Criteria for effective design, construction, and gene knockdown by shRNA vectors

BACKGROUND: RNA interference (RNAi) technology is a powerful methodology recently developed for the specific knockdown of targeted genes. RNAi is most commonly achieved either transiently by transfection of small interfering (si) RNA oligonucleotides, or stably using short hairpin (sh) RNA expressed from a DNA vector or virus. Much controversy has surrounded the development of rules for the design of effective siRNA oligonucleotides; and whether these rules apply to shRNA is not well characterized. RESULTS: To determine whether published algorithms for siRNA oligonucleotide design apply to shRNA, we constructed 27 shRNAs from 11 human genes expressed stably using retroviral vectors. We demonstrate an efficient method for preparing wild-type and mutant control shRNA vectors simultaneously using oligonucleotide hybrids. We show that sequencing through shRNA vectors can be problematic due to the intrinsic secondary structure of the hairpin, and we determine a strategy for effective sequencing by using a combination of modified BigDye chemistries and DNA relaxing agents. The efficacy of knockdown for the 27 shRNA vectors was evaluated against six published algorithms for siRNA oligonucleotide design. Our results show that none of the scoring algorithms can explain a significant percentage of variance in shRNA knockdown efficacy as assessed by linear regression analysis or ROC curve analysis. Application of a modification based on the stability of the 6 central bases of each shRNA provides fair-to-good predictions of knockdown efficacy for three of the algorithms. Analysis of an independent set of data from 38 shRNAs pooled from previous publications confirms these findings. CONCLUSION: The use of mixed oligonucleotide pairs provides a time and cost efficient method of producing wild type and mutant control shRNA vectors. The addition to sequencing reactions of a combination of mixed dITP/dGTP chemistries and DNA relaxing agents enables read through the intrinsic secondary structure of problematic shRNA vectors. Six published algorithms for siRNA oligonucleotide design that were tested in this study show little or no efficacy at predicting shRNA knockdown outcome. However, application of a modification based on the central shRNA stability should provide a useful improvement to the design of effective shRNA vectors

Modulational instability of solitary waves in non-degenerate three-wave mixing: The role of phase symmetries

We show how the analytical approach of Zakharov and Rubenchik [Sov. Phys. JETP {\bf 38}, 494 (1974)] to modulational instability (MI) of solitary waves in the nonlinear Schr\"oedinger equation (NLS) can be generalised for models with two phase symmetries. MI of three-wave parametric spatial solitons due to group velocity dispersion (GVD) is investigated as a typical example of such models. We reveal a new branch of neck instability, which dominates the usual snake type MI found for normal GVD. The resultant nonlinear evolution is thereby qualitatively different from cases with only a single phase symmetry.Comment: 4 pages with figure

N-band Observations of He 2-10: Unveiling the Dusty Engine of a Starburst Galaxy

To better understand the early stages of massive star cluster evolution we have obtained J,H,K', and N (10.8 micron) images of the nuclear region of the starburst galaxy He 2-10. The N-band images were obtained with the Gemini North telescope and reveal four of the five enshrouded clusters, or "ultradense HII regions" (UDHIIs), recently discovered in radio maps. None of these sources appears in either the optical HST images or the near-infrared images. They comprise about 60% of the total N-band flux from He 2-10 and, we suspect, a similar fraction of the total far infrared flux measured by IRAS. The inferred spectra of the UDHIIs are very similar to those of Galactic ultracompact HII regions and we have modeled the UDHIIs under the assumption that they are "scaled-up" versions of these objects. The bolometric luminosity of the brightest UDHII is estimated to be ~2x10^9 L_sun, and the total mass of the dust and gas is ~10^7 M_sun. The mass of the enshrouded stellar cluster must be > 2.5x10^6 M_sun and the age must be < 4.8x10^6 yr. All of the stellar clusters within the UDHIIs must have ages less than about 5x10^6 yr and masses greater than about 5x10^5 M_sun. The logarithmic ratios of the radio to far infrared flux densities for the individual UDHIIs, and He 2-10 as a whole, are significantly larger than the average for normal galaxies, but comparable to those for ultraluminous infrared galaxies. Large ratios for some starburst galaxies may indicate that a significant fraction of the far infrared flux arises from UDHIIs. If all of the far infrared flux from He 2-10 and other starbursts is produced by heavily obscured regions, the observed correlation between UV continuum slope and infrared-to-ultraviolet flux ratio in starbursts cannot be due primarily to UV reprocessing by dust in a foreground screen.Comment: 41 pages, 9 figure

Hierarchical Spherical Model from a Geometric Point of View

A continuous version of the hierarchical spherical model at dimension d=4 is investigated. Two limit distribution of the block spin variable X^{\gamma}, normalized with exponents \gamma =d+2 and \gamma =d at and above the critical temperature, are established. These results are proven by solving certain evolution equations corresponding to the renormalization group (RG) transformation of the O(N) hierarchical spin model of block size L^{d} in the limit L to 1 and N to \infty . Starting far away from the stationary Gaussian fixed point the trajectories of these dynamical system pass through two different regimes with distinguishable crossover behavior. An interpretation of this trajectories is given by the geometric theory of functions which describe precisely the motion of the Lee--Yang zeroes. The large--$N$ limit of RG transformation with L^{d} fixed equal to 2, at the criticality, has recently been investigated in both weak and strong (coupling) regimes by Watanabe \cite{W}. Although our analysis deals only with N=\infty case, it complements various aspects of that work.Comment: 27 pages, 6 figures, submitted to Journ. Stat. Phy

Schedule-selective biochemical modulation of 5-fluorouracil in advanced colorectal cancer – a phase II study

BACKGROUND: 5-fluorouracil remains the standard therapy for patients with advanced/metastatic colorectal cancer. Pre-clinical studies have demonstrated the biological modulation of 5-fluorouracil by methotrexate and leucovorin. This phase II study was initiated to determine the activity and toxicity of sequential methotrexate – leucovorin and 5-fluorouracil chemotherapy in patients with advanced colorectal cancer. METHODS: Ninety-seven patients with metastatic colorectal cancer were enrolled onto the study. Methotrexate – 30 mg/m(2) was administered every 6 hours for 6 doses followed by a 2 hour infusion of LV – 500 mg/m(2). Midway through the leucovorin infusion, patients received 5-fluorouracil – 600 mg/m(2). This constituted a cycle of therapy and was repeated every 2 weeks until progression. RESULTS: The median age was 64 yrs (34–84) and the Eastern Cooperative Group Oncology performance score was 0 in 37%, 1 in 55% and 2 in 8% of patients. Partial and complete responses were seen in 31% of patients with a median duration of response of 6.4 months. The overall median survival was 13.0 months. The estimated 1-year survival was 53.7%. Grade III and IV toxic effects were modest and included mucositis, nausea and vomiting. CONCLUSIONS: This phase II study supports previously reported data demonstrating the modest clinical benefit of 5-FU modulation utilizing methotrexate and leucovorin in patients with metastatic colorectal cancer. Ongoing studies evaluating 5-fluorouracil modulation with more novel agents (Irinotecan and/or oxaliplatin) are in progress and may prove encouraging

Carcinogenicity of cobalt, antimony compounds, and weapons-grade tungsten alloy

The complete evaluation of the carcinogenicity of cobalt, antimony compounds, and weapons-grade tungsten alloy will be published in Volume 131 of the IARC Monographs.[Excerpt] In March, 2022, a Working Group of 31 scientists from 13 countries met remotely at the invitation of the International Agency for Research on Cancer (IARC) to finalise their evaluation of the carcinogenicity of nine agents: cobalt metal (without tungsten carbide or other metal alloys), soluble cobalt(II) salts, cobalt(II) oxide, cobalt(II,III) oxide, cobalt(II) sulfide, other cobalt(II) compounds, trivalent antimony, pentavalent antimony, and weapons-grade tungsten (with nickel and cobalt) alloy. For cobalt metal and the cobalt compounds, particles of all sizes were included in the evaluation. These assessments will be published in Volume 131 of the IARC Monographs.1 Cobalt metal and soluble cobalt(II) salts were classified as “probably carcinogenic to humans” (Group 2A) based on “sufficient” evidence for cancer in experimental animals and “strong” mechanistic evidence in human primary cells. Cobalt(II) oxide and weapons-grade tungsten alloy were classified as “possibly carcinogenic to humans” (Group 2B) based on “sufficient” evidence in experimental animals. Trivalent antimony was classified as “probably carcinogenic to humans” (Group 2A), based on “limited” evidence for cancer in humans, “sufficient” evidence for cancer in experimental animals, and “strong” mechanistic evidence in human primary cells and in experimental systems. Cobalt(II,III) oxide, cobalt(II) sulfide, other cobalt(II) compounds, and pentavalent antimony were each evaluated as “not classifiable as to its carcinogenicity to humans” (Group 3).[...