The +4G Site in Kozak Consensus Is Not Related to the Efficiency of Translation Initiation

The optimal context for translation initiation in mammalian species is GCCRCCaugG (where R = purine and “aug” is the initiation codon), with the -3R and +4G being particularly important. The presence of +4G has been interpreted as necessary for efficient translation initiation. Accumulated experimental and bioinformatic evidence has suggested an alternative explanation based on amino acid constraint on the second codon, i.e., amino acid Ala or Gly are needed as the second amino acid in the nascent peptide for the cleavage of the initiator Met, and the consequent overuse of Ala and Gly codons (GCN and GGN) leads to the +4G consensus. I performed a critical test of these alternative hypotheses on +4G based on 34169 human protein-coding genes and published gene expression data. The result shows that the prevalence of +4G is not related to translation initiation. Among the five G-starting codons, only alanine codons (GCN), and glycine codons (GGN) to a much smaller extent, are overrepresented at the second codon, whereas the other three codons are not overrepresented. While highly expressed genes have more +4G than lowly expressed genes, the difference is caused by GCN and GGN codons at the second codon. These results are inconsistent with +4G being needed for efficient translation initiation, but consistent with the proposal of amino acid constraint hypothesis

Geometry-controlled kinetics

It has long been appreciated that transport properties can control reaction kinetics. This effect can be characterized by the time it takes a diffusing molecule to reach a target -- the first-passage time (FPT). Although essential to quantify the kinetics of reactions on all time scales, determining the FPT distribution was deemed so far intractable. Here, we calculate analytically this FPT distribution and show that transport processes as various as regular diffusion, anomalous diffusion, diffusion in disordered media and in fractals fall into the same universality classes. Beyond this theoretical aspect, this result changes the views on standard reaction kinetics. More precisely, we argue that geometry can become a key parameter so far ignored in this context, and introduce the concept of "geometry-controlled kinetics". These findings could help understand the crucial role of spatial organization of genes in transcription kinetics, and more generally the impact of geometry on diffusion-limited reactions.Comment: Submitted versio

Feasibility of Image-Guided Radiotherapy for Elderly Patients with Locally Advanced Rectal Cancer

PURPOSE: The study aims to assess the tolerance of elderly patients (70 years or older) with locally advanced rectal cancers to image-guided radiotherapy (IGRT). A retrospective review of 13 elderly patients with locally advanced rectal cancer who underwent preoperative chemoradiation using IGRT was performed. Grade 3-4 acute toxicities, survival, and long-term complications were compared to 17 younger patients (<70 years) with the same disease stage. RESULTS: Grade 3-4 hematologic toxicities occurred in 7.6% and 0% (p = 0.4) and gastrointestinal toxicities, and, in 15.2% and 5% (p = 0.5), of elderly and younger patients, respectively. Surgery was aborted in three patients, two in the elderly group and one in the younger group. One patient in the elderly group died after surgery from cardiac arrhythmia. After a median follow-up of 34 months, five patients had died, two in the elderly and three in the younger group. The 3-year survival was 90.9% and 87.5% (p = 0.7) for the elderly and younger group respectively. Two patients in the younger group developed ischemic colitis and fecal incontinence. There was no statistically significant difference in acute and late toxicities as well as survival between the two groups. CONCLUSIONS AND CLINICAL RELEVANCE: Elderly patients with locally advanced rectal cancers may tolerate preoperative chemoradiation with IGRT as well as younger patients. Further prospective studies should be performed to investigate the potential of IGRT for possible cure in elderly patients with locally advanced rectal cancer

Design and Pre-Clinical Evaluation of a Universal HIV-1 Vaccine

BACKGROUND: One of the big roadblocks in development of HIV-1/AIDS vaccines is the enormous diversity of HIV-1, which could limit the value of any HIV-1 vaccine candidate currently under test. METHODOLOGY AND FINDINGS: To address the HIV-1 variation, we designed a novel T cell immunogen, designated HIV(CONSV), by assembling the 14 most conserved regions of the HIV-1 proteome into one chimaeric protein. Each segment is a consensus sequence from one of the four major HIV-1 clades A, B, C and D, which alternate to ensure equal clade coverage. The gene coding for the HIV(CONSV) protein was inserted into the three most studied vaccine vectors, plasmid DNA, human adenovirus serotype 5 and modified vaccine virus Ankara (MVA), and induced HIV-1-specific T cell responses in mice. We also demonstrated that these conserved regions prime CD8(+) and CD4(+) T cell to highly conserved epitopes in humans and that these epitopes, although usually subdominant, generate memory T cells in patients during natural HIV-1 infection. SIGNIFICANCE: Therefore, this vaccine approach provides an attractive and testable alternative for overcoming the HIV-1 variability, while focusing T cell responses on regions of the virus that are less likely to mutate and escape. Furthermore, this approach has merit in the simplicity of design and delivery, requiring only a single immunogen to provide extensive coverage of global HIV-1 population diversity

Preclinical and post-treatment changes in the HCC-associated serum proteome

SELDI-based proteomic profiling of body fluids is currently in widespread use for cancer biomarker discovery. We have successfully used this technology for the diagnosis of hepatocellular carcinoma (HCC) in hepatitis C patients and now report its application to serial serum samples from 37 hepatitis C patients before development of HCC, with HCC and following radiofrequency ablation of the tumour. As with alpha-fetoprotein, an accepted biomarker for HCC, we hypothesised that HCC-associated proteomic features would ‘return to normal' following successful treatment and the primary aim of our study was to test this hypothesis. Several SELDI peaks that changed significantly during HCC development were detected but they did not reverse following treatment. These data may be interpreted to suggest that the characteristic SELDI profile is not linearly related to tumour burden but may result from the progression of underlying liver disease or from the emergence of precancerous lesions. β2-Microglobulin, a protein previously reported to be markedly elevated in patients with HCV related HCC, was also the most significantly HCC associated proteomic feature (m/z 11720) in this study

Identification of serum biomarkers for colon cancer by proteomic analysis

Colorectal cancer (CRC) is often diagnosed at a late stage with concomitant poor prognosis. Early detection greatly improves prognosis; however, the invasive, unpleasant and inconvenient nature of current diagnostic procedures limits their applicability. No serum-based test is currently of sufficient sensitivity or specificity for widespread use. In the best currently available blood test, carcinoembryonic antigen exhibits low sensitivity and specificity particularly in the setting of early disease. Hence, there is great need for new biomarkers for early detection of CRC. We have used surface-enhanced laser desorbtion/ionisation (SELDI) to investigate the serum proteome of 62 CRC patients and 31 noncancer subjects. We have identified proteins (complement C3a des-arg, α1-antitrypsin and transferrin) with diagnostic potential. Artificial neural networks trained using only the intensities of the SELDI peaks corresponding to identified proteins were able to classify the patients used in this study with 95% sensitivity and 91% specificity

The cellular source for APOBEC3G's incorporation into HIV-1

<p>Abstract</p> <p>Background</p> <p>Human APOBEC3G (hA3G) has been identified as a cellular inhibitor of HIV-1 infectivity. Viral incorporation of hA3G is an essential step for its antiviral activity. Although the mechanism underlying hA3G virion encapsidation has been investigated extensively, the cellular source of viral hA3G remains unclear.</p> <p>Results</p> <p>Previous studies have shown that hA3G forms low-molecular-mass (LMM) and high-molecular-mass (HMM) complexes. Our work herein provides evidence that the majority of newly-synthesized hA3G interacts with membrane lipid raft domains to form Lipid raft-associated hA3G (RA hA3G), which serve as the precursor of the mature HMM hA3G complex, while a minority of newly-synthesized hA3G remains in the cytoplasm as a soluble LMM form. The distribution of hA3G among the soluble LMM form, the RA LMM form and the mature forms of HMM is regulated by a mechanism involving the N-terminal part of the linker region and the C-terminus of hA3G. Mutagenesis studies reveal a direct correlation between the ability of hA3G to form the RA LMM complex and its viral incorporation.</p> <p>Conclusions</p> <p>Together these data suggest that the Lipid raft-associated LMM A3G complex functions as the cellular source of viral hA3G.</p

A Biobrick Library for Cloning Custom Eukaryotic Plasmids

Researchers often require customised variations of plasmids that are not commercially available. Here we demonstrate the applicability and versatility of standard synthetic biological parts (biobricks) to build custom plasmids. For this purpose we have built a collection of 52 parts that include multiple cloning sites (MCS) and common protein tags, protein reporters and selection markers, amongst others. Importantly, most of the parts are designed in a format to allow fusions that maintain the reading frame. We illustrate the collection by building several model contructs, including concatemers of protein binding-site motifs, and a variety of plasmids for eukaryotic stable cloning and chromosomal insertion. For example, in 3 biobrick iterations, we make a cerulean-reporter plasmid for cloning fluorescent protein fusions. Furthermore, we use the collection to implement a recombinase-mediated DNA insertion (RMDI), allowing chromosomal site-directed exchange of genes. By making one recipient stable cell line, many standardised cell lines can subsequently be generated, by fluorescent fusion-gene exchange. We propose that this biobrick collection may be distributed peer-to-peer as a stand-alone library, in addition to its distribution through the Registry of Standard Biological Parts (http://partsregistry.org/)

Immunogenic Comparison of Chimeric Adenovirus 5/35 Vector Carrying Optimized Human Immunodeficiency Virus Clade C Genes and Various Promoters

Adenovirus vector-based vaccine is a promising approach to protect HIV infection. However, a recent phase IIb clinical trial using the vector did not show its protective efficacy against HIV infection. To improve the vaccine, we explored the transgene protein expression and its immunogenicity using optimized codon usage, promoters and adaptors. We compared protein expression and immunogenicity of adenovirus vector vaccines carrying native or codon usage-optimized HIV-1 clade C gag and env genes expression cassettes driven by different promoters (CMV, CMVi, and CA promoters) and adapters (IRES and F2A). The adenovirus vector vaccine containing optimized gag gene produced higher Gag protein expression and induced higher immune responses than the vector containing native gag gene in mice. Furthermore, CA promoter generated higher transgene expression and elicited higher immune responses than other two popularly used promoters (CMV and CMVi). The second gene expression using F2A adaptor resulted in higher protein expression and immunity than that of using IRES and direct fusion protein. Taken together, the adenovirus vector containing the expression cassette with CA promoter, optimized HIV-1 clade C gene and an F2A adaptor produced the best protein expression and elicited the highest transgene-specific immune responses. This finding would be promising for vaccine design and gene therapy

Seizures in 204 comatose children: incidence and outcome

Purpose: Seizures are common in comatose children, but may be clinically subtle or only manifest on continuous electroencephalographic monitoring (cEEG); any association with outcome remains uncertain. Methods: cEEG (one to three channels) was performed for a median 42 h (range 2–630 h) in 204 unventilated and ventilated children aged $\leq$ 15 years (18 neonates, 61 infants) in coma with different aetiologies. Outcome at 1 month was independently determined and dichotomized for survivors into favourable (normal or moderate neurological handicap) and unfavourable (severe handicap or vegetative state). Results: Of the 204 patients, 110 had clinical seizures (CS) before cEEG commenced. During cEEG, 74 patients (36 %, 95 % confidence interval, 95 % CI, 32–41 %) had electroencephalographic seizures (ES), the majority without clinical accompaniment (non-convulsive seizures, NCS). CS occurred before NCS in 69 of the 204 patients; 5 ventilated with NCS had no CS observed. Death (93/204; 46 %) was independently predicted by admission Paediatric Index of Mortality (PIM; adjusted odds ratio, aOR, 1.027, 95 % CI 1.012–1.042; p 3 % fast, aOR 5.43, 95 % CI 1.90–15.6; excess slow with <3 % fast, aOR 8.71, 95 % CI 2.58–29.4; low amplitude, 10th centile <9 $\mu$V, aOR 3.78, 95 % CI 1.23–11.7; and burst suppression, aOR 10.68, 95 % CI 2.31–49.4) compared with normal cEEG, as well as absence of CS at any time (aOR 2.38, 95 % CI 1.18–4.81). Unfavourable outcome (29/111 survivors; 26 %) was independently predicted by the presence of ES (aOR 15.4, 95 % CI 4.7–49.7) and PIM (aOR 1.036, 95 % CI 1.013–1.059). Conclusion: Seizures are common in comatose children, and are associated with an unfavourable outcome in survivors. cEEG allows the detection of subtle CS and NCS and is a prognostic tool