Synthesis, antioxidant and neuroprotective analysis of diversely functionalized α-aryl-N-alkyl nitrones as potential agents for ischemic stroke therapy.

Herein, we report the synthesis, antioxidant and biological evaluation of 32 monosubstituted α-arylnitrones derived from α-phenyl-tert-butyl nitrone (PBN) in the search for neuroprotective compounds for ischemic stroke therapy, trying to elucidate the structural patterns responsible for their neuroprotective activity. Not surprisingly, the N-tert-butyl moiety plays beneficious role in comparison to other differently N-substituted nitrone groups. It seems that electron donor substituents at the ortho position and electron withdrawing substituents at the meta position of the aryl ring induce good neuroprotective activity. As a result, (Z)-N-tert-butyl-1-(2-hydroxyphenyl)methanimine oxide (21a) and (Z)-N-tert-butyl-1-(2-(prop-2-yn-1-yloxy)phenyl)methanimine oxide (24a) showed a significant increase in neuronal viability in an experimental ischemia model in primary neuronal cultures, and induced neuroprotection and improved neurodeficit score in an in vivo model of transient cerebral ischemia. These results showed that nitrones 21a and 24a are new effective small and readily available antioxidants, and suitable candidates for further structure optimization in the search for new phenyl-derived nitrones for the treatment of ischemic stroke and related diseases.

Development of Pharmacological Strategies with Therapeutic Potential in Ischemic Stroke.

Acute ischemic stroke constitutes a health challenge with great social impact due to its high incidence, with the social dependency that it generates being an important source of inequality. The lack of treatments serving as effective neuroprotective therapies beyond thrombolysis and thrombectomy is presented as a need. With this goal in mind, our research group's collaborative studies into cerebral ischemia and subsequent reperfusion concluded that there is a need to develop compounds with antioxidant and radical scavenger features. In this review, we summarize the path taken toward the identification of lead compounds as potential candidates for the treatment of acute ischemic stroke. Evaluations of the antioxidant capacity, neuroprotection of primary neuronal cultures and in vivo experimental models of cerebral ischemia, including neurological deficit score assessments, are conducted to characterize the biological efficacy of the various neuroprotective compounds developed. Moreover, the initial results in preclinical development, including dose-response studies, the therapeutic window, the long-term neuroprotective effect and in vivo antioxidant evaluation, are reported. The results prompt these compounds for clinical trials and are encouraging regarding new drug developments aimed at a successful therapy for ischemic stroke.

Bacterial molecular machinery in the Martian cryosphere conditions.

The exploration of Mars is one of the main objectives of space missions since the red planet is considered to be, or was in the past, potentially habitable. Although the surface of Mars is now dry and arid, abundant research suggests that water covered Mars billions of years ago. Recently, the existence of liquid water in subglacial lakes has been postulated below the South pole of Mars. Until now, experiments have been carried out on the survival of microorganisms in Martian surface conditions, but it remains unknown how their adaptation mechanisms would be in the Martian cryosphere. In this work, two bacterial species (Bacillus subtilis and Curtobacterium flacumfaciens) were subjected to a simulated Martian environment during 24 h using a planetary chamber. Afterward, the molecular machinery of both species was studied to investigate how they had been modified. Proteomes, the entire set of proteins expressed by each bacterium under Earth (named standard) conditions and Martian conditions, were compared using proteomic techniques. To establish this evaluation, both the expression levels of each protein, and the variation in their distribution within the different functional categories were considered. The results showed that these bacterial species followed a different strategy. The Bacillus subtilis resistance approach consisted of improving its stress response, membrane bioenergetics, degradation of biomolecules; and to a lesser extent, increasing its mobility and the formation of biofilms or resistance endospores. On the contrary, enduring strategy of Curtobacterium flacumfaciens comprised of strengthening the cell envelope, trying to protect cells from the extracellular environment. These results are especially important due to their implications for planetary protection, missions to Mars and sample return since contamination by microorganisms would invalidate the results of these investigations.

Dihydropyrimidinase-Related Protein 2 Is a New Partner in the Binding between 4E-BP2 and eIF4E Related to Neuronal Death after Cerebral Ischemia.

Transient cerebral ischemia induces neuronal degeneration, followed in time by secondary delayed neuronal death that is strongly correlated with a permanent inhibition of protein synthesis in vulnerable brain regions, while protein translational rates are recovered in resistant areas. In the translation-regulation initiation step, the eukaryotic initiation factor (eIF) 4E is a key player regulated by its association with eIF4E-binding proteins (4E-BPs), mostly 4E-BP2 in brain tissue. In a previous work, we identified dihydropyrimidinase-related protein 2 (DRP2) as a 4E-BP2-interacting protein. Here, using a proteomic approach in a model of transient cerebral ischemia, a detailed study of DRP2 was performed in order to address the challenge of translation restoration in vulnerable regions. In this report, several DRP2 isoforms that have a specific interaction with both 4E-BP2 and eIF4E were identified, showing significant and opposite differences in this association, and being differentially detected in resistant and vulnerable regions in response to ischemia reperfusion. Our results provide the first evidence of DRP2 isoforms as potential regulators of the 4E-BP2-eIF4E association that would have consequences in the delayed neuronal death under ischemic-reperfusion stress. The new knowledge reported here identifies DRP2 as a new target to promote neuronal survival after cerebral ischemia.

The hidden microbial ecosystem in the perennial ice from a Pyrenean ice cave.

Over the last years, perennial ice deposits located within caves have awakened interest as places to study microbial communities since they represent unique cryospheric archives of climate change. Since the beginning of the twentieth century, the temperature has gradually increased, and it is estimated that by the end of this century the increase in average temperature could be around 4.0°C. In this context of global warming the ice deposits of the Pyrenean caves are undergoing a significant regression. Among this type of caves, that on the Cotiella Massif in the Southern Pyrenees is one of the southernmost studied in Europe. These types of caves house microbial communities which have so far been barely explored, and therefore their study is necessary. In this work, the microbial communities of the Pyrenean ice cave A294 were identified using metabarcoding techniques. In addition, research work was carried out to analyze how the age and composition of the ice affect the composition of the bacterial and microeukaryotic populations. Finally, the in vivo effect of climate change on the cellular machinery that allow microorganisms to live with increasing temperatures has been studied using proteomic techniques.

Improving the Efficacy of Quinolylnitrones for Ischemic Stroke Therapy, QN4 and QN15 as New Neuroprotective Agents after Oxygen-Glucose Deprivation/Reoxygenation-Induced Neuronal Injury.

In our search for new neuroprotective agents for stroke therapy to improve the pharmacological profile of the compound quinolylnitrone QN23, we have prepared and studied sixteen new, related and easily available quinolylnitrones. As a result, we have identified compounds QN4 and QN15 as promising candidates showing high neuroprotection power in a cellular experimental model of ischemia. Even though they were found to be less active than our current lead compound QN23, QN4 and QN15 provide an improved potency and, particularly for QN4, an expanded range of tolerability and improved solubility compared to the parent compound. A computational DFT-based analysis has been carried out to understand the antioxidant power of quinolylnitrones QN23, QN4 and QN15. Altogether, these results show that subtle, simple modifications of the quinolylnitrone scaffold are tolerated, providing high neuroprotective activity and optimization of the pharmacological potency required for an improved design and future drug developments in the field.

Postischemic Neuroprotection of Aminoethoxydiphenyl Borate Associates Shortening of Peri-Infarct Depolarizations.

Brain stroke is a highly prevalent pathology and a main cause of disability among older adults. If not promptly treated with recanalization therapies, primary and secondary mechanisms of injury contribute to an increase in the lesion, enhancing neurological deficits. Targeting excitotoxicity and oxidative stress are very promising approaches, but only a few compounds have reached the clinic with relatively good positive outcomes. The exploration of novel targets might overcome the lack of clinical translation of previous efficient preclinical neuroprotective treatments. In this study, we examined the neuroprotective properties of 2-aminoethoxydiphenyl borate (2-APB), a molecule that interferes with intracellular calcium dynamics by the antagonization of several channels and receptors. In a permanent model of cerebral ischemia, we showed that 2-APB reduces the extent of the damage and preserves the functionality of the cortical territory, as evaluated by somatosensory evoked potentials (SSEPs). While in this permanent ischemia model, the neuroprotective effect exerted by the antioxidant scavenger cholesteronitrone F2 was associated with a reduction in reactive oxygen species (ROS) and better neuronal survival in the penumbra, 2-APB did not modify the inflammatory response or decrease the content of ROS and was mostly associated with a shortening of peri-infarct depolarizations, which translated into better cerebral blood perfusion in the penumbra. Our study highlights the potential of 2-APB to target spreading depolarization events and their associated inverse hemodynamic changes, which mainly contribute to extension of the area of lesion in cerebrovascular pathologies.

Preclinical Characterization of Antioxidant Quinolyl Nitrone QN23 as a New Candidate for the Treatment of Ischemic Stroke.

Nitrones are encouraging drug candidates for the treatment of oxidative stress-driven diseases such as acute ischemic stroke (AIS). In a previous study, we found a promising quinolylnitrone, QN23, which exerted a neuroprotective effect in neuronal cell cultures subjected to oxygen-glucose deprivation and in experimental models of cerebral ischemia. In this paper, we update the biological and pharmacological characterization of QN23. We describe the suitability of intravenous administration of QN23 to induce neuroprotection in transitory four-vessel occlusion (4VO) and middle cerebral artery occlusion (tMCAO) experimental models of brain ischemia by assessing neuronal death, apoptosis induction, and infarct area, as well as neurofunctional outcomes. QN23 significantly decreased the neuronal death and apoptosis induced by the ischemic episode in a dose-dependent manner and showed a therapeutic effect when administered up to 3 h after post-ischemic reperfusion onset, effects that remained 11 weeks after the ischemic episode. In addition, QN23 significantly reduced infarct volume, thus recovering the motor function in a tMCAO model. Remarkably, we assessed the antioxidant activity of QN23 in vivo using dihydroethidium as a molecular probe for radical species. Finally, we describe QN23 pharmacokinetic parameters. All these results pointing to QN23 as an interesting and promising preclinical candidate for the treatment of AIS.

Phosphorylation of Eukaryotic Initiation Factor 4G1 (eIF4G1) at Ser1147 Is Specific for eIF4G1 Bound to eIF4E in Delayed Neuronal Death after Ischemia.

Ischemic strokes are caused by a reduction in cerebral blood flow and both the ischemic period and subsequent reperfusion induce brain injury, with different tissue damage depending on the severity of the ischemic insult, its duration, and the particular areas of the brain affected. In those areas vulnerable to cerebral ischemia, the inhibition of protein translation is an essential process of the cellular response leading to delayed neuronal death. In particular, translation initiation is rate-limiting for protein synthesis and the eukaryotic initiation factor (eIF) 4F complex is indispensable for cap-dependent protein translation. In the eIF4F complex, eIF4G is a scaffolding protein that provides docking sites for the assembly of eIF4A and eIF4E, binding to the cap structure of the mRNA and stabilizing all proteins of the complex. The eIF4F complex constituents, eIF4A, eIF4E, and eIF4G, participate in translation regulation by their phosphorylation at specific sites under cellular stress conditions, modulating the activity of the cap-binding complex and protein translation. This work investigates the phosphorylation of eIF4G1 involved in the eIF4E/eIF4G1 association complex, and their regulation in ischemia-reperfusion (IR) as a stress-inducing condition. IR was induced in an animal model of transient cerebral ischemia and the results were studied in the resistant cortical region and in the vulnerable hippocampal CA1 region. The presented data demonstrate the phosphorylation of eIF4G1 at Ser1147, Ser1185, and Ser1231 in both brain regions and in control and ischemic conditions, being the phosphorylation of eIF4G1 at Ser1147 the only one found in the eIF4E/eIF4G association complex from the cap-containing matrix (m7GTP-Sepharose). In addition, our work reveals the specific modulation of the phosphorylation of eIF4G1 at Ser1147 in the vulnerable region, with increased levels and colocalization with eIF4E in response to IR. These findings contribute to elucidate the molecular mechanism of protein translation regulation that underlies in the balance of cell survival/death during pathophysiological stress, such as cerebral ischemia.

Differential Association of 4E-BP2-Interacting Proteins Is Related to Selective Delayed Neuronal Death after Ischemia.

Cerebral ischemia induces an inhibition of protein synthesis and causes cell death and neuronal deficits. These deleterious effects do not occur in resilient areas of the brain, where protein synthesis is restored. In cellular stress conditions, as brain ischemia, translational repressors named eukaryotic initiation factor (eIF) 4E-binding proteins (4E-BPs) specifically bind to eIF4E and are critical in the translational control. We previously described that 4E-BP2 protein, highly expressed in brain, can be a molecular target for the control of cell death or survival in the reperfusion after ischemia in an animal model of transient cerebral ischemia. Since these previous studies showed that phosphorylation would not be the regulation that controls the binding of 4E-BP2 to eIF4E under ischemic stress, we decided to investigate the differential detection of 4E-BP2-interacting proteins in two brain regions with different vulnerability to ischemia-reperfusion (IR) in this animal model, to discover new potential 4E-BP2 modulators and biomarkers of cerebral ischemia. For this purpose, 4E-BP2 immunoprecipitates from the resistant cortical region and the vulnerable hippocampal cornu ammonis 1 (CA1) region were analyzed by two-dimensional (2-D) fluorescence difference in gel electrophoresis (DIGE), and after a biological variation analysis, 4E-BP2-interacting proteins were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Interestingly, among the 4E-BP2-interacting proteins identified, heat shock 70 kDa protein-8 (HSC70), dihydropyrimidinase-related protein-2 (DRP2), enolase-1, ubiquitin carboxyl-terminal hydrolase isozyme-L1 (UCHL1), adenylate kinase isoenzyme-1 (ADK1), nucleoside diphosphate kinase-A (NDKA), and Rho GDP-dissociation inhibitor-1 (Rho-GDI), were of notable interest, showing significant differences in their association with 4E-BP2 between resistant and vulnerable regions to ischemic stress. Our data contributes to the first characterization of the 4E-BP2 interactome, increasing the knowledge in the molecular basis of the protection and vulnerability of the ischemic regions and opens the way to detect new biomarkers and therapeutic targets for diagnosis and treatment of cerebral ischemia.

Privileged Quinolylnitrones for the Combined Therapy of Ischemic Stroke and Alzheimer's Disease.

Cerebrovascular diseases such as ischemic stroke are known to exacerbate dementia caused by neurodegenerative pathologies such as Alzheimer's disease (AD). Besides, the increasing number of patients surviving stroke makes it necessary to treat the co-occurrence of these two diseases with a single and combined therapy. For the development of new dual therapeutic agents, eight hybrid quinolylnitrones have been designed and synthesized by the juxtaposition of selected pharmacophores from our most advanced lead-compounds for ischemic stroke and AD treatment. Biological analyses looking for efficient neuroprotective effects in suitable phenotypic assays led us to identify MC903 as a new small quinolylnitrone for the potential dual therapy of stroke and AD, showing strong neuroprotection on (i) primary cortical neurons under oxygen-glucose deprivation/normoglycemic reoxygenation as an experimental ischemia model; (ii), neuronal line cells treated with rotenone/oligomycin A, okadaic acid or ß-amyloid peptide Aß25-35, modeling toxic insults found among the effects of AD.

Proteomic analysis of plasma proteins of high-flux haemodialysis and on-line haemodiafiltration patients reveals differences in transthyretin levels related with anaemia.

A large proportion of end-stage renal disease (ESRD) patients under long-term haemodialysis, have persistent anaemia and require high doses of recombinant human erythropoietin (rhEPO). However, the underlying mechanisms of renal anaemia have not been fully elucidated in these patients. In this study, we will be focusing on anaemia and plasma proteins in ESRD patients on high-flux haemodialysis (HF) and on-line haemodiafiltration (HDF), to investigate using two proteomic approaches if patients undergoing these treatments develop differences in their plasma protein composition and how this could be related to their anaemia. The demographic and biochemical data revealed that HDF patients had lower anaemia and much lower rhEPO requirements than HF patients. Regarding their plasma proteomes, HDF patients had increased levels of a protein highly similar to serotransferrin, trypsin-1 and immunoglobulin heavy constant chain alpha-1, and lower levels of alpha-1 antitrypsin, transthyretin, apolipoproteins E and C-III, and haptoglobin-related protein. Lower transthyretin levels in HDF patients were further confirmed by transthyretin-peptide quantification and western blot detection. Since ESRD patients have increased transthyretin, a protein that can aggregate and inhibit transferrin endocytosis and erythropoiesis, our finding that HDF patients have lower transthyretin and lower anaemia suggests that the decrease in transthyretin plasma levels would allow an increase in transferrin endocytosis, contributing to erythropoiesis. Thus, transthyretin could be a critical actor for anaemia in ESRD patients and a novel player for haemodialysis adequacy.

Characterization of a CholesteroNitrone (ISQ-201), a Novel Drug Candidate for the Treatment of Ischemic Stroke.

Nitrones have a well-recognized capacity as spin-traps and are considered powerful free radical scavengers, which are two important issues in hypoxia-induced oxidative stress and cell death in brain ischemia. Consequently, nitrones have been proposed as therapeutic agents in acute ischemic stroke (AIS). In this paper, we update the biological and pharmacological characterization of ISQ-201, a previously identified cholesteronitrone hybrid with antioxidant and neuroprotective activity. This study characterizes ISQ-201 as a neuroprotective agent against the hypoxia-induced ischemic injury. Transitory four-vessel occlusion and middle cerebral artery occlusion (tMCAO) were used to induce cerebral ischemia. Functional outcomes were determined using neurofunctional tests. Infarct area, neuronal death, and apoptosis induction were evaluated. In addition, ISQ-201 reactivity towards free radicals was studied in a theoretical model. ISQ-201 significantly decreased the ischemia-induced neuronal death and apoptosis, in a dose-dependent manner, showing its therapeutic effect when administered up until 6 h after post-ischemic reperfusion onset, effects that remained after 3 months from the ischemic episode. Furthermore, ISQ-201 significantly reduced infarct volume, leading to recovery of the motor function in the tMCAO model. Finally, the theoretical study confirmed the reactivity of ISQ-201 towards hydroxyl radicals. The results reported here prompted us to suggest ISQ-201 as a promising candidate for the treatment of AIS.

Taxonomic and functional characterization of a microbial community from a volcanic englacial ecosystem in Deception Island, Antarctica.

Glaciers are populated by a large number of microorganisms including bacteria, archaea and microeukaryotes. Several factors such as solar radiation, nutrient availability and water content greatly determine the diversity and abundance of these microbial populations, the type of metabolism and the biogeochemical cycles. Three ecosystems can be differentiated in glaciers: supraglacial, subglacial and englacial ecosystems. Firstly, the supraglacial ecosystem, sunlit and oxygenated, is predominantly populated by photoautotrophic microorganisms. Secondly, the subglacial ecosystem contains a majority of chemoautotrophs that are fed on the mineral salts of the rocks and basal soil. Lastly, the englacial ecosystem is the least studied and the one that contains the smallest number of microorganisms. However, these unknown englacial microorganisms establish a food web and appear to have an active metabolism. In order to study their metabolic potentials, samples of englacial ice were taken from an Antarctic glacier. Microorganisms were analyzed by a polyphasic approach that combines a set of -omic techniques: 16S rRNA sequencing, culturomics and metaproteomics. This combination provides key information about diversity and functions of microbial populations, especially in rare habitats. Several whole essential proteins and enzymes related to metabolism and energy production, recombination and translation were found that demonstrate the existence of cellular activity at subzero temperatures. In this way it is shown that the englacial microorganisms are not quiescent, but that they maintain an active metabolism and play an important role in the glacial microbial community.

Novel Quinolylnitrones Combining Neuroprotective and Antioxidant Properties.

We describe here the preparation, neuroprotective analysis, and antioxidant capacity of 11 novel quinolylnitrones (QN). The neuroprotective analysis of QN1-11 in an oxygen-glucose deprivation model, in primary neuronal cultures, has been determined, allowing us to identify QN6 as a very potent neuroprotective agent, showing significant high value at 0.5 and 10 µM (86.2%), a result in good agreement with the observed strong hydroxyl radical scavenger of QN6.

New Quinolylnitrones for Stroke Therapy: Antioxidant and Neuroprotective ( Z)- N- tert-Butyl-1-(2-chloro-6-methoxyquinolin-3-yl)methanimine Oxide as a New Lead-Compound for Ischemic Stroke Treatment.

We describe herein the synthesis and neuroprotective capacity of an array of 31 compounds comprising quinolyloximes, quinolylhydrazones, quinolylimines, QNs, and related heterocyclic azolylnitrones. Neuronal cultures subjected to oxygen-glucose deprivation (OGD), as experimental model for ischemic conditions, were treated with our molecules at the onset of recovery period after OGD and showed that most of these QNs, but not the azo molecules, improved neuronal viability 24 h after recovery. Especially, QN ( Z)- N-tert-butyl-1-(2-chloro-6-methoxyquinolin-3-yl)methanimine oxide (23) was shown as a very potent neuroprotective agent. Antioxidant analysis based on the ability of QN 23 to trap different types of toxic radical oxygenated species supported and confirmed its strong neuroprotective capacity. Finally, QN 23 showed also neuroprotection induction in two in vivo models of cerebral ischemia, decreasing neuronal death and reducing infarct size, allowing us to conclude that QN 23 can be considered as new lead-compound for ischemic stroke treatment.

A comprehensive evaluation of omega-3 fatty acid supplementation in cystic fibrosis patients using lipidomics.

The evaluation of the benefits of omega-3 fatty acid supplementation in humans requires the identification and characterization of suitable biomarkers of its incorporation in the body. The reference method for the evaluation of omega-3, gas chromatography, is difficult to apply in clinical practice because of its low throughput and does not provide information about the incorporation of specific fatty acids in lipid species and the potential effects of supplementation on lipid classes. We used a quantitative lipidomic approach to follow the incorporation of omega-3 fatty acids into plasma lipids in cystic fibrosis patients (n=50) from a randomized controlled clinical trial after the supplementation of seaweed oil enriched with docosahexaenoic acid (DHA). Lipidomic analysis accurately showed the distribution of fatty acids in different lipid classes after omega-3 supplementation, and the performance in determining the compliance to supplementation was similar to that of gas chromatography coupled to mass spectrometry. Twelve months after fatty acid supplementation, DHA was predominantly incorporated into highly unsaturated cholesteryl esters (110.9±16.2 vs. 278.6±32.6 µM, mean±S.E.M.) and phosphatidylcholine (142.4±11.9 vs. 272.9±21.4 µM) and, to a lesser extent, into phosphatidylethanolamine (9.4±0.8 vs. 15.5±1.5 µM) and triglycerides (0.4±0.04 vs. 1.1±0.12 µM). In addition, a technique was developed for the fast measurement of the DHA/arachidonic acid ratio to simplify the follow-up of nutritional intervention with DHA-enriched foods. We conclude that lipidomics is a suitable approach for monitoring the incorporation of omega-3 fatty acids in nutritional studies.

Quinolinyl Nitrone RP19 Induces Neuroprotection after Transient Brain Ischemia.

There is a need to develop additional effective therapies for ischemic stroke. Nitrones, which were first developed as reactive oxygen species (ROS)-trapping compounds, have been proposed as neuroprotective agents for ischemic stroke, a ROS-related disorder. The previous reported ROS-trapping compound, quinolyl nitrone RP19, is here being assayed to induce neuroprotection to ischemia-reperfusion injury in three experimental ischemia models: (i) oxygen-glucose deprivation (OGD) on primary neuronal cultures; (ii) transient global cerebral ischemia in four-vessel occlusion model; and (iii) transient focal cerebral ischemia in middle cerebral artery occlusion (tMCAO) model. RP19 (50 µM) induced long-term neuroprotection at 5 days of recovery after OGD in primary neuronal cultures, evaluated by cell viability assay, and decreased both ROS formation and lipid peroxidation upon recovery after OGD. Furthermore, treatment of animals with RP19 at the onset of reperfusion after either global or focal ischemia, at the dose range that was demonstrated to be neuroprotective in neuronal cultures, decreased neuronal death and apoptosis induction, reduced the size of infarct, and improved the neurological deficit scores after 48 h or 5 days of reperfusion after ischemia. The molecule proposed, quinolyl nitrone RP19, induced substantial neuroprotection on experimental ischemia in neuronal cells, and against ischemic injury following transient brain ischemia in treated animals. This molecule may have potential therapeutic interest in ischemic stroke and to reduce the reoxygenation-induced injury after induced reperfusion.

Corrigendum: Melatonin and Nitrones As Potential Therapeutic Agents for Stroke.

[This corrects the article on p. 281 in vol. 8, PMID: 27932976.].

A novel ATTR L32V mutation causes familial amyloid polyneuropathy in a Bolivian family.

We report a new transthyretin (ATTR) gene c.272C>G mutation and variant protein, p.Leu32Val, in a kindred of Bolivian origin with a rapid progressive peripheral neuropathy and cardiomyopathy. Three individuals from a kindred with peripheral nerve and cardiac amyloidosis were examined. Analysis of the TTR gene was performed by Sanger direct sequencing. Neuropathologic examination was obtained on the index patient with mass spectrometry study of the ATTR deposition. Direct DNA sequence analysis of exons 2, 3, and 4 of the TTR gene demonstrated a c.272 C>G mutation in exon 2 (p.L32V). Sural nerve biopsy revealed massive amyloid deposition in the perineurium, endoneurium and vasa nervorum. Mass spectrometric analyses of ATTR immunoprecipitated from nerve biopsy showed the presence of both wild-type and variant proteins. The observed mass results for the wild-type and variant proteins were consistent with the predicted values calculated from the genetic analysis data. The ATTR L32V is associated with a severe course. This has implications for treatment of affected individuals and counseling of family members.