Tumour Enzyme Affinity Mediated Peptide Molecular Crowding for Targeted Disruption of Hyperactivated Glucose Uptake

An unconventional environment-responsive molecular crowding via specific binding between small molecule peptide inhibitor derivatives and overexpressed tumour enzyme has been developed. Assemblies of such short peptides selectively localize on tumour...

Using Network Analytics to Improve Targeted Disruption of Police Misconduct

Research into police misconduct traditionally considers the correlates and antecedents of misconduct among individual officers, as a means of disruption or prevention. However, more recently, deviance among police has been considered through network perspectives. This study considered 7755 allegations of misconduct accrued by 1495 officers from the Baltimore Police Department between January 2015 to January 2020. A social network analysis was employed to consider the characteristics and differences of misconduct networks between assignments and to identify key officers within these networks. Findings suggested that the misconduct networks of patrol assignments functioned marginally different to investigations or specialist duties. Discrete communities of misconduct were identified within each assignment, including a small number of officers that were particularly important to supporting these networks. This study holds practical implications for the identification and disruption of misconduct networks among law enforcement agencies.

E2f2 Attenuates Apoptosis of Activated T Lymphocytes and Protects from Immune-Mediated Injury through Repression of Fas and FasL

Targeted disruption of E2f2 in mice causes T-cell hyperactivation and a disproportionate cell cycle entry upon stimulation. However, E2f2−/− mice do not develop a lymphoproliferative condition. We report that E2f2 plays a Fas-dependent anti-apoptotic function in vitro and in vivo. TCR-stimulated murine E2f2−/− T cells overexpress the proapoptotic genes Fas and FasL and exhibit enhanced apoptosis, which is prevented by treatment with neutralizing anti-FasL antibodies. p53 pathway is activated in TCR-stimulated E2f2−/− lymphocytes, but targeted disruption of p53 in E2f2−/− mice does not abrogate Fas/FasL expression or apoptosis, implying a p53-independent apoptotic mechanism. We show that E2f2 is recruited to Fas and FasL gene promoters to repress their expression. in vivo, E2f2−/− mice are prone to develop immune-mediated liver injury owing to an aberrant lymphoid Fas/FasL activation. Taken together, our results suggest that E2f2-dependent inhibition of Fas/FasL pathway may play a direct role in limiting the development of immune-mediated pathologies.

Cellular and Molecular Engineering of Glycan Sialylation in Heterologous Systems

Glycans have been shown to play a key role in many biological processes, such as signal transduction, immunogenicity, and disease progression. Among the various glycosylation modifications found on cell surfaces and in biomolecules, sialylation is especially important, because sialic acids are typically found at the terminus of glycans and have unique negatively charged moieties associated with cellular and molecular interactions. Sialic acids are also crucial for glycosylated biopharmaceutics, where they promote stability and activity. In this regard, heterogenous sialylation may produce variability in efficacy and limit therapeutic applications. Homogenous sialylation may be achieved through cellular and molecular engineering, both of which have gained traction in recent years. In this paper, we describe the engineering of intracellular glycosylation pathways through targeted disruption and the introduction of carbohydrate active enzyme genes. The focus of this review is on sialic acid-related genes and efforts to achieve homogenous, humanlike sialylation in model hosts. We also discuss the molecular engineering of sialyltransferases and their application in chemoenzymatic sialylation and sialic acid visualization on cell surfaces. The integration of these complementary engineering strategies will be useful for glycoscience to explore the biological significance of sialic acids on cell surfaces as well as the future development of advanced biopharmaceuticals.

Deletion of Mocos induces xanthinuria with obstructive nephropathy and major metabolic disorders in mice

Background: Xanthinuria type II is a rare autosomal purine disorder. This recessive defect of purine metabolism remains an underrecognized disorder. Methods: Mice with targeted disruption of the molybdenum cofactor sulfurase (Mocos) gene were generated to enable an integrated understanding of purine disorders and evaluate pathophysiological functions of this gene found in large number of pathways and known to be associated with autism. Results: Mocos deficient mice die with 4 weeks of age due to renal failure of distinct obstructive nephropathy with xanthinuria, xanthine deposits, cystic tublular dilatation, Tamm Horsfall (uromodulin) protein deposits, tubular cell necrosis with neutrophils and occasionally hypdronephrosis with urolithiasis. Obstructive nephropathy is associated with moderate interstitial inflammatory and fibrotic responses, anemia, reduced detoxification systems and important alterations of the metabolism of purines, amino acids and phospholipids.Conversely, heterozygous mice expressing reduced MOCOS protein are healthy with no apparent pathology. Conclusions: Mocos deficient mice develop a lethal obstructive nephropathy associated with profound metabolic changes. Studying MOCOS functions may provide important clues about the underlying pathogenesis of xanthinuria and other diseases requiring early diagnosis

Targeted Disruption of the Inhibitor of DNA Binding 4 (Id4) Gene Alters Photic Entrainment of the Circadian Clock

Inhibitor of DNA binding (Id) genes comprise a family of four helix–loop–helix (HLH) transcriptional inhibitors. Our earlier studies revealed a role for ID2 within the circadian system, contributing to input, output, and core clock function through its interaction with CLOCK and BMAL1. Here, we explore the contribution of ID4 to the circadian system using a targeted disruption of the Id4 gene. Attributes of the circadian clock were assessed by monitoring the locomotor activity of Id4−/− mice, and they revealed disturbances in its operation. Id4-mutant mice expressed a shorter circadian period length, attenuated phase shifts in responses to continuous and discrete photic cues, and an advanced phase angle of entrainment under a 12:12 light:dark cycle and under short and long photoperiods. To understand the basis for these properties, suprachiasmatic nucleus (SCN) and retinal structures were examined. Anatomical analysis reveals a smaller Id4−/− SCN in the width dimension, which is a finding consistent with its smaller brain. As a result of this feature, anterograde tracing in Id4−/− mice revealed retinal afferents innovate a disproportionally larger SCN area. The Id4−/− photic entrainment responses are unlikely to be due to an impaired function of the retinal pathways since Id4−/− retinal anatomy and function tested by pupillometry were similar to wild-type mice. Furthermore, these circadian characteristics are opposite to those exhibited by the Id2−/− mouse, suggesting an opposing influence of the ID4 protein within the circadian system; or, the absence of ID4 results in changes in the expression or activity of other members of the Id gene family. Expression analysis of the Id genes within the Id4−/− SCN revealed a time-of-day specific elevated Id1. It is plausible that the increased Id1 and/or absence of ID4 result in changes in interactions with bHLH canonical clock components or with targets upstream and/or downstream of the clock, thereby resulting in abnormal properties of the circadian clock and its entrainment.

In Vitro Inhibition of Influenza Virus Using CRISPR/Cas13a in Chicken Cells

Advances in the field of CRISPR/Cas systems are expanding our ability to modulate cellular genomes and transcriptomes precisely and efficiently. Here, we assessed the Cas13a-mediated targeted disruption of RNA in chicken fibroblast DF1 cells. First, we developed a Tol2 transposon vector carrying the Cas13a-msGFP-NLS (pT-Cas13a) transgene, followed by a stable insertion of the Cas13a transgene into the genome of DF1 cells to generate stable DF1-Cas13a cells. To assess the Cas13a-mediated functional knockdown, DF1-Cas13a cells were transfected with the combination of a plasmid encoding DsRed coding sequence (pDsRed) and DsRed-specific crRNA (crRNA-DsRed) or non-specific crRNA (crRNA-NS). Fluorescence-activated cell sorting (FACS) and a microscopy analysis showed reduced levels of DsRed expression in cells transfected with crRNA-DsRed but not in crRNA-NS, confirming a sequence-specific Cas13a mediated mRNA knockdown. Next, we designed four crRNAs (crRNA-IAV) against the PB1, NP and M genes of influenza A virus (IAV) and cloned in tandem to express from a single vector. DF1-Cas13a cells were transfected with plasmids encoding the crRNA-IAV or crRNA-NS, followed by infection with WSN or PR8 IAV. DF1 cells transfected with crRNA-IAV showed reduced levels of viral titers compared to cells transfected with crRNA-NS. These results demonstrate the potential of Cas13a as an antiviral strategy against highly pathogenic strains of IAV in chickens.