Identity, pathogenicity and genetic diversity of Lasiodiplodia associated with stem-end rot of avocado in China.

Stem-end rot (SER) causes brown necrotic lesions in the pulp near the base of the fruit pedicel and is one of the most devastating postharvest diseases of avocados in all avocado growing regions of the world. China's avocado industry is growing very rapidly, and the planting area is expanding, but little is known about the pathogens and genetic diversity of avocado SER. To determine the causal agents of SER, avocado fruits were sampled from the main avocado-producing areas in China during 2020 and 2021. Fungal isolates were obtained from SER symptomatic avocado fruits and identified by morphology combined with phylogenetic analysis of internal transcribed spacer (ITS), translation elongation factor 1-α (EF1-α) and ß-tubulin (TUB2) gene sequences. All 101 isolates belonged to Lasiodiplodia spp., and four Lasiodiplodia species were identified, namely L. pseudotheobromae (59.41%), L. theobromae (24.75%), L. mahajangana (7.92%), L. euphorbiaceicola (1.98%), and six others are classified as Lasiodiplodia sp. (5.94%). There were only slight morphological differences in colonies and conidia of these four species of Lasiodiplodia. The pathogenicity tests showed symptoms of SER, and the 92.08% of the isolates exhibited a high level of virulence on avocado (disease index > 70), related to the disease severity on avocado fruit. All tested isolates grew well under conditions from 23 to 33℃. There was a significant difference in mycelial growth between the four species of Lasiodiplodia after treatment with high temperature or low temperature. L. pseudotheobromae growth was the fastest at 13 to 18℃, but was the lowest at 38℃ (P < 0.05). Red pigment could be produced by all tested isolates after culturing for 7 days at 38℃. The mycelial growth rate was the fastest on PDA medium, and the slowest on OMA medium but promoted spore formation (P < 0.05). In addition, was determined the genetic diversity of Lasiodiplodia pathogenic species associated with SER collected from avocado, mango, guava and soursop fruits was determined. A total of 74 isolates were clustered into 4 main ISSR groups by unweighted pair-group method with arithmetic mean (UPGMA) analysis, and the classification of this group was related to the host. Extensive diversity was detected in the Lasiodiplodia populations. The diverse geographical origins and host species significantly influenced the population differentiation, and most of the genetic variation occurred within populations (P < 0.001). This is the first study to identify the major pathogens of avocado SER in China and to survey their occurrence, pathogenicity and include a comparative analysis of genetic diversity with Lasiodiplodia spp. causing SER on other fruit hosts. Collectively, the Lasiodiplodia species complex affecting avocado showed high pathogenicity and diversity, while L. pseudotheobromae was the most frequently isolated species in China. The results of this study provide insights into the aspects of epidemic of SER disease caused by Lasiodiplodia species, which will help in developing strategies for the management and control of stem end-rot in avocado.

Slope aspect determines the abundance and composition of nitrogen-cycling microbial communities in an alpine ecosystem.

Slope aspect is an important topographic feature that can influence local environmental conditions. While strong effects of slope aspect on aboveground and belowground communities have been frequently elucidated, how slope aspect affects soil nitrogen (N) cycling microbes remains unclear. Here, we characterized the communities of soil N-cycling microbes on south- and north-facing slopes in an alpine ecosystem, by quantifying (qPCR) and high-throughput sequencing six genes involved in N-fixation (nifH), nitrification (archaeal and bacterial amoA) and denitrification (nirK, nirS and nosZ). We found that the abundance, diversity and community composition of major N-cycling microbes differed dramatically between the two slope aspects, and these variances could be well explained by the aspect-driven differences in environmental conditions, especially soil temperature and moisture. The response patterns of different N-cycling groups to slope aspect were much inconsistent, especially for those with similar functions (i.e. ammonia-oxidizing archaea vs. bacteria, nirK- vs. nirS-reducers), indicating strong niche differentiation between these counterparts. We also observed strong preferences and distinct co-occurrence patterns of N-cycling microbial taxa for the two slope aspects. These findings highlight the importance of slope aspect in determining the abundance, species distribution and community structure of N-cycling microbes, and consequently influencing N-cycling processes and ecosystem functioning.

In vitro study of injury on human bronchial epithelial cells caused by gunpowder smog.

Smog inhalation is associated with acute respiratory symptoms in exposed victims. However, despite the evidence from cell injury caused by smog, a stable and practical apparatus used to treat cells with smog is necessary. The aim of this study is to develop a cell research platform of smoke inhalation injury. In the smog-generation device, a wireless electromagnetic heater was used to ignite gunpowder and generate smog. The quality of black powder was checked by the black powder burn rate, and experimental smog was indirectly checked by the amount of cell damage. The temperature and humidity were set at 37 °C ± 1 °C and ≥95% in the smog-cells reaction chamber, respectively. Factors including gunpowder dosages, smog-exposure time, the cell density, modes of exposure, volumes of smog, test durations, volumes of the cell culture medium and combustion velocity were measured. Coefficient variation of different batches of gunpowder and smog were less than 4% and 9%, respectively. With larger gunpowder dosage and longer exposure time, cell injury appeared to increase. When cells were cultured in 4 × 10(4)/well density in culture medium (1 mL/well), exposed to more than 10 L smog with filter screens above plates, detected after 24 h culture in cell incubator and gunpowder burned out within 5 s, smog had the best effect on cell injury. In conclusion, the experimental device can produce test smog stably and safely. The apparatus treating cells with smog can induce cell injury effectively, and the injury is positively correlated with smog concentration and exposure time.

Cdk5 activation promotes Cos-7 cells transition towards neuronal-like cells.

Objectives: Cyclin-dependent kinase 5 (Cdk5) activity is specifically active in neurogenesis, and Cdk5 and neocortical neurons migration related biomarker are expressed in Cos-7 cells. However, the function of Cdk5 on the transformation of immortalized Cos-7 cells into neuronal-like cells is not clear. Methods: Cdk5 kinase activity was measured by [γ-32P] ATP and p81 phosphocellulose pads based method. The expression of neuron liker markers was evaluated by immunofluorescence, real-time PCR, Western blot, and Elisa. Results: P35 overexpression upregulated Cdk5 kinase activity in Cos-7 cells. p35 mediated Cdk5 expression promoted the generation of nerite-like outgrowth. Compared with the empty vector, p35-induced Cdk5 activation resulted in time-dependent increase in neuron-like marker, including Tau, NF-H, NF-H&M, and TuJ1. Tau-5 and NF-M exhibited increased expression at 48 h while TuJ1 was only detectable after 96 h in p35 expressed Cos-7 cells. Additionally, the neural cell biomarkers exhibited well colocation with p35 proteins. Next-generation RNA sequence showed that p35 overexpression significantly upregulated the level of nerve growth factor (NGF). Gene set enrichment analysis showed significant enrichment of multiple neuron development pathways and increased NGF expression after p35 overexpression. Conclusion: p35-mediated Cdk5 activation promotes the transformation of immortalized Cos-7 cells into neuronal-like cells by upregulating NGF level.

Platelet-to-albumin ratio is a potential biomarker for predicting diabetic nephropathy in patients with type 2 diabetes.

Aim: To evaluate whether platelet-to-albumin ratio (PAR) can predict diabetic nephropathy (DN) in type 2 diabetes mellitus (T2DM). Materials & methods: A total of 140 patients with T2DM and 40 healthy individuals were enrolled retrospectively. T2DM patients were divided into three groups based on the urinary albumin-to-creatinine ratio, PAR was compared and receiver operating characteristic curve was constructed to evaluate the predictive value of PAR in DN in T2DM. Results: There was a significant increase of PAR in DN among T2DM patients and PAR was positively correlated with serum creatinine, retinol-conjugated protein and ß2-microglobulin. Moreover, PAR was a risk factor for DN in T2DM patients, which predicted DN in T2DM with high sensitivity and specificity. Conclusion: PAR can be a potential candidate to predict DN in T2DM.

Phenotypical screening on metastatic PRCC-TFE3 fusion translocation renal cell carcinoma organoids reveals potential therapeutic agents.

PURPOSE: Translocation renal cell carcinoma (tRCC) is a subtype that occurs predominantly in children and young individuals. Metastatic tRCC occurring in young patients is more aggressive than that occurring in older patients, and there are still no effective therapies. Organoids can mimic original tissues and be assessed by high-throughput screening (HTS). We aimed to utilize patient-derived organoids and HTS to screen drugs that can be repurposed for metastatic tRCC with PRCC-TFE3 fusion. METHODS: Tumor tissues were obtained from treatment-naïve metastatic tRCC patients who underwent surgery. Histopathology and fluorescence in situ hybridization (FISH) confirmed the tRCC. Organoids derived from the dissected tissues were cultured and verified by FISH and RNA-seq. HTS was performed to seek promising drugs, and potential mechanisms were explored by RNA-seq and cell-based studies. RESULTS: We successfully established a metastatic tRCC organoid with PRCC-TFE3 fusion, a common fusion subtype, and its characteristics were verified by histopathology, FISH, and RNA-seq. An HTS assay was developed, and the robustness was confirmed. A compound library of 1816 drugs was screened. Eventually, axitinib, crizotinib, and JQ-1 were selected for further validation and were found to induce cell cycle arrest and apoptosis. RNA-seq analyses of posttreatment organoids indicated that crizotinib induced significant changes in autophagy-related genes, consistent with the potential pathogenesis of tRCC. CONCLUSIONS: We established and validated organoids derived from tissues dissected from a patient with metastatic tRCC with PRCC-TFE3 fusion and achieved the HTS process for the first time. Crizotinib might be a targeted therapy worthy of exploration in the clinic for metastatic tRCC with PRCC-TFE3 fusion. Such organoid and HTS assays may represent a promising model system in translational research assisting in the development of clinical strategies.

TFP5-Mediated CDK5 Activity Inhibition Improves Diabetic Nephropathy via NGF/Sirt1 Regulating Axis.

Diabetic nephropathy (DN) is one of the leading causes of chronic kidney disease (CKD), during which hyperglycemia is composed of the major force for the deterioration to end-stage renal disease (ESRD). However, the underlying mechanism triggering the effect of hyperglycemia on DN is not very clear and the clinically available drug for hyperglycemia-induced DN is in need of urgent development. Here, we found that high glucose (HG) increased the activity of cyclin-dependent kinase 5 (CDK5) dependent on P35/25 and which upregulated the oxidative stress and apoptosis of mouse podocytes (MPC-5). TFP5, a 25-amino acid peptide inhibiting CDK5 activity, decreased the secretion of inflammation cytokines in serum and kidney, and effectively protected the kidney function in db/db mouse from hyperglycemia-induced kidney injuries. In addition, TFP5 treatment decreased HG-induced oxidative stress and cell apoptosis in MPC-5 cells and kidney tissue of db/db mouse. The principal component analysis (PCA) of RNA-seq data showed that MPC-5 cell cultured under HG, was well discriminated from that under low glucose (LG) conditions, indicating the profound influence of HG on the properties of podocytes. Furthermore, we found that HG significantly decreased the level of NGF and Sirt1, both of which correlated with CDK5 activity. Furthermore, knockdown of NGF was correlated with the decreased expression of Sirt1 while NGF overexpression leads to upregulated Sirt1 and decreased oxidative stress and apoptosis in MPC-5 cells, indicating the positive regulation between NGF and Sirt1 in podocytes. Finally, we found that K252a, an inhibitor of NGF treatment could undermine the protective role of TFP5 on hyperglycemia-induced DN in db/db mouse model. In conclusion, the CDK5-NGF/Sirt1 regulating axis may be the novel pathway to prevent DN progression and TFP5 may be a promising compound to improved hyperglycemia induced DN.

Receptor interacting protein 3 suppresses vascular smooth muscle cell growth by inhibition of the phosphoinositide 3-kinase-Akt axis.

Proliferation of vascular smooth muscle cells (VSMCs) is a primary mechanism underlying cardiovascular proliferative disorders. Phosphoinositide 3-kinase (PI3K)-Akt (or protein kinase B) axis has been assigned at the center of pathways that regulate cell proliferation. Here we demonstrate that enhanced PI3K-Akt signaling by mitogenic stimulation or arterial injury profoundly elevates expression of receptor interacting protein 3 (RIP3) in primary cultured rat VSMCs and in vivo and that the up-regulation of RIP3 leads to VSMC growth arrest and apoptosis via inhibiting the PI3K-Akt signaling pathway, thereby alleviating balloon injury-induced neointimal formation. Specifically, mitogenic stimulation with platelet-derived growth factor-BB or angiotensin II leads to a profound increase in RIP3 expression, which is abolished by inhibition of PI3K or Akt, and increased PI3K-Akt signaling by expression of a constitutively active PI3K mutant also elevates RIP3 expression. Importantly, adenoviral overexpression of RIP3 not only triggers apoptosis but also causes cell cycle arrest at G(1)/G(0) phases that is associated with suppressed Akt activation. In sharp contrast, RIP3 gene silencing enhances serum- and platelet-derived growth factor-induced cell proliferation and Akt activation. In vivo adenoviral gene delivery of rat RIP3 (rRIP3) increased apoptosis and reduced VSMC proliferation, thus, effectively alleviating balloon injury-induced neointimal formation. The growth-suppressive and pro-apoptotic effects are independent of rRIP3 Ser/Thr kinase activity, because overexpression of a kinase-inactive mutant of rRIP3, similar to its wild type, is sufficient to induce growth arrest and apoptosis. These findings reveal a novel growth-suppressive action of RIP3, marking RIP3 as an important factor to prevent excessive mitogenic stimulation- or injury-induced vascular smooth muscle cells hyperplasia.

Halofuginone Sensitizes Lung Cancer Organoids to Cisplatin via Suppressing PI3K/AKT and MAPK Signaling Pathways.

Lung cancer is the leading cause of cancer death worldwide. Cisplatin is the major DNA-damaging anticancer drug that cross-links the DNA in cancer cells, but many patients inevitably develop resistance with treatment. Identification of a cisplatin sensitizer might postpone or even reverse the development of cisplatin resistance. Halofuginone (HF), a natural small molecule isolated from Dichroa febrifuga, has been found to play an antitumor role. In this study, we found that HF inhibited the proliferation, induced G0/G1 phase arrest, and promoted apoptosis in lung cancer cells in a dose-dependent manner. To explore the underlying mechanism of this antitumor effect of halofuginone, we performed RNA sequencing to profile transcriptomes of NSCLC cells treated with or without halofuginone. Gene expression profiling and KEGG analysis indicated that PI3K/AKT and MAPK signaling pathways were top-ranked pathways affected by halofuginone. Moreover, combination of cisplatin and HF revealed that HF could sensitize the cisplatin-resistant patient-derived lung cancer organoids and lung cancer cells to cisplatin treatment. Taken together, this study identified HF as a cisplatin sensitizer and a dual pathway inhibitor, which might provide a new strategy to improve prognosis of patients with cisplatin-resistant lung cancer.

Nuclear Ca2+ sparks and waves mediated by inositol 1,4,5-trisphosphate receptors in neonatal rat cardiomyocytes.

Dynamic nuclear Ca(2+) signals play pivotal roles in diverse cellular functions including gene transcription, cell growth, differentiation, and apoptosis. Here we report a novel nuclear Ca(2+) regulatory mechanism mediated by inositol 1,4,5-trisphosphate receptors (IP(3)Rs) around the nucleus in developing cardiac myocytes. Activation of IP(3)Rs by alpha(1)-adrenergic receptor (alpha(1)AR) stimulation or by IP(3) application (in saponin-permeabilized cells) increases Ca(2+) spark frequency preferentially in the region around the nucleus in neonatal rat ventricular myocytes. A nuclear enrichment of IP(3)R distribution supports the higher responsiveness of Ca(2+) release in this particular region. Strikingly, we observed "nuclear Ca(2+)waves" that engulf the entire nucleus without spreading into the bulk cytosol. alpha(1)AR stimulation enhances the occurrence of nuclear Ca(2+) waves and confers them the ability to trigger cytosolic Ca(2+) waves via IP(3)R-dependent pathways. This finding accounts, at least partly, for a profound frequency-dependent modulation of global Ca(2+) oscillations during alpha(1)AR stimulation. Thus, IP(3)R-mediated Ca(2+) waves traveling in the nuclear region provide active, autonomous regulation of nuclear Ca(2+) signaling, which provides for not only the local signal transduction, but also a pacemaker to drive global Ca(2+) transient in the context of alpha(1)AR stimulation in developing cardiac myocytes.

Direct coupling between arachidonic acid-induced Ca2+ release and Ca2+ entry in HEK293 cells.

Arachidonic acid (AA) modulates intracellular Ca2+ signaling via Ca2+ release or/and Ca2+ entry. However, the mechanism underlies either process is unknown; nor is it clear as to whether the two processes are mechanistically linked. By using Fura2/AM, we found that AA induced mobilization of internal Ca2+ store and an increment in Ca2+, Mn2+ and Ba2+ influx in HEK293 cells. The AA-mediated Ca2+ signaling was not due to AA metabolites, and insensitive to capacitative Ca2+ entry inhibitors. Interestingly, isotetrandrine and Gd3+ inhibited both AA-induced Ca2+ release and Ca2+ entry in a concentration-dependent manner without affecting Ca2+ discharge caused by carbachol, caffeine, or thapsigargin. Additionally, similar pattern of inhibition was observed with tetracaine treatment. More importantly, the three compounds exhibited almost equal potent inhibition of AA-initiated Ca2+ release as well as Ca2+ influx. Therefore, this study, for the first time, provides evidence for a direct coupling between AA-mediated Ca2+ release and Ca2+ entry.

Induction of adhesion molecule expression in co-culture of human bronchial epithelial cells and neutrophils suppressed by puerarin via down-regulating p38 mitogen-activated protein kinase and nuclear factor κB pathways.

OBJECTIVE: In this study, we aimed to investigate the expressions of adhesion molecules on human bronchial epithelial cells and neutrophils in co-culture system, assess the effects of puerarin on suppressing these adhesion molecules expressions, and explore the roles of two crucial signal-transduction elements p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappa B (NF-κB) in modulating adhesion molecules expressions. METHODS: Neutrophils and BEAS-2B cells (one human bronchial epithelial cell line) were co-cultured, and adhesion molecules expressions on cell surface were detected using flow cytometry. The mRNA levels of adhesion molecules were assessed by real-time quantitative polymerase chain reaction (real-time qPCR). Phosphorylated p38 MAPK and inhibitor κB were analyzed by Western blot. RESULTS: In co-culture system, adhesion molecules expressions on BEAS-2B cells and neutrophils were enhanced significantly (P<0.05). Correspondingly, the mRNA levels of adhesion molecules were also increased greatly. Moreover, the pretreatment of peurarin obviously suppressed adhesion molecules expressions on cell surface. Furthermore, phosphorylated p38 MAPK and inhibitor κB in BEAS-2B cells and neutrophils were elevated in co-culture system, but decreased significantly after upon the treatment of peurarin (P<0.05). CONCLUSIONS: Coculture boosted the interactions between human bronchial epithelial cells and neutrophils mimicking airway inflflammation, whereas peurarin decreased the expression of adhesion molecules on cell surface by suppressing the activities of p38 MAPK and NF-κB pathways, and exhibiting its anti-inflflammation activity.

Effect of puerarin on the release of interleukin-8 in co-culture of human bronchial epithelial cells and neutrophils.

OBJECTIVE: To investigate the effect of puerarin on interleukin (IL)-8 mRNA expression and the protein release in the co-culture of human bronchial epithelial (BEAS-2B) cells and human neutrophils. METHODS: BEAS-2B cells and neutrophills were cultured separately and co-cultured with puerarin (50, 100, and 200 µg/mL) for a predetermined time. Cytokines in culture supernatant were evaluated by protein array and IL-8 quantified by enzyme-linked immunosorbent assay (ELISA). IL-8 mRNA expression was evaluated by real-time quantitative polymerase chain reaction (real-time qPCR). RESULTS: The co-culture of BEAS-2B cells and neutrophils exhibited synergistic effects on IL-8 mRNA expression in BEAS-2B cells, but not in neutrophils after 12 h incubation (P<0.01), as compared with that in BEAS-2B cells or neutrophils alone. IL-8 protein release in the culture supernatant was obviously elevated when BEAS-2B cells were co-cultured with human neutrophils as compared with that in the supernatant of BEAS-2B cells or neutrophils alone after incubated for 2, 6, 12, and 18 h (P<0.01). Treatment with puerarin could significantly down-regulate the expression of IL-8 mRNA in BEAS-2B cells and IL-8 release in the supernatant of the co-culture of BEAS-2B cells and neutrophils (P<0.01). CONCLUSION: Puerarin could exhibit anti-inflammatory activity by suppressing IL-8 production from the co-culture of human bronchial epithelial cells and neutrophils.

Role of inositol 1,4,5-trisphosphate receptors in alpha1-adrenergic receptor-induced cardiomyocyte hypertrophy.

AIM: Intracellular Ca2+ plays pivotal roles in diverse cellular functions, including gene transcription that underlies cardiac remodeling during stress responses. However, the role of inositol 1,4,5-trisphosphate receptors (IP3Rs) in the mediation of cardiac intracellular Ca2+ and hypertrophic growth remains elusive. Prior work with neonatal rat ventricular myocytes suggests that activation of IP3Rs may be linked to a1 adrenergic receptor (alpha1AR) increased stereotyped Ca2+ spark occurrence and global Ca2+ oscillations. Thus, we hypothesized that Ca2+ release through IP3Rs was necessary for alpha1AR-stimulated cardiac hypertrophy. METHODS: We used myoinositol 1,4,5-trisphosphate hexakis (butyryloxymethyl) ester (IP3BM), a membrane-permeant ester of IP3, to activate IP3Rs directly, and Fluo 4/AM to measure intracellular Ca2+ signaling. RESULTS: IP3BM (10 micromol x L(-1)) mimicked the effects of phenylephrine, a selective agonist of alpha1AR, in increments in local Ca2+ spark release (especially in the perinuclear area) and global Ca2+ transient frequencies. More importantly, IP3R inhibitors, 2-aminoethoxydiphenyl borate and Xestospongin C, abolished the IP3BM-induced Ca2+ responses, and significantly suppressed alpha1AR-induced cardiomyocyte hypertrophy assayed by cell size, [3H] leucine incorporation and atrial natriuretic factor gene expression, during sustained (48 h) phenylephrine stimulation. CONCLUSION: These results, therefore, provide cellular mechanisms that link IP3R signaling to alpha1AR-stimulated gene expression and cardiomyocyte hypertrophy.