Detection of live Salmonella enterica in fresh-cut vegetables by a TaqMan-based one-step reverse transcription real-time PCR.

Fresh-cut produce is at greater risk of Salmonella contamination. Detection and early warning systems play an important role in reducing the dissemination of contaminated products. One-step Reverse Transcription Polymerase Chain Reaction (RT-qPCR) targeting Salmonella tmRNA with or without a 6-h enrichment was evaluated for the detection of Salmonella in fresh-cut vegetables after 6-h storage. LOD of one-step RT-qPCR was 1·0 CFU per ml (about 100 copies tmRNA per ml) by assessed 10-fold serially diluted RNA from 106 CFU per ml bacteria culture. Then, one-step RT-qPCR assay was applied to detect viable Salmonella cells in 14 fresh-cut vegetables after 6-h storage. Without enrichment, this assay could detect 10 CFU per g for fresh-cut lettuce, cilantro, spinach, cabbage, Chinese cabbage and bell pepper, and 102 CFU per g for other vegetables. With a 6-h enrichment, this assay could detect 10 CFU per g for all fresh-cut vegetables used in this study. Moreover, this assay was able to discriminate viable cells from dead cells. This rapid detection assay may provide potential processing control and early warning method in fresh-cut vegetable processing to strengthen food safety assurance. SIGNIFICANCE AND IMPACT OF THE STUDY: Significance and Impact of the Study: Fresh-cut produce is at greater risk of Salmonella contamination. Rapid detection methods play an important role in reducing the dissemination of contaminated products. One-step RT-qPCR assay used in this study could detect 10 CFU per g Salmonella for 14 fresh-cut vegetables with a 6-h short enrichment. Moreover, this assay was able to discriminate viable cells from dead cells. This rapid detection assay may provide potential processing control and early warning method in fresh-cut vegetable processing to strengthen food safety assurance.

Tumor cell-activated CARD9 signaling contributes to metastasis-associated macrophage polarization.

Macrophages are critical immune effector cells of the tumor microenvironment that promote seeding, extravasation and persistent growth of tumor cells in primary tumors and metastatic sites. Tumor progression and metastasis are affected by dynamic changes in the specific phenotypes of macrophage subpopulations; however, the mechanisms by which tumor cells modulate macrophage polarization remain incompletely understood. Caspase recruitment domain-containing protein 9 (CARD9) is a central adaptor protein of innate immune responses to extracellular pathogens. We report that increased CARD9 expression is primarily localized in infiltrated macrophages and significantly associated with advanced histopathologic stage and the presence of metastasis. Using CARD9-deficient (CARD9(-/-)) mice, we show that bone marrow-derived CARD9 promotes liver metastasis of colon carcinoma cells. Mechanistic studies reveal that CARD9 contributes to tumor metastasis by promoting metastasis-associated macrophage polarization through activation of the nuclear factor-kappa B signaling pathway. We further demonstrate that tumor cell-secreted vascular endothelial growth factor facilitates spleen tyrosine kinase activation in macrophages, which is necessary for formation of the CARD9-B-cell lymphoma/leukemia 10-mucosa-associated lymphoid tissue lymphoma translocation protein 1 complex. Taken together, our results indicating that CARD9 is a regulator of metastasis-associated macrophages will lead to new insights into evolution of the microenvironments supporting tumor metastasis, thereby providing targets for anticancer therapies.

GAG-augmented polysaccharide hydrogel: a novel biocompatible and biodegradable material to support chondrogenesis.

The quality of articular cartilage engineered using a cell-polymer construct depends, in part, on the chemical composition of the biomaterial and whether that biomaterial can support the chondrocytic phenotype. Acknowledging the supportive influence of tissue-specific matrix molecules on the chondrocytic phenotype, we have combined chondroitin sulfate-A (CSA) and chitosan, a glycosaminoglycan (GAG) analog, to develop a novel biomaterial to support chondrogenesis. Chitosan is a polycationic repeating monosaccharide of beta-1,4-linked glucosamine monomers with randomly located N-acetyl glucosamine units. Chitosan may be combined with the polyanionic CSA such that ionic crosslinking results in hydrogel formation. Bovine primary articular chondrocytes, when seeded onto a thin layer of CSA-chitosan, form discrete, focal adhesions to the material and maintain many characteristics of the differentiated chondrocytic phenotype, including round morphology, limited mitosis, collagen type II, and proteoglycan production. Our findings suggest CSA-chitosan may be well suited as a carrier material for the transplant of autologous chondrocytes or as a scaffold for the tissue engineering of cartilage-like tissue.

Binding of A/T-rich DNA by three high mobility group-like domains in c-Abl tyrosine kinase.

The c-Abl tyrosine kinase has been shown previously to bind DNA. Using polymerase chain reaction-based binding site-selection methods, no consensus high affinity binding site for c-Abl was found. Instead, oligonucleotides with runs of A/T sequences were isolated, and purified c-Abl was shown to bind A/T-containing oligonucleotides better than those without A/T sequences. DNA binding of c-Abl was dependent on three high mobility group 1-like boxes (HLBs), which bound cooperatively to the A/T-rich oligonucleotides. To distinguish binding to A/T sequences per se from binding to nonspecific DNA with a bend at the A/T-rich region, two oligonucleotides were compared for binding to c-Abl. Both oligonucleotides contained A/T sequences. In one, the A/T motif was part of an 80-mer duplex DNA. In another, the A/T motif was in the duplex arm of an 80-mer "bubble DNA" containing an internal unpaired 20-mer region to provide a flexible hinge. Interestingly, the HLBs of c-Abl bound better to the oligonucleotide containing the bubble, suggesting a higher affinity for bent DNA rather than A/T sequences per se. Taken together, these observations define a new class of DNA binding domains, the HLBs, which do not bind DNA with a high degree of sequence specificity, but may selectively bind to bent DNA or to sequences that are easier to distort.

cDNA cloning and characterization of rat salivary glycoproteins. Novel members of the proline-rich-protein multigene families.

The cDNAs for two glycoproteins, the 158-kDa submandibular glycoprotein (SGP158) and the 200-kDa parotid glycoprotein (PGP200), have been cloned from rat submandibular and parotid glands, respectively. Both cDNAs encode for identical proteins with repeating peptides -Asp-Gln-Gly-(Asn)-Gln-Thr-Gln-Pro-Arg-Pro-Pro-His-Pro-. A full-length cDNA encoding SGP158 was obtained using the strategy of anchor-PCR, and a full-length cDNA of PGP200 was prepared using RNA-PCR. Sequence analysis of the cDNAs revealed that SGP158 and PGP200 are identical proteins with 23 repeating peptides. Twenty-one peptides contain potential N-glycosylation sites and these two glycoproteins differ only in their glycosylation patterns. Southern-blot analysis showed that a single-copy gene encodes both mRNAs. PGP200 is constitutively expressed, but the synthesis of SGP158 is totally dependent upon treatment of animals with the beta-agonist isoproterenol. The first 106-nucleotide sequence of cDNAs for PGP200 and SGP158, which corresponds to the 5'-untranslated region and sequence encoding the signal peptide, is highly conserved when compared with proline-rich protein and glutamine-rich protein gene sequences. Based on the nucleotide sequences of exon I, a phylogenetic tree was constructed for 35 members of these multigene families. The tree fits with the generally recognized phylogeny of mammalian orders. We propose that exon I sequences of the proline-rich protein and glutamine-rich protein multigene families are relatively new and are possibly generated through exon shuffling during evolution.

[Superior sulcus cancer of the lung--report on 45 patients].

From 1960 to 1980, 45 patients with superior sulcus cancer of the lung were seen in our hospital. 39 were male and 6 were female. The youngest was 28 years old and the eldest was 75. The presenting symptoms and signs were back and shoulder pain in 32 patients, compression of the brachial plexus in 17, Horner's syndrome in 12, supraclavicular mass in 11, superior cava vena obstruction in 4, shadow in the apex in 45, destruction of the rib in 19 (10 in the second rib), destruction of the adjacent vertebra in 8 (5 in T3) and destruction of the clavicle in 1. In 18 patients proved by pathology and cytology, 8 were adenocarcinoma, 3 squamous cell carcinoma, 3 undifferentiated and 4 unclassified carcinoma. 27 patients were diagnosed by X-ray films. 40 patients were admitted for treatment. 3 recieved chemotherapy alone and all of them died in one year. Of the 6 treated by radiation plus chemotherapy, 3 survived for one year but all died in two years. 2 were treated by radiation plus operation and none survived for more than 3 years. 29 were treated by radiation only. The 1, 3 and 5 year survival rates were 51%, 13.7% and 6.9%. Destruction of the rib or the adjacent vertebral body was irrelative to the prognosis but the presence of supraclavicular mass reduced the survival rate. Cause of death was local recurrence in 48% (13/27) and distant metastasis in 48% (13/27). The authors suggest that radical en bloc resection together with radiation the worth further study.

[Carcinoma of esophagus treated by 8MV X-ray and checked by simulator].

From January 1, 1978 to December 31, 1983, 570 patients with carcinoma of esophagus were treated by 8 MV X-ray and checked by the Philips simulator. In order to assess their value, a series of 3,798 patients reported previously was used for comparison. To further evaluate the effect of 8 MV X-ray and simulator separately, a series of 154 patients was treatment by non-8 MV X-ray during the same period. The results showed that the 1 and 3 year survival rates of 8 MV X-ray series were better than the 3,798 series (P less than 0.01). It means that after the use of simulator and 8 MV X-ray the survival rates were improved. As compared to the radiation other than 8 MV X-ray, there was no difference between 8 MV X-ray and non-8 MV X-ray series, which means that the improvement may have been due to the better localization by the simulator. Having more importance, no radiation myelitis was found after the use of simulator. The sex, age, length, location, X-ray type, NSD and causes of failure were compared in these groups.

[Irradiation of recurrent esophageal carcinoma].

81 patients with recurrent esophageal carcinoma after radical radiotherapy were irradiated from Mar. 1958 to Dec. 1973. The results were compared with those of 137 patients with untreated postirradiation recurrences in the same period. Of these 81 patients, 21 failed to complete the second course due to esophageal perforation (8 cases), fatal hemorrhage (4 cases) and deterioration of the general condition (9 cases). The improvement rate of symptoms was 19.75% and that of barium meal was 44.44%. The mean survival of the re-irradiation group was 6.59 +/- 4.66 months in contrast to that of the control group, 4.51 +/- 4.40 months. There was significant difference between these two groups (P less than 0.01). It is shown that the total dose of re-irradiation has no significant effect on the survival. the survival of patients receiving 4,000 rad or less was 5.92 +/- 4.53 months and that of patients receiving more than 4,000 rad was 7.86 +/- 4.72 months (P greater than 0.05). There is no statistical significance in between. We believe that re-irradiation is capable of relieving the symptoms and prolonging survival. It should be selectively used in the patients with recurrent esophageal carcinoma after radical radiotherapy.