Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss

Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting. .     

Tetrodotoxin block of A-fibre afferents from skin and muscle - a tool to study pure C-fibre effects in the spinal cord

The properties of tetrodotoxin (TTX)-resistant C-fibre afferents of the dorsal roots were tested in Sprague-Dawley rats. Dorsal roots (L4-L6) were blocked with TTX (0.5-1 micro M) and the amplitude of the first response of the dorsal horn superficial interneurones (cord dorsum potential, CDP) to electrical stimulation of peripheral C-fibres in combination with natural noxious stimulation was taken as measure for intact conductivity of different kinds of noxious input by means of the C-fibre refractory period. After blockade of dorsal roots with TTX, formerly masked CDPs from muscle C-fibre afferents were uncovered. Noxious pressure to the gastrocnemius soleus muscle belly and noxious pinch to the calcanean tendon proved to be TTX resistant and therefore was propagated centrally. For cutaneous heat nociceptors it could also be shown that conductivity was intact after blockade of the dorsal roots with TTX. However, we could not exclude the TTX resistance of non-nociceptive receptors of muscle or skin. Nevertheless, blockade of afferents with TTX together with suitable stimulation techniques proves to be a reliable method to investigate central effects from C-fibre afferents without contaminating effects from A-fibres in the rat.     

Identification of Nordic Berries with Beneficial Effects on Cognitive Outcomes and Gut Microbiota in High-Fat-Fed Middle-Aged C57BL/6J Mice

High-fat diets are associated with neuronal and memory dysfunction. Berries may be useful in improving age-related memory deficits in humans, as well as in mice receiving high-fat diets. Emerging research has also demonstrated that brain health and cognitive function may be related to the dynamic changes in the gut microbiota. In this study, the impact of Nordic berries on the brain and the gut microbiota was investigated in middle-aged C57BL/6J mice. The mice were fed high-fat diets (60%E fat) supplemented with freeze-dried powder (6% dwb) of bilberry, lingonberry, cloudberry, blueberry, blackcurrant, and sea buckthorn for 4 months. The results suggest that supplementation with bilberry, blackcurrant, blueberry, lingonberry, and (to some extent) cloudberry has beneficial effects on spatial cognition, as seen by the enhanced performance following the T-maze alternation test, as well as a greater proportion of DCX-expressing cells with prolongation in hippocampus. Furthermore, the proportion of the mucosa-associated symbiotic bacteria Akkermansia muciniphila increased by 4–14 times in the cecal microbiota of mice fed diets supplemented with lingonberry, bilberry, sea buckthorn, and blueberry. These findings demonstrate the potential of Nordic berries to preserve memory and cognitive function, and to induce alterations of the gut microbiota composition.     

Dietary antioxidant intake in patients at risk for second primary cancer

OBJECTIVES: To analyze dietary antioxidant intake for head and neck cancer patients at risk for development of second primary cancers. STUDY DESIGN: Prospective observational study. METHODS: Twenty-four patients underwent three random, unscheduled, 24-hour dietary recalls over a 15-day period within 6 to 60 months after successful treatment for stage I or II oral cavity squamous cell carcinoma. RESULTS: The study sample had a lower mean daily dietary intake of fruits and vegetables and antioxidant nutrients, including vitamins A, C, E, and total carotenes than age- and sex-matched historic control subjects (all P <.05 except vitamin A). A positive linear correlation was noted between daily servings of F&V and dietary intake of vitamins A, C, E, and total carotenoids (all P <.05 except vitamin A). Compared to current recommendations, the study sample had lower mean daily dietary intake of vitamins A, C, and E (P =.81,.06, and <.01) and servings of fruits and vegetables (P <.01). When vitamin supplements were included in the analysis, mean daily intake exceeded recommended dietary allowance (RDA) for vitamins A, C, and E (all P <.05). CONCLUSION: This study suggests that patients treated for early-stage oral cavity carcinoma, at risk for second primary cancers, have a statistically significant deficiency in dietary (food) sources of antioxidant nutrients when compared with both historic control subjects and current recommendations. Vitamin supplementation significantly exceeded current RDAs. Because increased fruit and vegetable intake, but not vitamin supplementation exceeding RDA, is associated with reduced cancer risk, physicians may consider recommending at least five daily servings of fruits and vegetables as an alternative to vitamin supplementation.