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Abstract: Pteridine reductase 1 (PTR1, EC 1.5.1.33) is a NADPH dependent short-chain reductase (SDR) responsible for the salvage of pterins in the protozoan parasite Leishmania. This enzyme acts as a metabolic bypass for drugs targeting dihydrofolate reductase, therefore, for successful antifolate chemotherapy to be developed against Leishmania, it must target both enzyme activities. Based on homology model drawn on recombinant pteridine reductase isolated from a clinical isolate of L. donovani, we carried out molecular modeling and docking studies with two compounds of dihydrofolate reductase specificity showing promising antileishmanial activity in vitro. Both the inhibitors appeared to fit well in the active pocket revealing the tight binding of the carboxylic acid ethyl ester group of pyridine moiety to pteridine reductase and identify the important interactions necessary to assist the structure based development of novel pteridine reductase inhibitors. [Copyright &y& Elsevier]

The expression vector pTNF-GFP containing the 2351 bp 5′ flanking region of Japanese flounder tumor necrosis factor (TNF) gene was linked with the green fluorescence protein (GFP) gene and was introduced into YO-K cells derived from Japanese flounder kidney and HINAE cells derived Japanese flounder embryos. YO-K cells and HINAE cells were incubated with three concentrations (250, 500, 1000 μg/ml) of lipopolysaccharide (LPS) at 20 °C for 24 h. The number of cells expressing GFP, as well as the amount of GFP protein was increased by LPS stimulation in both cell lines. GFP mRNA transcription was also induced by LPS stimulation in both YO-K cells and HINAE cells after 1 h stimulation. In YO-K cells, expression level of GFP decreased gradually from 3 to 6 h post-stimulation, while a reverse trend was observed in HINAE cells. A deletion assay of TNF gene promoter showed that the 5′ flanking region, -1783 to -1300 bp, containing cis-acting regulatory elements mediated LPS induction. An electrophoretic mobility shift assay using 2 fragments (-1783 to -1541 bp and -1540 to -1300 bp) revealed that only LPS-stimulated nuclear extracts bound to the -1540 to -1300 bp fragment. These results suggest that transcription of the TNF gene promoter in homologous cultured cells exhibited an inducible pattern and was regulated under the control of the immune system. [Copyright &y& Elsevier]

Human immunodeficiency virus type 1 (HIV-1) Tat protein plays an important role in HIV-associated neuropathogenesis. Astrocytosis and neuron death are two hallmarks of HIV-1 infection of the central nervous system (CNS). However, whether there is a direct link between Tat expression, astrocytosis and subsequent neuron death is not known. In this study, we expressed Tat in astrocytes and examined Tat effects on astrocyte function and subsequent neuronal survival. The results showed that Tat expression resulted in a significant increase in glial fibrillary acidic protein (GFAP) expression, a cellular marker of astrocyte activation or astrocytosis. The GFAP promoter-driven reporter gene assay showed that Tat transactivated GFAP expression at the transcriptional level. Furthermore, Tat expression markedly impaired glutamate uptake by astrocytes. Importantly, cell culture supernatants from Tat-expressing astrocytes induced dramatic neuron death. Taken together, these data provide evidence for the first time to directly link Tat expression in astrocytes to astrocytosis, astrocyte dysfunction, and subsequent neuron death. In addition, these data suggest that astrocyte dysfunction contributes, at least in part, to Tat neurotoxicity and subsequently HIV-associated neuropathogenesis. [Copyright &y& Elsevier]

Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine involved in inflammation and immune responses as well as in growth factor-dependent cell proliferation, cell cycle, angiogenesis, and tumorigenesis. Several studies have documented MIF expression in the sera following hepatic resection or in the course of liver cancer progression, but there is a paucity of information regarding the effect of MIF on hepatoma cells and relating mechanisms. In this paper, by [3H] thymidine incorporation, we found that exogenously added MIF could promote the proliferation of HepG2 in a dose-dependent manner. Hepatopoietin (HPO), as a liver-specific regeneration augmenter, could be induced by the expression of MIF in hepatoma cells. The activity of HPO promoter was increased, and its levels were enhanced after MIF was overexpressed in hepatoma cells. The similarities between HPO and MIF in structure and action led us to investigate their interaction and the inducing biological significance. Using yeast two-hybrid identification, we found that HPO interacted with MIF in yeast cells, and their binding ability was higher than that between HPO and JAB1 (Jun activation domain binding protein) or MIF and JAB1 in yeast cells. Their interaction was further verified by His pull-down assay in vitro and coimmunoprecipitation experiment in vivo. They were colocalized in the cytoplasm. Both HPO and MIF could bind to JAB1 and modulate the AP-1 pathway. When HPO and MIF were cotransfected into HepG2 cells, the binding activity of MIF to JAB1 was reduced, and the activity of AP-1 was improved. In contrast, MIF overexpressed in HepG2 was unable to interfere with the binding activity of HPO to JAB1, but its potentiation on AP-1 activity was reduced significantly. Taken together, these results indicate that MIF plays an important role in the proliferation of hepatoma cells, and the effect of MIF is in concert with HPO. [Copyright &y& Elsevier]

αA- and αB-crystallins are distinct antiapoptotic regulators. Regarding the antiapoptotic mechanisms, we have previously demonstrated that under staurosporine treatment, HαA- and HαB-crystallins can interact with Bax and Bcl-XS, proapoptotic members of the Bcl-2 family, to sequester their translocation into mitochondria, and thus prevent the staurosporine-induced apoptosis. In the present study, we further compared the anti-apoptotic mechanisms of HαA- and HαB-crystallin in preventing human lens epithelial cells from UVA-induced apoptosis. UVA-irradiation of human lens epithelial cells turned on the apoptotic death program. Moreover, associated with the activation of the death program, UVA also activated the RAF/MEK/ERK signaling pathway. In contrast, p38 kinase and JNK1/2 signaling pathways were not activated. Inhibition of the RAF/MEK/ERK pathway by a dominant negative mutant RAF1 greatly attenuated UVA-induced apoptosis. Expression of the exogenous human αB-crystallin prevented UVA-induced activation of RAF/MEK/ERK pathway and thus substantially abrogated UVA-induced apoptosis. In contrast, expression of the exogenous human αA-crystallin did not prevent UVA-induced activation of RAF/MEK/ERK pathway. Instead, it activated AKT kinase pathway to promote survival and thus counteracted the UVA-induced apoptosis. Together, our results for the first time reveal that by regulating multiple signaling pathways the two α-crystallins can prevent stress-induced apoptosis through different mechanisms. [Copyright &y& Elsevier]

Mst3, a human Ste20-like protein kinase, has been recently demonstrated to undergo a caspase-mediated cleavage during apoptosis. The proteolytic cleavage of the C-terminus of Mst3 caused nuclear translocation of its kinase domain. This work provides evidence that Mst3 may contain a bipartite-like nuclear localization sequence (NLS) at the C-terminus of its kinase domain (residues 278–292). The removal of NLS from the kinase domain of Mst3 led to the cytoplasmic accumulation of EGFP-Mst3Δ277. The presence of nuclear exporting signals in the Mst3 was also demonstrated by leptomycin B-treatment and serial deletion of the C-terminal regulatory domain of Mst3. A nuclear export signal was also postulated to be in the regions of amino acids 335–386. In conclusion, Mst3 contains both NLS and NES signals, which may cooperate to control the subcellular distribution of Mst3. [Copyright &y& Elsevier]

We have recently shown that the avian reovirus non-structural protein μNS forms cytoplasmic inclusions in transfected cells and recruits σNS to these structures. In the present study we further demonstrate that μNS mediates the association of the major core protein λA, but not of σA or σC, with inclusions, indicating that the recruitment of viral proteins into avian reovirus factories has specificity. Thus, some proteins appear to be initially recruited to factories by association with μNS, whereas others are recruited subsequently through interaction with as-yet-unknown factors. We next used metabolic pulse-chase radiolabeling combined with cell fractionation and antibody immunoprecipitation to study the recruitment of newly synthesized viral polypeptides into viral factories and virus particles. The results of this combined approach revealed that avian reovirus morphogenesis is a complex and temporally controlled process that takes place exclusively within globular viral factories that are not microtubule-associated. Our findings further suggest that cores are assembled within the first 30 minutes after the synthesis of their polypeptide components, and that reovirion morphogenesis is completed over the next 30 minutes by the subsequent addition of outer capsid proteins. [Copyright &y& Elsevier]

The time-specific requirement of Rho proteins for the S phase progression of vascular endothelial cells was determined by reversibly introducing inhibitor proteins with a cell-penetrating peptide. We found evidence of the reversibility of protein transduction. The removal of extracellular protein caused the transduced protein to decay in a manner sensitive to low temperatures. The time required for a 50% decay correlated with the protein size. The time-specific transduction of the inhibitor proteins uncovered a critical period requiring Rho proteins in the G1–S transition phase. Reversible protein transduction may thus be a powerful tool to investigate the time-specific role of signaling proteins. [Copyright &y& Elsevier]

p23 is a regulatory co-chaperone of heat shock protein (Hsp) 90, but can also act as a general molecular chaperone by itself. Using novel point mutations of p23 that disrupt its interaction with Hsp90 we found its co-chaperone function to be required for its inhibitory effect on glucocorticoid receptor (GR). The C-terminal region of p23, which is required for its chaperone activity, is dispensable for inhibition of GR. Importantly, similar results were obtained with a constitutively active GR. Thus, the action of p23 on the nuclear stage of GR regulation requires its Hsp90 co-chaperone function, but not its chaperone activity. [Copyright &y& Elsevier]

Human Bfl-1 is an anti-apoptotic Bcl-2 family member. Here, we found that Bfl-1 was converted into a potent death-promoting protein by green fluorescent protein (GFP) fusion with its N-terminus. The transient expression of GFP-Bfl-1 induced cytochrome c release and triggered apoptosis in 293T cells, which depended on the mitochondrial localization of GFP-Bfl-1. Apoptosis induced by GFP-Bfl-1 was significantly blocked by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone, but was not blocked by either Bcl-xL or Bfl-1. Our findings provide a useful model for understanding the structural basis of Bcl-2 family proteins that act in an opposite way despite sharing structural similarity between anti-apoptotic and pro-apoptotic proteins. [Copyright &y& Elsevier]

ARC is a caspase recruitment domain-containing molecule that plays an important role in the regulation of apoptosis. We examined ARC expression during neuronal cell death following ischemic injury in vivo and in vitro. After exposure to transient global ischemic conditions, the expression of ARC was substantially reduced in the CA1 region of hippocampus in a time-dependent manner with concomitant increase of TUNEL-positive cells. Quantitative analysis using Western blotting exhibited that most of ARC protein disappeared in the cultured hippocampal neurons exposed to hypoxia for 12 h and showing 60% cell viability. Forced expression of ARC in the primary cultures of hippocampal neurons or B103 neuronal cells significantly reduced hypoxia-induced cell death. Further, the C-terminal P/E rich region of ARC was effective to attenuate hypoxic insults. These results suggest that down-regulation of ARC expression in hippocampal neurons may contribute to neuronal death induced by ischemia/hypoxia. [Copyright &y& Elsevier]

Sedlin is an evolutionarily conserved protein encoded by the causative gene SEDL for spondyloepiphyseal dysplasia tarda. Nevertheless, how Sedlin mutations cause the disease remains unknown. Here, the intracellular chloride channel protein CLIC1 was shown to associate with Sedlin by yeast two-hybrid screening. Green fluorescence protein-CLIC1 readily co-immunoprecipitated with FLAG-Sedlin. In addition, both proteins colocalized extensively in cytoplasmic vesicular/reticular structures in COS-7 cells, suggesting their interaction at intracellular membranous organelles. Sedlin also associated with CLIC2 in yeast two-hybrid assays. The link between Sedlin and the intracellular chloride channels is the first step to understand their functional interplays. [Copyright &y& Elsevier]

The SufI protein and the trimethylamine N-oxide reductase (TorA) are the two best-characterized prototype proteins exported by the Escherichia coli TAT system. Whereas SufI does not contain cofactors, TorA is a molybdo-enzyme and the acquisition of the molybdo-cofactor is a prerequisite for its translocation. The overproduction of each protein leads to the saturation of its translocation, but it was unknown if the overproduction of one substrate could saturate the TAT apparatus and block thus the translocation of other TAT substrates. Here, we showed that the overproduction of SufI saturated only its own translocation, but had no effect of the translocation of TorA and other TAT substrate analyzed. To dissect the saturation mechanism of TorA translocation, we shortened by about one-third of the TorA protein and removed nine consensus molybdo-cofactor-binding ligands. Like SufI, the truncated TorA (TorA502) did not contain cofactor and would not compete with the full length TorA for molybdo-cofactor acquisition. The overproduction of TorA502 completely inhibited the export of the full length TorA and dimethyl sulfoxide (DMSO) reductase, but had no effect on the translocation of SufI, nitrate-induced formate dehydrogenase and hydrogenase-2. Importantly, deletion of the twin-arginine signal peptide of TorA502 abolished the inhibitory effect. Moreover, the overproduction of the TorA signal peptide fused to the green fluorescence protein (GFP) was sufficient to block the TorA translocation. These results demonstrated that the twin-arginine signal peptide of the TorA protein specifically inhibits the translocation of a subset of TAT substrates, probably at the step of their targeting to the TAT apparatus. [Copyright &y& Elsevier]

KyoT, a LIM protein interacting with transcription factor RBP-J, repressed the RBP-J-mediated transcriptional activation by Notch by competing with it for binding to RBP-J. To identify other KyoT interacting proteins, the yeast two-hybrid screening using KyoT2 as the bait protein was performed. A novel gene encoding KBP (KyoT Binding Protein) was found to associate with KyoT2 in the yeast two-hybrid method and in vitro glutathione S-transferase (GST) pull-down and in vivo immunoprecipitation assay. Differential splicing gave rise to three transcripts of the KBP gene and they encoded predicted proteins of 210, 183 and 183 amino acids, respectively. Multiple-tissue Northern blot analysis showed that KBP was expressed in almost all tissues. Furthermore, transfection experiments with green fluorescence protein (GFP) expression vectors into COS-7 cells revealed that three kinds of full-length KBP fused to the GFP were localized in the whole cell. Moreover, it was found by yeast two-hybrid screening that the binding site of KyoT2 on KBP was the LIM domain. KBP had no effect on Notch signaling pathway. Taken together, we have isolated a novel protein, KBP, which may be involved in the functional regulation of KyoT. [Copyright &y& Elsevier]

Toll-like receptors (TLRs) are activated by microbial structures. To investigate the mechanisms of TLR activation, the 10 human TLRs were expressed as chimeras with the integrin αv and β5 subunits. Co-expression of the αv-TLR and β5-TLR chimeras in 293T cells generated 10 TLR homodimers, but only TLR4/4 could effectively activate NF-κB. TLR4 monomers also activated NF-κB but it was enhanced upon homodimerization. The TLR homodimers showed differential surface/intracellular expression. In TLR heterodimers, only TLR2/1 and TLR2/6 were potent in NF-κB activation. NF-κB activation by TLR2/1, TLR2/6 and the TLR4 monomer, but not TLR4/4, was completely inhibited by dominant negative MyD88, suggesting that TLR4 homodimers and monomers could activate NF-κB through different mechanisms. [Copyright &y& Elsevier]

Cells regulate gene expression using networks of transcription interactions; it is of interest to discover the principles that govern the dynamical behavior of such networks. An important characteristic of these systems is the rise-time: the delay from the initiation of production until half maximal product concentration is reached. Here we employ synthetic gene circuits in Escherichia coli to measure the rise-times of non-self-regulated and of negatively autoregulated transcription units. Non-self-regulated units have a rise-time of one cell-cycle. We demonstrate experimentally that negative autoregulation feedback (also termed autogenous control) reduces the rise-time to about one fifth of a cell-cycle. This agrees with an analytical solution of a mathematical model for negative autoregulation. This may help in understanding the function of negative autoregulation, which appears in over 40% of known transcription factors in E. coli. [Copyright &y& Elsevier]

Abnormal retention of ΔF508 CFTR (cystic fibrosis transmembrane conductance regulator) in the endoplasmic reticulum is a major cause of cystic fibrosis (CF). We show that calnexin Δ185–520 but not calnexin can partially reverse the mislocalization of ΔF508 CFTR. This 256-amino acid protein has neither the transmembrane domain nor the P domain of calnexin. Calnexin Δ185–520 interacted with CFTR directly, and was secreted into the extracellular compartment over time. Forty-eight hours after transfection into CHO cells, calnexin Δ185–520 increased the conversion of immature ΔF508 CFTR into mature ΔF508 CFTR. In immortalized human CF cell lines expressing ΔF508 CFTR, a halide efflux assay showed that calnexin Δ185–520 partially restored CFTR function. These data indicate that calnexin Δ185–520 may give a clue to develop the therapeutic way of cystic fibrosis with ΔF508 CFTR. [Copyright &y& Elsevier]

We have studied the cellular localization and the DNA-binding capability of human peptidyl-prolyl cis/trans isomerase hPar14. The cellular expression pattern shows an uneven distribution of the protein between cytoplasm and nucleus. To determine the nuclear localization of hPar14 in vivo the molecule was fused to green fluorescent protein and expressed in human HeLa cells. Deletion mutants of hPar14 were used to restrict a sequence, necessary for nuclear targeting, to Ser7-Lys14 of the N terminus of the protein. DNA–cellulose affinity experiments were performed to demonstrate that hPar14, which is present in the nuclear fraction, could bind to double-stranded native DNA in vitro. On the basis of homologies and similarities of hPar14 to members of the high-mobility group proteins, double-stranded DNA constructs were developed and tested for their hPar14 binding affinity in fluorescence titration assays. The protein binds preferentially to bent A-tract sequences. The binding interface of the protein was determined by 1-D and 2-D NMR studies of the complex of unlabeled DNA and uniformly 15N-labeled hPar14(1–131). Experiments with a truncated hPar14(25–131) showed that the unstructured N-terminal 25 amino acid residues are necessary for high-affinity binding to DNA. These findings in connection with sequence and structural homologies of hPar14 to members of the HMGB/HMGN protein family suggest a function of hPar14 in cell-cycle regulation or gene transcription. [Copyright &y& Elsevier]

Zinc-fingers and homeoboxes 1 (ZHX1) is a protein which interacts with the activation domain of the A subunit of nuclear factor-Y. To analyze the physiological role(s) of ZHX1, we searched ZHX1-interacting protein(s) using a yeast two-hybrid system. The rat counterpart of ZHX1 cDNAs was cloned from an ovarian granulosa cell complementary DNA (cDNA) library, indicating that ZHX1 is able to form a homodimer. An analysis of the nucleotide sequence and its deduced amino acid sequence show that rat ZHX1 consists of 873 amino acid residues. Northern blot analysis shows that ZHX1 messenger RNA is expressed ubiquitously and that the level in the ovary are not regulated by gonadotropins. Furthermore, transfection experiments with green fluorescence protein (GFP) expression vectors into human embryonic kidney HEK293 cells reveal that full-length ZHX1 fused to the GFP is localized in the nuclei. Thus, we report on the molecular cloning, expression and characterization of full-length rat ZHX1 cDNA. [Copyright &y& Elsevier]

The Neuropeptide Y (NPY) family of neuropeptides exert their function through a family of heptahelical G-protein coupled receptors regulating essential physiological processes. A 97 base pair intron (intron IV) intervenes the coding sequence of the human NPY Y1 receptor (hY1) gene and was found frequently retained at variable levels in poly A+ mRNA isolated from multiple human tissues. When included in hY1 expression vectors, either in its natural position or 5′ of the hY1 cDNA, intron IV mediated a significant increase of both hY1 mRNA and corresponding functional receptor protein in transfected mammalian cells, implying an in vivo regulatory function of the endogenous intron. Our results further indicate that the nuclear history of the hY1 pre-mRNA influence ectopic hY1 production through post-transcriptional mechanisms and argues against intron IV acting as a transcriptional enhancer as well as the possibility that a putative hY1 related 5TM accessory protein encoded by the non-spliced hY1 mRNA would facilitate hY1 production on a post-translational level. [Copyright &y& Elsevier]

The fruiting body of Polysphondylium pallidum is composed of whorls of branches along the axis of a primary stalk. In the course of fruiting body formation, the interval between neighboring whorls and the number and the spacing of branches in a whorl are highly regulated. In this study, using restriction enzyme mediated integration mutagenesis, we have obtained a mutant (strain M6226) with thicker and aberrant primary stalk. The gene responsible for the mutant phenotype, confirmed by homologous recombination, encodes an open reading frame with 383 aa residues (46.3 kDa) and was named thick and aberrant stalk A (tasA). TasA is highly homologous to Dictyostelium discoideum cyclic adenosine 3′,5′-monophosphate receptors. A tasA transcript is expressed strictly at the late aggregation stage. Cells expressing a tasA::gfp fusion DNA are localized at the posterior region of the primary sorogen where secondary sorogens and branches originate. This result indicates the existence of ‘prebranch’ and ‘pretrunk’ regions in P. pallidum instead of the prespore and prestalk regions in D. discoideum. The analyzes of the gene disruptant and chimeric fruiting bodies also suggests that TasA affects the normal morphogenesis of the primary stalk and the process of cell differentiation into prebranch cells, but not into spore or stalk cells directly. [Copyright &y& Elsevier]

The physiological role of mono-ADP-ribosyl transferase (Arr) of Mycobacterium smegmatis , which inactivates rifampicin, remains unclear. An earlier study reported increased expression of arr during oxidative stress and DNA damage. This suggested a role for Arr in the oxidative status of the cell and its associated effect on DNA damage. Since reactive oxygen species (ROS) influence oxidative status, we investigated whether Arr affected ROS levels in M. smegmatis . Significantly elevated levels of superoxide and hydroxyl radical were found in the mid-log phase (MLP) cultures of the arr knockout strain ( arr -KO) as compared those in the wild-type strain (WT). Complementation of arr -KO with expression from genomically integrated arr under its native promoter restored the levels of ROS equivalent to that in WT. Due to the inherently high ROS levels in the actively growing arr- KO, rifampicin resisters with rpoB mutations could be selected at 0 hr of exposure itself against rifampicin, unlike in the WT where the resisters emerged at 12 th hr of rifampicin exposure. Microarray analysis of the actively growing cultures of arr -KO revealed significantly high levels of expression of genes from succinate dehydrogenase I and NADH dehydrogenase I operons, which would have contributed to the increased superoxide levels. In parallel, expression of specific DNA repair genes was significantly decreased, favouring retention of the mutations inflicted by the ROS. Expression of several metabolic pathway genes also was significantly altered. These observations revealed that Arr was required for maintaining a gene expression profile that would provide optimum levels of ROS and DNA repair system in the actively growing M. smegmatis ., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Intestinal toxicity induced by chemotherapeutics has become an important reason for the interruption of therapy and withdrawal of approved agents. In this study, we demonstrated that chemotherapeutics-induced intestinal damage were commonly characterized by the sharp upregulation of tryptophan (Trp)-kynurenine (KYN)-kynurenic acid (KA) axis metabolism. Mechanistically, chemotherapy-induced intestinal damage triggered the formation of an interleukin-6 (IL-6)-indoleamine 2,3-dioxygenase 1 (IDO1)-aryl hydrocarbon receptor (AHR) positive feedback loop, which accelerated kynurenine pathway metabolism in gut. Besides, AHR and G protein-coupled receptor 35 (GPR35) negative feedback regulates intestinal damage and inflammation to maintain intestinal integrity and homeostasis through gradually sensing kynurenic acid level in gut and macrophage, respectively. Moreover, based on virtual screening and biological verification, vardenafil and linagliptin as GPR35 and AHR agonists respectively were discovered from 2388 approved drugs. Importantly, the results that vardenafil and linagliptin significantly alleviated chemotherapy-induced intestinal toxicity in vivo suggests that chemotherapeutics combined with the two could be a promising therapeutic strategy for cancer patients in clinic. This work highlights GPR35 and AHR as the guardian of kynurenine pathway metabolism and core component of defense responses against intestinal damage., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this article., (© 2021 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

In the present study, we have established a novel transgenic mouse and transgenic rats with dual reporters of EGFP and ELuc. In these transgenic (Tg) rodents, both GFP fluorescent and luciferase luminescent signals were ubiquitously detected in the heart, liver, kidney and testis, while only the GFP signal was detected in the brain. This expression system is based on a P2A linked EGFP/ELuc protein allowing both signals to be generated simultaneously. Microscopy experiments, FCM, and luciferase assays showed strong expression in freshly isolated ADSCs from Tg rodents upon transplantation of Tg rat-derived ADSCs into wild-type-mice. The ELuc transgene signal was observed and traced in vivo , and EGFP positive cells could be recovered from ELuc positive tissues in engraftment sites of wild-type mice for multiple analysis. These dual reporter Tg rodents are a useful reconstituted model system of regenerative medicine and are a valuable tool to study stem cells.

The physiological and metabolic processes of host plants are manipulated and remodeled by phytopathogenic fungi during infection, revealed obvious signs of biotrophy of the hemibiotrophic pathogen. As we known that effector proteins play key roles in interaction of hemibiotrophic fungi and their host plants. BAS4 (biotrophy-associated secreted protein 4) is an EIHM (extrainvasive hyphal membrane) matrix protein that was highly expressed in infectious hyphae. In order to study whether BAS4 is involved in the transition of rice blast fungus from biotrophic to necrotrophic phase, The susceptible rice cultivar Lijiangxintuanheigu (LTH) that were pre-treated with prokaryotic expression product of BAS4 and then followed with inoculation of the blast strain, more serious blast disease symptom, more biomass such as sporulation and fungal relative growth, and lower expression level of pathogenicity-related genes appeared in lesion of the rice leaves than those of the PBS-pretreated-leaves followed with inoculation of the same blast strain, which demonstrating that BAS4 invitro changed rice defense system to facilitate infection of rice blast strain. And the susceptible rice cultivar (LTH) were inoculated withBAS4-overexpressed blast strain, we also found more serious blast disease symptom and more biomass also appeared in lesion of leaves inoculated with BAS4-overexpressed strain than those of leaves inoculated with the wild-type strain, and expression level of pathogenicity-related genes appeared lower in biotrophic phase and higher in necrotrophic phase of infection, indicating BAS4 maybe in vivo regulate defense system of rice to facilitate transition of biotrophic to necrotrophic phase. Our data demonstrates that BAS4 in vitro and in vivo participates in transition from the biotrophic to the necrotrophic phase of Magnaporthe oryzae .

Background & Aims: Ileal bile acid absorption is mediated by uptake via the apical sodium-dependent bile acid transporter (ASBT), and export via the basolateral heteromeric organic solute transporter α-β (OSTα-OSTβ). In this study, we investigated the cytotoxic effects of enterocyte bile acid stasis in Ostα -/- mice, including the temporal relationship between intestinal injury and initiation of the enterohepatic circulation of bile acids., Methods: Ileal tissue morphometry, histology, markers of cell proliferation, gene, and protein expression were analyzed in male and female wild-type and Ostα -/- mice at postnatal days 5, 10, 15, 20, and 30. Ostα -/- Asbt -/- mice were generated and analyzed. Bile acid activation of intestinal Nrf2-activated pathways was investigated in Drosophila ., Results: As early as day 5, Ostα -/- mice showed significantly increased ileal weight per length, decreased villus height, and increased epithelial cell proliferation. This correlated with premature expression of the Asbt and induction of bile acid-activated farnesoid X receptor target genes in neonatal Ostα -/- mice. Expression of reduced nicotinamide adenine dinucleotide phosphate oxidase-1 and Nrf2-anti-oxidant responsive genes were increased significantly in neonatal Ostα -/- mice at these postnatal time points. Bile acids also activated Nrf2 in Drosophila enterocytes and enterocyte-specific knockdown of Nrf2 increased sensitivity of flies to bile acid-induced toxicity. Inactivation of the Asbt prevented the changes in ileal morphology and induction of anti-oxidant response genes in Ostα -/- mice., Conclusions: Early in postnatal development, loss of Ostα leads to bile acid accumulation, oxidative stress, and a restitution response in ileum. In addition to its essential role in maintaining bile acid homeostasis, Ostα-Ostβ functions to protect the ileal epithelium against bile acid-induced injury. NCBI Gene Expression Omnibus: GSE99579.

The primary cells that participate in islet transplantation are the endocrine cells. However, in the islet microenvironment, the endocrine cells are closely associated with the neurovascular tissues consisting of the Schwann cells and pericytes, which form sheaths/barriers at the islet exterior and interior borders. The two cell types have shown their plasticity in islet injury, but their roles in transplantation remain unclear. In this research, we applied 3-dimensional neurovascular histology with cell tracing to reveal the participation of Schwann cells and pericytes in mouse islet transplantation. Longitudinal studies of the grafts under the kidney capsule identify that the donor Schwann cells and pericytes re-associate with the engrafted islets at the peri-graft and perivascular domains, respectively, indicating their adaptability in transplantation. Based on the morphological proximity and cellular reactivity, we propose that the new islet microenvironment should include the peri-graft Schwann cell sheath and perivascular pericytes as an integral part of the new tissue.

Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing.