A Highlights from MBoC Selection: The rod domain is not essential for the function of plectin in maintaining tissue integrity

Epidermolysis bullosa simplex associated with late-onset muscular dystrophy (EBS-MD) is an autosomal recessive disorder resulting from mutations in the plectin gene. The majority of these mutations occur within the large exon 31 encoding the central rod domain and leave the production of a low-level rodless plectin splice variant unaffected. To investigate the function of the rod domain, we generated rodless plectin mice through conditional deletion of exon 31. Rodless plectin mice develop normally without signs of skin blistering or muscular dystrophy. Plectin localization and hemidesmosome organization are unaffected in rodless plectin mice. However, superresolution microscopy revealed a closer juxtaposition of the C-terminus of plectin to the integrin β4 subunit in rodless plectin keratinocytes. Wound healing occurred slightly faster in rodless plectin mice than in wild-type mice, and keratinocytes migration was increased in the absence of the rod domain. The faster migration of rodless plectin keratinocytes is not due to altered biochemical properties because, like full-length plectin, rodless plectin is a dimeric protein. Our data demonstrate that rodless plectin can functionally compensate for the loss of full-length plectin in mice. Thus the low expression level of plectin rather than the absence of the rod domain dictates the development of EBS-MD.     

Kinetic and thermodynamic analysis of the inhibitory effects of maltose,glucose, and related carbohydrates on wheat β-amylase

Inhibition of wheat β-amylase (WBA) by glucose and maltose was studied by kinetics and thermodynamics. The inhibitory effects of fructose, difructose, sucrose, trehalose, cellobiose, acarbose, and 1-deoxynojirimycin on WBA were also evaluated. The half maximal inhibitory concentrations (IC₅₀) of acarbose, maltose and glucose were 0.06 ± 0.01 M, 0.22 ± 0.09 M, and 1.41 ± 0.17 M, respectively. The inhibitor constant (K_i) and the thermodynamic parameters such as changes in Gibbs energy (ΔG), enthalpy (ΔH), and entropy (ΔS) of the dissociation reactions of the WBA-glucose and WBA-maltose complexes were temperature and pH-dependent. The dissociation reactions were endothermic and enthalpy-driven. Both glucose and maltose behaved as competitive inhibitors at pH 3.0 and 5.4 at a temperature of 25 °C with respective K_i values of 0.33 ± 0.02 M and 0.12 ± 0.03 M. In contrast, both sugars exhibited uncompetitive inhibition at pH 9 at a temperature of 25 °C with K_i values of 0.21 ± 0.03 M for glucose and 0.11 ± 0.04 M for maltose. The pH-dependence of the inhibition type and K_i values indicate that the ionizing groups of WBA influence drastically the interaction with these carbohydrates. This evidence enables us to consider temperature and pH in the WBA-catalyzed hydrolysis to manipulate the inhibition by end-product, maltose, and even by glucose.     

Influence of growth conditions on production and activity of mesenterocin 5 by a strain of Leuconostoc mesenteroides

The effects of various parameters on production and activity of mesenterocin 5, a bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides UL5, were investigated. Titres of bacteriocin and minimum inhibitory concentration values were determined by a critical dilution micromethod, using a sensitive strain of Listeria ivanovii as an indicator. Production of the antimicrobial compound was optimal at 37 and 40°C after 9 h of incubation, and was maximized in an aerobic fermentor maintained at pH 5.0. Tween 80 was a major factor in increasing mesenteroxin 5 production and specific production. Large quantities of bacteriocin could be obtained in whey and in whey permeate supplemented with yeast extract in the presence of the surfactant (0.1%). Most of the Listeria strains tested including L. monocytogenes were highly sensitive to the bacteriocin in the pH range 5.5 to 6.0 and at a temperature of 20 to 25°C.     

GeneXpert MTB/RIF Assay for the Diagnosis of Tuberculous Lymphadenitis on Concentrated Fine Needle Aspirates in High Tuberculosis Burden Settings

Introduction

The diagnosis of tuberculous lymphadenitis (TBL) remains challenging. The routinely used methods (cytology and smear microscopy) have sub-optimal sensitivity. Recently, WHO recommends GeneXpert to be used as the initial diagnostic test in patients suspected of having extra-pulmonary tuberculosis (EPTB). However, this was a conditional recommendation due to very low-quality evidence available and more studies are needed. In this study we evaluated the performance of Xpert for the diagnosis of TBL on concentrated fine needle aspirates (FNA) in Southwest Ethiopia.

Methods

FNA was collected from presumptive TBL cases. Two smears were prepared from each aspirate and processed for cytology and conventional microscopy. The remaining aspirate was treated with N-acetyl-L-cysteine-NaOH and centrifuged for 15minutes at 3000g. The concentrated sediment was used for culture and Xpert test. Capilia TB-Neo test was used to differentiate M. tuberculosis complex (MTBC) from non-tuberculous mycobacteria (NTM). Composite bacteriological methods (culture and/or smear microscopy) were considered as a reference standard.

Result

Out of 143 enrolled suspects, 64.3% (92/143) were confirmed TBL cases by the composite reference standard (CRS). Xpert detected M. tuberculosis complex (MTBC) in 60.1% (86/143) of the presumptive TBL cases. The sensitivity of Xpert compared to CRS was 87.8% [95% CI: 81.0–94.5] and specificity 91.1% [95% CI: 82.8–99.4]. The sensitivity was 27.8% for smear microscopy and 80% for cytology compared to CRS. Cytology showed the lowest specificity (57.8%). Xpert was positive in 4 out of 45 culture- and smear-negative cases. Among 47 cytomorphologically non-TBL cases, 15 were positive on Xpert. More than half of Xpert-positive cases were in the range of very low cut-off threshold values (28<Ct<38). Resistance to rifampicin was identified in 4.7% (4/86) of Xpert-positive cases.

Conclusion

Xpert test showed a high sensitivity and specificity for the diagnosis of TBL on concentrated FNA samples. In addition, Xpert offered rapid detection of rifampicin-resistant M. tuberculosis strains from lymph node aspirates.     

Dysfunction of the Scn8a Voltage-gated Sodium Channel Alters Sleep Architecture,Reduces Diurnal Corticosterone Levels,and Enhances Spatial Memory

Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of transient depolarizing currents and play a critical role in the electrical signaling between neurons. A null mutation in the VGSC gene SCN8A, which encodes the transmembrane protein Na_v1.6, was identified previously in a human family. Heterozygous mutation carriers displayed a range of phenotypes, including ataxia, cognitive deficits, and emotional instability. A possible role for SCN8A was also proposed in studies examining the genetic basis of attempted suicide and bipolar disorder. In addition, mice with a Scn8a loss-of-function mutation (Scn8a^med-Tg/+) show altered anxiety and depression-like phenotypes. Because psychiatric abnormalities are often associated with altered sleep and hormonal patterns, we evaluated heterozygous Scn8a^med-jo/+ mutants for alterations in sleep-wake architecture, diurnal corticosterone levels, and behavior. Compared with their wild-type littermates, Scn8a^med-jo/+ mutants experience more non-rapid eye movement (non-REM) sleep, a chronic impairment of REM sleep generation and quantity, and a lowered and flattened diurnal rhythm of corticosterone levels. No robust differences were observed between mutants and wild-type littermates in locomotor activity or in behavioral paradigms that evaluate anxiety or depression-like phenotypes; however, Scn8a^med-jo/+ mutants did show enhanced spatial memory. This study extends the spectrum of phenotypes associated with mutations in Scn8a and suggests a novel role for altered sodium channel function in human sleep disorders.     

Dysregulation of inflammatory responses by chronic circadian disruption

Circadian rhythms modulate nearly every mammalian physiological process. Chronic disruption of circadian timing in shift work or during chronic jet lag in animal models leads to a higher risk of several pathologies. Many of these conditions in both shift workers and experimental models share the common risk factor of inflammation. In this study, we show that experimentally induced circadian disruption altered innate immune responses. Endotoxemic shock induced by LPS was magnified, leading to hypothermia and death after four consecutive weekly 6-h phase advances of the light/dark schedule, with 89% mortality compared with 21% in unshifted control mice. This may be due to a heightened release of proinflammatory cytokines in response to LPS treatment in shifted animals. Isolated peritoneal macrophages harvested from shifted mice exhibited a similarly heightened response to LPS in vitro, indicating that these cells are a target for jet lag. Sleep deprivation and stress are known to alter immune function and are potential mediators of the effects we describe. However, polysomnographic recording in mice exposed to the shifting schedule revealed no sleep loss, and stress measures were not altered in shifted mice. In contrast, we observed altered or abolished rhythms in the expression of clock genes in the central clock, liver, thymus, and peritoneal macrophages in mice after chronic jet lag. We conclude that circadian disruption, but not sleep loss or stress, are associated with jet lag-related dysregulation of the innate immune system. Such immune changes might be a common mechanism for the myriad negative health effects of shift work.     

Trait diversity, heritability and genetic advance in selected germplasm lines of tef

The genetic improvement of the Ethiopian cereal, tef, Eragrostis tef (Zucc.) Trotter, depends upon the variability in the indigenous germplasm. A bi-replicated randomized complete block field experiment was, therefore, carried out at Debre Zeit and Alem Tena in Ethiopia during the 1996 main season to study the pheno-morphic and agronomic trait diversity in 320 tef germplasm lines. All of the 17 traits assessed showed substantial (p < or = 0.001) variation among the lines. Genotypes and locations interacted significantly (p < or = 0.05) on 11 of the traits. At about 50% similarity level, the tef lines grouped into six major clusters of nine to 243 lines. Five principal components (PCs) extracted about 71% of the entire variation of the lines. About 28% of the total variance explained by the first PC was due chiefly to variation in main shoot culm length, diameters of the two basal culm internodes, panicle length and grain yield/panicle. About 16% of the whole variance explained by the second PC originated mainly from variation in the length of the first and second basal culm internodes, grain yield/plant, and peduncle length. The third PC accounting for about 12% of the entire variance resulted largely from variation in harvest index and shoot phytomass yield/plant. Across traits, the phenotypic and genotypic coefficients of variation varied in that order from about 2% for grain yield/panicle to 58% for number of fertile tillers/plant, and from less than 1% for diameters of the two basal culm internodes and grain yield/panicle to 35% for panicle length. Estimates of broad sense heritability and genetic advance (as ratio of the mean) were highest for panicle length (71%) and number of fertile tillers/plant (21%), respectively. But both of these were lowest for the second basal culm internode diameter (< 1%). Overall, the study confirmed that tef is a highly versatile crop with broad trait diversity in the germplasm, and this offers ample opportunities for improvement through breeding.     

Iron-Dependent Regulation of Hepcidin in Hjv?/? Mice: Evidence That Hemojuvelin Is Dispensable for Sensing Body Iron Levels

Hemojuvelin (Hjv) is a bone morphogenetic protein (BMP) co-receptor involved in the control of systemic iron homeostasis. Functional inactivation of Hjv leads to severe iron overload in humans and mice due to marked suppression of the iron-regulatory hormone hepcidin. To investigate the role of Hjv in body iron sensing, Hjv−/− mice and isogenic wild type controls were placed on a moderately low, a standard or a high iron diet for four weeks. Hjv−/− mice developed systemic iron overload under all regimens. Transferrin (Tf) was highly saturated regardless of the dietary iron content, while liver iron deposition was proportional to it. Hepcidin mRNA expression responded to fluctuations in dietary iron intake, despite the absence of Hjv. Nevertheless, iron-dependent upregulation of hepcidin was more than an order of magnitude lower compared to that seen in wild type controls. Likewise, iron signaling via the BMP/Smad pathway was preserved but substantially attenuated. These findings suggest that Hjv is not required for sensing of body iron levels and merely functions as an enhancer for iron signaling to hepcidin.