Polymer formulated self-amplifying RNA vaccine is partially protective against influenza virus infection in ferrets.

COVID-19 has demonstrated the power of RNA vaccines as part of a pandemic response toolkit. Another virus with pandemic potential is influenza. Further development of RNA vaccines in advance of a future influenza pandemic will save time and lives. As RNA vaccines require formulation to enter cells and induce antigen expression, the aim of this study was to investigate the impact of a recently developed bioreducible cationic polymer, pABOL for the delivery of a self-amplifying RNA (saRNA) vaccine for seasonal influenza virus in mice and ferrets. Mice and ferrets were immunized with pABOL formulated saRNA vaccines expressing either haemagglutinin (HA) from H1N1 or H3N2 influenza virus in a prime boost regime. Antibody responses, both binding and functional were measured in serum after immunization. Animals were then challenged with a matched influenza virus either directly by intranasal inoculation or in a contact transmission model. While highly immunogenic in mice, pABOL-formulated saRNA led to variable responses in ferrets. Animals that responded to the vaccine with higher levels of influenza virus-specific neutralizing antibodies were more protected against influenza virus infection. pABOL-formulated saRNA is immunogenic in ferrets, but further optimization of RNA vaccine formulation and constructs is required to increase the quality and quantity of the antibody response to the vaccine.

Impact of mutations within the [Fe-S] cluster or the lipoic acid biosynthesis pathways on mitochondrial protein expression profiles in fibroblasts from patients.

Lipoic acid (LA) is the cofactor of the E2 subunit of mitochondrial ketoacid dehydrogenases and plays a major role in oxidative decarboxylation. De novo LA biosynthesis is dependent on LIAS activity together with LIPT1 and LIPT2. LIAS is an iron­sulfur (Fe-S) cluster-containing mitochondrial protein, like mitochondrial aconitase (mt-aco) and some subunits of respiratory chain (RC) complexes I, II and III. All of them harbor at least one [Fe-S] cluster and their activity is dependent on the mitochondrial [Fe-S] cluster (ISC) assembly machinery. Disorders in the ISC machinery affect numerous Fe-S proteins and lead to a heterogeneous group of diseases with a wide variety of clinical symptoms and combined enzymatic defects. Here, we present the biochemical profiles of several key mitochondrial [Fe-S]-containing proteins in fibroblasts from 13 patients carrying mutations in genes encoding proteins involved in either the lipoic acid (LIPT1 and LIPT2) or mitochondrial ISC biogenesis (FDX1L, ISCA2, IBA57, NFU1, BOLA3) pathway. Ten of them are new patients described for the first time. We confirm that the fibroblast is a good cellular model to study these deficiencies, except for patients presenting mutations in FDX1L and a muscular clinical phenotype. We find that oxidative phosphorylation can be affected by LA defects in LIPT1 and LIPT2 patients due to excessive oxidative stress or to another mechanism connecting LA and respiratory chain activity. We confirm that NFU1, BOLA3, ISCA2 and IBA57 operate in the maturation of [4Fe-4S] clusters and not in [2Fe-2S] protein maturation. Our work suggests a functional difference between IBA57 and other proteins involved in maturation of [Fe-S] proteins. IBA57 seems to require BOLA3, NFU1 and ISCA2 for its stability and NFU1 requires BOLA3. Finally, our study establishes different biochemical profiles for patients according to their mutated protein.

A novel flexible cuff-like microelectrode for dual purpose, acute and chronic electrical interfacing with the mouse cervical vagus nerve.

OBJECTIVE: Neural reflexes regulate immune responses and homeostasis. Advances in bioelectronic medicine indicate that electrical stimulation of the vagus nerve can be used to treat inflammatory disease, yet the understanding of neural signals that regulate inflammation is incomplete. Current interfaces with the vagus nerve do not permit effective chronic stimulation or recording in mouse models, which is vital to studying the molecular and neurophysiological mechanisms that control inflammation homeostasis in health and disease. We developed an implantable, dual purpose, multi-channel, flexible 'microelectrode' array, for recording and stimulation of the mouse vagus nerve. APPROACH: The array was microfabricated on an 8 µm layer of highly biocompatible parylene configured with 16 sites. The microelectrode was evaluated by studying the recording and stimulation performance. Mice were chronically implanted with devices for up to 12 weeks. MAIN RESULTS: Using the microelectrode in vivo, high fidelity signals were recorded during physiological challenges (e.g potassium chloride and interleukin-1ß), and electrical stimulation of the vagus nerve produced the expected significant reduction of blood levels of tumor necrosis factor (TNF) in endotoxemia. Inflammatory cell infiltration at the microelectrode 12 weeks of implantation was limited according to radial distribution analysis of inflammatory cells. SIGNIFICANCE: This novel device provides an important step towards a viable chronic interface for cervical vagus nerve stimulation and recording in mice.

Cracking the neural code, treating paralysis and the future of bioelectronic medicine.

The human nervous system is a vast network carrying not only sensory and movement information, but also information to and from our organs, intimately linking it to our overall health. Scientists and engineers have been working for decades to tap into this network and 'crack the neural code' by decoding neural signals and learning how to 'speak' the language of the nervous system. Progress has been made in developing neural decoding methods to decipher brain activity and bioelectronic technologies to treat rheumatoid arthritis, paralysis, epilepsy and for diagnosing brain-related diseases such as Parkinson's and Alzheimer's disease. In a recent first-in-human study involving paralysis, a paralysed male study participant regained movement in his hand, years after his injury, through the use of a bioelectronic neural bypass. This work combined neural decoding and neurostimulation methods to translate and re-route signals around damaged neural pathways within the central nervous system. By extending these methods to decipher neural messages in the peripheral nervous system, status information from our bodily functions and specific organs could be gained. This, one day, could allow real-time diagnostics to be performed to give us a deeper insight into a patient's condition, or potentially even predict disease or allow early diagnosis. The future of bioelectronic medicine is extremely bright and is wide open as new diagnostic and treatment options are developed for patients around the world.

Evolution of psychosocial factors at work in a French region.

BACKGROUND: Psychosocial factors at work (PFW) can be defined as all non-physicochemical occupational risks. Several epidemiological models have been proposed to measure PFW, but one of the most widely used is Karasek's model. AIMS: To determine whether psychosocial factors, evaluated by Karasek's questionnaire, had increased in a cohort of workers. METHODS: A random sample of workers in the Pays de la Loire region of France, who could be considered representative of the region's population of salaried workers, filled in a self-administered questionnaire, including Karasek's self-administered questionnaire, in 2002-05 and 2007-09. Karasek's questionnaire can be used to study three psychosocial dimensions (psychological demand, decision latitude and social support in the workplace) in workers in order to define two high-risk situations for their health: 'Job Strain' and 'Iso Strain'. Changes in job strain and iso strain among workers were studied according to the workers' sociodemographic characteristics and their working conditions. RESULTS: In this sample of 2049 workers, the proportion with iso strain increased between the two periods from 12 to 16%, P < 0.001, mainly among manual workers. Deterioration of Karasek indicators was mainly explained by an increase of the 'low social support' dimension (38 versus 49%, P < 0.001). Working conditions such as temporary employment of colleagues and high perceived physical exertion were associated with higher PFW. CONCLUSIONS: This study, based on a quantitative and collective model, showed deterioration of working team environments and increased risk for individual mental health in this cohort of French workers in recent years.

Contraception and screening for cervical and breast cancer in neuromuscular disease: a retrospective study of 50 patients monitored at a clinical reference centre.

OBJECTIVE: To analyse contraceptive methods and the extent of screening for breast and cervical cancer in women with neuromuscular disease, compare these results with data and guidelines for the general population and determine the environmental and attitudinal barriers encountered. PATIENTS AND METHODS: A retrospective, descriptive study in a population of female neuromuscular disease patients (aged 20 to 74) monitored at a clinical reference centre. RESULTS: Complete datasets were available for 49 patients. Seventy percent used contraception (hormonal contraception in most cases). Sixty-eight percent had undergone screening for cervical cancer at some time in the previous 3 years and 100% of the patients over 50 had undergone a mammography. Architectural accessibility and practical problems were the most common barriers to care and were more frequently encountered by wheelchair-bound, ventilated patients. CONCLUSIONS: In general, the patients had good access to contraceptive care and cervical and breast cancer screening. However, specific measures may be useful for the most severely disabled patients.

Management of low back pain in primary care prior to multidisciplinary functional restoration: a retrospective study of 72 patients.

OBJECTIVE: Chronic low back pain is a major socioeconomic health issue, due to the high direct (healthcare) and indirect (sick leave) costs. The aim of the present study was to describe the primary care management of low back pain patients prior to their inclusion in a multidisciplinary functional restoration network. METHODS: A descriptive, retrospective, questionnaire-based survey of the general practitioners dealing with 72 low back pain patients. RESULTS: Patients had been monitored by their general practitioner for an average of four years, with a mean frequency of eight appointments per year per patient. Ninety-three percent and 60% of the patients had been referred to a rheumatologist and a surgeon, respectively. Ninety-eight percent had had lumbar radiographies, 80% had undergone a computed tomography scan and 64% had undergone magnetic resonance imaging. The most commonly prescribed medications were anti-inflammatories and first- or second-line analgesics. Thirty percent had already received morphine analgesics and 50% had taken antidepressants. Thirty-two percent had undergone lumbar surgery. Physiotherapy was frequently reported and, indeed, 6% of patients had participated in over 100 sessions. Total sick leave averaged 8.25 months over the study's follow-up period. CONCLUSION: The time interval before referral to a multidisciplinary care team is long and so GPs should be encouraged and helped to organize this process earlier. It is also essential to determine factors which predict progression to chronic LBP.

[Effect of the antioxidants on NO-dependent induction of heme oxigenase 1 gene in U937 monocytes].

Previously it was shown that thiol antioxidants are potent inhibitors of the NO-dependent induction of heme oxygenase 1 (HOX-1) gene. However, the mechanism of HOX-1 gene down-regulation by thiol antioxidants and underlying signaling pathway remain unclear. In this study we have examined, whether the scavenging of reactive oxygen and reactive nitrogen species (ROS and RNS) is the major cause for thiol-mediated suppression of the HOX-1 induction by NO. Further, to identify the ROS family members implicated in the HOX-1 induction, we also exposed cells to various non-thiol antioxidants: dimethyl sulfoxide, dimetylthiourea, sodium salicylate, sodium formate, uric acid, catalase, and superoxide dismutase. A partial inhibition of HOX-1 induction occurred in the presence of non-polar hydroxyl radical scavengers, dimethyl sulfoxide and dimetylthiourea. The other non-thiol antioxidants were ineffective towards HOX-1 expression. Then, in order to determine, whether RNS scavenging is implicated in the HOX-1 down-regulation by thiol antioxidants, we took advantage of the capacity of suboptimal concentrations of the NO scavenger PTIO (2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide) to oxidize NO to nitrosating species. We showed that simultaneous cell treatment with NO donor and PTIO significantly enhanced the rate of the HOX-1 gene NO-dependent induction indicating that RNS are mediators of HOX-1 gene transcriptional activation. Thiol antioxidants completely suppressed PTIO stimulatory action. These findings imply that inhibitory action of thiol antioxidants is mediated by RNS scavenging. The study provides an approach for pharmacologycal modulation of cell response to NO and its derivatives through the use of antioxidants.

Microarray analysis of differential gene expression in lead-exposed astrocytes.

The toxic metal lead is a widespread environmental health hazard that can adversely affect human health. In an effort to better understand the cellular and molecular consequences of lead exposure, we have employed cDNA microarrays to analyze the effects of acute lead exposure on large-scale gene expression patterns in immortalized rat astrocytes. Our studies identified many genes previously reported to be differentially regulated by lead exposure. Additionally, we have identified novel putative targets of lead-mediated toxicity, including members of the family of calcium/phospholipid binding annexins, the angiogenesis-inducing thrombospondins, collagens, and tRNA synthetases. We demonstrate the ability to distinguish lead-exposed samples from control or sodium samples solely on the basis of large-scale gene expression patterns using two complementary clustering methods. We have confirmed the altered expression of candidate genes and their encoded proteins by RT-PCR and Western blotting, respectively. Finally, we show that the calcium-dependent phospholipid binding protein annexin A5, initially identified as a differentially regulated gene by our microarray analysis, is directly bound and activated by nanomolar concentrations of lead. We conclude that microarray technology is an effective tool for the identification of lead-induced patterns of gene expression and molecular targets of lead.

Emerging technologies for large-scale screening of human tissues and fluids in the study of severe psychiatric disease.

Neuropsychiatric diseases such as schizophrenia and bipolar disorder are major causes of morbidity throughout the world. Despite extensive searches, no single gene, RNA transcript, or protein has been found which can, on its own, account for these disorders. Recently, the availability of genomic tools such as cDNA microarrays, serial analysis of gene expression (SAGE) and large-scale sequencing of cDNA libraries has allowed researchers to assay biological samples for a large number of RNA transcripts. Similarly, proteomic tools allow for the quantitation of a large number of peptides and proteins. These methods include two-dimensional electrophoresis and surface-enhanced laser desorption/ionization (SELDI). We have initiated experiments which apply these techniques to the comparison of RNAs and proteins expressed in clinical samples obtained from individuals with psychiatric diseases and controls. These methods have the potential to identify pathways that are involved in the pathogenesis of complex psychiatric disorders. The characterization of these pathways may allow for the development of new methods for the diagnosis and treatment of schizophrenia, bipolar disorder, and other human psychiatric diseases.

Synaptotagmin I is a molecular target for lead.

Lead poisoning can cause a wide range of symptoms with particularly severe clinical effects on the CNS. Lead can increase spontaneous neurotransmitter release but decrease evoked neurotransmitter release. These effects may be caused by an interaction of lead with specific molecular targets involved in neurotransmitter release. We demonstrate here that the normally calcium-dependent binding characteristics of the synaptic vesicle protein synaptotagmin I are altered by lead. Nanomolar concentrations of lead induce the interaction of synaptotagmin I with phospholipid liposomes. The C2A domain of synaptotagmin I is required for lead-mediated phospholipid binding. Lead protects both recombinant and endogenous rat brain synaptotagmin I from proteolytic cleavage in a manner similar to calcium. However, lead is unable to promote the interaction of either recombinant or endogenous synaptotagmin I and syntaxin. Finally, nanomolar concentrations of lead are able to directly compete with and inhibit the ability of micromolar concentrations of calcium to induce the interaction of synaptotagmin I and syntaxin. Based on these findings, we conclude that synaptotagmin I may be an important, physiologically relevant target of lead.

Nitric oxide-inducible expression of heme oxygenase-1 in human cells. Translation-independent stabilization of the mRNA and evidence for direct action of nitric oxide.

Expression of heme oxygenase-1 (HO-1) in mammalian cells contributes to resistance to various types of free radical damage. Nitric oxide (NO) induces HO-1 in many cell types, but the specific contribution of transcriptional or post-transcriptional effects to this induction have remained unresolved. Here we show that the extent of HO-1 mRNA expression in IMR-90 and HeLa cells depends on the rate of NO delivery, and that the induction occurs more slowly in HeLa than in human fibroblast (IMR-90) cells. We used a specific NO scavenger (2-(4-carboxylphenyl)-4,4,5,5-tetramethylimidazolin-1-oxyl 3-oxide) that completely prevented the inducible expression of HO-1 by NO, pointing to direct signaling action of NO in this induction. By inhibiting transcription during the NO exposure, we have confirmed that NO treatment activates a mechanism that stabilizes HO-1 mRNA. The increase in the HO-1 mRNA half-life in IMR-90 cells was directly correlated with increasing rates of NO release. We also show here that the stabilization of the HO-1 message does not require de novo protein synthesis. Collectively, these results show that stabilization of HO-1 mRNA can be finely tuned to the NO exposure, and that the effect in human fibroblasts is mediated by a pre-existing protein.

Induction of vascular endothelial growth factor in human astrocytes by lead. Involvement of a protein kinase C/activator protein-1 complex-dependent and hypoxia-inducible factor 1-independent signaling pathway.

The mechanism(s) underlying lead neurotoxicity are not fully elucidated. cDNA expression microarray analysis identified lead-sensitive genes in immortalized human fetal astrocytes (SV-FHA). Of the represented genes expressed, vascular endothelial growth factor (VEGF) was one of the most sensitive. Lead induced VEGF mRNA 3-fold and VEGF protein approximately 2-fold with maximum mRNA induction following incubation with 10 micrometer lead acetate for 24 h. Phorbol 12-myristate 13-acetate (PMA), a potent protein kinase C (PKC) activator, increased VEGF mRNA 2-fold and PKC inhibition by GF-109203 completely blocked VEGF induction by lead. Expression of dominant-negative PKC-epsilon, but not PKC-alpha, completely inhibited VEGF mRNA induction by lead. Lead activated the transcription factor AP-1 and increased AP-1-dependent luciferase expression >2-fold. Transfection of cells with a c-jun dominant-negative effectively inhibited both AP-1 activation and VEGF mRNA induction by lead. Hypoxia-inducible factor 1 (HIF-1) activity in SV-FHAs was moderately increased by lead (86%) and PMA (96%). Pretreatment with GF-109203 completely inhibited these effects of lead and PMA. However, lead did not alter HIF-1-dependent luciferase expression and a HIF-1alpha dominant-negative had no effects on the induction of VEGF mRNA by lead. These findings indicate that lead induces VEGF expression in SV-FHAs via a PKC/AP-1-dependent and HIF-1-independent signaling pathway.

High throughput analysis of gene expression in the human brain.

The human brain is thought to have the greatest complexity of gene expression of any region of the body, reflecting the diverse functions of neurons and glia. Studies of gene expression in the human brain may yield fundamental information about the phenotype of brain cells in different stages of development, in different brain regions, and in different physiological and pathological states. As the human genome project nears completion, several technological advances allow the analysis of thousands of expressed genes in a small brain sample. This review describes available sources of human brain material, and several high throughput techniques used to measure the expression of thousands of genes. These techniques include expressed sequence tag (EST) sequencing of cDNA libraries; differential display; subtractive hybridization; serial analysis of gene expression (SAGE); and the emerging technology of high density DNA microarrays. Measurement of gene expression with microarrays and other technologies has potential applications in the study of human brain diseases, including cognitive disorders for which animal models are typically not available. Gene expression measurements may be used to identify genes that are abnormally regulated as a secondary consequence of a disease state, or to identify the response of brain cells to pharmacological treatments.

Effects of lead on gene expression.

Lead poisoning is a worldwide, environmental health-hazard that affects children and adults. In this review we discuss the effects of lead on gene expression due to both general and specific mechanisms. In particular we focus on the ability of lead to substitute for biologically essential metals such as calcium and zinc in metal-binding domains of cytoplasmic enzymes, nuclear transcription factors and other proteins. The binding of lead to these proteins causes an alteration of their activity resulting in aberrant expression of their own genes and in some cases their target genes. Finally, we discuss the impact of microarray technology on the study of the genome-wide effects of lead and other toxicants on gene expression.

DRAGON: Database Referencing of Array Genes Online.

UNLABELLED: "Database Referencing of Array Genes ONline" or "DRAGON" is a web-accessible database that aids in the analysis of differential gene expression data as a biological annotation tool. Users of DRAGON can submit data sets containing large lists of genes and then choose particular characteristics that DRAGON supplies for all genes on the list rapidly and simultaneously. AVAILABILITY: The DRAGON database is available for queries on the DRAGON web site www.kennedykrieger.org/pevsnerlab/dragon.htm. CONTACT: pevsner@kennedykrieger.org or cbouton@jhmi.edu

Nitrosative and oxidative modulation of iron regulatory proteins.

Cytokine-driven nitric oxide (NO) synthase II provides cells with effectors for reactions at redox-sensitive site(s) of proteins. Iron regulatory proteins (IRP1 and IRP2), two post-transcriptional regulators of gene expression, are particularly sensitive to NO synthesis and to oxidative stress. IRP1 possesses a redox-active Fe-S cluster and can also exhibit aconitase activity. IRP2 has no Fe-S cluster but exhibits several redox-sensitive cysteine residues. Under proper redox conditions, both IRPs bind to iron-responsive elements in the untranslated region of mRNAs encoding proteins involved in iron metabolism and energy production. This review describes and compares the effects of NO, peroxynitrite, and reactive oxygen species on these two chemosensitive proteins.

Cytonuclear disequilibria in wild populations of rabbit (Oryctolagus cuniculus L.) suggest unequal allele turnover rates at the b locus (IGKC1).

DNA sequence comparisons suggest that evolutionary rates at the rabbit IGKC1 locus can differ among allelic lineages. Here we address the question of whether population turnover rates can vary among IGKC1 alleles. We studied the distribution of sixteen IGKC1 (or b-locus) allotypes in areas comprising the aboriginal species range (Iberian peninsula). Rabbits in this area belong to one of two distantly related mitochondrial lineages (mtDNA types) A and B. In the more recent distribution area of the species, all rabbits belong to the mtDNA type B lineage, and IGKC1 alleles b4 and b5 comprise over 90% of the gene pool. These two alleles are also predominant in areas of mtDNA type B prevalence within the Iberian range. However, in areas of mtDNA type A prevalence, the b4 and b5 allotypes are rare or absent; they apparently have been replaced by serologically related, but distinct, 'endemic' variants. The cytonuclear disequilibria were highly significant, also within the subsample consisting of populations from Spain. These observations suggest that allelic persistence times for the predominant IGKC1 lineages could be shorter than the divergence time of the major mtDNA lineages A and B. In contrast, the relative gene frequencies of the IGKC1 allele b9 were similar among the type A and type B rabbits; it was present in most populations at low frequency. In consequence, persistence times of the b9 allele appear to be longer than the divergence time of lineages A and B. The data reported here are in agreement with the DNA sequence data, providing further proof that the molecular clock can run at different rates among allelic lineages at the rabbit IGKC1 locus.

Thioredoxin activation of iron regulatory proteins. Redox regulation of RNA binding after exposure to nitric oxide.

Iron regulatory proteins (IRP1 and IRP2) are redox-sensitive RNA-binding proteins that modulate the expression of several genes encoding key proteins of iron metabolism. IRP1 can also exist as an aconitase containing a [4Fe-4S] cluster bound to three cysteines at the active site. We previously showed that biosynthesis of nitric oxide (NO) induces the transition of IRP1 from aconitase to apoprotein able to bind RNA. This switch is also observed when cytosolic extracts are exposed to NO donors. However, the activation of IRP1 under these conditions is far from maximal. In this study we examined the capacity of physiological reducing systems to cooperate with NO in the activation of IRP1. Cytosolic extracts from the macrophage cell line RAW 264.7 or purified IRP1 were incubated with NO donors and subsequently exposed to glutathione or to thioredoxin (Trx), a strong protein disulfide reductase. Trx was the most effective, inducing a 2-6-fold enhancement of the RNA binding activity of NO-treated IRP1. Furthermore, the effect of NO on IRP1 from cytosolic extracts was abolished in the presence of anti-Trx antibodies. We also studied the combined effect of NO and Trx on IRP2, which exhibits constitutive RNA binding activity. We observed an inhibition of IRP2 activity following exposure to NO donors which was restored by Trx. Collectively, these results point to a crucial role of Trx as a modulator of IRP activity in situations of NO production.