Fang J et al. (2023) Commun Biol "Ribo-On and Ribo-Off tools using a self-cleaving ribozyme allow manipulation of endogenous gene expression in C. elegans."

Liu X, Wang Y, Fang J, Zou W, Wang J, Chen B

[Commun Biol, 2023]

Investigating gene function relies on the efficient manipulation of endogenous gene expression. Currently, a limited number of tools are available to robustly manipulate endogenous gene expression between "on" and "off" states. In this study, we insert a 63 bp coding sequence of T3H38 ribozyme into the 3' untranslated region (UTR) of C. elegans endogenous genes using the CRISPR/Cas9 technology, which reduces the endogenous gene expression to a nearly undetectable level and generated loss-of-function phenotypes similar to that of the genetic null animals. To achieve conditional knockout, a cassette of loxP-flanked transcriptional termination signal and ribozyme is inserted into the 3' UTR of endogenous genes, which eliminates gene expression spatially or temporally via the controllable expression of the Cre recombinase. Conditional endogenous gene turn-on can be achieved by either injecting morpholino, which blocks the ribozyme self-cleavage activity or using the Cre recombinase to remove the loxP-flanked ribozyme. Together, our results demonstrate that these ribozyme-based tools can efficiently manipulate endogenous gene expression both in space and time and expand the toolkit for studying the functions of endogenous genes.

Fang J et al. (2017) Front Pharmacol "Quantitative and Systems Pharmacology 3. Network-Based Identification of New Targets for Natural Products Enables Potential Uses in Aging-Associated Disorders."

Wu T, Ma H, Wang Q, Cheng F, Wu J, Wu Q, Gao L, Fang J

[Front Pharmacol, 2017]

Aging that refers the accumulation of genetic and physiology changes in cells and tissues over a lifetime has been shown a high risk of developing various complex diseases, such as neurodegenerative disease, cardiovascular disease and cancer. Over the past several decades, natural products have been demonstrated as anti-aging interveners via extending lifespan and preventing aging-associated disorders. In this study, we developed an integrated systems pharmacology infrastructure to uncover new indications for aging-associated disorders by natural products. Specifically, we incorporated 411 high-quality aging-associated human genes or human-orthologous genes from mus musculus (MM), saccharomyces cerevisiae (SC), caenorhabditis elegans (CE), and drosophila melanogaster (DM). We constructed a global drug-target network of natural products by integrating both experimental and computationally predicted drug-target interactions (DTI). We further built the statistical network models for identification of new anti-aging indications of natural products through integration of the curated aging-associated genes and drug-target network of natural products. High accuracy was achieved on the network models. We showcased several network-predicted anti-aging indications of four typical natural products (caffeic acid, metformin, myricetin, and resveratrol) with new mechanism-of-actions. In summary, this study offers a powerful systems pharmacology infrastructure to identify natural products for treatment of aging-associated disorders.

Fang K et al. (2001) J Antibiot (Tokyo) "New biotransformation products of nemadectins."

Schlingmann G, Fang K, Enos A, Carter GT

[J Antibiot (Tokyo), 2001]

Selected nemadectins (formerly LL-F28249 series) have been fed to a panel of microorganisms with the aim of generating new derivatives. In addition to products resulting from the oxidation of the terminal methyl group (C-29), a unique phosphorylated nemadectin was isolated. The phosphate group was determined to be at C-23 by HMBC between phosphorus and H-23. Milbemycin or nemadectin derivatives with natural substituents involving the 23-hydroxyl group were hitherto unknown.

Fang EF et al. (2017) J Vis Exp "In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice."

Hou Y, Poulton J, Fang EF, Dombi E, Cordonnier SA, Spangler RD, Fivenson EM, Bohr VA, Kerr JS, Tavernarakis N, Kassahun H, Nilsen H, Palikaras K, Sun N

[J Vis Exp, 2017]

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

Fang Z et al. (2020) J Food Biochem "The antioxidant capacity evaluation of polysaccharide hydrolyzates from pumpkin using Caenorhabditis elegans model."

Wang W, Lu A, Fang Z, Wang G, Zhang Y, Hao X, Xiao B, Jiang W, Li J, Tan J

[J Food Biochem, 2020]

Our previous study has optimized the acid hydrolysis process of pumpkin polysaccharides (PPe) with scavenging ability based on central composite design. The aim of this study was to explore the in vivo-antioxidant ability of PPe and pumpkin polysaccharides acid-hydrolysis (PPe-S) using Caenorhabditis elegans. In composition analysis, the constituents of total sugar, protein, uronic acid, and sulfur groups in PPe-S were 87.03+/-1.21%, 1.25+/-0.78%, 37.61+/-0.97%, and 0.14+/-0.04%, respectively. Besides, results of antioxidant ability showed that PPe and PPe-S could reduce the oxidative stress (OS) induced by methyl viologen, extend lifespan of worms, and reduce reactive oxygen species (ROS) level under oxidative conditions significantly (p<.05). Furthermore, PPe and PPe-S could enhance the stress-resistance related antioxidant enzymes including catalase (CAT) and superoxide dismutase (SOD) significantly (p<.05). Moreover, the antioxidant effect of PPe-S was superior to PPe at the concentration of 4.0mg/ml. In summary, this study demonstrated that the derived hydrolyzates from PPe had protective effects on the damage induced by the generation of intracellular free radical agents. PRACTICAL APPLICATIONS: OS plays an important role in the pathogenesis of metabolic diseases, including type 2 diabetes. It is widely acknowledged that diabetes and its complications pose a threat to human's health, and the number of people with diabetes will expand to 640 million in the 2040year. Current studies have shown that all diabetes drugs have a kind of side effects. Fortunately, researchers have found and confirmed that plant-derived polysaccharide had a notable hypoglycemic effect via reducing the OS level in cell and tissue, and could decrease the diabetes symptoms as well. In this study, we proved that the polysaccharide derived from pumpkin could effectively ameliorate the OS level in C. elegans, including decreasing the damage of biofilm and ROS level. Therefore, our study shows that there is a high potential for pumpkin-derived polysaccharide and its hydrolyzates to be a bioactive component to prevent diabetes. In other words, this research can be applied to diabetes prevention and other diseases induced by OS.

Fang Z et al. Indian J Med Microbiol "A recombinant plasmid of composite cysteine proteinase inhibitor/glyceraldehyde-3-phosphate dehydrogenase gene of periodic Brugia malayi functions on DNA immunity in the host."

Lu SJ, Wang YY, Fang H, Fang Z, Xu Q, Wu JQ

[Indian J Med Microbiol]

OBJECTIVES: Both cysteine proteinase inhibitors (CPIs) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) play important roles in the pathogenesis of parasites and their relationship with the hosts. We constructed a new eukaryotic recombinant expression plasmid pcDNA3.1(+)-BmCPI/BmGAPDH of periodic Brugia malayi for investigation of the DNA vaccine-elicited immune responses. MATERIALS AND METHODS: We cloned a gene encoding the CPIs and GAPDH from periodic B. malayi into vector pcDNA3.1. The composited plasmid or the control was injected into the tibialis anterior muscle of the hind leg in BALB/c mice, respectively. The target genes were detected by reverse transcription-polymerase chain reaction in muscle tissues. The stimulation index (SI) of T-lymphocyte proliferation and the levels of interferon-gamma (INF-g) and interleukin-4 ( IL-4) in serum were detected by thiazolyl blue tetrazolium blue and enzyme-linked immunosorbent assays. RESULTS: The pcDNA3.1(+)-BmCPI/BmGAPDH was amplified from muscle tissues of the mice after immunisation. The SI of the immunised group was significantly higher than that of the two control groups (P < 0.05). The levels of INF-g and IL-4 of pcDNA3.1(+)-BmCPI/BmGAPDH group were both higher than those of the two control groups (P < 0.05). The level of INF-g of pcDNA3.1(+)-BmCPI/BmGAPDH group was significantly higher than that of pcDNA3.1(+)-BmCPI/CpG group (P < 0.05). CONCLUSIONS: We conclude that the recombinant plasmid pcDNA3.1(+)-BmCPI/BmGAPDH could elicit specific humoural and cellular immune responses in mice.

Fang Z et al. (2012) Indian J Med Microbiol "Construction of a recombinant plasmid harbouring the glyceraldenyde-3-phosphate dehydrogenase gene of periodic Brugia malayi and observation on DNA immunity."

Xu B, Fang Z, Zhang S, Fang H, Tong H, Lu S

[Indian J Med Microbiol, 2012]

PURPOSE: Controlling and eliminating lymphatic filariasis will require further research of preventative measures and implementation. Parasite is dependent on glycolysis for ATP production. The glycolytic enzyme glyceraldenyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glycolysis and therefore is either a potential target for anti-parasite drug development or a vaccine candidate. Therefore, we tried to investigate the DNA vaccine-elicited immune responses in BALB/c mice. MATERIALS AND METHODS: We cloned a gene encoding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from periodic Brugia malayi into vector pcDNA3.1. Mice were injected at a dosage of 100 g recombinant plasmid DNA with CpG intramuscular injection and immunized three times at 2-week intervals. pcDNA3.1 and normal saline were used as control. The tissue of muscles at the 4 weeks after the third injection was collected and target genes were detected using RT-PCR. The humoral responses elicited in mice by inoculation with the recombinant plasmid pcDNA3.1-BmGAPDH were detected using a standard ELISA. Two weeks after the third immunization, stimulation index (SI) was measured using the MTT method and the level of secreted IL-4 and INF-g were detected using ELISA. RESULTS: Specific gene fragment coding GAPDH was amplified and the recombinant plasmid pcDNA3.1-BmGAPDH was constructed. Post-challenge sera from the mice immunized with the DNA vaccine had specific antibody titres of 1:1600 to 1:6400, and the highest titre was observed in the mice that were inoculated by pcDNA3.1-BmGAPDH/CpG at 6 weeks. At 4 weeks after immunization, the spleens of the mice were obviously enlarged. The proliferation of spleen T lymphocytes seen on the MTT assay was higher in the pcDNA3.1-BmGAPDH group than in the control group (P value <0.05). The levels of IL-4 and INF- in serums from the immunized mice were significantly higher than that of the control (P value <0.05). CONCLUSIONS: We conclude that the recombinant eukaryotic plasmid pcDNA3.1-BmGAPDH could elicit humoral and cellular immune responses in mice.

McGhee JD (1987) Biochemistry "Purification and characterization of a carboxylesterase from the intestine of the nematode Caenorhabditis elegans."

McGhee JD

[Biochemistry, 1987]

The major intestinal esterase from the nematode Caenorhabditis elegans has been purified to essential homogeneity. Starting from whole worms, the overall purification is 9000-fold with a 10% recovery of activity. The esterase is a single polypeptide chain of Mr 60,000 and is stoichiometrically inhibited by organophosphates. Substrate preferences and inhibition patterns classify the enzyme as a carboxylesterase (EC 3.1.1.1), but the physiological function is unknown. The sequence of 13 amino acid residues at the esterase N- terminus has been determined. This partial sequence shows a surprisingly high degree of similarity to the N-terminal sequence of two carboxylesterases recently isolated from Drosophila mojavensis [Pen, J., van Beeumen, J., & Beintema, J. J. (1986) Biochem. J. 238, 691-699].

Murakami S et al. (1999) Curr Biol "Life extension and stress resistance in Caenorhabditis elegans modulated by the tkr-1 gene."

Johnson TE, Murakami S

[Curr Biol, 1999]

In this Brief Communication, which appeared in the 14 September 1998 issue of Current Biology, the UV dose was reported erroneously. The dose reported was 20 J/m2 but the actual dose used was 0.4 J/cm2. Also, the gene formally referred to as tkr-1 has since been renamed old-1 (overexpression longevity determination).

Ramos CG et al. (2014) J Bacteriol "Retraction for Ramos et al., MtvR is a global small noncoding regulatory RNA in Burkholderia cenocepacia."

Feliciano JR, da Costa PJ, Ramos CG, Grilo AM, Leitao JH

[J Bacteriol, 2014]

Volume 195, no. 16, p. 35143523, 2013. A number of problems related to images published in this paper have been brought to our attention. Figure 1D contains duplicated images in lanes S and LE, and Fig. 4D and 6B contain images previously published in articles in this journal and in Microbiology and Microbial Pathogenesis, i.e., the following: C. G. Ramos, S. A. Sousa, A. M. Grilo, J. R. Feliciano, and J. H. Leitao, J. Bacteriol. 193:15151526, 2011. doi:10.1128/JB.01374-11. S. A. Sousa, C. G. Ramos, L. M. Moreira, and J. H. Leitao, Microbiology 156:896908, 2010. doi:10.1099/mic.0.035139-0. C. G. Ramos, S. A. Sousa, A. M. Grilo, L. Eberl, and J. H. Leitao, Microb. Pathog. 48:168177, 2010. doi: 10.1016/j.micpath.2010.02.006. Therefore, we retract the paper. We deeply regret this situation and apologize for any inconvenience to the editors and readers of Journal of Bacteriology, Microbial Pathogenesis, and Microbiology.