First Report of Leaf Spot Disease Caused by Alternaria brassicae on Camptotheca acuminata in Sichuan, China.

Camptotheca acuminata (C. acuminata), is belongs to a monotypic genus endemic to southwestern China and listed as the first class national protected plant in China in 1999 (Wen, et al. 2020). Camptothecin, isolated from the wood and bark of C. acuminata Decne, which exhibits clinical effects in various cancer treatments (Pommier, et al. 2006; Kang, et al. 2021). In October 2021, we investigated leaf spot disease occurrence on C. acuminata (FigS1.A) with 80% incidence in Beichuan County, Sichuan Province of China. Leaf symptoms were randomly distributed on the adaxial surfaces and consisted of punctate spots of alternating light gray and dark brown in the early stage of onset (FigS1. B, C). As the disease progressed, these spots expanded irregularly shaped regions of necrotic tissue, and gray-white mildew layers can be seen on the front and back of the lesions in a humid environment. Infected tissues from symptomatic leaves disinfected in 75% ethanol for 45 s, and with 0.1% HgCl2 for 1 min, rinsed then plated on potato dextrose agar (PDA) medium supplemented with ampicillin and carbenicillin (50 µg/ml each). Plates were incubated for 3 days at 25°C. Then prepared by transferring hyphal tips from the edges of these colonies onto fresh PDA medium for subculture. Aerial hyphae had a cotton-like appearance with white to pale gray color (FigS1.D). Conidia were present in long chains, with conidiophores being present in clusters or in isolation (FigS1E), with 1-6 transverse septa, 0-3 oblique and longitudinal septa and an ellipsoidal to obpyriform structure, measuring 10.0-50.9 µm in length and 5.6-11.8 µm in width (n = 20) (FigS1E, G). On the basis of conidial and cultural characteristics, the fungus was consistent with those of members of the Alternaria genus (Simmons, 2008). To confirm this tentative identification, DNA was extracted from isolate XS9, the internal transcribed spacer rDNA regions (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1), partial RNA polymerase II largest subunit (RPB2) genes were amplified with primers pairs ITS1/ITS4 (White et al.1990), GDF/GDR (Templeton et al.), TEF-728F/TEF-986R (Carbone & Kohn 1999) and RPB2-5F2/RPB2-7cR (Sung et al. 1990; Liu et al. 1999), Bt-2a/Bt-2b (Glass and Donaldson 1995) respectively. The resulting sequences were deposited in GenBank (ITS, OP113690; GAPDH, OP120953; TEF, OP120952; RPB2, OP120954). Further phylogenetic analyses of isolate XS9 revealed it to cluster in the A. brassicae clade with 97% bootstrap support. Pathogenicity identification of isolate XS9 was carried out on the detached leaves. The pure agar plugs (as control) or spraying water on the leaf surface were inoculated on detached leaves, the controls remained healthy after 8 days (FigS1.H-J). but the leaves inoculated with other the mycelium plugs (Fig S1K, L) or the conidia suspension (2×105 conidia/mL) of isolate XS9 was sprayed on the detached leaves (Fig S1M, N), both showed brown necrotic lesions that are similar to the symptoms observed in the field. The pathogen was reisolated and confirmed to be A. brassicae. To our knowledge, this is the first report of leaf spot disease caused by A. brassicae on C. acuminata in China. Leaf spot disease causes the branches and leaves of camptotheca acuminata to wither and even the whole plant to die. To ensure the protection of the irreplaceable species, effective measures should be taken to prevent the spread of the leaf spot disease.

First Report Davidia involucrate Baill Leaf Blight Caused by Nigrospora oryzae in Sichuan, China.

Davidia involucrata Baill. (D. involucrate), also known as dove tree, is listed as the first class national protected plant in China and the only extant member of the Davidiaceae family (Fu & Jin 1992). Referred to by the terms 'living fossil' and 'giant panda' owing to its evolutionary status as a Tertiary relic and its native distribution, D. involucrate exhibits substantial ornamental and academic value (Fang & Song 1975; Wu et al. 2004). A small rounded head inflorescence beneath its big white bracts have a unique charm to catch people's attention, thus they were cultivated in many areas of the world as an ornamental plant (Claßen-Bockhoff & Arndt 2018). In September 2021, dove trees in Meigu country (N 28°33', E 103°14'), Sichuan Province, China were found to appear symptoms of leaf blight of unknown origin. This blight disease incidence was 90% in a survey of 30 D. involucrata trees. Early symptoms appeared as circular, necrotic tissue that developed into circular or irregular spots (FigS1. A). Five leaves exhibiting typical symptoms of this form of leaf blight were excised from the margin between diseased and healthy tissue. These pieces were then treated for 40 s with 75% ethanol for surface sterilization, followed by treatment with 5% NaClO for 2 min, rinsed then plated on potato dextrose agar (PDA) medium supplemented with carbenicillin and ampicillin (each 50 µg/mL), and incubated in the dark for 4 days at 28°C. Pure cultures were then prepared by transferring hyphal tips from the edges of these colonies onto fresh PDA plates, with isolate LW11 being selected as a representative isolate for causal pathogen characterization. These cultured colonies were initially white before turning grayish-black over time (FigS1. B). Conidia were single-celled, black, spherical or oblate and ranged from 10 - 16.0 µm in diameter (mean = 12.5 ± 0.43 µm, n = 40) (FigS1. C), with conidia being present at the tip of conidiophores on hyaline vesicles. These morphological traits were found to align well with those of Nigrospora oryzae (Wang et al. 2017). To confirm this tentative identification, gDNA was extracted from isolate LW11, followed by the amplification of the internal transcribed spacer (ITS) region, beta-tubulin (TUB2), and translation elongation factor 1-alpha (TEF1) sequences with the respective ITS1/ITS4 (White et al., 1990), Bt-2a/Bt-2b (Glass & Donaldson 1995), and TEF1-728F/EF1-986R primer pairs (Carbone et al. 1999). Fragments of 577 bp, 442 bp, and 309 bp were obtained. A maximum likelihood bootstrapping approach (1000 bootstrap replicates) was used to construct a phylogenetic tree based on a combination of the ITS, TUB, and TEF1-α sequences, indicating that isolate LW11 clustered with other N. oryzae isolates (FigS2). The ITS, TUB, and TEF1-α sequences from isolate LW11 were deposited in GenBank with the accession numbers OL659284, OL685345, and OL685347, respectively. The pathogenicity test was performed by detached D. involucrate leaves with mycelial plugs, with the other half instead being inoculated using pure agar plugs as a negative control. Following incubation for 5 days, black lesions were evident on leaves inoculated with mycelial plugs (FigS1. F; FigS1, DE) but not on control leaves (FigS1. F). This report is the first to our knowledge of D. involucrata leaf blight by N.oryzae in China or anywhere else in the world. Further research is thus needed to better manage the spread of this disease with the goal of protecting this living fossil species.

Rhythmic cilia changes support SCN neuron coherence in circadian clock.

The suprachiasmatic nucleus (SCN) drives circadian clock coherence through intercellular coupling, which is resistant to environmental perturbations. We report that primary cilia are required for intercellular coupling among SCN neurons to maintain the robustness of the internal clock in mice. Cilia in neuromedin S-producing (NMS) neurons exhibit pronounced circadian rhythmicity in abundance and length. Genetic ablation of ciliogenesis in NMS neurons enabled a rapid phase shift of the internal clock under jet-lag conditions. The circadian rhythms of individual neurons in cilia-deficient SCN slices lost their coherence after external perturbations. Rhythmic cilia changes drive oscillations of Sonic Hedgehog (Shh) signaling and clock gene expression. Inactivation of Shh signaling in NMS neurons phenocopied the effects of cilia ablation. Thus, cilia-Shh signaling in the SCN aids intercellular coupling.

LUBAC regulates ciliogenesis by promoting CP110 removal from the mother centriole.

Primary cilia transduce diverse signals in embryonic development and adult tissues. Defective ciliogenesis results in a series of human disorders collectively known as ciliopathies. The CP110-CEP97 complex removal from the mother centriole is an early critical step for ciliogenesis, but the underlying mechanism for this step remains largely obscure. Here, we reveal that the linear ubiquitin chain assembly complex (LUBAC) plays an essential role in ciliogenesis by targeting the CP110-CEP97 complex. LUBAC specifically generates linear ubiquitin chains on CP110, which is required for CP110 removal from the mother centriole in ciliogenesis. We further identify that a pre-mRNA splicing factor, PRPF8, at the distal end of the mother centriole acts as the receptor of the linear ubiquitin chains to facilitate CP110 removal at the initial stage of ciliogenesis. Thus, our study reveals a direct mechanism of regulating CP110 removal in ciliogenesis and implicates the E3 ligase LUBAC as a potential therapy target of cilia-associated diseases, including ciliopathies and cancers.

APOE genotype-function relationship: evidence of -491 A/T promoter polymorphism modifying transcription control but not type 2 diabetes risk.

BACKGROUND: The apolipoprotein E gene (APOE) coding polymorphism modifies the risks of Alzheimer's disease, type 2 diabetes, and coronary heart disease. Aside from the coding variants, single nucleotide polymorphism (SNP) of the APOE promoter has also been shown to modify the risk of Alzheimer's disease. METHODOLOGY/PRINCIPAL FINDINGS: In this study we investigate the genotype-function relationship of APOE promoter polymorphism at molecular level and at physiological level: i.e., in transcription control of the gene and in the risk of type 2 diabetes. In molecular studies, the effect of the APOE -491A/T (rs449647) polymorphism on gene transcription was accessed by dual-luciferase reporter gene assays. The -491 A to T substitution decreased the activity (p<0.05) of the cloned APOE promoter (-1017 to +406). Using the -501 to -481 nucleotide sequence of the APOE promoter as a 'bait' to screen the human brain cDNA library by yeast one-hybrid system yielded ATF4, an endoplasmic reticulum stress response gene, as one of the interacting factors. Electrophoretic-mobility-shift assays (EMSA) and chromatin immuno-precipitation (ChIP) analyses further substantiated the physical interaction between ATF4 and the APOE promoter. Over-expression of ATF4 stimulated APOE expression whereas siRNA against ATF4 suppressed the expression of the gene. However, interaction between APOE promoter and ATF4 was not -491A/T-specific. At physiological level, the genotype-function relationship of APOE promoter polymorphism was studied in type 2 diabetes. In 630 cases and 595 controls, three APOE promoter SNPs -491A/T, -219G/T (rs405509), and +113G/C (rs440446) were genotyped and tested for association with type 2 diabetes in Hong Kong Chinese. No SNP or haplotype association with type 2 diabetes was detected. CONCLUSIONS/SIGNIFICANCE: At molecular level, polymorphism -491A/T and ATF4 elicit independent control of APOE gene expression. At physiological level, no genotype-risk association was detected between the studied APOE promoter SNPs and type 2 diabetes in Hong Kong Chinese.

[Meta analysis on effect of local application of chlorhexidine with scaling and root planing].

PURPOSE: Meta analysis was used to assess the effect of local drug delivery system of chlorhexidine (CHX) as an adjunct to scaling and root planing(SRP) versus SRP alone in patients with chronic periodontitis. METHODS: The selected studies of randomized controlled trials(RCTs) were pooled from six major electronic database up to May,2009 as CHX plus SRP versus SRP with at least 3 months of follow-up. Several English full texts were hand searched. Outcome measures were probing depth(PD) reduction and clinical attachment level (CAL)gain. RESULTS: Seven studies that met inclusion criteria were entered into the Meta analysis. A significant mean reduction of PD in patients with CHX plus SRP was observed, but there were no significant difference in CAL between the two treatment groups. CONCLUSIONS: Chlorhexidine as an adjunct to SRP could reduce probing depth in the treatment of chronic periodontitis. Supported by Research Project of Education Bureau of Liaoning Province(Grant No.20061021).

[Suppression of murine EAE by triptolide is related to downregulation of INF-gamma and upregulation of IL-10 secretion in splenic lymphocytes].

AIM: To explore the therapeutic effectivity and the possible mechanism of triptolide (Tri) on experimental autoimmune encephalomyelitis (EAE). METHODS: All female C57BL/6 mice were randomly divided into EAE group (28), Tri treated group (20) and adjuvant group (18). Mice in EAE and treated groups were immunized with myelin oligodendrocyte glycoprotein peptides 35-55 (MOG(35-55);), adjuvant group was injected at the same time, but instead of MOG(35-55); with normal saline. Tri was intraperitoneally injected in the dosage of 100 microg/(kg.d) in treated group on day 5 post-immunization (p.i.), and mice in EAE and adjuvant group injected with normal saline as control. The clinical feature and pathological changes were observed and the splenic lymphocytes were prepared on days 18-20 p.i. The cell cultures were divided into the control group (only 200 microL of cell suspension) and the experimental group (cell suspension in the presence of 10 mg/L MOG(35-55);). Then all of them were inoculated in 96-well flat-bottom plates under 37 degrees Celsius, 50 mL/L CO(2);. After 48 h, the proliferation assay was determined by MTT, and the supernatants were harvested for the detection of INF-gamma, IL-17, IL-10 and IL-4 by ELISA. RESULTS: Tri treatment showed an significantly protective action on EAE. After the intervention of Tri, the levels of IL-10 were increased (P<0.05), but the secretion of INF-gamma and proliferation response of splenic lymphocytes induced by MOG(35-55); were statistically significantly inhibited(P<0.05 and P<0.01, respectively). There were no influences on the amount of IL-17 and IL-4 by Tri. CONCLUSION: Tri is an effective drug in suppressing murine EAE. This suppression is supposed to be related to downregulation of INF-gamma and upregulation of IL-10 secretion in splenic lymphocytes.