Studies on the survival of enterohemorrhagic and environmental Escherichia coli strains in wastewater and in activated sludges from dairy sewage treatment plants

Survival of Escherichia coli O157:H7 strain isolated from milk in Poland and an environmental E. coli strain in wastewater from Garwolin and ?owicz dairies and in activated sludges from dairy sewage treatment plants as well as in dairy wastewater with activated sludges was examined. Environmental materials were contaminated with about 10(8) of target bacteria/ml of sample. The experiments were performed under temperature conditions typical of autumn-winter (6 degrees) and spring-summer (24 degrees C) seasons. It was found that the non-pathogenic E. coli strain survived longer in all media than the enterohemorrhagic serotype. E. coli O157:H7 bacteria were not detected (in direct plating method) in activated sludges after 21-28 days; in dairy wastewater as well as in wastewater with activated sludges after 21-25 days. These periods for environmental E. coli strain were 35-42 days (activated sludges), 25-28 days (wastewater with activated sludges). At higher temperature environmental E. coli were not detected in wastewater from ?owicz dairy sewage treatment plant after 25 days, but the bacteria were still present in wastewater from Garwolin dairy sewage tratment plant after 34 days. The obtained results show that the lack of environmental E. coli bacteria (as a indicator bacteria of fecal contamination) in dairy wastewater and in dairy wastewater with activated sludges could indicate the absence of pathogenic E. coli bacteria. Prolonged existence of the enterohemorrhagic serotype in activated sludges shows the need to treat activated sludges prior to the utilization of these materials as fertilizer.     

MMP19 is essential for T cell development and T cell-mediated cutaneous immune responses

Matrix metalloproteinase-19 (MMP19) affects cell proliferation, adhesion, and migration in vitro but its physiological role in vivo is poorly understood. To determine the function of MMP19, we generated mice deficient for MMP19 by disrupting the catalytic domain of mmp19 gene. Although MMP19-deficient mice do not show overt developmental and morphological abnormalities they display a distinct physiological phenotype. In a model of contact hypersensitivity (CHS) MMP19-deficient mice showed impaired T cell-mediated immune reaction that was characterized by limited influx of inflammatory cells, low proliferation of keratinocytes, and reduced number of activated CD8(+) T cells in draining lymph nodes. In the inflamed tissue, the low number of CD8(+) T cells in MMP19-deficient mice correlated with low amounts of proinflammatory cytokines, especially lymphotactin and interferon-inducible T cell alpha chemoattractant (I-TAC). Further analyses showed that T cell populations in the blood of immature, unsensitized mice were diminished and that this alteration originated from an altered maturation of thymocytes. In the thymus, thymocytes exhibited low proliferation rates and the number of CD4(+)CD8(+) double-positive cells was remarkably augmented. Based on the phenotype of MMP19-deficient mice we propose that MMP19 is an important factor in cutaneous immune responses and influences the development of T cells.     

Rare,high-affinity mouse anti-PD-1 antibodies that function in checkpoint blockade,discovered using microfluidics and molecular genomics

Conventionally, mouse hybridomas or well-plate screening are used to identify therapeutic monoclonal antibody candidates. In this study, we present an alternative to hybridoma-based discovery that combines microfluidics, yeast single-chain variable fragment (scFv) display, and deep sequencing to rapidly interrogate and screen mouse antibody repertoires. We used our approach on six wild-type mice to identify 269 molecules that bind to programmed cell death protein 1 (PD-1), which were present at an average of 1 in 2,000 in the pre-sort scFv libraries. Two rounds of fluorescence-activated cell sorting (FACS) produced populations of PD-1-binding scFv with a mean enrichment of 800-fold, whereas most scFv present in the pre-sort mouse repertoires were de-enriched. Therefore, our work suggests that most of the antibodies present in the repertoires of immunized mice are not strong binders to PD-1. We observed clusters of related antibody sequences in each mouse following FACS, suggesting evolution of clonal lineages. In the pre-sort repertoires, these putative clonal lineages varied in both the complementary-determining region (CDR)3K and CDR3H, while the FACS-selected PD-1-binding subsets varied primarily in the CDR3H. PD-1 binders were generally not highly diverged from germline, showing 98% identity on average with germline V-genes. Some CDR3 sequences were discovered in more than one animal, even across different mouse strains, suggesting convergent evolution. We synthesized 17 of the anti-PD-1 binders as full-length monoclonal antibodies. All 17 full-length antibodies bound recombinant PD-1 with K_D < 500 nM (average = 62 nM). Fifteen of the 17 full-length antibodies specifically bound surface-expressed PD-1 in a FACS assay, and nine of the antibodies functioned as checkpoint inhibitors in a cellular assay. We conclude that our method is a viable alternative to hybridomas, with key advantages in comprehensiveness and turnaround time.     

Prescribed “Breaks” as a Means to Disrupt Weight Control Efforts

Objective: Research on long‐term maintenance of weight loss is hampered by the fact that relapse typically does not occur until after 6 months. We sought to determine whether relapses could be induced earlier by intentionally interrupting the momentum of weight loss during a treatment program and thus provide a model for weight maintenance research. Research Methods and Procedures: Subjects (N = 142) were recruited at two centers and randomly assigned to a control group, a long break group (LB), or a short break group (SB). The control group received a standard behavioral weight loss program with 14 weekly sessions; the LB and SB groups received the same. However, the LB group took a 6‐week break after lesson 7, and the SB group took a 2‐week break after lessons 3, 6, and 9. Participants were instructed to stop all weight loss efforts during breaks. Results: Participants seemed to take breaks as prescribed, with interruptions in self‐monitoring, dietary adherence, and self‐weighing (but not in exercise). However, participants quickly resumed these behaviors after the break. Breaks produced a slowing of weight loss or slight regain, but weight losses during the breaks were not significantly different from the control group. Importantly, overall weight losses (0 to 5 months or 0 to 11 months) did not differ between conditions. Discussion: This study was not successful in developing a method to experimentally produce weight loss relapses. However, the finding that prescribed breaks do not have adverse effects may have clinical application.     

Direct evidence for the intracellular localization of Hsp104 in Saccharomyces cerevisiae by immunoelectron microscopy

To reveal the intracellular localization of Hsp104 in the yeast Saccharomyces cerevisiae before and after heat-shock, we performed immunoelectron microscopy after immunogold labeling with anti-Hsp104 antibody. At normal temperature (25 degrees C), a small amount of Hsp104 was located in the cytoplasm and nucleus. On exposure to mild heat-shock at 40 degrees C, protein aggregates appeared in the cytoplasm and nucleus, and Hsp104 increased around the aggregates with increasing time of the mild heat-shock treatment. Moreover, at lethal heat-shock temperature (51 degrees C) for 20 min after mild heat treatment at 40 degrees C, the intracellular localization of Hsp104 and intracellular structures were similar to those of the mild heat-shocked cells. However, in the lethally heat-shocked cells, certain intracellular structures were destroyed, and Hsp104 was not expressed. In the hsp104 null mutant strain Deltahsp104 which was treated at 40 degrees C, Hsp104 was not localized around the aggregates. Additionally, in the Deltahsp104 strain, even mild heat-shocked cells at 37 degrees C or 40 degrees C, showed destruction of intracellular structure compared to the wild-type strain. Our data suggest the following: (1) Hsp104 is associated closely with protein aggregates during heat-shock treatment, (2) Hsp104 is important for maintenance of the intracellular structure under lethal heat-shock conditions, (3) acquisition of thermotolerance depends on the amount of Hsp104 produced during mild heat-shock treatment.     

Pseudomonas syringae pv. tomato DC3000 HopPtoM (CEL ORF3) is important for lesion formation but not growth in tomato and is secreted and translocated by the Hrp type III secretion system in a chaperone-dependent manner

Pseudomonas syringae pv. tomato DC3000 is a pathogen of tomato and Arabidopsis that injects virulence effector proteins into host cells via a type III secretion system (TTSS). TTSS-deficient mutants have a Hrp- phenotype, that is, they cannot elicit the hypersensitive response (HR) in non-host plants or pathogenesis in host plants. Mutations in effector genes typically have weak virulence phenotypes (apparently due to redundancy), but deletion of six open reading frames (ORF) in the DC3000 conserved effector locus (CEL) reduces parasitic growth and abolishes disease symptoms without affecting function of the TTSS. The inability of the DeltaCEL mutant to cause disease symptoms in tomato was restored by a clone expressing two of the six ORF that had been deleted: CEL ORF3 (HopPtoM) and ORF4 (ShcM). A DeltahopPtoM::nptII mutant was constructed and found to grow like the wild type in tomato but to be strongly reduced in its production of necrotic lesion symptoms. HopPtoM expression in DC3000 was activated by the HrpL alternative sigma factor, and the protein was secreted by the Hrp TTSS in culture and translocated into Arabidopsis cells by the Hrp TTSS during infection. Secretion and translocation were dependent on ShcM, which was neither secreted nor translocated but, like typical TTSS chaperones, could be shown to interact with HopPtoM, its cognate effector, in yeast two-hybrid experiments. Thus, HopPtoM is a type III effector that, among known plant pathogen effectors, is unusual in making a major contribution to the elicitation of lesion symptoms but not growth in host tomato leaves.     

Proline dehydrogenase and pyrroline-5-carboxylate reductase from pumpkin cotyledons

Activity of proline dehydrogenase and pyrroline-5-carboxylate reductase was greatest after 5 and 7 days germination in green and etiolated cotyledons respectively of pumpkin (Cucurbita moschata Poir. cv. Dickinson Field). The ratio of pyrroline-5-carboxylate reductase to proline dehydrogenase activity was constant throughout germination. Both enzymes were purified 30-fold but the ratio pyrroline-5-carboxylate reductase—proline dehydrogenase activity was constant throughout purification. However, this ratio decreased with storage, especially in purified preparations. Both enzymes were stable at high temperature and the ratio pyrroline-5-carboxylate reductase—proline dehydrogenase remained unchanged on heating. Proline dehydrogenase and pyrroline-5-carboxylate reductase were inhibited by sodium bisulfite and cysteine. ATP, ADP and NADP caused inhibition of both enzymes. Proline dehydrogenase utilized NAD but not NADP. Pyrroline-5-carboxylate reductase had a 2.5-fold greater activity with NADH than NADPH. Most of the data presented suggest that proline dehydrogenase and pyrroline-5-carboxylate reductase activities occur on the same protein molecule.     

Crystal packing effects on protein loops

The effects of crystal packing on protein loop structures are examined by (1) a comparison of loops in proteins that have been crystallized in alternate packing arrangements, and (2) theoretical prediction of loops both with and without the inclusion of the crystal environment. Results show that in a minority of cases, loop geometries are dependent on crystal packing effects. Explicit representation of the crystal environment in a loop prediction algorithm can be used to model these effects and to reconstruct the structures, and relative energies, of a loop in alternative packing environments. By comparing prediction results with and without the inclusion of the crystal environment, the loop prediction algorithm can further be used to identify cases in which a crystal structure does not represent the most stable state of a loop in solution. We anticipate that this capability has implications for structural biology.     

Site-specific recombination in <Emphasis Type="Italic">Arabidopsis</Emphasis> plants promoted by the Integrase protein of coliphage HK022

The gene encoding the wild type Integrase protein of coliphage HK022 was integrated chromosomally and expressed in Arabidopsis thaliana plants. Double-transgenic plants cloned with the int gene as well as with a T-DNA fragment carrying the proper att sites in a tandem orientation showed that Int catalyzed a site-specific integration reaction (attP × attB) as well as a site-specific excision reaction (attL × attR). The reactions took place without the need to provide any of the accessory proteins that are required by Int in the bacterial host. When expressed in tobacco plants a GFP-Int fusion exhibits a predominant nuclear localization.These authors contributed equally to this work     

Caenorhabditis elegans OSR-1 regulates behavioral and physiological responses to hyperosmotic environments

The molecular mechanisms that enable multicellular organisms to sense and modulate their responses to hyperosmotic environments are poorly understood. Here, we employ Caenorhabditis elegans to characterize the response of a multicellular organism to osmotic stress and establish a genetic screen to isolate mutants that are osmotic stress resistant (OSR). In this study, we describe the cloning of a novel gene, osr-1, and demonstrate that it regulates osmosensation, adaptation, and survival in hyperosmotic environments. Whereas wild-type animals exposed to hyperosmotic conditions rapidly lose body volume, motility, and viability, osr-1(rm1) mutant animals maintain normal body volume, motility, and viability even upon chronic exposures to high osmolarity environments. In addition, osr-1(rm1) animals are specifically resistant to osmotic stress and are distinct from previously characterized osmotic avoidance defective (OSM) and general stress resistance age-1(hx546) mutants. OSR-1 is expressed in the hypodermis and intestine, and expression of OSR-1 in hypodermal cells rescues the osr-1(rm1) phenotypes. Genetic epistasis analysis indicates that OSR-1 regulates survival under osmotic stress via CaMKII and a conserved p38 MAP kinase signaling cascade and regulates osmotic avoidance and resistance to acute dehydration likely by distinct mechanisms. We suggest that OSR-1 plays a central role in integrating stress detection and adaptation responses by invoking multiple signaling pathways to promote survival under hyperosmotic environments.