RESUMEN
Chondroitin/dermatan sulfate (CS/DS) proteoglycans are indispensable for animal development and homeostasis but the large number of enzymes involved in their biosynthesis have made CS/DS function a challenging problem to study genetically. In our study, we generated loss-of-function alleles in zebrafish genes encoding CS/DS biosynthetic enzymes and characterized the effect on development in single and double mutants. Homozygous mutants in chsy1, csgalnact1a, csgalnat2, chpfa, ust and chst7, respectively, develop to adults. However, csgalnact1a-/- fish develop distinct craniofacial defects while the chsy1-/- skeletal phenotype is milder and the remaining mutants display no gross morphological abnormalities. These results suggest a high redundancy for the CS/DS biosynthetic enzymes and to further reduce CS/DS biosynthesis we combined mutant alleles. The craniofacial phenotype is further enhanced in csgalnact1a-/-;chsy1-/- adults and csgalnact1a-/-;csgalnact2-/- larvae. While csgalnact1a-/-;csgalnact2-/- was the most affected allele combination in our study, CS/DS is still not completely abolished. Transcriptome analysis of chsy1-/-, csgalnact1a-/- and csgalnact1a-/-;csgalnact2-/- larvae revealed that the expression had changed in a similar way in the three mutant lines but no differential expression was found in any of fifty GAG biosynthesis enzymes identified. Thus, zebrafish larvae do not increase transcription of GAG biosynthesis genes as a consequence of decreased CS/DS biosynthesis. The new zebrafish lines develop phenotypes similar to clinical characteristics of several human congenital disorders making the mutants potentially useful to study disease mechanisms and treatment.
Asunto(s)
Dermatán Sulfato , Pez Cebra , Animales , Sulfatos de Condroitina/metabolismo , Dermatán Sulfato/genética , Dermatán Sulfato/metabolismo , Glicosiltransferasas/genética , Fenotipo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
NDST1 (glucosaminyl N-deacetylase/N-sulfotransferase) is a key enzyme in heparan sulfate (HS) biosynthesis, where it is responsible for HS N-deacetylation and N-sulfation. In addition to the full length human enzyme of 882 amino acids, here designated NDST1A, a shorter form containing 825 amino acids (NDST1B) is synthesized after alternative splicing of the NDST1 mRNA. NDST1B is mostly expressed at a low level, but increased amounts are seen in several types of cancer where it is associated with shorter survival. In this study, we aimed at characterizing the enzymatic properties of NDST1B and its effect on HS biosynthesis. Purified recombinant NDST1B lacked both N-deacetylase and N-sulfotransferase activities. Interestingly, HEK293 cells overexpressing NDST1B synthesized HS with reduced sulfation and altered domain structure. Fluorescence resonance energy transfer-microscopy demonstrated that both NDST1A and NDST1B had the capacity to interact with the HS copolymerase subunits EXT1 and EXT2 and also to form NDST1A/NDST1B dimers. Since lysates from cells overexpressing NDST1B contained less NDST enzyme activity than control cells, we suggest that NDST1B works in a dominant negative manner, tentatively by replacing the active endogenous NDST1 in the enzyme complexes taking part in biosynthesis.
Asunto(s)
Heparitina Sulfato , Sulfotransferasas , Aminoácidos/genética , Células HEK293 , Heparitina Sulfato/química , Humanos , Mutación , Sulfotransferasas/metabolismoRESUMEN
The evolution of mobile technology is moving at a very fast pace. Smartphones are currently considered a primary communication platform where people exchange voice calls, text messages and emails. The human-smartphone interaction, however, is generally optimized for sighted people through the use of visual cues on the touchscreen, e.g., typing text by tapping on a visual keyboard. Unfortunately, this interaction scheme renders smartphone technology largely inaccessible to visually impaired people as it results in slow typing and higher error rates. Apple and some third party applications provide solutions specific to blind people which enables them to use Braille on smartphones. These applications usually require both hands for typing. However, Brailling with both hands while holding the phone is not very comfortable. Furthermore, two-handed Brailling is not possible on smartwatches, which will be used more pervasively in the future. Therefore, we develop a platform for one-handed Brailing consisting of a custom keyboard called BrailleEasy to input Arabic or English Braille codes within any application, and a BrailleTutor application for practicing. Our platform currently supports Braille grade 1, and will be extended to support contractions, spelling correction, and more languages. Preliminary analysis of user studies for blind participants showed that after less than two hours of practice, participants were able to type significantly faster with the BrailleEasy keyboard than with the standard QWERTY keyboard.