Computational analysis of genetic loci required for amphid structure and functions and their possibly corresponding microRNAs in C. elegans

Objective To examine the important roles of microRNAs （miRNAs） in regulating amphid structure and function, we performed a computational analysis for the genetic loci required for the sensory perception and their possibly corresponding miRNAs in C. elegans. Methods Total 55 genetic loci required for the amphid structure and function were selected. Sequence alignment was combined with E value evaluation to investigate and identify the possible corresponding miRNAs. Results Total 30 genes among the 55 genetic loci selected have their possible corresponding regulatory miRNA（s）, and identified genes participate in the regulation of almost all aspects of amphid structure and function. In addition, our data suggest that both the amphid structure and the amphid functions might be regulated by a series of network signaling pathways. Moreover, the distribution of miRNAs along the 3＇ untranslated region （UTR） of these 30 genes exhibits different patterns. Conclusion We present the possible miRNA-mediated signaling pathways involved in the regulation of chemosensation and thermosensation by controlling the corresponding sensory neuron and interneuron functions. Our work will be useful for better understanding of the miRNA-mediated control of the chemotaxis and thermotaxis in C. elegans.

原位杂交检测mex-3 mRNA在C.elegans野生型胚胎发育中的分布

Caenorhabditiselegans是发育生物学中的重要模式生物,也是全基因序列已知的唯一多细胞动物.许多基因参与C.elegans的细胞命运决定和细胞谱系发育.mex 3是C.elegans早期胚胎发育中细胞命运决定的重要基因.本文报告采用原位杂交技术检测mex 3mRNA在C.elegans野生型胚胎发育中的分布,发现mex 3mRNA在生殖腺和卵母细胞的细胞质都有广泛而丰富的分布,从受精开始mex 3mRNA的分布开始发生了变化,从均一分布的模式开始进入不对称的分布模式,在2细胞阶段mex 3mRNA仅在前部的大卵裂球分布很多、染色较深,后部的小卵裂球低水平分布,染色较浅,在4细胞的早期胚胎中只在前部的两个卵裂球有较丰富的分布,后部的2个细胞低水平分布.到8细胞和16细胞阶段,只有后部的一个卵裂球检测到mRNA的分布,此后的胚胎中未检测到mRNA的分布.

细菌介导的RNA干扰对C.elegans中par-3基因的作用

设计并构建了针对par-3基因的发夹RNA载体,将构建好的质粒转入大肠杆菌HT115,25℃喂食Caenorhabditiselegans(C.elegans)野生型虫体,24h后观察par-3(RNA干扰)C.elegans的胚胎发育情况。结果显示通过喂食形成发夹结构dsRNA的细菌可以对C.elegans中par-3基因进行RNA干扰,干扰率可以达到60%以上。干扰后的早期胚胎发育丧失第一次卵裂的不对称性,第二次卵裂的纺锤体方向发生改变,与par-3突变体的观察结果一致,为进一步在mex-3转基因虫体中通过RNA干扰研究基因表达打下了基础。

Involvement of genes required for synaptic function in aging control in C.elegans

Objective To identify new genes required for neurosecretory control of aging in C. elegans. Methods In view of the importance of nervous system in aging regulation, we performed the screen for genes involved in the aging regulation from genetic loci encoding synaptic proteins by lifespan assay and accumulation of lipofuscin autofluorescence. We further investigated the dauer formation phenotypes of their corresponding mutants and whether they were possibly up-regulated by the insulin-like signaling pathway. Results The genetic loci of unc-10, syd-2, hlb-1, dlk-1, mkk-4, scd- 2, snb-1, ric-4, nrx-1, unc-13, sbt-1 and unc-64 might be involved in the aging control. In addition, functions of unc-10, syd-2, hlb-1, dlk-1, mkk-4, scd-2, snb-1, ric-4 and nrx-1 in regulating aging may be opposite to those of unc-13, sbt-1 and unc-64. The intestinal autofluorescence assay further indicated that the identified long-lived and short-lived mutants were actually due to the suppressed or accelerated aging. Among the identified genes, syd-2, hlb-1, mkk-4, scd-2, snb-1, ric-4 and unc-64 were also involved in the control of dauer formation. Moreover, daf-2 mutation positively regulated the expression of syd-2 and hlb-1, and negatively regulated the expression of mkk-4, nrx-1, ric-4, sbt-1, rpm-1, unc-10, dlk- 1 and unc-13. The daf-16 mutation positively regulated the expression of syd-2 and hlb-1, and negatively regulated the expression of mkk-4, nrx-1, sbt-1, rpm-1, unc-10, dlk-1 and unc-13. Conclusion These data suggest the possibly important status of the synaptic transmission to the animal＇ s life-span control machinery, as well as the dauer formation control.

Screening for genetic loci affecting the active zone formation in C. elegans

Objective To screen and identify genetic loci affecting the active zone formation in C. elegans. Methods A SYD-2：：GFP reporter was constructed and used as an active zone marker for forward genetic screen to identify genetic loci affecting the active zone formation. Results Eight isolated mutant alleles were characterized from 15,000 haploid genomes. The SYD-2：：GFP phenotypes of these mutants are mainly reflected as the changes of number, morphology, distribution of puncta and the gaps appearance. Some mutants also exhibit visible behavioral or physical phenotypes, and aldicarb resistant or sensitive phenotypes. Conclusion These mutants provide the opportunity for further systematic research on the active zone formation and the neurotransmission.

ATP depletion is the major cause of MPP^+ induced dopamine neuronal death and worm lethality inα-synuclein transgenic C.elegans

Objective To investigate the toxic effect of environmental neurotoxin MPP^＋ to C.elegans and identify the mechanisms that cause the toxicity.Methods Humanα-synuclein transgenic C.elegans was used as the animal model,the toxic effect of MPP^＋ to dopamine（DA）neurons and the lifespan of worms was tested.The worms were feed with OP50 to determine whether ATP increase can rescue the worm from toxicity.ATP level and aberrant protein accumulation were analyzed in the MPP^＋ treated worms with or without OP50 addition.Results We found that MPP^＋ induced DA cell death and worm lethality,which could be prevented by OP50 treatment.OP50 exerted the protective effect by up-regulating ATP level,even though it also induced accumulation ofα-synuclein.Despite the undefined role of protein aggregation to the cell death,our results showed that the toxicity of MPP^＋ was mainly caused by the ATP depletion in theα-synuclein transgenic C.elegans.Conclusion MPP^＋ could induce DA neuronal death and worm lethality inα-synuclein transgenic C.elegans;Compared with the aggregation ofα-synuclein,the major cause of MPP^＋ toxicity appeared due to ATP depletion.

Assessment of locomotion behavioral defects induced by acute toxicity from heavy metal exposure in nematode Caenorhabditis elegans

Locomotion behaviors are susceptible to disruption by a broad spectrum of chemicals and environmental stresses. However, no systematic testing of locomotion behavior defects induced by metal exposure has been conducted in the model organism of nematode Caenorhabditis elegans. In this study, the acute toxicity from heavy metal exposure on the locomotion behaviors was analyzed in nematodes. Endpoints of head thrash, body bend, forward turn, backward turn, and Omega/U turn were chosen to evaluate the locomotio...

Computational analysis of genetic loci required for synapse structure and function and their corresponding microRNAs in C. elegans

Objective To elucidate the important functions of microRNAs （miRNAs） in regulating synaptic assembly and function, we performed a computational analysis for the genetic loci required for the synaptic structure and function and their corresponding miRNAs in C. elegans. Methods Total 198 genetic loci required for the synaptic structure and function were selected. Sequence alignment was combined with E value evaluation to investigate and identify the possible corresponding miRNAs. Results Total 163 genes among the 198 genetic loci selected have their possibly corresponding regulatory miRNA （s）, which covered most of the important genetic loci required for the synaptic structure and function. Moreover, only 22 genes among the analyzed 38 genetic loci encoding synaptic proteins have more possibility to under the control of non-coding RNA genes. In addition, the distribution of miRNAs along the 3＇ untranslated region （UTR） of these 22 genes exhibits different patterns. Condusion Here we provide the computational screen and analysis results for the genetic loci required for synaptic structure and function and their possible corresponding miRNAs. These data will be useful for the further attempt to systematically determine the roles of miRNAs in synaptic assembly and function regulation in worms.

Phenotypic and Behavioral Defects Induced by Iron Exposure Can Be Transferred to Progeny in Caenorhabditis elegans

Objective Previous work has showed that excess iron accumulation is harrnftd to reproduction and even promotes death; however, whether the multiple biological toxicity of iron （Fe） exposure could be transferred to progeny remains unknown. The present study used Caenorhabditis elegans to analyze the multiple toxicities of iron exposure and their possible transferable properties. Methods Three concentrations of iron sulfate solution （2.5μmol/L, 75μmol/L, and 200 μmol/L） were used. The endpoints of lifespan, body size, generation time, brood size, head thrash and body bend frequencies, and chemotaxis plasticity were selected to investigate Fe toxicity and its effect on progeny in Caenorhabditis elegans. Results The Fe toxicity could cause multiple biological defects in a dose-dependent manner by affecting different endpoints in nematodes. Most of the multiple biological defects and behavior toxicities could be transferred from Fe-exposed Caenorhabditis elegans to their progeny. Compared to the parents, no recovery phenotypes were observed for some of the defects in the progeny, such as body bend frequency and life span. We further summarized the defects caused by Fe exposure into 2 groups according to their transferable properties. Conclusion Our results suggest that Fe exposure could cause multiple biological defects, and most of these severe defects could be transferred from Fe exposed nematodes to their progeny.

C.elegans as a model system for Parkinson disease

Parkinson disease(PD) is characterized by the selective loss of dopaminergic neurons in the substantia nigra. Although investigation in mammalian animal models of PD has enhanced our understanding of PD, the complexity of the mammalian nervous system and our inability to visualize DA neurons in vivo restricts the advances in elucidating the molecular mechanisms of PD. Conservation between C. elegans and mammals in genomic, biosynthetic and metabolic pathways as well as the advantages of observing DA neurons morphology in vivo and the ease of transgenic and genetic manipulation make C. elegans an excellent model organism for PD.

High uric acid model in Caenorhabditis elegans

To establish experimental high uric acid model in C.elegans.Hypoxanthine,adenine,xanthine,and uric acid were used to treat C.elegans and then hyperuricemic C.elegans was evaluated by allopurinol.Hyperuricemic C.elegans were obtained after normal worms were treated by xanthine(0.25 mg/mL,18 h).For hyperuricemic worms,there was a statistically significant increase in the uric acid level(p<0.001)and a lower drug damage(p>0.05).Moreover,the model was proved to keep a high uric acid level for up to 12 h.After given allopurinol(0.25 mg/mL,12 h),the uric acid of hyperuricemic C.elegans had a significant reduction by 15%.Furthermore,xanthine oxidase activity in hyperuricemic C.elegans showed a statistically significant increase(p<0.001),which resulted in a raised uric acid content.A high uric acid model with low drug damage and high efficiency and stability was established in C.elegans after simply xanthine treatment.

A Comprehensive Understanding of Dietary Effects on C.elegans Physiology

Diet has been shown to play an important role in human physiology.It is a predominant exogenous factor regulating the composition of gut microbiota,and dietary intervention holds promise for treatment of diseases such as obesity,type 2 diabetes,and malnutrition.Furthermore,it was reported that diet has significant effects on physiological processes of C.elegans,including reproduction,fat storage,and aging.To reveal novel signaling pathways responsive to different diets,C.elegans and its bacterial diet were used as an interspecies model system to mimic the interaction between host and gut microbiota.Most signaling pathways identified in C.elegans are highly conserved across different species,including humans.A better understanding of these pathways can,therefore,help to develop interventions for human diseases.In this article,we summarize recent achievements on molecular mechanisms underlying the response of C.elegans to different diets and discuss their relevance to human health.

Particle selectivity of filtering by C. elegans

A nematode Caenorhabditis elegans(C. elegans) is a filter feeder, which draws a suspension of bacteria and separates bacteria from the solvent by using pharyngeal pumping motions and specific mouth parts. This mechanism has not been fully understood. We investigated the mechanism of filtering of bacteria in the pharynx of C. elegans by visualization by fluorescent particles and dyed E. coli. We succeeded in quantifying the selectivity of bacteria-sized particles by C. elegans. The most accumulated particles were those of 0.5 μm in diameter. The quantity of accumulated particles of 0.2 μm or 1.0 μm in diameter was about one third of that of particles of 0.5μm in diameter. The least accumulated particles were those of 0.05 μm in diameter. These results suggest that the pharyngeal structures of C. elegans would be suitable for eating bacteria because the size of bacteria ingested by C. elegans worms is about 0.5 μm in diameter. We also succeeded in visualizing pharyngeal structures and pumping motions and flow in the pharynx. We found that there were phase differences in the motions among procorpus, metacorpus and isthmus. This result suggests filtering would occur at the two tips of procorpus and isthmus by the phase differences. We found that bacteria-sized particles and bacteria were flowed and trapped in the channels, which existed along the central lumen from tip of procorpus to isthmus. From our results, we proposed the novel mechanism of filtering of bacteria through the channels for flowing and trapping. In future, this selective filtering mechanism of C. elegans would be applied to development of microfluidic filtration devices for medical and biological equipment.

Realgar transforming solution is involved of JNK, P38 MAPK pathway in down-regulating Ras/MAPK pathway in Caenorhabditis elegans

OBJECTIVE To investigate the effect and the mechanisms of realgar transforming solution(RTS)on down-regulating over activated ras.METHODS we used the optimizing technical processes to obtain the RTS,and eval⁃uate the mechanisms of RTS on down-regulating overactivated ras in Caenorhabditis elegans.RESULTS We found that the mRNA level of let60 and lin45 significantly decreased following exposure to RTS,but mRNA levels of mpk1 were not statistically significant in let60/ras(gf)mutant.RTS together with sorafenib(RAF inhibitors)significantly enhanced the activity of RTS on down-regulating overactivated ras more than RTS only,but 50μmol·L^-1 PD98059,a specific ERK inhibitor did not.Lin45 gene RNAi decreased the ability of RTS on down-regulating overactivated ras significantly,but mpk1 gene RNAi did not,indicating that the activity of RTS on down-regulating overactivated ras mainly through targeting to lin45/raf.In addition,RTS significantly increased mRNA level of pmk1/p38 and jnk1/jnk in let-60/ras(gf)mutant,50μmol·L^-1 SB203580(p38 inhibitor)and SP600125(JNK inhibitor)significantly attenuated the effects of RTS on down-regulating overactivated ras in some degree,and pmk1,jnk1 gene RNAi displayed the similar results,suggesting that P38 and JNK/MAPK pathways in some degree were involved in the effects of RTS on down-regulating overactivated ras in C.ele⁃gans.CONCLUSION Realgar transforming solution down-regulate the Ras/MAPK pathway through JNK and P38 MAPK pathways.

P. fluorescens and B. megaterium Effect on the Lifespan of Mutant-Type dpy-11 and Wild-Type of C. elegans

The research question being studied in this paper is how do different types of bacteria as food (Pseudomonas fluorescens and Bacillus megaterium) affect the lifespan of Caenorhabditis elegans in dpy-11 mutant-type and wild-type? P. fluorescens and B. megaterium will be the two pathogens that will be tested on two different types of C. elegans: mutant-type dpy-11 and wild-type. From the analysis of primary articles studying these pathogens, it can be concluded that P. fluorescens and B. megaterium are decent contenders for allowing C. elegans to grow and possibly extend the lifespan of it. P. fluorescens will allow the lifespan of the two types of nematodes to be longer. Additionally, the mu-tant-type dpy-11 of C. elegans will have a much longer lifespan, even double, compared to that of the wild-type. The results showed P. fluorescens had a longer lifespan than B. megaterium but not as long as C. elegans’ main food source, E. coli. C. elegans mutant dpy-11 had a longer lifespan than the wild-type. Furthermore, there were no C. elegans present in the B. megaterium wild-type plates.

Overexpression of Kdm6b induces testicular differentiation in a temperature-dependent sex determination system

In reptiles,such as the red-eared slider turtle(Trachemys scripta elegans),gonadal sex determination is highly dependent on the environmental temperature during embryonic stages.This complex process,which leads to differentiation into either testes or ovaries,is governed by the finely tuned expression of upstream genes,notably the testis-promoting gene Dmrt1 and the ovary-promoting gene Foxl2.Recent studies have identified epigenetic regulation as a crucial factor in testis development,with the H3K27me3 demethylase KDM6B being essential for Dmrt1 expression in T.s.elegans.However,whether KDM6B alone can induce testicular differentiation remains unclear.In this study,we found that overexpression of Kdm6b in T.s.elegans embryos induced the male development pathway,accompanied by a rapid increase in the gonadal expression of Dmrt1 at 31°C,a temperature typically resulting in female development.Notably,this sex reversal could be entirely rescued by Dmrt1 knockdown.These findings demonstrate that Kdm6b is sufficient for commitment to the male pathway,underscoring its role as a critical epigenetic regulator in the sex determination of the red-eared slider turtle.

层粘蛋白α链基因突变对神经组织发育影响的研究

目的研究层粘蛋白α链在神经系统发育中的作用。方法用乙基甲磺酸盐(ethyl m ethane-su lphonate,EMS)诱发线虫C.elegans层粘蛋白α链基因突变,通过DNA测序确定突变部位,用在C.elegans神经系统特异表达的启动子和绿色荧光蛋白报告基因构建的表达载体,注入生殖腺来观察后代正常和突变体的神经组织。结果诱导层粘蛋白α链基因突变产生四种突变体,其中两种即第803位和第2074位的突变形成终止密码,为无义突变,绿色荧光蛋白标记的神经纤维荧光图象显示呈锯齿状,且有纤维断裂。另外两种分别位于第98位、第1415位突变,是错义突变,二者引起的神经纤维改变不明显。结论层粘蛋白α链基因第803位和第2074位的无义突变引起神经纤维异常发育。

线虫脂肪沉积中组织蛋白酶B的表达及功能研究

文章利用高糖/高胆固醇饮食诱导了线虫,油红O染色结果表明线虫脂肪沉积显著增加。为了研究组织蛋白酶B在诱导线虫过程中的作用,利用实时荧光定量聚合酶链式反应(polymerase chain reaction,PCR)研究了组织蛋白酶B同源基因的表达。选取其中的关键基因利用RNA干扰(RNA interference,RNAi)技术,研究其在线虫脂肪沉积中的作用。结果表明:在线虫脂肪沉积改变后,组织蛋白酶B同源基因表达随之改变;而利用RNA干扰组织蛋白酶B关键基因cpr-3的表达,线虫的脂肪沉积未受影响。因此,组织蛋白酶B同源基因可能参与线虫脂肪沉积改变后的生理变化,而未直接参与线虫脂肪沉积的调控。

Hemicentins:What have we learned from worms?

Hemicentins are conserved extracellular matrix proteins discovered in Caenorhabditis elegans, with orthologs in all vertebrate species including human and mouse. Hemicentins share a single, highly conserved amino-terminal von Willebrand A domain, followed by a long （〉40） stretch of immunoglobulin repeats, multiple tandem epidermal growth factors and a fibulin-like carboxy-terminal module. C. elegans has a single hemicentin gene that has pleiotropic functions in transient cell contacts that are required for cell migration and basement membrane invasion and in stable contacts at hemidesmosome-mediated cell junctions and elastic fiber-like structures. Here, we summarize what is known about the function ofhemicentin in C. elegans and discuss implications for hemicentin function in other species.

The clinical antiprotozoal drug nitazoxanide and its metabolite tizoxanide extend Caenorhabditis elegans lifespan and healthspan

The drugs extending healthspan in clinic have always been searched.Nitazoxanide is an FDA-approved clinical antiprotozoal drug.Nitazoxanide is rapidly metabolized to tizoxanide after absorption in vivo.Our previous studies find that nitazoxanide and its metabolite tizoxanide induce mild mitochondrial uncoupling and activate cellular AMPK,oral nitazoxanide protects against experimental hyperlipidemia,hepatic steatosis,and atherosclerosis.Here,we demonstrate that both nitazoxanide and tizoxanide extend the lifespan and healthspan of Caenorhabditis elegans through Akt/AMPK/sir 2.1/daf16 pathway.Additionally,both nitazoxanide and tizoxanide improve high glucose-induced shortening of C.elegans lifespan.Nitazoxanide has been a clinical drug with a good safety profile,we suggest that it is a novel anti-aging drug.