Developmental validation of a microRNA panel using quadratic discriminant analysis for the classification of seven forensically relevant body fluids.

Body fluid identification is an important step in the forensic DNA workflow, and more advanced methods, such as microRNA (miRNA) analysis, have been research topics within the community over the last few decades. We previously reported a reverse transcription-quantitative PCR (RT-qPCR) panel of eight miRNAs that could classify blood, menstrual secretions, feces, urine, saliva, semen, and vaginal secretions through analysis of differential gene expression. The purpose of this project was to evaluate this panel in a larger population size, develop a more statistically robust analysis method and perform a series of developmental validation studies. Each of the eight miRNA markers was analyzed in > 40 donors each of blood, menstrual secretions, feces, urine, saliva, semen, and vaginal secretions. A 10-fold cross-validated quadratic discriminant analysis (QDA) model yielded the highest classification accuracy of 93% after eliminating miR-26b and miR-1246 from the panel. Accuracy of body fluid predictions was between 84% and 100% when various population demographics and samples from the same donor over multiple time periods were evaluated, but the assay demonstrated limited scope and reduced accuracy when mixed body fluid samples were tested. Limit of detection was found to be less than 104 copies/µL across multiple commercially available RT-qPCR analysis methods. These data suggest that miR-200b, miR-320c, miR-10b, and miR-891a, when normalized to let-7 g and let-7i, can consistently and robustly classify blood, feces and urine, but additional work is important to improve classification of saliva, semen, and female intimate secretions before implementation in forensic casework.

Ribosome collisions induce mRNA cleavage and ribosome rescue in bacteria.

Ribosome rescue pathways recycle stalled ribosomes and target problematic mRNAs and aborted proteins for degradation1,2. In bacteria, it remains unclear how rescue pathways distinguish ribosomes stalled in the middle of a transcript from actively translating ribosomes3-6. Here, using a genetic screen in Escherichia coli, we discovered a new rescue factor that has endonuclease activity. SmrB cleaves mRNAs upstream of stalled ribosomes, allowing the ribosome rescue factor tmRNA (which acts on truncated mRNAs3) to rescue upstream ribosomes. SmrB is recruited to ribosomes and is activated by collisions. Cryo-electron microscopy structures of collided disomes from E. coli and Bacillus subtilis show distinct and conserved arrangements of individual ribosomes and the composite SmrB-binding site. These findings reveal the underlying mechanisms by which ribosome collisions trigger ribosome rescue in bacteria.

Gain-of-function mutations in beet DODA2 identify key residues for betalain pigment evolution.

The key enzymatic step in betalain biosynthesis involves conversion of l-3,4-dihydroxyphenylalanine (l-DOPA) to betalamic acid. One class of enzymes capable of this is 3,4-dihydroxyphenylalanine 4,5-dioxygenase (DODA). In betalain-producing species, multiple paralogs of this gene are maintained. This study demonstrates which paralogs function in the betalain pathway and determines the residue changes required to evolve a betalain-nonfunctional DODA into a betalain-functional DODA. Functionalities of two pairs of DODAs were tested by expression in beets, Arabidopsis and yeast, and gene silencing was performed by virus-induced gene silencing. Site-directed mutagenesis identified amino acid residues essential for betalamic acid production. Beta vulgaris and Mirabilis jalapa both possess a DODA1 lineage that functions in the betalain pathway and at least one other lineage, DODA2, that does not. Site-directed mutagenesis resulted in betalain biosynthesis by a previously nonfunctional DODA, revealing key residues required for evolution of the betalain pathway. Divergent functionality of DODA paralogs, one clade involved in betalain biosynthesis but others not, is present in various Caryophyllales species. A minimum of seven amino acid residue changes conferred betalain enzymatic activity to a betalain-nonfunctional DODA paralog, providing insight into the evolution of the betalain pigment pathway in plants.

Advances in the MYB-bHLH-WD Repeat (MBW) Pigment Regulatory Model: Addition of a WRKY Factor and Co-option of an Anthocyanin MYB for Betalain Regulation.

Flavonoids are secondary metabolites derived from the general phenylpropanoid pathway and are widespread throughout the plant kingdom. The functions of flavonoids are diverse, including defense against phytopathogens, protection against UV light damage and oxidative stress, regulation of auxin transport and allelopathy. One of the most conspicuous functions of flavonoids has long attracted the attention of pollinators and scientist alike: the vivid shades of red, pink, orange, blue and purple on display in the flowers of angiosperms. Thus, flavonoid pigments have perhaps been the most intensely studied phenylpropanoids. From Mendel to McClintock and up to the present, studies centered on flavonoid pigments have resulted in some of the most important scientific discoveries of the last 150 years, including the first examples of transcriptional regulation in plants. Here we focus on the highly conserved MYB-bHLH-WD repeat (MBW) transcriptional complex model for the regulation of the flavonoid pigment pathway. We will survey the history of the MBW model spanning the last three decades, highlighting the major findings that have contributed to our current understanding. In particular, recent discoveries regarding WRKY protein control of the flavonoid pigment pathway and its relationship to the MBW complex will be emphasized. In addition, we will discuss recent findings about the regulation of the beet betalain pigment pathway, and how a MYB member of the MBW complex was co-opted to regulate this chemically unrelated but functionally equivalent pathway.

TTG2 controls the developmental regulation of seed coat tannins in Arabidopsis by regulating vacuolar transport steps in the proanthocyanidin pathway.

The brown color of Arabidopsis seeds is caused by the deposition of proanthocyanidins (PAs or condensed tannins) in their inner testa layer. A transcription factor complex consisting of TT2, TT8 and TTG1 controls expression of PA biosynthetic genes, just as similar TTG1-dependent complexes have been shown to control flavonoid pigment pathway gene expression in general. However, PA synthesis is controlled by at least one other gene. TTG2 mutants lack the pigmentation found in wild-type seeds, but produce other flavonoid compounds, such as anthocyanins in the shoot, suggesting that TTG2 regulates genes in the PA biosynthetic branch of the flavonoid pathway. We analyzed the expression of PA biosynthetic genes within the developing seeds of ttg2-1 and wild-type plants for potential TTG2 regulatory targets. We found that expression of TT12, encoding a MATE type transporter, is dependent on TTG2 and that TTG2 can bind to the upstream regulatory region of TT12 suggesting that TTG2 directly regulates TT12. Ectopic expression of TT12 in ttg2-1 plants partially restores seed coat pigmentation. Moreover, we show that TTG2 regulation of TT12 is dependent on TTG1 and that TTG1 and TTG2 physically interact. The observation that TTG1 interacts with TTG2, a WRKY type transcription factor, proposes the existence of a novel TTG1-containing complex, and an addendum to the existing paradigm of flavonoid pathway regulation.

Efficacy of Intravenous Immunoglobulin in the Treatment of Persistent BK Viremia and BK Virus Nephropathy in Renal Transplant Recipients.

BK virus associated nephropathy (BKVN) can cause clinically significant viral infections in renal transplant recipients, leading to allograft dysfunction and loss. The usual management of BKVN involves reduction of immunosuppression and the addition of leflunomide, quinolones, and cidofovir, but the rate of graft loss remains high. The aim of this study was to assess the impact of treatment with intravenous immunoglobulin (IVIG) on the outcome of BKVN in renal transplant recipients. Upon diagnosis of BKVN, patients remained on anti-polyomavirus treatment consisting of reduction of immunosuppression and the use of leflunomide therapy. Treatment with IVIG was given only to patients who did not respond to 8 weeks of the adjustment of immunosuppression and leflunomide. All 30 patients had persistent BK viremia and BKVN with their mean BK viral loads higher than the baseline (range 15,000 - 2 millions copies/mL). Mean peak BK load was 205,314 copies/mL compared to 697 copies/mL after one year follow-up. Twenty-seven patients (90%) had positive responses in clearing viremia. The actuarial patient and graft survival rates after 12 months were 100% and 96.7%, respectively. IVIG administration appeared to be safe and effective in treating BK viremia and BKVN and in preventing graft loss in patients who had inadequate response to immunosuppression reduction and leflunomide therapy.