Synthesis and Antiproliferative Evaluation of 2-Deoxy-N-glycosylbenzotriazoles/imidazoles.

A series of 2-deoxy-2-iodo-α-d-mannopyranosylbenzotriazoles was synthesized using the benzyl, 4,6-benzylidene and acetyl protected D-glucal in the presence of N-iodosuccinimide (NIS). Subsequent removal of the iodine at the C-2 position using tributyltin hydride under free radical conditions afforded the 2-deoxy-α-d-glucopyranosylbenzotriazoles in moderate to high yields. This method was extended to the preparation of substituted 2-deoxy-ß-d-glucopyranosylimidazoles as well. The stereoselectivity of the addition reaction and the effect of the protecting group and temperature on anomer distribution of the benzotriazole series were also investigated. The anticancer properties of the newly synthesized compounds were evaluated in a series of viability studies using HeLa (human cervical adenocarcinoma), human breast and lung cancer cell lines. The N-[3,4,6-tri-O-benzyl-2-deoxy-α-d-glucopyranosyl]-1H-benzotriazole and the N-[3,4,6-tri-O-acetyl-2-deoxy-α-d-glucopyranosyl]-2H-benzotriazole were found to be the most potent cancer cell inhibitors at 20 µM concentrations across all four cell lines.

Methamphetamine facilitates pulmonary and splenic tissue injury and reduces T cell infiltration in C57BL/6 mice after antigenic challenge.

Methamphetamine (METH) is a strong addictive central nervous system stimulant. METH abuse can alter biological processes and immune functions necessary for host defense. The acquisition and transmission of HIV, hepatitis, and other communicable diseases are possible serious infectious consequences of METH use. METH also accumulates extensively in major organs. Despite METH being a major public health and safety problem globally, there are limited studies addressing the impact of this popular recreational psychostimulant on tissue adaptive immune responses after exposure to T cell dependent [ovalbumin (OVA)] and independent [lipopolysaccharide (LPS)] antigens. We hypothesized that METH administration causes pulmonary and splenic tissue alterations and reduces T cell responses to OVA and LPS in vivo, suggesting the increased susceptibility of users to infection. Using a murine model of METH administration, we showed that METH causes tissue injury, apoptosis, and alters helper and cytotoxic T cell recruitment in antigen challenged mice. METH also reduces the expression and distribution of CD3 and CD28 molecules on the surface of human Jurkat T cells. In addition, METH decreases the production of IL-2 in these T-like cells, suggesting a negative impact on T lymphocyte activation and proliferation. Our findings demonstrate the pleotropic effects of METH on cell-mediated immunity. These alterations have notable implications on tissue homeostasis and the capacity of the host to respond to infection.

Nuclear Localization Is Not Required for Tip60 Tumor Suppressor Activity in Breast and Lung Cancer Cells.

The Tip60 lysine acetyltransferase is a tumor suppressor in most cancers but an oncogene in prostate and gastric cancer. Tip60 is commonly found in the nucleus, where it acetylates proteins involved in transcription, DNA repair, and chromatin; however, it has also been shown to acetylate cytoplasmic targets. In this study, we investigated the relationship between Tip60 localization and breast and lung cancer. In cell fractionation experiments, cancer-derived cell lines showed a shift from nuclear to cytoplasmic endogenous Tip60 compared with cell lines derived from normal cells. With immunofluorescence, we observed four different localization patterns of overexpressed Tip60 and found that cancer cells had increased cytoplasmic localization of Tip60 compared with HEK-293 cells. The addition of a nuclear localization signal (NLS) increased the number of cells containing nuclear Tip60, whereas mutation of a putative endogenous NLS increased the number of cells with cytoplasmic Tip60. Overexpression of Tip60 increased cancer cell line sensitivity to paclitaxel regardless of changes in localization. These results suggest that dysregulation of Tip60 in breast and lung cancer is not limited to reduced expression but may also involve subcellular localization.

A multiplex serologic platform for diagnosis of tick-borne diseases.

Tick-borne diseases are the most common vector-borne diseases in the United States, with serology being the primary method of diagnosis. We developed the first multiplex, array-based assay for serodiagnosis of tick-borne diseases called the TBD-Serochip. The TBD-Serochip was designed to discriminate antibody responses to 8 major tick-borne pathogens present in the United States, including Anaplasma phagocytophilum, Babesia microti, Borrelia burgdorferi, Borrelia miyamotoi, Ehrlichia chaffeensis, Rickettsia rickettsii, Heartland virus and Powassan virus. Each assay contains approximately 170,000 12-mer linear peptides that tile along the protein sequence of the major antigens from each agent with 11 amino acid overlap. This permits accurate identification of a wide range of specific immunodominant IgG and IgM epitopes that can then be used to enhance diagnostic accuracy and integrate differential diagnosis into a single assay. To test the performance of the TBD-Serochip, we examined sera from patients with confirmed Lyme disease, babesiosis, anaplasmosis, and Powassan virus disease. We identified a wide range of specific discriminatory epitopes that facilitated accurate diagnosis of each disease. We also identified previously undiagnosed infections. Our results indicate that the TBD-Serochip is a promising tool for a differential diagnosis not available with currently employed serologic assays for TBDs.

Seroprevalence of Babesia microti in Individuals with Lyme Disease.

INTRODUCTION: Babesiosis is an emerging tick-borne disease (TBD) caused by Babesia microti, an intracellular parasite of red blood cells. Currently, it is the highest ranked pathogen transmitted by blood transfusion. Most healthy individuals infected with B. microti are asymptomatic, but may be at risk for chronic infection. Similar to Lyme disease transmitted by Borrelia burgdorferi, B. microti is spread by Ixodes scapularis ticks. The rate of coinfection with these TBDs in humans is unclear as most studies have focused their prevalence in ticks or rodent reservoirs. MATERIALS AND METHODS: In this study, we aimed to determine the seroprevalence of B. microti infection in individuals who tested positive for Lyme disease. Serum samples obtained from 130 subjects in New York were tested by immunofluorescence assay (IFA) for the presence of IgM and IgG antibodies against B. microti. RESULTS: Overall, 26.9% of the serum samples tested were positive for IgM and IgG antibodies against B. microti, suggesting exposure to TBD. Individuals who tested positive for Lyme disease as determined by two-tiered serological testing and the presence of both IgM and IgG antibodies directed against B. burgdorferi, were significantly increased for antibodies directed against B. microti (28.6%; p < 0.05), suggesting the possibility of coinfection with both TBDs. In contrast, the Lyme disease-negative control group had only 6.7% of samples seropositive for B. microti. CONCLUSIONS: These findings suggest the need for more extensive studies investigating infection rates with multiple TBDs in areas where they are endemic and further support for the need to implement an FDA-approved screening test for blood products to help prevent transfusion-transmitted babesiosis.

Correlations between quality ratings of skilled nursing facilities and multidrug-resistant urinary tract infections.

BACKGROUND: The purpose of this study was to determine risk factors for the acquisition of urinary tract infections (UTIs) and multidrug-resistant organisms (MDROs) in residents of skilled nursing facilities (SNFs). METHODS: Using the informational database provided by the Centers for Medicare and Medicaid Services (CMS), a retrospective logistic regression was performed on 1,523 urine cultures from 12 SNFs located in Long Island, New York. RESULTS: Of the 1,142 positive urine cultures, Escherichia coli was most prevalent. Additionally, 164 (14.4%) of the UTIs were attributed to an MDRO. In multivariate logistic regression, sex and overall quality rating predicted the occurrence of UTIs, whereas identification of MDROs was dependent on the level of nursing care received. The mean predicted probability of UTIs and receipt of contaminated samples was inversely dependent on the facility's rating, where the likelihood increased as overall quality ratings decreased. CONCLUSIONS: The CMS's quality rating system may provide some insight into the status of infection control practices in SNFs. The results of this study suggest that potential consumers should focus on the overall star ratings and the competency of the nursing staff in these facilities rather than on individual quality measures.

A comparative analysis of vaccine administration in urban and non-urban skilled nursing facilities.

BACKGROUND: The U.S. population is aging at an unprecedented rate, resulting in an increased demand for skilled nursing facilities (SNFs) and long-term care. Residents of these facilities are at a high risk for pneumococcal disease or severe influenza-related illnesses and death. For these reasons, the Centers for Medicare and Medicaid Services use influenza and pneumococcal vaccination rates as a quality measure in the assessment of SNFs, as complications related to these infections increase morbidity and mortality rates. METHODS: Disparities have been reported amongst vaccination with increased rates in urban areas as compared to their non-urban counterparts. Statistical analyses were performed to compare influenza and pneumococcal vaccination in urban and non-urban SNFs to determine variables that may influence vaccination status. RESULTS: Of the 15,639 nursing homes included in the study, 10,107 were in urban areas, while 5532 were considered non-urban. We found the percent of eligible and willing residents with up-to-date influenza and pneumococcal vaccinations increased with overall five-star ratings of SNFs. Somewhat paradoxically, although urban SNFs had higher mean overall five-star ratings, they showed lower rates of influenza and pneumococcal vaccination compared to non-urban SNFs. Ordinary least squares regression analysis comparing overall ratings, type of ownership, and geographic location by region yielded statistically significant results in which the overall rating, ownership-type and certificate-type favored urban SNFs (p < 0.001). CONCLUSIONS: This is the first systematic and comparative analysis to use the Nursing Home Compare database to assess vaccine administration of urban and non-urban SNFs. The findings of this study may be used to encourage the development of programs to improve vaccination rates and the quality of care in these facilities.

UIM domain-dependent recruitment of the endocytic adaptor protein Eps15 to ubiquitin-enriched endosomes.

BACKGROUND: Eps15 is an endocytic adaptor protein that stimulates clathrin-mediated endocytosis. Among other interactions, Eps15 binds ubiquitin via UIM domains, recruiting ubiquitinated cargo into clathrin-coated vesicles. In EGF-treated cells, Eps15 also localizes to endosomes. The basis of this localization is not known. RESULTS: We show that accumulation of ubiquitinated cargo can recruit Eps15 to endosomes via UIM domain interactions. First, treatment of SK-Br-3 breast cancer cells, which overexpress the EGFR family member ErbB2, with geldanamycin to promote receptor ubiquitination and endosomal transport, recruited FLAG-Eps15 to endosomes. Two in-frame ubiquitin constructs, PM-GFP-Ub (retained in endosomes after endocytosis), and GFP-FYVE-UbΔGG (targeted directly to endosomes) also recruited Eps15 to endosomes, as did slowing endosome maturation with constitutively-active Rab5-Q79L. Endosomal recruitment required the UIM domains, but not the N-terminal EH domains or central coiled-coil domains, of Eps15. Silencing of the endosomal Eps15 binding partner Hrs did not affect recruitment of Eps15 to ubiquitin-enriched endosomes. In fact, Hrs silencing itself modestly recruited Eps15 to endosomes, probably by accumulating endogenous ubiquitinated cargo. Eps15 silencing did not affect lysosomal degradation of ubiquitinated ErbB2; however, GFP-FYVE-UbΔGG overexpression inhibited internalization of EGFR and transferrin receptor. CONCLUSIONS: We show for the first time that ubiquitin is sufficient for Eps15 recruitment to endosomes. We speculate that Eps15 recruitment to ubiquitin-rich endosomes may reduce the level of Eps15 at the plasma membrane, slowing endocytosis to allow time for processing of ubiquitinated cargo in endosomes.

Regulation of Ack1 localization and activity by the amino-terminal SAM domain.

BACKGROUND: The mechanisms that regulate the activity of the nonreceptor tyrosine kinase Ack1 (activated Cdc42-associated kinase) are poorly understood. The amino-terminal region of Ack1 is predicted to contain a sterile alpha motif (SAM) domain. SAM domains share a common fold and mediate protein-protein interactions in a wide variety of proteins. Here, we addressed the importance of the Ack1 SAM domain in kinase activity. RESULTS: We used immunofluorescence and Western blotting to show that Ack1 deletion mutants lacking the N-terminus displayed significantly reduced autophosphorylation in cells. A minimal construct comprising the N-terminus and kinase domain (NKD) was autophosphorylated, while the kinase domain alone (KD) was not. When expressed in mammalian cells, NKD localized to the plasma membrane, while KD showed a more diffuse cytosolic localization. Co-immunoprecipitation experiments showed a stronger interaction between full length Ack1 and NKD than between full length Ack1 and KD, indicating that the N-terminus was important for Ack1 dimerization. Increasing the local concentration of purified Ack1 kinase domain at the surface of lipid vesicles stimulated autophosphorylation and catalytic activity, consistent with a requirement for dimerization and trans-phosphorylation for activity. CONCLUSIONS: Collectively, the data suggest that the N-terminus of Ack1 promotes membrane localization and dimerization to allow for autophosphorylation.

Cancer-associated mutations activate the nonreceptor tyrosine kinase Ack1.

Ack1 is a nonreceptor tyrosine kinase that participates in tumorigenesis, cell survival, and migration. Relatively little is known about the mechanisms that regulate Ack1 activity. Recently, four somatic missense mutations of Ack1 were identified in cancer tissue samples, but the effects on Ack1 activity, and function have not been described. These mutations occur in the N-terminal region, the C-lobe of the kinase domain, and the SH3 domain. Here, we show that the cancer-associated mutations increase Ack1 autophosphorylation in mammalian cells without affecting localization and increase Ack1 activity in immune complex kinase assays. The cancer-associated mutations potentiate the ability of Ack1 to promote proliferation and migration, suggesting that point mutation is a mechanism for Ack1 deregulation. We propose that the C-terminal Mig6 homology region (MHR) (residues 802-990) participates in inhibitory intramolecular interactions. The isolated kinase domain of Ack1 interacts directly with the MHR, and the cancer-associated E346K mutation prevents binding. Likewise, mutation of a key hydrophobic residue in the MHR (Phe(820)) prevents the MHR-kinase interaction, activates Ack1, and increases cell migration. Thus, the cancer-associated mutation E346K appears to destabilize an autoinhibited conformation of Ack1, leading to constitutively high Ack1 activity.