Despite the successful application of LNA/2’OMe-FISH procedures for bacteria detection, there is a lack of knowledge on the properties that affect hybridization. Such information is crucial for the rational design of protocols. Hence, this work aimed to evaluate the effect of three essential factors on the LNA/2’OMe hybridization step—hybridization temperature, NaCl concentration and type and concentration of denaturant (formamide, ethylene carbonate and urea). This optimization was performed for 3 Gram-negative bacteria ( Escherichia coli , Pseudomonas aeruginosa and Citrobacter freundii ) and 2 Gram-positive bacteria ( Enterococcus faecalis and Staphylococcus epidermidis ), employing the response surface methodology and a Eubacteria probe. In general, it was observed that a high NaCl concentration is beneficial (from 2 M to 5 M), regardless of the denaturant used. Urea, formamide and ethylene carbonate are suitable denaturants for LNA/2’OMe-FISH applications; but urea provides higher fluorescence intensities among the different bacteria, especially for gram-positive bacteria and for P . aeruginosa . However, a unique optimal protocol was not found for all tested bacteria. Despite this, the results indicate that a hybridization solution with 2 M of urea and 4 M of NaCl would be a proper starting point. Furthermore, a hybridization temperature around 62°C, for 14 bp probes with LNA monomers at every third position of 2′OMe and 64% of GC content, should be use in initial optimization of new LNA/2’OMe-FISH protocols.

Hereditary amyloidosis associated with transthyretin (ATTRv), is a rare autosomal dominant disease characterized by length-dependent symmetric polyneuropathy that has gait impairment as one of its consequences. The gait pattern of V30M ATTRv amyloidosis patients has been described as similar to that of diabetic neuropathy, associated with steppage, but has never been quantitatively characterized. In this study we aim to characterize the gait pattern of patients with V30M ATTRv amyloidosis, thus providing information for a better understanding and potential for supporting diagnosis and disease progression evaluation. We present a case series in which we conducted two gait analyses, 18 months apart, of five V30M ATTRv amyloidosis patients using a 12-camera, marker based, optical system as well as six force platforms. Linear kinematics, ground reaction forces, and angular kinematics results are analyzed for all patients. All patients, except one, showed a delayed toe-off in the second assessment, as well as excessive pelvic rotation, hip extension and external transverse rotation and knee flexion (in stance and swing phases), along with reduced vertical and mediolateral ground reaction forces. The described gait anomalies are not clinically quantified; thus, gait analysis may contribute to the assessment of possible disease progression along with the clinical evaluation.

No abstract