A human breast tumor cell line (BT-474) that supports mouse mammary tumor virus replication

Summary A human breast tumor cell line BT-474 derived from an invasive ductal carcinoma was experimentally infected in vitro with a mouse mammary tumor virus from the RIII strain (RIII-MuMTV). The virus that replicated in the human cells was characterized as a mouse virus by immunofluorescence, electron microscopy and the presence of a specific RNA-directed DNA polymerase. The cells themselves were human as per the karyotype and isoenzyme migration patterns. It is concluded that human cells are susceptible to the mouse mammary tumor virus and can, eventually, support its replication. This work was supported by USPHS Grant CA-08515 from the National Cancer Institute and by NIH Contract N01-CP-81003.     

The effect of 5-Iodouracil on the growth and biosynthetic processes of bacteriophage T4td8 in the absence of light

5-Iodouracil (IUra)-substituted progeny bacteriophage T4td8 were grown under conditions such that, upon CsCl equilibrium isopycnic gradient centrifugation, progeny with density distributions about the median similar to that of unsubstituted phage are obtained. In the absence of light a monotonie relationship exists between decreasing progeny viability and increasing percent IUra substitution. IUra is equivalent to thymine as a growth factor on a molar basis, and at concentrations of IUra plus thymine above that required for maximum particle production, the percent IUra substitution in phage DNA is determined by the mole fraction of IUra in the medium. The lethal effects of 5-iodo-2'-deoxyuridine (IdUrd) and IUra are equivalent, and are not produced by a direct effect on the phage particles. At equivalent percent substitution in phage DNA the order of lethality is IUra > 5-bromouracil (BrUra) > 5-chlorouracil (ClUra). There is no interference with the transfer of thymine from host cell to progeny phage by the presence of IUra in the medium, and IUra affects neither the time of lysis nor the content of phage DNA in the infected cells.     

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.     

Listeria monocytogenes induces host DNA damage and delays the host cell cycle to promote infection

Listeria monocytogenes (Lm) is a human intracellular pathogen widely used to uncover the mechanisms evolved by pathogens to establish infection. However, its capacity to perturb the host cell cycle was never reported. We show that Lm infection affects the host cell cycle progression, increasing its overall duration but allowing consecutive rounds of division. A complete Lm infectious cycle induces a S-phase delay accompanied by a slower rate of DNA synthesis and increased levels of host DNA strand breaks. Additionally, DNA damage/replication checkpoint responses are triggered in an Lm dose-dependent manner through the phosphorylation of DNA-PK, H2A.X, and CDC25A and independently from ATM/ATR. While host DNA damage induced exogenously favors Lm dissemination, the override of checkpoint pathways limits infection. We propose that host DNA replication disturbed by Lm infection culminates in DNA strand breaks, triggering DNA damage/replication responses, and ensuring a cell cycle delay that favors Lm propagation.     

Lagenidium myophilum infection in the coonstripe shrimp,Pandalus hypsinotus

A fungal infection occurred in juvenile coonstripe shrimps,Pandalus hypsinotus, cultured at Hokkaido Institute of Mariculture, Hokkaido, Japan. The fungus was identified asLagenidium myophilum, the same fungus that had previously been isolated from the abdominal muscle of adult northern shrimps,Pandalus borealis, and larvae of the coonstripe shrimp. Histopathologically, numerous nonseptate hyphae were observed in the lesions, and melanized hemocytes were present within the blackened areas. The optimum temperature for growth of the present strain was 25–30°C, and the optimum NaCl concentration for growth was 0.5–1.0%. Its biological characteristics were compared with those ofLagenidium myophilum isolated from diseased larval coonstripe shrimp and adult northern shrimp. The fungus was pathogenic toward shrimps of the genusPandalus, which live in deep sea areas. The fungus could infect shrimps at various stages, from larva to adult.     

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species^1-5, as well as the wealth of information available regarding the insect''s immune system^6-8, and the pending genome sequence⁹ make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter¹⁰. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking¹¹ and oral toxicity assays¹². Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides⁷; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR¹³. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes⁶. To analyze these responses, injected insects can be dissected and visualized by microscopy^{13, 14}.     

Evaluation of the efficiency of using Salmonella Kentucky and Escherichia coli O119 bacteriophages in the treatment and prevention of salmonellosis and colibacillosis in broiler chickens

Phage therapy is considered an alternative modality in the treatment of different bacterial diseases. However, their therapeutic and preventive roles against infections caused by Salmonella Kentucky and Escherichia coli O119 were of little attention. In this study, two phages were isolated, characterized and assessed for their potential therapeutic and preventive roles against S. Kentucky and E. coli O119 infections in broilers. Commercial 1-day-old arboacres broiler chicks were assigned to seven groups: Group Ӏ was as a negative control, groups (П and Ш) were assigned as positive controls by the challenge of S. Kentucky and E. coli O119, respectively. The remaining four groups (IV, V, VI and VII) were administrated with five repeated phage doses to determine the effect of multiple doses. Phages were administrated in groups (IV and VI) after challenging with S. Kentucky and E. coli O119, respectively to assess their therapeutic role; moreover, their preventive role was evaluated through administration in groups (V and VII) before challenging with S. Kentucky and E. coli O119, respectively. Sampling was done from different organs at three time points and revealed that phage-treated groups had lower colony forming units of S. Kentucky and E. coli. Our results suggest that bacteriophages are efficient in the treatment and prevention of salmonellosis and colibacillosis in broiler farms.     

Identification of dynamic signatures associated with smoking‐related squamous cell lung cancer and chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a risk factor for the development of lung cancer. The aim of this study was to identify early diagnosis biomarkers for lung squamous cell carcinoma (SQCC) in COPD patients and to determine the potential pathogenetic mechanisms. The GSE12472 data set was downloaded from the Gene Expression Omnibus database. Differentially co‐expressed links (DLs) and differentially expressed genes (DEGs) in both COPD and normal tissues, or in both SQCC + COPD and COPD samples were used to construct a dynamic network associated with high‐risk genes for the SQCC pathogenetic process. Enrichment analysis was performed based on Gene Ontology annotations and Kyoto Encyclopedia of Genes and Genomes pathway analysis. We used the gene expression data and the clinical information to identify the co‐expression modules based on weighted gene co‐expression network analysis (WGCNA). In total, 205 dynamic DEGs, 5034 DLs and one pathway including CDKN1A, TP53, RB1 and MYC were found to have correlations with the pathogenetic progress. The pathogenetic mechanisms shared by both SQCC and COPD are closely related to oxidative stress, the immune response and infection. WGCNA identified 11 co‐expression modules, where magenta and black were correlated with the “time to distant metastasis.” And the “surgery due to” was closely related to the brown and blue modules. In conclusion, a pathway that includes TP53, CDKN1A, RB1 and MYC may play a vital role in driving COPD towards SQCC. Inflammatory processes and the immune response participate in COPD‐related carcinogenesis.