FUNCTIONAL AND MOLECULAR EVOLUTION OF THE PUF FAMILY

The modification of transcriptional regulation is a well-documented evolutionary mechanism in both plants and animals, but post-transcriptional controls have received less attention. The derived hermaphrodite of C. elegans has regulated spermatogenesis in an otherwise female body. PUF family RNA-binding proteins FBF-1 and FBF-2 limit XX spermatogenesis by repressing the male-promoting proteins FEM-3 and GLD-1. For my dissertation research, I examine the function of PUF homologs from other Caenorhabditis species, with emphasis on C. briggsae, which evolved selfing convergently. C. briggsae lacks a bona fide fbf-1/2 ortholog, but two members of the related PUF-2 subfamily, Cbr-puf-2 and Cbr-puf-1.2, do have a redundant germline sex determination role. Surprisingly, this is to promote, rather than limit, hermaphrodite spermatogenesis. I provide genetic, molecular, and biochemical evidence that Cbr-puf-2 and Cbr-puf-1.2 repress Cbr-gld-1 by a conserved mechanism. However, Cbr-gld-1 acts to limit, rather than promote, XX spermatogenesis. As with gld-1, no sex determination function for fbf or puf-2 orthologs is observed in gonochoristic Caenorhabditis. These results indicate that PUF family genes were coopted for sex determination in each hermaphrodite via their long-standing association with gld-1, and that their precise sex-determining roles depend on the species-specific context in which they act. Finally, I document non-redundant roles for Cbr-puf-2 in several aspects of somatic development. I show Cbr-puf-2 is required for reliable embryonic development, and that it is essential for vulval development and normal progression from early larval stage. I provide evidence suggesting that this latter role is related to pharyngeal muscle physiology. Thus, recently duplicated PUF paralogs, while redundant for some roles, can also rapidly acquire distinct non-redundant functions. This is consistent with theoretical models for the preservation of gene duplicates

Pulsed nanoelectrospray ionisation mass spectrometry

Pulsed nanoelectrospray ionisation (pulsed nESI) is an emerging technique for ionising volatile biomolecules for sensitive detection by mass spectrometry (MS). In conventional nESI, direct current high voltage (DC HV) is applied to the analyte solution resulting in the formation of ions with high sensitivity, low sample consumption and low limits of detection. Alternatively, in pulsed nESI, molecules are ionized by using a pulsed high voltage square waveform. During the course of this research project, another group reported that pulsed nESI can be used to measure peptide and protein ions by applying short, repetitive pulses of high voltage to the nESI emitter. However, the performance of such an ion source was not compared to conventional nESI. Thus, the extent that pulsed nESI can be used to improve performance of MS was unclear. Here, pulsed nESI is reported in which the voltage is rapidly pulsed from 0 to up to ~3 kV with a rise time of low to sub-nanoseconds and with duty cycles ranging from 1-50% corresponding to pulse durations of 9 to 450 μs. By use of pulsed nESI, the performance of MS for the detection of many different classes of molecules can be improved in terms of decreasing background chemical noise and enhancing the sensitivity for analytes ranging from small molecules to whole proteins. Specifically, pulses that are less than ~450 μs can be used to decrease the background chemical noise and increase signal-to-background chemical noise ratio by up to 93% and 691% in pulsed nESI MS for six test analytes compared to conventional nESI. Pulsed nESI can also be used for native mass spectrometry to improve signal-to- background chemical noise ratios. Overall, pulsed nESI can be used to significantly improve the performance of MS

Genetically modified adenoviral vector with the protein transduction domain of Tat improves gene transfer to CAR-deficient cells

The transduction efficiency of Ad (adenovirus) depends, to some extent, on the expression level of CAR (coxsackievirus and Ad receptor) of a target cell. The low level of CAR on the cell surface is a potential barrier to efficient gene transfer. To overcome this problem, PTD.AdeGFP (where eGFP is enhanced green fluorescent protein) was constructed by modifying the HI loop of Ad5 (Ad type 5) fibre with the Tat (trans-activating) PTD (protein transduction domain) derived from HIV. The present study showed that PTD.AdeGFP significantly improved gene transfer to multiple cell types deficient in expression of CAR. The improvement in gene transfer was not the result of charge-directed binding between the virus and the cell surface. Although PTD.AdeGFP formed aggregates, it infected target cells in a manner different from AdeGFP aggregates precipitated by calcium phosphate. In addition, PTD.AdeGFP was able to transduce target cells in a dynamin-independent pathway. The results provide some new clues as to how PTD.AdeGFP infects target cells. This new vector would be valuable in gene-function analysis and for gene therapy in cancer

An Integrative Pharmacology Model for Decoding the Underlying Therapeutic Mechanisms of Ermiao Powder for Rheumatoid Arthritis

As a systemic inflammatory arthritis disease, rheumatoid arthritis (RA) is complex and hereditary. Traditional Chinese medicine (TCM) has evident advantages in treating complex diseases, and a variety of TCM formulas have been reported that have effective treatment on RA. Clinical and pharmacological studies showed that Ermiao Powder, which consists of Phellodendron amurense Rupr. (PAR) and Atractylodes lancea (Thunb.) DC. (ALD), can be used in the treatment of RA. Currently, most studies focus on the anti-inflammatory mechanism of PAR and ALD and are less focused on their coordinated molecular mechanism. In this research, we established an integrative pharmacological strategy to explore the coordinated molecular mechanism of the two herbs of Ermiao Powder in treating RA. To explore the potential coordinated mechanism of PAR and ALD, we firstly developed a novel mathematical model to calculate the contribution score of 126 active components and 85 active components, which contributed 90% of the total contribution scores that were retained to construct the coordinated functional space. Then, the knapsack algorithm was applied to identify the core coordinated functional components from the 85 active components. Finally, we obtained the potential coordinated functional components group (CFCG) with 37 components, including wogonin, paeonol, ethyl caffeate, and magnoflorine. Also, functional enrichment analysis was performed on the targets of CFCG to explore the potential coordinated molecular mechanisms of PAR and ALD. The results indicated that the CFCG could treat RA by coordinated targeting to the genes involved in immunity and inflammation-related signal pathways, such as phosphatidylinositol 3‑kinase/protein kinase B signaling pathway, mitogen-activated protein kinase signaling pathway, tumor necrosis factor signaling pathway, and nuclear factor-kappa B signaling pathway. The docking and in vitro experiments were used to predict the affinity and validate the effect of CFCG and further confirm the reliability of our method. Our integrative pharmacological strategy, including CFCG identification and verification, can provide the methodological references for exploring the coordinated mechanism of TCM in treating complex diseases and contribute to improving our understanding of the coordinated mechanism

Fast Green FCF Attenuates Lipopolysaccharide-Induced Depressive-Like Behavior and Downregulates TLR4/Myd88/NF-κB Signal Pathway in the Mouse Hippocampus

Depression is a common neuropsychiatric disorder and new anti-depressive treatments are still in urgent demand. Fast Green FCF, a safe biocompatible color additive, has been suggested to mitigate chronic pain. However, Fast green FCF’s effect on depression is unknown. We aimed to investigate Fast green FCF’s effect on lipopolysaccharide (LPS)-induced depressive-like behavior and the underlying mechanisms. Pretreatment of Fast green FCF (100 mg/kg, i.p. daily for 7 days) alleviated depressive-like behavior in LPS-treated mice. Fast green FCF suppressed the LPS-induced microglial and astrocyte activation in the hippocampus. Fast green FCF decreased the mRNA and protein levels of Toll-like receptor 4 (TLR4) and Myeloid differentiation primary response 88 (Myd88) and suppressed the phosphorylation of nuclear factor-κB (NF-κB) in the hippocampus of LPS-treated mice. Fast green FCF also downregulated hippocampal tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, but did not alter the level of the brain-derived neurotrophic factor (BDNF) in the hippocampus of LPS-treated mice. The molecular docking simulation predicts that Fast green FCF may interact with TLR4 and interrupt the formation of the TLR4-MD2 complex. In conclusion, the anti-depressive action of Fast green FCF in LPS-treated mice may involve the suppression of neuroinflammation and the downregulation of TLR4/Myd88/NF-κB signal pathway in mouse hippocampus. Our findings indicate the potential of Fast green FCF for controlling depressive symptoms