Aythya fuligula) and the more-resistant mallard (Anas platyrhynchos)—were serially inoculated, first with 2014 H5N8 and after 9 months with 2016 H5N8. For both species, a control group of birds was first sham inoculated and after 9 months inoculated with 2016 H5N8. Subsequent infection with the more-virulent 2016 H5N8 caused no clinical signs in tufted ducks that had previously been infected with 2014 H5N8 (n = 6) but caused one death in tufted ducks that had been sham inoculated (n = 7). In mallards, 2016 H5N8 infection caused significant body weight loss in previously sham-inoculated birds (n = 8) but not in previously infected birds (n = 7).Highly pathogenic avian influenza viruses (HPAIVs) of the Goose/Guangdong (Gs/Gd) lineage are an emerging threat to wild birds. In the 2016–2017 H5N8 outbreak, unexplained variability was observed in susceptible species, with some reports of infected birds dying in high numbers and other reports of apparently subclinical infections. This experimental study was devised to test the hypothesis that previous infection with a less-virulent HPAIV  provides long-term immunity against subsequent infection with a more-virulent HPAIV . Therefore, two species of wild ducks—the more-susceptible tufted duck  in tufted ducks and 78% (7/9) in mallards after 1 month to 33% (2/6) in tufted ducks and 29% (2/7) in mallards after 9 months. This finding helps predict the expected impact that an HPAIV outbreak may have on wild bird populations, depending on whether they are immunologically naive or have survived previous infection with HPAIV. Anseriformes offer HPAIVs the great evolutionary advantage to travel via their migratory routes and the opportunity to change their genetical pool by reassorting with circulating low-pathogenicity avian influenza viruses (LPAIVs) (Highly pathogenic avian influenza viruses (HPAIVs) of the Goose/Guangdong (Gs/Gd) lineage are an emerging threat to wild birds –11. Sinc–(LPAIVs) , 12.Anas penelope, and mallards, Anas platyrhynchos) can be infected with 2014 H5N8 virus without clinical or pathological evidence of disease (–Aythya fuligula) and Eurasian wigeons  may have died (Branta leucopsis) were the most affected species  and mallards  provides a long-term immunity against a subsequent infection with a more-virulent HPAIV . To test this hypothesis, two groups of wild ducks  were serially inoculated, first with 2014 H5N8 and after 9 months with 2016 H5N8. For both species, a control group of birds was first sham inoculated and after 9 months inoculated with 2016 H5N8. We used species that differed in their susceptibility to disease from HPAIV infection, with the mallard being less susceptible and the tufted duck being more susceptible. We had planned to include the Eurasian wigeon as another less-susceptible species, but we could not source sufficient birds. The timing of the inoculations was planned to correspond with the autumn peak of AIV infections, to better mimic the field dynamics of the outbreaks. We hypothesized that all mallards, both sham inoculated and 2014 H5N8-inoculated, and possibly 2014 H5N8-inoculated tufted ducks would survive the infection with 2016 H5N8. Conversely, we hypothesized that the sham-inoculated tufted ducks would not survive the infection with 2016 H5N8, because they lacked protective immunity. The sera of the inoculated ducks were tested with a hemagglutination inhibition (HI) assay against the two HPAIVs used in the experiment and against a more recent HPAIV from clade 2.3.4.4b to assess the breadth of the immune response of the birds.t test, P < 0.002) of the infected mallards  compared to the control mallards    but not according to RT-PCR  based on reverse transcription-PCR (RT-PCR) and 94% (16/17) based on virus isolation . There wllards”) . Pharyngllards”) . For bot< 0.001)  to D. Vio RT-PCR  and B. HAt 1 month p.i., 87% (7/8) H5-mallards and 100% H5-tufted ducks had detectable H5-specific and nucleoprotein (NP)-specific antibodies . Just beAfter the second inoculation, 100% (13/13) of control birds  became infected, based on both RT-PCR and virus isolation; 50% (3/6) of the H5-H5 mallards became infected based on RT-PCR and 33% (2/6) were infected based on virus isolation; 85% (6/7) of the H5-H5 tufted ducks became infected based on RT-PCR and 28% (2/7) were infected based on virus isolation .There was no significant loss of body weight of H5-H5 tufted ducks compared to the sham-H5 tufted ducks . One of t test, P < 0.01) of sham-H5-mallards compared to the H5-H5-mallards, without any other clinical signs of disease. However, both groups of mallards, in particular H5-H5-mallards, had established a strong dominance hierarchy (dominant birds had priority access to food and water compared to subordinate birds) that may have interfered with body weight evaluation.There was a significant loss of body weight (paired P < 0.001)  and for sham-H5-tufted ducks versus H5-H5-tufted ducks . Pharyngeal excretion exceeded cloacal excretion in the different groups. Cloacal excretion was scarce, and there was no statistically significant difference in cloacal excretion between groups, according to virus isolation. Cloacal excretion was scarce for H5-H5-mallards and H5-H5-tufted ducks, and cloacal excretion was statistically higher for sham-H5-mallards versus H5-H5-mallards  and for sham-H5-tufted ducks versus H5-H5-tufted ducks , according to RT-PCR. Concentration of virus in drinking water was scarce for the different groups except that for sham-H5-tufted ducks, according to virus titration; virus concentration in the water was statistically higher for sham-H5-tufted ducks versus H5-H5-tufted ducks , according to the RT-PCR.Pharyngeal excretion significantly differed between groups  . The meaFifteen days after the second inoculation, all the birds had detectable serum NP-specific and H5-specific antibodies against the 2014 and 2016 H5N8 strains  and B. SThis study showed that ducks infected with a less-virulent HPAIV 2014 H5N8) developed a long-term immunity that was protective against a subsequent infection with a more-virulent HPAIV (2016 H5N8). This finding is consistent with the previously demonstrated long-term protective effect of LPAIV infection and short-term protective effect of HPAIV infection , 20 and 14 H5N8 dPrior infection with the less-virulent 2014 H5N8 protected against clinical signs from the more-virulent 2016 H5N8 infection nearly 1 year later. After inoculation with 2014 H5N8, both mallards and tufted ducks became infected and excreted infectious virus. In accordance with our hypothesis, all mallards, both sham inoculated and 2014-H5N8 inoculated, as well as 2014-H5N8-inoculated tufted ducks, survived the infection with 2016 H5N8. The 2014-H5N8-infected mallards showed detectable clinical signs of disease , and one of the 2014-H5N8-infected tufted ducks showed clinical signs of disease consisting of mild neurological signs. After inoculation with the more-virulent 2016 H5N8, control mallards showed weight loss and control tufted ducks manifested increased mortality, although less than we had hypothesized, with only a 14% (1/7) mortality rate. This incongruence could be due to the fact that birds in our study were all in good health preinfection and may have had a greater chance of survival compared to free-range wild birds .Pharyngeal viral excretion was higher than cloacal excretion, in accordance with previous experimental studies in these and other wild bird species –15. Infe–Antibody responses in ducks are still only partially understood . Studies24–Mallards were our model for less susceptible species and a comparison group for elaborating on the dynamics of the 2016 H5N8 outbreak in the Eurasian wigeon population. All mallards survived the two infections, and control mallards excreted more virus postinfection than control tufted ducks. The mallard excretion pattern of 2014 H5N8 was similar to those described by Keawcharoen et al. (in 2008) for 2005 H5N1 and by van den Brand et al. (in 2018) for 2014 H5N8 . In conc29–Aythya ferina, a diving duck species) was asymptomatic , that were not reported infected during previous HPAIV outbreaks had an unusually high number of deaths related to the infection in 2020–2021  will have higher chances of surviving a subsequent infection with a more-virulent HPAIV in the following influenza outbreak. The effect of this implication is suggested in the trend of the 2020–2021 HPAIV H5 outbreak. During this outbreak, high mortality in duck populations was not reported, which could be related to the long-term protective effect of their exposure to previous HPAIVs . Conversely, goose species, like the Barnacle goose . The viruses were propagated by two passages in Madin-Derby canine kidney (MDCK) cells. The harvested supernatant had a titer of 1 × 107 median tissue culture infectious dose (TCID50)/mL and was diluted with phosphate-buffered saline (PBS) to 1 × 106 TCID50/0.1 mL. These viruses were chosen because they both circulated in wild birds during the correspondent outbreaks and were expected to reproduce the field infections more realistically. All experiments with HPAIVs (2014 H5N8 and 2016 H5N8) were performed under biosafety level 3 (BSL3) conditions.The two HPAIVs used in this study for the serial inoculation of the wild ducks were 2014 H5N8 (A/Eurasian Wigeon/NL/EMC-1/2014_2.3.4.4c), isolated from the feces of a nonsymptomatic wild Eurasian wigeon, and 2016 H5N8 (A/Eurasian Wigeon/NL/4/2016_2.3.4.4b), isolated from a dead wild Eurasian wigeon. Full-length hemagglutinin (HA) and neuraminidase (NA) sequences and full genome sequences for these two virus isolates were obtained by Sanger sequencing, and sequences were deposited in a public database , A/Mallard/Sweden/49/2002 (non A/goose/Guangdong/1/1996 H5), A/muscovy-duck/Vietnam/NCVD-KA426/2013 (clade 1.1.2), A/duck/GizA/15292S/2015 (clade 2.2.1.2), A/Nepal/19FL1997/2019 (clade 2.3.2.1a), A/duck/Vietnam/NCVD-1584/2012 (clade 2.3.2.1e), and A/Guangdong/18SF020/2018 (clade 2.3.4.4h). These antigens were available in-house at the Viroscience Department of the Erasmus Medical Center as recombinant virus stocks propagated in MDCK cells, which contained the H5 HA of the respective viral strain without a multibasic cleavage site, in the background of seven gene segments of A/Puerto Rico/8/1934.Aythya fuligula) and one species of dabbling duck . All ducks used for the infection experiments were captive bred. Birds were 4 to 5 months of age at the time of first inoculation. Blood samples, cloacal swabs, and pharyngeal swabs were collected from all ducks 1 week before inoculation. Sera were analyzed, as described below, by using a commercially available influenza A virus antibody ELISA kit for the detection of antibodies against nucleoprotein (Idexx) according to the manufacturer’s instructions . Each day, a qualified veterinarian assessed all birds for clinical signs of disease. Body weights, water samples, and cloacal and pharyngeal swabs were collected daily for the first 7 days and every 2 days thereafter. After inoculation, ducks were weighed and means and standard deviations for relative weight loss (compared to the body weight at the day of inoculation) were calculated.For the infection with 2014 H5N8 (first inoculation), nine mallards and eight tufted ducks were housed in two negatively pressurized isolator units . The metPharyngeal and cloacal swabs were collected using sterile cotton swabs, and each swab was placed in 1 mL virus transport medium , 15. EacDuring the time between the two inoculations, three mallards and one tufted duck from the H5-infected groups and one mallard from the sham-inoculated group died from causes unrelated to HPAIV infection .6 TCID50 HPAIV 2016 H5N8 in 0.1 mL (second inoculation). The birds were monitored and sampled using the established methods. On day 15 p.i., all surviving birds were euthanized.Nine months after infection with 2014 H5N8, six mallards and seven tufted ducks from the infected group and seven mallards and six tufted ducks from the naive group were inoculated intrachoanally with 1 × 10RNA isolation and RT-PCR were performed as described previously , 15. BriVibrio cholerae filtrate containing receptor-destroying enzyme and subsequently inactivated at 56°C for 1 h and adsorbed with 10% turkey erythrocytes for 1 h at 4°C. Twofold serum dilutions of the serum samples were prepared in round-bottom 96-well plates, starting at a dilution of at 1:20; thus, the minimal detectable HI titer was 10. To each well containing 50 μL of serum dilution, 25 μL of PBS with 4 hemagglutinating units of virus was added to each well. Following incubation for 30 min at 37°C, 25 μL of 1% turkey erythrocytes was added to each well. Plates were incubated for 1 h at 4°C before reading the HI titer, which was determined as the reciprocal value of the highest serum dilution which completely inhibited erythrocyte agglutination.After the first inoculation, blood samples were taken monthly and tested for serum antibodies. Serum samples were tested for the presence of H5-specific and NP-specific antibodies. H5-specific antibodies were detected by using an HI test performed as described previously, using in-house turkey erythrocytes . BrieflyNP-specific antibodies were detected by using a commercial blocking ELISA . Idexx bELISA is a high-throughput method for NP antibody quantitation in various host species –25, 28. –Autopsies and tissue sampling were performed for all the birds, either those ill from HPAIV infection or those euthanized at the end of the experiment. After fixation of tissue samples in 10% neutral buffered formalin and embedding in paraffin, two sequential tissue sections were processed for histology with hematoxylin eosin staining or for immunohistochemistry with a monoclonal antibody against nucleoprotein of influenza A virus as the primary antibody for detection of influenza viral antigen , 15. TheThe study was approved by an independent animal experimentation ethical review committee and by the Dutch government (Stichting DEC consult) .http://platform.gisaid.org).All data are available in the main text and in the GISAID database (


Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme essential for NADPH production and protecting cells, especially red blood cells, from free radicals. The oxidative stress from drugs, chemicals, and infections can induce red blood cell hemolysis in G6PD deficiency patients, causing a genetic disorder.This study aims to provide more information on G6PD deficiency prevalence and the G6PD variants in the southern Thai population.Five hundred and twenty healthy subjects in 14 provinces in the southern part of Thailand participated in the study. EDTA-blood samples were collected for a hematological parameters study, G6PD deficiency screening, and a molecular study for G6PD mutation. G6PD deficiency screening was tested using a fluorescent spot test. The types of G6PD mutation were identified by the allele-specific PCR method.The prevalence of G6PD deficiency in southern Thailand was 6.1% (14/228) in males and 9.6% (28/292) in females. Two homozygous and 26 heterozygous G6PD deficiencies were found in females. G6PD Viangchan (871G>A) was the most common variant with 43%, followed by G6PD Mahidol (487G>A), 24% with an allele frequency of 0.025 and 0.012, respectively. Uncharacterized mutations existed in three samples. The study volunteers had anemia in 36.6% (107/292) females and 7.5% (17/228) males. Among G6PD deficiency subjects, only ten partial G6PD deficiency females had mild anemia.This study suggests that the prevalence of G6PD deficiency in southern Thailand aligns with that of other parts of Thailand. Newborn screening for G6PD deficiency is recommended for personal information and medical reference to prevent acute hemolysis from oxidative stressors. Plasmodium vivax, and Plasmodium ovalae, and reduces the transmission of Plasmodium malariae infection. The G6PD deficiency in persons with a malarial infection cannot be treated with a certain amount of primaquine because it may cause hemolytic anemia (Glucose-6-phosphate dehydrogenase (G6PD) is an essential enzyme in the pentose phosphate pathway, a step of the nicotinamide adenine dinucleotide phosphate (NADPH) generation . NADPH rc anemia . The stuIn Thailand, the prevalence of G6PD deficiency is in the range of 3–18% . The mosA cross-sectional study was conducted on 520 healthy volunteers  in 14 provinces in the south of Thailand between July 2016 and August 2019. The interested persons were announced and informed of the recruitment criteria stating that the volunteer and their parents would be domiciled in southern Thailand. The number of volunteers from each province was calculated depending on the population in that province and the ratio of the total volunteers. Five hundred twenty volunteers were selected randomly. They were briefly informed and filled in their information in the questionnaire. They signed a consent form before the following processes. The 2.5 mL peripheral blood was drawn from each person and collected in ethylenediaminetetraacetic acid (EDTA) collection tubes without hemolysis. The blood sample of each subject was divided into 1.5 mL for G6PD deficiency screening and hematological parameters testing and 1.0 mL for DNA extraction for gene mutation analysis. The Human Research Committee of the Ethics Research Council, Walailak University (16/031 and WUEC-16-055-01) approved this research and informed consent. Hematological parameters were tested using Sysmex XN-1000 automated hematology analyzer . The parameters included RBC count (RBC), hemoglobin (HBG), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red blood cell distribution width (RDW).G6PD deficiency screening was tested using a Fluorescent spot test (FST) . The protocol was clearly described in the previous study . The G6PGenomic DNA was extracted from peripheral blood leukocytes using a Geneaid kit . The DNA concentration of each sample was quantified with the NanoDrop spectrophotometer . The G6PD mutations in G6PD-deficient and partially deficient samples were identified using multiplex allele-specific PCR, DiaPlexC™ G6PD Genotyping Kit (Asian type) . The kit allows screening of eight mutations of Asian types, including Union (1360C>T), Canton (1376G>T), Kaiping (1388G>A), Viangchan (871G>A), Mahidol (487G>A), Mediterranean (563C>T), Coimbra (592C>T) and Vanua Lava (383T>C). The PCR reaction comprised 12.5 μL of 2X Multiplex PCR Smart mix (G6PD-Asian type), 2 μL of primer mixture (G6PD-Asian type), nuclease-free water, and DNA template (25–50 ng). PCR thermocycling conditions were as follows: initial denaturation at 95 °C for 15 min; followed by 30 cycles of 30 s at 95 °C, 30 s at 60 °C, and 40 s at 72 °C; followed by a final extension at 72 °C for 5 min. PCR products of each sample and DNA marker were analyzed by 3% agarose gel electrophoresis for the variants. After staining the gel with ethidium bromide for 5 min, the sizes of PCR products were detected under UV light with an ultraviolet transilluminator. Finally, PCR-RFLP was used for secondary confirmation of heterozygous female, hemizygous male, and homozygous female in the first three common G6PD variants  .https://infogram.com/).Data were analyzed using SPSS v.17 software. Hematological parameters were calculated for the mean, SD, median, and prevalence. The Kolmogorov-Smirnov test was used to test the normality of the data. The statistical difference in hematological parameters between the groups was calculated using the T-Test Independent statistics for normal distribution and the Mann-Whitney U test for non-normal distribution. The 100% stacked column was conducted using Infogram (p > 0.05) existed in hematological parameters of G6PD deficiency, G6PD partial, and normal G6PD  of the G6PD Canton  were analyzed for eight types of G6PD gene mutation by DiaPlexC™ G6PD Genotyping Kit (Asian type) and further confirmation by PCR-RFLP in the first three common mutations. PCR-RFLP with D Canton  in additectively . FiguresThe results disclosed that the most common G6PD variant in this population was G6PD Viangchan , followed by G6PD Mahidol , G6PD Canton , G6PD Union , G6PD Vanua Lava , and G6PD Kaiping . Uncharacterized mutations existed in three samples . Among GVXV). In the first case, a 19-year-old from Ranong province, her complete blood count was as follows: red cell count 4.82 × 1012/L, Hb level 13.2 g/dL, hematocrit 39.5%, MCV 82.0 fL, MCH 27.4 pg, MCHC 33.4 g/dL, and RDW 13.5%. The second case was a 20-year-old from Songkhla province; her complete blood count was as follows: red cell count 4.02 × 1012/L, Hb level 12.0 g/dL, hematocrit 36.6%, MCV 86.1 fL, MCH 28.9 pg, MCHC 33.5 g/dL, and RDW 14.7 % as in G6PD deficiency is the most common enzyme deficiency worldwide . The preP. vivax and P. ovale patients ; lane 7 G6PD Kaiping (557 bp); lane 8 G6PD Viangchan (501 bp); lane 9 G6PD Mahidol (337 bp) and lane 10 no detectable mutation by PCR used in this study.The pattern of PCR products of G6PD deficiency cases was analyzed by 3% gel electrophoresisClick here for additional data file.10.7717/peerj.14208/supp-3Supplemental Information 3The pattern of PCR products of G6PD partial deficiency cases analyzed by 3% gel electrophoresis. Lane 1, standard marker; lane 2, mutant type control; lane 3, wild type control; lane 4, non-template control; lane 5, G6PD normal; lane 6, G6PD Vanua Lava (154 bp); lane 7, G6PD Viangchan (501 bp); lane 8, G6PD Mahidol (337 bp); lane 9, Canton (681 bp); lane 10, G6PD Union (803 bp) and lane 11, no detectable mutation by PCR used in this study.Click here for additional data file.


P2RX7 risk alleles to assess the genetic impact on P2X7 function in AIBL and ADNI cohorts. No significant association was identified between the P2RX7 gene and AD, indicating that P2X7 downregulation in AD is likely caused by environmental changes rather than genetic factors. In conclusion, the downregulation of P2X7 and integrins at pre-clinical stage of AD indicates altered pro-inflammatory responses, phagocytic functions, and migrating capabilities of circulating monocytes in early AD pathogenesis. Our study not only improves our understanding of peripheral immune involvement in early stage of AD but also provides more insights into novel biomarker development, diagnosis, and prognosis of AD.Alzheimer’s disease (AD) has shown altered immune responses in the periphery. We studied P2X7 (a proinflammatory receptor and a scavenger receptor) and two integrins, CD11b and CD11c, on the surface of circulating leukocytes and analysed their associations with Aβ-PET, brain atrophy, neuropsychological assessments, and cerebrospinal fluid (CSF) biomarkers. Total 287 age-matched, sex-balanced participants were recruited in a discovery cohort and two validation cohorts through the AIBL study and studied using tri-colour flow cytometry. Our results demonstrated reduced expressions of P2X7, CD11b, and CD11c on leukocytes, particularly monocytes, in Aβ +ve cases compared with Aβ −ve controls. P2X7 and integrin downregulation was observed at pre-clinical stage of AD and stayed low throughout disease course. We further constructed a polygenic risk score (PRS) model based on 12  The carclear Aβ . These gclear Aβ ,4. Theseclear Aβ ,6, makinRecent genome-wide association studies (GWAS) of AD have discovered over 130 genomic loci associated with AD, demonstrating their roles in microglial phagocytosis of Aβ, particularly phagocytic receptors and endolysosomal network of the innate immune system ,7. In adITGAM, or αM) and CD11c , which are integrins responsible for phagocytosis mediated by complement system and the recruitment and migration of monocytes [1-42, total tau (T-tau), and tau phosphorylated at threonine 181 (P-tau181P) all reflect the natural history of AD in the three major stages [This study aimed at understanding the peripheral immune involvement in AD, regarding the phagocytic function and pro-inflammatory responses of innate immune cells. Our group has studied P2X purinoceptor 7 (P2X7) for decades, which is a dual-functional purinergic receptor that acts as an ATP-induced pro-inflammatory ion channel and a scavenger receptor responsible for phagocytosing apoptotic cells and debris . Additioonocytes . Given tonocytes , so T lyonocytes  and posionocytes , the perr stages . Therefo+CD16− classical monocytes, CD14dimCD16+ non-classical monocytes, and CD14+CD16+ intermediate monocytes were all reduced in Aβ +ve cases compared with Aβ −ve controls with effect sizes of −0.487, −0.503, and −0.514, respectively  study  and we uectively . Also, Pectively A. This cectively B–D, in wectively . While tectively B–D. Moreectively E–G. Regaiomarker H–J.1-42/T-tau and Aβ1-42/P-tau181P ratios in CSF compared with P2X7-high CN +ve individuals  of P2X7 on classical monocytes B. The grividuals A,B. Moreividuals C,D.++ neutrophils were also reduced significantly in Aβ +ve cases compared with Aβ −ve controls with effect sizes of −0.581 and −0.625, respectively . By categorising the validation cohort B into CN −ve, CN +ve, MCI− ve, and dementia groups, the percentage of HLA-DR− monocytes were significantly higher in dementia individuals compared with MCI −ve individuals , which were associated with neurodegeneration and P2X7 function. A total of 12 neurodegeneration-related SNPs were selected from our previous functional studies and literature, including 12 SNPs in P2RX7 and one SNP in P2RX4 (P2RX7 SNPs by our group and literature (n = 919) and the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database (n = 819). By excluding samples with missing demographics or genotyping data, 900 AIBL participants and 786 ADNI participants remained for PRS analysis. The characteristics of P2RX7 and P2RX4 SNPs in the AIBL and the ADNI cohort were summarised in P2RX7 and P2RX4 SNPs were associated with the clinical diagnosis. The only SNP that reached statistical significance was rs17525809 (V76A) in the AIBL cohort (p = 0.05). Subsequently, we calculated two PRS depending on the associated functions of these SNPs. The summary of PRS-pore and PRS-phago in each cohort and the combined cohort were presented in We developed a PRS comprised of 12 in P2RX4 . Among tin P2RX4 . Tradititerature . Given tterature . PRS-phaterature . A totalDysregulated immune responses have been raised as a possible contributor to AD pathogenesis and this study compared P2X7 expressions on adaptive and innate immune-related cells between Aβ +ve cases and Aβ −ve controls. We demonstrated globally reduced P2X7 expressions on peripheral leukocytes in Aβ +ve cases, including lymphocytes, monocytes, and neutrophils, as previously reported by our group . Leukocy1-42/T-tau ratio, higher CSF Aβ1-42/P-tau181P ratio, and better cognition (higher EM). They were likely to present faster expanding rate of ventricle and faster shrinking rate of hippocampus. AD progression is characterised by low CSF Aβ1-42, high CSF Tau, high CSF pTau181, low CSF Aβ1-42/Tau ratios [1-42/T-tau ratio or high EM. It suggested that CN individuals with low P2X7 level may undergo uncharacterised pathological changes and progress into the pre-clinical stage of AD soon, but more investigations are required to elucidate P2X7 downregulation in AD pathogenesis and progress. This pre-clinical P2X7 downregulation may be associated with its function as a scavenger receptor responsible for innate phagocytosis in serum-free environment [Following recognising differential P2X7 expressions in individuals with different PET-Aβ status, we further compared P2X7 expressions by including both PET-Aβ status and clinical symptoms. In the discovery cohort, due to the lack of prodromal AD patients (MCI −ve), only three groups were categorised: CN individuals (CN −ve), pre-clinical AD patients (CN +ve), and AD patients with dementia (Dementia: MCI +ve and AD). P2X7 expressions on monocytes and neutrophils were significantly lower in pre-clinical AD patients compared with CN individuals. P2X7 downregulation may happen early at the pre-clinical stage of AD and stayed stable throughout disease course, but the underlying mechanism remains unknown. Interestingly, P2X7 expressions on lymphocytes and monocytes resembled bimodal distribution, in which over half of CN individuals expressed high level of P2X7, while the others expressed low level of P2X7. Compared with CN individuals with high level of P2X7, CN individuals with low level of P2X7 were characterised by higher CSF Aβu ratios , and lowironment . In the ironment . Howeverironment . This suironment . Whether2 integrin subunit, CD18, to form complement receptor 3 (CR3) and 4 (CR4) respectively, mediating complement-coated particle phagocytosis [Integrin downregulation in Aβ +ve cases was also observed, particularly on professional phagocytes—monocytes and neutrophils. CD11b and CD11c play important roles in the recruitment and migration of mononuclear phagocytes, cell-cell contact formation, and immune cell signalling . They alocytosis . Severalocytosis , but our+CD16− classical monocytes, CD14dimCD16+ non-classical monocytes, and CD14+CD16+ intermediate monocytes to study their associations with AD. Progression of AD can be characterised by low CSF Aβ1-42, high CSF T-tau, high CSF P-tau181P, and low CSF Aβ1-42/T-tau ratio [+ monocytes to investigate adaptive immunity in AD progression. Our discovery cohort illustrated significantly fewer HLA-DR+ monocytes in AD cases with dementia compared with CN individuals, as supported by more HLA-DR− monocytes in the AD cases with dementia. Previous study showed reduced HLA-DR+CD14+ monocytes and increased HLA-DR+CD16+ monocytes during AD progression. HLA-DR+ monocytes were further associated with brain atrophy, neuropsychological estimates, and leukocyte surface P2X7 expressions. These observations further consolidate the involvement of both innate and adaptive immunity in AD, suggesting compromised phagocytic capacity at the pre-clinical stage of AD and altered antigen presentation ability of professional mononuclear phagocytes at the dementia stage of AD. Moreover, reduced CD16− monocytes  and increased CD16+ monocytes  in the blood circulation of AD patients were also noted and whether this phenomenon is associated with infiltrative monocytes into the CNS awaits further investigation.Given that our discoveries of P2X7 and integrins converge on innate immunity and monocytes, we further dissected monocyte population into CD14au ratio . As AD pau ratio . Intermeau ratio . The infau ratio . FurtherP2RX7 and P2RX4 genes that are known to inhibit or promote the pore formation function or phagocytic function of P2X7. However, no significant association was found between the P2RX7 gene and AD. This observation is reasonable because no P2RX7 SNP has been identified as a risk allele in AD GWAS [To further assess the association between P2X7 function and AD, we selected 12 SNPs in 1-42, Tau, and pTau181. However, CSF data were scarce, and we need to collect more CSF measurements to validate our findings.All participants were recruited in Perth and Melbourne through the AIBL study, and the ethnic background was limited to 99.9% Caucasians who were fluent in English. The application of our findings to general population would require more investigations by involving participants with various ethnic backgrounds. Second, our study showed interesting associations with CSF measurements of Aβn = 88), a validation stage A for the P2X7 study (n = 111), and a validation stage B for the HLA-DR study (n = 88)  . The par® tube  and was kept on ice during transportation. Processing of whole blood was completed within three hours after collection.Peripheral whole blood was collected via venepuncture between 8:00 a.m. and 10:30 a.m. following overnight fasting. Whole blood was kept in EDTA anti-coagulant VacutainerThis study was approved by the Eastern Health Research and Ethics Committee  and conducted according to Declaration of Helsinki principles. All participants and patient caregivers completed written informed consent. All clinical and demographic information was masked until the collection of all laboratory measurements.n = 919) and the ADNI database (n = 819). ADNI was initiated in 2003 in the United States and Canada, aiming at the development of standardized imaging procedure and biomarker assessment in normal, preclinical, and prodromal patients with AD. The demographics and characteristics of these two datasets were summarised in A total of 1738 participants were recruited from the AIBL database  and DAKO . The anti-human P2X7 monoclonal antibodies were produced from the L4 clone in house  and conj®) tubes with pre-mixed antibody cocktails. An autofluorescence tube containing only whole blood and an IgG isotype control  tube were prepared for each AIBL sample. Titration of each antibody was determined by saturation tests. Blood/antibody mixture was incubated for 15 min at room temperature with gentle shake, followed by incubating with 2 mL of BD FACS Lysing solution (Cat#555899) for another 15 min. 2 mL of PBS was added to each FACS® tubes, followed by centrifuging at 1400 rpm for 3 min. Supernatant was discarded and leukocytes were resuspended into 200 uL of PBS. Leukocytes were then analysed using FACSCaliburTM (BD Biosciences) and flow results were primarily analysed using FlowJo software .Cell surface staining was carried out following the BD standard protocol. Aliquots of 100 µL of fresh whole blood were added into fluorescence-activated cell sorting  for Aβ1-42, total Tau, and pTau181.The collection, processing, measurement, and long-term storage of CSF samples had been previously described . CSF wasAs a longitudinal study, most participants of AIBL have undergone long-term follow-up concerning brain imaging, cognitive assessments, and other pathological examinations in 18-month intervals. Most participants in this study had 2–6 visits to AIBL clinics, which enabled researchers to investigate progressive changes of their pathological tests. The yearly slopes of PET-Aβ scans, MRI, EM, and PACC were calculated from at least two time points using the Excel “Slope” function.t-test using the oneway.test function in R. In discovery cohort , the subsequent comparisons of receptors between CN −ve, CN +ve, and demented cases were determined by one-way ANOVA using oneway.test function in R. Its post-hoc tests were calculated by Dunnett’s test using the DunnettTest in R. In validation cohort, the comparisons between CN +ve and CN −ve and the comparisons between demented AD cases and prodromal AD cases were calculated by t-test using the oneway.test function in R. The correlations between receptors and Aβ burden, brain volumes, neuropsychological assessments, and CSF measurements of Aβ1-42, T-tau, and P-taau181P were determined by calculating Pearson product-moment correlation coefficients (r) using the cor function in R. After dissecting the CN controls by mean P2X7 MFI, two-group comparisons were determined by student t-test using oneway.test in R. All statistical plots were generated using the ggplot function in R. R version 3.6.3 (29 February 2020)—“Holding the Windsock” Copyright (C) 2020 The R Foundation for Statistical Computing Platform: x86_64-w64-mingw32/x64 (64-bit).Before conducting statistical analysis, qqPlot and barlett.test in R were used to assess normality and homogeneity of variances, respectively. Sample characteristics of age, sex, APOE ε4 carriers, year of education, and neuropsychological assessments were compared using one-way ANOVA (continuous variables) and chi-square test (categoric variables). The comparisons of leukocyte receptors were first performed between Aβ +ve cases and Aβ −ve controls by https://pngu.mgh.harvard.edu/purcell/plink/ accessed on 12 October 2013). A total of 12 neurodegeneration-related P2RX7 and P2RX4 SNPs were selected from our previous functional studies and literature, including 11 SNPs in P2RX7 and one SNP in P2RX4 (P2RX7 SNPs by our group and literature (Data was available across 919 individuals in AIBL database and 809 individuals in ADNI database. Prior to data analysis, data from each databased were QC’ed separately using PLINK (1.9, Shaun Purcell, in P2RX4 . The claterature . Two PRSterature . The secterature .We identified the downregulation of P2X7, CD11b, and CD11c on peripheral leukocyte surface at the pre-clinical stage of AD and they were further associated with brain atrophy, cognition decline, and CSF biomarkers of AD. Their significant associations with the current diagnostic standards of AD strengthened their involvement in early stage of disease course, suggesting that the dysfunction of pro-inflammatory responses, phagocytic functions, and migrating abilities of circulating phagocytes may happen at the pre-clinical stage of AD and stay compromised throughout disease course. On the contrary, the antigen presentation function of circulating monocytes may be altered at the dementia stage of AD. Our results consolidate that AD is a systemic disease modulated by both central and peripheral immune responses, in which altered innate immune responses may happen at asymptomatic stage of AD while altered adaptive immune responses happen at symptomatic stage of AD. Most interestingly, low level of P2X7 expression on monocytes might indicate patients who had faster shrinking rate of hippocampus but normal Aβ burden and cognitive function. Given that altered immune responses had been raised as a strong contributor to AD progression, as supported by many genetic studies, our study deepens the understanding of the peripheral immune dysfunction at the pre-clinical stage of AD. This not only improves the understanding of the role of immunity in AD pathogenesis, but also provides novel insights into biomarker discovery. Leukocyte surface expression of immune-related receptors, such as P2X7 and integrins, might be promising biomarkers of AD, which might facilitate the diagnosis and prognosis of pre-clinical AD patients. Given the over 60 microglial-specific AD risk genes identified by AD GWAS, it is promising to study these immune-related markers on professional phagocytes to improve our understanding of immune involvement in AD and to provide more insights into biomarker discovery and therapeutic development.


We found that the antibodies had broad reactivity across the American and Eurasian N6 lineages, but relatively little binding to the H5N6 NA. Several of the antibodies exhibited strong NA inhibition activity and some also showed activity in the antibody dependent cellular cytotoxicity reporter assay and neutralization assay. In addition, we generated escape mutant viruses for six monoclonal antibodies and found mutations on the lateral ridge of the NA. Lastly, we observed variable protection in H4N6 mouse challenge models when the antibodies were given prophylactically.Influenza A viruses are a diverse species that include 16 true hemagglutinin (HA) subtypes and 9 true neuraminidase (NA) subtypes. While the antigenicity of many HA subtypes is reasonably well studied, less is known about NA antigenicity, especially when it comes to non-human subtypes that only circulate in animal reservoirs. The N6 subtype NAs are mostly found in viruses infecting birds. However, they have also been identified in viruses that infect mammals, such as swine and seals. More recently, highly pathogenic H5N6 subtype viruses have caused rare infections and mortality in humans. Here, we generated murine mAbs to the N6 NA, characterized their breadth and antiviral properties  Influenza A viruses are categorized based on 16 true hemagglutinin (HAs) subtypes and 9 true neuraminidase (NAs) subtypes which are all found in the avian reservoir , 3. HAs vice versa. Some N6 isolates have also been found to be resistant to NA inhibitors, which is a concern , A/redhead/Alberta/192/2002 , A/duck/England/1956 , A/gull/Maryland/704/1977 , A/black-legged kittiwake/Quebec/02838-1/2009 , A/ring-billed gull/Quebec/02434-1/2009 , A/mallard/Alberta/125/1999 , A/blue-winged teal/Illinois/10OS1546/2010  and A/blue-winged teal/Wisconsin/402/1983  were obtained from the Biodefense and Emerging Infections Research Resources Repository (BEI Resources). The viruses were propagated in 10 day old embryonated chicken eggs (Charles River Laboratories) at 37°C for 2 days. The viral titers were determined by conducting a standard plaque assay on Madin Darby canine kidney (MDCK) cells as described earlier , 50.MDCK cells were cultivated in Dulbecco’s Modified Eagle’s Medium  containing 1% penicillin/streptomycin  antibiotic solution, 10% fetal bovine serum  and 1% hydroxyethylpiperazine ethane sulfonic acid (HEPES). SP2/0-Ag14 myeloma cells were propagated in complete DMEM supplemented with 1% L-glutamine (Gibco).Trichoplusia ni) were grown in serum-free Express Five media (Gibco) containing 1% L-glutamine and 1% penicillin/streptomycin antibiotics mix. Sf9 cells (Spodoptera frugiperda), adapted from the cell line ATCC CRL-1711, were maintained in Trichoplusia ni medium – Fred Hink  supplemented with 1% penicillin/streptomycin antibiotics mix, 1% pluronic F-68 (Sigma-Aldrich) and 10% fetal bovine serum. In order to passage the baculoviruses, the media was switched to 3% TNM-FH .High Five cells  of the respective N6 protein from the strains A/duck/Czechoslovakia/1956 (H4N6), A/Shenzen/1/2016 (H5N6), A/swine/Missouri/A01727926/2015 (H4N6), A/Caspian seal/Russia/T1/2012 (H4N6) and A/duck/Zhejiang/D9/2013 (H4N6) as well as for the N9 of A/Anhui/1/2013 (H7N9) were cloned into a baculovirus shuttle vector, containing an N-terminal signal peptide, followed by a hexahistidine purification tag, a VASP (vasodilator-stimulated phosphoprotein) tetramerization domain and a thrombin cleavage site . The bacNinety-six well flat bottom plates  were coated with 50 µl/well of 2 µg/ml recombinant protein in 1x coating buffer (Seracare) at 4°C overnight. The following day, the plate was washed three times with 100 µl/well of 0.1% Tween 20 (TPBS) to remove residual coating solution and then incubated with 100 µl/well of 3% milk dissolved in TPBS for 1h at room temperature to avoid non-specific binding. The blocking solution was removed and the primary antibody added at a starting concentration of 30 µg/ml in 1% milk/TPBS. The antibodies were serially diluted 1:3 and the plates were incubated for 1 hour at room temperature (RT). An anti-Lassa antibody (KL-AV-1A12 ) was incvia the intraperitoneal route with A/swine/Missouri/A01727926/2015 N6 antigen in 100 µl PBS, the preparation was used 21 days later for an intranasal immunization. Three weeks later, the mice were boosted with A/Shenzhen/1/2016 N6 antigen in a volume of 50 µl. After three weeks, one mouse received a final boost intraperitoneally with 100 µg of N6 antigen from A/duck/Zhejiang/D9/2013 (H4N6) adjuvanted with 10 µg of poly (I:C) (In vivogen). Three days after the final boost, the mouse was euthanized and the spleen removed. The spleen was washed with PBS and then flushed with serum-free DMEM (with 1% penicillin/streptomycin) to obtain the splenocytes. The splenocytes were fused with SP2/0-Ag14 myeloma cells in a ratio of 5:1 using pre-warmed polyethylene glycol . The cells were grown on semi-solid selection & cloning medium with hypoxanthine-aminopterin-thymidine  for 10 days and were then expanded to a 96-well cell culture plate.Mouse mAbs were produced using hybridoma technology, as previously described , 55. Brivia ELISA against recombinant NA of A/duck/Zhejiang/D9/2013 (H4N6). Reactive clones were tested using the Pierce rapid antibody isotyping kit (Life Technologies). Only IgG heavy-chain expressing clones were continuously expanded. The selected hybridoma clones were first expanded in Clonacell-HY Medium E and then switched to Hybridoma SFM media (Gibco) supplemented with 1% penicillin/streptomycin. The antibodies were then purified as previously described  of 3.0 for 16h using serum free minimal essential media , supplemented with penicillin/streptomycin, HEPES (Gibco), glutamine  and sodium bicarbonate . The cells were fixed with 3.7% paraformaldehyde (PFA)/PBS for 1h at RT. The plate was the blocked for 1h with 3%milk/PBS. The antibodies were diluted to 30 µg/ml in 1% milk/PBS and incubated for 1h at RT. An anti-Lassa antibody  was giv50 to the virus mixture and the cells were then incubated for 2 days at 37°C. The cell supernatant was collected and used to continue the passaging of the virus. The concentration of the mAbs was doubled with every passage. This procedure was repeated until an IC50 concentration of 128 was reached. The presence of the virus within the samples was confirmed by performing an immunostaining of the cell layer after every passage. The same virus was passaged in parallel completely without mAbs as well as together with an irrelevant antibody [anti-Lassa KL-AV-1A12  of 1 with the virus A/duck/Czechoslovakia/1956 (H4N6) in 1xMEM as well as 1 ug/ml TPCK-treated trypsin. The respective mAbs were added at a concentration of 1xIC-AV-1A12 ], to conOnce the last passage was reached, the viruses were plaque purified and injected, together with the respective mAb, in 10-day old embryonated chicken eggs, to propagate the virus. The eggs were incubated for 2 days at 37°C. RNA was extracted from the allantoic fluid by using the Direct-zol RNA isolation kit from Zymo research. The purified RNA was used to deep-sequence the escape viruses.The tetrameric head domain of the NA protein of A/duck/England/1/1956  was visualized using PyMOL. The structure is shown in top down, bottom up and side view to highlight the individual mutations identified through epitope mapping. One of the monomers has been highlighted in light grey.Ten mAbs with strong reactivity to the N6 NA of A/swine/Missouri/A01727926/2015 were identified as a result of the hybridoma fusion , plaque reduction neutralization (PRNT) and antibody-dependent cellular cytotoxicity (ADCC) reporter assays. NI was measured against A/duck/Czechoslovakia/1956 , A/swine/Missouri/A01727926/2015  and A/black-legged kittiwake/Quebec/02838-1/2009 . MAbs 2E8, 3C6 and 3D5 showed the strongest inhibition of A/duck/Czechoslovakia/1956 followed by 1G9, 1A5, 3F10, 1D4, 3E1, 3B5 and finally 3C6 , we also performed plaque reduction neutralization assays (PRNTs) to measure both reduction of plaque size (a common feature of anti-NA mAbs) and reduction of plaque numbers . All mAbs reduced plaque size to various degrees, mostly in accordance with their NI activity  by subjecting A/duck/Czechoslovakia/1956 to increasing antibody pressure by passaging the virus with increasing amounts of antibody as described before , 21. An via the intraperitoneal route two at 5 mg/kg of mAb and then challenged 2 hours later intranasally with 5 times the 50% lethal dose (mLD50) of A/duck/Czechoslovakia/1956 . Weight loss and survival was then monitored for 14 days. Protection from A/duck/Czechoslovakia/1956 was variable, with almost no weight loss and no mortality observed with 1A5, 3C6 and 3F10  had strong and two had moderate (1A5 and 3F10) NI activity. 1A5 had strong activity in the PRNT assay, but 3C6 did not have much activity here. However, 3C6 had outstanding activity in the ADCC reporter assay where 1A5 and 3F10 had only moderate activity against the challenge virus. It therefore appears that different in vitro activities are associated with in vivo protection. Of note, some of the antibodies which did less well in vivo were amongst the ones which performed best in the NI assay. While our work provides first insights into the antigenicity of the N6 NA, much more work is needed to better understand N6 NA immunogenicity, epitopes and antigenic drift.In this study, we are shedding first light on antigenicity of the N6 NA. As expected, we found significant cross-reactivity of the isolated mAbs. This could be due to our immunization regimen which might have favored cross-reactive epitopes but it may also be a consequence of the low level of antigenic drift for most avian derived N6 NAs. The characterized mAbs showed strong NI activity against isolates from the Eurasian and the North American lineage and protected – to various degrees – against challenge with a historic Eurasian lineage H4N6 strain. We also mapped the putative epitopes of six of the mAbs through generating escape mutants. Interestingly, all escape mutations were in close proximity on the lateral surface of the NA. This patch is reminiscent of the binding side of CD6 and other N1 specific and B NA specific antibodies that may span two monomers , 18, 61.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All animal procedures were performed in accordance with protocols approved by the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee (IACUC)SS, FA and JC performed experiments. SS and FK analyzed data. SS and FK wrote the manuscript. All authors read and edited the manuscript.This work was supported in part by the National Institute of Allergy and Infectious Disease (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract (HHSN272201400008C) and unrestricted institutional funds.We thank Parnavi Desai for excellent technical support.FK is listed as inventor on patent applications regarding NA-based vaccines and NA-based therapeutic mAbs. These patent applications have been filed by the Icahn School of Medicine at Mount Sinai and Washington University. Furthermore, FK is consulting for Pfizer, Merck, Third Rock Ventures and Avimex, and had past research support from Pfizer and GSK and current support from Toscana Life Sciences (TLS) Foundation.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Exserohilum turcicum, poses a grave threat to maize production worldwide. The resistance gene in A619Ht3, discovered decades ago, is an important genetic resource for NCLB control. By using a pair of near-isogenic lines (NILs) A619Ht3 and A619, together with the resistant and susceptible bulks derived from the cross of A619Ht3 and L3162 lines, we initially detected a Ht3-like (Ht3L) locus in bin 8.06 that was closely associated with NCLB resistance. We then performed five rounds of fine-mapping, which ultimately delimited the Ht3L locus to a 577-kb interval flanked by SNP markers KA002081 and KA002084. Plants homozygous for the Ht3L/Ht3L genotype exhibited an average reduction in diseased leaf area (DLA) by 16.5% compared to plants lacking Ht3L locus. The Ht3L locus showed extensive variation in genomic architecture among different maize lines and did not appear to contain any genes encoding canonical cell wall-associated kinases against NCLB. Moreover, the Ht3L locus was located ∼2.7 Mb away from the known Htn1 locus. We speculate that the Ht3L locus may contain a bona fide Ht3 gene or a novel NCLB resistance gene closely linked to Ht3. In practice, the Ht3L locus is a valuable resource for improving maize resistance to NCLB.Northern corn leaf blight (NCLB), caused by the fungal pathogen  Exserohilum turcicum (Zea mays L.) grown in areas with high humidity and moderate temperatures is more prone to NCLB outbreaks. During the grain-filling period, NCLB causes leaf necrosis and thus abolishes photosynthetic output, leading to lower grain yield (Northern corn leaf blight (NCLB), caused by the hemibiotrophic fungus turcicum , is one in yield . The devHelminthosporium turcium resistance l (Ht1), was identified in the inbred line ‘GE440’ and ‘Ladyfinger’ popcorn in 1959; this locus shows a partially dominant inheritance pattern and maps to chromosome 2L   show different symptoms, indicating that resistance mechanisms conferred by these genes are not equivalent  . Ht2 andtn1 gene . At the ormation . This obuivalent .qNLB1.06, was anchored to a 3.6-Mb region and narrowed down to two putative candidate genes by joint linkage and association mapping   at the Htn1 locus is the only resistance QTL against NCLB that has been cloned through map-based cloning (Previous studies have shown that quantitative trait loci (QTLs) for resistance to NCLB are dispersed over all 10 chromosomes in maize . Althoug mapping . The QTL (CSSLs) . Single- (CSSLs) . A total (CSSLs) . To date cloning . Further cloning .Htn1 locus is effective against most prevalent NCLB races, NCLB isolates with virulence in Htn1-bearing plants have been reported  (Triticum aestivum) (Ht3-like (Ht3L) locus in the resistant line A619Ht3 by combining NIL analysis, BSA and sequential QTL fine-mapping. Our results lay the foundation for the future map-based cloning of the causal gene at the Ht3L locus. The molecular markers on or adjacent to Ht3L described here can also be used for MABC to improve maize resistance to NCLB in breeding programs.Bulked-segregant analysis (BSA) is a rapid, technically simple method to identify markers linked to specific genes , which h sativa) , wheat (estivum)  and maizestivum) . Anotherestivum) . Many questivum) . In the Ht3 and A619 was obtained from the Eastern Cereal and Oilseed Research Centre of Agriculture and Agri-Food Canada. While A619Ht3 is highly resistant to NCLB, A619 is highly susceptible to NCLB, and both NILs share ∼98.84% genomic homozygosity (Ht3 (donor parent) and L3162 (recurrent parent), multiple backcross populations were developed for initial QTL detection and fine-mapping. In 2016, the two parental lines (A619Ht3 and L3162) and their BC2F1 backcross population comprising 362 individuals were grown at the Shenyang experimental station  in China for artificial inoculation. Twenty-nine highly susceptible and 29 highly resistant plants were selected to form the susceptible and resistant bulks, respectively, for initial mapping of the Ht3L locus. Subsequently, the BC3F1 progeny from recombination events in the mapped Ht3L region were identified and backcrossed to L3162  to produce BC4F1 progeny for fine-mapping. In the following five rounds of fine-mapping from 2017 to 2021, newly-occurring recombinants were screened in Shenyang and then backcrossed twice to L3162 in Shenyang and Hainan to generate progeny. All offspring were investigated for NCLB resistance at the Shenyang experimental station. A619Ht3 and L3162 were planted annually in each experimental block as resistant and susceptible controls, respectively.The pair of near-isogenic lines (NILs) A619zygosity . The eliExserohilum turcicum  was cultured on potato dextrose agar (PDA) medium for 15 days at 25°C and then stored in the dark at room temperature. Plant materials were artificially inoculated at the V8-V10 leaf stages with ∼5 mL of spore suspension (1 × 105-106 conidia per mL in 0.02% [v/v] Tween 20) per plant. Spray irrigation was then performed for 2 days following inoculation, three to four times a day, to maintain a humid environment. Four weeks after inoculation, NCLB lesions were prominent and suitable for first scoring, followed by two more scorings over the next 14 days. Diseased leaf area (DLA) was examined for three leaves (ear leaf and its upper and lower leaves) based on standards listed in Ht3 and A619) and the two parental lines (A619Ht3 and L3162). The Maize6K Chip was used to genotype each plant in the selected susceptible and resistant bulks. Kompetitive Allele-Specific PCR (KASP) markers, developed in the target Ht3L region, were subjected to automated genotyping with the QuantStudio 12 K Flex Real-Time PCR System (Applied Biosystems by Life Technologies).Fresh leaf tissue at the five-leaf seedling stage was harvested for high-throughput extraction of genomic DNA in 96-well plates. Plant genomic DNA was extracted according to the method described previously . The MaiHt3L. The average Δ(SNP-index) value of the SNPs located in each genomic interval was calculated using a sliding window with a window size of 10 Mb and 10-kb steps. The distribution of Δ(SNP-index) along the chromosomes was plotted using R software. SNP markers in the candidate Ht3L regions were also used to conduct regional association analyses. For each SNP marker, the significant difference in SNP-index between two bulks was determined by chi-squared test.The single-nucleotide polymorphism (SNP) index was calculated based on the heterozygosity at each SNP within a given bulk. For each SNP, the Δ(SNP-index) value was calculated using the following formula: Δ(SNP-index) = SNP-index in the resistant bulk – SNP-index in the susceptible bulk . The Δ by inoculating designated physiological races of Exserohilum turcicum  in growth chamber. The results showed that A619Ht3 exhibits susceptible to races 3 and 123N, while resistant to races 1 and 2 (Ht3 locus in OH43 and HZS genetic backgrounds (Ht3 as Ht3-like (Ht3L) to distinguish it from the recently reported Ht3 allele that is identical to Ht2 and allelic to Htn1  potentially covering the Ht3L locus. We detected three segments with stretches of polymorphisms between the two NILs in bins 3.08 (6 SNPs), 7.03/7.04 (12 SNPs) and 8.06 (12 SNPs) (Ht3L regions.The NILs A619 and A619esistant . We geno12 SNPs) , which wHt3L locus, we selected the susceptible line L3162 that is genetically distinct from A619Ht3 to generate mapping populations. We genotyped A619Ht3 and L3162 with the Maize3K Chip, which returned 493 SNPs distributed over all maize chromosomes, numbers sufficient for an initial mapping of Ht3L  for each SNP in the resistant and susceptible bulks. This analysis revealed a prominent peak on chromosome 8 .From a BCmosome 8 . We also.03/7.04 . By contbin 8.06 . Taken tHt3L region to saturate the target region. Accordingly, we retrieved the SNP markers in the Ht3L region between A619Ht3 and L3162 from the genotype data of the Maize6K Chip. We then converted all SNPs located in the Ht3L region into KASP markers and tested them against A619Ht3 and L3162. Totally, we obtained 18 effective KASP markers for fine-mapping  to detect recombination events in the 11.23-Mb Ht3L region from the BC2F1 population obtained in 2016. We identified seven BC2F1 recombinants, which we backcrossed twice to the susceptible parent L3162 to produce their corresponding BC4F1 populations. With an additional five newly-developed KASP markers , we determined the precise recombination breakpoint for each recombinant. To this end, we grew 1,678 plants from the BC4F1 progeny in the field and scored them for their extent of diseased leaf area (DLA) in Shenyang in 2017  from heterozygous (A619Ht3/L3162) BC4F1 plants. With the both genotypic and phenotypic data, an average DLA value can be estimated for both homozygous and heterozygous genotypes in each BC4F1 progeny. Significant difference in DLA between two genotypes indicated the presence of the Ht3L locus in the A619Ht3 donor segment; otherwise, there is no Ht3L locus. As shown, recombinants I to V showed a significant difference (P < 0.05) in their DLA values between homozygous and heterozygous genotypes in their BC4F1 progeny, indicating that they carried the Ht3L locus in the A619Ht3 donor segment (P > 0.05), and thus lacked the Ht3L locus. Recombinants V (with Ht3L) and VII (without Ht3L) defined the left boundary of the mapping interval to marker A007452, while recombinants I (with Ht3L) and VI (without Ht3L) marked the right boundary with marker A007455. The resulting interval spanned a region of 2.17 Mb (AGPv5)  .Ht3L locus with high-density markers. Thus, we backcrossed recombinants I and V to L3162 twice to produce their BC6F1 progeny. We also identified another three new recombinants within the 2.17-Mb region from the BC4F1 progeny, which, together with a heterozygous plant (as a positive control), were backcrossed twice to L3162 to produce BC6F1 progeny. We genotyped the five resulting BC6F1 progeny consisting of 847 individuals, grown in the field for NCLB testing in 2018, with 11 markers spanning the 2.17-Mb region, including seven newly-developed markers. We also tested the markers against the heterozygous Ht3L region to distinguish homozygous from heterozygous BC6F1 progeny. We detected a significant difference (P < 0.05) in DLA for recombinants I, II and III, together with the positive control .From the BCnt IV in  shows a e region . The oth10F1 plants corresponding to seven recombinants in Shenyang for further fine-mapping. Of them, two new recombinants (IV and VI in the P > 0.05) in DLA between the BC10F1 homozygous and heterozygous genotypes, indicating the A619Ht3 donor region lacked Ht3L. Based on these two recombinants, we moved the right boundary from marker KA002085 to marker KA002084. Phenotypic scorings for NCLB severity in the field indicated that the progeny from recombinants I-III showed a significant difference (P < 0.05) in DLA between the homozygotes and heterozygotes in their BC10F1 progeny, whereas the progeny from recombinants IV–VII did not show this difference. Thus, we ultimately anchored Ht3L to a 577-kb interval flanked by markers KA002081 and KA002084 (AGPv5).In the summer of 2020, we planted 3,200 BC10F1 progeny, we selfed heterozygous recombinants to develop three pairs of NILs, which were planted in the summer of 2021 to evaluate their DLA values. The first two pairs of NILs .From the BC did not . These rHt3L/ht3l genotype as the difference in the DLA values between heterozygous and homozygous genotypes for each of the BC4F1, BC6F1, BC8F1 and BC10F1 progeny. For the ht3l/ht3l and Ht3L/ht3l genotypes, we estimated the average DLA values to be 20.3% and 16.5% in BC4F1, 29.4% and 21.6% in BC6F1, 40.5% and 32.1% in BC8F1 and 42.2% and 35.9% in BC10F1, respectively  and resistant NILs (with Ht3L/Ht3L) were 40.4% and 23.9%, respectively. Thus, the genetic effect of the homozygous Ht3L/Ht3L genotype was 16.5% , a zinc knuckle (CCHC-type) protein (Zm00001eb361470), two violaxanthin de-epoxidases (Zm00001eb361480 and Zm00001eb361490), a brassinosteroid-insensitive protein (Zm00001eb361520), a potassium channel KAT protein (Zm00001eb361550) and a phosphatidylinositol kinase (Zm00001eb361560).Bin 8.06 is associated with several tructure . Within t report . Three oHt3L region from 25 sequenced founders of the NAM population (see text footnote 1). Of these 25 lines, seven were highly susceptible and another ten were highly resistant to NCLB, based on a previous report  in bin 8.06, flanked by the SNP markers PZE-108095959 and PZE-108110343. Notably, this 11.23-Mb segment overlapped with the known location of the Htn1 locus. After five rounds of fine-mapping, we delineated the Ht3L locus to a 577-kb interval. To our surprise, the Ht3L locus was located ∼2.7 Mb away from Htn1  with two loci (Ht3L and Htn1) did not display a lower DLA score than those BC2F1 recombinants (No. V) only harboring the Ht3L locus. The lack of phenotypic variation associated with the Htn1 locus between A619Ht3 and L3162 thus indicated that both lines have the same susceptible Htn1 allele. The other possibility is that Htn1 may lose its NCLB-resistance function due to the presence of an unidentified suppressor or the absence of a co-receptor in the L3162 background. For example, a dominant suppressor inhibiting the expression of Ht2 was found in lines related to ‘B14’  the 577-kb Ht3L locus interval may contain a bona fide Ht3 gene that is located ∼2.7 Mb away from the known Ht2/Ht3/Htn1 locus; or 2) A619Ht3 harbors a Ht3 gene that is allelic to Htn1, as claimed by Ht3L locus must contain a novel NCLB resistance gene. In this alternative scenario, we speculate that A619Ht3 is unlikely to harbor another resistant allele at the reported Ht2/Ht3 or Htn1 loci, as only one resistance QTL was detected in our mapping populations. Since the detailed genomic sequence of the parental line A619Ht3 is not currently available, we are still uncertain about the relationship between the currently mapped Ht3L locus and the reported Ht2/Ht3/Htn1 locus. In addition, the genetic backgrounds clearly have pronounced effects on Ht3L-conferred NCLB resistance, as we observed wide variation in DLA scores between homozygotes and heterozygotes in the fine-mapping progeny of different parental recombinants.We detected extensive genomic variation within the 577-kb n1 locus . In fielExserohilum turcicum shows clear physiological differentiation as a function of the resistance performance of inbred lines and differences of climatic conditions. Moreover, the distribution of physiological races is also complex. Twelve physiological races have been identified in China, of which races 0 and 1 are dominant  haplotypes into seven elite maize inbred lines exhibited enhanced resistance to maize stalk rot (Ht3L locus for NCLB resistance. Considering the substantial annual yield loss caused by NCLB, the Ht3L locus will be valuable in future breeding programs of NCLB-resistant maize.dominant . Becausetalk rot . The curHt3L to an interval of 577kb via five rounds of sequential fine-mapping. The homozygous Ht3L/Ht3L genotype reduced DLA by 16.5% compared to that in lines without Ht3L. Our results will facilitate the cloning of the causative gene underlying the Ht3L locus and accelerate application of Ht3L in the breeding of NCLB-resistant maize varieties.Mining and utilizing resistant loci/genes can greatly promote the development of resistant varieties, which will decrease yield losses and improve grain quality. With the availability of high-density SNP markers, we delimited The original contributions presented in this study are included in the article/MJ and YW designed the experiments. JM, XL, XQ, XG, and MJ were responsible for the field tests. YG, YZ, and XL conducted genotyping. MZ and YG analyzed the data. MZ, JM, and MJ wrote the manuscript. MJ and YW supervised the project. MZ, JM, and MJ revised the manuscript in response to the reviewers. All authors read and approved the manuscript.


CD36) , scavenger receptor class B type 1 (SCARB1) (rs5888), and major facilitator superfamily domain containing the 2a (MFSD2A)  on the relationship between circulating lipids in aging subjects with T2DM.Lipid metabolism disorder commonly happens in subjects with Type 2 diabetes mellitus (T2DM) which may be linked to genetic variants of lipid metabolism-related genes. However, few studies have explored the relationship between lipid metabolism-related gene polymorphism and serum lipid profile in aging subjects with T2DM. The present study was designed to explore the impact of genetic polymorphism of cluster determinant 36  was used to compare the means of serum lipids between the groups.205 T2DM patients and 205 age and gender matched control subjects were recruited. Information on demographic characteristics was collected by using a self-administered questionnaire. Fasting venous blood samples were taken for lipid-related gene genotyping and serum lipid profile measurement. The Chi-square test was used to compare percentage differences and to calculate CD36 rs1054516 and MFSD2A rs12072037 were correlated with serum TC level. In control group, CD36 rs1049673 was correlated with serum HDL-C level. Meanwhile, T2DM subjects with MFSD2A rs12083239 (CG), MFSD2A rs4233508 (TT), and MFSD2A rs12072037 (AA) had higher TG level than control subjects. T2DM subjects with CD36 rs1049673 , CD36 rs1054516 (CT), CD36 rs2151916 , SCARB1 rs5888 (GG), MFSD2A rs12083239 , MFSD2A rs4233508 (TT), and MFSD2A rs12072037  had lower HDL-C level than control subjects. T2DM subjects with MFSD2A rs12072037 (AA) had lower LDL-C level than control subjects. In dominant model, major genotype (GG) of SCARB1 gene was associated with the risk of T2DM .In T2DM group, CD36 , SCARB1 (rs5888), and MFSD2A  were associated with serum lipids in T2DM subjects. The SCARB1 rs5888 major genotype (GG) was a protective factor for T2DM. Large scale cohort study is required to determine the relationship between lipid metabolism-related gene polymorphism, serum lipid profile and T2DM in aging subjects.The genetic polymorphism of  Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease that commonly happens in the middle-aged and aged population. The typical clinical characteristics of T2DM are the disorder of glucose and lipid metabolism, manifested by increased plasma the level of glucose and insulin and compensatory insulin secretion insufficiency , 2. MoreCD36 gene is highly polymorphic and it locates on the q11.2 region of human chromosome 7, has 15 exons (SCARB1 gene locates on chromosome 12 and consists of 13 exons and 12 introns (MFSD2A) gene locates on chromosome 1, 14.3 kb in length and consists of 14 exons and 13 introns. MFSD2A is extensively expressed in brain, liver and brown fat tissues. MFSD2A mainly participants in the cellular uptake of n-3 polyunsaturated fatty acids (n-3 PUFAs) (Lipid metabolism is involved with various FFA transporters. Scavenger Receptor (SR-B), as a member of the FFA transporter system, was reported directly to participate in lipid metabolism through mediating lipoprotein uptake. SR-B includes the subtypes of fatty acid translocase cluster determinant 36 (CD36) and scavenger receptor class B type 1 (SCARB1). The 15 exons . CD36 is3 PUFAs) , 9.mir-143/145  level in serum, liver, and brown adipose tissue  and gender matched control subjects were recruited in Nanyuan Community  and Wulituo Community . There were 73 men and 132 women in each group. Diabetes patients were determined according to guidelines for the prevention and control of type 2 diabetes in China , with typical symptoms and fasting blood glucose ≥ 7.0 mmol/L or postprandial blood glucose ≥ 11.1 mmol/L. The study protocol was approved by the Committee on Medical Ethics of Capital Medical University (No. 2012SY23). The study procedures followed the ethical standards of the Helsinki Declaration of 1975. Informed consent was signed by all participants.2).Participants underwent a general information survey, medical history survey and physical examination. Information on demographic characteristics , lifestyle factors , and medical history of chronic disease  were collected by using a self-administered questionnaire. The nurses of the community medical service center documented anthropometric measures (height and weight). Body mass index (BMI) was calculated as weight (kg)/height (mThe fasting venous blood (5 mL) was collected from all the participants in the morning. After centrifuging at 1,500 g for 15 min, the plasma was separated and used for lipid parameter measurement. ILAB600 clinical chemistry analyzer  was applied for glucose (GLU), total cholesterol (TC) and triglyceride (TG) measurement. High-density lipoprotein cholesterol (HDL-C) was determined by a commercially available assay from the Instrumentation Laboratory , and low-density lipoprotein cholesterol (LDL-C) was calculated according to the Friedewald formula . All samCD36 , SCARB1 (rs5888), MFSD2A  was detected by SNPscan genotyping assay according to the method descripted by previous study  was used to extract DNA from the whole blood sample. The genetic polymorphism of us study .SD). Chi-square test was used to compare the differences in percentages and assess Hardy-Weinberg equilibrium. The allele and genotype frequency were calculated from the observed genotypic counts. The effect of different SNPs on the risk of T2DM in different gene models by using logistic regression. Multiple linear regression was applied to ascertain the correlation between alleles or genotypes and serum lipids. General linear model (GLM) was used to compare the means of the detected parameters between the groups. Potential confounding factors, including sex, age, BMI, hyperlipidemia, hypertension, coronary heart disease, atherosclerosis, smoking, alcohol drinking and physical activity, were adjusted during data analysis. The power was analyzed by using Gpower 3.1 software. P < 0.05 was considered as significant.All data were statistically analyzed by SPSS 26.0 software . Graphs were prepared by GraphPad Prism 8.0 (GraphPad Software). Continuous variables were represented as mean ± standard deviation (P > 0.05). The number of subjects with hyperlipidemia, hypertension, coronary heart disease and atherosclerosis in the T2DM group was significantly higher than that in the control group (P < 0.05). There was no significant difference in lifestyle  and stroke between groups. Compared with the control subjects, the T2DM subjects had higher plasma GLU and lower HDL-C levels (P < 0.001). There was no significant difference in TC, TG and LDL-C levels between groups.The demographic characteristics and plasma lipid levels of the participants are shown in P < 0.001). No association was found between T2DM risk and TC, TG, or LDL-C levels.As shown in P > 0.05). No significant statistical difference was found in the allele frequency of SCARB1 rs5888 between T2DM and the control groups. However, the difference of SCARB1 rs5888 genotype frequency was detected between T2DM and control groups. Compared to the control group, a higher percentage of subjects with heterozygote (G/A) but a lower percentage of subjects with major (G/G) or minor (A/A) genotype were found in the T2DM group (P < 0.05). There was no significant difference in the allele and genotype frequency of the other SNPs between T2DM and control subjects (P > 0.05).The allele and genotype frequency of the lipid metabolism-related genes in the T2DM and control groups are shown in SCARB1 rs5888 negatively correlated with the risk of T2DM in aging subjects with the OR value of 0.636  in dominant model. There was no statistical association between other SNPs and the risk of T2DM in aging subjects.As shown in CD36 and MFSD2A in T2DM subjects and control subjects (P < 0.05). The allele and genotype of CD36 rs1054516 were associated with TC in the T2DM population (P < 0.05). In T2DM subjects, plasma TC level also was MFSD2A rs12072037 genotype related (P < 0.05); and LDL-C level was CD36 rs1054516 genotype related (P < 0.05). The allele and genotype of CD36 rs1049673 were associated with HDL-C in the control subjects (P < 0.05).The result of multiple linear regression analysis between alleles or genotypes and plasma lipids is shown in CD36 rs1054516 and MFSD2A rs12072037 was found in T2DM subjects. Subjects with CD36 rs1054516 minor genotype (CC) had higher TC level than subjects with major genotype (TT) (P < 0.05); and the subjects with MFSD2A rs12072037 minor genotype (AA) had lower TC level than the major allele  carrier (P < 0.05). In control group, subjects with CD36 rs1049673 minor genotype (GG) had higher HDL-C level than major genotype (CC) (P < 0.05). There was no difference of serum TC, TG, HDL-C and LDL-C levels between groups according to different genotypes (P > 0.05).Firstly, the difference of serum lipid was compared between T2DM and control groups according to different genotypes. As shown in MFSD2A rs12083239 (CG), MFSD2A rs4233508 (TT) and MFSD2A rs12072037 (AA) displayed higher TG level than the control subjects (P < 0.05). Serum HDL-C level was associated with the genetic polymorphism of CD36, SCARB1 and MFSD2A. T2DM patients with CD36 rs1049673 , CD36 rs1054516 (CT), CD36 rs2151916 , SCARB1 rs5888 (GG), MFSD2A rs12083239 , MFSD2A rs4233508 (TT) and MFSD2A rs12072037  genotypes had lower HDL-C level than the control ones (P < 0.05). And the T2DM subjects with MFSD2A rs12072037 (AA) genotype showed lower LDL-C level than the control subjects (P < 0.05).Secondly, the serum lipids between subjects with different genotypes were compared according to whether they were T2DM patients. As shown in Studies found that elevation of insulin during insulin resistance affects the lipid metabolism , 28, andThe association between insulin resistance and low circulating HDL-C level was found in T2DM patients –33. ElevSCARB1 rs4238001 GA genotype is significantly increased as compared with those carrying GG after ingestion of monounsaturated fatty acids  genotype in the T2DM patients than the control ones. The association of SCARB1 rs5888 genetic polymorphism with the risk of T2DM was found in the dominant model, which was demonstrated by a decreased risk for T2DM in subjects with the major genotype (GG) as compared with the minor allele carrier  . Studies reported that genetic variation of the SCARB1 gene affected SCARB1 gene expression, resulting in the change of circulating lipids profile, especially the level of HDL (SCARB1 rs5888 CT genotype had significantly lower serum TG levels than the subjects with CC genotype (P < 0.05)  was significantly lower than that in control subjects. These results indicated that the major genotype (GG) of SCARB1 rs5888 may play a protective role in T2DM through affecting plasma HDL-C level.The close relationship between T2DM and abnormal lipid metabolism, and the role of lipid metabolism-related gene variants in the relationship between serum lipid and diabetes are worthy of exploration. Pérez-Martínez et al. reported that the insulin sensitivity of T2DM patients carrying l of HDL . Smalins < 0.05) . In our CD36 rs3211938 genotype as well as the minor alleles of CD36 SNPs  with clinical profiles in T2DM patients has been reported in previous study  on increasing body weight, BMI and the risk of T2DM was also demonstrated  (CD36 genetic variants are associated with serum TG and HDL-C levels in Chinese population. Subjects with rs3211848 minor genotype (AA) had higher TG level than those with major genotype (GG), and subjects with rs1054516 minor allele carrier  had lower HDL-C level than those with major genotype (TT) (CD36 rs1054516 genotype and HDL-C in T2DM patients. While the T2DM subjects carrying the minor genotype (CC) of CD36 rs1054516 showed increased TC level than the subjects carrying the major genotype (TT). In contrast, in control group, increased plasma HDL-C level was observed in the subjects with the minor genotype (GG) of CD36 rs1049673 as compared with the subjects carrying the major genotype (CC). Moreover, the serum HDL-C level of T2DM patients with CD36 rs1049673 , CD36 rs1054516 (CT), CD36 rs2151916  genotypes was lower than the control subjects. These results indicated that CD36 genetic variants mainly affect the circulating lipid profile through the regulation of TC and HDL-C, and the T2DM subjects with CD36 rs1049673, rs1054516, and rs2151916 genetic variants are prone to have dyslipidemia.Significant relationship between the us study . The imp= 0.007) . Study hype (TT) . In our MFSD2A gene knockout mice showed slimmer bodies, increased energy expenditure, and reduced serum TG level , MFSD2A rs4233508 (TT), and MFSD2A rs12072037 (AA) displayed increased plasma TG level in comparison with control subjects. Besides, the T2DM subjects with MFSD2A rs12083239 , MFSD2A rs4233508 (TT), and MFSD2A rs12072037  genotypes showed lower HDL-C level than control subjects. These results indicated that the MFSD2A genetic variants mainly affect circulating lipids through regulating the TG and HDL-C levels, and T2DM subjects with MFSD2A rs12083239, rs4233508, and rs12072037 genetic variants are prone to have abnormal circulating TG and HDL-C levels.The study reported that fasting could induce the hepatic MFSD2A mRNA expression in a β-adrenergic signaling-dependent manner in experimental animals . AdditioTG level , suggestSCARB1 gene were associated with an increased risk of T2DM, which demonstrated that, for healthy carriers of this gene, regularly monitor of circulating lipids  and blood glucose levels might be benefit to reduce the risk of T2DM. Therefore, for patients with the SNPs mentioned above, more attention should be paid to lipid management to reduce the risk of dyslipidemia-based complications. This will provide a scientific basis for clinicians to efficiently prevent and treat T2DM and dyslipidemia according to individual’s genetic background.In this study, we found that subjects with minor allele genotypes  of the There are several potential limitations in this study. Firstly, the study was conducted in Chinese population, the difference in lifestyle, living conditions and diet pattern makes the extrapolation of our conclusion to other population should be with caution. Secondly, environmental and genetic factors are regulators of an individual’s lipid profile, the interaction between environment and genetic factors were not measured in our study, and this should be taken into consideration during decomposing the relationship between genetic factors, serum lipids and T2DM in the future. Thirdly, the treatment and management of the T2DM subjects were another potential confounding factor, which should be considered in future large-scale cohort studies. Finally, the relatively small sample size is another limitation of the study, and population-based large scale cohort studies are needed to verify the accurate relationship between lipid metabolism-related gene polymorphism, lipids and T2DM in aging subjects in the future.SCARB1 are associated with an increased risk of T2DM. Plasma TC, HDL-C and TG levels were related to CD36, SCARB1 and MFSD2A genetic polymorphism in T2DM subjects. Large-scale population-based cohort studies are needed to reveal the biological interactional mechanism between lipid metabolism-related gene polymorphism, serum lipid level and T2DM in aging subjects.In summary, our data implicated the association of lipid metabolism-related gene genetic variation, serum lipid level with T2DM in aging subjects. Genetic variants of The original contributions presented in this study are included in the article/The studies involving human participants were reviewed and approved by the Committee on Medical Ethics of Capital Medical University. The patients/participants provided their written informed consent to participate in this study.LY designed the study. XW, JX, XM, YG, and HY conducted the investigation and collected the blood sample. XW performed the data statistical analysis. XW, LY, and SZ drafted the manuscript. All authors reviewed the manuscript, read, and approved the final manuscript.


Disruptor of telomeric silencing 1 (DOT1) was first identified in yeast (DOT1p) and is the sole methyltransferase responsible for histone three lysine 79 (H3K79) mono-, di-, and tri-methylation. Mammalian DOT1 (DOT1-like protein or DOT1L) has been implicated in many cellular processes, such as cell cycle progression, DNA damage response, and development. A notable developmental process reliant on DOT1L function is normal hematopoiesis, as DOT1L knockout leads to impairment in blood lineage formation. Aberrant activity of DOT1L has been implicated in hematopoietic malignancies as well, especially those with high expression of the homeobox (HOX) genes, as genetic or pharmacological DOT1L inhibition causes defects in leukemic transformation and maintenance. Recent studies have uncovered methyltransferase-independent functions and a novel mechanism of DOT1L function. Here, we summarize the roles of DOT1L in normal and malignant hematopoiesis and the potential mechanism behind DOT1L function in hematopoiesis, in light of recent discoveries. Disruptor of telomeric silencing 1 (DOT1 or KMT4) was first identified in yeast as a regulator of telomeric silencing, as overexpression of DOT1 decreases silencing at and around telomeric regions . Yeast DStudies show DOT1L is important for cellular processes including cell cycle progression, DNA damage repair, and transcriptional regulation (Reviewed in ). DOT1L DOT1L constitutive knockout (KO) is embryonic-lethal in mice . By embrA constitutive DOT1L methyltransferase mutant (DOT1L-MM N241D) mouse model was recently characterized, showing potential non-enzymatic roles of DOT1L in hematopoiesis during early development. Similar to DOT1L KO mice, DOT1L-MM embryos died before E13.5 , but shof/f Cre-ER, postnatal Dot1l deletion resulted in pancytopenia, BM hypocellularity, and reductions of HSPC and mature cells in all lineages during steady-state hematopoiesis  resulted in anemia, neutropenia, lymphopenia, and reduced BM cellularity with significant reductions in HSPC compartments. Older mice showed partial chimerism due to non-deleted clones . Using topoiesis . These eopoiesis . Repeateopoiesis . One stuopoiesis . Similaric cells . Overallic cells . Based oic cells , and datic cells , and expic cells , suggestDOT1L has also been implicated in hematopoietic malignancies, especially in leukemias harboring Mixed Lineage Leukemia gene (MLL) rearrangements  and such crosstalk is conserved from yeast to metazoan. Recent cryo-electron microscopy (Cryo-EM) studies of DOT1L bound to ubiquitinated nucleosomes showed that DOT1L interacts with H2Bub through a C-terminal hydrophobic helix. Additional contact is made with an arginine anchor within DOT1L and an H2A/H2B acidic patch in both poised and active DOT1L complexes. In the active complex, binding of H4 tail to a cleft in DOT1L positions the catalytic center above H3K79 and induces conformation changes in H3 to reposition the inaccessible H3K79, inserting the side chain into the active site . FurtherBeyond their role in transcription elongation, H3K79me2/3 marks have been found in functionally active enhancers . GodfreyDOT1L has been implicated in many important processes including cell cycle, transcription regulation, DNA damage repair, and general development. In normal hematopoiesis, DOT1L is required for both embryonic and adult hematopoiesis by regulating genes important in HSC differentiation and proliferation, such as HOX/MEIS and FLT3. Recent findings show that DOT1L may function in a methyltransferase-independent manner in primitive erythropoiesis , in a yeHOX and FLT3 genes. Recent research has provided a rationale to target DOT1L outside of MLLr leukemia, including in NPM1 (Aberrant activity of DOT1L is implicated in many hematopoietic malignancies, particularly MLL-rearranged leukemias. The canonical target genes of DOT1L in leukemia are similar to those in normal hematopoiesis, including  in NPM1  and cohe in NPM1 . Researc in NPM1 . Given tRecent reports have advanced our understanding of the molecular mechanism of DOT1L function. Two studies have shown that DOT1L does not play a major role in transcription elongation. Instead, one points to its role in transcription termination checkpoint control, which is independent of its enzymatic function , and the


Here, we adjusted the microstructure of the pristine bulk-g-C3N4 (PCN) and further loaded silver (Ag) nanoparticles. Abundant Ag nanoparticles were grown on the thin-layer g-C3N4 nanosheets (CNNS), and the Ag nanoparticles decorated g-C3N4 nanosheets (Ag@CNNS) were successfully synthesized. The thin-layer nanosheet-like structure was not only beneficial for the loading of Ag nanoparticles but also for the adsorption and activation of reactants via exposing more active sites. Moreover, the surface plasmon resonance (SPR) effect induced by Ag nanoparticles enhanced the absorption of visible light by narrowing the band gap of the substrate. Meanwhile, the composite band structure effectively promoted the separation and transfer of carriers. Benefiting from these merits, the Ag@CNNS reached a superior hydrogen peroxide (H2O2) yield of 120.53 μmol/g/h under visible light irradiation in pure water (about 8.0 times higher than that of PCN), significantly surpassing most previous reports. The design method of manipulating the microstructure of the catalyst combined with the modification of metal nanoparticles provides a new idea for the rational development and application of efficient photocatalysts.As a promising metal-free photocatalyst, graphitic carbon nitride (g-C Herein, by tuning the microstructure of pristine bulk-g-C3N4 (PCN), thin-layer porous g-C3N4 nanosheet (CNNS) was designed and synthesized via a continuous thermal etching and ultrasonic etching method. Considering that silver (Ag) is a noble metal with low price and high crustal abundance [3N4 nanosheets (Ag@CNNS). The preparation process of Ag@CNNS could be schematically illustrated in 2O2 evolution (120.53 μmol/g/h) under visible light irradiation. The unique properties of Ag@CNNS accelerate the efficiency of the whole artificial photocatalytic H2O2 production.Although Teng et al.  proposedbundance ,38, withbundance , Ag nano4N2O, 99%), sodium borohydride , potassium iodide , potassium phthalate monobasic , silver nitrate solution  and ethanol  were all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China. All the chemical reagents were used without further purification. Deionized water was used throughout the entire experiment.Urea  and 2 mL of NaBH4 solution (0.2 mg/mL) sequentially, and kept stirring at 70 °C for 2 h. Finally, the obtained products were washed with deionized water and ethanol several times, respectively, and dried at 80 °C for 12 h.Ag@CNNS was synthesized using a chemical reduction method. In detail, 60 mg of CNNS was placed into 30 mL of deionized water and ultrasonicated for 30 min to achieve uniform dispersion, followed by the addition of 30 mL of ethanol. Then, added with 0.6 mL of AgNOα radiation (λ = 0.15418 nm) at room temperature. The specific surface areas of all samples were measured according to the Brunner-Emmet-Teller (BET) method on a BELSORP-max II. The UV–vis absorption spectra (UV-vis) and UV-vis diffuse reflectance spectroscopy (UV-DRS) of the samples were carried out on a HITACHI U-3900H spectrophotometer (wavelength range of 300–700 nm) and HITACHI UH4150 spectrophotometer , respectively. Photoluminescence (PL) spectra were measured on a Hitachi F-7000 fluorescence spectrometer with an excitation wavelength of 375 nm. Transmission electron microscope (TEM) images were collected using an FEI Titan Themis G2 microscope operated at an accelerating voltage of 300 kV. The powder X-ray diffraction (XRD) patterns of the samples were collected on a Bruker, D8 ADVANCE with Cu K2O2 production by the as-prepared catalysts was evaluated using a 300 W Xe lamp  without the absence of any sacrificial agent. Here, 25 mg of each photocatalyst was added to 25 mL of deionized water (1 mg/mL) in a specific reactor. Ultrasonic treatment for 30 min to make the powder completely dispersed, and then O2 was bubbled through the solution to make the O2 saturated. Next, maintain the reaction solution at 25 °C under a circulating water system and keep the photocatalyst dispersed under magnetic stirring, while irradiating the reaction solution with 400 nm cutoff film (λ ≥ 400 nm) to start the photoreaction test. During the photoreaction progress, 1.5 mL of the reaction solution was taken out to remove the photocatalyst every 15 min. The photocatalytic H2O2 were measured by iodometry [8H5KO4 aqueous solutions and 0.5 mL of 0.4 mol/L KI aqueous solution were added to 1 mL of the obtained supernatant and then kept for 60 min. The following reaction occurs in the mixed solution: H2O2 + 3I− + 2H+ → I3− + 2H2O, the generated I3− has a strong absorption peak around 350 nm, from which the amount of H2O2 produced during the photocatalytic reaction can be calculated by UV–vis spectroscopy. 2O2 and the UV-vis absorption intensity of different H2O2 concentrations by iodimetry. The concentrations of Hodometry . 0.5 mL The electrochemical impedance spectroscopy (EIS) and Mott–Schottky plots were performed on an electrochemical workstation (CHI600A) using a three-electrode setup with ITO glasses covered by the as-prepared photocatalysts as working electrodes, a Pt wire as the counter electrode, and Ag/AgCl electrode as the reference electrode. The conversion relationship between the measured potentials (vs. Ag/AgCl) and reversible hydrogen electrode (RHE) at 25 °C is as follows (Equation (3)): 4 to reduce Ag(Ⅰ) into Ag nanoparticles, which can be observed in the TEM images. 2 physisorption measurements (Transmission electron microscopy (TEM) was performed to observe the structure and morphology of as-prepared samples. urements  showed tThe microstructure of the photocatalysts was further investigated via powder X-ray diffraction (XRD). As shown in 2 on the surface of photocatalyst  to reveal the optical absorption properties of the samples. As shown in catalyst f.To systematically investigate the separation, transfer, and recombination behavior of photogenerated carriers during the catalytic progress, steady-state photoluminescence (PL) and electrochemical impedance spectroscopy (EIS) were carried out. As shown in 2O2 by PCN, CNNS, and Ag@CNNS was further evaluated in pure water . 2O2 yields of various catalysts during the evaluation process. As shown in 2O2 generation rate reaching 120.53 μmol/g/h, about 8.0 times higher than that of PCN (15.40 μmol/g/h). It could be interpreted as follows: (1) The nanosheet-like structure exposed more surface for reactant adsorption and activation. (2) The SPR effect of Ag nanoparticles narrowed the band gap and promoted the absorption of visible light. (3) A Schottky barrier was formed at the interface between Ag nanoparticles and the substrate to capture the photogenerated electrons, thus realizing the effective separation of photogenerated electron-hole pairs. Considering the stability in practical applications, a cycling test has been carried out. As shown in 2O2 yield could be observed. Ag nanoparticles were still uniformly and firmly dispersed on the g-C3N4 nanosheets (2O2 production fully confirmed that Ag@CNNS has excellent cyclability and stability. Furthermore, the Ag@CNNS was also evaluated under a dark condition and nitrogen atmosphere, respectively. No H2O2 could be detected, suggesting the importance of visible light and O2 in the photoredox progress [3N4-based photocatalysts for H2O2 production in recent years (2O2 production.The photocatalytic production of Hnosheets , indicatf PCN 15. μmol/g/hnt years . Based o3N4 via microstructure control and noble metal nanoparticle modification. The introduction of Ag nanoparticles not only enhanced the light absorption properties but also realized the effective capture of photogenerated electrons. Besides, the thin-layer nanosheet-like structure not only accelerated the transfer of charges but also promoted the adsorption and activation of reactants. As a result, the photocatalytic H2O2 yield of the resulted Ag@CNNS reached 120.53 μmol/g/h, about 8.0 times higher than that of PCN, significantly improving photocatalytic activity. This work provides a promising guide for the rational design of efficient g-C3N4-based photocatalysts for H2O2 production.In summary, we successfully optimized the optical properties, band structure and carrier transfer behavior of the pristine bulk-g-C