The CDKN2A/p16INK4a 5′UTR sequence and translational regulation: impact of novel variants predisposing to melanoma

Many variants of uncertain functional significance in cancer susceptibility genes lie in regulatory regions, and clarifying their association with disease risk poses significant challenges. We studied 17 germline variants (nine of which were novel) in the CDKN2A 5′UTR with independent approaches, which included mono and bicistronic reporter assays, Western blot of endogenous protein, and allelic representation after polysomal profiling to investigate their impact on CDKN2A mRNA translation regulation. Two of the novel variants (c.‐27del23, c.‐93‐91delAGG) were classified as causal mutations (score ≥3), along with the c.‐21C>T, c.‐34G>T, and c.‐56G>T, which had already been studied by a subset of assays. The novel c.‐42T>A as well as the previously described c.‐67G>C were classified as potential mutations (score 1 or 2). The remaining variants (c.‐14C>T, c.‐20A>G, c.‐25C>T+c.‐180G>A, c.‐30G>A, c.‐40C>T, c.‐45G>A, c.‐59C>G, c.‐87T>A, c.‐252A>T) were classified as neutral (score 0). In conclusion, we found evidence that nearly half of the variants found in this region had a negative impact on CDKN2A mRNA translation, supporting the hypothesis that 5′UTR can act as a cellular Internal Ribosome Entry Site (IRES) to modulate p16^INK4a translation.     

THE EFFECT OF BREATHING OXYGEN ON THE METABOLISM OF THE RAT BRAIN UNDER NORMAL AND ISCHAEMIC CONDITIONS

Abstract—

• 1 Breathing oxygen (1 atm.) for 2 hr increased the glycogen content of the rat brain from 3·38 to 4·35 μmoles glucosyl residues/g wet wt. At the same time the glucose and lactate concentrations were significantly decreased.
• 2 Both under normal conditions and when breathing oxygen, the sum (glycogen + glucose) × 2 + lactate, with which the balance of carbohydrate breakdown and lactate formation was assessed, was 13·5 μmoles/g wet wt.
• 2 Oxygen breathing effected a significant decrease in this sum after an ischaemic period of 1–15 min. In the control group breathing normal air, the sum, after all periods of ischaemia, ranged from 98 to 106 per cent of the starting value.
• 3 An increased partial pressure of oxygen did not change the breakdown rate of the high-energy phosphate compounds. This result was not consistent with an oxidation of the carbohydrates which were missing in the balance. It is probable that other metabolites, which were not tested for, accumulated.
• 5 0 We failed to find any indication of storage of oxygen which the ischaemic brain could use for oxidative energy production.

     

XRCC1 deficiency influences the cytotoxicity and the genomic instability induced by Me-lex,a specific inducer of N3-methyladenine

Me-lex is a sequence-specific alkylating agent synthesized to preferentially (>90%) generate N3-methyladenine (3-mA) in the minor groove of double-strand DNA, in A-T rich regions. In this paper we investigated the effect of XRCC1 deficiency in the processing of 3-mA adducts generated by Me-lex, through the molecular analysis of the Hprt mutations and the evaluation of cytogenetic end points such as sister chromatid exchanges (SCEs), micronuclei (MN) and nucleus fragmentation. EM-C11 cells, deficient in XRCC1 activity, showed a 2.5-fold higher sensitivity to the toxicity of Me-lex compared to the DNA repair proficient parental CHO-9 cells, but were not hyper mutable. The spontaneous mutation spectrum at the Hprt locus generated in EM-C11 cells revealed a high percentage of genomic deletions. After Me-lex treatment, the percentage of genomic deletions did not increase, but a class of mutations which appeared to target regulatory regions of the gene significantly increased (p = 0.0277), suggesting that non-coding Hprt genomic sequences represent a strong target for the rare mutations induced by Me-lex. The number of SCEs per chromosome increased 3-fold above background in 50 μМ Me-lex treated CHO-9 cells, while at higher Me-lex concentrations a sharp increase in the percentage of MN and fragmented nuclei was observed. In EM-C11 cells the background level of SCEs (0.939 ± 0.182) was approximately 10-fold higher than in CHO-9 (0.129 ± 0.027) and higher levels of multinucleated cells and MN were also found. In EM-C11, even low doses of Me-lex (25 μM) led to a significant increase in genomic damage. These results indicate that XRCC1 deficiency can lead to genomic instability even in the absence of an exogenous genotoxic insult and low levels of Me-lex-induced lesions, i.e., 3-mA and/or a BER intermediate, can exacerbate this instability.     

Exclusive Decoration of Simian Immunodeficiency Virus Env with High-Mannose Type N-Glycans Is Not Compatible with Mucosal Transmission in Rhesus Macaques

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelope (Env) proteins are extensively decorated with N-glycans, predominantly of the high-mannose type. However, it is unclear how high-mannose N-glycans on Env impact viral spread. We show that exclusive modification of SIV Env with these N-glycans reduces viral infectivity and abrogates mucosal transmission, despite increasing viral capture by immune cell lectins. Thus, high-mannose N-glycans have opposed effects on SIV infectivity and lectin reactivity, and a balance might be required for efficient mucosal transmission.     

Multiple blood feeding in mosquitoes shortens the Plasmodium falciparum incubation period and increases malaria transmission potential

Many mosquito species, including the major malaria vector Anopheles gambiae, naturally undergo multiple reproductive cycles of blood feeding, egg development and egg laying in their lifespan. Such complex mosquito behavior is regularly overlooked when mosquitoes are experimentally infected with malaria parasites, limiting our ability to accurately describe potential effects on transmission. Here, we examine how Plasmodium falciparum development and transmission potential is impacted when infected mosquitoes feed an additional time. We measured P. falciparum oocyst size and performed sporozoite time course analyses to determine the parasite’s extrinsic incubation period (EIP), i.e. the time required by parasites to reach infectious sporozoite stages, in An. gambiae females blood fed either once or twice. An additional blood feed at 3 days post infection drastically accelerates oocyst growth rates, causing earlier sporozoite accumulation in the salivary glands, thereby shortening the EIP (reduction of 2.3 ± 0.4 days). Moreover, parasite growth is further accelerated in transgenic mosquitoes with reduced reproductive capacity, which mimic genetic modifications currently proposed in population suppression gene drives. We incorporate our shortened EIP values into a measure of transmission potential, the basic reproduction number R₀, and find the average R₀ is higher (range: 10.1%–12.1% increase) across sub-Saharan Africa than when using traditional EIP measurements. These data suggest that malaria elimination may be substantially more challenging and that younger mosquitoes or those with reduced reproductive ability may provide a larger contribution to infection than currently believed. Our findings have profound implications for current and future mosquito control interventions.     

Populations of a widespread hexacoral have trophic plasticity and flexible syntrophic interactions across the Indo-Pacific Ocean

Coral Reefs - Benthic cnidarians are suspension feeders that ingest items ranging from particulate organic matter to macrozooplankton. Additionally, many species receive photosynthetic products...     

Development of the red algal parasite Vertebrata aterrimophila sp. nov. (Rhodomelaceae,Ceramiales) from New Zealand

Parasitic red algae grow only on other red algae and have over 120 described species. Developmental studies in red algal parasites are few, although they have shown that secondary pit connections formed between parasite and host and proposed that this was an important process in successful parasitism. Furthermore, it was recorded that the transfer of parasite nuclei by these secondary pit connections led to different host cell effects. We used developmental studies to reconstruct early stages and any host cell effects of a parasite on Vertebrata aterrima. A mitochondrial marker (cox1) and morphological observations (light and fluorescence microscopy) were used to describe this new red algal parasite as Vertebrata aterrimophila sp. nov. Early developmental stages show that a parasite spore connects via secondary pit connections with a pericentral host cell after cuticle penetration. Developmental observations revealed a unique connection cell that grows into a ‘trunk-like’ structure. Host cell transformation after infection by the parasite included apparent increases in both carbohydrate concentrations and nuclear size, as well as structural changes. Analyses of molecular phylogenies and reproductive structures indicated that the closest relative of V. aterrimophila is its host, V. aterrima. Our study shows a novel developmental parasite stage (‘trunk-like’ cell) and highlights the need for further developmental studies to investigate the range of developmental patterns and host effects in parasitic red algae.