Selection of Reference Genes for RT-qPCR Studies in Different Organs of Rice Cultivar BRS AG Submitted to Recurrent Saline Stress.

Quantitative real-time polymerase chain reactions (RT-qPCR) have become one of the most widely used methods for analyzing gene expression, provided suitable reference genes are available to normalize the data. RNA was isolated from leaves, grain, rachises and sheaths of rice (Oryza sativa L. cv. BRS AG) submitted to different saline stress events for seven days, and expression analysis was carried out by RT-qPCR. Expression levels of ten candidate reference genes were assessed, actin11 (ACT11), ubiquitin conjugating enzyme E2 (UBC-E2), eukaryotic elongation factor1-a (Eef-1a), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), B-tubulin (B-Tub), eukaryotic initiation factor 4a (Eif-4-a), ubiquitin10 (UBQ10), ubiquitin5 (UBQ5), aquaporin TIP41 (TIP41-like). Gene expression stability was calculated using the common statistical algorithms geNorm, BestKeeper and ?Ct method, NormFinder and RefFinder. The most stably expressed genes were UBC2E and GAPDH for leaves, UBQ5 and UBQ10 for sheaths, TIP41 and UBQ10 for rachises, and TIP41 and cyclophilin for grain. Gene expression of triose phosphate translocator (TPT1), ADP-glucose transporter (BT1-1), choline monooxygenase (CMO) was used to validate the selected reference genes. The results highlighted the importance of using suitable reference gene to normalize gene expression data in rice plants

MEIS proteins as partners of the TLX1/HOX11 oncoprotein

Aberrant expression of the TLX1/. HOX11 proto-oncogene is associated with a significant subset of T-cell acute lymphoblastic leukemias (T-ALL). Yet the manner in which TLX1 contributes to oncogenesis is not fully understood. Since, typically, interactions of HOX and TALE homeodomain proteins are determinant of HOX function, and HOX/MEIS co-expression has been shown to accelerate some leukemias, we systematically examined whether TLX1 interacts with MEIS and PBX proteins. Here, we report that TLX1 and MEIS proteins both interact and are co-expressed in T-ALL, and suggest that co-operation between TLX1 and MEIS proteins may have a significant role in T-cell leukemogenesis

Target validation of the inosine monophosphate dehydrogenase (IMPDH) gene in Cryptosporidium using Phylomer® peptides

Cryptosporidiosis, a gastroenteric disease characterised mainly by diarrheal illnesses in humans and mammals is caused by infection with the protozoan parasite Cryptosporidium. Treatment options for cryptosporidiosis are limited, with the current therapeutic nitazoxanide, only partly efficacious in immunocompetent individuals. The parasite lacks de novo purine synthesis, and is exclusively dependant on purine salvage from its host. Inhibition of the inosine 5' monophosphate dehydrogenase (IMPDH), a purine salvage enzyme that is essential for DNA synthesis, thereby offers a potential drug target against this parasite. In the present study, a yeast-two-hybrid system was used to identify Phylomer peptides within a library constructed from the genomes of 25 phylogenetically diverse bacteria that targeted the IMPDH of Cryptosporidium parvum (IMPcp) and Cryptosporidium hominis (IMPch). We identified 38 unique interacting Phylomers, of which, 12 were synthesised and screened against C. parvum in vitro. Two Phylomers exhibited significant growth inhibition (81.2-83.8% inhibition; P < 0.05), one of which consistently exhibited positive interactions with IMPcp and IMPch during primary and recapitulation yeast two-hybrid screening and did not interact with either of the human IMPDH proteins. The present study highlightsthe potential of Phylomer peptides as target validation tools for Cryptosporidium and other organisms and diseases because of their ability to bind with high affinity to target proteins and disrupt function

A platform for discovery of functional cell-penetrating peptides for efficient multi-cargo intracellular delivery

Cell penetrating peptides (CPPs) offer great potential to deliver therapeutic molecules to previously inaccessible intracellular targets. However, many CPPs are inefficient and often leave their attached cargo stranded in the cell’s endosome. We report a versatile platform for the isolation of peptides delivering a wide range of cargos into the cytoplasm of cells. We used this screening platform to identify multiple “Phylomer” CPPs, derived from bacterial and viral genomes. These peptides are amenable to conventional sequence optimization and engineering approaches for cell targeting and half-life extension. We demonstrate potent, functional delivery of protein, peptide, and nucleic acid analog cargos into cells using Phylomer CPPs. We validate in vivo activity in the cytoplasm, through successful transport of an oligonucleotide therapeutic fused to a Phylomer CPP in a disease model for Duchenne’s muscular dystrophy. This report thus establishes a discovery platform for identifying novel, functional CPPs to expand the delivery landscape of druggable intracellular targets for biological therapeutics

β-Lactamase Tools for Establishing Cell Internalization and Cytosolic Delivery of Cell Penetrating Peptides

The ability of cell penetrating peptides (CPPs) to deliver biologically relevant cargos into cells is becoming more important as targets in the intracellular space continue to be explored. We have developed two assays based on CPP-dependent, intracellular delivery of TEM-1 &beta;-lactamase enzyme, a functional biological molecule comparable in size to many protein therapeutics. The first assay focuses on the delivery of full-length &beta;-lactamase to evaluate the internalization potential of a CPP sequence. The second assay uses a split-protein system where one component of &beta;-lactamase is constitutively expressed in the cytoplasm of a stable cell line and the other component is delivered by a CPP. The delivery of a split &beta;-lactamase component evaluates the cytosolic delivery capacity of a CPP. We demonstrate that these assays are rapid, flexible and have potential for use with any cell type and CPP sequence. Both assays are validated using canonical and novel CPPs, with limits of detection from &lt;500 nM to 1 &micro;M. Together, the &beta;-lactamase assays provide compatible tools for functional characterization of CPP activity and the delivery of biological cargos into cells

Beyond structural bioinformatics for genomics with dynamics characterization of an expanded KRAS mutational landscape

Current capabilities in genomic sequencing outpace functional interpretations. Our previous work showed that 3D protein structure calculations enhance mechanistic understanding of genetic variation in sequenced tumors and patients with rare diseases. The KRAS GTPase is among the critical genetic factors driving cancer and germline conditions. Because KRAS-altered tumors frequently harbor one of three classic hotspot mutations, nearly all studies have focused on these mutations, leaving significant functional ambiguity across the broader KRAS genomic landscape observed in cancer and non-cancer diseases. Herein, we extend structural bioinformatics with molecular simulations to study an expanded landscape of 86 KRAS mutations. We identify multiple coordinated changes strongly associated with experimentally established KRAS biophysical and biochemical properties. The patterns we observe span hotspot and non-hotspot alterations, which can all dysregulate Switch regions, producing mutation-restricted conformations with different effector binding propensities. We experimentally measured mutation thermostability and identified shared and distinct patterns with simulations. Our results indicate mutation-specific conformations, which show potential for future research into how these alterations reverberate into different molecular and cellular functions. The data we present is not predictable using current genomic tools, demonstrating the added functional information derived from molecular simulations for interpreting human genetic variation