Engineering adeno-associated virus vectors for gene therapy.

Adeno-associated virus (AAV) vector-mediated gene delivery was recently approved for the treatment of inherited blindness and spinal muscular atrophy, and long-term therapeutic effects have been achieved for other rare diseases, including haemophilia and Duchenne muscular dystrophy. However, current research indicates that the genetic modification of AAV vectors may further facilitate the success of AAV gene therapy. Vector engineering can increase AAV transduction efficiency (by optimizing the transgene cassette), vector tropism (using capsid engineering) and the ability of the capsid and transgene to avoid the host immune response (by genetically modifying these components), as well as optimize the large-scale production of AAV.

Minimal residual disease monitoring in childhood Philadelphia chromosome-positive acute lymphoblastic leukemia: prognostic significance and correlation between multiparameter flow cytometry and real-time quantitative polymerase chain reaction

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The extraordinary rearrangement of mitochondrial genome of the wheat pest, Aptinothrips stylifer and the mitochondrial phylogeny of Thripidae (Thysanoptera).

The mitochondrial gene order in Thysanoptera is notably distinct and highly rearranged, with each species exhibiting its own unique arrangement. To elucidate the relationship between gene rearrangements and phylogeny, the complete mitochondrial genome (mitogenome) of the wheat pest, Aptinothrips stylifer, was sequenced and assembled, spanning a total length of 16,033 bp. Compared with the ancestral arthropod mitogenome, significant rearrangement differences were evident in A. stylifer, whereas the gene order between A. stylifer and Anaphothrips obscurus was similar. Phylogenetic trees were reconstructed based on all 13 protein-coding gene sequences using Bayesian inference and maximum-likelihood methods, both yielding similar topological structures. Notably, A. stylifer was robustly clustered with A. obscurus, affirming its classification within Anaphothrips genus group. This exemplifies the potential correlation between gene rearrangements and phylogeny in the Thripidae family. Additionally, the mitogenome of A. stylifer exhibited several atypical features, including: (1) Three putative control regions (CRs) in close proximity, with CR2 and CR3 displaying partial similarity, and CR1 differing in base composition; (2) Two transfer RNAs (tRNAs), trnS1 and trnV, lacking the DHU arm; (3) Two ribosomal RNA (rRNA) genes inverted and positioned distant from each other; (4) Negative AT and GC skew (AT skew = -0.001, GC skew = -0.077); (5) One transposition (nad6), one inverse transposition (trnQ), four inversions (trnF, trnH, trnC, and gene block nad1-trnL1-rrnL-trnV-rrnS), and four tandem duplication random loss events; and (6) Two protein-coding genes, nad2 and atp8, terminated with an incomplete stop codon "T".

Nonintubated spontaneous ventilation versus intubated mechanical ventilation anesthesia for video-assisted thoracic surgery in terms of perioperative complications and practitioners' workload assessments: a pilot randomized control study.

BACKGROUND: The use of nonintubated video-assisted thoracoscopic surgery (NI-VATS) has been increasingly reported to yield favourable outcomes. However, this technology has not been routinely used because its advantages and safety have not been fully confirmed. The aim of this study was to assess the safety and feasibility of nonintubated spontaneous ventilation (NI-SV) anesthesia compared to intubated mechanical ventilation (I-MV) anesthesia in VATS by evaluating of perioperative complications and practitioners' workloads. METHODS: Patients who underwent uniportal VATS were randomly assigned at a 1:1 ratio to receive NI-SV or I-MV anesthesia. The primary outcome was the occurrence of intraoperative airway intervention events, including transient MV, conversion to intubation and repositioning of the double-lumen tube. The secondary outcomes included perioperative complications and modified National Aeronautics and Space Administration Task Load Index (NASA-TLX) scores from anesthesiologists and surgeons. RESULTS: Thirty-five patients in each group were enrolled in the intention-to-treat analysis. The incidence of intraoperative airway intervention events was greater in the NI-SV group than in the I-MV group (12 [34.3%] vs. 3 [8.6%]; OR = 0.180; 95% CI = 0.045-0.710; p = 0.009). No significant difference was found in the postoperative pulmonary complications between the groups (p > 0.05). The median of the anesthesiologists' overall NASA-TLX score was 37.5 (29-52) when administering the NI-SV, which was greater than the 25 (19-34.5) when the I-MV was administered (p < 0.001). The surgeons' overall NASA-TLX score was comparable between the two ventilation strategies (28 [21-38.5] vs. 27 [20.5-38.5], p = 0.814). CONCLUSION: The NI-SV anesthesia was feasible for VATS in the selected patients, with a greater incidence of intraoperative airway intervention events than I-MV anesthesia, and with more surgical effort required by anesthesiologists. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR2200055427. https://www.chictr.org.cn/showproj.html?proj=147872 was registered on January 09, 2022.

[Application of automatic slide-dropping instrument in bone marrow chromosomal karyotyping].

OBJECTIVE: To explore the application of an automatic slide-dropping instrument in bone marrow chromosomal karyotyping. METHODS: The effects of manual and automatic dropping methods under different environmental humidity were retrospectively analyzed, and the repeatability of the automatic dropping method was analyzed. RESULTS: No statistical difference was found between the results of automatic and manual dropping methods under the optimum ambient humidity and high humidity (P > 0.05). At low humidity, there was a statistical difference between the two methods (P < 0.05). With regard to the repeatability, the coefficient of variations of the automatic dropping method for the number of split phases, the rate of good dispersion and the rate of overlap were all lower than those of the manual dropping method. A statistical difference was also found in the number of split phases (P < 0.05) but not in the discrete excellent rate and overlapping rate between the two methods (P > 0.05). CONCLUSION: Better effect can be obtained by the automatic dropping instrument. It is suggested to gradually replace manual work with machine.

Combined gene therapy via VEGF and mini-dystrophin synergistically improves pathologies in temporalis muscle of dystrophin/utrophin double knockout mice.

Duchenne muscular dystrophy (DMD) is a severe X-linked inherited muscular disorder characterized by the loss of dystrophin. We have previously shown that monogene therapy using the mini-dystrophin gene improves muscle function in DMD. However, chronic inflammation plays an important role in progressive muscle degeneration in DMD as well. Vascular endothelial growth factor (VEGF) has been used to enhance muscle vasculature, reduce local inflammation and improve DMD muscle function. Temporalis muscles are the key skeletal muscles for mastication and loss of their function negatively affects DMD patient quality of life by reducing nutritional intake, but little is known about the pathology and treatment of the temporalis muscle in DMD. In this work, we tested the hypothesis that the combined delivery of the human mini-dystrophin and human VEGF genes to the temporalis muscles using separate recombinant adeno-associated viral (rAAV) vectors will synergistically improve muscle function and pathology in adult male dystrophin/utrophin double-knockout (mdx/utrn+/-) mice. The experimental mice were divided into four groups including: dystrophin + VEGF combined, dystrophin only, VEGF only and PBS control. After 2 months, gene expression and histological analysis of the temporalis muscles showed a synergistic improvement in temporalis muscle pathology and function coincident with increased restoration of dystrophin-associated protein complexes and nNOS in the dystrophin + VEGF combined group. We also observed significantly reduced inflammatory cell infiltration, central nucleation, and fibrosis in the dystrophin + VEGF combined group. We have demonstrated the efficacy of combined rAAV-mediated dystrophin and VEGF treatment of temporalis muscles in a DMD mouse model.

Impact of the International Consensus Classification of myelodysplastic syndromes.

The impact of the 2022 International Consensus Classification (ICC) of myelodysplastic syndromes (MDS) needs study. We analysed data from 989 MDS subjects classified using the 2016 World Health Organization (WHO) criteria to determine the impact of the new proposal. Our analyses suggested the ICC criteria of MDS-SF3B1 identifies a more homogenous disease entity than the WHO 2016 criteria of myelodysplastic syndromes with ring sideroblasts (MDS-RS). MDS, not otherwise specified with single lineage dysplasia (MDS, NOS-SLD) patients had a better prognosis than MDS, NOS with multilineage dysplasia (MDS, NOS-MLD) patients. MDS with mutated TP53 and MDS/acute myeloid leukaemia with mutated TP53 patients had the briefest survivals. These data support the ICC of MDS, which allows more accurate diagnoses and risk stratification.

[Genetic analysis of two cases with MYC "negative" Burkitt lymphoma].

OBJECTIVE: To carry out combined genetic analysis on two patients suspected for Burkitt lymphoma to facilitate their diagnosis and treatment. METHODS: G banded karyotyping and interphase and metaphase fluorescence in situ hybridization (FISH) were used to detect the specific sites of chromosomes by using separate and fusion probes. RESULTS: The separate probe showed no presence of MYC gene abnormality, while fusion probe confirmed the IGH::MYC translocation in the samples. Combined with the clinical features and pathological characteristics, the two patients were finally diagnosed with Burkitt lymphoma, which was confirmed by targeted capture next generation sequencing. CONCLUSION: The separate probe for the MYC gene has some shortcomings and should be used together with dual fusion probe to improve the accuracy of diagnosis.

Interfacially Super-Assembled Benzimidazole Derivative-Based Mesoporous Silica Nanoprobe for Sensitive Copper (II) Detection and Biosensing in Living Cells.

Mesoporous silica nanoparticles (MSNs) functionalized with benzimidazole-derived fluorescent molecules (DHBM) are fabricated via a feasible interfacial superassembly strategy for the highly sensitive and selective detection of Cu2+ . DHBM-MSN exhibits an obvious quenching effect on Cu2+ in aqueous solutions, and the detection limit can be as low as 7.69×10-8  M. The DHBM-MSN solid-state sensor has good recyclability, and the silica framework can simultaneously improve the photostability of DHBM. Two mesoporous silica nanoparticles with different morphologies were specially designed to verify that nanocarriers with different morphologies do not affect the specific detectionability. The detection mechanism of the fluorescent probe was systematically elucidated by combining experimental results and density function theory calculations. Moreover, the detection system was successfully applied to detect Cu2+ in bovine serum, juice, and live cells. These results indicate that the DHBM-MSN fluorescent sensor holds great potential in practical and biomedical applications.

Purine nucleoside analogs plus rituximab are an effective treatment choice for hairy cell leukemia-variant.

Both characteristics and optimal treatment strategy for hairy cell leukemia-variant (HCL-v) remain elusive due to its rarity. We retrospectively analyzed the clinical features of HCL-v and the efficacy of first-line treatment options in a large Chinese cohort. In this study, we recruited 33 HCL-v patients (23 males and 10 females) with a median age of 59 years (range, 34-79 years). The chief complaints included abdominal mass and relative signs (67%) and abnormal complete blood count (27%). Immunophenotyping showed monoclonal B-cells positive for pan B-cell antigens and CD11c, weakly positive for CD103 and CD200, while negative for CD5, CD10, CD25, CD123, and annexin A1. No BRAF V600E mutation was detected, but TP53 abnormality was recurrent. Treatment choices included interferon-α (IFN-α) in 11 patients, chlorambucil (CLB) in 5 patients, single purine nucleoside analogs (PNA) in 3 patients, PNA plus rituximab (PNA + R) in 9 patients, and others in 3 patients. Four patients who received IFN-α or CLB treatment also underwent splenectomy. Patients who received PNA + R had a higher complete response rate (88% versus 5%, P < 0.001) and longer progression-free survival (PFS, 3-year PFS rate 42% [95% CI 1-84] vs. 16% [95% CI 3-40], P = 0.042) than those who received other regimens. Overall, HCL-v is an indolent lymphoma with unique characteristics. The PNA + R regimen is the preferred choice in the first-line treatment for HCL-v.

Myeloid Neoplasm with PCM1-PDGFRB Transcript Responded to Low-Dose Imatinib: One Case Report with Literature Review.

Through an RNA-seq analysis of an adult patient with unclassifiable myelodysplastic/myeloproliferative neoplasms (MDS/MPN-U), we identified a rare PDGFRB fusion partner gene, PCM1. Conventional chromosome karyotype analysis showed abnormal clones of t(5;8)(q32;p22), and fluorescence in situ hybridization (FISH) confirmed rearrangement of the PDGFRB gene. Reverse transcription PCR (RT-PCR) and Sanger sequencing further confirmed that exon 30 of the PCM1 gene was fused with exon 11 of PDGFRB in frame, and the fusion event was accompanied by a 14 bp deletion of exon 11 of PDGFRB. After low-dose imatinib treatment, the patient achieved complete molecular remission. This study not only broadens the understanding of myeloid/lymphoid neoplasms with PDGFRB rearrangement but also reflects the vital role of RNA-seq in identifying PDGFRB rearrangements.

Inhibition of antigen presentation during AAV gene therapy using virus peptides.

The clinical trial using adeno-associated virus (AAV) vector delivery of mini-dystrophin in patients with Duchenne Muscular Dystrophy (DMD) demonstrated a cytotoxic lymphocyte (CTL) response targeting the transgene product. These mini-dystrophin-specific T-cells have the potential to clear all transduced muscle, presenting the general gene therapy concern of overcoming the CTL response to foreign proteins that provide therapeutic benefit. In this study, we exploited a natural immunosuppression strategy employed by some viruses that results in CTL evasion only in transduced cells. After transfection of the plasmids encoding viral peptides and ovalbumin, which includes the immune-domain epitope SIINFEKL, several viral small peptides (ICP47 and US6) inhibited the SIINFEKL peptide presentation. A single AAV vector genome that consisted of either transgene AAT fused with SIINFEKL epitope and, separately, ICP47 expressed from different promoters or a single fusion protein with ICP47 linked by a furin cleavage peptide (AATOVA-ICP47) decreased antigen presentation. Compared with AAV/AATOVA in which decreased AAT expression was observed at late time points, persistent transgene expression was obtained after systemic administration of AAV/AATOVA-ICP47 vectors in mice. We extended this strategy to DMD gene therapy. After administration of AAV vector encoding human mini-dystrophin fusion protein with ICP47 into mdx mice, a lower mini-dystrophin-specific CTL response was induced. Importantly, the ICP47 fusion to mini-dystrophin inhibited CTLs mediated cytotoxicity. Although demonstrated herein using AAT and mini-dystrophin transgenes in an AAV context, the collective results have implications for all gene therapy applications resulting in foreign peptides by immune suppression in only genetically modified cells.

Occurrence and genomic characterization of ESBL-producing Escherichia coli ST29 strains from swine with abundant virulence genes.

Food-production animals were considered to be a major reservoir of antimicrobial-resistant bacteria and clinically relevant pathogens. The potential of commensal Escherichia coli from pigs as a source of opportunistic pathogens associated with extraintestinal infections in humans needs to be assessed. In this study, 13 E. coli isolates from an intensive pig farm in China were analyzed using whole genome sequencing followed by in-depth in silico analysis. Genomic analysis showed comprehensive antimicrobial resistance profiles, with each isolate carrying between 4 and 22 antimicrobial resistance genes. Although these E. coli isolates were assigned to low-virulence phylogroup A and B1, 31 different virulence genes were detected at least once in the 13 sequenced isolates. Extraintestinal pathogenic E. coli-associated virulence genes, including iss, iha, tsh and iroN, were found in commensal E. coli isolates in this study. Of note, a large number of virulence genes (n = 22) were identified in ESBL-producing E. coli sequence type (ST) 29 isolates. Our study revealed the presence of comprehensive antimicrobial resistance and virulence gene profiles in commensal E. coli isolates of pigs. The emerged ESBL-producing E. coli ST 29 isolates harboring a high abundance of VAGs highlighted that this new clonal linage may pose a threat to public health.

IgH translocation with undefined partners is associated with superior outcome in multiple myeloma patients.

BACKGROUND: In multiple myeloma (MM), impact of specific chromosomal translocations involving IgH (14q21 locus, including t(4;14), t(11;14), and t(14;16)) has been explored extensively. However, over 15% MM patients harboring IgH translocation with undefined partners have long been ignored. METHODS: A prospective non-randomized cohort study with a total of 715 newly-diagnosed MM cases was conducted, 13.6% of whom were t(14;undefined) positive. The whole cohort was divided into four groups: no IgH split (47.7%); t(14;undefined) (13.6%); t(11;14) (17.6%); and t(4;14) or t(14;16) group (21.1%). RESULTS: Median OS for the four groups was 84.2, not reached (NR), 58.7, and 44.2 months, respectively, with P values for t(14;undefined) vs no IgH split, t(11;14), and t(4;14)/t(14;16) groups of 0.197, 0.022, and 0.001, respectively. In bortezomib-based group, the survival advantage gained by t(14;undefined) group was much more significant compared to t(11;14) and t(4;14)/t(14;16) groups. Importantly, t(14;undefined) turned out to be an independent predictive factor for longer OS of MM patients in multivariate analysis, especially in the context of bortezomib treatment. Similar results were also observed in the PUMCH external validation cohort. CONCLUSION: Collectively, our data confirmed and externally validated the favorable prognosis of the t(14;undefined) groups, especially in the era of novel agents.

Synthesis of difluoroalkylated 2-azaspiro[4.5]decane derivatives via copper-catalyzed difluoroalkylation/dearomatization of N-benzylacrylamides.

An efficient method for the synthesis of difluoroalkylated 2-azaspiro[4.5]decanes via copper-catalyzed difluoroalkylation of N-benzylacrylamides with ethyl bromodifluoroacetate has been established. The reaction experienced a tandem radical addition and dearomatizing cyclization process. In addition, the resultant products can be smoothly converted into a difluoroalkylated quinolinone and saturated spirocyclohexanone scaffold.

Comparison of Bispectral Index-Guided Individualized Anesthesia with Standard General Anesthesia on Inadequate Emergence and Postoperative Delirium in Elderly Patients Undergoing Esophagectomy: A Retrospective Study at a Single Center.

BACKGROUND Elderly patients are susceptible to general anesthetics, with a higher bispectral index (BIS) at loss of consciousness (LOC) achieved by propofol infusion compared with young patients. Overexposure to general anesthetics can have adverse effects such as inadequate emergence and postoperative delirium (PD). This study aimed to compare the effects of BIS-guided individualized anesthesia with standard general anesthesia on emergence and delirium after esophagectomy. MATERIAL AND METHODS Data on 161 elderly patients undergoing esophagectomy for cancer were retrospectively obtained from electronic medical records. We performed propensity score matching analysis between patients receiving individualized anesthesia (BIS value maintained at about 10 less than the value at LOC) and those receiving standard anesthesia (BIS value maintained at 40-60). In addition, we conducted univariate and multivariate logistic -analyses in the entire cohort. RESULTS Patients receiving individualized anesthesia had higher BIS values and a lower propofol requirement during surgery than those receiving standard general anesthesia (P<0.05). The overall incidences of inadequate emergence and PD were 37.9% and 18.0% (n=161), respectively. Logistic regression analysis revealed that the independent risk factors for PD were organic brain disease (odds ratio [OR] 6.308; 95% confidence interval [CI] 2.458-16.187) and inadequate emergence (OR 4.063; 95% CI 1.645-10.033). CONCLUSIONS BIS-guided individualized anesthesia (lighter) does not reduce inadequate emergence or PD compared with standard general anesthesia in elderly patients undergoing esophagectomy. Independent risk factors for PD include organic brain disease and inadequate emergence.

Superior human hepatocyte transduction with adeno-associated virus vector serotype 7.

Although therapeutic outcomes have been achieved in hemophilia patients after delivery of clotting factor genes to the liver using adeno-associated virus (AAV) vectors, it is well known that the preclinical results generated from hemophilia animal models have not been directly predictive of successful translation in humans. To address this discrepancy humanized mouse models have recently been used to predict AAV transduction efficiency for human hepatocytes. In this study we evaluated AAV vector transduction from several serotypes in human liver hepatocytes xenografted into chimeric mice. After systemic administration of AAV vectors encoding a GFP transgene in humanized mice, the liver was harvested for either immunohistochemistry staining or flow cytometry assay for AAV human hepatocyte transduction analysis. We observed that AAV7 consistently transduced human hepatocytes more efficiently than other serotypes in both immunohistochemistry assay and flow cytometry analysis. To better assess the future application of AAV7 for systemic administration in the treatment of hemophilia or other liver diseases, we analyzed the prevalence of neutralizing antibodies (NAbs) to AAV7 in sera from healthy subjects and patients with hemophilia. In the general population, the prevalence of NAbs to AAV7 was lower than that of AAV2 or AAV3B. However, a higher prevalence of AAV7 NAbs was found in patients with hemophilia. In summary, results from this study suggest that AAV7 vectors should be considered as an effective vehicle for human liver targeting in future clinical trials.

Effectiveness and safety of chemotherapy with cytokine-induced killer cells in non-small cell lung cancer: A systematic review and meta-analysis of 32 randomized controlled trials.

BACKGROUND AIMS: Cytokine-induced killer (CIK) cells are the most commonly used cellular immunotherapy for multiple tumors. To further confirm whether chemotherapy with CIK cells improves clinical effectiveness and to reveal its optimal use in non-small cell lung cancer (NSCLC), we systematically reevaluated all relevant studies. METHODS: We collected all studies about chemotherapy with CIK cells for NSCLC from the Medline, Embase, Web of Science, China National Knowledge Infrastructure Database (CNKI), Chinese Scientific Journals Full-Text Database (VIP), Wanfang Data, China Biological Medicine Database (CBM), Cochrane Central Register of Controlled Trials (CENTRAL), Chinese clinical trial registry (Chi-CTR), World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) and U.S. clinical trials. We evaluated their quality according to the Cochrane evaluation handbook of randomized controlled trials (RCTs) (version 5.1.0), extracted the data using a standard data extraction form, synthesized the data using meta-analysis and finally rated the evidence quality using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. RESULTS: Thirty-two RCTs with 2250 patients were included, and most trials had unclear risk of bias. The merged risk ratios values and their 95% confidence intervals of meta-analysis for objective response rate, disease control rate, 1- and 2-year overall survival rates, 1- and 2-year progression-free survival rates were as following: 1.45 (1.31-1.61), 1.26 (1.16-.37), 1.42 (1.23-1.63), 2.06 (1.36-3.12), 1.93 (1.38-2.69) and 3.30 (1.13-9.67). Compared with chemotherapy alone, all differences were statistically significant. CIK cells could increase the CD3+ T cells, CD3+ CD4+ T cells, NK cells and the ratio of CD4+/CD8+ T cells. The chemotherapy with CIK cells had a lower risk of hematotoxicity, gastrointestinal toxicity, liver injury and a higher fever than that of chemotherapy alone. The evidence quality was "moderate" to "very low." CONCLUSIONS: The available moderate evidences indicate that chemotherapy with CIK cells, especially autologous CIK cells, can significantly improve the tumor responses, 1- and 2-year overall and progression-free survival rates in patients with advanced NSCLC. This treatment does have a high risk of fever. The optimal use may be treatment with one or two cycles and in combination with vinorelbine and cisplatin, paclitaxel and cisplatin, or docetaxel and cisplatin.