Identification and Characterization of Nine Novel X-Chromosomal Short Tandem Repeats on Xp21.1, Xq21.31, and Xq23 Regions

The application of X-chromosomal short tandem repeats (X-STRs) has been recognized as a powerful tool in complex kinship testing. To support further development of X-STR analysis in forensic use, we identified nine novel X-STRs, which could be clustered into three linkage groups on Xp21.1, Xq21.31, and Xq23. A multiplex PCR system was built based on the electrophoresis. A total of 198 unrelated Shanghai Han samples along with 168 samples from 43 families was collected to investigate the genetic polymorphism and forensic parameters of the nine loci. Allele numbers ranged from 5 to 12, and amplicon sizes ranged from 146 to 477 bp. The multiplex showed high values for the combined power of discrimination (0.99997977 in males and 0.99999999 in females) and combined mean exclusion chances (0.99997918 and 0.99997821 in trios, 0.99984939 in duos, and 0.99984200 in deficiency cases). The linkage between all pairs of loci was estimated via Kosambi mapping function and linkage disequilibrium test, and further investigated through the family study. The data from 43 families strongly demonstrated an independent transmission between LGs and a tight linkage among loci within the same LG. All these results support that the newly described X-STRs and the multiplex system are highly promising for further forensic use.

CRISPRi as a Tool to Repress Multiple Copies of Extracellular Polymeric Substances (EPS)-Related Genes in the Cyanobacterium Synechocystis sp. PCC 6803

The use of the versatile cyanobacterial extracellular polymeric substances (EPS) for biotechnological/biomedical applications implies an extensive knowledge of their biosynthetic pathways to improve/control polymer production yields and characteristics. The multiple copies of EPS-related genes, scattered throughout cyanobacterial genomes, adds another layer of complexity, making these studies challenging and time-consuming. Usually, this issue would be tackled by generating deletion mutants, a process that in cyanobacteria is also hindered by the polyploidy. Thus, the use of the CRISPRi multiplex system constitutes an efficient approach to addressing this redundancy. Here, three putative Synechocystis sp. PCC 6803 kpsM homologues (slr0977, slr2107, and sll0574) were repressed using this methodology. The characterization of the 3-sgRNA mutant in terms of fitness/growth and total carbohydrates, released and capsular polysaccharides, and its comparison with previously generated single knockout mutants pointed towards Slr0977 being the key KpsM player in Synechocystis EPS production. This work validates CRISPRi as a powerful tool to unravel cyanobacterial complex EPS biosynthetic pathways expediting this type of studies.

Development of a multiplex system for determining 11 genetic markers of predisposition to obesity

A system has been developed to identify 11 genetic markers associated with the risk of obesity: rs10852521, rs11075990, rs1121980, rs1421085, rs1477196, rs17817449, rs3751812, rs7206790, rs8047395, rs9940128 (FTO gene) and rs1137101 (LEPR gene) by minisequencing (SNaPshot analysis). The conditions for carrying out the amplification and minisequencing reactions, as well as the compositions of the reaction mixtures, were optimized so that the analysis was carried out for all 11 markers simultaneously. The resulting system was tested and showed a high degree of reproducibility and sensitivity required for the detection of these polymorphisms.

Development and Validation of a Forensic Multiplex System With 38 X-InDel Loci

In the present study, a novel multiplex system, AGCU X-InDel 38 kit, was designed to amplify 38 X-InDel markers and amelogenin in a single Polymerase Chain Reaction (PCR). To demonstrate the suitability and efficiency for forensic applications, a series of validation experiments were conducted, including sensitivity, species specificity, reproducibility, stability, case samples, balance of peak height, size precision, as well as allele frequency and forensic parameter analysis. The results showed that AGCU X-InDel 38 kit was capable to get full profiles even with 62.5 pg of template DNA, and full profiles can be obtained when hematin concentration ≤25 μmol/L, or hemoglobin concentration ≤50 μmol/L, showing good tolerance to six common inhibitors. Moreover, the analyzed case samples indicated that AGCU X-InDel 38 kit had better performance for degraded and trace DNA samples. The 200 unrelated males from Guangdong Han population showed that the combined PDMale and PDFemale were both more than 0.999999999, and the combined MECKrüger, MECKishida, and MECDesmarais Duo were 0.999369481, 0.999999917, and 0.999941556, respectively. Robust discrimination capability of this novel multiplex system could be demonstrated through the high values of forensic parameters. In conclusion, AGCU X-InDel 38 kit is sensitive, precise, reproducible, and highly informative and could be used as a complementary tool for complex and challenging kinship cases.

X-chromosomal STR based genetic polymorphisms and demographic history of Sri Lankan ethnicities and their relationship with global populations

A new 16 X-short tandem repeat (STR) multiplex PCR system has recently been developed for Sr Lankans, though its applicability in evolutionary genetics and forensic investigations has not been thoroughly assessed. In this study, 838 unrelated individuals covering all four major ethnic groups (Sinhalese, Sri Lankan Tamils, Indian Tamils and Moors) in Sri Lanka were successfully genotyped using this new multiplex system. The results indicated a high forensic efficiency for the tested loci in all four ethnicities confirming its suitability for forensic applications of Sri Lankans. Allele frequency distribution of Indian Tamils showed subtle but statistically significant differences from those of Sinhalese and Moors, in contrast to frequency distributions previously reported for autosomal STR alleles. This suggest a sex biased demographic history among Sri Lankans requiring a separate X-STR allele frequency database for Indian Tamils. Substantial differences observed in the patterns of LD among the four groups demand the use of a separate haplotype frequency databases for each individual ethnicity. When analysed together with other 14 world populations, all Sri Lankan ethnicities except Indian Tamils clustered closely with populations from Indian Bhil tribe, Bangladesh and Europe reflecting their shared Indo-Aryan ancestry.

Profiling Complement System Components in Primary CNS Vasculitis

Complement activation has been implicated in the pathogenesis of many vasculitic syndromes such as anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides. Using an array-based multiplex system, we simultaneously quantified serum and CSF levels of activated and regulatory complement system proteins in patients with primary CNS vasculitis (PACNS; n = 20) compared to patients with non-inflammatory conditions (n = 16). Compared to non-inflammatory controls, levels of C3a, C5a, and SC5b-9, indicative for general activation of the complement system, of C4a, specific for the activation of the classical pathway, Ba and Bb, reflective for alternative complement activation as well as concentrations of complement-inhibitory proteins factor H and factor I were unchanged in patients with PACNS. Our study does not support the hypothesis that complement activation is systemically increased in patients with PACNS.