Arriving at a correlation between the flagellar arrangement and multicellularity

Cilia and flagella are organelles of motility that enable cells to swim or move liquid over its surface. An exhaustive literature survey for the presence of the organelle in organisms across phyla showed that most animal cells harbor cilia in contrast to very few fungal cells. While this was not unexpected, it was the position and arrangement of this organelle in each cell that intrigued our attention. Natural selection might have favored motility over chemotaxis;and it would have done so to evolve a stable structure that could have undergone an optimization process requiring a precise geometry in the shape of cells and the structure that would help cells to move. The positioning of such a structure would play a pre-dominant role in optimal motility. It is now known that the flagellar position of a cell is a genetically distinct trait, occasionally used in phylogeny of bacteria, distributed in distinguishing patterns over cellular surface, but basically are of two types, either polar (one flagellum arising from one pole per cell) or peritrichous (lateral flagella distributed over the entire cell surface). Irrespective of the cellular habitat, flagella origin, ultrastructure and proteome, the present investigation surveyed 26 sub-types of flagellar arrangements from as many species as possible. A peculiar pattern ensued-Prokaryotes harbored predominantly polar and peritrichous types;eukaryotes showed a mere change of the peritrichous one. These numbers when used to create a Similarity tree depicted a similarity distance of 14 between the Eubacteria and Archaebacteria forming the first neighborhood;Protozoans, Algae, Fungi, Plantae and Animalia formed a second neighborhood. We offer a working hypothesis for this pattern and the gradual shift in the flagellar arrangement from polar, peritrichous, sub-apical, and apical to lateral throughout evolution.

DYNAMIC STUDIES ON FLAGELLAR REGENERATION IN DUNALIELLA SALINA

Flagella,the basic locomotive organ in algae,as well as bacteria and some cells of animals or high plants,would be damaged in the well stirred mass culture due to the strong forces caused by the fast mixing impellers. The dynamic regeneration of the flagella in deflagellated Dunaliella salina was studied microscopically by using a bench top flat bottom photobioreactor. The results showed that 90 minutes was necessary for the repair of flagella,after which half of the cells became motile as their flagella generated within 120 minutes and nearly all of the cells could swim freely within 180 minutes.

Therapeutic MUC1-Based Cancer Vaccine Expressed in Flagella-Efficacy in an Aggressive Model of Breast Cancer

MUC1, a tumor-associated antigen overexpressed in many carcinomas, represents a candidate of choice for cancer immunotherapy. Flagella-based MUC1 vaccines were tested in therapeutic setting in two aggressive breast cancer models, comprising the implantation of the 4T1-MUC1 cell line in either Balb/c, or Human MUC1 transgenic mice in which spontaneous metastases occurs. Recombinant flagella carrying only 7 amino acid of MUC1 elicited therapeutic activity, affecting both the growth of established growing tumors and the number of metastases. Higher therapeutic activity was achieved with an additional recombinant flagella designed with the SYFPEITHI algorithm. The vaccines triggered a Th1 response against MUC1 with no evident autoimmune response towards healthy MUC1-expressing tissues. Recombinant flagella carrying a 25-residue fragment of MUC1, induced the most effective response, as evidenced by a significant reduction of both the size and growth rate of the tumor as well as by the lower number of metastases, and expanding life span of vaccinated mice.

Organization, expression and evolution of flagellar genesin <i>Rhodobacter sphaeroides </i>2.4.1

Rhodobacter sphaeroides 2.4.1 belongs to the?-3 subdivision of the Proteobacteria. It possesses a multipartite genome structure consisting of two circular chromosomes, andit displays a wide range of metabolic diversity.Approximately 40 flagellar proteins are required for structure, assembly, and regulation of the flagellum formation in most bacterial species. R. sphaeroidescontains two flagellar gene clusters (fla1 and fla2),which encode 38 and 21 proteins, respectively. Thirty-six of these genes exist in duplicate gene-pairs.A combination of genome analysis, phylogenetic analysis and mRNA expression analysis were employed to examine the conservation of structure, function and evolution of fla1 and fla2 in R. sphaeroides. The results demonstrated that fla2, which was shared among members of ?-Proteobacteria, is native toR. sphaeroides, while fla1 was horizontally transferred from a member of ?-Proteobacteria.In addition, genes located in fla1 are expressed over several growth conditions, but those in fla2 are barely expressed.

Micro-robot design based on swimming mechanism of bacterial flagella

Nanomachines are controllable machines at the nano meter or molecular scale that are composed of nano-scale components.They have their own mechanochemistry,dynamics,workspace,and usability and are composed of nature's building blocks:namely proteins,DNA,and other compounds.Some bacteria(i.e.Escherichia coli)swim by rotating helical flagella.The structure and motion character of the flagellum are introduced.Through the study,a micro-robot was designed and its mechanical structure was explained in this paper.In the future,the bionic micro-robot is expected tobe built,which can travel inside the human body and carry out a host of complex operations such as minimally invasive surgery,highly localized drug delivery,and screening for diseases that are in their very early stages.It is important to medicine and could be applied in other areas,including space exploration,electronics and military.

A novel homozygous frameshift variant in DNAH8 causes multiple morphological abnormalities of the sperm flagella in a consanguineous Pakistani family

Multiple morphological abnormalities of the sperm flagella(MMAF)is a severe form of asthenozoospermia categorized by immotile spermatozoa with abnormal flagella in ejaculate.Whole-exome sequencing(WES)is used to detect pathogenic variants in patients with MMAF.In this study,a novel homozygous frameshift variant(c.6158_6159insT)in dynein axonemal heavy chain 8(DNAH8)from two infertile brothers with MMAF in a consanguineous Pakistani family was identified by WES.Reverse transcription-polymerase chain reaction(RT-PCR)confirmed DNAH8 mRNA decay in these patients with the DNAH8 mutation.Hematoxylin–eosin staining and transmission electron microscopy revealed highly divergent morphology and ultrastructure of sperm flagella in these patients.Furthermore,an immunofluorescence assay showed the absence of DNAH8 and a reduction in its associated protein DNAH17 in the patients'spermatozoa.Collectively,our study expands the phenotypic spectrum of patients with DNAH8-related MMAF worldwide.

In sight on multiple morphological abno rmalities of sperm flagella in male infertility:what is new?

The syndrome of multiple morphological abnormalities of the sperm flagella(MMAF)is a specific kind of asthenoteratozoospermia with a mosaic of flagellar morphological abnormalities(absent,short,bent,coiled,and irregular flagella).MMAF was proposed in 2014 and has attracted increasing attention;however,it has not been clearly understood.In this review,we elucidate the definition of MMAF from a systematical view,the difference between MMAF and other conditions with asthenoteratozoospermia or asthenozoospermia(such as primary mitochondrial sheath defects and primary ciliary dyskinesia),the knowledge regarding its etiological mechanism and related genetic findings,and the clinical significance of MMAF for intracytoplasmic sperm injection and genetic coun sell ng.This review provides the basic kno wledge for MMAF and puts forward some suggestions for further investigations.

Absence of murine CFAP61 causes male infertility due to multiple morphological abnormalities of the flagella

Impaired flagellar development and impaired motility of sperm is a cause of infertility in males. Several genes, including those of the AKAP, CCDC, CFAP, and DNAH families, among others, are involved in the‘‘multiple morphological abnormalities of the flagella"(MMAF) phenotype;these are the most common causes of male infertility. The Cilia-and flagella-associated protein(CFAP) family includes six members reported to cause MMAF phenotypes: CFAP43, CFAP44, CFAP69, CFAP65, CFAP70, and CFAP251. Here, we found that cilia-and flagella-associated protein 61(Cfap61) is highly expressed specifically in murine testes and show that the Cfap61-knockout male mice demonstrate MMAF phenotype, including sperm with short, coiled, and irregular flagella. Deletion of Cfap61 resulted in severe morphological and behavior abnormalities in sperm, reduced total sperm counts, impaired sperm motility, and led to male infertility.Notably, absence of Cfap61 impaired sperm flagella ultrastructural abnormalities on account of numerous distortions in multiple flagellum components. Immunostaining experiments in wild-type mice and healthy adult humans indicated that Cfap61 is initially localized at the neck of sperm, where it potentially functions in flagellum formation, and is later localized to the midpiece of the sperm. Thus, our study provides compelling evidence that dysregulation of Cfap61 affects sperm flagellum development and induces male infertility in mice. Further investigations of the CFAP61 gene in humans alongside clinical evidence showing MMAF phenotype in humans should contribute to our understanding of developmental processes underlying sperm flagellum formation and the pathogenic mechanisms that cause male infertility.

Novel mutation in ODF2 causes multiple morphological abnormalities of the sperm flagella in an infertile male

Numerous genes have been associated with multiple morphological abnormalities of the sperm flagella(MMAF),which cause severe asthenozoospermia and lead to male infertility,while the causes of approximately 50%of MMAF cases remain unclear.To reveal the genetic causes of MMAF in an infertile patient,whole-exome sequencing was performed to screen for pathogenic genes,and electron microscope was used to reveal the sperm flagellar ultrastructure.A novel heterozygous missense mutation in the outer dense fiber protein 2(ODF2)gene was detected,which was inherited from the patient’s mother and predicted to be potentially damaging.Transmission electron microscopy revealed that the outer dense fibers were defective in the patient’s sperm tail,which was similar to that of the reported heterozygous Odf2 mutation mouse.Immunostaining of ODF2 showed severe ODF2 expression defects in the patient’s sperm.Therefore,it was concluded that the heterozygous mutation in ODF2 caused MMAF in this case.To evaluate the possibility of assisted reproductive technology(ART)treatment for this patient,intracytoplasmic sperm injection(ICSI)was performed,with the help of a hypo-osmotic swelling test and laser-assisted immotile sperm selection(LAISS)for available sperm screening,and artificial oocyte activation with ionomycin was applied to improve the fertilization rate.Four ICSI cycles were performed,and live birth was achieved in the LAISS-applied cycle,suggesting that LAISS would be valuable in ART treatment for MMAF.

常染色体显性遗传2型多囊肾病基因参与精子尾部组装的作用研究

目的研究常染色体显性遗传2型多囊肾病基因(PKD2)在精子尾部组装过程中的作用。方法回顾分析1例携带PKD2基因突变的非梗阻性无精症(NOA)患者的临床资料,Sanger测序验证该基因突变,对睾丸组织进行HE和免疫荧光染色;通过对正常人精子涂片进行免疫荧光染色观察PKD2在精子的定位,利用人视网膜色素上皮(hRPE)细胞检测PKD2在纤毛发育过程中的定位。结果该NOA患者PKD2基因存在c.1571T>A(P.I524N)杂合突变,其精子细胞尾部发育缺陷;PKD2主要定位于精子鞭毛起始部,与参与精子尾部组装及发育的重要蛋白Ac-Tubulin及γ-Tubulin存在共定位,在hRPE细胞纤毛发育过程中PKD2主要定位于纤毛的基体。结论本研究报道了1例全新的PKD2杂合突变的NOA患者病例,结果提示PKD2可能参与精子尾部的起始组装,为进一步研究精子鞭毛组装的机制提供了基础,PKD2基因c.1571T>A(P.I524N)杂合突变可能是无精症诊疗的新靶点。

Characterization of the flagellar biosynthesis regulatory gene flbD in Azospirillum brasilense

A flagellar gene cluster fragment includingflbD of Azospirillum brasilense was cloned and sequenced. TheflbD mutant strain was found to be nonmotile——losingboth polar and lateral flagella (Fla-Laf-). Motility and fla-gella were regained by complementation with plasmid-borne multicopy flbD, but altered with larger swarming circle and fewer lateral flagella on the semisolid plate. This result indicated that FlbD plays an important role in the regulation of both polar and lateral flagellar biosynthesis in A. brasilense.

海洋假交替单胞菌端生和侧生鞭毛对生物膜形成的影响

海洋假交替单胞菌广泛分布于各种海洋环境中,能分泌多种生物活性物质和胞外酶类,多具有较强的生物膜形成能力。这些生物膜具有诱导矿化和吸引腐蚀生物幼体附着的功能,在海洋生态系统的能量和元素循环中扮演特殊角色。细菌的鞭毛系统介导细菌的运动行为,在营养的获取以及生物膜和浮游状态转化过程中发挥作用。有研究表明,鞭毛也是生物膜基质的有机组分,但目前海洋假交替单胞菌鞭毛系统在生物膜形成中的作用尚不清楚。本文以南海表层来源的假交替单胞菌(Pseudoalteromonas sp.SCSIO 11900)和深海沉积物来源的近源假交替单胞菌(Pseudoalteromonas.sp.SM9913)为研究对象,分别选取了编码鞭毛不同组分的基因进行敲除,研究鞭毛缺失对细胞游动性和生物膜形成能力的影响。结果表明,无论来自表层还是深海的假交替单胞菌都使用端生鞭毛维持细胞游动性,而且其缺失促进生物膜的形成。此外,深海来源的SM9913还具有侧生鞭毛。尽管这种鞭毛有助于提高细胞游动性,却不影响细菌的生物膜形成能力。这些端生鞭毛系统在近缘海洋假交替单胞菌中广泛分布,可能为该属菌株在不同海洋环境中获取营养、定殖及适应环境的过程中发挥重要作用。

布鲁氏菌鞭毛和毒力因子及疫苗研究进展

布鲁氏菌病是一种重要的人兽共患病,在全球范围内造成了巨大的经济损失和公共卫生问题。近年来,我国布鲁氏菌病仍没有得到很好的控制,目前尚无国际上认可的人用布鲁氏菌疫苗,动物布鲁氏菌疫苗在近几十年布鲁氏菌病防控中起着重要作用,但仍存在一些不足。论文综述了布鲁氏菌鞭毛的组成结构和鞭毛蛋白的作用与功能、毒力因子和疫苗研发等方面的研究进展,为后续布鲁氏菌致病性的分子机制研究和疫苗研发提供参考。

特殊类型畸精子症行卵细胞胞质内单精子注射的治疗结局分析

目的:特殊类型畸精子症是男性不育中罕见病和单基因遗传疾病,本文探讨其卵细胞胞质内单精子注射(ICSI)的治疗结局。方法:回顾性分析2011年1月至2021年1月于江苏省人民医院生殖医学中心实施ICSI治疗的特殊类型畸精子症患者,根据精子形态分为三组(圆头精子症、无头精子症和精子鞭毛多发形态异常),三组患者基因检测均发现致病或可能致病的基因突变。比较三组患者间的临床和实验室数据(年龄、BMI、精子参数、成熟卵子数、受精率、优质胚胎率、临床妊娠率、活产率和自然流产率)差异。结果:三组共34对患者夫妇,三组患者间年龄,BMI,MII卵子数差别无统计学意义(P>0.05),圆头精子症的精子浓度和精子活力显著高于其他两组(P<0.01),圆头精子症组的受精率显著低于其他两组(P<0.01)。三组共34个取卵周期,其中4个取卵周期未形成可移植胚胎,形成可移植胚胎的30个取卵周期在第一个胚胎移植周期中获得22例临床妊娠,最终活产20例,自然流产2例。无头精子症和精子鞭毛多发形态异常两组的临床妊娠率和活产率显著高于圆头精子症组(P<0.01),三组间自然流产率没有统计学差异(P>0.05)。结论:圆头精子症患者实施人工卵母细胞激活依然有部分患者完全不受精或受精率低下。精子鞭毛多发形态异常和无头精子症患者可以通过ICSI取得相对较好的妊娠结局。

头花蓼对幽门螺杆菌粘附定植的影响

目的观察头花蓼水提物对幽门螺杆菌(Helicobacter pylori,H.pylori)粘附定植的影响。方法采用尿素酶活性比色法定量检测头花蓼水提物作用前后幽门螺杆菌尿素酶活性的影响;运用半固体布氏琼脂平板法检测不同浓度头花蓼对幽门螺杆菌鞭毛运动的影响;利用细胞爬片革兰染色观察不同浓度头花蓼作用后幽门螺杆菌在GES-1细胞表面粘附的变化并计算各组抑制率;采用Real-time PCR技术检测头花蓼作用前后相关基因ureB、ureE、ureF、flaA、flaB、babA、alpA、alpB的mRNA转录水平。结果头花蓼水提物对幽门螺杆菌尿素酶活性具有抑制作用,抑制率为44.90±0.289。头花蓼浓度为1/2MIC、1/4MIC、1/8MIC分别作用后幽门螺杆菌菌膜晕圈直径显著小于对照组,分别为(5.67±0.55)mm,(8.5±0.50)mm,(11.67±1.53)mm,差异有统计学意义(P<0.05);不同浓度头花蓼水提物均能抑制细菌粘附胃上皮细胞,各浓度组与对照组比较,差异有统计学意义(P<0.05)。头花蓼作用后,与对照组比较,下调了尿素酶结构基因ureB,尿素酶活性基因ureE、ureF、鞭毛相关基因flaB、flaA以及粘附素基因babA、alpA、alpB mRNA的转录水平,差异均具有统计学意义(P<0.05)。结论头花蓼对幽门螺杆菌具有明显的抑制作用,是通过抑制幽门螺杆菌尿素酶活性,减缓幽门螺杆菌的鞭毛动力,影响幽门螺杆菌对胃粘膜上皮细胞的粘附等方面发挥其抑菌作用。

细菌鞭毛研究概况及进展

鞭毛是细菌的一种特殊结构,约半数的杆菌、极少数球菌和所有的螺旋菌及弧菌都有鞭毛。鞭毛与细菌的运动有关,并在感染与免疫以及分类鉴定等方面发挥重要的作用,受到细菌研究者的高度重视。从细菌鞭毛的结构以及它在细菌致病性和免疫中的作用最新研究进展加以概述,以供细菌研究者参考。

青岛海域大面积聚集漂浮浒苔的显微观测

对近期在青岛海域大面积聚集漂浮的浒苔(Enteromorpha prolifera(Muell)J.Ag.)进行常规显微镜观察,发现细胞内存在大小约1μm的可动的颗粒状结构。分别采用鞭毛染色和扫描电镜观察的方法对其进行了初步研究,均未发现此颗粒状可动结构具有鞭毛。用透射电镜对浒苔细胞的超微结构特征进行了观察,显示浒苔细胞壁致密而厚,每个浒苔细胞有1个片状叶绿体,贴近于细胞壁分布,淀粉粒含量丰富,但依然未发现胞内颗粒状结构具有鞭毛。

雌核发育银鲫和两性生殖彩鲫精子蛋白组份的比较研究

对雌核发育银鲫和两性融合彩鲫精子蛋白组份进行了比较分析。通过分级抽提得到精子的不同组份精浆、精头的膜、鞭毛和脱膜精头等 ,然后经不同的凝胶电泳系统 ,比较分析了银鲫精子和其两性亲缘种彩鲫精子相应组份可溶性蛋白成份的差异。研究表明 ,经分级抽提的银鲫精子和彩鲫精子的各个组份都含有其特定的蛋白谱带。精浆蛋白在两种鱼之间和两种鱼的不同个体之间都存在一定差异。精头膜、鞭毛和脱膜精头的可溶性蛋白在同种鱼不同个体间高度一致 ,但在两种鱼之间表现出差异。两种鱼精头膜的可溶性蛋白在SDS PAGE电泳图谱上基本一致 ,而在非变性聚丙烯酰胺凝胶电泳图谱上则具有各自的特征性谱带。鞭毛可溶性蛋白的SDS PAGE分析在雌核发育银鲫中揭示出一条特异的蛋白带。脱膜精头的可溶性蛋白在SDS PAGE电泳图谱上差异明显 ,存在几条特征性蛋白带 ,并经Acid UreaPAGE系统分析 ,证实这些特征性蛋白为碱性蛋白。这些发现为进一步鉴定雌核发育银鲫雄鱼精子的特异性蛋白和揭示其分子机制打下了基础。

小鼠精原细胞体外分化为精子细胞的研究

目的探讨在辅以外源生殖激素混合培养的小鼠睾丸细胞中，精原细胞向精子细胞的转化。方法采用7～8d龄小鼠睾丸组织，以组合酶消化法制备睾丸细胞，在含重组卵泡刺激素（r-FSH）和睾酮的培养基中进行体外培养；定期观察细胞的生长和形态变化；用分子生物学和流式细胞技术对生长各阶段细胞进行分析。结果培养5d即可观察到形态与大小类似于圆形精子细胞，7d后可见带有鞭毛与变形的精子细胞；逆转录聚合酶链反应（RT-PCR）结果显示，培养前细胞的睾丸特异蛋白激酶（TESK1）与鱼精蛋白2（Prm2）mRNA表达阴性，培养9与17d细胞TESK1与Prm2 mRNA表达均阳性；DNA倍体分析显示，培养5d细胞有单倍体峰出现，并随培养天数增加而增加。结论在体外混合培养的小鼠睾丸细胞中加入外源生殖激素可以使精原细胞转化为精子细胞。

鞭毛对F18^+大肠杆菌体外生物被膜形成影响的研究

为研究鞭毛在F18+大肠杆菌(F18+E.coli)生物被膜(BF)形成过程中的作用,本研究利用λ-Red同源重组系统构建了F18+E.coli鞭毛素编码基因(fliC)缺失突变株(F18+△fliC)。以野生株为对照,通过在细胞表面形成典型微菌落试验以及BF的定性和定量试验,比较F18+△fliC菌株在体外形成BF能力的变化。吉姆萨染色结果表明,F18+△fliC菌株在IPEC-J2细胞表面形成典型微菌落数减少。BF体外试管培养定性试验和结晶紫定量试验均表明,F18+△fliC菌株形成BF的能力下降。本研究表明鞭毛在F18+E.coli体外BF形成过程中发挥了重要的作用,为进一步研究F18+E.coli的致病机制提供了实验依据。