Pm57 from Aegilops searsii encodes a tandem kinase protein and confers wheat powdery mildew resistance.

Powdery mildew is a devastating disease that affects wheat yield and quality. Wheat wild relatives represent valuable sources of disease resistance genes. Cloning and characterization of these genes will facilitate their incorporation into wheat breeding programs. Here, we report the cloning of Pm57, a wheat powdery mildew resistance gene from Aegilops searsii. It encodes a tandem kinase protein with putative kinase-pseudokinase domains followed by a von Willebrand factor A domain (WTK-vWA), being ortholog of Lr9 that mediates wheat leaf rust resistance. The resistance function of Pm57 is validated via independent mutants, gene silencing, and transgenic assays. Stable Pm57 transgenic wheat lines and introgression lines exhibit high levels of all-stage resistance to diverse isolates of the Bgt fungus, and no negative impacts on agronomic parameters are observed in our experimental set-up. Our findings highlight the emerging role of kinase fusion proteins in plant disease resistance and provide a valuable gene for wheat breeding.

The SPL transcription factor TaSPL6 negatively regulates drought stress response in wheat.

Environmental stresses, such as heat and drought, severely affect plant growth and development, and reduce wheat yield and quality globally. Squamosa promoter binding protein-like (SPL) proteins are plant-specific transcription factors that play a critical role in regulating plant responses to diverse stresses. In this study, we cloned and characterized TaSPL6, a wheat orthologous gene of rice OsSPL6. Three TaSPL6 homoeologs are located on the long arms of chromosomes 4A, 5B, and 5D, respectively, and share more than 98% sequence identity with each other. The TaSPL6 genes were preferentially expressed in roots, and their expression levels were downregulated in wheat seedlings subjected to heat, dehydration, and abscisic acid treatments. Subcellular localization experiments showed that TaSPL6 was localized in the nucleus. Overexpression of TaSPL6-A in wheat resulted in enhanced sensitivity to drought stress. The transgenic lines exhibited higher leaf water loss, malondialdehyde and reactive oxygen species (ROS) content, and lower antioxidant enzyme activities after drought treatment than wild-type plants. Gene silencing of TaSPL6 enhanced the drought tolerance of wheat, as reflected by better growth status. Additionally, RNA-seq and qRT-PCR analyses revealed that TaSPL6-A functions by decreasing the expression of a number of genes involved in stress responses. These findings suggest that TaSPL6 acts as a negative regulator of drought stress responses in plants, which may have major implications for understanding and enhancing crop tolerance to environmental stresses.

Structural mechanism of R2D2 and Loqs-PD synergistic modulation on DmDcr-2 oligomers.

Small interference RNAs are the key components of RNA interference, a conserved RNA silencing or viral defense mechanism in many eukaryotes. In Drosophila melanogaster, Dicer-2 (DmDcr-2)-mediated RNAi pathway plays important roles in defending against viral infections and protecting genome integrity. During the maturation of siRNAs, two cofactors can regulate DmDcr-2's functions: Loqs-PD that is required for dsRNA processing, and R2D2 that is essential for the subsequent loading of siRNAs into effector Ago2 to form RISC complexes. However, due to the lack of structural information, it is still unclear whether R2D2 and Loqs-PD affect the functions of DmDcr-2 simultaneously. Here we present several cryo-EM structures of DmDcr-2/R2D2/Loqs-PD complex bound to dsRNAs with various lengths by the Helicase domain. These structures revealed that R2D2 and Loqs-PD can bind to different regions of DmDcr-2 without interfering with each other. Furthermore, the cryo-EM results demonstrate that these complexes can form large oligomers and assemble into fibers. The formation and depolymerization of these oligomers are associated with ATP hydrolysis. These findings provide insights into the structural mechanism of DmDcr-2 and its cofactors during siRNA processing.

Digital Transformation and Green Innovation of Chinese Firms: The Moderating Role of Regulatory Pressure and International Opportunities.

The digitalization of business processes has increasingly challenged conventional wisdom in corporate green innovation. This empirical paper studies the timely but theoretically underexplored relationship between digital transformation and green innovation in a developing country context. Given that firms' digital transformation shifts organizational structures toward decentralization, we employ a digital perspective to analyze organizational coordination, control, and learning mechanisms and propose that digital transformation positively affects corporate green innovation. Moreover, drawing on structural contingency theory, we demonstrate that such effects can be strengthened by external contingencies, specifically regulatory pressure and international opportunities. Using a dataset of Chinese listed firms, we find empirical support for our hypotheses. Our study is one of the first to examine how firms can leverage organizational digital transformation to enhance their green innovation performance and thus provides new insights into the drivers of sustainable practices for firms in developing countries.

Value of 3D Printed PLGA Scaffolds for Cartilage Defects in Terms of Repair.

Objective: To examine the poly (lactic-co-glycolic acid) and sodium alginate (SA) scaffolds produced by 3D printing technology, access the healing morphology of bones following PLGA/SA implantation within rat cartilage, and examine osteogenesis-related factors in rat serum to determine the efficacy of PLGA/SA scaffolds in healing animal cartilage injuries. To identify the potential of this material to repair a tissue engineering osteochondral injury. Methods: Polylactic acid-glycolic acid copolymer and sodium alginate were used as raw materials to create PLGA/SA scaffolds. We observed the scaffold's macrostructure and microstructure, and the scaffold's microstructure was observed through a scanning electron microscope (SEM). The mechanical toughness of a stent was assessed using a biomechanical device. Hematoxylin-eosin staining revealed immune rejection after embedding the scaffolds under the skin of SD rats. The CCK-8 cell proliferation test kit was used to measure cell proliferation. An experimental model of cartilage injury in the knee joint was created in rats. Rats were used to establish an experimental model of cartilage damage in the knee joint. 120 female rats aged 5 weeks were chosen at random from the pool and divided into the experimental and control groups. They were all completely anesthetized with an anesthetic before having the lateral skin of the knee articular cartilage incised. Implanted PLGA/SA scaffolds were not used in the control group and only in the experiment group. Both groups of rats had their muscles and skin sutured and covered in plaster bandages. On the third, seventh, fourteenth, twenty-first, twenty-eighth, and thirty-fifth days after the procedure, the two groups of rats were divided into groups. At various stages, bone tissue, blood samples, and cartilage were examined and evaluated. Immunohistochemistry was used to identify the local bone morphogenetic protein-2 (BMP2). Results: (1) PLGA/SA was successfully used to build an artificial cartilage scaffold. (2) Macroscopic and SEM observation results showed the material had increased density and numerous microvoids on the surface. (3) The result of the biomechanical test showed that the PLGA/SA scaffold had superior biomechanical characteristics. (4) The stent did not exhibit any noticeable immunological rejection, according to the results of the subcutaneous embedding experiment performed on rats. (5) The CCK-8 data demonstrated that as the cell development time rose, the number of cells gradually increased. However, there was not statistically significant difference between the growth of the cells in the scaffold extract and the control group (P > 0.05). (6) A successful rat model based on a cartilage defect of the medial knee joint has been built. (7) Observations of specimens revealed that the experimental group's bone tissue score was higher than that of the control group. (8) Using immunohistochemistry, it was found that the experimental group's BMP2 expression was higher on the 7th, 14th, and 28th days than it was in the control group (P < 0.05). Conclusion: Strong mechanical and biological properties are present in stable, biodegradable PLGA/SA scaffolds that mimic cartilage. We demonstrated that the cartilage biomimetic PLGA/SA scaffold may repair cartilage and prevent negative reactions such as osteoarthritis in rat knee cartilage, making it suitable as a cartilage scaffolding material for tissue engineering. The PLGA/SA scaffold was also able to promote BMP2 expression in the bone healing zone when inserted into a knee cartilage lesion. Improved cartilage damage is the outcome of BMP2's promotion of bone formation and restriction of bone resorption in the bone healing zone.

Mapping of the novel powdery mildew resistance gene Pm2Mb from Aegilops biuncialis based on ph1b-induced homoeologous recombination.

KEY MESSAGE: A novel powdery mildew resistance gene Pm2Mb from Aegilops biuncialis was transferred into common wheat and mapped to chromosome 2MbL bin FL 0.49-0.66 by molecular cytogenetic analysis of 2Mb recombinants. Aegilops biuncialis, a wild relative of common wheat, is highly resistant to powdery mildew. Previous studies identified that chromosome 2Mb in Chinese Spring (CS)-Ae. biuncialis 2Mb disomic addition line TA7733 conferred high resistance to powdery mildew, and the resistance gene was temporarily designated as Pm2Mb. In this study, a total of 65 CS-Ae. biuncialis 2Mb recombinants were developed by ph1b-induced homoeologous recombination and they were grouped into 12 different types based on the presence of different markers of 2Mb-specificity. Segment sizes and breakpoints of each 2Mb recombinant type were further characterized using in situ hybridization and molecular marker analyses. Powdery mildew responses of each type were assessed by inoculation of each 2Mb recombinant-derived F2 progenies using the isolate E05. Combined analyses of in situ hybridization, molecular markers and powdery mildew resistance data of the 2Mb recombinants, the gene Pm2Mb was cytologically located to an interval of FL 0.49-0.66 in the long arm of 2Mb, where 19 2Mb-specific markers were located. Among the 65 2Mb recombinants, T-11 (T2DS.2DL-2MbL) and T-12 (Ti2DS.2DL-2MbL-2DL) contained a small 2MbL segment harboring Pm2Mb. Besides, a physical map of chromosome 2Mb was constructed with 70 2Mb-specific markers in 10 chromosomal bins and the map showed that submetacentric chromosome 2Mb of Ae. biuncialis was rearranged by a terminal intrachromosomal translocation. The newly developed 2Mb recombinants with powdery mildew resistance, the 2Mb-specific molecular markers and the physical map of chromosome 2Mb will benefit wheat disease breeding as well as fine mapping and cloning of Pm2Mb.

Wheat heat shock factor TaHsfA2d contributes to plant responses to phosphate deficiency.

Phosphate (Pi) availability has become a major constraint limiting crop growth and production. Heat shock factors (Hsfs) play important roles in mediating plant resistance to various environmental stresses, including heat, drought and salinity. However, whether members of the Hsf family are involved in the transcriptional regulation of plant responses to Pi insufficiency has not been reported. Here, we identified that TaHsfA2d, a member of the heat shock factor family, was strongly repressed by Pi deficiency. Overexpressing TaHsfA2d-4A in Arabidopsis results in significantly enhanced sensitivity to Pi deficiency, evidenced by increased anthocyanin content, decreased proliferation and elongation of lateral roots, and reduced Pi uptake. Furthermore, RNA-seq analyses showed that TaHsfA2d-4A functions through up-regulation of a number of genes involved in stress responses and flavonoid biosynthesis. Collectively, these results provide evidence that TaHsfA2d participates in the regulation of Pi deficiency stress, and that TaHsfA2d could serve as a valuable gene for genetic modification of crop tolerance to Pi starvation.

Structural insights into dsRNA processing by Drosophila Dicer-2-Loqs-PD.

Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA-silencing mechanism in many eukaryotes1,2. In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), has an important role in generating 21 bp siRNA duplexes from long double-stranded RNAs (dsRNAs)3,4. ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes5,6. Here we report the cryo-electron microscopy structures of Dcr-2-Loqs-PD in the apo state and in multiple states in which it is processing a 50 bp dsRNA substrate. The structures elucidated interactions between Dcr-2 and Loqs-PD, and substantial conformational changes of Dcr-2 during a dsRNA-processing cycle. The N-terminal helicase and domain of unknown function 283 (DUF283) domains undergo conformational changes after initial dsRNA binding, forming an ATP-binding pocket and a 5'-phosphate-binding pocket. The overall conformation of Dcr-2-Loqs-PD is relatively rigid during translocating along the dsRNA in the presence of ATP, whereas the interactions between the DUF283 and RIIIDb domains prevent non-specific cleavage during translocation by blocking the access of dsRNA to the RNase active centre. Additional ATP-dependent conformational changes are required to form an active dicing state and precisely cleave the dsRNA into a 21 bp siRNA duplex as confirmed by the structure in the post-dicing state. Collectively, this study revealed the molecular mechanism for the full cycle of ATP-dependent dsRNA processing by Dcr-2-Loqs-PD.

Development and Characterization of Triticum aestivum-Aegilops longissima 6Sl Recombinants Harboring a Novel Powdery Mildew Resistance Gene Pm6Sl.

Powdery mildew of wheat is a foliar disease that is spread worldwide. Cultivation of resistant varieties is the most effective, economical, and environmentally friendly strategy to curb this disease. Powdery mildew resistance genes (Pm) are the primary resources for resistance breeding, and new Pm genes are in constant demand. Previously, we identified Aegilops longissima chromosome 6Sl#3 as a carrier of powdery mildew resistance and designated the resistance gene as Pm6Sl. Here, we reported the design of 24 markers specific to 6Sl#3 on the basis of the full-length cDNA sequences of 6Sl#3 donor Ae. longissma accession TA1910, and the development of wheat-Ae. longissima 6Sl#3 introgression stocks by ph1b-induced homoeologous recombination. Further, 6Sl#3 introgression lines were identified and characterized by integration analysis of powdery mildew responses, in situ hybridization, and molecular markers and Pm6Sl was mapped to a distal interval of 42.80 Mb between markers Ael58410 and Ael57699 in the long arm of 6Sl#3. Two resistant recombinants, R43 (T6BS.6BL-6Sl#3L) and T27 (Ti6AS.6AL-6Sl#3L-6AL), contained segments harboring Pm6Sl with less than 8% of 6Sl#3 genomic length, and two markers were diagnostic for Pm6Sl. This study broadened powdery mildew resistance gene resources for wheat improvement and provided a fundamental basis for fine mapping and cloning of Pm6Sl to further understand its molecular mechanism of disease resistance.

Characterization of the Wheat Heat Shock Factor TaHsfA2e-5D Conferring Heat and Drought Tolerance in Arabidopsis.

Environmental stresses, especially heat and drought, severely limit plant growth and negatively affect wheat yield and quality worldwide. Heat shock factors (Hsfs) play a central role in regulating plant responses to various stresses. In this study, the wheat heat shock factor gene TaHsfA2e-5D on chromosome 5D was isolated and functionally characterized, with the goal of investigating its role in responses to heat and drought stresses. Gene expression profiling showed that TaHsfA2e-5D was expressed constitutively in various wheat tissues, most highly in roots at the reproductive stage. The expression of TaHsfA2e-5D was highly up-regulated in wheat seedlings by heat, cold, drought, high salinity, and multiple phytohormones. The TaHsfA2e-5D protein was localized in the nucleus and showed a transcriptional activation activity. Ectopic expression of the TaHsfA2e-5D in yeast exhibited improved thermotolerance. Overexpression of the TaHsfA2e-5D in Arabidopsis results in enhanced tolerance to heat and drought stresses. Furthermore, RT-qPCR analyses revealed that TaHsfA2e-5D functions through increasing the expression of Hsp genes and other stress-related genes, including APX2 and GolS1. Collectively, these results suggest that TaHsfA2e-5D functions as a positive regulator of plants' responses to heat and drought stresses, which may be of great significance for understanding and improving environmental stress tolerance in crops.

PBMC transcriptomics identifies immune-metabolism disorder during the development of HBV-ACLF.

OBJECTIVE: Hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) pathophysiology remains unclear. This study aims to characterise the molecular basis of HBV-ACLF using transcriptomics. METHODS: Four hundred subjects with HBV-ACLF, acute-on-chronic hepatic dysfunction (ACHD), liver cirrhosis (LC) or chronic hepatitis B (CHB) and normal controls (NC) from a prospective multicentre cohort were studied, and 65 subjects (ACLF, 20; ACHD, 10; LC, 10; CHB, 10; NC, 15) among them underwent mRNA sequencing using peripheral blood mononuclear cells (PBMCs). RESULTS: The functional synergy analysis focusing on seven bioprocesses related to the PBMC response and the top 500 differentially expressed genes (DEGs) showed that viral processes were associated with all disease stages. Immune dysregulation, as the most prominent change and disorder triggered by HBV exacerbation, drove CHB or LC to ACHD and ACLF. Metabolic disruption was significant in ACHD and severe in ACLF. The analysis of 62 overlapping DEGs further linked the HBV-based immune-metabolism disorder to ACLF progression. The signatures of interferon-related, neutrophil-related and monocyte-related pathways related to the innate immune response were significantly upregulated. Signatures linked to the adaptive immune response were downregulated. Disruptions of lipid and fatty acid metabolism were observed during ACLF development. External validation of four DEGs underlying the aforementioned molecular mechanism in patients and experimental rats confirmed their specificity and potential as biomarkers for HBV-ACLF pathogenesis. CONCLUSIONS: This study highlights immune-metabolism disorder triggered by HBV exacerbation as a potential mechanism of HBV-ACLF and may indicate a novel diagnostic and treatment target to reduce HBV-ACLF-related mortality.

Hippo/Mst1 overexpression induces mitochondrial death in head and neck squamous cell carcinoma via activating ß-catenin/Drp1 pathway.

Mammalian Ste20-like kinase 1 (Mst1) is associated with cell apoptosis. In the current study, we explored the regulatory effects of Mst1 on squamous cell carcinoma of the head and neck (SCCHN) in vitro. SCCHN Cal27 cells and Tu686 cells were transfected with adenovirus-loaded Mst1 to detect the role of Mst1 in cell viability. Then, siRNA against Drp1 was transfected into cells to evaluate the influence of mitochondrial fission in cancer survival. Our data illustrated that Mst1 overexpression promoted SCCHN Cal27 cell and Tu686 cell death via activating mitochondria-related apoptosis. Cells transfected with adenovirus-loaded Mst1 have increased expression of DRP1 and higher DRP1 promoted mitochondrial fission. Active mitochondrial fission mediated mitochondrial damage, as evidenced by increased mitochondrial oxidative stress, decreased mitochondrial energy production, and reduced mitochondrial respiratory complex function. Moreover, Mst1 overexpression triggered mitochondria-dependent cell apoptosis via DRP1-related mitochondrial fission. Further, we found that Mst1 overexpression controlled mitochondrial fission via the ß-catenin/DRP1 pathways; inhibition of ß-catenin and/or knockdown of DRP1 abolished the pro-apoptotic effects of Mst1 overexpression on SCCHN Cal27 cells and Tu686 cells, leading to the survival of cancer cells in vitro. In sum, our results illustrate that Mst1/ß-catenin/DRP1 axis affects SCCHN Cal27 cell and Tu686 cell viability via controlling mitochondrial dynamics balance. This finding identifies Mst1 activation might be an effective therapeutic target for the treatment of SCCHN.

Structural analysis of Phytophthora suppressor of RNA silencing 2 (PSR2) reveals a conserved modular fold contributing to virulence.

Phytophthora are eukaryotic pathogens that cause enormous losses in agriculture and forestry. Each Phytophthora species encodes hundreds of effector proteins that collectively have essential roles in manipulating host cellular processes and facilitating disease development. Here we report the crystal structure of the effector Phytophthora suppressor of RNA silencing 2 (PSR2). PSR2 produced by the soybean pathogen Phytophthora sojae (PsPSR2) consists of seven tandem repeat units, including one W-Y motif and six L-W-Y motifs. Each L-W-Y motif forms a highly conserved fold consisting of five α-helices. Adjacent units are connected through stable, directional linkages between an internal loop at the C terminus of one unit and a hydrophobic pocket at the N terminus of the following unit. This unique concatenation results in an overall stick-like structure of PsPSR2. Genome-wide analyses reveal 293 effectors from five Phytophthora species that have the PsPSR2-like arrangement, that is, containing a W-Y motif as the "start" unit, various numbers of L-W-Y motifs as the "middle" units, and a degenerate L-W-Y as the "end" unit. Residues involved in the interunit interactions show significant conservation, suggesting that these effectors also use the conserved concatenation mechanism. Furthermore, functional analysis demonstrates differential contributions of individual units to the virulence activity of PsPSR2. These findings suggest that the L-W-Y fold is a basic structural and functional module that may serve as a "building block" to accelerate effector evolution in Phytophthora.

A Dynamic Model for Predicting Outcome in Patients with HBV Related Acute-On-Chronic Liver Failure.

INTRODUCTION AND AIM: Accurately predicting the prognosis of individual patient is crucial in the management of ACLF. We aimed to establish a specific prognostic model for HBV-related ACLF patients treated with nucleoside analog (NA). MATERIAL AND METHODS: We prospectively collected 205 ACLF cases diagnosed according to the APASL criteria. A dynamic prognostic model based on APASL criteria was established and validated. To demonstrate that the model is also applicable to those within EASL criteria, we divided the patients into two groups: met APASL criteria only (group A, n = 123); met both APASL and EASL criteria (group B, n = 82). Its prognostic accuracy was also compared with chronic liver failure-sequential organ failure assessment (CLIF-SOFA) score in group B. RESULTS: The model is: R = 0.94 x Bilirubin + 0.53 x evolution of Bilirubin - 0.45 x PT-A - 0.22 x evolution in PT-A -0.1 x PLT + 10 x anti-HBe. The area under receiver operating characteristic curve (AUC) of the model for predicting 90-day mortality was 0.86, which was significantly higher than that of model for end stage liver disease(MELD), MELD-Na, CLIF-SOFA, ΔMELD (7d) and ΔMELD-Na (7d), ΔCLIF- SOFA(7d) (all p < 0.01). The AUC of our model in the validation group was 0.79 which was superior to MELD (0.45) CLIF-SOFA (0.53) score in group B patients (p < 0.01). CONCLUSION: In conclusion, the model was superior to the conventional methods in predicting the outcomes of patients with HBV related ACLF treated with NA. It is the first description of a novel prognostic model using consecutive data in patients with HBV-induced acute-on-chronic liver failure (ACLF) treated by nucleoside analogs.

YTHDF2 destabilizes m(6)A-containing RNA through direct recruitment of the CCR4-NOT deadenylase complex.

Methylation at the N6 position of adenosine (m(6)A) is the most abundant RNA modification within protein-coding and long noncoding RNAs in eukaryotes and is a reversible process with important biological functions. YT521-B homology domain family (YTHDF) proteins are the readers of m(6)A, the binding of which results in the alteration of the translation efficiency and stability of m(6)A-containing RNAs. However, the mechanism by which YTHDF proteins cause the degradation of m(6)A-containing RNAs is poorly understood. Here we report that m(6)A-containing RNAs exhibit accelerated deadenylation that is mediated by the CCR4-NOT deadenylase complex. We further show that YTHDF2 recruits the CCR4-NOT complex through a direct interaction between the YTHDF2 N-terminal region and the SH domain of the CNOT1 subunit, and that this recruitment is essential for the deadenylation of m(6)A-containing RNAs by CAF1 and CCR4. Therefore, we have uncovered the mechanism of YTHDF2-mediated degradation of m(6)A-containing RNAs in mammalian cells.

Targeted delivery of tumor antigens to activated dendritic cells via CD11c molecules induces potent antitumor immunity in mice.

PURPOSE: CD11c is an antigen receptor predominantly expressed on dendritic cells (DC), to which antigen targeting has been shown to induce robust antigen-specific immune responses. To facilitate targeted delivery of tumor antigens to DCs, we generated fusion proteins consisting of the extracellular domain of human HER or its rat homologue neu, fused to the single-chain fragment variable specific for CD11c (scFv(CD11c)-HER2/neu). EXPERIMENTAL DESIGN: Induction of cellular and humoral immune responses and antitumoral activity of the fusion proteins admixed with DC-activating CpG oligonucleotides (scFv(CD11c)-HER2/neu(CpG)) were tested in transplantable HER2/neu-expressing murine tumor models and in transgenic BALB-neuT mice developing spontaneous neu-driven mammary carcinomas. RESULTS: Vaccination of BALB/c mice with scFv(CD11c)-HER2(CpG) protected mice from subsequent challenge with HER2-positive, but not HER2-negative, murine breast tumor cells, accompanied by induction of strong HER2-specific T-cell and antibody responses. In a therapeutic setting, injection of scFv(CD11c)-HER2(CpG) caused rejection of established HER2-positive tumors. Importantly, antitumoral activity of such a fusion protein vaccine could be reproduced in immunotolerant BALB-neuT mice, where scFv(CD11c)-neu(CpG) vaccination significantly protected against a subsequent challenge with neu-expressing murine breast tumor cells and markedly delayed the onset of spontaneous mammary carcinomas. CONCLUSIONS: CD11c-targeted protein vaccines for in vivo delivery of tumor antigens to DCs induce potent immune responses and antitumoral activities and provide a rationale for further development of this approach for cancer immunotherapy.

Tumor-targeted PE38KDEL delivery via PEGylated anti-HER2 immunoliposomes.

We previously reported the development of PE38KDEL-loaded anti-HER2 poly(lactic-co-glycolic acid) (PLGA) nanoparticles that bind and internalize in HER2-overexpressing breast cancer cells, enabling potent anti-tumor activity. To overcome the problems associated with the short half-lives of this drug delivery system, we have constructed PE38KDEL-loaded anti-HER2 PEGylated liposomes (PE-HER-liposomes). PE-HER-liposomes were constructed with Fab' of recombinant humanized anti-HER2 monoclonal antibody (anti-HER2 Fab') covalently linked to PEGylated liposomes containing PE38KDEL (PE-liposomes). We attached anti-HER2 Fab' to the terminus of PEG (polyethylene glycol) on PEGylated liposomes. Incorporation of pyridylthiopropionoylamino-PEG-distearoylphosphatidylethanolamine (PDP-PEG-DSPE) into PEGylated liposomes followed by mild thiolysis of the PDP groups resulted in the formation of reactive thiol groups at the periphery of the liposomes. Efficient attachment of maleimide-derivatized anti-HER2 Fab' took place under mild conditions. The characterization of PE-HER-liposomes, such as particle size, was evaluated by dynamic light-scattering detector. The Micro BCA method was used to determine the encapsulation efficiency of PE38KDEL and the quantity of conjugated Fab'. Flow cytometry and confocal microscopy showed that PE-HER-liposomes possessed receptor-specific binding and internalization for HER2-overexpressing SK-BR3 cells. Remarkably, PE-HER-liposomes were more cytotoxic than non-targeted PE-liposomes in HER2-overexpressing breast cancer cells. In conclusion, PE-HER-liposomes could serve as a promising therapeutic candidate for the treatment of HER2-overexpressing breast cancers.

[Survival analysis on liver failure patients treated with an artificial liver support system].

OBJECTIVE: To evaluate the efficacy of artificial liver support system (ALSS) in the treatment of liver failure patients. METHODS: This is a prospective, multi-center, controlled, large sample clinic trial. 518 patients with liver failure from 5 hospitals were studied and followed. All the patients received similar pharmacological manipulation according to one and the same protocol but were divided into an ALSS treatment group and a control group without ALSS treatment. The ALSS treatment procedures included plasma exchange, molecular adsorbent recirculating system (MARS), plasma exchange plus hemofiltration and other combined nonbioartificial methods. The analysis of survival time was computed using the Kaplain-Maier method, and comparison among groups was done using Log-Rank, Breslow and/or the Tarone-Ware test. RESULTS: Survival time of acute liver failure patients was prolonged from 4.0+/-0.2 days to 8.0+/-0.4 days (P=0.004). ALSS was shown to be two times more effective. ALSS increased the survival time of acute on chronic (A on C) liver failure patients from 27.0+/-1.6 days to 39.0+/-4.0 days (P less than 0.01). In addition, it increased the survival time of the patients in the middle and end stage of subacute liver failure and A on C liver failure, but had no significant effects on early stage patients. The survival time of middle stage patients was 38.0+/-17.5 days in the control group vs 66.0+/-18.6 days in the ALSS group (P less than 0.05). The survival time of end stage patients of the control group and the ALSS group was 18.0+/-4.0 days vs 26.0+/-2.5 days (P less than 0.01). CONCLUSIONS: Multi ALSS treatment is more effective than the standard medicinal liver care treatment. Multi-ALSS treatment could increase survival time of patients suffering from acute liver failure or A on C liver failure, especially in their middle and end stages. It is important and necessary to treat these patients with ALSS.