A novel method for DNA delivery into bacteria using cationic copolymers

Amphiphilic copolymers have a wide variety of medical and biotechnological applications, including DNA transfection in eukaryotic cells. Still, no polymer-primed transfection of prokaryotic cells has been described. The reversible addition-fragmentation chain transfer (RAFT) polymer synthesis technique and the reversible deactivation radical polymerization variants allow the design of polymers with well-controlled molar mass, morphology, and hydrophilicity/hydrophobicity ratios. RAFT was used to synthesize two amphiphilic copolymers containing different ratios of the amphiphilic poly[2-(dimethyl-amino) ethyl methacrylate] and the hydrophobic poly [methyl methacrylate]. These copolymers bound to pUC-19 DNA and successfully transfected non-competent Escherichia coli DH5α, with transformation efficiency in the range of 10³ colony-forming units per µg of plasmid DNA. These results demonstrate prokaryote transformation using polymers with controlled amphiphilic/hydrophobic ratios.     

Gene-disruption of the entomopathogenic fungus Beauveria bassiana incubated with dsRNA

The species of Beauveria bassiana is widely used for the management of agricultural insect pests. In this study, we integrated egfp-double-stranded RNA (dsRNA) to a previously generated egfp-expressing B. bassiana transformant (Bb-egfp#3) using a protoplast integration method. The Bb-egfp#3 protoplast was mixed with the dsRNA under PEG/CaCl₂ conditions and liquid-cultured in Sabouraud dextrose broth for 5 days. A control culture followed the same procedure without dsRNA. Bb-egfp#3/egfp-dsRNA cultures showed very low fungal growth (OD₆₃₀ = 0.2) compared to the control culture, Bb-egfp#3 only (OD₆₃₀ = 1.1). Screening of possible transformants on Sabouraud dextrose agar revealed a transformant T3, without egfp signal. T3 was confirmed as B. bassiana through sequencing of conserved genes and insect bioassays. Interestingly, the genomic egfp fragment of T3 was disrupted, and the egfp signal was not detected over four subcultures, which was also confirmed by RNA-seq of Bb-egfp#3 and T3. This study provides an interesting observation that protoplast integration with dsRNA could possibly generate significantly reduced gene expression in B. bassiana and it is stable across several generations.     

Exploring the stage-specific roles of Tcf-1 in T cell development and malignancy at single-cell resolution

Tcf-1 (encoded by Tcf7) not only plays critical roles in promoting T cell development and differentiation but also has been identified as a tumor suppressor involved in preventing T cell malignancy. However, the comprehensive mechanisms of Tcf-1 involved in T cell transformation remain poorly understood. In this study, Tcf7^fl/fl mice were crossed with Vav-cre, Lck-cre, or Cd4-cre mice to delete Tcf-1 conditionally at the beginning of the HSC, DN2–DN3, or DP stage, respectively. The defective T cell development phenotypes became gradually less severe as the deletion stage became more advanced in distinct mouse models. Interestingly, consistent with Tcf7^−/− mice, Tcf7^fl/flVav-cre mice developed aggressive T cell lymphoma within 45 weeks, but no tumors were generated in Tcf7^fl/flLck-cre or Tcf7^fl/flCd4-cre mice. Single-cell RNA-seq (ScRNA-seq) indicated that ablation of Tcf-1 at distinct phases can subdivide DN1 cells into three clusters (C1, C2, and C3) and DN2–DN3 cells into three clusters (C4, C5, and C6). Moreover, Tcf-1 deficiency redirects bifurcation among divergent cell fates, and clusters C1 and C4 exhibit high potential for leukemic transformation. Mechanistically, we found that Tcf-1 directly binds and mediates chromatin accessibility for both typical T cell regulators and proto-oncogenes, including Myb, Mycn, Runx1, and Lyl1 in the DN1 phase and Lef1, Id2, Dtx1, Fyn, Bcl11b, and Zfp36l2 in the DN2–DN3 phase. The aberrant expression of these genes due to Tcf-1 deficiency in very early T cells contributes to subsequent tumorigenesis. Thus, we demonstrated that Tcf-1 plays stage-specific roles in regulating early thymocyte development and transformation, providing new insights and evidence for clinical trials on T-ALL leukemia.     

跟随病例式教学在培养口腔医学生思维模式中的作用研究#br#

目的    比较跟随病例式教学和分学科教学在培养口腔医学生思维模式中的作用。方法    选择中国医科大学口腔临床专业97期本科生62人和98期本科生60人,分别采用跟随病例式教学模式（跟随病例式教学组）和分学科教学模式（分学科教学组）。临床实习结束后分别对两组本科生进行理论-临床思维转换、批判性思维能力评估。结果    在理论-临床思维转换能力评估中,跟随病例式教学组本科生在临床接诊、病例汇报方面的成绩优于分学科教学组（均P < 0.05）;在批判性思维能力评估中,跟随病例式教学组本科生在寻找真理、分析能力、求知欲、认知成熟等方面,相较于分学科教学组本科生具有更加明显的正性批判性思维倾向（均P < 0.05）。结论    采用跟随病例式教学可能更有助于培养口腔医学生理论-临床思维转换能力和批判性思维能力。     

Well‐differentiated papillary mesothelioma,possibly giving rise to diffuse malignant mesothelioma: A case report

Well‐differentiated papillary mesothelioma (WDPM) is a distinct subtype of mesothelial tumor from diffuse malignant mesothelioma (DMM), with an uncertain malignant potential. The relationship between WDPM and DMM, with regard to the ability of the former to develop into the latter, is also unknown. A 58‐year‐old woman, diagnosed with a rectal carcinoid tumor, underwent removal of the lymph nodes via the abdomen in 2004. A large number of white miliary nodules were identified on the mesentery and peritoneum, which were histologically diagnosed as WDPM. No further therapy was administered, but the patient was followed‐up using imaging methods. Seven years later, an abdominal wall mass was discovered using positron emission tomography‐computed tomography, and a laparotomy biopsy was performed. DMM was diagnosed, because mesothelioma with extended invasion had been histologically identified. Mesothelioma similar to papillary proliferation was present on the outer layer of the peritoneum, and an infiltrating lesion with continuous restiform or solid‐like structures was noted. WDPM was believed to have undergone malignant transformation. Compared to DMM, WDPM has a good prognosis and is considered a benign or borderline neoplasm. Our findings suggest that WDPM does have malignant potential, however, because histological findings indicated a malignant transformation of WDPM to DMM.     

Histological transformation of non-small cell lung cancer: Clinical analysis of nine cases

BACKGROUNDHistological transformation is one of the numerous mechanisms of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). Given its rarity, the underlying transformational mechanisms, clinical features, and therapeutic prognoses are only studied through limited case reports.AIMTo analyze the clinical characteristics and underlying mechanisms in non-small cell lung cancer (SCLC) patients with histological transformation after treatment with EGFR-TKIs.METHODSWe retrospectively investigated nine patients diagnosed with non-SCLC transforming to SCLC, large-cell neuroendocrine carcinoma (LCNEC), or squamous cell carcinoma on re-biopsy after first- or third-generation EGFR-TKIs.RESULTSThe median age of nine patients was 60 years. Among them, six patients had the EGFR 19del mutation, one had the L858R mutation, and one had wild-type EGFR. The level of plasma NSE was measured in six patients with SCLC or LCNEC transformation when transformation occurred, and five patients had elevated plasma NSE levels. All patients received standard chemotherapy after transformation with the exception of one patient who received chemotherapy and anlotinib.CONCLUSIONTumor re-biopsy should be performed routinely when EGFR-TKI therapy fails in lung cancer patients to avoid ignoring histological transformation and to select a subsequent therapeutic strategy. The transformed tumor retained the original EGFR mutation, indicating that histological transformation represents an evolution from the initial tumor.     

Acoustic properties of fracture of dental restorative materials and endocrown restorations under quasi-static loading

Objectives(1) To investigate the properties of acoustic emission (AE) signals from fracture of dental materials and endocrown restorations during quasi-static compression using the continuous wavelet transform analysis. (2) To determine by the parameters of AE signals in which structural element of tooth-endocrown system the fracture starts.MethodsFive restorative materials (zirconium dioxide (Prettau zirconia), ceramics (IPS e.max Press), metal ceramics (GC Initial MC+Nicrallium N2 BCS), composite resin (Nano Q), luting cement (Relyx™ U200)) were manufactured into discs, 13 mm diameter, 5 mm thick, which were divided into 5 groups (Group B, C, D, E, F, respectively) and included into Type 1. Twenty-five extracted human molars were divided into 5 groups and included into Type 2: Group A (control, no restoration); BE (tooth restored by zirconium dioxide endocrowns); CE (restored by ceramic endocrowns); DE (restored by metal ceramic endocrowns); EE (restored by composite resin endocrowns). An increasing load was applied to the center of the samples with a hard steel ball until a fracture occurred. The loading rate was 0.12 mm/min. Two-channel AE detection system was used to record the AE activity during testing. The parameters including the peak frequency, bandwidth, duration time and energy of AE signals were analyzed.ResultsThe restorative materials (Type 1) differ from each other by acoustic properties such as the peak frequency main bands and energy of AE signals. The detected AE signals correspond to the different fracture types. The dominant fracture mechanism of Group C, E and F specimens was the macrocracking (brittle fracture), and of Group D specimens was the microcracking, which alternated with the plastic deformation and macrocracking. For the number of events corresponding to the brittle fracture, statistically significant differences (p < 0.05) were found between all group pairs. AE signals were not released during compression test of the zirconium dioxide samples, thus the fracture was not detected.All the AE signals detected during compression test of the different endocrown systems (Type 2) had two peak frequency bands. The fracture of Groups A, BE, CE and DE samples characterized by alternating the micro- and macrocracking with plastic deformation, and the brittle fracture was the dominant fracture mechanism. The restorations of Group EE fractured without plastic deformation. For the number of events corresponding to the brittle fracture, statistically insignificant differences (p > 0.05) were found between Groups A vs CE and A vs EE, and statistically significant differences (p < 0.05) were found between Groups A vs BE and A vs DE.SignificanceThe initiation of fracture in the restorations with endocrowns made from different materials begins with the tooth damage. The metal ceramic endocrown restoration showed the highest fracture resistance, what is especially important for clinical use.     

整合素αvβ3抑制剂Cyclo-RGDfK对纤连蛋白诱导乳腺上皮细胞间质转化的影响

目的本研究探讨整合素αvβ3抑制剂(Cyclo-RGDfK)对纤连蛋白(fibronectin,FN)诱导人乳腺上皮细胞MCF-10A上皮间质转化的影响。方法体外传代培养MCF-10A细胞,采用FN诱导建立上皮间质转化(EMT)模型。Western blot实验检测αv integrin、β3 integrin、上皮标志物E-cadherin和间充质标志物N-cadherin、Vimentin的蛋白表达;划痕修复实验和Transwell小室实验检测细胞的迁移和侵袭能力。结果 Western blot结果显示FN作用48 h后可明显增强αv integrin、β3 integrin、N-cadherin、Vimentin的蛋白表达,下调E-cadherin蛋白表达,在给予Cyclo-RGDfK作用后,FN诱导的N-cadherin和Vimentin表达上调和E-cadherin表达下调被明显逆转;划痕修复实验和Transwell小室实验结果表明,FN能显著增强MCF-10A细胞迁移和侵袭能力,Cyclo-RGDfK能显著抑制FN的诱导作用。结论 Cyclo-RGDfK能抑制FN诱导乳腺上皮细胞MCF-10A的EMT过程,降低细胞的侵袭和迁移能力,其可能是治疗乳腺癌转移的潜在药物。     

脑梗死后微血管渗透性改变及出血性转化的预测

目的探讨缺血性脑梗死后急性期和亚急性期微血管渗透性改变及脑梗死后出血性转化(hemorrhagic transformation,HT)的预测。材料与方法收集缺血性脑梗死病人43例,急性期10例,亚急性期33例,进行常规MRI及DCE-MRI扫描。利用药代动力学模型计算容积转运常数Ktrans,比较每一个病人梗死区和对侧正常脑组织的Ktrans值以及出血组和非出血组的Ktrans值有无差异,比较不同时期梗死区强化对预测HT的意义。结果所有病人梗死区Ktrans值较对侧正常脑组织明显增高(P0.05)。10例在急性期有脑实质强化的病人都有继发出血,在亚急性期和慢性期15例病人有继发出血,18例没有继发出血,Fisher's精确检验有统计学意义(P0.05)。与亚急性期HT或没有HT的病人相比,急性期有HT组病人的Ktrans值明显增高(P0.05),但是亚急性期HT和非HT的病人Ktrans值比较没有统计学差异(P0.05)。结论急性期脑实质强化对HT的预测有更高的特异性,且渗透性比亚急性期更高。早期脑实质强化及HT与毛细血管内皮的紧密连接和基底膜损伤有关。后期脑实质强化及HT与侧枝循环的建立有关,DCE-MRI可以定量评估血脑屏障的渗透性,对进一步研究HT的分型提供更科学的依据。