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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure or effective treatment in which TAR DNA Binding Protein of 43 kDa (TDP-43) abnormally accumulates into misfolded protein aggregates in affected neurons. It is widely accepted that protein misfolding and aggregation promotes proteotoxic stress. The molecular chaperones are a primary line of defense against proteotoxic stress, and there has been long-standing interest in understanding the relationship between chaperones and aggregated protein in ALS. Of particular interest are the heat shock protein of 70 kDa (Hsp70) family of chaperones. However, defining which of the 13 human Hsp70 isoforms is critical for ALS has presented many challenges. To gain insight into the specific Hsp70 that modulates TDP-43, we investigated the relationship between TDP-43 and the Hsp70s using proximity-dependent biotin identification (BioID) and discovered several Hsp70 isoforms associated with TDP-43 in the nucleus, raising the possibility of an interaction with native TDP-43. We further found that HspA5 bound specifically to the RNA-binding domain of TDP-43 using recombinantly expressed proteins. Moreover, in a Drosophila strain that mimics ALS upon TDP-43 expression, the mRNA levels of the HspA5 homologue (Hsc70.3) were significantly increased. Similarly we observed upregulation of HspA5 in prefrontal cortex neurons from human ALS patients. Finally, overexpression of HspA5 in Drosophila rescued TDP-43-induced toxicity, suggesting that upregulation of HspA5 may have a compensatory role in ALS pathobiology., (© 2022. The Author(s).)

The voltage-gated sodium NaV1.7 channel, critical for sensing pain, has been actively targeted by drug developers; however, there are currently no effective and safe therapies targeting NaV1.7. Here, we tested whether a different approach, indirect NaV1.7 regulation, could have antinociceptive effects in preclinical models. We found that preventing addition of small ubiquitin-like modifier (SUMO) on the NaV1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked NaV1.7 functions and had antinociceptive effects in rodents. In silico targeting of the SUMOylation site in CRMP2 (Lys374) identified >200 hits, of which compound 194 exhibited selective in vitro and ex vivo NaV1.7 engagement. Orally administered 194 was not only antinociceptive in preclinical models of acute and chronic pain but also demonstrated synergy alongside other analgesics—without eliciting addiction, rewarding properties, or neurotoxicity. Analgesia conferred by 194 was opioid receptor dependent. Our results demonstrate that 194 is a first-in-class protein-protein inhibitor that capitalizes on CRMP2-NaV1.7 regulation to deliver safe analgesia in rodents.

Tar DNA binding (TDP)-43 proteinopathy, typically described as cytoplasmic accumulation of highly modified and misfolded TDP-43 molecules, is characteristic of several neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) and limbic-predominant age-related TDP-43 encephalopathy (LATE). TDP-43 proposed proteinopathies include homeostatic imbalance between nuclear and cytoplasmic localization, aggregation of ubiquitinated and hyper-phosphorylated TDP-43, and an increase in protein truncation of cytoplasmic TDP-43. Given the therapeutic interest of targeting TDP-43, this review focuses on the current landscape of strategies, ranging from biologics to small molecules, that directly target TDP-43. Antibodies, peptides and compounds have been designed or found to recognize specific TDP-43 sequences but alleviate TDP-43 toxicity through different mechanisms. While two antibodies described here were able to induce degradation of pathological TDP-43, the peptides and small molecules were primarily designed to reduce aggregation of TDP-43. Furthermore, we discuss promising emerging therapeutic targets., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

In this study, we targeted the N-terminal domain (NTD) of transactive response (TAR) DNA binding protein (TDP-43), which is implicated in several neurodegenerative diseases. In silico docking of 50K compounds to the NTD domain of TDP-43 identified a small molecule (nTRD22) that is bound to the N-terminal domain. Interestingly, nTRD22 caused allosteric modulation of the RNA binding domain (RRM) of TDP-43, resulting in decreased binding to RNA in vitro . Moreover, incubation of primary motor neurons with nTRD22 induced a reduction of TDP-43 protein levels, similar to TDP-43 RNA binding-deficient mutants and supporting a disruption of TDP-43 binding to RNA. Finally, nTRD22 mitigated motor impairment in a Drosophila model of amyotrophic lateral sclerosis. Our findings provide an exciting way of allosteric modulation of the RNA-binding region of TDP-43 through the N-terminal domain.

Naringenin (2S)-5,7-dihydroxy-2-(4-hydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-4-one is a natural flavonoid found in fruits from the citrus family. Because (2S)-naringenin is known to racemize, its bioactivity might be related to one or both enantiomers. Computational studies predicted that (2R)-naringenin may act on voltage-gated ion channels, particularly the N-type calcium channel (CaV2.2) and the NaV1.7 sodium channel-both of which are key for pain signaling. Here we set out to identify the possible mechanism of action of naringenin. Naringenin inhibited depolarization-evoked Ca 2+ influx in acetylcholine-, ATP-, and capsaicin-responding rat dorsal root ganglion (DRG) neurons. This was corroborated in electrophysiological recordings from DRG neurons. Pharmacological dissection of each of the voltage-gated Ca 2+ channels subtypes could not pinpoint any selectivity of naringenin. Instead, naringenin inhibited NaV1.8-dependent and tetrodotoxin (TTX)-resistant while sparing tetrodotoxin sensitive (TTX-S) voltage-gated Na + channels as evidenced by the lack of further inhibition by the NaV1.8 blocker A-803467. The effects of the natural flavonoid were validated ex vivo in spinal cord slices where naringenin decreased both the frequency and amplitude of sEPSC recorded in neurons within the substantia gelatinosa. The antinociceptive potential of naringenin was evaluated in male and female mice. Naringenin had no effect on the nociceptive thresholds evoked by heat. Naringenin's reversed allodynia was in mouse models of postsurgical and neuropathic pain. Here, driven by a call by the National Center for Complementary and Integrative Health's strategic plan to advance fundamental research into basic biological mechanisms of the action of natural products, we advance the antinociceptive potential of the flavonoid naringenin.

Transactive response DNA binding protein (TDP-43) is a key player in neurodegenerative diseases. In this review, we have gathered and presented structural information on the different regions of TDP-43 with high resolution structures available. A thorough understanding of TDP-43 structure, effect of modifications, aggregation and sites of localization is necessary as we develop therapeutic strategies targeting TDP-43 for neurodegenerative diseases. We discuss how different domains as well as post-translational modification may influence TDP-43 overall structure, aggregation and droplet formation. The primary aim of the review is to utilize structural insights as we develop an understanding of the deleterious behavior of TDP-43 and highlight locations of established and proposed post-translation modifications. TDP-43 structure and effect on localization is paralleled by many RNA-binding proteins and this review serves as an example of how structure may be modulated by numerous compounding elements., (Copyright © 2019 François-Moutal, Perez-Miller, Scott, Miranda, Mollasalehi and Khanna.)

RNA dysregulation likely contributes to disease pathogenesis of amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. A pathological form of the transactive response (TAR) DNA binding protein (TDP-43) binds to RNA in stress granules and forms membraneless, amyloid-like TDP-43 aggregates in the cytoplasm of ALS motor neurons. In this study, we hypothesized that by targeting the RNA recognition motif (RRM) domains of TDP-43 that confer a pathogenic interaction between TDP-43 and RNA, motor neuron toxicity could be reduced. In silico docking of 50000 compounds to the RRM domains of TDP-43 identified a small molecule (rTRD01) that (i) bound to TDP-43's RRM1 and RRM2 domains, (ii) partially disrupted TDP-43's interaction with the hexanucleotide RNA repeat of the disease-linked c9orf72 gene, but not with (UG) 6 canonical binding sequence of TDP-43, and (iii) improved larval turning, an assay measuring neuromuscular coordination and strength, in an ALS fly model based on the overexpression of mutant TDP-43. Our findings provide an instructive example of a chemical biology approach pivoted to discover small molecules targeting RNA-protein interactions in neurodegenerative diseases.

TAR DNA-binding protein 43 (TDP-43) is a ubiquitously expressed nuclear protein that influences diverse cellular processes by regulating alternative splicing of RNA and microRNA biogenesis. It is also a pathological protein found in sporadic ALS and in the most common subtype of frontotemporal lobar degeneration with ubiquitinated inclusions (FLTD-U). TDP-43 has two tandem RNA-binding domains, RRM1 and RRM2. The NMR structure of TDP-43 was solved in the presence of UG-rich RNA sequences bound to the RRM1 and RRM2 domains. Here we report the backbone assignment of apo TDP-43. The chemical shift (HN, N, C, C α and C β ) analysis shows the predicted regions of secondary structure are in good agreement with those observed for TDP-43 in complex with RNA. However, our data show that the apo structure of TPD-43 has increased flexibility in the regions that would normally have been used to anchor the RNA bases. The backbone chemical shifts assignments will prove useful in the study of TDP-43 interaction with non-canonical RNA and RRM-binding proteins.

The process of creating a translation-competent mRNA is highly complex and involves numerous steps including transcription, splicing, addition of modifications, and, finally, export to the cytoplasm. Historically, much of the research on regulation of gene expression at the level of the mRNA has been focused on either the regulation of mRNA synthesis (transcription and splicing) or metabolism (translation and degradation). However, in recent years, the advent of new experimental techniques has revealed the export of mRNA to be a major node in the regulation of gene expression, and numerous large-scale and specific mRNA export pathways have been defined. In this chapter, we will begin by outlining the mechanism by which most mRNAs are homeostatically exported ("bulk mRNA export"), involving the recruitment of the NXF1/TAP export receptor by the Aly/REF and THOC5 components of the TREX complex. We will then examine various mechanisms by which this pathway may be controlled, modified, or bypassed in order to promote the export of subset(s) of cellular mRNAs, which include the use of metazoan-specific orthologs of bulk mRNA export factors, specific cis RNA motifs which recruit mRNA export machinery via specific trans-acting-binding factors, posttranscriptional mRNA modifications that act as "inducible" export cis elements, the use of the atypical mRNA export receptor, CRM1, and the manipulation or bypass of the nuclear pore itself. Finally, we will discuss major outstanding questions in the field of mRNA export heterogeneity and outline how cutting-edge experimental techniques are providing new insights into and tools for investigating the intriguing field of mRNA export heterogeneity.

Mutations of EXOSC3 have been linked to the rare neurological disorder known as Pontocerebellar Hypoplasia type 1B (PCH1B). EXOSC3 is one of three putative RNA-binding structural cap proteins that guide RNA into the RNA exosome, the cellular machinery that degrades RNA. Using RNAcompete, we identified a G-rich RNA motif binding to EXOSC3. Surface plasmon resonance (SPR) and microscale thermophoresis (MST) indicated an affinity in the low micromolar range of EXOSC3 for long and short G-rich RNA sequences. Although several PCH1B-causing mutations in EXOSC3 did not engage a specific RNA motif as shown by RNAcompete, they exhibited lower binding affinity to G-rich RNA as demonstrated by MST. To test the hypothesis that modification of the RNA-protein interface in EXOSC3 mutants may be phenocopied by small molecules, we performed an in-silico screen of 50 000 small molecules and used enzyme-linked immunosorbant assays (ELISAs) and MST to assess the ability of the molecules to inhibit RNA-binding by EXOSC3. We identified a small molecule, EXOSC3-RNA disrupting (ERD) compound 3 (ERD03), which ( i) bound specifically to EXOSC3 in saturation transfer difference nuclear magnetic resonance (STD-NMR), ( ii) disrupted the EXOSC3-RNA interaction in a concentration-dependent manner, and ( iii) produced a PCH1B-like phenotype with a 50% reduction in the cerebellum and an abnormally curved spine in zebrafish embryos. This compound also induced modification of zebrafish RNA expression levels similar to that observed with a morpholino against EXOSC3. To our knowledge, this is the first example of a small molecule obtained by rational design that models the abnormal developmental effects of a neurodegenerative disease in a whole organism.

Drug discovery campaigns directly targeting the voltage-gated sodium channel NaV1.7, a highly prized target in chronic pain, have not yet been clinically successful. In a differentiated approach, we demonstrated allosteric control of trafficking and activity of NaV1.7 by prevention of SUMOylation of collapsin response mediator protein 2 (CRMP2). Spinal administration of a SUMOylation incompetent CRMP2 (CRMP2 K374A) significantly attenuated pain behavior in the spared nerve injury (SNI) model of neuropathic pain, underscoring the importance of SUMOylation of CRMP2 as a pathologic event in chronic pain. Using a rational design strategy, we identified a heptamer peptide harboring CRMP2's SUMO motif that disrupted the CRMP2-Ubc9 interaction, inhibited CRMP2 SUMOylation, inhibited NaV1.7 membrane trafficking, and specifically inhibited NaV1.7 sodium influx in sensory neurons. Importantly, this peptide reversed nerve injury-induced thermal and mechanical hypersensitivity in the SNI model, supporting the practicality of discovering pain drugs by indirectly targeting NaV1.7 via prevention of CRMP2 SUMOylation. Here, our goal was to map the unique interface between CRMP2 and Ubc9, the E2 SUMO conjugating enzyme. Using computational and biophysical approaches, we demonstrate the enzyme/substrate nature of Ubc9/CRMP2 binding and identify hot spots on CRMP2 that may form the basis of future drug discovery campaigns disrupting the CRMP2-Ubc9 interaction to recapitulate allosteric regulation of NaV1.7 for pain relief.

To counteract the breakdown of genome integrity, eukaryotic cells have developed a network of surveillance pathways to prevent and resolve DNA damage. Recent data has recognized the importance of RNA binding proteins (RBPs) in DNA damage repair (DDR) pathways. Here, we describe Nol12 as a multifunctional RBP with roles in RNA metabolism and genome maintenance. Nol12 is found in different subcellular compartments-nucleoli, where it associates with ribosomal RNA and is required for efficient separation of large and small subunit precursors at site 2; the nucleoplasm, where it co-localizes with the RNA/DNA helicase Dhx9 and paraspeckles; as well as GW/P-bodies in the cytoplasm. Loss of Nol12 results in the inability of cells to recover from DNA stress and a rapid p53-independent ATR-Chk1-mediated apoptotic response. Nol12 co-localizes with DNA repair proteins in vivo including Dhx9, as well as with TOPBP1 at sites of replication stalls, suggesting a role for Nol12 in the resolution of DNA stress and maintenance of genome integrity. Identification of a complex Nol12 interactome, which includes NONO, Dhx9, DNA-PK and Stau1, further supports the protein's diverse functions in RNA metabolism and DNA maintenance, establishing Nol12 as a multifunctional RBP essential for genome integrity., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The nucleolus represents a highly multifunctional intranuclear organelle in which, in addition to the canonical ribosome assembly, numerous processes such as transcription, DNA repair and replication, the cell cycle, and apoptosis are coordinated. The nucleolus is further a key hub in the sensing of cellular stress and undergoes major structural and compositional changes in response to cellular perturbations. Numerous nucleolar proteins have been identified that, upon sensing nucleolar stress, deploy additional, non-ribosomal roles in the regulation of varied cell processes including cell cycle arrest, arrest of DNA replication, induction of DNA repair, and apoptosis, among others. The highly abundant proteins nucleophosmin (NPM1) and nucleolin (NCL) are two such factors that transit to the nucleoplasm in response to stress, and participate directly in the repair of numerous different DNA damages. This review discusses the contributions made by NCL and (or) NPM1 to the different DNA repair pathways employed by mammalian cells to repair DNA insults, and examines the implications of such activities for the regulation, pathogenesis, and therapeutic targeting of NPM1 and NCL.

The post-transcriptional addition of non-templated nucleotides to the 3' ends of RNA molecules can have a profound impact on their stability and biological function. Evidence accumulated over the past few decades has identified roles for polyadenylation in RNA stabilisation, degradation and, in the case of eukaryotic mRNAs, translational competence. By contrast, the biological significance of RNA 3' modification by uridylation has only recently started to become apparent. The evolutionary origin of eukaryotic RNA terminal uridyltransferases can be traced to an ancestral poly(A) polymerase. Here we review what is currently known about the biological roles of these enzymes, the ways in which their activity is regulated and the consequences of this covalent modification for the target RNA molecule, with a focus on those instances where uridylation has been found to contribute to RNA degradation. Roles for uridylation have been identified in the turnover of mRNAs, pre-microRNAs, piwi-interacting RNAs and the products of microRNA-directed mRNA cleavage; many mature microRNAs are also modified by uridylation, though the consequences in this case are currently less well understood. In the case of piwi-interacting RNAs, modification of the 3'-terminal nucleotide by the HEN1 methyltransferase blocks uridylation and so stabilises the small RNA. The extent to which other uridylation-dependent mechanisms of RNA decay are similarly regulated awaits further investigation. This article is part of a Special Issue entitled: RNA Decay mechanisms., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Using the foraging movements of an insectivorous bat, Myotis mystacinus, we describe temporal switching of foraging behaviour in response to resource availability. These observations conform to predictions of optimized search under the Lévy flight paradigm. However, we suggest that this occurs as a result of a preference behaviour and knowledge of resource distribution. Preferential behaviour and knowledge of a familiar area generate distinct movement patterns as resource availability changes on short temporal scales. The behavioural response of predators to changes in prey fields can elicit different functional responses, which are considered to be central in the development of stable predator–prey communities. Recognizing how the foraging movements of an animal relate to environmental conditions also elucidates the evolution of optimized search and the prevalence of discrete strategies in natural systems. Applying techniques that use changes in the frequency distribution of movements facilitates exploration of the processes that underpin behavioural changes.

Joubert syndrome (JBTS) is an autosomal recessive disorder characterized by cerebellum and brainstem malformations. Individuals with JBTS have abnormal breathing and eye movements, ataxia, hypotonia, and cognitive difficulty, and they display mirror movements. Mutations in the Abelson-helper integration site-1 gene (AHI1) cause JBTS in humans, suggesting that AHI1 is required for hindbrain development; however AHI1 may also be required for neuronal function. Support for this idea comes from studies demonstrating that the AHI1 locus is associated with schizophrenia. To gain further insight into the function of AHI1 in both the developing and mature central nervous system, we determined the spatial and temporal expression patterns of the gene products of AHI1 orthologs throughout development, in human, mouse, and zebrafish. Murine Ahi1 was distributed throughout the cytoplasm, dendrites, and axons of neurons, but was absent in glial cells. Ahi1 expression in the mouse brain was observed as early as embryonic day 10.5 and persisted into adulthood, with peak expression during the first postnatal week. Murine Ahi1 was observed in neurons of the hindbrain, midbrain, and ventral forebrain. Generally, the AHI1/Ahi1/ahi1 orthologs had a conserved distribution pattern in human, mouse, and zebrafish, but mouse Ahi1 was not present in the developing and mature cerebellum. Ahi1 was also observed consistently in the stigmoid body, a poorly characterized cytoplasmic organelle found in neurons. Overall, these results suggest roles for AHI1 in neurodevelopmental processes that underlie most of the neuroanatomical defects in JBTS, and perhaps in neuronal functions that contribute to schizophrenia., ((c) 2008 Wiley-Liss, Inc.)

This study employed transmission electron microscopy (TEM) to conduct research on the ultrastructure of watermelon (cultivar: Hazera SW1) mesocarp samples of different maturities. Micrographs from immature fruit showed incompletely formed chromoplasts. A combination of distinct pigment-bearing chromoplasts and incompletely formed chromoplasts was observed in mature watermelon micrographs. Electron micrographs showed chromoplasts changing from a less organized globular form in immature to a symmetrical form in mature to an asymmetrical form in overmature watermelons. This study furthers our understanding of watermelon physiology and the effect of maturity on compartmentalization of lycopene.

The use of bone grafts in the repair of defects has a long history of success, primarily with the use of autologous bone. With increasing technologic advances, researchers have been able to broaden the spectrum of grafting materials to allografts, xenografts, and synthetic materials, which provide the surgeon and patient with options, each with unique advantages. It is with the knowledge of each material that the clinician can present and suggest the best material and tailor treatment plans to fit each individual. In this article, we present an overview of the principles of bone grafting, the types of graft materials available, and an outlook to what the future holds in this area of medicine and dentistry.

The tau subunit of Escherichia coli DNA polymerase III holoenzyme interacts with the alpha subunit through its C-terminal Domain V, tau(C)16. We show that the extreme C-terminal region of tau(C)16 constitutes the site of interaction with alpha. The tau(C)16 domain, but not a derivative of it with a C-terminal deletion of seven residues (tau(C)16Delta7), forms an isolable complex with alpha. Surface plasmon resonance measurements were used to determine the dissociation constant (K(D)) of the alpha-tau(C)16 complex to be approximately 260 pM. Competition with immobilized tau(C)16 by tau(C)16 derivatives for binding to alpha gave values of K(D) of 7 muM for the alpha-tau(C)16Delta7 complex. Low-level expression of the genes encoding tau(C)16 and tau(C)16triangle up7, but not tau(C)16Delta11, is lethal to E. coli. Suppression of this lethal phenotype enabled selection of mutations in the 3' end of the tau(C)16 gene, that led to defects in alpha binding. The data suggest that the unstructured C-terminus of tau becomes folded into a helix-loop-helix in its complex with alpha. An N-terminally extended construct, tau(C)24, was found to bind DNA in a salt-sensitive manner while no binding was observed for tau(C)16, suggesting that the processivity switch of the replisome functionally involves Domain IV of tau.

Lycopene, a lipophilic antioxidant, plays a crucial role in biological systems. It may play an important role in human biological systems by providing protection against cardiovascular disease and some cancers and by boosting the immune system. The oxygen radical absorbance capacity (ORAC) has been validated as an index of antioxidant activity for many hydrophilic antioxidants but not for lycopene. This study validates the ORAC assay for different concentrations of lycopene in the presence of beta-cyclodextrin, a water-solubility enhancer. Lyc-O-Mato 6% extract was used as a source of lycopene for these experiments. Lycopene was extracted according to a standard spectrophotometric assay procedure in the presence of beta-cyclodextrin at concentrations of 0, 0.4, 0.8, and 1.6%, and the antioxidant activity of lycopene was measured with the ORAC assay. Experiments were conducted in quadruplicate and statistical pooled correlations analyzed. Statistical analysis showed a very high correlation (R2 = 0.99) between ORAC and ascorbic acid concentrations, validating this method. Lycopene concentration correlated poorly with ORAC (R2 = 0.33) in the absence of beta-cyclodextrin. Correlations improved with increasing levels of beta-cyclodextrin (R2 = 0.58 and 0.91 for 0.4 and 0.8% beta-cyclodextrin, respectively). A very high beta-cyclodextrin concentration (1.6%) decreased the correlation between ORAC and lycopene concentration. Inclusion of beta-cyclodextrin in the ORAC assay improves correlation between ORAC and lycopene concentration, thus expanding the scope of the ORAC assay to include an additional fat-soluble antioxidant.

Objective: To investigate the effect of gabapentin on subject self-report and physician-administered spasticity scales in individuals with multiple sclerosis., Design: Prospective, double-masked, placebo-controlled, crossover design., Setting: The Multiple Sclerosis Center at the Denver Veterans Affairs Medical Center., Intervention: Subjects were titrated to either 900 mg gabapentin orally three times a day or placebo over a 6-day period. Subjects underwent a 14-day washout and then were crossed over. No other changes were made to their medication regimen., Main Outcome Measures: The outcome measures were divided into two categories: subject self-report scales physician-administered scales. Subject self-report scales included the spasm frequency scale, spasm severity scale, interference with function scale, painful spasm scale, and global assessment scale. Physician-administered scales included the Modified Ashworth Scale, clonus scale, deep tendon reflexes, plantar stimulation response, and the Kurtzke Expanded Disability Status (EDSS) Scale. Digit Span and Digit Symbol subtests of the WAIS-R Intelligence Scale were administered to assess for possible impaired concentration. The Fatigue Impact Scale was administered to assess for changes in fatigue. The adjective generation technique was administered to assess for alterations in mood., Results: A statistically significant reduction in the impairment of spasticity was found in the gabapentin-treated subjects compared with placebo as measured by the self-report scales of the spasm severity scale, interference with function scale, painful spasm scale, and global assessment scale and by the physician-administered scales of the Modified Ashworth and plantar stimulation response. No significant difference was noted in the Digit Span, Digit Symbol, adjective generation technique, and EDSS., Conclusion: Gabapentin reduces the impairment of spasticity, compared with placebo, without the side effects of worsening concentration and fatigue.

To document the oral health of mid-nineteenth century frontiersmen, the dentitions of eight Seventh Cavalry troopers from the Little Bighorn Battlefield were examined for indications of disease. These observations are supplemented with historic records and compared with the oral health of other soldiers from the Indian Wars. The Seventh Cavalry troopers, despite their young ages, had a variety of oral problems, including abscesses, caries, periodontitis, attrition, antemortem tooth loss, and indications of tobacco use. One of the specimens had gold and tin foil fillings. Combining historic records and a dental approach to the archaeological specimens provides complementary insights which are unavailable otherwise from either of the individual fields.

Fillings found in the skull of a 7th Cavalry trooper killed 118 years ago in the Battle of Little Bighorn show sophisticated, well-executed dentistry. Some fillings are examples of early preventive dentistry, using a variety of materials and techniques typical of 19th-century cavity preparation. Dental care most likely was delivered by a private dentist or performed in part at a dental school in the East or Midwest, in a large city or at West Point. The logical, deductive process suggests an identification: Cpl. George Lell. Without additional corroborative documentation, however, this is speculation. Whoever he was, the cavalryman was older and shorter and had better oral care and health than most of the comrades who died with him in 1876. He used tobacco, most likely by chewing it. His remains provide a glimpse into the reality of life and death, dental care and hygiene of the mid-19th century.

A 77-year-old man who was undergoing comprehensive rehabilitation for a right subcapital femoral neck fracture presented with intermittent digital cyanosis of 3 days duration. He had no other cardiopulmonary signs or symptoms, therefore a diagnosis of Raynaud's Phenomenon was considered but ruled out because of no prior history of similar episodes or associated conditions. A ventilation-perfusion (V/Q) scan was obtained and was highly indicative of pulmonary embolus. Dopplers and impedance plethysmography (IPG) revealed a left lower extremity deep vein thrombosis. Heparinization resulted in cessation of the cyanotic episodes. Intermittent digital cyanosis as the sole presentation of pulmonary embolus has not been previously reported in humans. Pulmonary embolus is notoriously difficult to diagnose based on clinical signs and symptoms. Intermittent peripheral cyanosis should raise the index of suspicion for the possibility of pulmonary embolus.

Piriformis syndrome is a controversial cause of hip and low back pain whose etiology stems from anatomic irregularities of the piriformis muscle and surrounding structures of the posterior hip region. Although this syndrome has been described by many clinicians, the association of piriformis muscle dysfunction and pain in the hip region remains conjectural. The diagnosis is one of exclusion of other more common pain generators in the hip, pelvis, or low back.The authors of this article will review the anatomy of the region as it relates to piriformis muscle dysfunction and delineate the various clinical, radiological, and interventional procedures described in the literature to diagnose and treat this elusive syndrome.

We used a liposome lysis assay to measure antibodies against a panel of glycolipids. Antibodies to one or more compounds were detected in 34 of 46 patients with multiple sclerosis, 19 of 31 patients with systemic lupus erythematosus (SLE), and in the majority of patients with cranial trauma or cerebrovascular accidents. Antibodies against ganglioside GM1 and asialo GM1 were found most commonly, and they were frequently present in the same sera. The specificity of the antibodies was tested in four sera that contained antibodies to both glycolipids. The anti-GM1 antibodies cross-reacted with asialo GM1, but the converse was not true. Among patients whose sera contained antibodies to glycolipids, anti-asialo GM1 alone was more common in patients with SLE (7 of 17) than in multiple sclerosis (2 of 34; p = 0.004). Anti-GM1 alone was found in 9 of 34 patients with multiple sclerosis and 1 of 17 patients with SLE, a difference that was not statistically significant (0.135). No correlation was observed between the presence of anti-glycolipid antibodies and symptoms related to the nervous system in patients with SLE. Because of our inability to detect these antibodies by a solid phase immunoassay (ELISA), a comparison was made of the titers obtained with three monoclonal anti-glycolipid antibodies in the liposome lysis assay and ELISA. The ELISA was less sensitive in all instances, requiring from four to 1000 times as much antibody as the liposome lysis assay to give a positive test. We conclude that antibodies to glycolipids occur frequently in patients with multiple sclerosis, SLE, major cranial trauma, and cerebrovascular accidents. Their role in the initiation or perpetuation of inflammatory disease of the central nervous system has yet to be determined.

We have developed a high-performance liquid chromatographic (HPLC) procedure for the rapid separation of individual ganglioside components on a 5-micron porous silica gel column using a mixture of isopropanol-hexane-water with increasing water content and decreasing hexane content. Total ganglioside mixtures were first fractionated on DEAE-silica gel according to the number of sialic acid residues. Each fraction was then separated into individual ganglioside components by HPLC. Total elution time was less than 2 h. This procedure has been applied for the separation of major ganglioside components of human erythrocytes and beef brain and is highly reproducible.