Assessing and enhancing foldability in designed proteins

Recent advances in protein-design methodology have led to a dramatic increase in its reliability and scale. With these advances, dozens and even thousands of designed proteins are automatically generated and screened. Nevertheless, the success rate, particularly in design of functional proteins, is low and fundamental goals such as reliable de novo design of efficient enzymes remain beyond reach. Experimental analyses have consistently indicated that a major cause of design failure is inaccuracy and misfolding relative to the design model. To address this challenge, we describe complementary methods to diagnose and ameliorate suboptimal regions in designed proteins: first, we develop a Rosetta atomistic computational mutation scanning approach to detect energetically suboptimal positions in designs; second, we demonstrate that the AlphaFold2 ab initio structure prediction method flags regions that may misfold in designed enzymes and binders; and third, we focus FuncLib design calculations on suboptimal positions in a previously designed low-efficiency enzyme, thereby improving its catalytic efficiency by 330 fold. Thus, foldability analysis and enhancement may dramatically increase the success rate in design of functional proteins.

Mucopolysaccharidosis VI diagnosis by laboratory methods

Mucopolysaccharidosis type VI (MPS VI) results from a defect in arylsulfatase B (ARSB). There are several diagnostic methods using to identify patients; hence, we aimed to review these approaches and consider if one of them could be assigned as the gold standard method. The information of this study was obtained by searching through PubMed and Google scholar databases. In order to collect the most accurate and up to date data, we limited our research to papers in the time period between 2010 and 2017. We collected articles related to our research and extracted the most relevant and accurate data which included the steps of MPS VI diagnosis by routine laboratory approaches. We concluded that an all-inclusive diagnostic approach requires urinary glycosaminoglycan (GAG) analysis, enzyme activity analysis and molecular analysis by mutation scanning through polymerase chain reaction (PCR) and Sanger sequencing or alternative methods such as multiplex ligation-dependent probe amplification (MLPA), real-time polymerase chain reaction, array-comparative genomic hybridization (aCGH) and next generation sequencing (NGS). Reliable classification of patients with MPS VI is necessary for ongoing and future studies on treatments, outcomes and prenatal diagnoses (PNDs). The dependable characterization of patients would be achieved by biochemical techniques and enzymatic assay. However, if a molecular defect is previously identified in the family, PND via mutation scanning is possible.

Genetic analysis of three families with X-linked dominant hypophosphatemic rickets

Background Hypophosphatemic rickets, including familial hypophosphatemic vitamin D-resistant rickets, which commonly manifests in childhood, is generally hereditary. X-linked dominant hypophosphatemic rickets (XLH, MIM307800), caused by inactivating mutations in the PHEX gene, is the most common form. This study aimed to identify the gene mutations responsible for three cases of XLH and its clinical phenotype. Methods We conducted a genetic diagnosis and clinical phenotypic linkage analysis of three pedigrees with XLH. Three probands finally diagnosed as XLH were analyzed by next-generation sequencing (NGS). Sanger sequencing was used for mutation scanning in other family members. Results For the three patients with XLH, the age of onset ranged from 1.5 to 2 years and their heights were less than three standard deviations (SDs) below the median. The patients exhibited curved deformities in both lower limbs, hypophosphatemia, elevated serum FGF23 levels and elevated levels of blood alkaline phosphatase, with normal levels of blood parathyroid hormone (PTH) and calcium. X-ray analysis of the limbs and chest revealed characteristic rickets signs. Three candidate pathogenic mutations were identified in PHEX (NM_000444.5): c.433G>T (p.Glu145*, p.E145*) in exon 4, c.1735G>A (p.Gly579Arg, p.G579R) (rs875989883) in exon 17 and c.2245T>C (p.Trp749Arg, p.W749R) in exon 22. The nonsense mutation (p.E145*) in PHEX is novel and is predicted to cause a truncation of the encoded protein, resulting in loss of function. Conclusions The novel nonsense mutation (p.E145*) in PHEX is possibly involved in inherited XLH.

An optimized HRM method for diagnosis of G6PD deficiency in kinh Vietnamese via viangchan mutation

With Glucose-6-phosphate dehydrogenase (G6PD) deficiency being the most common enzyme disorder in human, there have been 184 discovered point mutations and several methods that have been applied for diagnosing this disease. However, these techniques often pose several major problems such as being time-consuming, low sensitivity and high cost. Recently, the High Resolution Melting (HRM) has been studied and proven to be effective for DNA genotyping, mutation scanning and sequence matching. Therefore, HRM has been chosen for diagnosing G6PD deficiency via Viangchan mutation in this study. In this study, a total of 56 dried blood spot samples (including six control samples which were known the exact genotype by sequencing and fifty unknown samples) were collected and extracted DNA by using QIAamp DNA Blood Mini Kit. Primers for HRM analysis were designed through by the Umelt software. Then HRM optimization was carried out for annealing temperature of primers (Ta) and MgCl2 concentration on six control samples. The optimized HRM protocol with 2.5 μM of MgCl2 and Ta at 62oC was applied for fifty G6PD samples and then comparing with ARMS-PCR genotyping results for the validation process. In the final step, genotyping results were confirmed by sequencing. In a results, both sensitivity and specificity of this technique reached 100%. Based on these favorable outcomes, this study has successfully optimized the HRM conditions for diagnosing fifty G6PD samples. It was such an essential precondition that showed HRM could be applied for other types of G6PD through other types of mutations such as Canton mutation or continues to be developed for HRM-Multiplex reactions.

Assessment for Melting Temperature Measurement of Nucleic Acid by HRM

High resolution melting (HRM), with a high sensitivity to distinguish the nucleic acid species with small variations, has been widely applied in the mutation scanning, methylation analysis, and genotyping. For the aim of extending HRM for the evaluation of thermal stability of nucleic acid secondary structures on sequence dependence, we investigated effects of the dye of EvaGreen, metal ions, and impurities (such as dNTPs) on melting temperature (Tm) measurement by HRM. The accuracy of HRM was assessed as compared with UV melting method, and little difference between the two methods was found when the DNA Tm was higher than 40°C. Both insufficiency and excessiveness of EvaGreen were found to give rise to a little bit higher Tm, showing that the proportion of dye should be considered for precise Tm measurement of nucleic acids. Finally, HRM method was also successfully used to measure Tms of DNA triplex, hairpin, and RNA duplex. In conclusion, HRM can be applied in the evaluation of thermal stability of nucleic acid (DNA or RNA) or secondary structural elements (even when dNTPs are present).