Blood Microbiota and Cancer: Cell Wall-Deficient L-Forms of Bacteria and Fungi as Cancer-Promoting Environment

In recent years, valuable experience and insights have been gained into L-forms (cell-wall-deficient variants) of bacteria and fungi and their disease-trigger potential in cases with chronic infections, autism spectrum disorders, autoimmune and neurodegenerative diseases. Based on the concept of “internal” blood microbiota, consisting of L-forms and its relevance to health and disease, the current study aims to outline the profile of dysbiotic disorders in three cancer patients (with endometrial cancer, breast cancer and acute myeloid leukemia), all in a phase before chemotherapy. Venous blood samples from the patients and from one control healthy person, were microbiologically studied. The used novel methodology of blood microbiota assessment was based on the following phases: isolation of L-forms, development and propagation, cultivation and conversion of L-forms into classical bacteria and fungi, as well as their identification with MALDI-TOF method. From the patients were isolated L-forms of opportunistic bacteria (Enterococcus faecalis, Esherichia coli, Enterobacter cloacae and Pseudomonas oryzihabitans) and fungi such as Rhodotorula mucilaginosa, Aspergillus fumigatus and Mucorales. In conclusion, the common feature found for the three cancer patients was the isolation from the blood of highly associated communities consisting of morphologically indistinguishable L-bodies, which through reversion in broth, were identified as distinct bacterial and fungal species. Unlike classic bacteria or fungi causing sepsis and bacteremia/fungemia, the presence of L-forms in blood is hidden, it does not demonstrate clinical signs nor it can be detected by conventional methods. It should be noted, however, that the dysbiotic blood microbiota shows unique and individual characteristics for the concrete cancer patient, correlates to the common state of the organism and tumor localization in the body, as well as it outlines the cancer promoting role of L-forms in processes of malignization, cancer genesis and progression.

Novel Approach to Microbiological Study of Chronic Inflammations at Upper Respiratory Tract: Research of Blood L-Form Microbiota

Background: The recognition of human blood microbiota, consisting of cell wall-deficient microbes (L-forms), is a major challenge today in the field of microbiology. There are accumulating data confirming the concept of “internal” blood L-form microbiota and its significance for health and diseases. Finding out whether the blood microbiota can be of diagnostic and prognostic importance for detection and evaluation of chronic infections anywhere in the body is a major objective. In the context of chronically infected upper respiratory tract (URT), the aim of the current study was to understand whether a local infection can be a source for entry of bacteria and fungi in the blood. Methods: Blood samples from six persons with chronic inflammations in URT diagnosed with hypertrophied adenoids, chronic sinusitis, nasal polyps, chronic naso-pharyngitis and one control healthy person were studied. Blood microbiota assessment methodology that be used, included three phases: 1) isolation of L-form cultures from blood-development and propagation;2) cultivation directed to conversion of L-forms into bacterial and fungal cultures;3) isolation of pure classical bacterial and fungal cultures and their identification by MALDI-TOF method. Results: From the patients were isolated L-forms of opportunistic bacteria (Streptococcus mitis, Roseomonas mucosa, Dermacoccus nishinomiyaensis, Enterococcus faecalis, Acinetobacter johnsonii, Pseudomonas putida, Staphylococcus aureus, Pseudomonas luteola, Enterobacter cloacae) and fungi such as Rhodotorula mucilaginosa, Aspergillus niger, Aspergillus fumigatus and Mucorales. Conclusion: The novel innovative methodology for assessment of blood L-form microbiota was successfully applied for detection of microbes responsible for chronic infections at URT.

Application of PCR-SSCP in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis

Objective: To study the relationship between the polymorphism of drug resistant gene rpoB and drug resistance against rifampicin(RFP) of M. tuberculosis L-forms, and to evaluate its clinical application. Methods: A total of 52 clinical isolated strains of M. tuberculosis L-forms were collected. rpoB gene polymorphism was analyzed by polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) and conventional antimicrobial susceptibility test (AST). Their results were compared. Results: AST results showed that 38 of 52 clinical isolated strains were drug resistance (73.08％),while PCR-SSCP indicated 65.38％ (32/52) rpoB gene polymorphism. There was no statistic significance(χ2= 2.4914) between the 2 methods. Conclusion:Combined the application of PCR-SSCP with AST in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis may have advantages at earlier diagnosis and guidance of clinical medications.

Detecting katG Drug Resistant Genetic Mutation among pneumoconiosis patients complicated with tuberculosis in Mycobacterium tuberculosis L-forms by PCR-SSCP

Objective：To study the relationship between mutation in the katG gene and drug resistance of INH in Mycobacterium tuberculosis L-forms among patients of pneumoconiosis complicated with pulmonary tuberculosis, and to explore the clinical application of PCR-SSCP. Methods： A total of 52 clinical isolated strains of Mycobacterium tuberculosis L-forms were collected. Mutation in the katG genes was detected by PCR-SSCP and traditional antimicrobial susceptibility test （AST）. Results： The results by AST showed that there were 40 persisters in 52 clinical isolated strains. The drug resistant rate was 76.92%（40/52）, and the gene mutation rate of katG was 57.70%（30/52）by PCR-SSCP, the difference was no quite significance （X^2 = 2.8507, P 〉 0.05）. The coincidence rate of two methods was 75.00% （30/40）. Conclusion： The detectionrate of katG resistant strains in Mycobacterium tuberculosis L-forms was high by PCR-SSCP. The combined application of PCR-SSCP and traditional antimicrobial susceptibility test can improve the detecting rate.

Detecting drug resistant genetic mutation among pneumoconiosis patients complicated with tuberculosis in Mycobacterium tuberculosis L-forms application of PCR-SSCP technique in Huainan mining district

Objective： To study the relationship between drug resistant genetic mutation and drug resistance in Mycobacterium tuberculosis L-form, discuss the internal relationship between drug resistances and drug-resistant related genes and explore the value of PCR- SSCP to clinical application. Methods： A total of 52 clinically isolated strains of tuberculosis L-form were collected among 97 pneumoconiosis patients complicated with tuberculosis. The gene mutations of katG, rpoB and rpsL were detected by PCR-SSCP, and the results were compared with those analyzed by traditional antimicrobial susceptibility test（AST）. Results： The gene muta- tion rates of katG, rpoB and rpsL by PCR-SSCP were respectively 57.70% （30/52）, 65.38% （32/52） and 40.38% （21/52）. The rate of reversion was 78.85%（41/52） and the result of drag-resistant genes was invariable. The results of AST showed that there were 40 （76.92%） multi-drug resistant strains in 52 clinically isolated strains. The number for three-drug resistant strain was 21 （40.38%） and that of two-drug resistant was 19（36.54%）, but only 12（23.08%） strains were one drug resistant. The rate of total drug-resistance was 100%, but there were 15 strains of allied mutation of three genes, 16 of two mutations and 6 of only one by PCR-SSCP. The coincidences were respectively 71.43%, 84.12% and 50.00%. Then there was no significant difference between the allied mutations of multi-drug resistant gene and the mutations of only one drug resistant gene （P 〉 0.05）. Conclusion： PCR-SSCP technique has a higher sensibility and specificity to detect the genes of katG, rpoB and rpsL in tuberculosis L-form among pneumoconiosis complicated with tuberculosis,and the detecting rate of two drug resistant strains and three drug resistant strains was higher. The combined application of PCR-SSCP and AST has advantages at earlier diagnosis and guidance of clinical medications.

Sequence analysis on drug-resistant rpoB gene of Mycobacterium tuberculosis L-form of isolated from pneumoconiosis workers

Objective:To study the characteristics of drug-resistant genetic mutation of rpoB on coal workers' pneumoconiosis complicated with L-form of Mycobacterium tuberculosis. Methods: A total of 42 clinical isolated strains of Mycobacterium tuberculosis L-forms were collected, including 31 drug-resistant strains. Their genomes DNA were extracted, the target genes were amplified by PCR, and the hot regions in the rpoB gene were analyzed by automated DNA sequenator. Results: No mutation of rpoB gene was identified in 11 rifampicin-sensitive strains while conformation changes were found in 31 rifampicin-resistant strains. The mutation rate was 93.55% (29/31) in resistant strains, mainly concentrated in codon 531 (51.6%, 16/31) and 526 (32.26%, 10/31). Base substitutions happened, including 27 unit point mutation and 2 two point mutation. The mutation of codon 516 that new found wasn't reported by internal and overseas scholars. Conclusion: The substitution of highly conserved amino acids encoded by rpoB gene results in the molecular mechanism responsible for rifampicin resistance in Mycobacterium tuberculosis L-forms. It also proves that rpoB gene is diversiform.

铜在狐尾藻中的积累及亚细胞分布和化学形态

为探索狐尾藻对重金属铜的积累和耐性机制,本研究通过水培试验,研究不同浓度铜处理(0、20、50 mg·L^(-1))对狐尾藻(Myriophyllum spicatum L.)生长生理特性以及叶片表皮细胞形态的影响,分析各器官中铜吸收转运及铜在各组织器官亚细胞中的分布和化学形态。结果表明:各浓度铜处理下狐尾藻均能存活,但铜浓度高于50 mg·L^(-1)时,狐尾藻根、茎、叶生物量相比对照(铜0 mg·L^(-1))处理降低53.48%、36.99%和32.22%。铜处理后,狐尾藻根、茎和叶铜含量分别为11.81~186.34、1.32~7.89、2.11~11.99 mg·kg^(-1),根系中铜含量均高于叶片和茎部。铜在狐尾藻中的亚细胞分布主要位于根、茎、叶的细胞壁部分(36.49%~49.61%、45.44%~49.92%、41.45%~55.92%),其次是可溶性组分(21.65%~25.99%、23.03%~27.65%、18.01%~34.63%)。狐尾藻中铜的赋存化学形态以盐酸提取态、醋酸提取态和乙醇提取态为主,所占比例为76.34%~86.67%,均是活性较低的形态。因此,狐尾藻是铜富集较好的植物,其根部的耐性大于茎、叶。铜以吸附态或蛋白质、果胶酸盐等低活性形态赋存于细胞壁或可溶性组分(液泡)中是狐尾藻积累和耐受铜的重要机制。

基于自由曲面和L-system的红掌生长模型

针对红掌花卉生长可视化中参数难以确定的问题进行研究,提出推导控制点的方法,建立红掌器官模型:采用Bezier曲线结合扫描成型的方法来构造叶柄模型,采用双三次NURBS曲线对佛焰苞进行建模;运用红掌的相关表型数据拟合生长函数,提出基于红掌生长规律的微分L-system,可有效模拟红掌的拓扑结构和生长过程;通过虚拟器官表示红掌器官的几何属性,降低微分L-system的复杂度。试验验证提出的方法对红掌各项生长指标拟合度可达0.89以上,并可对每个生长阶段的红掌进行模拟,能有效地对红掌的生长过程进行建模。

Maass形式的傅里叶系数在算术级数中的渐近性

自守形式理论是现代数论的重要课题.自守形式的傅里叶系数蕴含了深刻的数论性质,其在数论中有许多应用.设f是本原Maass尖形式,λ_(f)(n)是它在尖点无穷远处的第n个傅里叶系数.结合经典的解析方法和一些本原自守L-函数的性质,本文研究了全模群上Maass尖形式的傅里叶系数在算术级数上的分布性质,得到了相应的渐近公式.

L波段全国产化2.5 kW级脉冲模块设计

随着国防和民用技术的迅速发展,高性能的L波段脉冲模块成为雷达和通信领域的关键组件。文章介绍了一个全国产化的2.5 kW级L波段脉冲模块的设计流程。该模块采用先进的射频放大技术和脉冲形成网络设计,结合本土材料和工艺技术的优势,实现模块的高性能和低成本生产。仿真和实验验证表明,设计的模块满足了所有预期的技术参数,包括功率水平、脉冲宽度和效率,通过了严格的质量控制和可靠性评估。

短期使用城镇生活污泥产品后镉在国槐中的迁移和富集

为探索短期添加城镇生活污泥产品后,重金属镉(Cd)在常用绿化树种国槐体内的积累和转移机制,通过盆栽试验,研究污泥产品不同添加量(0、2、4 kg·m^(-2))和使用时间(40、80、120、160 d)对Cd在国槐根系和叶片中的质量分数及其亚细胞分布、Cd富集和转运的影响,比较国槐不同器官中Cd积累量以及Cd在根、叶中的化学形态。结果表明:污泥产品添加量、使用时间及其交互作用均对国槐根和叶中的Cd质量分数有显著影响(P<0.001)。使用污泥产品会抑制Cd在国槐中的富集和转运,在160 d时有所改善。在国槐根和叶中,Cd主要分布在细胞壁组分、核蛋白成分和可溶性组分中,两者总质量比超过90%,随使用时间增加,会使Cd在可溶性组分中的质量比有所增加,但细胞壁中Cd的质量比仍是最大,在160 d时,发现添加污泥产品会提高Cd在根、叶细胞壁组分中的质量比。国槐根系和叶片中Cd主要以NaCl和HAc提取态存在,分别占质量比82.6%~92.0%和56.0%~86.0%,均是活性较低的形态。国槐可以应对污泥产品低添加量带来的Cd胁迫,主要途径为细胞壁固定、液泡区隔化以及将Cd转化为低活性形态贮存,可考虑以2 kg·m^(-2)污泥产品添加量在国槐幼林中推广应用。

L-半胱氨酸对揭竹过程中腐竹品质的影响

在腐竹制作过程中添加不同浓度的L-半胱氨酸,替代亚硫酸盐类漂白剂用于改善腐竹色泽,并进一步研究其对揭竹过程中腐竹的成膜速率、产率、蒸煮损失率、机械特性、营养组成及感官品质的影响,为传统豆制品腐竹的质量提高和科学化生产提供理论依据。结果表明,L-半胱氨酸添加量为0.05%时腐竹的成膜速率及总产率最高,蒸煮损失率较小。添加量0.25%时,腐竹的L*值最大、机械特性以及感官得分最高,同时可以显著提高腐竹的成膜速率及产率。

磷对甘蓝型油菜幼苗镉吸收及土壤酶活性的影响

【目的】探究施磷条件下油菜幼苗各部位镉含量、土壤有效镉(DTPA-Cd)、土壤磷形态和土壤酶活性的变化,以期为镉污染土壤的修复与可持续利用提供参考。【方法】通过盆栽实验,研究不同镉含量(0.3、3.0、5.0 mg/kg)下,磷添加(KH2PO4,5.0、75.0、150.0 mg/kg)对苗期油菜根、茎、叶部镉含量,土壤pH,有效镉含量及酶活性的影响。【结果】高磷添加增加了苗期油菜的生物量和镉积累量。土壤镉含量为3.0和5.0 mg/kg时,磷添加量为150.0 mg/kg时,油菜植株生物量最高,分别为2.68和2.03 g;其镉积累量也最多,分别为164.00、165.62微克/盆。对于土壤pH、有效镉含量和磷形态含量而言,镉含量为3.0、5.0 mg/kg时,磷施加量由5.0 mg/kg增加至150.0 mg/kg时,土壤pH分别降低了0.11、0.06个单位,土壤矿物质磷(Ligand-P)含量分别降低了10.2%、10.3%,土壤生物有效磷(Bioavailable-P)含量分别增加了48.7%、35.9%,土壤有效镉(DTPA-Cd)含量降低了7.5%、15.2%。这表明磷添加量的增加降低了土壤pH,促进土壤矿物质磷的释放,增加生物有效磷的含量,降低了土壤有效镉含量。此外,土壤酶活性数据也表明,随着磷添加量的增加,土壤中β-1,4-N-乙酰氨基葡萄糖苷酶(NAG)、脲酶(UE)的活性显著提高,而酸性磷酸酶(ACP)、碱性磷酸酶(ALP)的活性显著降低。这表明磷添加有效缓解了油菜根际对磷的需求,提高了氮循环酶的活性。【结论】施磷增加了土壤活性磷含量、降低了土壤有效镉含量、提高了氮循环酶的活性从而优化油菜根际环境,促进了油菜的生长,进而增加了油菜对镉的积累。

Cd在地肤[Kochia scoparia(L.)Schrad.]中的化学形态及亚细胞分布

为探究镉(Cd)在地肤[Kochia scoparia(L.)Schrad.]茎部与根系中的亚细胞分布特征,在不同pH和不同Cd浓度胁迫下,对成熟期地肤生物量及Cd在地肤根系、茎部的积累状况和化学形态特征进行研究。结果表明:所有处理地肤茎部生物量均高于根系,当Cd添加量为1.5~3.0 mg·kg^(-1)时,T处理(pH 6.1)地肤总生物量高于TS处理(pH 5.0);地肤将58.09%~89.35%的Cd积累在茎部,所有处理地肤Cd的富集系数大小为茎>根系,表明地肤茎对Cd的积累能力强于根系,且在Cd添加量为1.5~9.0 mg·kg^(-1)时,T处理地肤茎Cd积累量高于TS处理;地肤根系和茎中超过85%的Cd贮存在细胞壁与液泡中,表明二者是地肤细胞中Cd区室化分布和解毒的重要场所;地肤根系细胞器Cd所占比例低于地肤茎部,这也是地肤将更多的Cd富集在茎部的一个重要原因;地肤茎和根系中均以移动性和毒性相对较低的醋酸提取态、氯化钠提取态及乙醇提取态Cd分配比例最大(T为82.96%~88.17%;TS为83.70%~89.70%),其中醋酸提取态含量最高(T为37.31%~56.24%;TS为40.98%~52.32%),氯化钠提取态与乙醇提取态Cd含量接近,这种Cd赋存形式是地肤降低Cd生物有效性和减少Cd毒害的一种重要防御机制。当土壤pH为6.1和Cd添加量为1.5 mg·kg^(-1)时,地肤对土壤Cd的解毒较好。研究表明,地肤具有对不同Cd胁迫水平的耐性,且对酸性土壤有较强的适生性,因此适用于湖南地区酸性农田土壤的修复治理。

球空间中极小超曲面上L^(p)调和1-形式的有限性定理

设M^(m)(m≥3)是空间S^(m+1)中的完备定向非紧极小超曲面,考虑子流M^(m)上L^(p)调和1-形式的有限性问题.如果极小超曲面M^(m)存在一个紧致子集Ω使得MΩ是稳定的,则称M^(m)具有有限的指数.首先,在M^(m)有有限指数的假设条件下,应用Bochner公式、Sobolev不等式及截断函数和指标迭代的方法,证得:如果2≤p≤2m/m-1,则M^(m)上L^(p)调和1-形式空间的维数有限.其次,记A为超曲面M^(m)的第二基本形式,M^(m)的全曲率定义为第二基本形式的L2模.在M^(m)全曲率有正上界的假设条件下(特别地,该正上界的取值仅依赖于子流形M^(m)的维数m),利用截断函数法,得到了M^(m)上L^(p)调和1-形式的有限性定理.特别地,令p=2,可进一步得到,在极小超曲面M^(m)具有有限指数或全曲率有正上界的假设条件下,M^(m)仅有有限多个非抛物端.

道路工程中L形混凝土预制挡土墙的应用

预制拼装技术属于市政工程行业中备受推崇的新兴技术,在预制拼装桥梁技术快速兴起进的过程中,现代化道路工程建设的预制化、模块化发展要求发生明显变化,预制挡土墙作为道路工程中的重要组成结构,需对其具体应用展开深入研究。对道路工程中L形混凝土预制挡土墙的应用展开研究,整理相关经验,说明该技术具有的现浇主要优势,并对后续快速化施工、现场人员数量控制以及场地占用比例进行详细说明并给出针对性发展建议,希望可以为同领域工作者提供合理参考。

胃癌中幽门螺杆菌L型感染与MIF、MMP9、VEGF表达的关系

目的探讨胃癌组织中幽门螺杆菌L型(Hp-L)感染与巨噬细胞移动抑制因子(MIF)、基质金属蛋白酶-9(MMP-9)和血管内皮生长因子(VEGF)表达之间的相互关系及临床意义。方法应用免疫组织化学染色法及革兰染色检测80例胃癌组织及50例癌旁正常组织中Hp-L感染情况,同时检测上述组织中MIF、MMP9、VEGF蛋白的表达;运用RT-PCR及Western blotting法检测30例新鲜胃癌组织及相应切缘组织中MIFmRNA及MIF、MMP9、VEGF蛋白的表达。结果 80例胃癌组织中经免疫组化和革兰染色同时阳性病例有57例,与对照组相比具有显著性差异(71.25%vs 14%,P<0.05);胃癌组织中Hp-L阳性组中MIF、MMP9、VEGF蛋白表达阳性率高于Hp-L阴性组(P<0.05),且Hp-L感染与MIF、MMP9和VEGF表达均呈正相关关系(r=0.598,r=0.292,r=0.341,P<0.05)。30例胃癌新鲜组织中MIF-mRNA表达(1.583±0.177)高于对照组(1.407±0.078),两者差异具有显著性(t=3.729,P=0.001);Western blotting法检测MIF、MMP9、VEGF蛋白在胃癌组织中的表达显著高于癌旁正常组织;胃癌组织中MIF、MMP9均与肿瘤浸润深度及淋巴结转移有关,VEGF蛋白表达与肿瘤浸润深度有关(P<0.05)。结论 Hp-L型感染是促进胃癌的侵袭与转移的重要因素之一,其机制与上调胃癌组织中MIF、MMP9、VEGF表达有关。

金葡菌及其L型感染荷瘤小鼠的实验研究

本文应用金黄色葡萄球菌及Ｌ型分别感染肿瘤和非肿瘤Ｃ＿（５７）ＢＬ／６Ｎ小鼠，病理学和细菌学检测发现细菌及Ｌ型感染种植肿瘤的小鼠组，肿瘤转移率和诱癌能力均显著提高。此外金葡菌及其Ｌ型感染还可引起小鼠多器官间质性炎，尤以肺、肝、肾、脑及生殖器官等为著。结果表明细菌及Ｌ型感染与肿瘤转移和肿瘤的发生可能有关。

结核分枝杆菌L型致病性的实验研究

以结核分枝杆菌稳定Ｌ型感染豚鼠，证明结核分枝杆菌变为Ｌ型后致病性减弱，发病时间延长，ＯＴ试验阴性，引起的病理变化主要表现为组织的间质性炎症，有干酪样坏死，而无典型结核结节形成。原因在于细菌变为Ｌ型后细胞壁缺损，磷脂减少，不足以刺激巨噬细胞转变为上皮样组胞与郎罕氏巨细胞，而形成结核结节。在诊断上容易造成误诊或漏诊。

人体病理组织中细菌L型的电镜观察

对9例细菌L型感染的人体病理组织进行透射电镜观察。结果显示:(1)L型可分布于组织间质或进入吞噬细胞、上皮细胞和癌细胞等细胞胞质;(2)L型具有多形态、大小不等、胞壁缺陷、电子密度低等特点,细胞胞质内的细菌L型尚有A、B两种形态区别;(3)L型在组织中形成了不完全箍缩分裂;(4)宿主细胞超微结构仅出现轻微改变。本结果与文献报道基本一致,提出了透射电镜下病理组织中细菌L型的形态特征及分布;并对L型感染与慢性炎症的关系等问题进行了讨论。