An Inducible,Isogenic Cancer Cell Line System for Targeting the State of Mismatch Repair Deficiency

The DNA mismatch repair system (MMR) maintains genome stability through recognition and repair of single-base mismatches and small insertion-deletion loops. Inactivation of the MMR pathway causes microsatellite instability and the accumulation of genomic mutations that can cause or contribute to cancer. In fact, 10-20% of certain solid and hematologic cancers are MMR-deficient. MMR-deficient cancers do not respond to some standard of care chemotherapeutics because of presumed increased tolerance of DNA damage, highlighting the need for novel therapeutic drugs. Toward this goal, we generated isogenic cancer cell lines for direct comparison of MMR-proficient and MMR-deficient cells. We engineered NCI-H23 lung adenocarcinoma cells to contain a doxycycline-inducible shRNA designed to suppress the expression of the mismatch repair gene MLH1, and compared single cell subclones that were uninduced (MLH1-proficient) versus induced for the MLH1 shRNA (MLH1-deficient). Here we present the characterization of these MMR-inducible cell lines and validate a novel class of rhodium metalloinsertor compounds that differentially inhibit the proliferation of MMR-deficient cancer cells.     

Prevalence of HIV,STIs, and Risk Behaviors in a Cross-Sectional Community- and Clinic-Based Sample of Men Who Have Sex with Men (MSM) in Lima,Peru

Background

Further research is necessary to understand the factors contributing to the high prevalence of HIV/STIs among men who have sex with men (MSM) in Peru. We compared HIV/STI prevalence and risk factors between two non-probability samples of MSM, one passively enrolled from an STI clinic and the other actively enrolled from community venues surrounding the clinic in Lima, Peru.

Methods

A total of 560 self-identified MSM were enrolled between May-December, 2007. 438 subjects enrolled from a municipal STI clinic and 122 subjects enrolled during community outreach visits. All participants underwent screening for HIV, syphilis, HSV-2, gonorrhoea, and chlamydia and completed a survey assessing their history of HIV/STIs, prior HIV testing, and sexual behavior.

Results

HIV prevalence was significantly higher among MSM enrolled from the clinic, with previously undiagnosed HIV identified in 9.1% compared with 2.6% of community participants. 15.4 % of all MSM screened were infected with ≥1 curable STI, 7.4% with early syphilis (RPR≥1∶16) and 5.5% with urethral gonorrhoea and/or chlamydia. No significant differences between populations were reported in prevalence of STIs, number of male sex partners, history of unprotected anal intercourse, or alcohol and/or drug use prior to sex. Exchange of sex for money or goods was reported by 33.5% of MSM enrolled from the clinic and 21.2% of MSM from the community (p = 0.01).

Conclusions

Our data demonstrate that the prevalence of HIV and STIs, including syphilis, gonorrhoea, and chlamydia are extremely high among MSM enrolled from both clinic and community venues in urban Peru. New strategies are needed to address differences in HIV/STI epidemiology between clinic- and community-enrolled samples of MSM.     

Copper promotes the migration of bone marrow mesenchymal stem cells via Rnd3-dependent cytoskeleton remodeling

The motility of mesenchymal stem cells (MSCs) is highly related to their homing in vivo, a critical issue in regenerative medicine. Our previous study indicated copper (Cu) might promote the recruitment of endogenous MSCs in canine esophagus defect model. In this study, we investigated the effect of Cu on the motility of bone marrow mesenchymal stem cells (BMSCs) and the underlying mechanism in vitro. Cu supplementation could enhance the motility of BMSCs, and upregulate the expression of hypoxia-inducible factor 1α (Hif1α) at the protein level, and upregulate the expression of rho family GTPase 3 (Rnd3) at messenger RNA and protein level. When Hif1α was silenced by small interfering RNA (siRNA), Cu-induced Rnd3 upregulation was blocked. When Rnd3 was silenced by siRNA, the motility of BMSCs was decreased with or without Cu supplementation, and Cu-induced cytoskeleton remodeling was neutralized. Furthermore, overexpression of Rnd3 also increased the motility of BMSCs and induced cytoskeleton remodeling. Overall, our results demonstrated that Cu enhanced BMSCs migration through, at least in part, cytoskeleton remodeling via Hif1α-dependent upregulation of Rnd3. This study provided an insight into the mechanism of the effect of Cu on the motility of BMSCs, and a theoretical foundation of applying Cu to improve the recruitment of BMSCs in tissue engineering and cytotherapy.     

Activation of Extracellular Signal-Regulated Kinase but Not of p38 Mitogen-Activated Protein Kinase Pathways in Lymphocytes Requires Allosteric Activation of SOS

Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cγ (PLCγ)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation.     

A Lipoprotein Receptor Cluster IV Mutant Preferentially Binds Amyloid-β and Regulates Its Clearance from the Mouse Brain

Soluble low density lipoprotein receptor-related protein-1 (sLRP1) binds ∼70% of amyloid β-peptide (Aβ) in human plasma. In Alzheimer disease (AD) and individuals with mild cognitive impairment converting to AD, plasma sLRP1 levels are reduced and sLRP1 is oxidized, which results in diminished Aβ peripheral binding and higher levels of free Aβ in plasma. Experimental studies have shown that free circulating Aβ re-enters the brain and that sLRP1 and/or its recombinant wild type cluster IV (WT-LRPIV) prevent Aβ from entering the brain. Treatment of Alzheimer APPsw^+/0 mice with WT-LRPIV has been shown to reduce brain Aβ pathology. In addition to Aβ, LRPIV binds multiple ligands. To enhance LRPIV binding for Aβ relative to other LRP1 ligands, we generated a library of LRPIV-derived fragments and full-length LRPIV variants with glycine replacing aspartic acid residues 3394, 3556, and 3674 in the calcium binding sites. Compared with WT-LRPIV, a lead LRPIV-D3674G mutant had 1.6- and 2.7-fold higher binding affinity for Aβ40 and Aβ42 in vitro, respectively, and a lower binding affinity for other LRP1 ligands (e.g. apolipoprotein E2, E3, and E4 (1.3–1.8-fold), tissue plasminogen activator (2.7-fold), matrix metalloproteinase-9 (4.1-fold), and Factor Xa (3.8-fold)). LRPIV-D3674G cleared mouse endogenous brain Aβ40 and Aβ42 25–27% better than WT-LRPIV. A 3-month subcutaneous treatment of APPsw^+/0 mice with LRPIV-D3674G (40 μg/kg/day) reduced Aβ40 and Αβ42 levels in the hippocampus, cortex, and cerebrospinal fluid by 60–80% and improved cerebral blood flow responses and hippocampal function at 9 months of age. Thus, LRPIV-D3674G is an efficient new Aβ clearance therapy.     

Neuraminidase-Inhibiting Antibody Is a Correlate of Cross-Protection against Lethal H5N1 Influenza Virus in Ferrets Immunized with Seasonal Influenza Vaccine

In preparing for the threat of a pandemic of avian H5N1 influenza virus, we need to consider the significant delay (4 to 6 months) necessary to produce a strain-matched vaccine. As some degree of cross-reactivity between seasonal influenza vaccines and H5N1 virus has been reported, this was further explored in the ferret model to determine the targets of protective immunity. Ferrets were vaccinated with two intramuscular inoculations of trivalent inactivated split influenza vaccine or subcomponent vaccines, with and without adjuvant, and later challenged with a lethal dose of A/Vietnam/1203/2004 (H5N1) influenza virus. We confirmed that vaccination with seasonal influenza vaccine afforded partial protection against lethal H5N1 challenge and showed that use of either AlPO₄ or Iscomatrix adjuvant with the vaccine resulted in complete protection against disease and death. The protection was due exclusively to the H1N1 vaccine component, and although the hemagglutinin contributed to protection, the dominant protective response was targeted toward the neuraminidase (NA) and correlated with sialic acid cleavage-inhibiting antibody titers. Purified heterologous NA formulated with Iscomatrix adjuvant was also protective. These results suggest that adjuvanted seasonal trivalent vaccine could be used as an interim measure to decrease morbidity and mortality from H5N1 prior to the availability of a specific vaccine. The data also highlight that an inducer of cross-protective immunity is the NA, a protein whose levels are not normally monitored in vaccines and whose capacity to induce immunity in recipients is not normally assessed.     

Simultaneous transfer, introgression, and genomic localization of genes for resistance to stem rust race TTKSK (Ug99) from Aegilops tauschii to wheat

Wheat production is currently threatened by widely virulent races of the wheat stem rust fungus, Puccinia graminis f. sp. tritici, that are part of the TTKSK (also known as ‘Ug99’) race group. The diploid D genome donor species Aegilops tauschii (2n = 2x = 14, DD) is a readily accessible source of resistance to TTKSK and its derivatives that can be transferred to hexaploid wheat, Triticum aestivum (2n = 6x = 42, AABBDD). To expedite transfer of TTKSK resistance from Ae. tauschii, a direct hybridization approach was undertaken that integrates gene transfer, mapping, and introgression into one process. Direct crossing of Ae. tauschii accessions with an elite wheat breeding line combines the steps of gene transfer and introgression while development of mapping populations during gene transfer enables the identification of closely linked markers. Direct crosses were made using TTKSK-resistant Ae. tauschii accessions TA1662 and PI 603225 as males and a stem rust-susceptible T. aestivum breeding line, KS05HW14, as a female. Embryo rescue enabled recovery of F₁ (ABDD) plants that were backcrossed as females to the hexaploid recurrent parent. Stem rust-resistant BC₁F₁ plants from each Ae. tauschii donor source were used as males to generate BC₂F₁ mapping populations. Bulked segregant analysis of BC₂F₁ genotypes was performed using 70 SSR loci distributed across the D genome. Using this approach, stem rust resistance genes from both accessions were located on chromosome arm 1DS and mapped using SSR and EST-STS markers. An allelism test indicated the stem rust resistance gene transferred from PI 603225 is Sr33. Race specificity suggests the stem rust resistance gene transferred from TA1662 is unique and this gene has been temporarily designated SrTA1662. Stem rust resistance genes derived from TA1662 and PI 603225 have been made available with selectable molecular markers in genetic backgrounds suitable for stem rust resistance breeding.     

Sensitivity of continental United States atmospheric budgets of oxidized and reduced nitrogen to dry deposition parametrizations

Reactive nitrogen (N_r) is removed by surface fluxes (air–surface exchange) and wet deposition. The chemistry and physics of the atmosphere result in a complicated system in which competing chemical sources and sinks exist and impact that removal. Therefore, uncertainties are best examined with complete regional chemical transport models that simulate these feedbacks. We analysed several uncertainties in regional air quality model resistance analogue representations of air–surface exchange for unidirectional and bi-directional fluxes and their effect on the continental N_r budget. Model sensitivity tests of key parameters in dry deposition formulations showed that uncertainty estimates of continental total nitrogen deposition are surprisingly small, 5 per cent or less, owing to feedbacks in the chemistry and rebalancing among removal pathways. The largest uncertainties (5%) occur with the change from a unidirectional to a bi-directional NH₃ formulation followed by uncertainties in bi-directional compensation points (1–4%) and unidirectional aerodynamic resistance (2%). Uncertainties have a greater effect at the local scale. Between unidirectional and bi-directional formulations, single grid cell changes can be up to 50 per cent, whereas 84 per cent of the cells have changes less than 30 per cent. For uncertainties within either formulation, single grid cell change can be up to 20 per cent, but for 90 per cent of the cells changes are less than 10 per cent.