Regulation of the Escherichia coli HipBA toxin-antitoxin system by proteolysis

Bacterial populations produce antibiotic-tolerant persister cells. A number of recent studies point to the involvement of toxin/antitoxin (TA) modules in persister formation. hipBA is a type II TA module that codes for the HipB antitoxin and the HipA toxin. HipA is an EF-Tu kinase, which causes protein synthesis inhibition and dormancy upon phosphorylation of its substrate. Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases. We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lon(-) background. These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis. Moreover, we demonstrated that degradation of HipB is dependent on the presence of an unstructured carboxy-terminal stretch of HipB that encompasses the last 16 amino acid residues. Further, substitution of the conserved carboxy-terminal tryptophan of HipB to alanine or even the complete removal of this 16 residue fragment did not alter the affinity of HipB for hipBA operator DNA or for HipA indicating that the major role of this region of HipB is to control HipB degradation and hence HipA-mediated persistence.     

Allele Copy Number and Underlying Pathology Are Associated with Subclinical Severity in Equine Type 1 Polysaccharide Storage Myopathy (PSSM1)

Equine type 1 polysaccharide storage myopathy (PSSM1), a common glycogenosis associated with an R309H founder mutation in the glycogen synthase 1 gene (GYS1), shares pathological features with several human myopathies. In common with related human disorders, the pathogenesis remains unclear in particular, the marked phenotypic variability between affected animals. Given that affected animals accumulate glycogen and alpha-crystalline polysaccharide within their muscles, it is possible that physical disruption associated with the presence of this material could exacerbate the phenotype. The aim of this study was to compare the histopathological changes in horses with PSSM1, and specifically, to investigate the hypothesis that the severity of underlying pathology, (e.g. vacuolation and inclusion formation) would (1) be higher in homozygotes than heterozygotes and (2) correlate with clinical severity. Resting and post-exercise plasma creatine kinase (CK) and aspartate aminotransferase (AST) enzyme activity measurements and muscle pathology were assessed in matched cohorts of PSSM1 homozygotes, heterozygotes or control horses. Median (interquartile range (IR)) resting CK activities were 364 (332–764) U/L for homozygotes, 301 (222–377) U/L for heterozygotes and 260 (216–320) U/L for controls, and mean (+/− SD) AST activity for homozygotes were 502 (+/116) U/L, for heterozygotes, 357 (+/−92) U/L and for controls, 311 (+/−64) U/L and were significantly different between groups (P = 0.04 and P = 0.01 respectively). Resting plasma AST activity was significantly associated with the severity of subsarcolemmal vacuolation (rho = 0.816; P = 0.01) and cytoplasmic inclusions (rho = 0.766; P = 0.01). There were fewer type 2× and more type 2a muscle fibres in PSSM1-affected horses. Our results indicate that PSSM1 has incomplete dominance. Furthermore, the association between plasma muscle enzyme activity and severity of underlying pathology suggests that physical disruption of myofibres may contribute to the myopathic phenotype. This work provides insight into PSSM1 pathogenesis and has implications for related human glycogenoses.     

Irrelevance of Microsatellite Instability in the Epidemiology of Sporadic Pancreatic Ductal Adenocarcinoma

Background and Aims

Pancreatic cancer risk is increased in Lynch syndrome (LS) patients with mismatch repair gene defects predisposing to colonic and extracolonic cancers with microsatellite instability (MSI). However, the frequency of MSI pancreatic cancers has never been ascertained in consecutive, unselected clinical series, and their contribution to the sporadic and inherited burden of pancreatic cancer remains to be established. Aims of the study were to determine the prevalence of MSI in surgically resected pancreatic cancers in a multicentric, retrospective study, and to assess the occurrence of pancreatic cancer in LS.

Methods

MS-status was screened by a panel of 5 mononucleotide repeats (Bat26, Bat25, NR-21, NR-24 and NR-27) in 338 consecutive pancreatic ductal adenocarcinoma (PDAC), resected at two Italian and one German referral centres. The personal history of pancreatic cancer was assessed in an independent set of 58 probands with LS and in 138 first degree relatives who had cancers.

Results

Only one PDAC (0.3%) showed MSI. This was a medullary type cancer, with hMLH1-deficiency, and no identified germ-line mutation but methylation of hMLH1. Pancreatic cancer occurred in 5 (2.5%) LS patients. Histological sampling was available for 2 cases, revealing PDAC in one case and an ampullary cancer in the other one.

Conclusions

MSI prevalence is negligible in sporadic, resected PDAC. Differently, the prevalence of pancreatic cancer is 2.5% in LS patients, and cancers other than PDAC may be encountered in this setting. Surveillance for pancreatic cancer should be advised in LS mutation carriers at referral centers.     

High sensitivity of intestinal CD8+ T cells to nucleotides indicates P2X7 as a regulator for intestinal T cell responses

The purinoreceptor P2X7 is expressed on subsets of T cells and mediates responses of these cells to extracellular nucleotides such as ATP or NAD(+). We identified P2X7 as a molecule highly up-regulated on conventional CD8alphabeta(+) and unconventional CD8alphaalpha(+) T cells of the intestinal epithelium of mice. In contrast, CD8(+) T cells derived from spleen, mesenteric lymph nodes, and liver expressed only marginal levels of P2X7. However, P2X7 was highly up-regulated on CD8(+) T cells from spleen and lymph nodes when T cells were activated in the presence of retinoic acid. High P2X7 expression on intestinal CD8(+) T cells as well as on CD8(+) T cells incubated with retinoic acid resulted in enhanced sensitivity of cells to extracellular nucleotides. Both cell populations showed a high level of apoptosis following incubation with NAD(+) and the ATP derivative 2',3'-O-(benzoyl-4-benzoyl)-ATP, and injection of NAD(+) caused selective in vivo depletion of intestinal CD8(+) T cells. Following oral infection with Listeria monocytogenes, P2X7-deficient mice showed similar CD8(+) T cell responses in the spleen, but enhanced responses in the intestinal mucosa, when compared with similarly treated wild-type control mice. Overall, our observations define P2X7 as a new regulatory element in the control of CD8(+) T cell responses in the intestinal mucosa.     

Requirements for effective antitumor responses of TCR transduced T cells

Adoptive transfer of TCR gene-modified T cells has been proposed as an attractive approach to target tumors for which it is difficult or impossible to induce strong tumor-specific T cell responses by vaccination. Whereas the feasibility of generating tumor Ag-specific T cells by gene transfer has been demonstrated, the factors that determine the in vivo effectiveness of TCR-modified T cells are largely unknown. We have analyzed the value of a number of clinically feasible strategies to enhance the antitumor potential of TCR modified T cells. These experiments reveal three factors that contribute greatly to the in vivo potency of TCR-modified T cells. First, irradiation-induced host conditioning is superior to vaccine-induced activation of genetically modified T cells. Second, increasing TCR expression through genetic optimization of TCR sequences has a profound effect on in vivo antitumor activity. Third, a high precursor frequency of TCR modified T cells within the graft is essential. Tumors that ultimately progress in animals treated with this optimized regimen for TCR-based adoptive cell transfer invariably display a reduced expression of the target Ag. This suggests TCR gene therapy can achieve a sufficiently strong selective pressure to warrant the simultaneous targeting of multiple Ags. The strategies outlined in this study should be of value to enhance the antitumor activity of TCR-modified T cells in clinical trials.     

The DNA deaminase activity of human APOBEC3G is required for Ty1, MusD, and human immunodeficiency virus type 1 restriction

Human APOBEC3G and several other APOBEC3 proteins have been shown to inhibit the replication of a variety of retrotransposons and retroviruses. All of these enzymes can deaminate cytosines within single-strand DNA, but the overall importance of this conserved activity in retroelement restriction has been questioned by reports of deaminase-independent mechanisms. Here, three distinct retroelements, a yeast retrotransposon, Ty1, a murine endogenous retrovirus, MusD, and a lentivirus, human immunodeficiency virus type 1 (HIV-1), were used to evaluate the relative contributions of deaminase-dependent and -independent mechanisms. Although human APOBEC3G can restrict the replication of all three of these retroelements, APOBEC3G lacking the catalytic glutamate (E259Q) was clearly defective. This phenotype was particularly clear in experiments with low levels of APOBEC3G expression. In contrast, purposeful overexpression of APOBEC3G-E259Q was able to cause modest to severe reductions in the replication of Ty1, MusD, and HIV-1(ΔVif). The importance of these observations was highlighted by data showing that CEM-SS T-cell lines expressing near-physiologic levels of APOBEC3G-E259Q failed to inhibit the replication of HIV-1(ΔVif), whereas similar levels of wild-type APOBEC3G fully suppressed virus infectivity. Despite the requirement for DNA deamination, uracil DNA glycosylase did not modulate APOBEC3G-dependent restriction of Ty1 or HIV-1(ΔVif), further supporting prior studies indicating that the major uracil excision repair system of cells is not involved. In conclusion, the absolute requirement for the catalytic glutamate of APOBEC3G in Ty1, MusD, and HIV-1 restriction strongly indicates that DNA cytosine deamination is an essential part of the mechanism.     

Quantitative protein microarrays for time-resolved measurements of protein phosphorylation

The quantitative analysis of signaling networks requires highly sensitive methods for the time-resolved determination of protein phosphorylation. For this reason, we developed a quantitative protein microarray that monitors the activation of multiple signaling pathways in parallel, and at high temporal resolution. A label-free sandwich approach was combined with near infrared detection, thus permitting the accurate quantification of low-level phosphoproteins in limited biological samples corresponding to less than 50,000 cells, and with a very low standard deviation of approximately 5%. The identification of suitable antibody pairs was facilitated by determining their accuracy and dynamic range using our customized software package Quantpro. Thus, we are providing an important tool to generate quantitative data for systems biology approaches, and to drive innovative diagnostic applications.     

RNA silencing movement in plants.

Higher eukaryotes have developed a mechanism of sequence-specific RNA degradation which is known as RNA silencing. In plants and some animals, similar to the nematode Caenorhabditis elegans, RNA silencing is a non-cell-autonomous event. Hence, silencing initiation in one or a few cells leads progressively to the sequence-specific suppression of homologous sequences in neighbouring cells in an RNA-mediated fashion. Spreading of silencing in plants occurs through plasmodesmata and results from a cell-to-cell movement of a short-range silencing signal, most probably 21-nt siRNAs (short interfering RNAs) that are produced by one of the plant Dicer enzymes. In addition, silencing spreads systemically through the phloem system of the plants, which also translocates metabolites from source to sink tissues. Unlike the short-range silencing signal, there is little known about the mediators of systemic silencing. Recent studies have revealed various and sometimes surprising genetic elements of the short-range silencing spread pathway, elucidating several aspects of the processes involved. In this review we attempt to clarify commonalities and differences between the individual silencing pathways of RNA silencing spread in plants.