Identification of proteins interacting with the human mismatch repair protein MLH1

Loss of expression of the human DNA mismatch repair (MMR) gene, hMLH1, is seen in a number of tumour cell lines resistant to a variety of cytotoxic drugs. The aim of this study was to identify other proteins that interact with hMLH1 to attempt to further elucidate its role in MMR and the engagement of downstream damage response pathways. A yeast two-hybrid system, an in vivo system for detecting protein-protein interactions was utilised for this purpose. Fifteen known and five unknown genes were identified as encoding proteins interacting with hMLH1. These included three known hMLH1 binding proteins, hMLH3, hPMS1 and MED1. Amongst the other genes identified was the proto-oncogene c-MYC, a gene previously implicated in genetic instability and apoptosis. Using in vitro derived mutants of c-MYC, it has been shown that hMLH1 interacts with the leucine-zipper domain of c-MYC. The effect of elevated c-MYC expression on functional MMR was examined. An inducible c-MYC expression system, Rat-1 fibroblasts expressing c-MYCERTM, a fusion of c-MYC to the hormone binding domain of the oestrogen receptor was utilised. Elevated expression of c-MYC did not effect the mismatch specific binding complex activity in these cells as measured in EMSA experiments. However c-MYC overexpression utilising the Rat-1 cMYCERTM system was shown to result in a mutator phenotype in these cells. The results suggest there may be a link between the mutator phenotype, induced through overexpression of c-MYC, and loss of MMR. Overexpression of c-MYC, which is associated with many cancers, may result in the sequestration of hMLH1 preventing functional MMR. The interaction between hMLH1 and c-MYC is proposed to act in a DNA damage response pathway which is disrupted upon aberrant c-MYC expression

Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast

<p>Abstract</p> <p>Background</p> <p>Loss of DNA mismatch repair (MMR) in humans, mainly due to mutations in the <it>hMLH1 </it>gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC). Because not all <it>MLH1 </it>alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, <it>in vivo </it>assays for functional characterization of <it>MLH1 </it>mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of <it>hMLH1 </it>mutations <it>in vivo</it>, based on co-expression of human MLH1 and PMS2 in yeast cells.</p> <p>Methods</p> <p>Yeast <it>MLH1 </it>and <it>PMS1 </it>genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested.</p> <p>Results</p> <p>The yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related <it>MLH1 </it>variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T) were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic.</p> <p>Conclusion</p> <p>Results of our <it>in vivo </it>yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of <it>MLH1 </it>variants in cancer patients found throughout the entire coding region of the gene.</p

Pathophysiology, causes, and management of non-traumatic spinal injury

Non-traumatic spinal cord injury (NTSCI) is at least as common as traumatic spinal cord injury (TSCI). It affects both sexes equally and an older population than TSCI. It is a devastating condition with immense functional implications for the individuals involved. There is a wide spectrum of aetiologies with varying pathophysiology and knowledge of these is important to avoid delay in diagnosis and time-critical treatment. The most common causes described in case series in developed countries are degenerative disc disease, canal stenosis, tumours, vascular diseases and inflammatory conditions. History and examination may help direct investigations, but magnetic resonance imaging is usually required. Management of NTSCI focuses on diagnosing and treating the precipitating cause, supportive management, and preventing complications. The outcomes of non-traumatic spinal cord injury are similar to those of traumatic spinal cord injury and depend on the grade and level of injury, pre-morbid status, and concurrent co-morbidities.</p

Folic acid fortification and public health: report on threshold doses above which unmetabolised folic acid appear in serum

Background All flour in the USA is fortified with folic acid at a level of 140 ?g/100 g which is estimated to supply an extra 100 ?g daily to the average diet. Some researchers have advocated that this be increased to double and even four times this amount. Based on previous research these higher levels are likely to lead to the appearance of unmetabolised vitamin in the circulation, which may have safety implications for sub-groups of the population. The UK and the Republic of Ireland will likely introduce mandatory fortification also in the next year or so. The aim of this study was to capture the short-term effect of folic acid fortification on unmetabolised folic acid in serum after chronic consumption of folic acid. Methods After pre-saturation with 400 ?g folic acid supplements daily for 14-weeks, healthy folate replete adults (n = 20) consumed folic acid fortified bread, at three different levels (400 ?g, 200 ?g, 100 ?g) over a period of one week each. The dose was administered in two-equal sized slices consumed at 09.00 hrs and 13.00 hrs. Serum samples for total folate and folic acid were collected at baseline, after 14-weeks of supplementation, and pre and post (at 1, 2, 3 and 4 hours) each dose tested. Results Unmetabolised folic acid was detected after the 14-week supplementation period. Folic acid was not detected in either the 200 ?g or 100 ?g (current US regime) doses tested but was present at the highest level (400 ?g) tested. Conclusion Our findings suggest that persons exposed to the current US fortification programme supplying an average of 100 ?g per day or less are unlikely to have unmetabolised folic acid in serum. It also seems that daily consumption of the higher level of 200 ?g or less is unlikely to be problematic. Increasing the level however to 400 ?g on the other hand is likely to lead to unmetabolised folic acid appearance

3D-printed procedural task trainer for the aspiration of penile corpus cavernosa in ischaemic priapism

Objective: The development and initial clinical assessment of a novel 3D-printed procedural task trainer for the aspiration of penile corpus cavernosa in ischaemic priapism. Methods: A task trainer for the aspiration of penile corpus cavernosa was designed and manufactured using commercially available 3D printing equipment. The trainer was assessed in two separate training sessions led by faculty investigators. Participants in the sessions were asked to complete a post-procedure survey with regards to the utility and realism of the task trainer. Results: The participants (n = 14) covered a broad spectrum of clinician types. The trainer was perceived by the participants as being anatomically realistic, and especially while under drapes provided a reasonable facsimile of real clinical setup. The trainer proved resilient to multiple attempts at aspiration by multiple participants. Conclusions: Participant and facilitator feedback indicates that the task trainer is a useful platform to train for what is a low frequency, but high stakes, procedure. Small numbers of participants preclude statistical rigour and certainty regarding overall performance of the trainer. However, the uniformity in the responses would suggest that this is indeed a task trainer that is ‘fit for purpose’

Double flap from amputated opposite lower limb for cover of plantar and dorsal surfaces of a crushed foot

Bilateral limb trauma poses many possibilities for management. In a situation of bilateral amputation, if the amputated limb is not salvageable or replantation is not advisable, the amputated limb can be used to harvest tissue for free tissue transfer to cover the amputation stump. We describe a case here in which we have used these principles