine, and 0.25% penicillin/streptomycin for 24 h at 37 °C. The bones were then incubated in 0.25% trypsin–EDTA for 15 min at 37 °C, followed by further incubation with 3 mg/ml type II collagenase (Sigma Aldrich) in Dulbecco’s modified essential medium (DMEM) for 2 h at 37 °C with gentle shaking. Cells were filtered through a 40 μm nylon mesh (BD Biosciences) and collected by centrifugation. The cells cultured in a 2:3 mixture of DMEM and Ham’s F12 (DMEM/F12) with 10% foetal bovine serum (FBS), and 0.25% l-glutamine. For starvation and treatment, DMEM/F12 with 1% FBS was used.Primary chondrocytes were prepared from the femur and tibia of postcoital day 15.5 ICR mouse embryos (E15.5) as previously described5 cells per well) and treated with TGFβ1 (10 ng/ml) or TGFβ1 plus calcium ionophore (2 mM) with or without ZDON (100 μM) for 24 h. Cell lysates were prepared with a lysis buffer . TG2 activity assays were performed using a TG2 Colorimetric Assay Kit according to the manufacturer's instructions .Primary chondrocytes were seeded in 6-well plates (5 × 105 cells/well) and transfected with expression constructs of β-catenin (Santa Cruz) and HA-Ubiquitin16. After 24 h, cells were incubated in the media containing TGFβ1 (10 ng/ml) or TGFβ1 plus calcium ionophore (2 mM) with or without ZDON (100 μM) for 20 h, followed by exposure to 10 μM MG132 for 5 h. Then, cell lysates were prepared with 20 mM HEPES, pH 7.9 buffer containing 300 mM KCl, 10% glycerol, 10% NP-40, 1 mM DTT and 1 × protease inhibitors. Lysates were precleared with Protein A Sepharose  for 30 min, precipitated with anti-β-catenin antibody for 18 h, incubated with Protein A Sepharose beads for 2 h at 4 ℃ with gentle shaking and subjected to Western blot analysis.ATDC5 cells were seeded in 6-well plates  tests were used to determine differences between means. All analyses were conducted using SPSS version 14.0 software . The results are presented as the mean ± S.D., and statistical significance was defined as p values of < 0.05.Mann–Whitney 17. To identify paracrine factors regulating TG2 expression, we treated primary chondrocytes with major growth factors known to be important in cartilage biology. TG2 was selectively induced by TGFβ1 in a dose-dependent manner in primary chondrocytes . TG2 was selectively expressed in the articular cartilage and growth plate of DMM-operated mice but not in the joint tissue of sham-operated mice Fig. a. The TGTo investigate the role of TG2 in OA, we surgically induced OA in 12-week-old male mice and administered weekly intra-articular injections of 10 μL of ZDON (200 μM), a cell-permeable, peptide-based inhibitor of TG2 activity. Eight weeks after DMM surgery, ZDON-treated mice showed less severe cartilage damage than control mice  or a calcium ionophore, A23187 (2 μM). Enzyme activity assays of TG2 showed that TGFβ1 increased the enzyme activity of TG2 compared to that of the control, but the activity was not suppressed by ZDON. Calcium ionophore treatment in the presence of TGFβ1 significantly increased TG2 activity compared to that of the TGFβ1 alone group, and the activity was suppressed by ZDON Fig. .Figure 3To elucidate the role of TG2 enzyme activity in chondrocyte signaling, we stimulated starved primary chondrocytes with the calcium ionophore (10 μM) in the presence of ZDON. The ZDON pretreatment significantly reduced total β-catenin level, and the same pattern was observed for the Ser33/37/Thr41-phosphorylated β-catenin. The inhibition of TG2 enzyme activity by ZDON significantly suppressed the Thr172 phosphorylation of AMPKa and Ser253 phosphorylation of FoxO3a. However, activation of p65, the major component of canonical NF-kB signaling, and mTOR was not affected by the calcium ionophore or ZDON  and TGFβ, and the inhibition of TGFβ in subchondral bone can attenuate the progression of surgically induced OA19. However, the genetic deletion of a key component of TGFβ signaling, including TGFβ receptor type II or Smad3, aggravates the severity of OA21. These conflicting results can be explained by the difference in the degree of TGFβ signaling blockade between a partial inhibition with chemicals or blocking antibody and total inhibition with a genetic model. The discrepancy could also result from the differential role of TGFβ according to the differentiation status of chondrocytes, namely, resting, proliferating and hypertrophic chondrocytes22. TGFβ may naturally have both anabolic and catabolic functions, and its overall effect can vary depending on the differentiation status of chondrocytes. Our study provides a possible explanation for the catabolic function of TGFβ via TG2 in subchondral bone. Active subchondral bone remodelling during early OA can increase both TGFβ and calcium levels in the subchondral milieu12. TGFβ can increase the expression of TG2 in hypertrophic chondrocytes in the neighbouring subchondral bone9, and this calcium-rich environment in subchondral bone may enhance the calcium-dependent enzyme activity of TG2.This study showed articular chondrocyte-specific expression of TG2 in the early stage of OA. In addition, TG2 was specifically induced by TGFβ in a dose-dependent manner Fig. . The act24, and the regulation of chondrogenic and hypertrophic differentiation27. TG2 has been confirmed to enhance canonical Wnt signaling in tumour and vascular smooth muscle cells (VSMCs)30. A recent study showed that TG2 does not affect the transcriptional level of β-catenin, but it increases the phosphorylation of glycogen synthase kinase (GSK)-3β, which was inactivated by phosphorylation28. This inhibition of GSK-3β results in the accumulation of β-catenin and enhancement of Wnt signalling28. Furthermore, TG2 directly interacts with low density lipoprotein related-protein 5 (LRP5) and activates canonical β-catenin signaling in VSMCs30. Interestingly, our study demonstrated that TG2 enhances canonical Wnt signaling through the increase in total β-catenin level by inhibition of its ubiquitination in primary chondrocytes (Fig. 32. To our knowledge, this is first evidence showing that TG2 enzyme activity is needed to maintain β-catenin stability and Wnt signaling in chondrocytes.Wnt signaling has been shown to play a key role in OA pathogenesis through the induction of proteases, especially MMP-1334. This functions to protect chondrocytes from cellular stress and consequently attenuate the progression of OA36. FoxO3a, a major downstream target molecule of AMPK, is also known to have a protective role in chondrocytes under metabolic stress conditions39. FoxO3a increases PGC1α expression to mediate mitochondrial biogenesis, induces the autophagic programme in an mTOR-dependent and independent manner, and enhances the production of endogenous antioxidants to maintain the cellular redox balance41. The inhibition of TG2 enzyme activity significantly suppressed the calcium ionophore-mediated phosphorylation of AMPK and FoxO3a, as well as the nuclear translocation of FoxO3a, in our study (Figs. AMPK is activated under metabolic stress conditions, such as hypoxia and nutrient deprivation, to induce cell-protective autophagy and to stabilize cellular metabolism by increasing glucose uptake and fatty acid oxidationdy Figs. . A calci42. Through MMP-13 induction, FoxO3a promotes atherosclerosis and degeneration of the arterial intima and medial layer42. FoxO3a-dependent MMP-13 induction was also observed in a melanoma cell line in serum deprivation conditions43. The current pathophysiologic mechanisms that are considered important in OA eventually converged to the production of MMP-134. Our data suggest that FoxO3a mediates the catabolic effect of TG2 through the induction of MMP-13.In addition to its effects on phosphorylation, TG2 increased MMP-13 expression through FoxO3a. The FoxO3a-mediated increase of MMP-13 was completely suppressed by inhibition of TG2 enzyme activity in this study. The ability of FoxO3a to enhance transcription of MMP-13 was highlighted in a recent study that showed FoxO3a directly bound to the MMP-13 promoter and induced MMP-13 promoter activity in VSMCs46. TG2 is known to constitutively activate NF-kB signaling through the enhanced degradation of IkBα via non-proteasomal and proteasomal degradation47. However, our data showed that the activation or inhibition of TG2 failed to affect NF-kB activation or nuclear translocation in primary chondrocytes (Figs. Several lines of evidence showed that canonical NF-kB signaling contributes to hypertrophic differentiation as well as MMP and nitric oxide production as upstream signaling of HIF-2α in OA chondrocyteses Figs.  and 5, sIn conclusion, TG2 mainly induced by TGFβ can stabilize β-catenin to enhance canonical Wnt signaling and promote the nuclear translocation of FoxO3a, which can be responsible for MMP-13 production Fig. . These fSupplementary Figures.


The sensitivity was 33.3% (3/9) in mucinous adenocarcinoma. The ADC value (1.31 ± 0.32 × 10−3 mm2/s) of adenocarcinoma was significantly higher than that (1.17 ± 0.29 × 10−3 mm2/s) of squamous cell carcinoma (p = 0.012), or (0.93 ± 0.14 × 10−3 mm2/s) of small cell carcinoma (p = 0.0095). The ADC value (1.91 ± 0.36 × 10−3 mm2/s) of mucinous adenocarcinoma was significantly higher than that (1.25 ± 0.25 × 10−3 mm2/s) of adenocarcinoma with mucin and that (1.24 ± 0.30 × 10−3 mm2/s) of other cell types. The ADC (1.11 ± 0.26 × 10−3 mm2/s) of lung cancer with necrosis was significantly lower than that (1.32 ± 0.33 × 10−3 mm2/s) of lung cancer without necrosis. The ADC of mucinous adenocarcinoma was significantly higher than those of adenocarcinoma of other cell types. The ADC of lung cancer was likely to decrease according to cell differentiation decreasing. The sensitivity of DWI for lung cancer was 92% and this result shows that DWI is valuable for the evaluation of lung cancer. Lung cancer could be evaluated qualitatively using DWI.For detecting malignant tumors, diffusion-weighted magnetic resonance imaging (DWI) as well as fluoro-2-deoxy-glucose positron emission tomography/computed tomography (FDG-PET/CT) are available. It is not definitive how DWI correlates the pathological findings of lung cancer. The aim of this study is to evaluate the relationships between DWI findings and pathologic findings. In this study, 226 patients with resected lung cancers were enrolled. DWI was performed on each patient before surgery. There were 167 patients with adenocarcinoma, 44 patients with squamous cell carcinoma, and 15 patients with other cell types. Relationships between the apparent diffusion coefficient (ADC) of DWI and the pathology were analyzed. When the optimal cutoff value (OCV) of ADC for diagnosing malignancy was 1.70 × 10 Lung cancer is one of the leading causes of cancer-related deaths and has many patterns of progression and treatment responses. As the imaging method of choice in tumor staging, fluoro-2-deoxy-glucose positron emission tomography/computed tomography (FDG-PET/CT) has been widely adopted. Its maximum standardized uptake value (SUVmax) presents glucose uptake and indicates how aggressive the cancer is. FDG-PET/CT is useful for differentiating malignant from benign pulmonary nodules . HoweverFor the last two decades, magnetic resonance imaging (MRI) of the staging of lung cancer has been narrowly available in the cases of chest wall invasion or mediastinum invasion of lung cancer partly due to the report of Webb et al.  of the RDWI possesses great potential for monitoring treatment response in cancer patients shortly after the initiation of radiotherapy . FunctioIn this article, we analyze relationships between DWI and the pathological findings of resected lung cancers and got information about DWI for the pathologic characteristics of lung cancer. Chest CT, FDG-PET/CT, DWI, ADC map, and pathologic hematoxylin and eosin stain are presented according to lepidic adenocarcinoma , mucinou−3 mm2/s [When the optimal cutoff value (OCV) of ADC for diagnosing malignancy was 1.70 × 10−3 mm2/s , the sen−3 mm2/s) of adenocarcinoma was significantly higher than that (1.17 ± 0.29 × 10−3 mm2/s) of squamous cell carcinoma (p = 0.012), or (0.93 ± 0.14 × 10−3 mm2/s) of small cell carcinoma (p = 0.0095). The ADC value (1.62 ± 0.40 × 10−3 mm2/s) of LCNEC was significantly higher than that (1.17 ± 0.29 × 10−3 mm2/s) of squamous cell carcinoma (p = 0.016), or (0.93 ± 0.14 × 10−3 mm2/s) of small cell carcinoma (p = 0.011).The ADC value by pathologic cell type of lung cancer is presented in −3 mm2/s) of mucinous adenocarcinoma was significantly higher than that (1.31 ± 0.28 × 10−3 mm2/s) of acinar adenocarcinoma (p < 0.0001), (1.29 ± 0.30 × 10−3 mm2/s) of papillary adenocarcinoma (p < 0.0001), (1.28 ± 0.28 × 10−3 mm2/s) of lepidic adenocarcinoma (p < 0.0001), (1.14 ± 0.20 × 10−3 mm2/s) of micropapillary adenocarcinoma (p = 0.0002), or (1.12 ± 0.20 × 10−3 mm2/s) of solid adenocarcinoma (p < 0.0001). The ADC value by pathologic subtype of adenocarcinoma is shown in −3 mm2/s) of mucinous adenocarcinoma was significantly higher than that (1.25 ± 0.25 × 10−3 mm2/s) of adenocarcinoma with mucin, or (1.24 ± 0.30 × 10−3 mm2/s) of other cell types (−3 mm2/s) of lung cancer with necrosis was significantly lower than that (1.32 ± 0.33 × 10−3 mm2/s) of lung cancer without necrosis (p = 0.0001) (−3 mm2/s in well differentiation (G1), 1.24 ± 0.28 × 10−3 mm2/s in moderate differentiation (G2), 1.12 ± 0.19 × 10−3 mm2/s in poor differentiation (G3), and 1.18 ± 0.43 × 10−3 mm2/s in undifferentiation (G4). There is a correlation between the decrease in ADC value and the decrease in cell differentiation. The ADC value (1.91 ± 0.36 × 10ll types . The ADC 0.0001) . For celA concise update of lung cancer staging, supported by the European Society of Thoracic Surgeons and the American College of Chest Physicians, expressed that DWI can differentiate benign from malignant lymph nodes and showed that diffusion MRI has equal sensitivity to PET-CT  but higher specificity (PET-CT 0.89 versus MRI 0.95) . In our In the Japanese lung cancer practice guidelines published in 2018 , MRI wasThe optimal cutoff value is very useful for distinguishing malignancy from benignity but would change based on the patients of a study. There are two articles which compare the diagnostic capability of DWI with that of FDG-PET/CT for pulmonary nodules and masses ,26: in oThe ADC value of adenocarcinoma was significantly higher than that of either squamous cell carcinoma or small cell carcinoma, which shows that the tissue cellularity of squamous cell carcinoma or small cell carcinoma would be higher than that of adenocarcinoma. Through DWI examination we realized that we could detect histopathological necrosis and mucinous areas in lung cancer and the ADC values could be related to the pathological structure. −3 mm2/s) of abscesses was significantly lower than that (2.118 × 10−3 mm2/s) of phlegmon (p < 0.001) and that (3.008 × 10−3 mm2/s) of edema (p < 0.01) [−3 mm2/s. As a result, the ADC of a lung cancer with necrosis became lower than that of a lung cancer without necrosis because the ADC value of the abscess was lower. If the ADC value of another tumor was lower than that of the abscess, the ADC of a tumor with necrosis would become higher than that of a tumor without necrosis. In DWI, 22% of benign lesions exhibited restricted diffusion in images with high b-values [One of the pulmonary lesions which presented restricted diffusion and lower ADC values in DWI was a pulmonary abscess with pathological necrosis. The heavily impeded mobility of pus may have been caused by its high cellularity and viscosity and shows the low ADC values . Abscess < 0.01) . In our b-values . The artOn the other hand, mucinous carcinomas were usually hypointense and showed higher ADC values, which could be misdiagnosed as benign lesions in DWI . MucinouDWI and FDG-PET/CT have their own advantages . DWI proWe should keep in mind that the study had three limitations. First, it was a retrospective study and was conducted at a single institution. Second, our ADC measurements were repeated three times and the minimum ADC value was obtained. There is no consensus for the optimal DWI techniques and image analysis procedure in the literature, including region of interest (ROI) size and placement. Third, in some cases, image quality could make calculating the true ADC value more difficult. This might be a limitation of this technique compared to PET. Further studies would be necessary to evaluate the performance of DWI for pathological characteristics.The study protocol for examining DWI and FDG-PET/CT in patients with lung cancer was approved by the ethical committee of Kanazawa Medical University . Written informed consent for MRI and a pathological examination of resected materials were obtained from each patient after discussing the risks and benefits of the examinations with their surgeons. There were three patients who were excluded due to lower imaging quality. Finally, 226 patients with primary lung cancer were enrolled in this study . They unThere were 167 adenocarcinomas, 44 squamous cell carcinomas, 5 small cell carcinomas, 3 large cell neuroendocrine carcinoma (LCNEC), 3 large cell carcinomas, and 4 carcinomas of other cell types. Three LCNECs were all combined LCNEC and adenocarcinoma. TNM classification and the lymph node stations of lung cancer were classified according to the new definition of UICC  7 . There wb value = 0 and 800 s/mm2; field of view, 350 mm; matrix size, 128 × 128. After image reconstruction, a two-dimensional (2D) round or elliptical region of interest (ROI) was drawn on the lesion which was detected visually on the ADC map with reference to T2-weighted or CT image by a radiologist (M.D.) with 25 years of MRI experience who was unaware of the patients’ clinical data. The procedures were repeated three times and the minimum ADC value was obtained. The radiologist (M.D.) and one pulmonologist (K.U.) with 28 years of experience evaluated the MRI data. They eventually reached the same consensus. The OCV of ADC for diagnosing malignancy in DWI was determined to be 1.70 × 10−3 mm2/s using the receiver operating characteristics curve as previously reported [All MR images were produced with a 1.5 T superconducting magnetic scanner  with two anterior six-channel body phased-array coils and two posterior spinal clusters (six-channels each). The conventional MR images consisted of a coronal T1-weighted spin-echo sequence and coronal and axial T2-weighted fast spin-echo sequences. DWIs using a single-shot echo-planar method were applied with a slice thickness of 6mm under SPAIR  with a respiratory triggered scan with the following parameter: TR/TE/flip angle, 3000–4500/65/90; diffusion gradient encoding in three orthogonal directions; reported . The parameters of pathological findings were adopted according to pathological reports: cell type, sub-cell type, T factor, N factor, pathological stage, cell differentiation, presence of mucin, and presence of necrosis. t test was applied for comparison of ADC values in several prognostic factors. The statistical analyses were performed using the computer software program StatView for Windows . A p value of <0.05 was considered statistically significant. The data is expressed as the mean ± standard deviation. A two-tailed Student-The sensitivity of DWI for lung cancer was 92% and DWI would be a useful tool for clinical diagnosis and evaluation of lung cancer. DWI would have an advantage in qualitative evaluation of lung cancer.