First report of Alternaria crassa causing yellow spots on Datura stramonium in China.

Datura stramonium L.(jimson weed) is an invasive weed in agricultural fields and a medicinal plant. In April 2022, a leaf disease on D. stramonium was observed in Zhanjiang (21.17 N, 110.18 E), Guangdong province, China. Early symptoms were small yellow spots on leaves. Later, the spots gradually expanded and turned becoming necrotic with a clear yellow halo and a white center. The disease incidence in the field was 85% (n = 50, about 1 ha). Twenty diseased leaves were collected from the field. The margin of the diseased tissues was cut into 2 mm × 2 mm pieces, surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, respectively, and rinsed twice with sterile water before isolation. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 ℃. After 2-day incubation, grayish fungal colonies appeared on the PDA, then pure cultures were produced by transferring hyphal tips to new PDA plates. Single-spore isolation method was used to recover pure cultures for three isolates (DSAC-1, DSAC-2, and DSAC-3). The isolates were morphologically identical . They colonies were gray to brownish black. Conidiophores were branched, brown. Conidia were brown, long ellipsoid, had 4-12 transverse and 0-3 longitudinal septa; measured within 67.5-127.8 (average = 105.6) × 12.5-27.8 (average = 20.4) µm (n = 30). Apical beak was longer than conidia body. measured within 40.5-423.5 (average = 365.2) × 2.5-5.8 (average = 3.2) µm (n = 30). Based on morphological characteristics, the three isolates were identified as Alternaria crassa (Sacc.) Rands (Simmons 2007). Molecular identification was performed using the colony polymerase chain reaction method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) to amplify internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase second largest subunit (RPB2) and translation elongation factor (TEF1) with primers of ITS1/ITS4, GDF1/GDR1, RPB2-5F2/fRPB2-7cR, and EF-1α-F/EF-1α-R, respectively (Walther et al. 2013; Woudenberg et al. 2015; Nishikawa and Nakashima. 2020). Amplicons of the isolates were sequenced and submitted to GenBank (ITS, ON430524-ON430526; GAPDH, ON500656-ON500658; RPB2, ON500659-ON500661; TEF1, ON500662-ON500664). The sequences were 100% identical with those of Alternaria crassa strain CBS 116647 upon BLAST analysis. The sequences were also concatenated for phylogenetic analysis by maximum likelihood. The isolates clustered with A. crassa (CBS 116647, CBS 116648, CBS CBS-110.38, and CBS_103.18 ). Thus, the fungus associated with leaf yellow spot on D. stramonium was identified as A. crassa. Pathogenicity tests were conducted in a greenhouse at 24 â„ƒ-30 â„ƒ with 80% relative humidity using 3 isolates. Individual plants were grown in pots (n = 5, 1 month old). The unwounded leaflets were inoculated using three isolates (DSAC-1, DSAC-2, and DSAC-3). The fungal mycelium on 5 mm-diameter PDA plugs were placed faced down to the leaves. Sterile PDA was used for mock inoculated comtrols.. The test was performed three times. Disease symptoms were observed on the leaves after 7 days, whereas the controls remained healthy. The pathogen was re-isolated from infected leaves and was morphologically identical to the original isolates, fulfilling Koch's postulates. A. crassa was reported causing leaf spot on D. stramonium in Algeria (Nabahat et al. 2020). To our knowledge, this report is the first report of A. crassa causing leaf yellow spot on D. stramonium in China. This pathogen possesses potential biocontrol properties on the invasive weed, while this study also provides an important reference for the control of the disease of the medicinal plant.

First report of Rhizoctonia solani AG-1 IA causing rice sheath blight of Oryza rufipogon in China.

Red rice (Oryza rufipogon Griff.) is a valuable source of important agronomic traits as well as genes for biotic and abiotic stress tolerance. In June 2020, rice sheath blight on O. rufipogon cv. Bin09 was observed in Zhanjiang (20.93N, 109.79E), China. Initial symptoms on sheaths were water-soaked and light green lesions. Then, the lesions gradually expanded into oval or cloud shaped lesions with a gray white center. The lesions coalesced, causing the entire sheath to become blighted. Disease incidence reached approximately 30% in the fields (10 ha) surveyed. Twenty sheaths with symptoms were collected and cut into pieces of 2 × 2 cm in size. They were surface-disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite (NaOCl) for 60 s, rinsed three times with sterile water, blotted dry on sterile paper, plated on potato dextrose agar (PDA), and incubated at 28°C in the dark for 4 days. Thirty-six pure cultures were obtained by transferring hyphal tips to new PDA plates, and three isolates (ORRS-1, ORRS-2, and ORRS-3) with similar morphological characteristics on PDA were selected as the representative isolates for study. Colony of isolate ORRS-1 was white initially, then turned brown with brown sclerotia. Septate hyphae were hyaline, smooth, and branched at right angles with a septum near the point of branching. Based on these morphological characteristics, the fungus was identified as Rhizoctonia solani Kuhn (Sneh et al. 1991). The isolates were deposited in the fungus collection of the Aquatic Organisms Museum of Guangdong Ocean University. For molecular identification, genomic DNA from each of the three isolates was extracted, and the internal transcribed spacer (ITS) region was amplified, and sequenced with the primer pair ITS5/ITS4 (White et al. 1990). The sequences were deposited in GenBank (accession nos. OP497977 to OP497979). The three isolates were 100% identical (716/716 bp; 716/716 bp; and 716/716 bp) with those of R. solani AG-1 IA (accession nos. KX674518, MK481078, and MK480532) through BLAST analysis. The phylogenetic tree grouped the three isolates within the R. solani AG-1 IA clade with high bootstrap support (99%) by the maximum likelihood method. A pathogenicity test was performed with these three isolates in a greenhouse at 24 to 30°C. Approximately 50 seedling of red rice cv. Bin09 were grown in each cup ( 250 ml in size with sterile soil 50 cm3). At the 3-leaf stage, plants in five cups were inoculated with each isolate by spraying a mycelial suspension (106 mycelial fragments/ml) until runoff. The mycelial suspension was prepared by adding sterile distilled water to the cultures and gently scraping the surface with a sterilized scalpel blade. Five plants sprayed with sterile water served as the controls. The test was conducted three times. Sheath blight was observed on the inoculated leaves after 15 days while no disease was observed in the control plants. Morphological characteristics and the ITS sequences of fungal isolates re-isolated from the diseased sheaths were identical to those of R. solani AG-1 IA. R. solani AG-1 IA is one of the most important plant pathogens worldwide, causing foliar diseases on maize, rice (O. sativa L.), and soybean (Joana et al. 2009). To our knowledge, this is the first report of R. solani AG-1 IA causing rice sheath blight of O. rufipogon in China (Farr and Rossman, 2022). With the spread of the pathogen on weedy populations of red rice, resistant races or pathotypes may evolve that could spread to cultivated rice.

First Report of Pseudocercospora paraguayensis Causing Leaf Spots on Bixa orellana from Chinese Mainland.

Bixa orellana L. is a traditional Chinese medicine. In December 2019, a leaf spot disease was observed on B. orellana from a field in Zhanjiang (21°18'12''N, 110°17'22''E), China. Disease incidence was around 85% (n = 100 investigated plants from about 30 hectares). Initial leaf spots were circular, and the center of the lesions was grayish-white with a purple black border. The coalescence of individual spots eventually led to leaf wilt. Ten symptomatic leaves from 10 plants were sampled. The margins of the samples were cut into 2 mm × 2 mm pieces, and the surfaces were disinfected with 75% ethanol for 30 sec, and 2% sodium hypochlorite for 60 sec. The the samples were then rinsed three times in sterile water, plated on potato dextrose agar (PDA), and incubated at 28 °C. Pure cultures were obtained by transferring the hyphal tips to new PDA plates. Three representative isolates (BOPP-1, BOPP-2, and BOPP-3) were used for further study. The colonies of isolates on PDA were dark olive green with off-white aerial mycelia after 7 days at 28 °C. Conidia were solitary, smooth to verrucous, olive to light brown, slightly curved, narrowly obclavate, apex obtuse, base obconic-truncate, had 2-4 septa, and 30.4-55.5×2.0-3.5 µm in size.. These morphological characteristics showed did not differ from the description of Pseudocercospora paraguayensis (Crous et al. 1997). For molecular identification, the internal transcribed spacer (ITS) region, the translation elongation factor 1-α (TEF1) gene, and actin (ACT) gene were amplified using primer pairs ITS1/ITS4 (White et al., 1990), EF1/EF2 ( O'Donnell et al. 1998), and ACT-512F/ACT-783R (Carbone and Kohn, 1999) and sequenced from DNA extracted from the three isolates, respectively. Sequences were deposited in GenBank under accession no. MZ363823-MZ363825 (ITS), MZ614954-MZ614956 (TEF1), and MZ614951-MZ614953 (ACT). A phylogenetic tree was generated on the basis of the concatenated data from the sequences of ITS, TEF1, and ACT that the three isolates were nested within the clade containing the type specimen of P. paraguayensis (CBS 111286) but not within P. bixae (the type specimen CPC 25244). Pathogenicity was tested through in vivo experiments. Inoculation and control seedlings (n = 5, 1-month-old) were sprayed with a spore suspension (1 × 105 per ml) of P. paraguayensis and sterile distilled water (control), respectively, until run-off (Fang. 1998). The plants were grown in pots in a greenhouse at 28°C, with at approximately 80% RH. The test was performed three times. Symptoms similar to those in the field were observed on the inoculated plants after two weeks. The control plants remained healthy. The fungus was re-isolated from the infected leaves and confirmed as the same isolates by morphological and comparison of ITS sequences with 100% identical to those of isolates. No original fungi were isolated from the control plants. A previous study reported that P. paraguayensis caused leaf spots on pistachio and eucalypts, and the fungus causing the leaf spots of B. orellana was redescribed as P. bixae (Crous et al. 2019). However, multilocus phylogenetic analyses differentiated P. paraguayensis from P. bixae. In the present study, P. paraguayensis was distinguished from P. bixae due to the absence of catenulate conidia and the presence of finely verruculose conidia (Crous et al. 2013). P. eucalypti as a synonyms was reported in Taiwan (www.MycoBank.org). The current study is the first to report P. paraguayensis causing leaf spots on B. orellana from Chinese Mainland. This finding will help to provide a scientific basis for the disease detection.

ßII-Spectrin (SPTBN1) suppresses progression of hepatocellular carcinoma and Wnt signaling by regulation of Wnt inhibitor kallistatin.

UNLABELLED: ßII-Spectrin (SPTBN1) is an adapter protein for Smad3/Smad4 complex formation during transforming growth factor beta (TGF-ß) signal transduction. Forty percent of SPTBN1(+/-) mice spontaneously develop hepatocellular carcinoma (HCC), and most cases of human HCC have significant reductions in SPTBN1 expression. In this study, we investigated the possible mechanisms by which loss of SPTBN1 may contribute to tumorigenesis. Livers of SPTBN1(+/-) mice, compared to wild-type mouse livers, display a significant increase in epithelial cell adhesion molecule-positive (EpCAM(+)) cells and overall EpCAM expression. Inhibition of SPTBN1 in human HCC cell lines increased the expression of stem cell markers EpCAM, Claudin7, and Oct4, as well as decreased E-cadherin expression and increased expression of vimentin and c-Myc, suggesting reversion of these cells to a less differentiated state. HCC cells with decreased SPTBN1 also demonstrate increased sphere formation, xenograft tumor development, and invasion. Here we investigate possible mechanisms by which SPTBN1 may influence the stem cell traits and aggressive behavior of HCC cell lines. We found that HCC cells with decreased SPTBN1 express much less of the Wnt inhibitor kallistatin and exhibit decreased ß-catenin phosphorylation and increased ß-catenin nuclear localization, indicating Wnt signaling activation. Restoration of kallistatin expression in these cells reversed the observed Wnt activation. CONCLUSION: SPTBN1 expression in human HCC tissues is positively correlated with E-cadherin and kallistatin levels, and decreased SPTBN1 and kallistatin gene expression is associated with decreased relapse-free survival. Our data suggest that loss of SPTBN1 activates Wnt signaling, which promotes acquisition of stem cell-like features, and ultimately contributes to malignant tumor progression.

Transcriptional regulation of STAT3 by SPTBN1 and SMAD3 in HCC through cAMP-response element-binding proteins ATF3 and CREB2.

The cytoskeletal protein Spectrin, beta, non-erythrocytic 1 (SPTBN1), an adapter protein to SMAD3 in TGF-ß signaling, may prevent hepatocellular carcinoma (HCC) development by downregulating the expression of signal transducer and activator of transcription 3 (STAT3). To elucidate the as yet undefined mechanisms that regulate this process, we demonstrate that higher levels of STAT3 transcription are found in livers of heterozygous SPTBN1(+/-) mice as compared to that of wild type mice. We also found increased levels of STAT3 mRNA, STAT3 protein, and p-STAT3 in human HCC cell-lines after knockdown of SPTBN1 or SMAD3, which promoted cell colony formation. Inhibition of STAT3 overrode the increase in cell colony formation due to knockdown of SPTBN1 or SMAD3. We also found that inhibition of SPTBN1 or SMAD3 upregulated STAT3 promoter activity in HCC cell-lines, which is dependent upon the cAMP-response element (CRE) and STAT-binding element (SBE) sites of the STAT3 promoter. Mechanistically, suppression of SPTBN1 and SMAD3 augmented the transcription of STAT3 by upregulating the CRE-binding proteins ATF3 and CREB2 and augmented the binding of those proteins to the regions within or upstream of the CRE site of the STAT3 promoter. Finally, in human HCC tissues, SPTBN1 expression correlated negatively with expression levels of STAT3, ATF3, and CREB2; SMAD3 expression correlated negatively with STAT3 expression; and the level of phosphorylated SMAD3 (p-SMAD3) correlated negatively with ATF3 and CREB2 protein levels. SPTBN1 and SMAD3 collaborate with CRE-binding transcription factors to inhibit STAT3, thereby preventing HCC development.

Renewable regeneration optic fiber glucose sensor based on succinylaminobenzenoboronic acid modified excessively tilted fiber grating.

BACKGROUND: Optical fiber sensors have been used to detect glucose owing to advantages such as low cost, small size, and ease of operation etc. phenylboronic acid is one of the commonly used receptors for glucose detection, however phenylboronic acid based regenerative optical fiber sensors are commonly cumulative regeneration, renewable regeneration sensor has been missing from the literature. RESULTS: In this work, instead of using phenylboronic acid, we synthesized succinylaminobenzenoboronic acid molecule (BPOA) by introducing a short chain containing carboxyl group at the other end of phenylboronic acid then covalently bonded BPOA on the surface of excessively tilted fiber grating (Ex-TFG). This provides a very stable platform for renewable regeneration and the regenerative buffer was also optimized. The proposed renewable regeneration method exhibited higher linearity and sensitivity (R2 = 0.9992, 8 pm/mM) in relative to the conventional cumulative regeneration method (R2 = 0.9718, 4.9 pm/mM). The binding affinity between BPOA and glucose was found to be almost constant over 140 bind/release cycles with a variation of less than 0.3 % relative standard deviation. SIGNIFICANCE: The regenerative and label-free sensing capacity of the proposed device provides a theoretical foundation for label-free saccharide detection and the development of wearable glucose monitoring devices based on fiber optic sensors.

Transforming growth factor-ß adaptor, ß2-spectrin, modulates cyclin dependent kinase 4 to reduce development of hepatocellular cancer.

UNLABELLED: Transforming growth factor beta (TGF-ß) is an important regulator of cell growth, and loss of TGF-ß signaling is a hallmark of carcinogenesis. The Smad3/4 adaptor protein ß2-spectrin (ß2SP) is emerging as a potent regulator of tumorigenesis through its ability to modulate the tumor suppressor function of TGF-ß. However, to date the role of the TGF-ß signaling pathway at specific stages of the development of hepatocellular carcinoma (HCC), particularly in relation to the activation of other oncogenic pathways, remains poorly delineated. Here we identify a mechanism by which ß2SP, a crucial Smad3 adaptor, modulates cyclin dependent kinase 4 (CDK4), cell cycle progression, and suppression of HCC. Increased expression of ß2SP inhibits phosphorylation of the retinoblastoma gene product (Rb) and markedly reduces CDK4 expression to a far greater extent than other CDKs and cyclins. Furthermore, suppression of CDK4 by ß2SP efficiently restores Rb hypophosphorylation and cell cycle arrest in G(1) . We further demonstrate that ß2SP interacts with CDK4 and Smad3 in a competitive and TGF-ß-dependent manner. In addition, haploinsufficiency of cdk4 in ß2sp(+/-) mice results in a dramatic decline in HCC formation compared to that observed in ß2sp(+/-) mice. CONCLUSION: ß2SP deficiency leads to CDK4 activation and contributes to dysregulation of the cell cycle, cellular proliferation, oncogene overexpression, and the formation of HCCs. Our data highlight CDK4 as an attractive target for the pharmacologic inhibition of HCC and demonstrate the importance of ß2sp(+/-) mice as a model of preclinical efficacy in the treatment of HCC.

Fusion of the CH1 domain of IgG1 to epidermal growth factor (EGF) prolongs its retention in the blood but does not increase tumor uptake.

An expression vector (pJW4) for a human epidermal growth factor (hEGF)-CH1 fusion protein was constructed by fusing the gene for hEGF with the gene for CH1 of murine IgG1 with/without a peptide linker sequence [(GGGGS)3] and inserting the recombinant gene into vector pGEX2T. Expression vector pGEX2T was transfected into E. coli (BL-21) and hEGF-CH1 expressed by induction of the lac Iq promotor with 50 microM isopropyl beta-D-thiogalactopyranoside (IPTG). hEGF- CH1 fused to glutathione S-transferase (GST) was isolated and purified by affinity chromatography. GST was cleaved using thrombin. SDS-PAGE demonstrated a protein with the expected M(r) (18 kDa) positive for hEGF by Western blot. hEGF-linker-CH1 exhibited preserved binding to A431 (2-3 x 10(6) EGFR/cell) and MDA-MB-468 breast cancer cells (1-2 x 10(6) EGFR/cell). hEGF-CH1 without the linker exhibited poor receptor binding. hEGF-linker-CH1 also exhibited strong binding to soluble EGFR equivalent to that of hEGF. The tumor and normal tissue distribution of hEGF-linker-CH1 labeled with 123I was compared with 123 I-hEGF at 24 h after i.v. injection to mice implanted with s.c. MDA-MB-468 xenografts. Fusion of hEGF with CH1 increased its retention in the blood 14-fold but did not significantly increase tumor uptake. Tumor/blood ratios were higher for hEGF than for hEGF-linker-CH1. We conclude that hEGF is more attractive than hEGF-linker-CH1 for imaging EGFR-positive tumors.

Construction and evaluation of the tumor imaging properties of 123I-labeled recombinant and enzymatically generated Fab fragments of the TAG-72 monoclonal antibody CC49.

Tumor-associated glycoprotein-72 (TAG-72) is overexpressed in a high percentage of epithelial cancers and has proven useful as a target for imaging and targeted radiotherapy. Our goal was to express a recombinant Fab (rFab) of the TAG-72 monoclonal antibody CC49 in Pichia pastoris and directly compare its tumor and normal tissue uptake and imaging properties with enzymatically generated Fab (eFab). In this study, the genes coding for CC49 Fab were cloned from hybridoma cells and expressed in P. pastoris. Fab was purified to homogeneity and its immunoreactivity toward bovine submaxillary mucin (TAG-72) confirmed by ELISA. The tumor and normal tissue localization of (123)I-CC49 rFab and eFab were compared in athymic mice bearing s.c. LS174T colon cancer or TAG-72-negative A375 melanoma xenografts. Results showed that pure and immunoreactive rFab of CC49 was produced and labeled with (123)I. At 24 h post i.v. injection (p.i.), tumor uptake for (123)I-rFab in LS174T xenografts was 6.0% ID/g which was 18-fold higher than in A375 tumors. Tumor-to-normal tissue ratios increased between 2 and 24 h and exceeded 5:1 at 24 h p.i. of (123)I-rFab. (123)I-rFab exhibited significantly lower liver uptake at 12 h p.i. and lower kidney uptake at 2 h p.i. than (123)I-eFab. LS174T tumors were imaged as early as 2 h after administration of (123)I-rFab. We conclude that CC49 rFab can be produced in a P. pastoris host system and accumulated at comparable levels as eFab in LS174T colon cancer xenografts in mice. The lower liver uptake of (123)I-rFab as compared with eFab suggests that it may be more useful for imaging liver lesions. No major effect, except for kidneys and liver, was observed on tumor and normal tissue uptake due to introduction of hexahistidine and FLAG affinity tags or peptide linkers in the scaffold of rFab.

Nuclear magnetic resonance-based dissection of a glycosyltransferase specificity for the mucin MUC1 tandem repeat.

The human glycoprotein MUC1 mucin plays a critical role in cancer progression. Breast, ovarian, and colon cancer cells often display unique cell-surface antigens corresponding to aberrantly glycosylated forms of the MUC1 tandem repeat. In this report, (15)N- and (13)C-labeled forms of a recombinant MUC1 construct containing five tandem repeats were used as substrates to define the order and kinetics of addition of N-acetylgalactosamine (GalNAc) moieties by a recombinant active form of the human enzyme UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase I (ppGalNAc-T1; residues 40-559). Heteronuclear NMR experiments were performed to assign resonances associated with the two serines (Ser5 and Ser15) and three threonines (Thr6, Thr14, and Thr19) present in the 20-residue long MUC1 repeat. The kinetics and order of addition of GalNAc moieties (Tn antigen) on the MUC1 construct by human ppGalNAc-T1 were subsequently dissected by NMR spectroscopy. Threonine 14 was shown to be rapidly glycosylated by ppGalNAc-T1 with an initial rate of 25 microM/min, followed by Thr6 (8.6 microM/min). The enzyme also modified Ser5 at a slower rate (1.7 microM/min), an event that started only after the glycosylation of Thr14 and Thr6 side chains was mostly completed. Ser15 and Thr19 remained unglycosylated by ppGalNAc-T1. Corresponding O-glycosylation sites within all five tandem repeats were simultaneously modified by ppGalNAc-T1, suggesting that each repeat behaves as an independent substrate unit. This study demonstrated that the hydroxyl oxygens of Thr14 and to a lesser extent Thr 6 represent the two dominant substrates modified by ppGalNAc-T1 within the context of a complex MUC1 peptide substrate. More importantly, the availability of defined isotopically labelled MUC1 glycopeptide substrates and the relative simplicity of their NMR spectra will facilitate the analysis of other transferases within the O-glycosylation pathways and the rational design of tumor-associated MUC1 antigens.