Mitofusins and OPA1 Mediate Sequential Steps in Mitochondrial Membrane Fusion

Mitochondrial fusion requires the coordinated fusion of the outer and inner membranes. Three large GTPases—OPA1 and the mitofusins Mfn1 and Mfn2—are essential for the fusion of mammalian mitochondria. OPA1 is mutated in dominant optic atrophy, a neurodegenerative disease of the optic nerve. In yeast, the OPA1 ortholog Mgm1 is required for inner membrane fusion in vitro; nevertheless, yeast lacking Mgm1 show neither outer nor inner membrane fusion in vivo, because of the tight coupling between these two processes. We find that outer membrane fusion can be readily visualized in OPA1-null mouse cells in vivo, but these events do not progress to inner membrane fusion. Similar defects are found in cells lacking prohibitins, which are required for proper OPA1 processing. In contrast, double Mfn-null cells show neither outer nor inner membrane fusion. Mitochondria in OPA1-null cells often contain multiple matrix compartments bounded together by a single outer membrane, consistent with uncoupling of outer versus inner membrane fusion. In addition, unlike mitofusins and yeast Mgm1, OPA1 is not required on adjacent mitochondria to mediate membrane fusion. These results indicate that mammalian mitofusins and OPA1 mediate distinct sequential fusion steps that are readily uncoupled, in contrast to the situation in yeast

Comparative transcriptomics of multidrug-resistant Acinetobacter baumannii in response to antibiotic treatments

Abstract Multidrug-resistant Acinetobacter baumannii, a major hospital-acquired pathogen, is a serious health threat and poses a great challenge to healthcare providers. Although there have been many genomic studies on the evolution and antibiotic resistance of this species, there have been very limited transcriptome studies on its responses to antibiotics. We conducted a comparative transcriptomic study on 12 strains with different growth rates and antibiotic resistance profiles, including 3 fast-growing pan-drug-resistant strains, under separate treatment with 3 antibiotics, namely amikacin, imipenem, and meropenem. We performed deep sequencing using a strand-specific RNA-sequencing protocol, and used de novo transcriptome assembly to analyze gene expression in the form of polycistronic transcripts. Our results indicated that genes associated with transposable elements generally showed higher levels of expression under antibiotic-treated conditions, and many of these transposon-associated genes have previously been linked to drug resistance. Using co-expressed transposon genes as markers, we further identified and experimentally validated two novel genes of which overexpression conferred significant increases in amikacin resistance. To the best of our knowledge, this study represents the first comparative transcriptomic analysis of multidrug-resistant A. baumannii under different antibiotic treatments, and revealed a new relationship between transposons and antibiotic resistance

The Clinical Utility of Zinc Transporter 8 Autoantibody Measurement in Diabetes

Maturity onset diabetes of the young (MODY) is caused by single gene mutations that are of autosomal dominant inheritance. Mutations are highly penetrant, and patients often develop a phenotype similar to type 1 or type 2 diabetes. Glucokinase, Hepatic nuclear factor 1a and 4a mutations consists of 80% of MODY cases. Approximately 1% of patients with diabetes have MODY, and it is often misdiagnosed. Diagnosis is important as patients with MODY often have a good prognosis and glycaemic control if they are treated appropriately. The aim of this thesis was to explore the use of islet autoantibodies, in particular a new autoantibody against Zinc Transporter 8, as biomarkers to identify MODY. A literature review of MODY and its important subtypes are discussed. It highlights the major mutation that cause MODY and the management of patients with MODY is also explored. Islet autoantibodies will also be reviewed in the same chapter, with a discussion on established autoantibodies and ZnT8 autoantibodies in relation to type 1 diabetes. Chapter 1 aims to investigate whether ZnT8 autoantibodies are similar to established autoantibodies against GAD and IA-2 as a biomarker in differentiating T1D patients from MODY patients. The prevalence of ZnT8 autoantibodies in MODY patients and the effect of disease duration on antibody prevalence and discriminative power would also be investigated. In Chapter 2, a study was performed to investigate whether islet autoantibodies are useful in the MODY referral setting in ruling out patients for genetic testing. This is a way to rationalise genetic testing at the Exeter molecular genetics referral service. Additionally, other biomarkers will also be investigated, namely C-peptide levels and Type 1 Diabetes Genetic Risk score. Results from the study will have implications to how MODY is diagnosed at the referral service. A discussion of the findings of each chapter, implications and plans for future research will be explored in chapter 3

Effect of D222G Mutation in the Hemagglutinin Protein on Receptor Binding, Pathogenesis and Transmissibility of the 2009 Pandemic H1N1 Influenza Virus

Influenza viruses isolated during the 2009 H1N1 pandemic generally lack known molecular determinants of virulence associated with previous pandemic and highly pathogenic avian influenza viruses. The frequency of the amino acid substitution D222G in the hemagglutinin (HA) of 2009 H1N1 viruses isolated from severe but not mild human cases represents the first molecular marker associated with enhanced disease. To assess the relative contribution of this substitution in virus pathogenesis, transmission, and tropism, we introduced D222G by reverse genetics in the wild-type HA of the 2009 H1N1 virus, A/California/04/09 (CA/04). A dose-dependent glycan array analysis with the D222G virus showed a modest reduction in the binding avidity to human-like (α2-6 sialylated glycan) receptors and an increase in the binding to avian-like (α2-3 sialylated glycan) receptors in comparison with wild-type virus. In the ferret pathogenesis model, the D222G mutant virus was found to be similar to wild-type CA/04 virus with respect to lethargy, weight loss and replication efficiency in the upper and lower respiratory tract. Moreover, based on viral detection, the respiratory droplet transmission properties of these two viruses were found to be similar. The D222G virus failed to productively infect mice inoculated by the ocular route, but exhibited greater viral replication and weight loss than wild-type CA/04 virus in mice inoculated by the intranasal route. In a more relevant human cell model, D222G virus replicated with delayed kinetics compared with wild-type virus but to higher titer in human bronchial epithelial cells. These findings suggest that although the D222G mutation does not influence virus transmission, it may be considered a molecular marker for enhanced replication in certain cell types.Centers for Disease Control and Prevention (U.S.)United States. National Institutes of Health (merit award R37 GM057073-13)Singapore-MIT Alliance for Research and Technolog

The past, present and future challenges in epilepsy related and sudden deaths and biobanking.

Awareness and research on epilepsy-related deaths (ERD), in particular Sudden Unexpected Death in Epilepsy (SUDEP), have exponentially increased over the last two decades. Most publications have focused on guidelines that inform clinicians dealing with these deaths, educating patients, potential risk factors and mechanisms. There is a relative paucity of information available for pathologists who conduct these autopsies regarding appropriate post-mortem practice and investigations. As we move from recognizing SUDEP as the most common form of ERD toward in-depth investigations into its causes and prevention, health professionals involved with these autopsies and post-mortem procedure must remain fully informed. Systematizing a more comprehensive and consistent practice of examining these cases will facilitate 1) more precise determination of cause of death, 2) identification of SUDEP for improved epidemiological surveillance (the first step for an intervention study), and 3) bio-banking and cell-based research. This article reviews how pathologists and healthcare professionals have approached ERD, current practices, logistical problems and areas to improve and harmonize. The main neuropathology, cardiac and genetic findings in SUDEP are outlined, providing a framework for best practices, integration of clinical, pathologic and molecular genetic investigations in SUDEP, and ultimately prevention

Nanotools for Neuroscience and Brain Activity Mapping

Neuroscience is at a crossroads. Great effort is being invested into deciphering specific neural interactions and circuits. At the same time, there exist few general theories or principles that explain brain function. We attribute this disparity, in part, to limitations in current methodologies. Traditional neurophysiological approaches record the activities of one neuron or a few neurons at a time. Neurochemical approaches focus on single neurotransmitters. Yet, there is an increasing realization that neural circuits operate at emergent levels, where the interactions between hundreds or thousands of neurons, utilizing multiple chemical transmitters, generate functional states. Brains function at the nanoscale, so tools to study brains must ultimately operate at this scale, as well. Nanoscience and nanotechnology are poised to provide a rich toolkit of novel methods to explore brain function by enabling simultaneous measurement and manipulation of activity of thousands or even millions of neurons. We and others refer to this goal as the Brain Activity Mapping Project. In this Nano Focus, we discuss how recent developments in nanoscale analysis tools and in the design and synthesis of nanomaterials have generated optical, electrical, and chemical methods that can readily be adapted for use in neuroscience. These approaches represent exciting areas of technical development and research. Moreover, unique opportunities exist for nanoscientists, nanotechnologists, and other physical scientists and engineers to contribute to tackling the challenging problems involved in understanding the fundamentals of brain function

Multi-agency collaboration in flood disaster management in sarawak Malaysia

In reducing the impact of current and future disasters so as to form a resilient future, disaster management deserves highest priority and plays a vital role in managing the risk of disaster. However, it cannot be addressed or undertaken by the government alone. It requires a strong supporting multi-agency collaboration to undertake various types of tasks at various stages of the disaster management cycle which has been mentioned as a key point of professionalism in disaster management activities. To accomplish a successful multi-agency coordination in Sarawak flood disaster management, collaboration among the relevant agencies plays a vital role for aim to reduce or avoid the potential losses from hazards, assure speedy and appropriate assistance to the victims of disaster as well as achieve rapid and effective recovery. Therefore, this paper reviews the concept of collaboration and collaboration of relevant agencies in disaster management through a case study in Sarawak, Malaysia. This paper is prepared by conducting a series of literature review in order to establish a foundation for a new insight of contribution to knowledge. Content analysis, a pure qualitative method is used to identify related agencies with disaster management in the state of Sarawak, Malaysia. The content analysis was conducted by reviewing content of 39 related agencies from Sarawak. It is envisaged that the findings of this study could be clinically useful for building a successful multi-agency coordination in Sarawak, Malaysia disaster management

The G1613A Mutation in the HBV Genome Affects HBeAg Expression and Viral Replication through Altered Core Promoter Activity

Infection of hepatitis B virus (HBV) causes acute and chronic hepatitis and is closely associated with the development of cirrhosis and hepatocellular carcinoma (HCC). Previously, we demonstrated that the G1613A mutation in the HBV negative regulatory element (NRE) is a hotspot mutation in HCC patients. In this study, we further investigated the functional consequences of this mutation in the context of the full length HBV genome and its replication. We showed that the G1613A mutation significantly suppresses the secretion of e antigen (HBeAg) and enhances the synthesis of viral DNA, which is in consistence to our clinical result that the G1613A mutation associates with high viral load in chronic HBV carriers. To further investigate the molecular mechanism of the mutation, we performed the electrophoretic mobility shift assay with the recombinant RFX1 protein, a trans-activator that was shown to interact with the NRE of HBV. Intriguingly, RFX1 binds to the G1613A mutant with higher affinity than the wild-type sequence, indicating that the mutation possesses the trans-activating effect to the core promoter via NRE. The trans-activating effect was further validated by the enhancement of the core promoter activity after overexpression of RFX1 in liver cell line. In summary, our results suggest the functional consequences of the hotspot G1613A mutation found in HBV. We also provide a possible molecular mechanism of this hotspot mutation to the increased viral load of HBV carriers, which increases the risk to HCC

Automatic Physiological Waveform Processing for fMRI Noise Correction and Analysis

Functional MRI resting state and connectivity studies of brain focus on neural fluctuations at low frequencies which share power with physiological fluctuations originating from lung and heart. Due to the lack of automated software to process physiological signals collected at high magnetic fields, a gap exists in the processing pathway between the acquisition of physiological data and its use in fMRI software for both physiological noise correction and functional analyses of brain activation and connectivity. To fill this gap, we developed an open source, physiological signal processing program, called PhysioNoise, in the python language. We tested its automated processing algorithms and dynamic signal visualization on resting monkey cardiac and respiratory waveforms. PhysioNoise consistently identifies physiological fluctuations for fMRI noise correction and also generates covariates for subsequent analyses of brain activation and connectivity