Berberine is effective and safe in the treatment of type 2 diabetes and dyslipidemia.

Background. Progressive multifocal leukoencephalopathy (PML) in natalizumab-treated MS patients is linked to JC virus (JCV) infection. JCV sequence variation and rearrangements influence viral pathogenicity and tropism. To better understand PML development, we analyzed viral DNA sequences in blood, CSF and/or urine of natalizumab-treated PML patients.Methods. Using biofluid samples from 17 natalizumab-treated PML patients, we sequenced multiple isolates of the JCV noncoding control region (NCCR), VP1 capsid coding region, and the entire 5 kb viral genome.Results. Analysis of JCV from multiple biofluids revealed that individuals were infected with a single genotype. Across our patient cohort, multiple PML-associated NCCR rearrangements and VP1 mutations were present in CSF and blood, but absent from urine-derived virus. NCCR rearrangements occurred in CSF of 100% of our cohort. VP1 mutations were observed in blood or CSF in 81% of patients. Sequencing of complete JCV genomes demonstrated that NCCR rearrangements could occur without VP1 mutations, but VP1 mutations were not observed without NCCR rearrangement.Conclusions. These data confirm that JCV in natalizumab-PML patients is similar to that observed in other PML patient groups, multiple genotypes are associated with PML, individual patients appear to be infected with a single genotype, and PML-associated mutations arise in patients during PML development.

Cytokine and growth factor receptor engagement leads to the rapid phosphorylation and activation of latent, cytosolic signal transducers and activators of transcription (STAT) proteins, which then translocate to the nucleus where they regulate transcriptional events from specific promoter sequences. STAT3 expression in particular has been associated with Abl, Src, and HTLV-1 transformation of normal cells. B-1 lymphocytes are self-renewing, CD5+ B cells that display a propensity for malignant transformation and are the normal counterpart to human chronic lymphocytic leukemias. Further, B-1 cells are characterized by aberrant intracellular signaling, including hyperresponsiveness to phorbol ester PKC agonists. Here we demonstrate that B-1 lymphocytes constitutively express nuclear activated STAT3, which is not expressed by unmanipulated conventional (B-2) lymphocytes. In contrast, STAT3 activation is induced in B-2 cells after antigen receptor engagement in a delayed fashion (after 3 h). Induction of STAT3 is inhibited by both the serine/threonine protein kinase inhibitor H-7 and the immunosuppressive drug rapamycin and requires de novo protein synthesis, demonstrating novel coupling between sIg and STAT proteins that differs from the classical paradigm for STAT induction by cytokine receptors. The inability of prolonged stimulation of conventional B-2 cells with anti-Ig, a treatment sufficient to induce CD5 expression, to result in sustained STAT3 activation suggests that STAT3 is a specific nuclear marker for B-1 cells. Thus, STAT3 may play a role in B cell antigen-specific signaling responses, and its constitutive activation is associated with a normal cell population exhibiting intrinsic proliferative behavior.

Two sets of experiments have been performed to test the DNA loop model of repression of the araBAD operon of Escherichia coli. First, dimethyl sulfate methylation protection measurements on normally growing cells show that the AraC regulatory protein occupies the aral site in the presence and absence of the inducer arabinose. Similarly, the araO2 site is shown to be occupied by AraC protein in the presence and absence of arabinose; however, its occupancy by AraC is greatly reduced when aral and adjacent sequences are deleted. Thus, AraC protein binds to araO2 cooperatively with some other component of the ara system located at least 60 base pairs away. Second, the mutational analysis presented here shows that the DNA components required for repression of araBAD are aral, araO2, and perhaps the araBAD operon RNA polymerase binding site.The L-arabinose operon of Escherichia coli has long been known to be positively regulated by AraC protein (1). Classical genetic (2-f: and biochemical (5-7) studies have also revealed that th;e) dRAD genes are negatively regulated by AraC and that a site required for this repression is located upstream from all the sites required for induction (2,8,10).Recently, a site required for repression, araO2, was found and was determined to lie 210 base pairs upstream from the AraC binding site required for induction, araI ( Fig. 1) (8). Surprisingly, the behaviors of strains with small deletions and insertions between the araBAD induction region and the araO2 site suggest that repression of the araBAD operon involves the formation of a DNA loop that brings araO2 near the induction region (8). Since repression of a constitutive araBAD promoter requires functional araO2 and araI sites as well as the presence of AraC protein (7) The results of genetic experiments were also as predicted by the DNA loop model. Mutations isolated solely on the basis that they reduced repression were found to lie in the araO2 and araI sites. However, repression-defective mutations also were found in the araBAD operon RNA polymerase binding site, raising the possibility of the involvement of RNA polymerase in repression. Mutations constructed in other known protein binding sites in the regulatory region did not interfere with repression. The results of these standard genetic experiments suggest that only the araI, araO2, and possibly RNA polymerase binding sites are involved in repression. These results strengthen previous results obtained by using "inverse genetics" in which regions of DNA are intentionally mutated and the results observed. MATERIALS AND METHODSMedia, Plasmids, Strains, and General Methods. Media and general methods were as described (11,12). Plasmid pTD3 (9) contains 440 base pairs of the araCBAD regulatory region on a HindIII/EcoRI fragment with PBAD driving galK of the pKO1 vector (13). The plasmid with the araCBAD induction region deleted and used for in vivo footprinting of araO2, pLH1, was made by deleting from to BamHI (position -46) of pTD3. AraO2 deletions of plasmids with araI ...

The araCBAD regulatory region of Escherichia coli contains two divergently oriented promoters and three sites to which AraC, the regulatory protein of the operon, can bind. This paper presents the results of in vivo dimethyl sulfate "footprinting" experiments to monitor occupancy of the three AraC sites and measurements of activity of the two promoters. These measurements were made both in the absence of the inducer arabinose and at various times after arabinose addition to growing cells containing the wild-type ara regulatory region or the regulatory region containing various deletions and point mutations. The data lead to the conclusion that two different DNA loops can form in the ara regulatory region. These loops are generated by AraC protein molecules binding to two different DNA sites and binding to each other. One of these loops predominates in the absence of arabinose and plays a major role in repressing activity of one of the promoters. Upon the addition of arabinose the amount of the first loop type, the repression loop, decreases and the amount of a second loop increases. Formation of this second loop precludes the counterproductive formation of the repression loop.AraC protein is a multifunctional regulatory protein of the genes required for uptake and catabolism of L-arabinose in Escherichia coli (1)(2)(3)(4). This paper concerns the inducing and repressing activities of AraC on the two promoters located in the regulatory region between the araC and araBAD genes. One, PBAD, serves the araBAD catabolic genes, and the other, Pc, serves the araC regulatory gene. In the presence of arabinose, AraC protein bound at the araI site, which is immediately adjacent to the RNA polymerase binding site of the PBAD promoter, stimulates transcription of the araBAD genes (Fig. 1) (8,9).A variety of experiments have led to the conclusion that looping occurs in the ara system (5-7, 10). Additionally, DNA looping appears to be a common mechanism for gene regulation, as a number of other systems also show one or more of the properties displayed by the ara system and can be interpreted to loop so that two proteins bound to separated sites on DNA are in direct contact (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Finally, the strength of the protein-DNA interactions and the proteinprotein interactions in some systems (14,16,20,22) permits loops generated on linear DNA to be observed in the electron microscope.Previous in vivo dimethyl sulfate methylation "footprinting" studies of the araCBAD operon revealed that in the absence of the two other AraC protein binding sites, araO2 binds AraC protein so weakly that it is virtually unoccupied (9). That is, the presence of the other sites, even though they are located >100 bp away, greatly increases AraC protein occupancy of araO2. Since AraC bound at araO2 must be interacting with one or both of the other sites to generate this binding-site cooperativity, either they are communicating by sending a signal along the DNA between the sites or, more likely, the DNA is lo...

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Natural killer (NK) cells and T cells are key effectors of antitumor immune responses and major targets of checkpoint inhibitors. In multiple cancer types, we find that the expression of Wnt signaling potentiator R-spondin genes (e.g., RSPO3) is associated with favorable prognosis and positively correlates with gene signatures of both NK cells and T cells. Although endothelial cells and cancer-associated fibroblasts comprise the R-spondin 3–producing cells, NK cells and T cells correspondingly express the R-spondin 3 receptor LGR6 within the tumor microenvironment (TME). Exogenous expression or intratumor injection of R-spondin 3 in tumors enhanced the infiltration and function of cytotoxic effector cells, which led to tumor regression. NK cells and CD8+ T cells independently and cooperatively contributed to R-spondin 3–induced control of distinct tumor types. The effect of R-spondin 3 was mediated in part through upregulation of MYC and ribosomal biogenesis. Importantly, R-spondin 3 expression enhanced tumor sensitivity to anti–PD-1 therapy, thereby highlighting new therapeutic avenues. Significance: Our study identifies novel targets in enhancing antitumor immunity and sensitizing immune checkpoint inhibition, which provides a rationale for developing new immunotherapies against cancers. It also offers mechanistic insights on Wnt signaling–mediated modulation of anticancer immunity in the TME and implications for a putative R-spondin–LGR6 axis in regulating NK-cell biology. This article is highlighted in the In This Issue feature, p. 2945