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Abstract Background An important step in the proteomics of solid tumors, including breast cancer, consists of efficiently extracting most of proteins in the tumor specimen. For this purpose, Radio-Immunoprecipitation Assay (RIPA) buffer is widely employed. RIPA buffer's rapid and highly efficient cell lysis and good solubilization of a wide range of proteins is further augmented by its compatibility with protease and phosphatase inhibitors, ability to minimize non-specific protein binding leading to a lower background in immunoprecipitation, and its suitability for protein quantitation. Results In this work, the insoluble matter left after RIPA buffer extraction of proteins from breast tumors are subjected to another extraction step, using a urea-based buffer. It is shown that RIPA and urea lysis buffers fractionate breast tissue proteins primarily on the basis of molecular weights. The average molecular weight of proteins that dissolve exclusively in urea buffer is up to 60% higher than in RIPA. Gene Ontology (GO) and Directed Acyclic Graphs (DAG) are used to map the collective biological and biophysical attributes of the RIPA and urea proteomes. The Cellular Component and Molecular Function annotations reveal protein solubilization preferences of the buffers, especially the compartmentalization and functional distributions. It is shown that nearly all extracellular matrix proteins (ECM) in the breast tumors and matched normal tissues are found, nearly exclusively, in the urea fraction, while they are mostly insoluble in RIPA buffer. Additionally, it is demonstrated that cytoskeletal and extracellular region proteins are more soluble in urea than in RIPA, whereas for nuclear, cytoplasmic and mitochondrial proteins, RIPA buffer is preferred. Extracellular matrix proteins are highly implicated in cancer, including their proteinase-mediated degradation and remodelling, tumor development, progression, adhesion and metastasis. Thus, if they are not efficiently extracted by RIPA buffer, important information may be missed in cancer research. Conclusion For proteomics of solid tumors, a two-step extraction process is recommended. First, proteins in the tumor specimen should be extracted with RIPA buffer. Second, the RIPA-insoluble material should be extracted with the urea-based buffer employed in this work.

Abstract Background Oxidoreductases are enzymes that catalyze many redox reactions in normal and neoplastic cells. Their actions include catalysis of the transformation of free, neutral oxygen gas into oxygen free radicals, superoxide, hydroperoxide, singlet oxygen and hydrogen peroxide. These activated forms of oxygen contribute to oxidative stress that modifies lipids, proteins, DNA and carbohydrates. On the other hand, oxidoreductases constitute one of the most important free radical scavenger systems typified by catalase, superoxide dismutase and glutathione peroxidase. In this work, proteomics, Gene Ontology mapping and Directed Acyclic Graphs (DAG) are employed to detect and quantify differential oxidoreductase enzyme expressions between HepG2 cells and normal human liver tissues. Results For the set of bioinformatics calculations whose BLAST searches are performed using the BLAST program BLASTP 2.2.13 [Nov-27-2005], DAG of the Gene Ontology's Molecular Function annotations show that oxidoreductase activity parent node of the liver proteome contains 331 annotated protein sequences, 7 child nodes and an annotation score of 188.9, whereas that of HepG2 cells has 188 annotated protein sequences, 3 child nodes and an annotation score of only 91.9. Overwhelming preponderance of oxidoreductases in the liver is additionally supported by the isomerase DAGs: nearly all the reactions described in the normal liver isomerase DAG are oxidoreductase isomerization reactions, whereas only one of the three child nodes in the HepG2 isomerase DAG is oxidoreductase. Upon normalization of the annotation scores to the parent Molecular Function nodes, oxidoreductases are down-regulated in HepG2 cells by 58%. Similarly, for the set of bioinformatics calculations whose BLAST searches are carried out using BLASTP 2.2.15 [Oct-15-2006], oxidoreductases are down-regulated in HepG2 cells by 56%. Conclusion Proteomics and Gene Ontology reveal, for the first time, differential enzyme activities between HepG2 cells and normal human liver tissues, which may be a promising new prognostic marker of Hepatocellular carcinoma. Two independent sets of bioinformatics calculations that employ two BLAST program versions, and searched different databases, arrived at essentially the same conclusion: oxidoreductases are down-regulated in HepG2 cells by approximately 57%, when compared to normal human liver tissues. Down-regulation of oxidoreductases in hepatoma is additionally supported by Gene Ontology analysis of isomerises.

Introduction: Numerous immunomodulatory treatments exist for multiple sclerosis (MS), including those that deplete immune cells (e.g. anti-CD20 medications), relocate immune cells (e.g. natalizumab, S1P modulators), or modulate immune subsets (e.g. fumarates). All disease-modifying treatments (DMTs) can increase infection risk which could worsen with prolonged use., Methods: This is a retrospective, single-center, observational cohort study. We analyzed medical records of adult people with MS who took natalizumab, S1P modulators, fumarates or anti-CD20 medications for over two years between January 2013 and April 2021 at Yale. We identified severe infections (requiring hospitalization) and mild infections (identified through outpatient antibiotic prescriptions or chart reference to "infection"). We used a zero-inflated negative binomial regression to assess the effects of DMT use, treatment duration, and patient characteristics on infection likelihood and frequency, while controlling for biologic sex, body mass index, ambulatory status, Charlson Comorbidity Index (CCI), diagnosis, disease modifying therapy and treatment duration., Results: 104 patients received natalizumab, 61 fumarates, 17 S1P modulators and 291 anti-CD20 medications, with significant baseline differences in age, diagnosis, duration of DMT use, and CCI. Mild infection rates did not differ across DMTs, but severe infections were more common in patients on fumarates. Patients with longer DMT duration or requiring a walking aid had higher mild infection rates, while those with progressive MS or on long-term fumarates had higher severe infection rates, even after controlling for other variables., Discussion: This study demonstrates how real-life practice patterns, patient factors and DMT choice can influence infection rates, differing from randomized trial patterns. Natalizumab appears safe over extended use, while fumarates were linked to more severe infections, potentially due to the clinical selection of patients with poorer baseline health. The duration of DMT use may predict mild infection rates., Competing Interests: Declaration of competing interest EEL: Consulting for Genentech, Novartis, TG Therapeutics, Bristol Myers Squibb, EMD Serono, Genzyme. Research support from Biogen, Genentech. CML: None. JJP: None, TH: None, (Copyright © 2024. Published by Elsevier B.V.)

Competing Interests: The authors report no relevant disclosures. Go to Neurology.org/NE for full disclosures.

The elimination of synapses during circuit remodeling is critical for brain maturation; however, the molecular mechanisms directing synapse elimination and its timing remain elusive. We show that the transcriptional regulator DVE-1, which shares homology with special AT-rich sequence-binding (SATB) family members previously implicated in human neurodevelopmental disorders, directs the elimination of juvenile synaptic inputs onto remodeling C. elegans GABAergic neurons. Juvenile acetylcholine receptor clusters and apposing presynaptic sites are eliminated during the maturation of wild-type GABAergic neurons but persist into adulthood in dve-1 mutants, producing heightened motor connectivity. DVE-1 localization to GABAergic nuclei is required for synapse elimination, consistent with DVE-1 regulation of transcription. Pathway analysis of putative DVE-1 target genes, proteasome inhibitor, and genetic experiments implicate the ubiquitin-proteasome system in synapse elimination. Together, our findings define a previously unappreciated role for a SATB family member in directing synapse elimination during circuit remodeling, likely through transcriptional regulation of protein degradation processes., (© 2023. The Author(s).)

The functional properties of neural circuits are defined by the patterns of synaptic connections between their partnering neurons, but the mechanisms that stabilize circuit connectivity are poorly understood. We systemically examined this question at synapses onto newly characterized dendritic spines of C. elegans GABAergic motor neurons. We show that the presynaptic adhesion protein neurexin/NRX-1 is required for stabilization of postsynaptic structure. We find that early postsynaptic developmental events proceed without a strict requirement for synaptic activity and are not disrupted by deletion of neurexin/nrx-1. However, in the absence of presynaptic NRX-1, dendritic spines and receptor clusters become destabilized and collapse prior to adulthood. We demonstrate that NRX-1 delivery to presynaptic terminals is dependent on kinesin-3/UNC-104 and show that ongoing UNC-104 function is required for postsynaptic maintenance in mature animals. By defining the dynamics and temporal order of synapse formation and maintenance events in vivo, we describe a mechanism for stabilizing mature circuit connectivity through neurexin-based adhesion., Competing Interests: The authors have declared that no competing interests exist.

Neuromodulators promote adaptive behaviors that are often complex and involve concerted activity changes across circuits that are often not physically connected. It is not well understood how neuromodulatory systems accomplish these tasks. Here, we show that the Caenorhabditis elegans NLP-12 neuropeptide system shapes responses to food availability by modulating the activity of head and body wall motor neurons through alternate G-protein coupled receptor (GPCR) targets, CKR-1 and CKR-2. We show ckr-2 deletion reduces body bend depth during movement under basal conditions. We demonstrate CKR-1 is a functional NLP-12 receptor and define its expression in the nervous system. In contrast to basal locomotion, biased CKR-1 GPCR stimulation of head motor neurons promotes turning during local searching. Deletion of ckr-1 reduces head neuron activity and diminishes turning while specific ckr-1 overexpression or head neuron activation promote turning. Thus, our studies suggest locomotor responses to changing food availability are regulated through conditional NLP-12 stimulation of head or body wall motor circuits., Competing Interests: SR, NB, RB, ML, JF, CL, DT, KA, LS, MA, IB, MF No competing interests declared, (© 2021, Ramachandran et al.)

Recurrent ischemic strokes are a cause of significant healthcare burdens globally. Patients with uncontrolled vascular risk factors are more likely to develop recurrent ischemic strokes. This study aims to compile information gained from current secondary prevention programs. A pre-defined literature search strategy was applied to PubMed, SCOPUS, CINAHL, and Google Scholar databases, and studies from 1997 to 2020 were evaluated for quality, study aims, and outcomes. The search produced 1175 articles (1092 after duplicates were removed) and titles were screened; 55 titles were retained for the full-text analysis. Of the remaining studies, 31 were retained for assessment, five demonstrated long-term effectiveness, eight demonstrated short-term effectiveness, and 18 demonstrated no effectiveness. The successful studies utilized a variety of different techniques in the categories of physical fitness, education, and adherence to care plans to reduce the risk of recurrent strokes. The lessons we learned from the current prevention programs included (1) offer tailored care for underserved groups, (2) control blood pressure, (3) provide opportunities for medication dosage titration, (4) establish the care plan prior to discharge, (5) invest in supervised exercise programs, (6) remove barriers to accessing care in low resource settings, and (7) improve the transition of care.

In neural circuits, individual neurons often make projections onto multiple postsynaptic partners. Here, we investigate molecular mechanisms by which these divergent connections are generated, using dyadic synapses in C. elegans as a model. We report that C. elegans nrx-1 /neurexin directs divergent connectivity through differential actions at synapses with partnering neurons and muscles. We show that cholinergic outputs onto neurons are, unexpectedly, located at previously undefined spine-like protrusions from GABAergic dendrites. Both these spine-like features and cholinergic receptor clustering are strikingly disrupted in the absence of nrx-1 . Excitatory transmission onto GABAergic neurons, but not neuromuscular transmission, is also disrupted. Our data indicate that NRX-1 located at presynaptic sites specifically directs postsynaptic development in GABAergic neurons. Our findings provide evidence that individual neurons can direct differential patterns of connectivity with their post-synaptic partners through partner-specific utilization of synaptic organizers, offering a novel view into molecular control of divergent connectivity., Competing Interests: AP, SR, CL, DO, JF, MA, ML, MF No competing interests declared, (© 2018, Philbrook et al.)

Establishing and maintaining the appropriate number of GABA synapses is key for balancing excitation and inhibition in the nervous system, though we have only a limited understanding of the mechanisms controlling GABA circuit connectivity. Here, we show that disrupting cholinergic innervation of GABAergic neurons in the C. elegans motor circuit alters GABAergic neuron synaptic connectivity. These changes are accompanied by reduced frequency and increased amplitude of GABAergic synaptic events. Acute genetic disruption in early development, during the integration of post-embryonic-born GABAergic neurons into the circuit, produces irreversible effects on GABAergic synaptic connectivity that mimic those produced by chronic manipulations. In contrast, acute genetic disruption of cholinergic signaling in the adult circuit does not reproduce these effects. Our findings reveal that GABAergic signaling is regulated by cholinergic neuronal activity, probably through distinct mechanisms in the developing and mature nervous system., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

An organism's ability to thrive in changing environmental conditions requires the capacity for making flexible behavioral responses. Here we show that, in the nematode Caenorhabditis elegans, foraging responses to changes in food availability require nlp-12, a homolog of the mammalian neuropeptide cholecystokinin (CCK). nlp-12 expression is limited to a single interneuron (DVA) that is postsynaptic to dopaminergic neurons involved in food-sensing, and presynaptic to locomotory control neurons. NLP-12 release from DVA is regulated through the D1-like dopamine receptor DOP-1, and both nlp-12 and dop-1 are required for normal local food searching responses. nlp-12/CCK overexpression recapitulates characteristics of local food searching, and DVA ablation or mutations disrupting muscle acetylcholine receptor function attenuate these effects. Conversely, nlp-12 deletion reverses behavioral and functional changes associated with genetically enhanced muscle acetylcholine receptor activity. Thus, our data suggest that dopamine-mediated sensory information about food availability shapes foraging in a context-dependent manner through peptide modulation of locomotory output.

Insulinoma-associated protein (IA)-2 and IA-2β are transmembrane proteins involved in neurotransmitter secretion. Mice with targeted disruption of both IA-2 and IA-2β (double-knockout, or DKO mice) have numerous endocrine and physiological disruptions, including disruption of circadian and diurnal rhythms. In the present study, we have assessed the impact of disruption of IA-2 and IA-2β on molecular rhythms in the brain and peripheral oscillators. We used in situ hybridization to assess molecular rhythms in the hypothalamic suprachiasmatic nuclei (SCN) of wild-type (WT) and DKO mice. The results indicate significant disruption of molecular rhythmicity in the SCN, which serves as the central pacemaker regulating circadian behavior. We also used quantitative PCR to assess gene expression rhythms in peripheral tissues of DKO, single-knockout, and WT mice. The results indicate significant attenuation of gene expression rhythms in several peripheral tissues of DKO mice but not in either single knockout. To distinguish whether this reduction in rhythmicity reflects defective oscillatory function in peripheral tissues or lack of entrainment of peripheral tissues, animals were injected with dexamethasone daily for 15 days, and then molecular rhythms were assessed throughout the day after discontinuation of injections. Dexamethasone injections improved gene expression rhythms in liver and heart of DKO mice. These results are consistent with the hypothesis that peripheral tissues of DKO mice have a functioning circadian clockwork, but rhythmicity is greatly reduced in the absence of robust, rhythmic physiological signals originating from the SCN. Thus, IA-2 and IA-2β play an important role in the regulation of circadian rhythms, likely through their participation in neurochemical communication among SCN neurons.

Research on the mechanisms underlying circadian rhythmicity and the response of brain and body clocks to environmental and physiological challenges requires assessing levels of circadian clock proteins. Too often, however, it is difficult to acquire antibodies that specifically and reliably label these proteins. Many of these antibodies also lack appropriate validation. The goal of this project was to generate and characterize antibodies against several circadian clock proteins. We examined mice and hamsters at peak and trough times of clock protein expression in the suprachiasmatic nucleus (SCN). In addition, we confirmed specificity by testing the antibodies on mice with targeted disruption of the relevant genes. Our results identify antibodies against PER1, PER2, BMAL1 and CLOCK that are useful for assessing circadian clock proteins in the SCN by immunocytochemistry.

The circadian clock in the mammalian hypothalamic suprachiasmatic nucleus (SCN) is entrained by the ambient light/dark cycle, which differentially acts to cause the clock to advance or delay. Light-induced changes in the rhythmic expression of SCN clock genes are believed to be a critical step in this process, but how the two entrainment modalities--advances vs. delays--engage the molecular clockwork remains incompletely understood. We investigated molecular substrates of photic entrainment of the clock in the SCN by stably entraining hamsters to T cycles (non-24-h light/dark cycles) consisting of a single 1-h light pulse repeated as either a short (23.33-h) or a long (24.67-h) cycle; under these conditions, the light pulse of the short cycle acts as "dawn," whereas that of the long cycle acts as "dusk." Analyses of the expression of the photoinducible and rhythmic clock genes Period 1 and 2 (Per1 and Per2) in the SCN revealed fundamental differences under these two entrainment modes. Light at dawn advanced the clock, advancing the onset of the Per1 mRNA rhythm and acutely increasing mRNA transcription, whereas light at dusk delayed the clock, delaying the offset of the Per2 mRNA rhythm and tonically increasing mRNA stability. The results suggest that the underlying molecular mechanisms of circadian entrainment differ with morning (advancing) or evening (delaying) light exposure, and such differences may reflect how entrainment takes place in nocturnal animals under natural conditions.

Aims: Vascular remodelling diseases are characterized by the presence of proliferative and apoptosis-resistant vascular smooth muscle cells (VSMC). There is evidence that pro-proliferative and anti-apoptotic states are characterized by metabolic remodelling (a glycolytic phenotype with hyperpolarized mitochondria) involving Akt pathway activation by circulating growth factors. Hypoxia-inducible factor-1 (HIF-1) is involved in different vascular diseases. Since this transcription factor is implicated in metabolic responses, we hypothesized that HIF-1 activity could be involved in vascular remodelling in response to arterial injury., Methods and Results: Our findings indicate that growth factors, such as platelet-derived growth factor (PDGF), activate the Akt pathway (measured by immunoblot) in human carotid artery VSMC. Activation of this pathway increased HIF-1 activation (measured by immunoblot), leading to increased glycolysis in VSMC. Expression and mitochondrial activity of hexokinase 2 (HXK2), a primary initiator of glycolysis, are increased during HIF-1 activation. The mitochondrial activity of HXK2 in VSMC led to the hyperpolarization of mitochondrial membrane potential (measured by tetramethylrhodamine methyl-ester perchlorate) and the suppression of apoptosis (measured by TUNEL assay and 3 activity), effects that are blocked by HIF-1 inhibition. Additionally, HIF-1 inhibition also decreased VSMC proliferation (proliferating cell nuclear antigen and Ki-67 assays). In vivo, we demonstrate that localized HIF-1 inhibition, using a dominant-negative HIF-1α adenoviral construct, prevented carotid artery post-injury remodelling in rats., Conclusion: We propose that HIF-1 is centrally involved in carotid artery remodelling in response to arterial injury and that localized inhibition of HIF-1 may be a novel therapeutic strategy to prevent carotid stenosis.

Vascular remodeling diseases (VRDs) are characterized by enhanced inflammation and proliferative and apoptosis-resistant vascular smooth muscle cells (VSMCs). The sustainability of this phenotype has been attributed in part to the activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). There is evidence that circulating cytokines can act as HIF-1 activators in a variety of tissues, including VSMCs. Increased circulating tumor necrosis factor (TNF) levels have been associated with vascular diseases, but the mechanisms involved remain unknown. We hypothesized that increased circulating levels of TNF promotes VRDs by the activation of HIF-1, resulting in VSMC proliferation and resistance to apoptosis. Circulating TNF levels were significantly increased in patients with vascular diseases (n = 19) compared with healthy donors (n = 15). Using human carotid artery smooth muscle cells (CASMCs), we demonstrated that TNF (100 ng/ml) activates HIF-1 (HIF-1α expression), leading to increased CASMC proliferation (Ki-67 and PCNA staining) and resistance to mitochondrial-dependent apoptosis [tetramethylrhodamine methyl ester perchlorate (TMRM), terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL), annexin-V staining]. In vivo, TNF inhibition using polyethylene glycol coupled with TNF membrane receptor 1 (PEGsTNFR1), a soluble TNF receptor inhibiting circulating TNF, prevented carotid artery postinjury media remodeling and neointima development in rats. This effect was associated with lowered HIF-1 activation and decreased CASMC proliferation. In conclusion, we demonstrate for the first time that the inhibition of the TNF/Akt/HIF-1 axis prevents vascular remodeling. TNF inhibitors may therefore represent new and interesting therapeutic tools against VRDs.

Vibrotactile stimulation has been used successfully to activate the human somatosensory pathway in functional magnetic resonance imaging (fMRI) experiments. The design and characterization of these devices are of particular interest in frequency discrimination tasks and investigations of the somatopic organization of sensory areas. However, few have investigated the utility of vibrotactile stimulation in a clinical context. We have previously demonstrated that vibrotactile stimulation can provide robust activations in areas targeted in stereotactic functional neurosurgical procedures used for tumour resection (i.e.: primary and secondary somatosensory areas) and subcortical targets for thalamic pain and movement disorders (i.e.: sensory thalamus). The main contribution of this manuscript is the presentation of the design, materials, construction, and validation of a novel vibrotactile stimulator intended for clinical use. The thalamic activations are also compared to a digital atlas in order to evaluate anatomical localization. The proposed stimulator was constructed entirely from non-ferromagnetic parts, uses compressed air to deliver stimulation using computer control, and stimulates the entirety of the hand and fingers to ensure robust somatosensory activations. In addition, this stimulator is constructed entirely from "off-the-shelf" parts and would be easily replicated due to the simplicity of design and the relatively small expense of the parts required. The device was tested by stimulating the right hand of 10 normal controls (5 females, 5 males, all right handed; age range: 25-42 years, mean: 30.9 years, standard deviation: 5.2 years) during an fMRI experiment. The results demonstrate significant single subject activations of primary and secondary somatosensory cortices and of the sensory thalamus.

Both casein kinase 1 delta (CK1delta) and epsilon (CK1epsilon) phosphorylate core clock proteins of the mammalian circadian oscillator. To assess the roles of CK1delta and CK1epsilon in the circadian clock mechanism, we generated mice in which the genes encoding these proteins (Csnk1d and Csnk1e, respectively) could be disrupted using the Cre-loxP system. Cre-mediated excision of the floxed exon 2 from Csnk1d led to in-frame splicing and production of a deletion mutant protein (CK1delta(Delta2)). This product is nonfunctional. Mice homozygous for the allele lacking exon 2 die in the perinatal period, so we generated mice with liver-specific disruption of CK1delta. In livers from these mice, daytime levels of nuclear PER proteins, and PER-CRY-CLOCK complexes were elevated. In vitro, the half-life of PER2 was increased by approximately 20%, and the period of PER2::luciferase bioluminescence rhythms was 2 h longer than in controls. Fibroblast cultures from CK1delta-deficient embryos also had long-period rhythms. In contrast, disruption of the gene encoding CK1epsilon did not alter these circadian endpoints. These results reveal important functional differences between CK1delta and CK1epsilon: CK1delta plays an unexpectedly important role in maintaining the 24-h circadian cycle length.

The circadian clock mechanism in the mouse is composed of interlocking transcriptional feedback loops. Two transcription factors, CLOCK and BMAL1, are believed to be essential components of the circadian clock. We have used the Cre-LoxP system to generate whole-animal knockouts of CLOCK and evaluated the resultant circadian phenotypes. Surprisingly, CLOCK-deficient mice continue to express robust circadian rhythms in locomotor activity, although they do have altered responses to light. At the molecular and biochemical levels, clock gene mRNA and protein levels in both the master clock in the suprachiasmatic nuclei and a peripheral clock in the liver show alterations in the CLOCK-deficient animals, although the molecular feedback loops continue to function. Our data challenge a central feature of the current mammalian circadian clock model regarding the necessity of CLOCK:BMAL1 heterodimers for clock function.

Combinations of disease modifying antirheumatic drugs (DMARDs) are increasingly being used in patients with early rheumatoid arthritis (RA) when long term results with sequential DMARD monotherapy are disappointing. Combination DMARD therapy may be more effective than monotherapy, and has no additional short term adverse events. The evidence for using combination DMARD therapy is still weak, however, and further trials are needed.

Prokineticin 2 (PK2) is a putative output molecule from the SCN. PK2 RNA levels are rhythmic in the mouse SCN, with high levels during the day, and PK2 administration suppresses nocturnal locomotor activity in rats. The authors examined the PK2 system in a diurnal rodent, Arvicanthis niloticus, to determine whether PK2 or PK2 receptors differ between diurnal and nocturnal species. The major transcript variant of A. niloticus PK2 (AnPK2) encodes a 26-residue signal peptide followed by the presumed mature peptide of 81 residues. Within the grass rat signal sequence, polymorphic sequences and amino acid substitutions were observed relative to mouse and laboratory rats, but the hydrophobic core and cleavage site of the signal sequence were preserved. The mature PK2 peptide is identical among A. niloticus, rat, and mouse. AnPK2 mRNA is rhythmically expressed in the SCN, with peak RNA levels occurring in the morning, preceding peaks of Per1 and Per2 as in mouse SCN. Analysis of prokineticin receptor 2 (PKR2) sequences revealed polymorphisms among the grass rats studied. PKR2 mRNA was expressed in the SCN and paraventricular nuclei of the thalamus and hypothalamus. While further analysis is necessary, there is no clear evidence indicating that a difference in the PK2 ligand/receptor system accounts for diurnality in this rodent species. These data contribute to a growing body of evidence suggesting that the key to diurnality lies downstream of the SCN in A. niloticus.

Objective: To examine whether dose escalation of intramuscular (IM) methotrexate (MTX) up to 45 mg/week improves disease control in patients who have active rheumatoid arthritis (RA) despite receiving conventional doses (15 mg/week) of IM MTX, and to obtain preliminary data on patient tolerability and adverse effects of higher doses of IM MTX., Methods: Patients >18 years of age who had active RA, defined as a European League Against Rheumatism (EULAR) Disease Activity Score in 28 joints (DAS28) of >3.2, and who had received 15-20 mg/week of oral MTX for at least 2 months were switched (week 0) to 15 mg/week of IM MTX for 6 weeks. Patients whose DAS28 remained >3.2 at both week 4 and week 6 were randomized, in a double-blind manner, either to continue to receive 15 mg/week IM MTX with monthly placebo escalation or to receive escalating doses of IM MTX monthly up to 45 mg/week. The dose of MTX or placebo was escalated every 4 weeks if the DAS28 was >2.5. Safety assessments and determination of the DAS28 were performed every 2 weeks and monthly, respectively. Disease activity parameters from the American College of Rheumatology (ACR) core disease activity set and health status as recorded on the Medical Outcomes Study Short-Form 12 were determined at baseline (week 0) and final assessment (week 22). The primary outcome was the percentage of patients in each group achieving a target DAS28 of <3.2. Secondary outcomes comprised the percentage of patients whose DAS28 improved by >1.2, the percentage of patients achieving a 20% improvement in the ACR core disease activity measures (ACR20), and the percentage of patients achieving a good response, a moderate response, or no response in accordance with the EULAR response criteria., Results: Sixty-four patients were eligible for entry and were switched from oral MTX to 15 mg/week IM MTX. At baseline, the mean +/- SD DAS28 was 5.6 +/- 0.88; after 6 weeks of IM MTX, the DAS28 had improved by a mean of 0.42 (95% confidence interval [95% CI] 0.15-0.69). At 6 weeks, 54 patients still had a DAS28 of >3.2 and were therefore eligible for randomization. By 22 weeks, 1 patient (3.7%) in each group achieved the primary outcome of a DAS28 <3.2 (95% CI for the difference between the groups -15% to +15%). Five patients (18.5%) in each group showed an improvement of >1.2 in the DAS28 (95% CI for the difference between the groups -18% to +18%). One patient (3.7%) in each group achieved an ACR20 response, but none achieved a good response as defined by the EULAR response criteria. One patient in each group had a serious adverse reaction; minor adverse reactions were more frequently reported in the dose escalation group., Conclusion: Switching from oral to parenteral MTX 15 mg/week results in a minor improvement in disease control. For patients with active RA receiving 15 mg/week IM MTX, increasing the dose up to 45 mg/week does not improve disease control. Higher doses of IM MTX were generally well tolerated and not associated with an increase in serious adverse reactions to the drug.

Objective: To examine the effect of waiting times on the health status of patients referred for a non-urgent rheumatology opinion., Methods: The study was a randomized controlled clinical study evaluating a 'fast track' appointment with a 6-week target waiting time against an 'ordinary' appointment in the main city out-patient clinic of the rheumatology service for the Lothian and Borders region (population approximately 1 million). Health status was measured using the SF12 physical and mental summary component T-scores and pain was measured with a 100 mm visual analogue pain scale. Secondary outcomes were health utility and perceived health both measured with the EuroQol instrument, mental health measured with the Hospital Anxiety and Depression scale, disability with the modified Health Assessment Questionnaire and economic costs measured from a societal perspective., Results: Mean waiting times were 43 days (sigma = +/-16) and 105 days (sigma = +/-51) for 'fast track' and 'ordinary' appointments, respectively. Both groups showed significant improvements in mean [95% confidence interval (CI)] scores for pain: 11 (7, 16)(P < 0.001); physical health status: 4 (2, 5) (P < 0.001); mental health status: 2 (0.1, 4) (P < 0.02); and health utility: 0.11 (0.07, 0.16) (P < 0.001) by the end of the 15-month period of the study, but there was no significant difference between either arm of the study., Conclusions: Rationing by delay was not detrimental to either mental or physical health and patients in both arms of the study showed significant and similar improvement in health by 15 months. Expenditure of resources on waiting times without regard to clinical outcomes is likely to be wasteful and additional resources should be directed at achieving the greatest clinical benefit. More research into effective methods of controlling demand and better identification of those who would benefit from access to specialist care is needed.

We describe an audit of 158 patients with RA treated with weekly methotrexate and 5 mg of folic acid 24 h later. Our aim was to assess the safety and efficacy of this regime in our hands compared with published clinical trials of methotrexate in RA, and to examine patient outcomes. Treatment improved ESR, but only 69% of patients continuing therapy for prolonged periods believed their arthritis to be better on treatment. Health Assessment Questionnaire and Hospital Anxiety and Depression questionnaire scores in prospectively studied patients were not significantly altered by treatment. Toxicity occurred frequently (59% in those continuing and 89% in those ceasing therapy) and cessation of therapy solely due to lack of efficacy was rare. The probability of patients continuing with methotrexate and folic acid after 1, 2, 3 and 4 yr was 87, 76, 74 and 74%, respectively, figures that are at the upper end of the reported range for methotrexate alone.

Health economic evaluation comprises the systematic appraisal of the costs, the benefits and the relative economic efficiency of different medical interventions. The first section of this paper outlines the techniques of economic analysis and how they relate to the efficient use of health resources. This is followed by a review of the health economics of rheumatoid arthritis as a model for the wider application of health economics in the field of rheumatology.

The aims of this pilot study, which compares day patient with inpatient care for management of active RA were (i) to test the feasibility of a trial protocol design including the method of randomization and the practicality of data collection, and (ii) to obtain preliminary information on economic cost and clinical outcome of these two methods of management. Twenty consecutive patients requiring admission for management of active RA were randomized to receive either day patient or inpatient care. All hospital, transport, community and indirect costs incurred over a 6-month period from recruitment were collected for each patient. Disease activity and clinical outcome were assessed using the Ritchie articular index, ESR, Health Assessment Questionnaire, Functional Independence Measure and Hospital Anxiety and Depression Scale. The trial protocol was found to be feasible and no patient allocated to the day patient group requested or required to be transferred to inpatient care. Day care was significantly cheaper than inpatient care despite higher transport costs; the total cost of treating 10 day patients was UK 10,272 pounds compared with 14,528 pounds for 10 inpatients. Clinical outcome was comparable in both groups for all parameters studied and there was no obvious detrimental effect on patients receiving day care. This pilot study demonstrates that day care is feasible and acceptable to patients with active RA. The preliminary data suggest that day care is substantially cheaper than inpatient care and does not apparently compromise clinical outcome.

A 38-yr-old liver transplant recipient presented with a monoarthritis caused by Mycobacterium avium complex (MAC). Diagnosis was not obtained with repeated microscopy and culture of SF aspirate, and required synovial biopsy.

The case is reported of a 63 year old man presenting with a rapidly destructive symmetrical polyarthritis and widespread papular nodular skin lesions, confirmed by a biopsy to be due to multicentric reticulohistiocytosis. Biventricular cardiac failure developed secondary to extensive myocardial infiltration with multicentric reticulohistiocytosis, a complication of this disease which has not previously been reported. The joint, skin, and cardiac manifestations of multicentric reticulohistiocytosis substantially regressed following resection of an associated squamous cell carcinoma. This report adds to the small amount of published work which suggests that multicentric reticulohistiocytosis can be a paraneoplastic disease that may respond to treatment directed at the underlying tumour.

Instrumental musicians are prone to a variety of occupationally determined upper limb problems that produce significant disability and loss of earnings. As the majority of these affect the musculoskeletal system in one way or another they assume a particular relevance to the practice of rheumatology. Recent advances in our understanding of the aetiology of these conditions are described together their mode of presentation, differential diagnosis, investigation and the therapeutic options available for them.

The flow cytometric crossmatch (FCXM) has been reported to be more sensitive and capable of detecting very low levels of antibodies than the normally used complement dependent cytotoxicity test. We studied both the two colour IgG T cell FCXM and CDC-XM in 146 renal allograft recipients, 111 primary and 35 regrafts, of which 26% (29/111) of 1st and 20% (7/35) of regrafts had a positive FCXM. There was no overall correlation between the FCXM results and early graft outcome in primary renal allografts. The FCXM did not appear to have any advantage over the CDC-XM in predicting graft outcome in unsensitized first grafts. In the small number of regrafts studied, a positive FCXM was associated with a higher degree of graft failure. FCXM can exhibit false negative results if sera are used solely neat although these prozone phenomena do not influence subsequent graft outcome.

The effect of high-dose ibuprofen, a nonsteroidal anti-inflammatory drug (NSAID), on the blood pressure of treated hypertensive patients was evaluated in a randomized, placebo-controlled, double-blind, crossover trial with 24-hour ambulatory blood pressure monitoring. Twelve middle-aged black women with essential hypertension, controlled with 50 mg hydrochlorothiazide per day, randomly received 3200 mg ibuprofen and a placebo for 8 days. Each treatment phase was separated by a 1-week washout period. Ambulatory blood pressure monitoring (ABPM), body weight, and 24-hour urinary excretion of sodium, creatinine, and prostaglandin E2 (PGE2) were determined at the end of each treatment phase. Mean (+/- SEM) 24-hour systolic and diastolic blood pressures were 122/85 (+/- 2.9/1.7) and 125/85 (+/- 3.0/1.1) during the placebo and ibuprofen phases, respectively. Mean ABPM during six consecutive 4-hour periods also revealed no significant differences between placebo and ibuprofen. We conclude that 3200 mg ibuprofen per day for up to 1 week induced little change in blood pressure in hypertensive who are receiving hydrochlorothiazide.

Mobilization of the multicopy plasmid NTP16, like that of ColE1, is promoted by a range of conjugal plasmids. However, the mechanisms employed for NTP16 mobilization differ between groups. Mobilization by the IncI1 plasmid R64 requires trans-acting products from NTP16 plus a cis-acting region of the small plasmid. In contrast, this system is used inefficiently by the F plasmid and instead, a high-frequency conduction process occurs. Analysis of exconjugant cells reveals that F-mediated mobilization of NTP16 frequently involves rearrangements of NTP16 DNA, promoted by the Tn1000 transposon of F and/or by the kanamycin resistance transposon (Tn4352) of NTP16. Possible mechanisms for the high-frequency F-mediated mobilization of NTP16 are discussed. The plasmid NTP1, which is closely related to NTP16, is also mobilized efficiently by R64. It is not however efficiently mobilized by F, demonstrating the requirement for the Tn4352 element, which is not present in this plasmid, for effective F-mediated transfer.

Phenylpropanolamine (PPA) is a sympathomimetic amine and component of many over-the-counter decongestants and anorectic agents. It has been reported to cause elevated blood pressure and even hypertensive crises. The pressor effects with therapeutic doses are not well established. We monitored the effects of acute and chronic PPA dosing using 24-hour ambulatory blood pressure recording as a sensitive method of monitoring blood pressure variability. Eighteen normotensive male subjects were randomly assigned to receive 75 mg PPA (sustained-release preparation) or placebo in a double-blind crossover design with blood pressure monitored on days 1 (D1) and 6 (D6) of each period. There was no significant difference in blood pressure when compared as either 2-hour intervals or 24-hour global means: (placebo) 116/68 (D1), 117/68 (D6); (PPA) 118/69 (D1), 119/69 (D6). Our results document the absence of pressor effect with PPA in therapeutic doses even with repeated measurements and further confirm the reproducibility of 24-hour blood pressure monitoring.

A functional and physical analysis of the multicopy plasmid NTP16 is presented. The plasmid-encoded drug resistance determinants are located, as are regions encoding the origin of replication, incompatibility functions, copy number determinants, and mobility functions. It is demonstrated that NTP16 probably arose from the closely related plasmid NTP1 by the acquisition of a novel kanamycin resistance transposon, Tn4352, followed by deletion of some NTP1 sequences. The incompatibility behavior of NTP16 derivatives indicates a system of control rather more complex than that which operates in ColE1. In addition to the RNA I/primer RNA system, the production of a further trans-acting product is demonstrated and its site of action located. A series of derivative plasmids have been created which may prove useful as vectors for genetic engineering.