Use of modified U1 snRNAs to inhibit HIV-1 replication

Control of RNA processing plays a central role in regulating the replication of HIV-1, in particular the 3′ polyadenylation of viral RNA. Based on the demonstration that polyadenylation of mRNAs can be disrupted by the targeted binding of modified U1 snRNA, we examined whether binding of U1 snRNAs to conserved 10 nt regions within the terminal exon of HIV-1 was able to inhibit viral structural protein expression. In this report, we demonstrate that U1 snRNAs complementary to 5 of the 15 regions targeted result in significant suppression of HIV-1 protein expression and viral replication coincident with loss of viral RNA. Suppression of viral gene expression is dependent upon appropriate assembly of a U1 snRNP particle as mutations of U1 snRNA that affect binding of U1 70K or Sm proteins significantly reduced efficacy. However, constructs lacking U1A binding sites retained significant anti-viral activity. This finding suggests a role for these mutants in situations where the wild-type constructs cause toxic effects. The conserved nature of the sequences targeted and the high efficacy of the constructs suggests that this strategy has significant potential as an HIV therapeutic

Diagnostics and correction of batch effects in large-scale proteomic studies: a tutorial.

Advancements in mass spectrometry-based proteomics have enabled experiments encompassing hundreds of samples. While these large sample sets deliver much-needed statistical power, handling them introduces technical variability known as batch effects. Here, we present a step-by-step protocol for the assessment, normalization, and batch correction of proteomic data. We review established methodologies from related fields and describe solutions specific to proteomic challenges, such as ion intensity drift and missing values in quantitative feature matrices. Finally, we compile a set of techniques that enable control of batch effect adjustment quality. We provide an R package, proBatch , containing functions required for each step of the protocol. We demonstrate the utility of this methodology on five proteomic datasets each encompassing hundreds of samples and consisting of multiple experimental designs. In conclusion, we provide guidelines and tools to make the extraction of true biological signal from large proteomic studies more robust and transparent, ultimately facilitating reliable and reproducible research in clinical proteomics and systems biology

The translocator protein (TSPO) is prodromal to mitophagy loss in neurotoxicity

Dysfunctional mitochondria characterise Parkinson's Disease (PD). Uncovering etiological molecules, which harm the homeostasis of mitochondria in response to pathological cues, is therefore pivotal to inform early diagnosis and therapy in the condition, especially in its idiopathic forms. This study proposes the 18 kDa Translocator Protein (TSPO) to be one of those. Both in vitro and in vivo data show that neurotoxins, which phenotypically mimic PD, increase TSPO to enhance cellular redox-stress, susceptibility to dopamine-induced cell death, and repression of ubiquitin-dependent mitophagy. TSPO amplifies the extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) signalling, forming positive feedback, which represses the transcription factor EB (TFEB) and the controlled production of lysosomes. Finally, genetic variances in the transcriptome confirm that TSPO is required to alter the autophagy-lysosomal pathway during neurotoxicity

Separation of blood microsamples by exploiting sedimentation at the microscale.

Microsample analysis is highly beneficial in blood-based testing where cutting-edge bioanalytical technologies enable the analysis of volumes down to a few tens of microliters. Despite the availability of analytical methods, the difficulty in obtaining high-quality and standardized microsamples at the point of collection remains a major limitation of the process. Here, we detail and model a blood separation principle which exploits discrete viscosity differences caused by blood particle sedimentation in a laminar flow. Based on this phenomenon, we developed a portable capillary-driven microfluidic device that separates blood microsamples collected from finger-pricks and delivers 2 µL of metered serum for bench-top analysis. Flow cytometric analysis demonstrated the high purity of generated microsamples. Proteomic and metabolomic analyses of the microsamples of 283 proteins and 1351 metabolite features was consistent with samples generated via a conventional centrifugation method. These results were confirmed by a clinical study scrutinising 8 blood markers in obese patients

The translocator protein (TSPO) is prodromal to mitophagy loss in neurotoxicity

Dysfunctional mitochondria characterise Parkinson’s Disease (PD). Uncovering etiological molecules, which harm the homeostasis of mitochondria in response to pathological cues, is therefore pivotal to inform early diagnosis and therapy in the condition, especially in its idiopathic forms. This study proposes the 18 kDa Translocator Protein (TSPO) to be one of those. Both in vitro and in vivo data show that neurotoxins, which phenotypically mimic PD, increase TSPO to enhance cellular redox-stress, susceptibility to dopamine-induced cell death, and repression of ubiquitin-dependent mitophagy. TSPO amplifies the extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) signalling, forming positive feedback, which represses the transcription factor EB (TFEB) and the controlled production of lysosomes. Finally, genetic variances in the transcriptome confirm that TSPO is required to alter the autophagy–lysosomal pathway during neurotoxicity

Mesenchymal stem cells lack efficacy in the treatment of experimental autoimmune neuritis despite in vitro inhibition of T-cell proliferation.

Mesenchymal stem cells have been demonstrated to ameliorate experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, prompting clinical trials in multiple sclerosis which are currently ongoing. An important question is whether this therapeutic effect generalises to other autoimmune neurological diseases. We performed two trials of efficacy of MSCs in experimental autoimmune neuritis (EAN) in Lewis (LEW/Han (M)Hsd) rats, a model of human autoimmune inflammatory neuropathies. No differences between the groups were found in clinical, histological or electrophysiological outcome measures. This was despite the ability of mesenchymal stem cells to inhibit proliferation of CD4+ T-cells in vitro. Therefore the efficacy of MSCs observed in autoimmune CNS demyelination models do not necessarily generalise to the treatment of other forms of neurological autoimmunity

AAV-mediated in vivo knockdown of luciferase using combinatorial RNAi and U1i

RNA interference (RNAi) has been successfully employed for specific inhibition of gene expression; however, safety and delivery of RNAi remain critical issues. We investigated the combinatorial use of RNAi and U1 interference (U1i). U1i is a gene-silencing technique that acts on the pre-mRNA by preventing polyadenylation. RNAi and U1i have distinct mechanisms of action in different cellular compartments and their combined effect allows usage of minimal doses, thereby avoiding toxicity while retaining high target inhibition. As a proof of concept, we investigated knockdown of the firefly luciferase reporter gene by combinatorial use of RNAi and U1i, and evaluated their inhibitory potential both in vitro and in vivo. Co-transfection of RNAi and U1i constructs showed additive reduction of luciferase expression up to 95% in vitro. We attained similar knockdown when RNAi and U1i constructs were hydrodynamically transfected into murine liver, demonstrating for the first time successful in vivo application of U1i. Moreover, we demonstrated long-term gene silencing by AAV-mediated transduction of murine muscle with RNAi/U1i constructs targeting firefly luciferase. In conclusion, these results provide a proof of principle for the combinatorial use of RNAi and U1i to enhance target gene knockdown in vivo

FimH Adhesin of Type 1 Fimbriae Is a Potent Inducer of Innate Antimicrobial Responses Which Requires TLR4 and Type 1 Interferon Signalling

Components of bacteria have been shown to induce innate antiviral immunity via Toll-like receptors (TLRs). We have recently shown that FimH, the adhesin portion of type 1 fimbria, can induce the innate immune system via TLR4. Here we report that FimH induces potent in vitro and in vivo innate antimicrobial responses. FimH induced an innate antiviral state in murine macrophage and primary MEFs which was correlated with IFN-β production. Moreover, FimH induced the innate antiviral responses in cells from wild type, but not from MyD88−/−, Trif−/−, IFN−α/βR−/− or IRF3−/− mice. Vaginal delivery of FimH, but not LPS, completely protected wild type, but not MyD88−/−, IFN-α/βR−/−, IRF3−/− or TLR4−/− mice from subsequent genital HSV-2 challenge. The FimH-induced innate antiviral immunity correlated with the production of IFN-β, but not IFN-α or IFN-γ. To examine whether FimH plays a role in innate immune induction in the context of a natural infection, the innate immune responses to wild type uropathogenic E. coli (UPEC) and a FimH null mutant were examined in the urinary tract of C57Bl/6 (B6) mice and TLR4-deficient mice. While UPEC expressing FimH induced a robust polymorphonuclear response in B6, but not TLR4−/− mice, mutant bacteria lacking FimH did not. In addition, the presence of TLR4 was essential for innate control of and protection against UPEC. Our results demonstrate that FimH is a potent inducer of innate antimicrobial responses and signals differently, from that of LPS, via TLR4 at mucosal surfaces. Our studies suggest that FimH can potentially be used as an innate microbicide against mucosal pathogens

Methodological advances in imaging intravital axonal transport.

Axonal transport is the active process whereby neurons transport cargoes such as organelles and proteins anterogradely from the cell body to the axon terminal and retrogradely in the opposite direction. Bi-directional transport in axons is absolutely essential for the functioning and survival of neurons and appears to be negatively impacted by both aging and diseases of the nervous system, such as Alzheimer's disease and amyotrophic lateral sclerosis. The movement of individual cargoes along axons has been studied in vitro in live neurons and tissue explants for a number of years; however, it is currently unclear as to whether these systems faithfully and consistently replicate the in vivo situation. A number of intravital techniques originally developed for studying diverse biological events have recently been adapted to monitor axonal transport in real-time in a range of live organisms and are providing novel insight into this dynamic process. Here, we highlight these methodological advances in intravital imaging of axonal transport, outlining key strengths and limitations while discussing findings, possible improvements, and outstanding questions