First Report of Phaeoacremonium minimum Associated with Grapevine Trunk Diseases in China

Grapevine trunk diseases (GTDs) are a complex of diseases that strongly affect grape productivity, wine quality, and vineyard longevity worldwide. GTDs occur in diverse regions, and in some areas disease agents are present in up to 100% of vines (Pintos et al. 2018). Recently, species belonging to Botryosphaeria and Diaporthe were identified as pathogens associated with GTDs in China (Dissanayake et al. 2015; Yan et al. 2013). During 2017 to 2018, interveinal leaf necrosis and wood white decay surrounded by brown necrosis in longitudinal section and black spots in cross section were observed in several grapevines (Cabernet) in Huailai County, Hebei Province, China. Disease incidence was up to 2 to 3% of the vineyard. Diseased samples (whole trunk and roots of 19-year-old vines) were collected and taken to the laboratory. Woody samples were cut into small chips, surface sterilized in 1.5% NaOCl for 3 min followed by 70% ethanol for 30 s, and rinsed three times with sterile distilled water (SDW). Once the samples were dried, they were placed onto PDA plates amended with ampicillin (0.1 g/liter). Plates were incubated at 25°C under dark conditions. After 14 days of incubation, hyphal tips of fungi growing from wood pieces were transferred onto new PDA plates and incubated until they produced conidia. One type of colony was consistently isolated from the discolored tissue, with honey brown mycelium, and producing a yellow pigment on PDA. Conidia were ellipsoid to allantoid, 3.8 to 6.2 μm long, and 1.6 to 3.2 μm wide (n = 50). Morphologically these isolates resembled species belonging to Phaeoacremonium (Mostert et al. 2006). For species confirmation, genomic DNA of three representative isolates (JZB3190001, JZB3190003, and JZB3190005) was extracted. PCR amplification was performed using two phylogenetic markers (actin and β-tubulin) amplified with primers ACT-513F/ACT-783R (Carbone and Kohn 1999) and T1/Bt2b (Glass and Donaldson 1995). The sequences obtained were deposited in GenBank under the accession numbers MK994188 to MK994193. Phylogenetic analysis was conducted using maximum likelihood in RAxML, which was accomplished using RAxML-HPC2 on XSEDE in the CIPRES Science Gateway platform (http://www.phylo.org/). In the phylogenetic tree, the isolates from the present study clustered together with Phaeoacremonium minimum (CBS 246.91), with 100 bootstrap values. Based on morphological characters and phylogenetic results, the species isolated in this study was identified as P. minimum (Tul. & C. Tul.) D. Gramaje, L. Mostert & Crous (Gramaje et al. 2015). The pathogenicity test was conducted on healthy, 2-month-old, rooted 'Furcal' grapevines that were grown in an inoculation chamber. Roots were washed using flow water and rinsed with SDW. The ends of SDW-treated roots (roughly 1 cm) were cut with sterilized scissors, and the roots and trunk base of Furcal vines were dipped in a 10⁶/ml P. minimum spore suspension for 30 min. Inoculated plants were immediately planted in individual pots, and 10 ml of spore suspension was added to the soil per pot. SDW was used as a control. Two P. minimum isolates (JZB3190001 and JZB3190003) were used to do the pathogenicity test, and 10 plants were inoculated with each isolate and the control water. Temperature of the inoculation chamber was controlled between 24 and 25°C, and humidity was maintained at 60%. After 114 days of inoculation, the inoculated plants developed black necrosis at the base of wood but did not show leaf necrosis, whereas the control plants showed no symptoms both in wood and leaves. Koch's postulates were confirmed by reisolating and identification based on cultural and morphological characters of the inoculated isolates. To our knowledge, this is the first report of P. minimum associated with GTDs in China. The results of this study will enhance the capability of controlling GTDs in China by correct identification of the causal organism

Trail of decryption of molecular research on Botryosphaeriaceae in woody plants

The family Botryosphaeriaceae is species rich and includes pathogens, saprobes and endophytes of economically important agricultural crops and plants. As pathogens, Botryosphaeriaceae species employ evolving defensive and counter-defensive strategies that enable them to infect their hosts. Molecular genetic results showed high genetic variability among Botryosphaeriaceae species, which could be a key determinant to their differing environmental roles on hosts and differences in their virulence. Improved technologies such as whole genome re-sequencing, integrated RNA-Seq, comparative and ab initio approaches for molecular genetics and mutation analysis have revealed possible virulence factors that might be involved in the pathogenicity of these fungi. Several genes involved in the pathogenicity processes of botryosphaeriaceous fungi have been identified and characterized. There are numerous reports on the involvement of phytotoxic metabolites in the pathogenicity of these taxa. Availability of whole genomes, genetic transformation and transcriptome analysis of some botryosphaeriaceous species have contributed towards identifying the biological functions of many genes in a fast and accurate way, enabling these fungi to be used as model organism for molecular research of plant opportunistic fungal pathogens in woody plants. The research on botryosphaeriaceous species will provide understanding of the infection mechanisms, and with designing control strategies against diseases caused by opportunistic fungal pathogens

Construction and evaluation of a transformant library of Lasiodiplodia theobromae generated through Restriction Enzyme-Mediated Integration

Grapevine dieback, caused by Lasiodiplodia theobromae, is an important trunk disease worldwide. Transformants of L. theobromae were generated in an attempt to identify potential pathogenicity-related genes. Lasiodiplodia theobromae strain JZB 0300251, a highly virulent isolate, was selected for the genetic transformation. Based on optimised conditions, the Restriction Enzyme-Mediated Integration (REMI) methodology was established in L. theobromae using pUCATPH (a plasmid carrying a hygromycin B marker). A total of 6,036 transformants were generated with four restriction enzymes, respectively and the transformant library was evaluated based on 200 randomly selected transformants. Mutants that exhibited various degrees of virulence and different growth rates were obtained. The study provides basic results that will lead to increased understanding of the role of the pathogenicity-related genes involved in the infection process of L. theobromae

Chronic Myeloid Leukemia Patients Sensitive and Resistant to Imatinib Treatment Show Different Metabolic Responses

The BCR-ABL tyrosine kinase inhibitor imatinib is highly effective for chronic myeloid leukemia (CML). However, some patients gradually develop resistance to imatinib, resulting in therapeutic failure. Metabonomic and genomic profiling of patients' responses to drug interventions can provide novel information about the in vivo metabolism of low-molecular-weight compounds and extend our insight into the mechanism of drug resistance. Based on a multi-platform of high-throughput metabonomics, SNP array analysis, karyotype and mutation, the metabolic phenotypes and genomic polymorphisms of CML patients and their diverse responses to imatinib were characterized. The untreated CML patients (UCML) showed different metabolic patterns from those of healthy controls, and the discriminatory metabolites suggested the perturbed metabolism of the urea cycle, tricarboxylic acid cycle, lipid metabolism, and amino acid turnover in UCML. After imatinib treatment, patients sensitive to imatinib (SCML) and patients resistant to imatinib (RCML) had similar metabolic phenotypes to those of healthy controls and UCML, respectively. SCML showed a significant metabolic response to imatinib, with marked restoration of the perturbed metabolism. Most of the metabolites characterizing CML were adjusted to normal levels, including the intermediates of the urea cycle and tricarboxylic acid cycle (TCA). In contrast, neither cytogenetic nor metabonomic analysis indicated any positive response to imatinib in RCML. We report for the first time the associated genetic and metabonomic responses of CML patients to imatinib and show that the perturbed in vivo metabolism of UCML is independent of imatinib treatment in resistant patients. Thus, metabonomics can potentially characterize patients' sensitivity or resistance to drug intervention

Orally Administered Crocin Protects Against Cerebral Ischemia/Reperfusion Injury Through the Metabolic Transformation of Crocetin by Gut Microbiota

Our pilot study suggested that orally administered crocin was hardly absorbed into circulatory system, but it was effective against cerebral ischemic/reperfusion (I/R) injury. The pharmacologically active component and targeting site of crocin remain elusive. In this study, the cerebral-protective effect of crocin was evaluated on a rat transient middle cerebral artery occlusion (MCAO) model. Our data showed that oral administration of crocin had better effectiveness in cerebral protection than an intravenous injection. Neither crocin nor its metabolite crocetin were determined in the brain of cerebral I/R rats, indicating a target site of periphery. Abundant crocetin was detected in plasma after oral administration instead of intravenous injection of crocin. Meanwhile, orally administered crocetin showed similar cerebral protection to that of crocin, but this exciting effect was not clearly observed by intravenous administration of crocetin, indicating the importance of crocetin in gut. Moreover, orally administered crocin showed less cerebral-protective effect in pseudo germ-free (pGF) MCAO rats. In vitro and in vivo experiments confirmed that crocin could be deglycosylated to crocetin in gut content of normal rats, rather than that of pGF rats, indicating that gut microbiota facilitated the transformation of crocin into crocetin, which played a key role in the activation of the pharmacological effect. Metabolomic study revealed that microbial-host co-metabolic molecules were significantly perturbed after oral administration of crocin, indicating a regulation on intestinal ecosystem. It was further suggested that gut microbiota may be the potential target of the cerebral-protective effect of crocin

CRL4 antagonizes SCFFbxo7-mediated turnover of cereblon and BK channel to regulate learning and memory

Intellectual disability (ID), one of the most common human developmental disorders, can be caused by genetic mutations in Cullin 4B (Cul4B) and cereblon (CRBN). CRBN is a substrate receptor for the Cul4A/B-DDB1 ubiquitin ligase (CRL4) and can target voltage- and calcium-activated BK channel for ER retention. Here we report that ID-associated CRL4CRBNmutations abolish the interaction of the BK channel with CRL4, and redirect the BK channel to the SCFFbxo7ubiquitin ligase for proteasomal degradation. Glioma cell lines harbouring CRBN mutations record density-dependent decrease of BK currents, which can be restored by blocking Cullin ubiquitin ligase activity. Importantly, mice with neuron-specific deletion of DDB1 or CRBN express reduced BK protein levels in the brain, and exhibit similar impairment in learning and memory, a deficit that can be partially rescued by activating the BK channel. Our results reveal a competitive targeting of the BK channel by two ubiquitin ligases to achieve exquisite control of its stability, and support changes in neuronal excitability as a common pathogenic mechanism underlying CRL4CRBN–associated ID

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat