Leptosphaerulina americana causing holing disease on tobacco in China.

Tobacco (Nicotiana tabacum) is one of the most important industrial crops in the world. Its leaves are the main raw material for cigarettes, but they are often threatened by fungal pathogens in the production process (Wang et al. 2022). From May to June 2022, a disease of tobacco (cv K326) (15% of plants) in a 0.3-ha field in Jingxi of Guangxi Province showed symptoms of local necrosis and perforation of middle and basal leaves (Fig S1). Pieces of leaf tissue (3 × 3 mm) were excised from the edge of the necrotic lesion of each plant, treated with 75% ethanol for 10 s, soaked in 2% NaClO solution for 1-2 min, rinsed with sterile water for three times, and then plated on potato dextrose agar（PDA）medium and incubated at 28°C. Isolate TJYA13 was used for subsequent studies. After 8 days, the colony margin was yellowish brown and irregular, the center was black and plicated. The isolate TJYA13 was incubated on oatmeal agar medium at 28°C for 4 days, and many pseudothecia were observed embedded on the surface of the medium. Pseudothecium was globose or subglobose, dark brown, and size was 184.7-304.7 µm × 187.5-340.5 µm (n=20). Ascospores were usually wrapped by the saccate ascus in pseudothecium, cylindrical or ellipsoidal, with 5-6 transverse septa, and size was 12.2-18.5 µm × 35.6-51.8 µm (n=80). The morphological characteristics of ascospores were consistent with a Leptosphaerulina species (Hou et al. 2020). For accurate identification, the genomic DNA of isolate TJYA13 was extracted with Ezup Column Fungi Genomic DNA Purification Kit (Sangon, Shanghai, China). The ITS region, 28s ribosomal RNA (LSU), ß-tubulin (TUB), and RNA polymerase II second largest subunit (RPB2) were amplified with primers ITS1/ITS4 (Gardes and Bruns 1993; White et al. 1990), LROR/LR7 (Rehner and Samuels 1994), Btub2Fd/Btub4Rd (Woudenberg et al. 2009), and RPB2-5F2/fRPB2-7cR (Liu et al. 1999), respectively and sequenced at Sangon Biotech (Sichuan, China). The sequences were deposited in GenBank (accession nos. OP926927, OP926933, OP939419, OP939422). The phylogenetic analysis grouped the isolate TJYA13 within the L. americana clade (Fig S2) (Hou et al. 2020). Pathogenicity of the isolate TJYA13 was verified on four healthy tobacco plants (cv K326). The mycelial plugs were inoculated on leaves sterilized with 75% ethanol, and control plants were inoculated with sterile PDA plugs. Plants were incubated at 28 ℃ and 78% humidity. After 10 days, the leaves inoculated with mycelial plugs had symptoms similar to those in the field, but there were no symptoms on the control leaves. L. americana were reisolated from the leaves inoculated with the mycelial plugs. To the best of our knowledge, this is the first report of L. americana causing holing disease on tobacco in China. This disease may reduce yields and lower quality of flue-cured tobacco leaf. Therefore, the emergence of tobacco holing disease should be noted to prevent potential damage to tobacco production in Guangxi. Reference 1. Hou L. W., et al. 2020. Stud. Mycol. 96: 309-396 2. Liu, Y. J., et al. 1999. Mol. Biol. Evol. 16:1799. 3. Rehner, S. A., and Samuels, G. J. 1994. Mycol. Res. 98:625. 4. Wang H. et al. 2022. Microorganisms. 10: 1890. 5. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. 6. Woudenberg, J. H. C., et al. 2009. Persoonia 22:56. The author(s) declare no conflict of interest. Funding: Funding was provided by Guangxi Zhuang Autonomous Region Tobacco Monopoly Bureau (grant no. 202,145,000,024,006). Tobacco (Nicotiana tabacum) is one of the most important industrial crops in the world. Its leaves are the main raw material for cigarettes, but they are often threatened by fungal pathogens in the production process (Wang et al. 2022). From May to June 2022, a disease of tobacco (cv K326) (15% of plants) in a 0.3-ha field in Jingxi of Guangxi Province showed symptoms of local necrosis and perforation of middle and basal leaves (Fig S1). Pieces of leaf tissue (3 × 3 mm) were excised from the edge of the necrotic lesion of each plant, treated with 75% ethanol for 10 s, soaked in 2% NaClO solution for 1-2 min, rinsed with sterile water for three times, and then plated on potato dextrose agar（PDA）medium and incubated at 28°C. Isolate TJYA13 was used for subsequent studies. After 8 days, the colony margin was yellowish brown and irregular, the center was black and plicated. The isolate TJYA13 was incubated on oatmeal agar medium at 28°C for 4 days, and many pseudothecia were observed embedded on the surface of the medium. Pseudothecium was globose or subglobose, dark brown, and size was 184.7-304.7 µm × 187.5-340.5 µm (n=20). Ascospores were usually wrapped by the saccate ascus in pseudothecium, cylindrical or ellipsoidal, with 5-6 transverse septa, and size was 12.2-18.5 µm × 35.6-51.8 µm (n=80). The morphological characteristics of ascospores were consistent with a Leptosphaerulina species (Hou et al. 2020). For accurate identification, the genomic DNA of isolate TJYA13 was extracted with Ezup Column Fungi Genomic DNA Purification Kit (Sangon, Shanghai, China). The ITS region, 28s ribosomal RNA (LSU), ß-tubulin (TUB), and RNA polymerase II second largest subunit (RPB2) were amplified with primers ITS1/ITS4 (Gardes and Bruns 1993; White et al. 1990), LROR/LR7 (Rehner and Samuels 1994), Btub2Fd/Btub4Rd (Woudenberg et al. 2009), and RPB2-5F2/fRPB2-7cR (Liu et al. 1999), respectively and sequenced at Sangon Biotech (Sichuan, China). The sequences were deposited in GenBank (accession nos. OP926927, OP926933, OP939419, OP939422). The phylogenetic analysis grouped the isolate TJYA13 within the L. americana clade (Fig S2) (Hou et al. 2020). Pathogenicity of the isolate TJYA13 was verified on four healthy tobacco plants (cv K326). The mycelial plugs were inoculated on leaves sterilized with 75% ethanol, and control plants were inoculated with sterile PDA plugs. Plants were incubated at 28 ℃ and 78% humidity. After 10 days, the leaves inoculated with mycelial plugs had symptoms similar to those in the field, but there were no symptoms on the control leaves. L. americana were reisolated from the leaves inoculated with the mycelial plugs. To the best of our knowledge, this is the first report of L. americana causing holing disease on tobacco in China. This disease may reduce yields and lower quality of flue-cured tobacco leaf. Therefore, the emergence of tobacco holing disease should be noted to prevent potential damage to tobacco production in Guangxi.

First Report of Colletotrichum truncatum Causing Anthracnose in Tobacco in China.

In July 2022, large spots were observed on the leaves of tobacco in Guangxi province, China, whose shape was round and elliptical or irregular. The margins of spots were brown or dark brown with a pale yellow centre and several small black fruiting bodies. The pathogen was isolated by tissue isolation. Diseased leaves collected were cut into small pieces, sterilized with 75% ethanol for 30s and 2% sodium hypochlorite (NaCIO) for 60s, and rinsed with sterile deionized water for three times. Each air-dried tissue segment was cultured on potato dextrose agar (PDA) and incubated at 28℃ for 5 to 7 days in the dark (Wang et al. 2022). A total of six isolates were isolated, with differences in colony shape, edge type and colony colour, and aerial mycelium morphology, with the colony shape round or subrounded, and the edge rounded crenate, dentate or sinuate. The color of the colony was initially light yellow, then gradually changed to yellow and dark yellow. After 3-4 days, white aerial mycelia gradually grew up, which was peony-like or covered the whole colony, thus the color of the colony appeared white, and then gradually changed to orange, gray or nearly black, and all six isolates rarely produced conidia, which was consistent with the description of previous reports(Mayonjo and Kapooria 2003, Feng et al. 2021, Xiao et al. 2018). Conidia were hyaline, aseptate, and falcate, with the size of 7.8 to 12.9 × 2.2 to 3.5 µm. For molecular identification, the colony PCR method was used to amplify the internal transcribed spacer(ITS), actin(ACT), chitin synthase(CHS), and beta-tubulin(TUB2) loci of the six isolates using primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and T1/Bt2b, respectively(Cheng et al. 2014). Partial sequences were amplified, sequenced, and uploaded to GenBank (GenBank accession Nos. OP484886，OP518265，OP518266，OP756065，OP756066, and OP756067 for ITS, OP620430 to OP620435 for ACT, OP620436 to OP620441 for CHS, and OP603924 to OP603929 for TUB2). These sequences had 99 to 100% similarity with C. truncatum isolates C-118(ITS), TM19(ACT), OCC69(CHS), and CBS 120709(TUB2) in GenBank. Homology matching was performed using BLAST and a phylogenetic tree was constructed using the Neighbor-Joining (NJ) method using MEGA (7.0) software based on ITS, ACT, CHS, and TUB2 sequences, which showed that all six isolates clustered in the same score as the C. truncatum. A pathogenicity test was performed with healthy tobacco infected with mycelial plugs (about 5 mm in diameter) of six isolates of C. truncatum from a 5-day-old culture, while negative controls on the other leaves were inoculated with sterile PDA plugs. All plants were placed in a greenhouse at 25℃ to 30℃ with 90% relative humidity. The experiment was conducted three times. Five days later, all inoculated leaves had diseased spots, whereas no symptoms appeared on negative controls. The same pathogen, C. truncatum, was identified from the inoculated leaves on the basis of morphological and molecular charchseristics as described above, fulfilling Koch's postulates. In this study, it is the first time to report that the anthracnose on tobacco was caused by C. truncatum. Thus, this work provides a foundation for controlling tobacco anthracnose in the future.

Computational insights into ligand-induced G protein and ß-arrestin signaling of the dopamine D1 receptor.

The dopamine D1 receptor (D1R), is a class A G protein coupled-receptor (GPCR) which has been a promising drug target for psychiatric and neurological disorders such as Parkinson's disease (PD). Previous studies have suggested that therapeutic effects can be realized by targeting the ß-arrestin signaling pathway of dopamine receptors, while overactivation of the G protein-dependent pathways leads to side effects, such as dyskinesias. Therefore, it is highly desirable to develop a D1R ligand that selectively regulates the ß-arrestin pathway. Currently, most D1R agonists are signaling-balanced and stimulate both G protein and ß-arrestin pathways, with a few reports of G protein biased ligands. However, identification and characterization of ß-arrestin biased D1R agonists has been a challenge thus far. In this study, we implemented Gaussian accelerated molecular dynamics (GaMD) simulations to provide valuable computational insights into the possible underlying molecular mechanism of the different signaling properties of two catechol and two non-catechol D1R agonists that are either G protein biased or signaling-balanced. Dynamic network analysis further identified critical residues in the allosteric signaling network of D1R for each ligand at different conformational or binding states. Some of these residues are crucial for G protein or arrestin signals of GPCRs based on previous studies. Finally, we provided a molecular design strategy which can be utilized by medicinal chemists to develop potential ß-arrestin biased D1R ligands. The proposed hypotheses are experimentally testable and can guide the development of safer and more effective medications for a variety of CNS disorders.

Description and complete mitochondrial genome of Atkinsoniella zizhongi sp. nov. (Hemiptera: Cicadellidae: Cicadellinae) from China and its phylogenetic implications.

A new species, Atkinsoniella zizhongi sp. nov. of the subfamily Cicadellinae, was described and illustrated from China. The new species is similar to A. nigrominiatula (Jacobi, 1944), A. limba Kuoh, 1991, A. dormana Li, 1992, A. peaka Yang, Meng et Li, 2017, and A. divaricata Yang, Meng et Li, 2017. But the characteristics of aedeagus and pygofer process can be used to distinguish them easily. The complete mitochondrial genome of the paratype was sequenced and assembled. The mitogenome of A. zizhongi sp. nov. was 16,483 bp in length, with an A+T content of 75.9%, containing 37 typical genes and a control region (CR). The gene order was consistent with the inferred insect ancestral mitochondrial genome. All of the PCGs were determined to have the typical stop codon TAA or TAG, while COX2 and ND5 ended with incomplete termination codons T and TA, respectively. In addition, phylogenetic trees were reconstructed based on PCGs and rRNAs using both the maximum likelihood (ML) and Bayesian inference (BI) methods. The results showed that the intergeneric and interspecific relationships within the subfamily Cicadellinae were completely consistent in all of the phylogenetic trees, except that the different interspecific relationships within the genus Bothrogonia were detected in the ML analysis based on the amino acid sequences. This study enriches the species diversity of Cicadellinae and further promotes research on its phylogeny.

Structure-Functional Selectivity Relationship Studies on A-86929 Analogs and Small Aryl Fragments toward the Discovery of Biased Dopamine D1 Receptor Agonists.

Dopamine regulates normal functions such as movement, reinforcement learning, and cognition, and its dysfunction has been implicated in multiple psychiatric and neurological disorders. Dopamine acts through D1- (D1R and D5R) and D2-class (D2R, D3R, and D4R) receptors and activates both G protein- and ß-arrestin-dependent signaling pathways. Current dopamine receptor-based therapies are used to ameliorate motor deficits in Parkinson's disease or as antipsychotic medications for schizophrenia. These drugs show efficacy for ameliorating only some symptoms caused by dopamine dysfunction and are plagued by debilitating side effects. Studies in primates and rodents have shown that shifting the balance of dopamine receptor signaling toward the arrestin pathway can be beneficial for inducing normal movement, while reducing motor side effects such as dyskinesias, and can be efficacious at enhancing cognitive function compared to balanced agonists. Several structure-activity relationship (SAR) studies have embarked on discovering ß-arrestin-biased dopamine agonists, focused on D2 partial agonists, noncatechol D1 agonists, and mixed D1/D2R dopamine receptor agonists. Here, we describe an SAR study to identify novel D1R ß-arrestin-biased ligands using A-86929, a high-affinity D1R catechol agonist, as a core scaffold to identify chemical motifs responsible for ß-arrestin-biased activity at both D1 and D2Rs. Most of the A-86929 analogs screened were G protein-biased, but none of them were exclusively arrestin-biased. Additionally, various small-fragment molecular probes displayed weak bias toward the ß-arrestin pathway. Continued in-depth SFSR (structure-functional selectivity relationship) studies informed by structure determination, molecular modeling, and mutagenesis studies will facilitate the discovery of potent and efficacious arrestin-biased dopamine receptor ligands.

Comparative Analysis of Mitochondrial Genomes among Twelve Sibling Species of the Genus Atkinsoniella Distant, 1908 (Hemiptera: Cicadellidae: Cicadellinae) and Phylogenetic Analysis.

The herbivorous leafhopper genus Atkinsoniella Distant, 1908 (Hemiptera: Cicadellidae: Cicadellinae), a large genus of subfamily Cicadellinae, consists of 98 valid species worldwide and 88 species recorded in China. Some species of the genus are very similar in morphological characteristics, so they are difficult to identify accurately. In this study, 12 mitochondrial genomes of Atkinsoniella species with similar morphological characteristics were first obtained through high-throughput sequencing, which featured a typical circular molecule of 15,034-15,988 bp in length. The arrangement and orientation of 37 genes were identical to those of typical Cicadellidae mitogenomes. The phylogenetic relationship within the subfamily Cicadellinae was reconstructed using maximum-likelihood (ML) and Bayesian inference (BI) methods based on three concatenated datasets. The topological structures of the six obtained phylogenetic trees were highly consistent. The results suggested that Atkinsoniella was recovered as a monophyletic group and emerged as a sister group with the monophyletic clade of Bothrogonia, Paracrocampsa (part), and Draeculacephala (part). The branches of the 12 newly sequenced species were clearly separated, with most nodes receiving strong support in all analyses. In addition, the key to the 12 Atkinsoniella species was provided to identify species according to morphological characteristics. This study further promotes research on the classification, genetics, evolution, and phylogeny of the genus Atkinsoniella and subfamily Cicadellinae.

Comprehensive Network Analysis Identified SIRT7, NTRK2, and CHI3L1 as New Potential Markers for Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IDD) is considered the basis of serious clinical symptoms, especially for low back pain (LBP). Therefore, it is essential to explore the regulatory role and diagnostic performance of dysregulated genes and potential drugs in IDD. Through WGCNA co-expression analysis, 36 co-expression modules were obtained. Among them, MidnightBlue and Red modules were the most related to IDD. Functional enrichment analysis showed that the Red module was mainly related to neutrophil activation and regulation of cytokine-mediated signaling pathway and apoptosis, whereas the MidnightBlue module was mainly related to extracellular matrix organization, bone development, extracellular matrix, extracellular matrix component, and other extracellular matrices. Furthermore, 356 genes highly related to the module were screened to construct a protein interaction network. Network degree distribution analysis showed that the known IDD-related genes had a higher degree of distribution. Enrichment analysis demonstrated that these genes were enriched in MAPK_SIGNALING_PATHWAY (FDR = 0.012), CHEMOKINE_SIGNALING_PATHWAY, and some other pathways. By constructing a disease-gene interaction network, three disease-specific genes were finally identified. Through combining with the drug-target gene interaction network, two potential therapeutic drugs, entrectinib and larotrectinib, were determined. Finally, based on these genes, the diagnostic model in the training dataset, test dataset, and verification dataset all showed a high diagnostic performance. The findings of this study contributed to the diagnosis of IDD and personalized treatment of IDD.

Host Deprivation Effects on the Functional Response and Parasitism Rate of Habrobracon hebetor (Hymenoptera: Braconidae) on Ephestia elutella (Lepidoptera: Pyralidae) in the Laboratory.

Habrobracon hebetor (Say) is an important biological control agent for lepidopteran pests of stored products. In this study, the age-specific functional response, paralysis rate, and parasitism rate of H. hebetor under different host deprivation treatments (PC: without host deprivation, used as the control, P1d: host deprivation, but the host was removed after 1 d contact, and PW: host deprivation from beginning) were evaluated at different larval densities (5, 10, 20, 40, and 80) of the Ephestia elutella (Hübner) at 28 ± 1°C, 75 ± 5% RH and 16:8 h L:D. Ages of parasitoid females used were 2, 5, 10, and 20 d old. The logistic regression results indicated that the functional response of H. hebetor females under different host deprivation treatments was type II. The longest handling time was observed in 20-d old females, while the shortest handling time and highest maximum attack rate (T/Th) were estimated at the age of 2 d in all treatments. The paralysis and parasitism rates of H. hebetor were the highest at 2, 5, and 10-d old in all treatments. The results of this study suggest that H. hebetor females up to 10-d old can be used as an efficient biological control agent against E. elutella. The data of this study can also be used to predict the efficacy of different aged H. hebetor females in controlling E. elutella populations.

Characterization of Two Complete Mitochondrial Genomes of Atkinsoniella (Hemiptera: Cicadellidae: Cicadellinae) and the Phylogenetic Implications.

The complete mitochondrial genomes of Atkinsoniella grahami and Atkinsoniella xanthonota were sequenced. The results showed that the mitogenomes of these two species are 15,621 and 15,895 bp in length, with A+T contents of 78.6% and 78.4%, respectively. Both mitogenomes contain 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and a control region (CR). For all PCGs, a standard start ATN codon (ATT, ATG, or ATA) was found at the initiation site, except for ATP8, for which translation is initiated with a TTG codon. All PCGs terminate with a complete TAA or TAG stop codon, except for COX2, which terminates with an incomplete stop codon T. All tRNAs have the typical cloverleaf secondary structure, except for trnS, which has a reduced dihydrouridine arm. Furthermore, these phylogenetic analyses were reconstructed based on 13 PCGs and two rRNA genes of 73 mitochondrial genome sequences, with both the maximum likelihood (ML) and Bayesian inference (BI) methods. The obtained mitogenome sequences in this study will promote research into the classification, population genetics, and evolution of Cicadellinae insects in the future.

Host deprivation effects on population performance and paralysis rates of Habrobracon hebetor (hymenoptera: Braconidae).

BACKGROUND: Habrobracon hebetor (Hymenoptera: Braconidae) is a gregarious ectoparasitoid that attacks the larvae of several species of pyralid and noctuid moths. The reproduction and population dynamics of parasitoids in general are affected by host deprivation. However, how host deprivation affects H. hebetor is unknown. The effect of host deprivation on the parental generation, life table parameters, and the paralysis rate of the F1 generation of H. hebetor were evaluated using the age-stage, two-sex life table under laboratory conditions. RESULTS: The results indicated that the greatest longevity and the least lifetime fecundity of the F0 generation occurred after 19 days of host deprivation (PW-20 treatment). The life table parameters (intrinsic rate of increase, r; finite rate of increase, λ; and net reproductive rate, R0 ) and the paralysis rate parameters (net paralysis rate, C0 ; transformation rate, Qp ; stable paralysis rate, ψ; and finite paralysis rate, ω) of F1 individuals after PW-20 treatment were significantly higher than those of individuals subjected to the control treatment (no host deprivation). However, no difference was detected between the two host deprivation treatments: host deprivation after 1 day of host contact and immediate host deprivation (PW treatment). CONCLUSION: Our results demonstrated that the effectiveness of H. hebetor did not decrease even during host deprivation for 19 days. Meanwhile, it was observed that mass rearing of the parasitoid could be improved by providing 10 individuals of 5th instar larvae of Ephestia elutella (Lepidoptera: Pyralidae) with a 20% honey-water solution. © 2020 Society of Chemical Industry.

Mitochondrial genome of Bolanusoides shaanxiensis (Cicadellidae: Typhlocybinae: Typhlocybini), with its phylogenetic analysis.

The mitochondrial genome of one leafhopper species Bolanusoides shaanxiensis was sequenced and annotated. The mitogenome is 15,724 bp in length, containing 37 typical genes and a control region. The A + T content of the whole mitogenome is 78.9%. Most of PCGs started with ATN and stopped with TAA, except for ATP8 started with TTG, COX2, COX3 and ND5 used incomplete T as stop codon. The phylogeny tree is monophyletic among 31 related species. The relationships of B. shaanxiensis and Typhlocyba sp. were closer than others. This study further enriched mitogenome database of the tribe Typhlocybini.

Role of AP-2α/TGF-ß1/Smad3 axis in rats with intervertebral disc degeneration.

OBJECTIVE: Studies have proposed the role of AP-2α in human disease. However, few have focused on its effects on intervertebral disc degeneration (IDD). This study intends to discuss the role of AP-2α in IDD by regulating TGF-ß1 and Smad3 expression. METHODS: The AP-2α and TGF-ß1 expression in IDD NP clinical samples was detected. Rat models of IDD were established by acupuncture. The rats were injected with AP-2α low expression adeno-associated virus or TGF-ß1 high expression adeno-associated virus to observe their effects on pathological damages, NP cell apoptosis, matrix metalloproteinase-2 (MMP-2), MMP-9, Smad3, Aggrecan and collagen (Col)-2 expression in NP tissues. The NP cells were isolated and transfected with silenced AP-2α or overexpressed TGF-ß1 vector to figure out their functions in growth, senescence and apoptosis. RESULTS: AP-2α and TGF-ß1 were upregulated in NP tissues of patients and rats with IDD. AP-2α silencing limited the activation of TGF-ß1 signaling pathway. Reduced AP-2α ameliorated pathological changes, declined MMP-2, MMP-9 and Smad3 expression and elevated Aggrecan and Col-2 expression in NP tissues of rats with IDD, and speeded up the growth and depressed senescence and apoptosis of NP cells of rats with IDD. Up-regulating TGF-ß1 weakened the effect of down-regulated AP-2α on NP tissues and cells in IDD. CONCLUSION: Collectively, our study demonstrates that knockdown of AP-2α restricts TGF-ß1 and Smad3 expression to promote proliferation and depress senescence and apoptosis of NP cells in rats with IDD.

Discovery of Mitochondrial Transcription Inhibitors Active in Pancreatic Cancer Cells.

Mitochondrial dysfunction is a hallmark of cancer cells and targeting cancer mitochondria has emerged as a promising anti-cancer therapy. Previously, we repurposed chlorambucil by conjugating it to a mitochondrial targeting triphenylphosphonium (TPP) group to design Mito-Chlor, a novel agent that acts on mitochondria DNA (mtDNA). Herein, we show that Mito-Chlor, but not chlorambucil, inhibits the nascent transcription of mtDNA. Clustering analysis of transcriptomic profile of our Bru-seq database led to the identification of another mitochondrial transcription inhibitor SQD1, which inhibits the proliferation of MIA PaCa-2 cells with an IC50 of 1.3 µM. Interestingly, Mito-Chlor reduces expression of mitochondrial proteins, interferes with mitochondria membrane potential, and impairs oxidative phosphorylation while SQD1 does not. Both compounds increased cellular and mitochondrial reactive oxygen species and stimulated similar signaling pathways in response to oxidative stress. As mitochondrial transcription inhibitors and redox modulators, SQD1 and Mito-Chlor are promising for the treatment of pancreatic cancer by blocking mitochondrial function.

Nicotiana tabacum seed endophytic communities share a common core structure and genotype-specific signatures in diverging cultivars.

Seed endophytes of crop plants have recently received increased attention due to their implications in plant health and the potential to be included in agro-biotechnological applications. While previous studies indicated that plants from the Solanaceae family harbor a highly diverse seed microbiome, genotype-specific effects on the community composition and structure remained largely unexplored. The present study revealed Enterobacteriaceae-dominated seed-endophytic communities in four Nicotiana tabacum L. cultivars originating from Brazil, China, and the USA. When the dissimilarity of bacterial communities was assessed, none of the cultivars showed significant differences in microbial community composition. Various unusual endophyte signatures were represented by Spirochaetaceae family members and the genera Mycobacterium, Clostridium, and Staphylococcus. The bacterial fraction shared by all cultivars was dominated by members of the phyla Proteobacteria and Firmicutes. In total, 29 OTUs were present in all investigated cultivars and accounted for 65.5% of the combined core microbiome reads. Cultivars from the same breeding line were shown to share a higher number of common OTUs than more distant lines. Moreover, the Chinese cultivar Yunyan 87 contained the highest number (33 taxa) of unique signatures. Our results indicate that a distinct proportion of the seed microbiome of N. tabacum remained unaffected by breeding approaches of the last century, while a substantial proportion co-diverged with the plant genotype. Moreover, they provide the basis to identify plant-specific endophytes that could be addressed for upcoming biotechnological approaches in agriculture.

Intervertebral disc degeneration in mice with type II diabetes induced by leptin receptor deficiency.

BACKGROUND: The leptin receptor-deficient knockout (db/db) mouse is a well-established model for studying type II diabetes mellitus (T2DM). T2DM is an important risk factor of intervertebral disc degeneration (IVDD). Although the relationship between type I diabetes and IVDD has been reported by many studies, few studies have reported the effects of T2DM on IVDD in db/db mice model. METHODS: Mice were separated into 3 groups: wild-type (WT), db/db, and IGF-1 groups (leptin receptor-deficient mice were treated with insulin-like growth factor-1 (IGF-1). To observe the effects of T2DM and glucose-lowering treatment on IVDD, IGF-1 injection was used. The IVD phenotype was detected by H&E and safranin O fast green staining among db/db, WT and IGF-1 mice. The levels of blood glucose and weight in mice were also recorded. The changes in the mass of the trabecular bone in the fifth lumbar vertebra were documented by micro-computed tomography (micro-CT). Tunnel assays were used to detect cell apoptosis in each group. RESULTS: The weight of the mice were 27.68 ± 1.6 g in WT group, which was less than 57.56 ± 4.8 g in db/db group, and 52.17 ± 3.7 g in IGF-1 injected group (P < 0.05). The blood glucose levels were also significantly higher in the db/db mice group. T2DM caused by leptin receptor knockout showed an association with significantly decreased vertebral bone mass and increased IVDD when compared to WT mice. The db/db mice induced by leptin deletion showed a higher percentage of MMP3 expression as well as cell apoptosis in IVDD mice than WT mice (P < 0.05), while IGF-1 treatment reversed this situation (P < 0.05). CONCLUSIONS: T2DM induced by leptin receptor knockout led to IVDD by increasing the levels of MMP3 and promoting cell apoptosis. IGF-1 treatment partially rescue the phenotype of IVDD induced by leptin receptor knockout.

Differences in fungicide resistance profiles and multiple resistance to a quinone-outside inhibitor (QoI), two succinate dehydrogenase inhibitors (SDHI), and a demethylation inhibitor (DMI) for two Stagonosporopsis species causing gummy stem blight of cucurbits.

BACKGROUND: Gummy stem blight (GSB) is a devastating disease of cucurbits that has been effectively managed with fungicide applications. However, the Stagonosporopsis spp. that cause GSB have rapidly evolved resistance to multiple classes of fungicides. To better understand the evolution and persistence of fungicide resistance in field populations, resistance profiles of unique and clonal genotypes of 113 Stagonosporopsis citrulli and 19 S. caricae isolates to four different fungicides were determined based on in vitro mycelial growth assays and molecular markers based on genes encoding fungicide targets. RESULTS: All 19 S. caricae isolates screened were resistant to tebuconazole and azoxystrobin, and sensitive to boscalid and fluopyram. All 113 S. citrulli isolates were sensitive to tebuconazole and sensitive to fluopyram, with one exception that was fluopyram-resistant. All isolates of S. citrulli except two were resistant to azoxystrobin. Phenotypic differences in response to boscalid were detected among S. citrulli isolates, but the phenotypes were not associated with multilocus genotypes (MLG) determined by 16 microsatellite loci. Additionally, isolates sharing the same MLG varied by SdhB genotype. A unique mutation of I229V in SdhB, a target of succinate dehydrogenase inhibitor fungicides, was detected for the fluopyram-resistant isolate of S. citrulli. CONCLUSION: Both the lack of association of fungicide resistance profiles with genetic similarity of isolates based on microsatellite loci and the finding that widely distributed MLG varied in fungicide resistance profiles suggest that independent evolutionary events for resistance to boscalid have likely occurred. Frequent genetic recombination within populations may be responsible for resistance to multiple fungicides. This study provides useful information for effectively managing both species of GSB fungi present in the southeastern USA and understanding the evolution of fungicide resistance within populations of plant-pathogenic fungi. © 2019 Society of Chemical Industry.

Synthesis of saccharin-glycoconjugates targeting carbonic anhydrase using a one-pot cyclization/deprotection strategy.

Carbonic anhydrase IX (CA IX) has been identified as a biomarker and drug target for several malignant tumors due to its role in cancer cell growth and proliferation. Simple cyclic sulfonamides, like saccharin (SAC), have shown up to a 60-fold selectivity towards CA IX over other ubiquitous CA isoforms, with greater selectivity obtained applying the "tail-approach" to derivatize SAC with a methylene triazole linker that connected to a "tail" beta glucoside. These modifications of SAC led to an increased selectivity of more than 1000-fold towards CA IX, whereas clinically available CA inhibitors show little to no isoform selectivity. As part of our interest in the development of new CA inhibitors, we found the existing synthetic protocol, which relies on a N-tert-butyl saccharin intermediate, to be problematic in the final deprotection steps. We therefore describe an alternative approach to the synthesis of these compounds featuring a gentle "one pot" deprotection/cyclization as the final synthetic step, and report new galactosyl and glucosyl conjugates with low to mid nM inhibition of CA IX.

The tea leaf microbiome shows specific responses to chemical pesticides and biocontrol applications.

The plant microbiome is known to be influenced by certain biotic as well as abiotic factors. Nevertheless, the drivers for specific changes in microbial community composition and structure are largely unknown. In the present study, the effects of chemical and biological treatments for plant protection on the indigenous microbiome of Camellia sinensis (L.) Kuntze were contrasted. Assessment of bacteria-specific ribosomal RNA gene fragment amplicons from a representative set of samples showed an increased microbial diversity in treated plants when compared to untreated samples. Moreover, distinct microbial fingerprints were found for plants subjected to a conventional pesticide treatment with lime sulfur as well as for plants that were biologically treated with a Piriformospora indica spore solution. The bacterial community of pesticide-treated plants was augmented by 11 taxa assigned to Proteobacteria and Actinobacteria. In contrast, plants from biological control treatments were augmented by 10 taxa representing a more diversified community enrichment and included members of Actionobacteria, Proteobacteria, Bacteroidetes, Planctomycetes, and Verrucomicrobia. Complementary, molecular quantification of fungi in the samples showed a significantly lower number of internal transcribed spacer copies in plants subjected to biological control treatments, indicating the highest efficiency against fungal pathogens. The overall results show that leaves that are used for tea production show distinct microbiome shifts that are elicited by common pest and pathogen management practices. These shifts in the microbial population indicate non-target effects of the applied treatments.

The complete mitochondrial genome sequences of two insect-tea producers in Pyralidae (Lepidoptera) from South China: Pyralis farinalis and Orthopygia glaucinalis.

Complete mitochondrial genomes of Pyralis farinalis and Orthopygia glaucinalis were sequenced, respectively. Both contains 13 protein-coding genes (PCGs), 22 tRNA, two rRNA genes, and one AT-rich region. Pyralis farinlis mitogenome was 15,204 bp, with 11,234 bp coding 3732 aa. The rRNA had 1004 bp LSU and 802 bp SSU. Mitogenome of O. glaucinalis was 15,032 bp, with 11,038 bp coding 3668 aa. The rRNA contained 1406 bp LSU and 814 bp SSU. All PCGs used TAN as stop codon, except for both ND4 and ND5 of O. glaucinalis. Phylogenetic relationship of both species was also shown with 13 references.

Microbiome-Guided Exploration of the Microbial Assemblage of the Exotic Beverage "Insect Tea" Native to Southwestern China.

Insect tea is a unique beverage that is native to Southwestern China and traditionally produced by local farmers in an elaborate process. It consists of insect larvae excrements that are commonly obtained from meal moths (Pyralis farinalis Linnaeus 1758) reared on a specific plant-based diet. We have reconstructed the whole production process under laboratory conditions in order to obtain microbiome-level insights into this uncommon beverage and to trace back the origin of the prevalent bacteria in the final product. The bacterial community composition was specific for each production stage, with a high proportion of Streptomycetacea, Pseudonocaridaceae, Enterococcaceae, and Enterobacteriaceae in the insect tea. A large proportion of the constituents was traced back to the producing insect (13.2%) and its excrements (43.8%), while the initial plant-based substrate for tea production was found to contribute only 0.6% of the traceable bacteria in the final product. Moreover, an enrichment of Enterobactericeae was observed during the analyzed process steps and verified with complementary analyses. The cultivation experiments indicated a high occurrence of viable bacteria in the tea at 2.7 × 105 ± 1.2 × 105 cfu g-1. The isolated bacteria included Bordetella petrii and Enterococcus spp. that were recovered from a commercial product. By implementing an integrative approach, the insect tea was shown to harbor a species-rich bacterial community that can be traced back to certain plant and insect microbiome constituents from distinct production steps. Moreover, the microbial profile of the insect tea was found to be unique for a food product so far and contained several bacterial groups that are considered from the current perspective as food contaminants or yet unreported in other beverages. Due to the high number of viable bacteria, the tea harbors a so far undescribed dynamic component that might have implications for human health.