Tandem bispecific CD123/CLL-1 CAR-T cells exhibit specific cytolytic effector functions against human acute myeloid leukaemia.

OBJECTIVES: The treatment of refractory and recurrent acute myeloid leukaemia (AML) is still a challenge with poor response rates and short survival times. In an attempt to solve this problem, we constructed a tandem bispecific chimeric antigen receptor (CAR) targeting CD123 and C-type lectin-like molecule 1 (CLL-1), two different AML antigens, and verified its cytotoxic effects in vitro. METHODS: We established and cultured K562 cell lines expressing both CD123 and CLL1 antigens. Single-target CAR-T cells specific to CD123 and CLL1 were engineered, alongside tandem CD123/CLL1 bispecific CAR-T cells. Flow cytometry was used to determine cell phenotypes, transfection efficiencies, cytokine release, and CAR-T-cell proliferation, and an lactate dehydrogenase assay was used to detect the cytotoxicity of CD123/CLL-1 bispecific tandem CAR-T cells in vitro. RESULTS: Two types of tandem CAR-T cells exhibited significant killing effects on CLL-1 + CD123+ leukaemia cell lines and primary AML tumour cells. The killing efficiency of tandem CAR-T cells in the case of single antigen expression is comparable to that of single target CAR-T cells. When faced with dual target tumour cells, dual target CAR-T cells significantly surpass single target CAR-T cells. CD123/CLL-1 CAR-T cells in tandem targeted and killed CD123- and CLL-1-positive leukaemia cell lines and released a large number of cytokines. CONCLUSIONS: CD123/CLL-1 CAR-T cells in tandem can simultaneously target CD123 and CLL-1 on AML cells, demonstrating a significant ability to kill single antigens and multi-target tumour cells. This suggests that CD123/CLL-1 CAR-T cells exhibit significant advantages in the expression of multiple antigens in a wide range of target cells, which may help overcome the challenges posed by tumour heterogeneity and evasion mechanisms.

The B56γ3-containing protein phosphatase 2A attenuates p70S6K-mediated negative feedback loop to enhance AKT-facilitated epithelial-mesenchymal transition in colorectal cancer.

BACKGROUND: Protein phosphatase 2A (PP2A) is one of the major protein phosphatases in eukaryotic cells and is essential for cellular homeostasis. PP2A is a heterotrimer comprising the dimeric AC core enzyme and a highly variable regulatory B subunit. Distinct B subunits help the core enzyme gain full activity toward specific substrates and contribute to diverse cellular roles of PP2A. PP2A has been thought to play a tumor suppressor and the B56γ3 regulatory subunit was shown to play a key tumor suppressor regulatory subunit of PP2A. Nevertheless, we uncovered a molecular mechanism of how B56γ3 may act as an oncogene in colorectal cancer (CRC). METHODS: Polyclonal pools of CRC cells with stable B56γ3 overexpression or knockdown were generated by retroviral or lentiviral infection and subsequent drug selection. Co-immunoprecipitation(co-IP) and in vitro pull-down analysis were applied to analyze the protein-protein interaction. Transwell migration and invasion assays were applied to investigate the role of B56γ3 in affecting motility and invasive capability of CRC cells. The sensitivity of CRC cells to 5-fluorouracil (5-FU) was analyzed using the PrestoBlue reagent assay for cell viability. Immunohistochemistry (IHC) was applied to investigate the expression levels of phospho-AKT and B56γ3 in paired tumor and normal tissue specimens of CRC. DataSets of TCGA and GEO were analyzed to investigate the correlation of B56γ3 expression with overall survival rates of CRC patients. RESULTS: We showed that B56γ3 promoted epithelial-mesenchymal transition (EMT) and reduced the sensitivity of CRC cells to 5-FU through upregulating AKT activity. Mechanistically, B56γ3 upregulates AKT activity by targeting PP2A to attenuate the p70S6K-mediated negative feedback loop regulation on PI3K/AKT activation. B56γ3 was highly expressed and positively correlated with the level of phospho-AKT in tumor tissues of CRC. Moreover, high B56γ3 expression is associated with poor prognosis of a subset of patients with CRC. CONCLUSIONS: Our finding reveals that the B56γ3 regulatory subunit-containing PP2A plays an oncogenic role in CRC cells by sustaining AKT activation through suppressing p70S6K activity and suggests that the interaction between B56γ3 and p70S6K may serve as a therapeutic target for CRC. Video Abstract.

Preliminary evaluation of SaCoVLM video laryngeal mask-guided intubation in airway management for anesthetized children.

BACKGROUNDS: To preliminary evaluate the application of novel SaCoVLM video laryngeal mask -guided intubation for anesthetized children. METHODS: One hundred twenty-four children with microtia (ages 5-15 years,) who required general intubation anaesthesia, were enrolled in the study. After induction of general anesthesia,guided tracheal intubation under direct vision of the SaCoVLM was performed. Our primary outcome was first-pass success rate of guided tracheal tube placement. Secondary outcome included glottic visualization grades, the first-attempt success rate of LMA placement, the time for LMA placement and time to endotracheal intubation as well as the time for LMA removal after successful intubation, the fiberoptic grade of laryngeal view, the baseline and postinduction hemodynamic parameters were also recorded,and the incidence 24 h complications after operation. RESULTS: The first-pass success rate of guided tracheal tube placement was 91.1% (95%CI = 1.04-1.14), the status of glottic visualization was classified: grade 1 in 27cases, grade 2 in 36 cases, grade 3 in 41 cases and grade 4 in 20 cases. The first success rate of LMA placement was 92.7% (95%CI = 1.03-1.13), the time for LMA insertion was 15.7 (±9.1) s, intubation time was 30.9 (±17.6) s and withdrawl time was 24.9 (±9.3) s. The incidence of postoperative sore throat at 2 h was 29%, and 16.1% at 24 h, without dysphagia and hypoxia. CONCLUSION: The SaCoVLM video laryngeal mask-guided intubation is feasible in children, with a high success rate, could be a new promising device to guide intubation in airway management. TRIAL REGISTRATION: This study was approved by the University's Institutional Review Board and written informed consent was obtained from all subjects participating in the trial. The trial was registered prior to patient enrollment at clinicaltrials.gov (ChiCTR2200061481, http://www.chictr.org.cn . Principal investigator: Juan Zhi; Date of registration: 26/06/2022.

First report of apple root rot caused by Phytopythium vexans in China.

Apple production is of great economic importance in the fruit industry of China, where Yunnan Province is considered as a major producing area. A survey was conducted to identify apple trees that were problematic from March to November 2020 in Yunnan Province. Symptoms included smaller yellowing leaves, fewer sprouts per branch, browning and necrosis of the roots and lower parts of the stem bark, and wilting. 20% to 45% of apple trees were found infected and randomly scattered in the surveyed orchards. A total of 110 soil samples were collected from the root area of symptomatic apple trees in Tuanjie Town of Kunming City, Zhaotong City, and Malong District of Qujing City in Yunnan Province. Two grams of each soil sample was suspended in 400 ml of sterile water for three days and each soil extract was baited with two apple leaves (Red Fuji's). Following the baiting, those leaves were cut into 10 pieces (5mm×5mm), surface-sterilized with 70% ethanol for 30 seconds, rinsed three times with sterile water, and then air-dried. Each leaf piece was placed in a Petri dish with the oatmeal agar medium containing PCNB 20 mg/ml, rifampicin 20 mg/ml, and then incubated at 25℃ in the dark for 3 days. A mycelial agar plug was picked from the edge of the colonies and transferred to a fresh Potato Dexrose Agar (PDA) plate. Seventy colonies with similar growing characteristics were isolated from the 110 soil samples. Three isolates were retained for further analysis and named XLD8-1, SD1, and YF2. After being cultivated on PDA plates and incubated at 25℃ in the dark for 4 days, their colonies were rose petal-type and white with dense aerial hyphae (Fig 1, A). In ten days of incubation, oogonium measuring 24.55 ± 1.9µm × 20.27 ± 2.3µm and sporangia measuring 21.65 ± 1.3µm × 19.35 ± 1µm were observed (Fig 1, C, D). The total DNA of the isolates was extracted and amplified using three pairs of primers, ITS1/ITS4 (White et al. 1990), LROR/LR7 (LSU) (Vilgalys R, et al. 1990), and FM58/FM66 (COXⅡ) (Martin F N. 2000). The sequences were uploaded to GenBank (Accession No. OL960234, OK037658, OK052604 for ITS, OL960388, OM838413, OM838314 for LSU, and OM962847, OM962848, OM962849 for COXⅡ). ITS sequences of the three isolates (XLD8-1, SD1, YF2) showed 99.87%,99.87%, 99.87% similar to Pp. vexans (Accession No. AB468784, AB468784, and AM701801). LSU sequences of the three isolates showed 99.92%, 99.72%, 100% similar to Pp. vexans (Accession No. EF426541, MT729990, and EF426541). COXⅡ sequences of the three isolates showed 100%, 99.81%, 99.81% similar to Pp. vexans (Accession No. GU133560). Based on the sequence similarity and morphology, the isolates were identified as Phytopythium vexans. Koch's postulates were conducted by wounding the bases of 3 apple seedlings (1-year-old Red Fuji's) with a cork borer. A plug of mycelium of the isolate XLD8-1 grown on PDA plates was placed on each wound (Fig 1, B). Controls were set up to use sterile agar plugs as an inoculum. Seedlings have incubated an incubator at 23-26°C under the alternating light and dark intervals, 12-hours of each. In 15 days, after were inoculated with XLD8-1 the roots and lower part of the stem bark of those seedlings became brownish and necrotic, and their epidermis was easily sloughed off (Fig 1, E-G). The pathogen isolated from the necrotic root tissues were identical to the isolate XLD8-1. Symptoms of apple growth decline caused by Pp. vexans were reported in Morocco (Jabiri Salma, et al. 2021). This experiment verified that Pp. vexans causes root rot of apple. In China, Fusarium sp. is usually considered the main pathogen causing apple root rot. However, the discovery of large numbers of apple trees that were infected by Pp. vexans in Yunnan Province and the confirmation of pathogenicity of Pp. vexans on apple seedlings have demonstrated for the first time that Pp. vexans could cause apple root rot as Fusarium spp does and become an incoming threat to the apple industry, which lays the foundation for study on the disease epidemiology and integrated management of apple root rot in China. References: Jabiri Salma, et al. 2021. Microorganisms, doi:10.3390/MICROORGANISMS9091916. Martin, F. N. 2000. Mycologia, 92(4), 711-727. Vilgalys R., et al. 1990. Journal of Bacteriology, 172:4238-4246 White, T. J., et al. 1990. PCR Protocols: a guide to methods and applications, 18: 315.

The inter-observer agreement in the assessment of carotid plaque neovascularization by contrast-enhanced ultrasonography: The impact of plaque thickness.

BACKGROUND: The interobserver agreement in the assessment of the grade of carotid plaque neovascularization by contrast-enhanced ultrasonography is poorly established. METHOD: We examined 140 carotid plaques in 66 patients (all patients had bilateral plaques, and 8 patients had 2 plaques on one side). We performed conventional and contrast-enhanced ultrasonography to analyze the presence of carotid plaque neovascularization, which was graded by two independent observers whose interobserver agreement (κ) was evaluated according to the thickness of carotid plaque. RESULTS: For all carotid plaques, the mean κ was 0.689 (95% confidence interval 0.604-0.774). It was 0.689 (0.569-0.808), 0.637 (0.487-0.787), and 0.740 (0.585-0.896), respectively for carotid plaques with maximal thickness <2 mm, from 2 mm to 3 mm, and >3 mm. CONCLUSION: The interobserver agreement for assessing carotid plaque neovascularization by using contrast-enhanced ultrasonography is substantial and acceptable for research purposes, regardless of the maximal thickness of the plaque.

Comparative genomics of rhizobia nodulating soybean suggests extensive recruitment of lineage-specific genes in adaptations.

The rhizobium-legume symbiosis has been widely studied as the model of mutualistic evolution and the essential component of sustainable agriculture. Extensive genetic and recent genomic studies have led to the hypothesis that many distinct strategies, regardless of rhizobial phylogeny, contributed to the varied rhizobium-legume symbiosis. We sequenced 26 genomes of Sinorhizobium and Bradyrhizobium nodulating soybean to test this hypothesis. The Bradyrhizobium core genome is disproportionally enriched in lipid and secondary metabolism, whereas several gene clusters known to be involved in osmoprotection and adaptation to alkaline pH are specific to the Sinorhizobium core genome. These features are consistent with biogeographic patterns of these bacteria. Surprisingly, no genes are specifically shared by these soybean microsymbionts compared with other legume microsymbionts. On the other hand, phyletic patterns of 561 known symbiosis genes of rhizobia reflected the species phylogeny of these soybean microsymbionts and other rhizobia. Similar analyses with 887 known functional genes or the whole pan genome of rhizobia revealed that only the phyletic distribution of functional genes was consistent with the species tree of rhizobia. Further evolutionary genetics revealed that recombination dominated the evolution of core genome. Taken together, our results suggested that faithfully vertical genes were rare compared with those with history of recombination including lateral gene transfer, although rhizobial adaptations to symbiotic interactions and other environmental conditions extensively recruited lineage-specific shell genes under direct or indirect control through the speciation process.

Genetic divergence of bradyrhizobium strains nodulating soybeans as revealed by multilocus sequence analysis of genes inside and outside the symbiosis island.

The genus Bradyrhizobium has been considered to be a taxonomically difficult group. In this study, phylogenetics and evolutionary genetics analyses were used to investigate divergence levels among Bradyrhizobium strains nodulating soybeans in China. Eleven genospecies were identified by sequence analysis of three phylogenetic and taxonomic markers (SMc00019, thrA, and truA). This was also supported by analyses of eight genes outside the symbiosis island ("off-island" genes; SMc00019, thrA, truA, fabB, glyA, phyR, exoN, and hsfA). However, seven genes inside the symbiosis island ("island" genes; nifA, nifH, nodC, nodV, fixA, trpD, and rhcC2) showed contrasting lower levels of nucleotide diversity and recombination rates than did off-island genes. Island genes had significantly incongruent gene phylogenies compared to the species tree. Four phylogenetic clusters were observed in island genes, and the epidemic cluster IV (harbored by Bradyrhizobium japonicum, Bradyrhizobium diazoefficiens, Bradyrhizobium huanghuaihaiense, Bradyrhizobium liaoningense, Bradyrhizobium daqingense, Bradyrhizobium sp. I, Bradyrhizobium sp. III, and Bradyrhizobium sp. IV) was not found in Bradyrhizobium yuanmingense, Bradyrhizobium sp. II, or Bradyrhizobium elkanii. The gene flow level of island genes among genospecies is discussed in the context of the divergence level of off-island genes.

Replicon-dependent differentiation of symbiosis-related genes in Sinorhizobium strains nodulating Glycine max.

In order to investigate the genetic differentiation of Sinorhizobium strains nodulating Glycine max and related microevolutionary mechanisms, three housekeeping genes (SMc00019, truA, and thrA) and 16 symbiosis-related genes on the chromosome (7 genes), pSymA (6 genes), and pSymB (3 genes) were analyzed. Five distinct species were identified among the test strains by calculating the average nucleotide identity (ANI) of SMc00019-truA-thrA: Sinorhizobium fredii, Sinorhizobium sojae, Sinorhizobium sp. I, Sinorhizobium sp. II, and Sinorhizobium sp. III. These species assignments were also supported by population genetics and phylogenetic analyses of housekeeping genes and symbiosis-related genes on the chromosome and pSymB. Different levels of genetic differentiation were observed among these species or different replicons. S. sojae was the most divergent from the other test species and was characterized by its low intraspecies diversity and limited geographic distribution. Intergenic recombination dominated the evolution of 19 genes from different replicons. Intraspecies recombination happened frequently in housekeeping genes and symbiosis-related genes on the chromosome and pSymB, whereas pSymA genes showed a clear pattern of lateral-transfer events between different species. Moreover, pSymA genes were characterized by a lower level of polymorphism and recombination than those on the chromosome and pSymB. Taken together, genes from different replicons of rhizobia might be involved in the establishment of symbiosis with legumes, but these symbiosis-related genes might have evolved differently according to their corresponding replicons.

Current understanding of genomic DNA of porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in swine and is also described as the modulator of host immunity that exacerbates the clinical outcome of many bacterial and viral infections. To date, it has caused increasingly larger losses in the pig industry worldwide. The genomic DNA of PCV2 is predicted to contain 11 open reading frames (ORFs) and at least seven potential ORFs-encoding proteins larger than 5 kDa. Currently, however, only five virally encoded proteins (Rep, Rep', Cap, ORF3, and ORF4 protein) have been identified in PCV2 replication. In the present review, we strive to discuss the current understanding of the genomic DNA of PCV2 with the purpose of providing insight into the scientific basis of the pathogenesis of PCV2 and the prevention of its infection.

Low-dose cytarabine and aclarubicin combined with granulocyte colony-stimulating factor for the treatment of relapsed or primary refractory acute lymphocytic leukemia: a retrospective study of 25 Chinese patients.

Despite improvements in treatment, the prognosis of relapsed or primary refractory acute lymphocytic leukemia (ALL) remains poor, and outcomes are worse in older adults with the short first complete remission (CR). Attainment of the second CR by salvage therapy would improve the survival of these patients and may enable them to undergo curative treatment with allogeneic hematopoietic stem cell transplantation. The fact that there are diverse salvage protocols for these adult patients but without a striking CR-induction efficacy indicates that efforts are still needed to indentify new effective reinduction regimens. In this study, the CAG regimen (cytarabine, 10 mg/m(2) subcutaneously every 12 h on days 1-14; aclarubicin, 5-7 mg/m(2) intravenously daily on days 1-8; and concurrent granulocyte colony-stimulating factor, 200 µg/m(2) /day subcutaneously) was administered to 25 patients with relapsed or refractory ALL, including 11 T-cell ALL (T-ALL) and 14 B-cell (B-ALL) patients (age range, 11-61 years; median age, 26 years), to assess its efficacy as a salvage therapy. One course of the CAG regimen resulted in an overall response [CR or partial remission (PR)] rate of 64%, a CR rate of 56% and generally mild adverse effects. An overall response was observed in all 11 T-ALL patients (10 CR and 1 PR) and 35.7% of B-ALL patients (p = 0.0009). The significant treatment potential of CAG regimen for relapsed or primary refractory ALL, especially for T-ALL patients, described in this report would prepare them for a second CR to pursue longer survival.

Bradyrhizobium daqingense sp. nov., isolated from soybean nodules.

Thirteen slow-growing rhizobial strains isolated from root nodules of soybean (Glycine max L.) grown in Daqing city in China were classified in the genus Bradyrhizobium based on 16S rRNA gene sequence analysis. Multilocus sequence analysis of IGS, atpD, glnII and recA genes revealed that the isolates represented a novel clade in this genus. DNA-DNA relatedness lower than 42.5 % between the representative strain CCBAU 15774(T) and the type strains of the closely related species Bradyrhizobium liaoningense USDA 3622(T), Bradyrhizobium yuanmingense CCBAU 10071(T) and Bradyrhizobium betae LMG 21987(T), further confirmed that this group represented a novel species. CCBAU 15774(T) shared seven cellular fatty acids with the three above-mentioned species, but the fatty acids 15 : 0 iso and summed feature 5 (18 : 2ω6,9c and/or 18 : 0 anteiso) were unique for this strain. The respiratory quinone in CCBAU 15774(T) was ubiquinone-10 and the cellular polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, cardiolipin and unknown aminolipid, polar lipid and phospholipid. In addition, some phenotypic features could be used to differentiate the novel group from the related species. On basis of these results, we propose the name Bradyrhizobium daqingense sp. nov., with CCBAU 15774(T) ( = LMG 26137(T) = HAMBI 3184(T) = CGMCC 1.10947(T)) as the type strain. The DNA G+C content of the type strain is 61.2 mol% (T(m)).

[Change and significance of Th22 cells in patients with aplastic anemia].

OBJECTIVE: To explore the proportion of Th22 cells in peripheral blood of patients with aplastic anemia (AA) and evaluate its significance. METHODS: From January 2011 to June 2012, a total of 47 AA patients were recruited and divided into 4 groups: severe aplastic anemia (SAA) pre-therapy (group A, n = 11), non-severe aplastic anemia (NSAA) pre-therapy (group B, n = 12), SAA post-therapy (group C, n = 12), NSAA post-therapy (group D, n = 12) and healthy donor controls (n = 12). The proportion of Th22 cells in peripheral blood of each group was evaluated by flow cytometry. The cytokines interleukin-22 (IL-22), transforming growth factor-ß (TGF-ß), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured by ELISA. And the level of IL-22 mRNA was examined by reverse transcription-PCR (RT-PCR). RESULTS: The percentage of Th22 cells and the level of IL-22, TNF-α, IL-6 and IL-22 mRNA in group A (4.3% ± 1.4%, (57 ± 17) ng/L, (497 ± 123) ng/L, (323 ± 88) ng/L, 1.65 ± 0.51) and group C (2.6% ± 0.6%, (34 ± 10) ng/L, (314 ± 79) ng/L, (187 ± 45) ng/L, 0.92 ± 0.28) were significantly higher than that in control group (1.2% ± 0.3%, (19 ± 6) ng/L, (228 ± 50) ng/L, (134 ± 26) ng/L, 0.47 ± 0.09,all P < 0.05). The percentage of Th22 cells and the level of IL-22, TNF-α , IL-6 and IL-22 mRNA in group A were higher than those in group C (all P < 0.05). NSAA patients had similar results. The percentage of Th22 cells and the level of IL-22, TNF-α , IL-6 and IL-22 mRNA in group A were higher than those in group B (all P < 0.05). But the level of TGF-ß in groups A and C were significantly lower than that in control group ((3.4 ± 1.1) and (5.8 ± 1.7) vs (9.7 ± 2.8) ng/L, P < 0.05). And the level of TGF-ß in group A was lower than that of group B (P < 0.05). CONCLUSIONS: The number of Th22 cells is elevated in AA patients. Th22 cells may be positively correlated with the development of AA. And a higher level of TNF-α, IL-6 and a lower level of TGF-ß promote the differentiation of Th22 cells.

[Comparison of Cookgas and Fastrach intubating laryngeal mask airway with fiberoptic bronchoscope in anticipated difficult airway management].

OBJECTIVE: To compare the clinical effectiveness of fiberoptic bronchoscope (FOB)-guided intubation through the Cookgas intubating laryngeal airway(CILA)and the Fastrach intubating laryngeal mask airway (FT-LMA) in the management of anticipated difficult airways. METHODS: Sixty patients with all three difficult intubation criterion (thyromental distance<60 mm, interincisor distance<35 mm, and Mallampati class 3 or 4) undergoing elective plastic surgery under general anesthesia were randomly allocated into CILA group (n=30) and FT-LMA group (n=30). After anesthesia being induced and CILA or FT-LMA being inserted, the patients were treated with FOB-guided intubation through CILA or FT-LMA. The success of the intubating laryngeal airway(ILA)insertion and FOB-guided intubation, the number of attempts, and the duration of the successful attempt were recorded. RESULTS: The ILA was inserted successfully in 30 patients from CILA group and 27 patients from FT-LMA group. Three failed cases in FT-LMA group were inserted successfully with CILA. In CILA group, the first FOB-guided intubation attempt succeeded in 26 patients, and 4 cases were intubated at the second attempt. In 27 patients of FT-LMA group, 20 cases were intubated successfully at the first attempt, 4 cases at the second attempt, and 3 cases failed; of these three failed patients, two patients were intubated smoothly with FOB through CILA at the first attempt, one was intubated by FOB via CILA at the second attempt. The duration of FT-LMA insertion [(35.3±12.8)s] was significantly longer when compared with CILA [(23.9±17.5)s] (P<0.05). However, the duration of FOB-guided intubation through CILA and FT-LMA [(48.6±13.5)s vs.(53.2±14.2)s] and the time of ILA removal [(40.4±10.2)s vs. (38.5±11.3)s] were not significantly different between these two groups (P>0.05). The adverse events during and after intubtion were not significantly different between these two groups. CONCLUSIONS: FOB-guided intubation through CILA and FT-LMA is safe and feasible for the management of anticipated difficult airways. However, in patients with severe scar contracture of face and neck and those with huge expander in neck, the CILA insertion and FOB-guided intubation via CILA is superior to FT-LMA.

[Clinical Observation of Venetoclax Combined with Demethylating Agents on the Treatment of Relapsed/Refractory Acute Myeloid Leukemia].

OBJECTIVE: To investigate the efficacy and safety of venetoclax (VEN) combined with demethylating agents (HMA) in the treatment of relapsed/refractory acute myeloid leukemia (R/R AML). METHODS: The clinical data of 26 adult R/R AML patients who received the combination of VEN with azacitidine (AZA) or decitabine (DAC) in Huai'an Second People's Hospital from February 2019 to November 2021 were retrospectively analyzed. The treatment response, adverse events as well as survival were observed, and the factors of influencing the efficacy and survival were explored. RESULTS: The overall response rate (ORR) of 26 patients was 57.7% (15 cases), including 13 cases of complete response (CR) and CR with incomplete count recovery (CRi) and 2 cases of partial response (PR). Among the 13 patients who got CR/CRi, 7 cases achieved CRm (minimal residual disease negative CR) and 6 cases did not, with statistically significant differences in overall survival (OS) and event-free survival (EFS) between the two groups (P=0.044, 0.036). The median OS of all the patients was 6.6 (0.5-15.6) months, and median EFS was 3.4 (0.5-9.9) months. There were 13 patients in the relapse group and refractory group, respectively, with response rate of 84.6% and 30.8% (P=0.015). The survival analysis showed that the relapse group had a better OS than the refractory group (P=0.026), but there was no significant difference in EFS (P=0.069). Sixteen patients who treated for 1-2 cycles and 10 patients who treated for more than 3 cycles achieved response rates of 37.5% and 90.0%, respectively (P=0.014), and patients treated for more cycles had superior OS and EFS (both P<0.01). Adverse effects were mainly bone marrow suppression, complicated by various degrees of infection, bleeding, and gastrointestinal discomfort was common, but these could be all tolerated by patients. CONCLUSION: VEN combined with HMA is an effective salvage therapy for patients with R/R AML and is well tolerated by patients. Achieving minimal residual disease negativity is able to improve long-term survival of patients.

In vitro inhibition of transmissible gastroenteritis coronavirus replication in swine testicular cells by short hairpin RNAs targeting the ORF 7 gene.

BACKGROUND: Transmissible gastroenteritis (TGE) is a highly contagious viral disease of swine, characterized by severe vomiting, diarrhea, and high mortality. Currently, the vaccines for it are only partially effective and no specific drug is available for treatment of TGE virus (TGEV) infection. RNA interference has been confirmed as a new approach for controlling viral infections. In this study, the inhibitory effect of short hairpin RNAs (shRNAs) targeting the ORF 7 gene of TGEV on virus replication was examined. RESULTS: Four theoretically effective sequences of TGEV ORF 7 gene were designed and selected for construction of shRNA expression plasmids. In the reporter assays, three of four shRNA expression plasmids were able to inhibit significantly the expression of ORF 7 gene and replication of TGEV, as shown by real-time quantitative RT-PCR analysis of viral ORF 7 and N genes and detection of virus titers (TCID50/ml). Stable swine testicular (ST) cells expressing the shRNAs were established. Observation of the cytopathic effect and apoptosis, as well as a cell proliferation assay demonstrated that the three shRNAs were capable of protecting ST cells against TGEV destruction, with high specificity and efficiency. CONCLUSIONS: Our results indicated that plasmid-transcribed shRNAs targeting the ORF 7 gene in the TGEV genome effectively inhibited expression of the viral target gene and viral replication in vitro. These findings provide evidence that the shRNAs have potential therapeutic application for treatment of TGE.

Bradyrhizobium huanghuaihaiense sp. nov., an effective symbiotic bacterium isolated from soybean (Glycine max L.) nodules.

In a survey of the biodiversity and biogeography of rhizobia associated with soybean (Glycine max L.) in different sites of the Northern (Huang-Huai-Hai) Plain of China, ten strains were defined as representing a novel genomic species in the genus of Bradyrhizobium. They were distinguished from defined species in restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene and the 16S-23S rRNA gene intergenic spacer (IGS). In BOX-PCR, these strains presented two patterns that shared 94% similarity, demonstrating that they were a homogenous group with limited diversity. In phylogenetic analyses of the 16S rRNA gene, IGS and housekeeping gene sequences, four representative strains formed a distant lineage within the genus Bradyrhizobium, which was consistent with the results of DNA-DNA hybridization. The strains of this novel group formed effective nodules with G. max, Glycine soja and Vigna unguiculata in cross-nodulation tests and harboured symbiotic genes (nodC and nifH) identical to those of reference strains of Bradyrhizobium japonicum, Bradyrhizobium liaoningense and 'Bradyrhizobium daqingense' originating from soybean, implying that the novel group may have obtained these symbiotic genes by lateral gene transfer. In analyses of cellular fatty acids and phenotypic features, some differences were found between the novel group and related Bradyrhizobium species, demonstrating that the novel group is distinct phenotypically from other Bradyrhizobium species. Based upon the data obtained, these strains are proposed to represent a novel species, Bradyrhizobium huanghuaihaiense sp. nov., with CCBAU 23303(T) ( = LMG 26136(T)  = CGMCC 1.10948(T)  = HAMBI 3180(T)) as the type strain. The DNA G+C content of strain CCBAU 23303(T) is 61.5 mol% (T(m)).

Mesorhizobium silamurunense sp. nov., isolated from root nodules of Astragalus species.

Four rhizobial strains representing a previously defined novel group in the genus Mesorhizobium and isolated from Astragalus species in China were further characterized using a polyphasic approach. Phylogenetic analysis of 16S rRNA gene sequences showed that these Gram-negative bacteria belonged to the genus Mesorhizobium, with Mesorhizobium plurifarium LMG 11892(T) as the closest neighbour sharing a sequence similarity of 99.8 %. Comparative sequence analysis of the atpD, recA, glnII, rpoB, nodC and nifH genes, SDS-PAGE of whole-cell soluble proteins, DNA-DNA hybridization, fatty acid profiles and a series of phenotypic and physiological tests differentiated the novel group from all recognized species of the genus Mesorhizobium. Based on the data obtained in the present and previous studies, this group represents a novel species within the genus Mesorhizobium, for which the name Mesorhizobium silamurunense sp. nov. is proposed. The type strain is CCBAU 01550(T) (= HAMBI 3029(T) = LMG 24822(T)), and could form effective nodules on Astragalus membranaceus, Astragalus adsurgens and Caragana intermedia, and ineffective nodules on Phaseolus vulgaris in cross-nodulation tests.

Classical swine fever virus NS5A protein localizes to endoplasmic reticulum and induces oxidative stress in vascular endothelial cells.

Classical swine fever virus (CSFV) causes a severe disease of pigs that is characterized by hemorrhage, disseminated intravascular coagulation, and leucopenia. Until now, the role of the nonstructural protein 5A (NS5A) produced by CSFV in the pathogenesis of CSF is not well known. In this study, we investigated the function of CSFV NS5A by examining its role in the induction of oxidative stress and related intracellular events. Stable swine umbilical vein endothelial cell lines expressing CSFV NS5A were established and showed that CSFV NS5A is localized to endoplasmic reticulum and induces oxidative stress associated with enhanced reactive oxygen species production. The expression of NS5A protein exerts different effects on the three major antioxidants. Particularly, it exhibits a significant increase in transcriptional activities of antioxidant proteins thioredoxin and peroxiredoxin-6, but accompanied by a concomitant decrease of antioxidant protein heme oxygenase-1. Further studies showed that cyclooxygenase-2, a pro-inflammatory protein related to oxidative stress, is up-regulated while anti-inflammatory protein peroxisome proliferator-activated receptor-γ, an important mediator in vascular functional regulation, is down-regulated in CSFV NS5A expressing cells. This study suggested that CSFV NS5A plays important roles in the induction of oxidative stress and inflammatory response in vascular endothelial cells. These findings provide novel information on the function of the poorly characterized CSFV NS5A and provide an insight into the mechanism by which CSFV NS5A can alter intracellular events associated with the viral infection.

miR-615 facilitates porcine epidemic diarrhea virus replication by targeting IRAK1 to inhibit type III interferon expression.

Porcine epidemic diarrhea virus (PEDV) in the Coronavirus family is a highly contagious enteric pathogen in the swine industry, which has evolved mechanisms to evade host innate immune responses. The PEDV-mediated inhibition of interferons (IFNs) has been linked to the nuclear factor-kappa B (NF-κB) pathway. MicroRNAs (miRNAs) are involved in virus-host interactions and IFN-I regulation. However, the mechanism by which the PEDV regulates IFN during PEDV infection has not yet been investigated in its natural target cells. We here report a novel mechanism of viral immune escape involving miR-615, which was screened from a high-throughput sequencing library of porcine intestinal epithelial cells (IECs) infected with PEDV. PEDV infection altered the profiles of miRNAs and the activities of several pathways involved in innate immunity. Overexpression of miR-615 increased PEDV replication, inhibited IFN expression, downregulated the NF-κB pathway, and blocked p65 nuclear translocation. In contrast, knockdown of miR-615 enhanced IFN expression, suppressed PEDV replication, and activated the NF-κB pathway. We further determined that IRAK1 is the target gene of miR-615 in IECs. Our findings show that miR-615 suppresses activation of the NF-κB pathway by suppressing the IRAK1 protein and reducing the generation of IFN-IIIs, which in turn facilitates PEDV infection in IECs. Moreover, miR-615 inhibited PEDV replication and NF-κB pathway activation in both IECs and MARC-145 cells. These findings support an important role for miR-615 in the innate immune regulation of PEDV infections and provide a novel perspective for developing new treatments.

Biodiversity and biogeography of rhizobia associated with soybean plants grown in the North China Plain.

As the putative center of origin for soybean and the second largest region of soybean production in China, the North China Plain covers temperate and subtropical regions with diverse soil characteristics. However, the soybean rhizobia in this plain have not been sufficiently studied. To investigate the biodiversity and biogeography of soybean rhizobia in this plain, a total of 309 isolates of symbiotic bacteria from the soybean nodules collected from 16 sampling sites were studied by molecular characterization. These isolates were classified into 10 genospecies belonging to the genera Sinorhizobium and Bradyrhizobium, including four novel groups, with S. fredii (68.28%) as the dominant group. The phylogeny of symbiotic genes nodC and nifH defined four lineages among the isolates associated with Sinorhizobium fredii, Bradyrhizobium elkanii, B. japonicum, and B. yuanmingense, demonstrating the different origins of symbiotic genes and their coevolution with the chromosome. The possible lateral transfer of symbiotic genes was detected in several cases. The association between soil factors (available N, P, and K and pH) and the distribution of genospecies suggest clear biogeographic patterns: Sinorhizobium spp. were superdominant in sampling sites with alkaline-saline soils, while Bradyrhizobium spp. were more abundant in neutral soils. This study clarified the biodiversity and biogeography of soybean rhizobia in the North China Plain.