Comparative genomic and transcriptome analyses of two Pectobacterium brasiliense strains revealed distinct virulence determinants and phenotypic features.

Potato soft rot caused by Pectobacterium spp. are devastating diseases of potato which cause severe economic losses worldwide. Pectobacterium brasiliense is considered as one of the most virulent species. However, the virulence mechanisms and pathogenicity factors of this strain have not been fully elucidated. Here, through pathogenicity screening, we identified two Pectobacterium brasiliense isolates, SM and DQ, with distinct pathogenicity levels. SM exhibits higher virulence compared to DQ in inducing aerial stem rot, blackleg and tuber soft rot. Our genomic and transcriptomic analyses revealed that SM encodes strain specific genes with regard to plant cell wall degradation and express higher level of genes associated with bacterial motility and secretion systems. Our plate assays verified higher pectinase, cellulase, and protease activities, as well as fast swimming and swarming motility in SM. Importantly, a unique endoglucanase S specific to SM was identified. Expression of this cellulase in DQ greatly enhances its virulence compared to wild type strain. Our study sheds light on possible determinants causing different pathogenicity of Pectobacterium brasiliense species with close evolutionary distance and provides new insight into the direction of genome evolution in response to host variation and environmental stimuli.

Species diversity of Pectobacterium spp. causing potato aerial stem rot in China.

Pectobacterium spp. are the primary causative agents of aerial stem rot in potatoes in China. A nationwide survey revealed the widespread occurrence of aerial stem rot in the northern, southern, and southwestern cultivation regions, with occurrence rates ranging from 1% to 60%. In total, 36 strains were isolated and identified at the species level using multi-locus sequence analysis of six housekeeping genes (rpoS, proA, gapA, icdA, gyrA, and mdh). Genome sequencing was conducted on one representative strain for each species, and further confirmation of their identities was achieved through ANI and isDDH analysis. Five Pectobacterium species were identified, namely Pectobacterium atrosepticum, Pectobacterium brasiliense, Pectobacterium carotovorum, Pectobacterium polaris and Pectobacterium punjabense, with P. atrosepticum and P. brasiliense being the most widely distributed. Pathogenicity tests demonstrated that, among the strains isolated in this study and those obtained from other studies, P. atrosepticum and P. brasiliense are also the most virulent species. To the best of our knowledge, this is the first nationwide study describing the diversity and distribution of Pectobacterium spp. affecting potatoes in China. The information gathered will be utilized for disease diagnosis and the development of pathogen-specific integrated pest management (IPM) strategies to protect potato production.

First Report of Pectobacterium parvum Causing Diseases on Potato in Inner Mongolia, China.

Soft rot enterobacterial plant pathogens Pectobacterium spp. and Dickeya spp. have caused devastating blackleg, aerial stem rot, and soft rot of potato tubers (Charkowski 2018). In August 2021, potato plants (cv. Xisen6# or Maiken1) with blackleg or aerial stem rot symptoms were observed in two commercial fields in Xinghe County, Ulanqab City, Inner Mongolia Autonomous Region, China. The plants were wilted, and the crown stem showed gradual degradation and browning. The disease incidence was around 3 to 7% and 20 to 25% in Xinghe Zhangyou Village (33 ha) and Bianjia Village (7 ha), respectively. Pathogens were isolated on crystal violet pectate agar (CVP) plates (Ge et al. 2018). Briefly, symptomatic stem tissues were surface sterilized in 75% ethanol, ground, then serial dilutions were cultured on CVP plates (Handique et al. 2022). The plates were incubated at 28oC for 2 days. Pure colonies of Pectobacterium spp. were obtained from the pits of CVP plates and sequenced for identification using the universal 16S rRNA gene primers 27F/1492R (Monciardini et al. 2002). Results of the comparison of 16S sequences against NCBI GenBank showed 100% sequence identity to P. parvum FN20211T (CP087392.1) type strain for the three colonies designated as ZRIMU1006 (1542/1542 bp), ZRIMU1019 (1542/1542 bp), and ZRIMU1020 (1542/1542 bp). Sequences were deposited under accession numbers OP941529, OP941525, OP941526, respectively. Additionally, six housekeeping gene sequences were used to confirm identification at the species level and were uploaded to GenBank: fusA (OP793177, OP793171, OP793172), gapA (OP793221, OP793216, OP793217), gyrB (OP793265, OP793259, OP793260), infB (OP793310, OP793304, OP793305), pgi (OP793355, OP793349, OP793350), and rplB (OP793400, OP793394, OP793395). Phylogenetic trees constructed using the MEGA X program of concatenated sequences of the housekeeping genes sequences show that the three isolates grouped with P. parvam FN20211T, confirming that they are P. parvam. Pathogenicity tests for stem rot were done by injecting a bacterial suspension into potato seedlings (cv. Favorita) grown from seed tubers. The tubers were planted in perlite potting mix and 3 weeks after emergence, a 100 µl bacterial suspension (105 CFU/ml) or sterile phosphate-buffered solution was injected at the stem base. The bacterial injection experiment was performed twice in a greenhouse with five plants inoculated per bacterial strain. Plants were covered with plastic bags to maintain 100% humidity at 25°C for 2 days. Seven days after inoculation, the inoculated area of the seedlings had rotted or turned black, while the controls remained symptomless. Symptomatic tissues from each strain were processed as above and placed on CVP plates to reisolate the Pectobacterium spp. 16S rRNA sequence analysis confirmed the bacteria to be similar in sequence to the inoculated strains, thus completing Koch's postulates. Soft rot development was performed by adding bacterial suspension (100 µl, 105 CFU/ml) on tuber slices. The infected tubers rotted, while the controls were symptomless. The vacuum infiltration method on tuber that is used to test pathogens for blackleg did not result in the development of blackleg symptoms. Briefly, five tubers were inoculated with the pathogen by vacuum infiltration and planted in potting mix. The plants showed wilting after emergence, but no blackleg symptoms were observed. Recently, multiple new Pectobacterium species including P. parmentieri, P. polaris, and P. punjabense, were identified to cause potato disease in different provinces of China (Cao et al. 2021; Handique et al. 2022). In China, P. parvum was first isolated from Brassica, and in the year 2022, it was reported to cause aerial stem-rot on potato in Hebei province in China (Wang et al. 2022). Inner Mongolia is a major potato-seed-producing province and the incidence of new strains of Pectobacterium in the province causing aerial stem rot and soft rot of tuber might cause a reduction in seed production. This report will draw attention to the management of P. parvum by the seed-producing companies in Inner Mongolia which distributes the seed throughout China.

MicroRNA-378 inhibits hepatocyte apoptosis during acute liver failure by targeting caspase-9 in mice.

BACKGROUND: Acute liver failure (ALF) is a severe and potentially lethal clinical syndrome. It has been demonstrated that micro ribonucleic acids (miRNAs) are crucial mediators of nearly all pathological processes, including liver disease. OBJECTIVE: The present study investigates the role of miR-378 in ALF. An ALF mouse model was induced using intraperitoneal injections of d-galactosamine/lipopolysaccharide (d-GalN/LPS). A hepatocyte cell line and miR-378 analogue were used in vitro to investigate the possible roles of miR-378 in ALF. METHODS: The expressions of miR-378 and predicted target genes were measured via reverse transcription-quantitative polymerase chain reaction and western blotting, and cell apoptosis was assayed using flow cytometry. RESULTS: Compared with mice in the control group, the mice challenged with d-GalN/LPS showed higher levels of alanine aminotransferase, aspartate aminotransferase, tumour necrosis factor-alpha and interleukin-6, more severe liver damage and increased numbers of apoptotic hepatocytes. Hepatic miR-378 was distinctly downregulated, while messenger RNA and protein levels of cysteinyl aspartate specific proteinase 9 (caspase-9) were upregulated in the ALF model. Furthermore, miR-378 was downregulated in d-GalN/TNF-induced hepatocyte cells, and miR-378 was found to inhibit hepatocyte apoptosis by targeting caspase-9. CONCLUSION: Together, the present results indicate that miR-378 is a previously unrecognised post-ALF hepatocyte apoptosis regulator and may be a potential therapeutic target in the context of ALF.

Design and construction strategies to improve covalent organic frameworks photocatalyst's performance for degradation of organic pollutants.

Covalent organic frameworks (COFs) are a class of crystalline porous organic polymers. In recent years, COFs have received extensive attention in the field of photocatalytic degradation due to their large specific surface area, good thermal and solvent stability, and diverse structures. This review studies the progress of COF in the field of photocatalytic degradation, and summarizes the strategies to improve the photocatalytic activity of covalent organic frameworks, including the designs of ligands and structures. In particular, the design and construction of the COF composites (COF/MOF, COF/g-C3N4, COF/metal semiconductor) are discussed. The photocatalytic mechanism is described in detail, and the prospect of COFs in photocatalytic degradation is prospected.

First Report of Streptomyces stelliscabiei Causing Potato Common Scab in Guizhou Province, China.

Potato (Solanum tuberosum L.) common scab can be caused by multiple pathogenic Streptomyces spp. worldwide. Potato tubers (cv. Favorita) with severe pitted common scab symptoms were observed at a small farm (2 hectares) during harvest in Anshun, Guizhou province in early May 2020. The disease incidence was around 10%, and symptomatic samples were collected to isolate the pathogen. Two isolates, ZR-IMU141 and ZR-IMU146 (Accession number MW995958 and MW995959 respectively), showed more than 99% sequence identity to S. stelliscabiei sequences (Accession No. HM018085). Five house-keeping genes for multi-locus sequence analyze (MLSA) of Streptomycetaceae were amplified, sequenced and uploaded to NCBI: atpD (MZ343164 and MZ343165), gyrB (MZ343162 and MZ343163), recA (MZ343166 and MZ343167), rpoB (MZ343168and MZ343169) and trpB (MZ343170 and MZ343171). All the genes show over 98% identity with S. stelliscabiei. Phylogenetic trees of 16S rRNA gene sequence and multi-locus sequence analysis (MLSA) were constructed. The two isolates contain pathogenicity genes txtAB, nec1, and tomA, which was confirmed by PCR. To complete Koch's postulates, 9 potato seedlings (cv. Favorita, 15 centimeters high), were transferred to new pots and inoculated with spore suspensions of ZR-IMU141 and ZR-IMU146 (104 CFU/ml), or water as a negative control. Two months later, potato tubers inoculated with either ZR-IMU141 or ZR-IMU146 exhibited typical symptoms of potato common scab, such as superficial or deep, raised, pitted, or polygonal lesions like the field symptoms, but the negative controls remained asymptomatic. The pathogens were reisolated from the lesions and confirmed identical to the original isolate by 16s rRNA gene sequences. To our knowledge, this is the first report of S. stelliscabiei causing potato common scab in Guizhou province, China. We believe that this report will draw attention to the study and management of the increased pool of scab pathogens in China.

First Report of Pectobacterium parmentieri Causing Blackleg on Potato in Inner Mongolia, China.

Blackleg on potato plants (Solanum tuberosum) is caused by Pectobacterium spp. and Dickeya spp. (Charkowski, 2018) worldwide. From June to August in both 2018 and 2019, cases of blackleg were investigated in potato-producing areas in Hulunbuir, Ulanqab, and Hohhot in Inner Mongolia, China. The total surveyed field area was about 200 hectares. The plants showed typical blackleg symptoms, such as black and stunted stems or curled leaves (Fig. S1), and the number of infected plants were counted. The disease showed an incidence of around 8%. Five diseased plants were collected from a 10 ha potato field with approximately 75,000 potato plants (cv. mainly Favorita and Xisen) per hectare. Two-centimeter-long samples of symptomatic stems were removed from the selected plants using a sterile scalpel. The surfaces of the samples were disinfected with 75% ethanol for 2 min. They were then rinsed with sterile distilled water and soaked in 5 ml sterile distilled water for 30 min. Aliquots of three tenfold dilutions of this solution were plated onto the crystal violet pectate agar (CVP) plate and incubated for 3 days at 28°C (Ge et al., 2018). A single bacterial colony that showed pitting on CVP plates (Fig. S2) was picked with a toothpick, streaked onto nutritional agar (She et al., 2013) to obtain pure colonies. Amplification of a 1.4-kb segment containing 16S rRNA gene was performed on the pure colonies using the universal primer set 27F/1492R (Monciardini et al., 2002). The amplicons were sequenced and submitted to the GenBank Nucleotide Basic Local Alignment Search Tool analysis. The 16S rRNA gene sequences of four isolates (GenBank accession numbers: MN626412, MN626449, MN625916, and MT235556) showed more than 99% sequence identity to Pectobacterium parmentieri type strain RNS 08-42-1A (NR_153752.1) (Fig. S3). Six housekeeping genes proA (MT427753-MT427756), gyrA (MT427757-MT427760), icdA (MT427761-MT427764), mdh (MT427765-MT427768), gapA (MT427769-MT427772), and rpoS (MT427773-MT427776) of these four isolates were amplified and sequenced (Ma et al., 2007, Waleron et al., 2008). All sequences showed 99% to 100% sequence identity with Pectobacterium parmentieri strains. Phylogenetic trees (Fig. S4) were constructed by multi-locus sequence analysis (MLSA) using MEGA 6.0 software (Tamura et al., 2013). The samples were tested against Koch's postulates on potato seedlings (cv. Favorita) by injecting 100 µl bacterial suspension (107 CFU/ml) or sterile phosphate buffered solution into the stems 2 cm above the base (Ge et al., 2018). The seedlings were incubated at 21°C and 80% humidity (She et al., 2013). Three to 5 days after inoculation, only infected seedlings showed similar symptoms as those observed in the field: the infected area turned black and rotten (Fig. S5). Bacterial colonies isolated from these symptomatic seedlings were identified using the same methods described above and were identified as inoculated Pectobacterium parmentieri strains. Blackleg on potato plants has been reported to be caused by Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum, and Pectobacterium carotovorum subsp. brasiliense in China (Zhao et al., 2018). To our knowledge, this is the first report of blackleg of potato caused by Pectobacterium parmentieri in Inner Mongolia, China. We believe that this report will draw attention to the identification of this pathogen, which is essential to disease management.

Heparanase induces necroptosis of microvascular endothelial cells to promote the metastasis of hepatocellular carcinoma.

Heparanase (HPSE) is a kind of multifunctional extracellular hydrolase, and related to metastasis of hepatocellular carcinoma (HCC). Endothelial necroptosis promotes the metastasis of cancer cells. It is not clear whether HPSE could mediate necroptosis of microvascular endothelial cells (MVECs) to promote HCC metastasis. Here we found HPSE expression was up-regulated in HCC tissues and its over-expression was correlated with multiple tumor foci, microvascular invasion, and poor outcome of HCC patients. Non-contact co-culture experiments showed high-expressed HPSE in HCC cells mediated the necroptosis of human umbilical vein endothelial cells (HUVECs) and elevated the expression levels of syndecan-1 (SDC-1) and tumor necrosis factor-α (TNF-α) in vitro. As a result of necroptosis, trans-endothelial migration (TEM) of HCC cells was increased. Conversely, both HPSE and SDC-1 knockdowns reversed necroptosis and decreased TNF-α expression level, while HPSE over-expression increased SDC-1 and TNF-α expression and aggravated necroptosis. Animal experiments found that the nude mice, intraperitoneally injected with HPSE high expressing HCC cells, had obvious necroptosis of MVECs and high intrahepatic metastasis rate, which could be relieved by inhibitor of necroptosis. Morever, HPSE elevated the expression levels of p38 mitogen-activated protein kinase (p38 MAPK) rather than nuclear factor kappa B in vitro. Our data suggest that HPSE induces necroptosis of MVECs to promote the metastasis of HCC by activating HPSE/SDC-1/TNF-α axis and p38 MAPK pathway.

Exosomes Derived from Dendritic Cells Attenuate Liver Injury by Modulating the Balance of Treg and Th17 Cells After Ischemia Reperfusion.

BACKGROUND/AIMS: The present study aimed to evaluate the effects as well as the underlying mechanisms of bone marrow-derived dendritic cells (BMDCs) and exosomes produced by BMDCs (DEXs) on hepatic ischemia-reperfusion (I/R) injury (IRI). METHODS: Primary hepatocytes were isolated and used to mimic the liver IR microenvironment. BMDCs were induced and characterized both biochemically with a flow cytometer (FCM) and biophysically with a microscope. Then, we exposed BMDCs to the supernatants from primary hepatocytes and evaluated the maturation of BMDCs by FCM. BMDCs were systemically injected into mice before liver IR via the tail vein, and the therapeutic effects were evaluated. The serum levels of transaminases (aspartate aminotransferase (AST) and alanine aminotransferase (ALT), inflammatory cytokines, and histological changes were respectively examined by ELISA, RT-qPCR and microscopy. Furthermore, we isolated DEXs by ultracentrifugation, characterized DEXs by transmission electron microscopy (TEM) and nanosight tracking analysis (NTA) and western blotting (WB), and then we co-cultured BMDCs/DEXs and naïve T cells and performed FCM, ELISA and confocal imaging. Moreover, we injected DEXs into mice prior to liver IR via the tail vein and examined its therapeutic effects by microscopy and ELISA. Finally, inhibitors of HSP70 (cmHSP70.1), PI3K (BKM120) and mTOR (Rapamycin) were used to investigate the role of HSP70 and the PI3K/mTOR axis in the effects of DEXs on naïve T cells by WB and FCM. RESULTS: Bone marrow cells were efficiently induced into dendritic cells (DCs) with typical DC characteristics. The supernatants from primary hepatocytes exposed to H/R upregulated DC maturation markers. After DC administration, liver IR injury was improved with histopathological scores and serum transaminases. Additionally, we found that the anti-inflammatory cytokines TGF-ß, Foxp3 and interleukin (IL)-10 were upregulated and that IL-17 was downregulated. Furthermore, confocal imaging revealed that the uptake of H/R-DEXs by naïve T cells was greater than that of DEXs derived from the control or negative group of BMDCs, and this increase was correlated with a significantly greater degree of differentiation of Tregs and Th17 cells. Moreover, H/R-DEXs administration improved liver function in mice after IR. Finally, the inhibition of HSP70, PI3K and mTOR completely abolished the effect of DEXs on naïve T cells. CONCLUSION: These results demonstrated that BMDCs and DEXs could alleviate hepatic I/R injury via modulating the balance between Tregs and Th17 cells. DEXs transported HSP70 into naïve T cells and stimulated the PI3K/mTOR axis to modulate the balance between Tregs and Th17 cells and protect the liver from IR injury.

A Self-Supported λ-MnO2 Film Electrode used for Electrochemical Lithium Recovery from Brines.

Lithium recovery from an aqueous resource was accelerated by electrochemically driving the transformation of MnIV /MnIII with a spinel λ-MnO2 film electrode. A λ-MnO2 electrode without binders or conductive additives is preferred for achieving a large capacity at high current density and long-term cycling capability. In this study, a film of Mn(OH)2 was first deposited on the surface of Pt or graphite substrates owing to alkalization near the cathode, then it was oxidized to a Mn3 O4 film by air, followed by being hydrothermally lithiated to LiMn2 O4 spinel and, finally, it was turned into the λ-MnO2 film electrode through potentiostatic delithiation. The results show that the charging/discharging electric capacity of the fabricated λ-MnO2 film electrode was up to ≈100 mAh g-1 at a current density of 50 mA g-1 in 30 mm Li+ aqueous solution, twice that of the λ-MnO2 powder electrode. Also, 82.3 % lithium capacity remained after 100 cycles of an electrochemically assisted lithium recovery process, indicating high availability and good stability of the λ-MnO2 spinel on the electrode. The energy consumption for each cycle is estimated to be approximately 1.55±0.09 J, implying that only 4.14 Wh is required for recovery of one mole of lithium ions by this method.

Mir-24 regulates hepatocyte apoptosis via BIM during acute liver failure.

Acuteliver failure (ALF) has a high mortality rate and is characterized by massive hepatocyte destruction. Although microRNAs (miRNAs) play an important role in manyliver diseases, the role of miRNAs in ALF development is unknown. In this study, the murine ALF model was induced by intraperitoneal injection of D-galactosamine/lipopolysaccharide (D-GalN/LPS). Compared with saline-treated mice, miR-24 was distinctly down-regulated post D-GalN/LPS challenge in vivo and D-galactosamine/tumor necrosis factor (D-GalN/TNF) challenge in vitro, which was confirmed by quantitative real-time polymerase chain reaction. Meanwhile, the mRNA and protein levels of the BH3-only-domain-containing protein BIM were upregulated after challenge both in vivo and in vitro. Previous studies have demonstrated that hepatocyte apoptosis is a distinguishing feature of D-GalN/LPS-associated liver failure. In this study, D-GalN/LPS-challenged mice showed higher alanine aminotransferase and aspartate aminotransferase levels, more severe liver damage, increased numbers of apoptotic hepatocytes and higher levels of caspase-3 compared with saline-treated mice. In D-GalN/TNF-treated BNLCL2 cells, miR-24 overexpression attenuated apoptosis.Furthermore, miR-24 overexpression reduced BIM mRNA and protein levels in vitro. Taken together, these findings demonstrate that miR-24 regulates hepatocyte apoptosis via BIM during ALF development, suggesting that miR-24 is a novel onco-miRNA that may provide potential therapeutic targets for ALF.

Interleukin-11 protects mouse liver from warm ischemia/reperfusion (WI/Rp) injury.

BACKGROUND: IL-11 is a multifunctional cytokine that belongs to the IL-6 family. Previous studies have demonstrated that IL-11 has underlying anti-inflammatory and anti-apoptotic properties. In this study, we evaluated the potential effects of IL-11 on mouse liver WI/Rp injury. METHODS: For in vivo experiments, mice were randomly divided into four main experimental groups (n=5 each): (1) normal group - anesthesia; (2) sham group- laparotomy; (3) I/R group- liver WI/Rp; and (4) IL-11 pretreatment (500µg/kg, tail vein injection) group- administration of RhIL-11 2h before liver WI/Rp induced in the same manner as in group 3. For in vitro experiments, cells were divided into two groups: (1) H/R group- H/R; and (2) IL-11 pretreatment group- pretreatment with RhIL-11 (2µg/mL for 12h) before the induction of H/R. For both groups, three periods of reoxygenation were examined (2h, 6h, and 12h). RESULTS: In the in vivo experiments, IL-11 protected mouse livers from WI/Rp by reducing liver enzyme levels and cellular degeneration. In the in vitro experiments, IL-11 significantly reduced hepatocyte apoptosis. In both the in vivo and in vitro experiments, IL-11 pre-treatment significantly reduced the expression of TNF-α and IL-1ß. In addition, NF-κB, a target of IL-11, was suppressed in macrophages after IL-11 pre-treatment. CONCLUSIONS: Pre-treatment with IL-11 protects mouse livers from WI/Rp injury by suppressing NF-kB activity.