The Critical Role of Equilibrative Nucleoside Transporter-2 in Modulating Cerebral Damage and Vascular Dysfunction in Mice with Brain Ischemia-Reperfusion.

BACKGROUND: Cerebral vascular protection is critical for stroke treatment. Adenosine modulates vascular flow and exhibits neuroprotective effects, in which brain extracellular concentration of adenosine is dramatically increased during ischemic events and ischemia-reperfusion. Since the equilibrative nucleoside transporter-2 (Ent2) is important in regulating brain adenosine homeostasis, the present study aimed to investigate the role of Ent2 in mice with cerebral ischemia-reperfusion. METHODS: Cerebral ischemia-reperfusion injury was examined in mice with transient middle cerebral artery occlusion (tMCAO) for 90 minutes, followed by 24-hour reperfusion. Infarct volume, brain edema, neuroinflammation, microvascular structure, regional cerebral blood flow (rCBF), cerebral metabolic rate of oxygen (CMRO2), and the production of reactive oxygen species (ROS) were examined following the reperfusion. RESULTS: Ent2 deletion reduced the infarct volume, brain edema, and neuroinflammation in mice with cerebral ischemia-reperfusion. tMCAO-induced disruption of brain microvessels was ameliorated in Ent2-/- mice, with a reduced expression of matrix metalloproteinases-9 and aquaporin-4 proteins. Following the reperfusion, the rCBF of the wild-type (WT) mice was quickly restored to the baseline, whereas, in Ent2-/- mice, rCBF was slowly recovered initially, but was then higher than that in the WT mice at the later phase of reperfusion. The improved CMRO2 and reduced ROS level support the beneficial effects caused by the changes in the rCBF of Ent2-/- mice. Further studies showed that the protective effects of Ent2 deletion in mice with tMCAO involve adenosine receptor A2AR. CONCLUSIONS: Ent2 plays a critical role in modulating cerebral collateral circulation and ameliorating pathological events of brain ischemia and reperfusion injury.

Intracellular vesicle clusters are organelles that synthesize extracellular vesicle-associated cargo proteins in yeast.

Extracellular vesicles (EVs) play important roles in cell-cell communication. In budding yeast (Saccharomyces cerevisiae), EVs function as carriers to transport cargo proteins into the periplasm for storage during glucose starvation. However, intracellular organelles that synthesize these EV-associated cargo proteins have not been identified. Here, we investigated whether cytoplasmic organelles-called intracellular vesicle clusters (IVCs)-serve as sites for the synthesis of proteins targeted for secretion as EV-associated proteins. Using proteomics, we identified 377 IVC-associated proteins in yeast cells grown under steady-state low-glucose conditions, with the largest group being involved in protein translation. Isolated IVCs exhibited protein synthesis activities that required initiation and elongation factors. We have also identified 431 newly synthesized proteins on isolated IVCs. Expression of 103Q-GFP, a foreign protein with a long polyglutamine extension, resulted in distribution of this protein as large puncta that co-localized with IVC markers, including fructose-1,6-bisphosphatase (FBPase) and the vacuole import and degradation protein Vid24p. We did not observe this pattern in cycloheximide-treated cells or in cells lacking VID genes, required for IVC formation. The induction of 103Q-GFP on IVCs adversely affected total protein synthesis in intact cells and on isolated IVCs. This expression also decreased levels of EV-associated cargo proteins in the extracellular fraction without affecting the number of secreted EVs. Our results provide important insights into the functions of IVCs as sites for the synthesis of EV-associated proteins targeted for secretion to the periplasm.

Which Fruits and Vegetables Should Be Excluded from a Low-Salicylate Diet? An Analysis of Salicylic Acid in Foodstuffs in Taiwan.

BACKGROUND: Eliminating pseudoallergens is an important element of managing chronic spontaneous urticaria (CSU). Salicylic acid (SA) is a primary pseudoallergen in plant-based foodstuffs. Current dietary recommendations are not applicable in East Asia because data on the SA content of many vegetables and fruits commonly consumed in this region are lacking. METHODS: We therefore determined the concentration of free SA in 79 popular vegetables and fruits frequently consumed in Taiwan using gas chromatography-mass spectrometry. RESULTS: The SA content ranged from 0.09 to 2.3 mg/kg in the fresh vegetables examined, and from 0.01 to 0.48 mg/kg in the fruits. CONCLUSIONS: Data regarding the SA content of East Asian vegetables and fruits could help CSU patients limit their pseudoallergen consumption.

Vps15p regulates the distribution of cup-shaped organelles containing the major eisosome protein Pil1p to the extracellular fraction required for endocytosis of extracellular vesicles carrying metabolic enzymes.

BACKGROUND INFORMATION: Exosomes are small vesicles secreted from virtually every cell from bacteria to humans. Saccharomyces cerevisiae is a model system to study trafficking of small vesicles in response to changes in the environment. When yeast cells are grown in low glucose, vesicles carrying gluconeogenic enzymes are present as free vesicles and aggregated clusters in the cytoplasm. These vesicles are also secreted into the periplasm and account for more than 90% of total extracellular organelles, while less than 10% are larger 100-300 nm structures with unknown functions. When glucose is added to glucose-starved cells, secreted vesicles are endocytosed and then targeted to the vacuole. Recent secretomic studies indicated that more than 300 proteins involved in diverse biological functions are secreted during glucose starvation and endocytosed during glucose re-feeding. We hypothesised that extracellular vesicles are internalised using novel mechanisms independent of clathrin-mediated endocytosis. RESULTS: Our results showed that vesicles carrying metabolic enzymes were endocytosed at a fast rate, whereas vesicles carrying the heat shock protein Ssa1p were endocytosed at a slow rate. The PI3K regulator Vps15p is critical for the fast internalisation of extracellular vesicles. VPS15 regulates the distribution of the 100-300 nm organelles that contain the major eisosome protein Pil1p to the extracellular fraction. These Pil1p-containing structures were purified and showed unique cup-shape with their centres deeper than the peripheries. In the absence of VPS15, PIL1 or when PIL1 was mutated, the 100-300 nm structures were not observed in the extracellular fraction and the rapid internalisation of vesicles was impaired. CONCLUSIONS: We conclude that VPS15 regulates the distribution of the 100-300 nm Pil1p-containing organelles to the extracellular fraction required for fast endocytosis of vesicles carrying metabolic enzymes. This work provides the first evidence showing that Pil1p displayed unique distribution patterns in the intracellular and extracellular fractions. This work also demonstrates that endocytosis of vesicles is divided into a fast and a slow pathway. The fast pathway is the predominant pathway and is used by vesicles carrying metabolic enzymes. Cup-shaped Pil1p-containing structures are critical for the rapid endocytosis of vesicles into the cytoplasm. SIGNIFICANCE: This work provides the first evidence showing that Pil1p displayed unique distribution patterns in the intracellular and extracellular fractions. This work also demonstrates that endocytosis of vesicles is divided into a fast and a slow pathway. The fast pathway is the predominant pathway and is used by vesicles carrying metabolic enzymes. Cup-shaped Pil1p-containing structures are critical for the rapid endocytosis of vesicles into the cytoplasm.

Vid28 protein is required for the association of vacuole import and degradation (Vid) vesicles with actin patches and the retention of Vid vesicle proteins in the intracellular fraction.

Gluconeogenic enzymes are induced when Saccharomyces cerevisiae are starved of glucose. However, when glucose is added to prolonged starved cells, these enzymes are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. The Vid pathway is linked to the nonclassical secretory and internalizing pathways. In prolonged starved cells, substantial amounts of the key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) are in the extracellular fraction (periplasm). However, when glucose is added to glucose-starved cells, levels of extracellular FBPase decrease rapidly. Ultrastructural studies indicate that FBPase is in Vid/endosomes following glucose addition, suggesting that FBPase is internalized in response to glucose refeeding. Under the same conditions, the majority of Vid vesicle proteins are in the intracellular fraction. In yeast, actin polymerization is involved in endocytosis. Vid vesicles associate with actin patches initially, and they dissociate later. Here, we show that VID28 plays a critical role in the association of Vid vesicles with actin patches and the retention of Vid vesicle proteins in the intracellular fraction. Vid28p was distributed to Vid vesicles and interacted with other Vid vesicle proteins. Vid28p contains an Armadillo (ARM) domain required for FBPase degradation. When VID28 was deleted or when the ARM domain was mutated, Vid vesicles failed to co-localize with actin patches, and Vid vesicle proteins appeared in the extracellular fraction. We suggest that the ARM domain is required for the association of Vid vesicles with actin patches and the retention of Vid vesicle proteins in the intracellular fraction.

Glucose induces rapid changes in the secretome of Saccharomyces cerevisiae.

BACKGROUND: Protein secretion is a fundamental process in all living cells. Proteins can either be secreted via the classical or non-classical pathways. In Saccharomyces cerevisiae, gluconeogenic enzymes are in the extracellular fraction/periplasm when cells are grown in media containing low glucose. Following a transfer of cells to high glucose media, their levels in the extracellular fraction are reduced rapidly. We hypothesized that changes in the secretome were not restricted to gluconeogenic enzymes. The goal of the current study was to use a proteomic approach to identify extracellular proteins whose levels changed when cells were transferred from low to high glucose media. RESULTS: We performed two iTRAQ experiments and identified 347 proteins that were present in the extracellular fraction including metabolic enzymes, proteins involved in oxidative stress, protein folding, and proteins with unknown functions. Most of these proteins did not contain typical ER-Golgi signal sequences. Moreover, levels of many of these proteins decreased upon a transfer of cells from media containing low to high glucose media. Using an extraction procedure and Western blotting, we confirmed that the metabolic enzymes (glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, glucose-6-phosphate dehydrogenase, pyruvate decarboxylase), proteins involved in oxidative stress (superoxide dismutase and thioredoxin), and heat shock proteins (Ssa1p, Hsc82p, and Hsp104p) were in the extracellular fraction during growth in low glucose and that the levels of these extracellular proteins were reduced when cells were transferred to media containing high glucose. These proteins were associated with membranes in vesicle-enriched fraction. We also showed that small vesicles were present in the extracellular fraction in cells grown in low glucose. Following a transfer from low to high glucose media for 30 minutes, 98% of these vesicles disappeared from the extracellular fraction. CONCLUSIONS: Our data indicate that transferring cells from low to high glucose media induces a rapid decline in levels of a large number of extracellular proteins and the disappearance of small vesicles from the extracellular fraction. Therefore, we conclude that the secretome undergoes dynamic changes during transition from glucose-deficient to glucose-rich media. Most of these extracellular proteins do not contain typical ER signal sequences, suggesting that they are secreted via the non-classical pathway.

"It'll never end, I'll never go": Representation of Caregiving in Samuel Beckett's Endgame and Footfalls.

Research on the unrepresentability of death in Samuel Beckett's oeuvre abound in Beckett scholarship, but little attention has been given to the artist's representation of caregiving to the dying in his plays. With reference to Martin Heidegger's concept of care and Albert Camus's idea of the absurd, this article analyzes Endgame (1957) and Footfalls (1976) by attending to Beckett's dramatic representation of caregiving as undergirded by a sense of its absurdity. The almost 20-year gap between the writing of both plays highlights the development of an understanding that this sense of absurdity is never about the caregiver's questioning of one's obligation to the dependent but about how one chooses to respond to caregiving as an absurd predicament. The pertinence of such a representation of caregiving by Beckett lies in its poignant articulation of a complex experience that is often left unexpressed by caregivers who prioritize their dependent loved ones over themselves.

Vps34p is required for the decline of extracellular fructose-1,6-bisphosphatase in the vacuole import and degradation pathway.

When Saccharomyces cerevisiae are starved of glucose for a prolonged period of time, gluconeogenic enzymes such as fructose-1,6-bisphosphatase (FBPase), malate dehydrogenase, isocitrate lyase, and phosphoenolpyruvate carboxykinase are induced. However, when glucose is added to prolonged-starved cells, these enzymes are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. The Vid pathway merges with the endocytic pathway to remove intracellular and extracellular proteins simultaneously. Ultrastructural and cell extraction studies indicate that substantial amounts of FBPase were in the extracellular fraction (periplasm) during glucose starvation. FBPase levels in the extracellular fraction decreased after glucose re-feeding in wild-type cells. The decline of FBPase in the extracellular fraction was dependent on the SLA1 and ARC18 genes involved in actin polymerization and endocytosis. Moreover, the reduction of extracellular FBPase was also dependent on the VPS34 gene. VPS34 encodes the PI3 kinase and is also required for the Vid pathway. Vps34p co-localized with actin patches in prolonged-starved cells. In the absence of this gene, FBPase and the Vid vesicle protein Vid24p associated with actin patches before and after the addition of glucose. Furthermore, high levels of FBPase remained in the extracellular fraction in the Δvps34 mutant during glucose re-feeding. When the Asn-736 residue of Vps34p was mutated and when the C-terminal 11 amino acids were deleted, mutant proteins failed to co-localize with actin patches, and FBPase in the extracellular fraction did not decrease as rapidly. We suggest that VPS34 plays a critical role in the decline of extracellular FBPase in response to glucose.

Comparative proteomic analysis of transition of saccharomyces cerevisiae from glucose-deficient medium to glucose-rich medium.

BACKGROUND: When glucose is added to Saccharomyces cerevisiae grown in non-fermentable carbon sources, genes encoding ribosomal, cell-cycle, and glycolytic proteins are induced. By contrast, genes involved in mitochondrial functions, gluconeogenesis, and the utilization of other carbon sources are repressed. Glucose also causes the activation of the plasma membrane ATPase and the inactivation of gluconeogenic enzymes and mitochondrial enzymes. The goals of this study were to use the iTRAQ-labeling mass spectrometry technique to identify proteins whose relative levels change in response to glucose re-feeding and to correlate changes in protein abundance with changes in transcription and enzymatic activities. We used an experimental condition that causes the degradation of gluconeogenic enzymes when glucose starved cells are replenished with glucose. Identification of these enzymes as being down-regulated by glucose served as an internal control. Furthermore, we sought to identify new proteins that were either up-regulated or down-regulated by glucose. RESULTS: We have identified new and known proteins that change their relative levels in cells that were transferred from medium containing low glucose to medium containing high glucose. Up-regulated proteins included ribosomal subunits, proteins involved in protein translation, and the plasma membrane ATPase. Down-regulated proteins included small heat shock proteins, mitochondrial proteins, glycolytic enzymes, and gluconeogenic enzymes. Ach1p is involved in acetate metabolism and is also down-regulated by glucose. CONCLUSIONS: We have identified known proteins that have previously been reported to be regulated by glucose as well as new glucose-regulated proteins. Up-regulation of ribosomal proteins and proteins involved in translation may lead to an increase in protein synthesis and in nutrient uptake. Down-regulation of glycolytic enzymes, gluconeogenic enzymes, and mitochondrial proteins may result in changes in glycolysis, gluconeogenesis, and mitochondrial functions. These changes may be beneficial for glucose-starved cells to adapt to the addition of glucose.

Predictors of Response to Oral Medications and Low-Histamine Diet in Patients with Chronic Urticaria.

BACKGROUND: Chronic urticaria (CU) is comprised of diverse phenotypes, and thus, a shift towards a precision medical approach is warranted in its management. METHODS: This study enrolled 78 patients with CU. Serum erythrocyte sedimentation rate, hemoglobin, hematocrit, eosinophil count, IgE, antinuclear antibody (ANA), and serum diamine oxidase (DAO) levels of the patients were measured and were compared according to the patient's response to second-generation antihistamines (sgAH), corticosteroids, leukotriene receptor antagonist (LTRA), H2 blockers, and low-histamine diet. RESULTS: Age- and sex-adjusted logistic regression analysis showed that patients with duration of CU > 3 years (adjusted odd ratio [aOR] = 4.39) and a DAO level < 10 U/mL (aOR = 3.90) were significantly associated with a good sgAH response. Age > 50 years (aOR = 0.02), duration of chronic urticaria > 3 years (aOR =0.06), and an ANA titer ≥ 1 : 80 (aOR = 0.03) were significantly and inversely associated with corticosteroid response. A low-histamine diet response was significantly associated with LTRA response (aOR = 67.29). In addition, a DAO level < 5.4 U/mL (aOR = 71.95) was significantly associated with H2 blocker response. Furthermore, concomitant angioedema (aOR = 10.56), multiple food triggers (aOR = 11.69), and a DAO level < 5.4 U/mL (aOR = 3.78) were significantly associated with a low-histamine diet response. Conversely, dermatographic urticaria and a hematocrit level < 36% were significantly and inversely associated with low-histamine diet response. CONCLUSIONS: Several promising biomarkers were identified in this study to predict the efficacy of chronic urticaria treatment. DAO could be a novel biomarker for predicting the efficacy not only of dietary intervention but also for antagonists of H1 and H2 receptors.

The vacuole import and degradation pathway utilizes early steps of endocytosis and actin polymerization to deliver cargo proteins to the vacuole for degradation.

When glucose is added to yeast cells that are starved for 3 days, fructose-1,6-bisphosphatase (FBPase) and malate dehydrogenase 2 are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. In this study, we examined the distribution of FBPase at the ultrastructural level. FBPase was observed in areas close to the plasma membrane and in cytoplasmic structures that are heterogeneous in size and density. We have isolated these intracellular structures that contain FBPase, the Vid vesicle marker Vid24p, and the endosomal marker Pep12p. They appeared irregular in size and shape. In yeast, actin polymerization plays an important role in early steps of endocytosis. Mutants that affect actin polymerization inhibited FBPase degradation, suggesting that actin polymerization is important for FBPase degradation. Both FBPase and malate dehydrogenase 2 were associated with actin patches. Vid vesicle proteins such as Vid24p or Sec28p were also at actin patches, although they dissociated from these structures at later time points. We propose that Vid24p and Sec28p are present at actin patches during glucose starvation. Cargo proteins arrive at these sites following the addition of glucose, and the endocytic vesicles then pinch off from the plasma membrane. Following the fusion of endosomes with the vacuole, cargo proteins are then degraded in the vacuole.

The TOR complex 1 is distributed in endosomes and in retrograde vesicles that form from the vacuole membrane and plays an important role in the vacuole import and degradation pathway.

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced when Saccharomyces cerevisiae are starved of glucose. However, when glucose is added to cells that have been starved for 3 days, FBPase is degraded in the vacuole. FBPase is first imported to Vid (vacuole import and degradation) vesicles, and these vesicles then merge with the endocytic pathway. In this report we show that two additional gluconeogenic enzymes, isocitrate lyase and phosphoenolpyruvate carboxykinase, were also degraded in the vacuole via the Vid pathway. These new cargo proteins and FBPase interacted with the TORC1 complex during glucose starvation. However, Tor1p was dissociated from FBPase after the addition of glucose. FBPase degradation was inhibited in cells overexpressing TOR1, suggesting that excessive Tor1p is inhibitory. Both Tco89p and Tor1p were found in endosomes coming from the plasma membrane as well as in retrograde vesicles forming from the vacuole membrane. When TORC1 was inactivated by rapamycin, FBPase degradation was inhibited. We suggest that TORC1 interacts with multiple cargo proteins destined for the Vid pathway and plays an important role in the degradation of FBPase in the vacuole.

Association between clinical phenotypes of dermatomyositis and polymyositis with myositis-specific antibodies and overlap systemic autoimmune diseases.

ABSTRACT: The aim of this study was to evaluate the association between clinical phenotypes of dermatomyositis (DM) and polymyositis (PM) with myositis-specific antibodies (MSAs), and overlap diagnosis of systemic autoimmune diseases.This cross-sectional study was conducted on 67 patients with DM and 27 patients with PM recruited from a regional hospital in southern Taiwan. Clinical phenotypes of DM and PM were assessed and MSAs were measured using a commercial line blot assay. The association of clinical phenotypes of DM and PM with MSAs and overlap diagnosis of systemic autoimmune diseases was performed using univariate and multiple logistic regression analyses.Clinically, patients with DM and PM and overlap diagnosis of systemic sclerosis were associated with a higher risk of interstitial lung diseases (ILDs) (odds ratio [OR] = 6.73; P = .048), Raynaud phenomenon (OR = 7.30; P = .034), and malignancy (OR = 350.77; P = .013). The risk of malignancy was also associated with older age (OR 1.31; P = .012), and male patients were associated with a higher risk of fever. For MSAs, anti-aminoacyl-tRNA synthetase antibodies were associated with ILD, antinuclear antibody were associated with a lower risk of arthritis, anti-transcription intermediary factor 1-gamma antibodies were associated with milder symptoms of muscle weakness, anti-Ku antibodies were associated with overlap diagnosis of systemic lupus erythematosus, and anti-Ro52 antibodies were associated with the development of Raynaud phenomenon and Sjögren syndrome.MSAs and overlap diagnosis of systemic sclerosis were significantly associated with clinical phenotypes of DM and PM. Physicians should be vigilant for malignancy in older DM and PM patients with overlap diagnosis of systeic sclerosis. The possibility of developing ILD in patients with overlap diagnosis of systemic sclerosis or serum positivity of anti-aminoacyl-tRNA synthetase antibodies should be considered.

The Shape of Vesicle-Containing Organelles Is Critical for Their Functions in Vesicle Endocytosis.

Exosomes are small vesicles secreted by a variety of cell types under physiological and pathological conditions. When Saccharomyces cerevisiae are grown in low glucose, small vesicles carrying more than 300 proteins with diverse biological functions are secreted. Upon glucose addition, secreted vesicles are endocytosed that requires cup-shaped organelles containing the major eisosome protein Pil1p at the rims. We aim to identify genes that regulate the function of cup-shaped organelles in vesicle endocytosis. In cells lacking either VID27 or VID21, Pil1p distribution was altered and cup-shaped organelles became elongated with narrower openings. Change in shape reduced the number of vesicles in the deeper areas and impaired vesicle endocytosis. Vid21p and Vid27p were localized to vesicle clusters and interacted with other Vid proteins. In the absence of these genes, these vesicles failed to aggregate and were secreted. Vid21p and Vid27p are required for the aggregation and retention of vesicles that contain Vid proteins in the cytoplasm. Increased vesicles near the plasma membrane in mutant strains correlate with an increased Pil1p movement resulting in the fusion of cup-shaped organelles. We conclude that the shape of vesicle-containing organelles is critical for their functions in vesicle endocytosis.

Comparison of sensitivity of transcarpal median motor conduction velocity and conventional conduction techniques in electrodiagnosis of carpal tunnel syndrome.

OBJECTIVE: To compare the sensitivity of median wrist-palm motor conduction velocity (W-P MCV) with those of standard sensory conduction techniques in the electrodiagnosis of carpal tunnel syndrome (CTS). METHODS: This study included 280 consecutively suspected CTS patients (360 hands) referred for evaluation and 150 volunteers who served as controls. We determined and calculated (1) median W-P MCV, (2) median motor distal latencies (DL) and median sensory DL for (3) thumb (D1), (4) index (D2) and (5) ring finger (D4), (6) median wrist-palm sensory conduction velocity (W-P SCV) and sensory conduction time (W-P SCT) for index finger and sensory latency differences between (7) median-radial (M-R) for thumb and (8) median-ulnar (M-U) nerves for ring finger. The normal limits were calculated from the median of normal controls +/-2.5 standard deviations. The sensitivities of each test were determined and compared. RESULTS: Among the 360 hands with suspected CTS, 32 hands (8.9%) had normal electrodiagnostic studies and 328 (91.1%) had at least one abnormal electrodiagnostic study. Among the 328 hands with abnormalities, 234 (65%) had abnormal motor DL and 294 (81.7%) had abnormal W-P MCV. The sensitivity was 80.3% for D1, 72.5% for D2, 76.7% for D4, 86.7% for M-R (specificity, 98.7%), 87.2% for M-U (specificity, 96.7%), 80.8% for sensory W-P SCT and 73.6% for W-P SCV. CONCLUSIONS: W-P MCV is a valuable motor conduction technique for the diagnosis of CTS and it is confirmed again that W-P MCV is equal to or more sensitive than W-P SCV and W-P SCT. Furthermore, the findings of the present study are in agreement with the conventional wisdom that internal comparison of latency differences between median and ulnar or radial nerves is the best method for a diagnosis of patients with suspected CTS. Therefore, we recommend that CTS patients be studied according to the following steps: (1) routine sensory and motor DL, (2) if step 1 is negative, then perform and determine W-P MCV or SCT. This may increase the diagnostic yield of 10%, (3) if step 2 is negative, measure the M-U or MR. These are the final and more sensitive techniques in making a diagnosis with an additional diagnostic yield of 10%. SIGNIFICANCE: We provide the evidence of W-P MCV that could be a standard technique for electrodiagnosis of CTS. Furthermore, we make a reasonable flow chart and recommendation for electrodiagnosis of CTS for electromyographers.

Yeast as a Model System to Study Trafficking of Small Vesicles Carrying Signal-less Proteins In and Out of the Cell.

Exosomes are small vesicles that are released from a variety of cells and are involved in cell-to-cell communication. In humans, exosomes are detected in the plasma, urine, saliva, and cerebrospinal fluid. These vesicles carry multiple cargo proteins, as well as microRNA that affect the transcription of target genes. During cancer progression, secretion of exosomes increases. As such, proteins and microRNAs released from exosomes in cancer patients can be used as biomarkers for cancer diagnosis and progression. Yeast is an excellent model system to study trafficking of small vesicles in and out of the cells. When yeast cells are grown in low glucose, small vesicles transport gluconeogenic enzymes to different locations. In the cytoplasm, they exist as free vesicles and aggregated clusters. They are also secreted and account for more than 90% of total organelles in the extracellular fraction. When glucose is added to prolongedstarved cells, extracellular vesicles are endocytosed resulting in a dramatic decrease in vesicles in the extracellular fraction and the appearance of vesicle clusters in the cytoplasm. Internalization also leads to a rapid decline in protein levels for vesicle-associated proteins in the extracellular fraction. Using large-scale proteomics/secretomics, more than 300 proteins that were secreted into the extracellular fraction/periplasm were identified. They showed distribution patterns similar to gluconeogenic enzymes. They were secreted during glucose starvation and their levels in the extracellular fraction decreased following glucose re-feeding. Multiple techniques have been used to study the biogenesis, clustering, secretion, and internalization of vesicles in yeast.

Does retrograde axonal atrophy really occur in carpal tunnel syndrome patients with normal forearm conduction velocity?

OBJECTIVE: The cause of decreased median forearm motor conduction velocity (FMCV) in carpal tunnel syndrome (CTS) is best ascribed to retrograde axonal atrophy (RAA); however, the relationships between the occurrence of RAA and electrophysiological or clinical severity remains controversial. We attempt to determine whether RAA really occurs in CTS patients with normal median FMCV and to investigate any relationships between RAA and severity of compression at the wrist. METHODS: Consecutive CTS patients were enrolled and age-matched volunteers served as controls. We performed conventional nerve conduction studies (NCS) and measured median and ulnar distal motor latencies (DML), FMCV, compound muscle action potential (CMAP) amplitudes, distal sensory latencies (DSL), and sensory nerve action potential (SNAP) amplitudes. Furthermore, palmar median stimulation was done to calculate the wrist-palm motor conduction velocity (W-P MCV). Patients included for analysis should have normal FMCV and needle examination. We compared each electrodiagnostic parameters between the patient group and controls. RESULTS: The mean+/-SD of the W-P MCV for patients and controls were 33.26+/-6.74 and 52.14+/-5.85 m/s and those of median FMCV were 55.26+/-3.56 and 57.82+/-3.9 m/s, respectively. There was a significant reduction in the W-P MCV (36.2%, P<0.00001), significant decrease in the median FMCV (4.43%, P<0.00001) and SNAP amplitudes, and an increase of the DML and DSL in the patient group (P<0.00001) compared to the controls; however, there were no differences in median and ulnar CMAP amplitudes, ulnar FMCV and DML between the controls and patients. CONCLUSIONS: RAA and relatively slowed median FMCV do occur in CTS patients with normal median FMCV, regardless of severity of clinical manifestations and electrophysiological abnormalities. SIGNIFICANCE: This article provides new information for research of the electrophysiological changes of the proximal nerve part at distal injury.

The cause of slowed forearm median conduction velocity in carpal tunnel syndrome: a Palmar stimulation study.

OBJECTIVES: To elucidate the etiopathogenesis of decreased forearm median motor conduction velocity (FMMCV) in carpal tunnel syndrome (CTS), we used segmental stimulation at the palm, wrist and antecubital fossa to determine conduction block at wrist and calculate and compare the segmental median motor conduction velocity (MMCV) to determine the pathogenesis. BACKGROUND: The cause of the decreased FMMCV in CTS remains unclear. Animal models have supported retrograde axonal atrophy as the cause. Some authors believe standard FMMCV, calculated by subtracting the distal latency, may not represent an exact assessment of FMMCV but rather the velocity of small fibers that persist throughout the carpal tunnel. SUBJECTS AND METHODS: Patients with clinical symptoms and signs of CTS which had been confirmed with standard electrodiagnosis, were included. The patients were divided into two groups: one with reduced FMMCV <50m/s (Group I, n=20) and the other with normal FMMCV>50m/s (Group II, n=40). Age-matched volunteers served as controls (n=60). We used palm, wrist and antecubital stimulation, and recorded compound muscle action potential (CMAP) amplitudes at the abductor pollicis brevis (APB) muscle. Based on a ratio of the CMAP amplitudes obtained from wrist and palm stimulation (W/P ratio) and the latency differences, we calculated the W/P ratio and the across wrist MMCV (AWMMCV) and FMMCV and compared and correlated them between two patient groups. RESULTS: There was no difference in median motor and sensory distal latency between Groups I and II. CMAP and sensory nerve action potential amplitudes were reduced in Group I compared with Group II, but the difference was only marginally significant. Four patients had a significant reduction of the W/P ratio in Group I, compared with 7 patients in Group II, which did not reach a significance. Sixteen patients (80%) in Group I demonstrated no conduction block. Furthermore, Group I showed significantly decreased FMMCV when compared with Group II; however, AWMMCV was not significantly reduced in Group I, suggesting that decreased FMMCV does not result from a decrease in AWMMCV. CONCLUSIONS: There was no significant motor conduction block and no correlation of the FMMCV and AWMMCV in CTS patients with a decrease of FMMCV, suggesting retrograde axonal atrophy, and not selective conduction block of the large fibers at the wrist, is the direct cause of decreased FMMCV in CTS.

Does direct measurement of forearm mixed nerve conduction velocity reflect actual nerve conduction velocity through the carpal tunnel?

OBJECTIVES: The purpose of this study was to determine whether forearm (wrist-elbow) mixed nerve conduction velocity (W-Emix) represents the actual nerve conduction velocity (CV) of nerve fibers passing through the carpal tunnel. BACKGROUND: W-Emix is presumed to reflect the actual forearm CV through the carpal tunnel. However, it has been argued that W-Emix chiefly originates from the nerve fibers passing outside the carpal tunnel. Therefore, the direct measurement of W-Emix cannot be used to assess retrograde axonal atrophy in carpal tunnel syndrome (CTS). SUBJECTS AND METHODS: Thirty patients with clinical signs and symptoms of CTS were recruited and the diagnosis was confirmed with standard electrodiagnosis. Fifty age-matched volunteers served as control. Recording electrodes were placed over the elbow and index finger for mixed nerve and sensory nerve conduction studies, respectively. Stimulation was applied at the palm and wrist for the measurement of mixed nerve wrist-palm CV (W-Pmix), wrist-elbow CV (W-Emix), and elbow-palm CV (E-Pmix). Stimulation was applied at the elbow, wrist, and palm for the measurement of wrist-elbow sensory CV (W-Esen), wrist-palm CV (W-Psen), and elbow-palm CV (E-Psen). Comparisons were made between W-Pmix and W-Psen, W-Emix and W-Esen, and E-Pmix and E-Psen. RESULTS: Correlations between W-Emix and W-Esen, E-Pmix and E-Psen, and W-Pmix and W-Psen were good in the control. In the patient group, there was a strong positive correlation between W-Pmix and W-Psen, and between E-Pmix and E-Psen. However, W-Esen correlated weakly with W-Emix, suggesting that W-Emix chiefly represents the CV of fibers passing outside the carpal tunnel. Therefore, the direct measurement of W-Emix cannot be used to assess retrograde axonal atrophy. Furthermore, the reduction in W-Psen was more marked than the reduction in W-Esen, implying that a conduction block at the wrist is the least likely cause of proximal slowing in CTS. CONCLUSIONS: W-Emix does not reflect the actual CV of the nerve fibers passing through the carpal tunnel. In addition, retrograde axonal atrophy appears to be the primary cause of decreased forearm CV in CTS.

Forearm mixed nerve conduction velocity: questionable role in the evaluation of retrograde axonal atrophy in carpal tunnel syndrome.

The objective of this study was to determine whether forearm mixed nerve conduction velocity (Fmix) reflects the real conduction velocity of forearm motor nerve (Fmot) and forearm sensory nerve (Fsen) fibers passing through the carpal tunnel. Forearm mixed nerve conduction velocity is presumed to be indicative of the conduction velocity of the median nerve over the forearm. Therefore, Fmix is used widely to assess the causes of slowing forearm conduction velocity in carpal tunnel syndrome. However, some authors claim that Fmix comes chiefly from the undamaged fibers in carpal tunnel syndrome, and thus cannot replace Fmot or Fsen in the evaluation of retrograde axonal atrophy. Patients with clinical symptoms and signs of carpal tunnel syndrome confirmed with standard electrodiagnosis were included. Age-matched volunteers served as control subjects. Conduction velocities across the wrist and over the forearm were measured, including those of the wrist sensory (Wsen), wrist motor (Wmot), and wrist mixed nerves (Wmix); and forearm mixed (Fmix), forearm motor (Fmot), and forearm sensory nerves (Fsen). The authors compared and correlated Wsen, Wmot, and Wmix; and Fmix, Fmot, and Fsen respectively. The mean values of Wsen, Wmot, Wmix, Fmix, Fmot, and Fsen of the control subjects less those of corresponding conduction velocity of carpal tunnel syndrome patients were designated Wsen N, Wmot N, Wmix N, Fmix N, Fmot N, and Fsen N respectively and were compared and correlated again. Wrist motor nerve conduction velocity, Wsen, and Wmix were significantly lower in carpal tunnel syndrome patients, and Fmot and Fsen but not Fmix were reduced significantly when compared with control subjects. Mean wrist sensory nerve conduction velocity, Wmot N, and Wmix N; and Fsen N and Fmot N showed good correlation except for Fmix N, suggesting that Fmix reflects the conduction velocity of undamaged fibers in carpal tunnel syndrome. Forearm mixed nerve conduction velocity cannot replace Fmot or Fsen in the assessment of retrograde axonal atrophy in carpal tunnel syndrome. In the disease state, Fmix possibly represents the conduction velocity of the palmar cutaneous branch.