Comparative investigation of the pathogenicity of three Mycobacterium tuberculosis mutants defective in the synthesis of p-hydroxybenzoic acid derivatives

p-Hydroxybenzoic acid derivatives (p-HBADs) are glycoconjugates secreted by all Mycobacterium tuberculosis isolates whose contribution to pathogenicity remains to be determined. The pathogenicity of three transposon mutants of M. tuberculosis deficient in the biosynthesis of some or all forms of p-HBADs was studied. Whilst the mutants grew similarly to the wild-type strain in macrophages and C57BL/6 mice, two of the mutants induced a more severe and diffuse inflammation in the lungs. The lack of production of some or all forms of p-HBADs in these two mutants also correlated with an increased secretion of the pro-inflammatory cytokines tumour-necrosis factor alpha, interleukin 6 and interleukin 12 in vivo. We propose that the loss of production of p-HBADs by tubercle bacilli results in their diminished ability to suppress the pro-inflammatory response to infection and that this ultimately provokes extensive pulmonary lesions in the C57BL/6 model of tuberculosis infection.     

Microarray analysis of Bacillus cereus group virulence factors

Bacillus cereus, B. thuringiensis and B. anthracis are closely related medically and economically important bacterial species that belong to the B. cereus group. Members of the B. cereus group carry genes encoding several important virulence factors, including enterotoxins, phospholipases and exotoxins. Since it is difficult to differentiate among B. cereus group members, and because Bacillus virulence factors are very important for pathogenesis, we explored the use of microarray-based detection of virulence factor genes as a tool for strain identification and for determining virulence. Our method requires an initial multiplex PCR amplification step, followed by identification of the PCR amplicons by hybridization to an oligonucleotide microarray containing genes for all three types of Bacillus virulence factors including B. anthracis virulence factors. The DNA chip described here contains 21 identical arrays used for analysis of seven samples in triplicates. Using the arrays, we found that virulence factors are present in several combinations in the strains analyzed. This work also demonstrates the potential of oligonucleotide microarrays for medical, food safety and biodefense analysis of microbial pathogens.     

A parametric study of wind chill equivalent temperatures by a dimensionless steady-state analysis

A first order analytical approximation of steady-state heat conduction in a hollow cylinder exchanging heat at its external surface by convection with a cold and windy environment is presented. The model depicts the thermal behavior of certain body elements, e.g., head/face, when exposed to such environments. The results are presented by dimensionless parameters and facilitate the estimation of wind chill equivalent temperatures (WCETs). The effects of several variables on determining WCETs were studied using specific examples, leading to the following generalizations: (1) the conditions assumed for "calm" wind speed appear to be a dominant factor in determining WCET; (2) the effects, on both (skin) surface temperature and on WCET, of a 1°C change in environmental temperature appear to be more pronounced than those of a 1 m/s change in wind speed; (3) similarly, predicted WCETs are more sensitive to the geometrical dimensions assumed for the modeled entity than they are to wind speeds; and (4) tissue thermal conductivity, the angle at which the convective heat transfer coefficient is measured relative to wind direction, and the factor used to establish "effective" wind speeds in the domain occupied by humans relative to reported values, all seem to have relatively small effects on the determination of WCET. These conclusions strongly suggest, among other things, that for any given combination of environmental conditions, wind chill indices may best be presented as ranges rather than as single values. This seems to apply even when worst-case scenarios are considered. Also emphasized is the need for careful and realistic selection of all the parameter values used in the determination of WCETs.     

Wind-chill-equivalent temperatures: regarding the impact due to the variability of the environmental convective heat transfer coefficient

The wind-chill index (WCI), developed in Antarctica in the 1940s and recently updated by the weather services in the USA and Canada, expresses the enhancement of heat loss in cold climates from exposed body parts, e.g., face, due to wind. The index provides a simple and practical means for assessing the thermal effects of wind on humans outdoors. It is also used for indicating weather conditions that may pose adverse risks of freezing at subfreezing environmental temperatures. Values of the WCI depend on a number of parameters, i.e, temperatures, physical properties of the air, wind speed, etc., and on insolation and evaporation. This paper focuses on the effects of various empirical correlations used in the literature for calculating the convective heat transfer coefficients between humans and their environment. Insolation and evaporation are not included in the presentation. Large differences in calculated values among these correlations are demonstrated and quantified. Steady-state wind-chill-equivalent temperatures (WCETs) are estimated by a simple, one-dimensional heat-conducting hollow-cylindrical model using these empirical correlations. Partial comparison of these values with the published “new” WCETs is presented. The variability of the estimated WCETs, due to different correlations employed to calculate them, is clearly demonstrated. The results of this study clearly suggest the need for establishing a “gold standard” for estimating convective heat exchange between exposed body elements and the cold and windy environment. This should be done prior to the introduction and adoption of further modifications to WCETs and indices. Correlations to estimate the convective heat transfer coefficients between exposed body parts of humans in windy and cold environments influence the WCETs and need to be standardized.     

AMPK activation regulates apoptosis, adipogenesis, and lipolysis by eIF2alpha in adipocytes

AMP-activated protein kinase (AMPK) is a metabolic master switch regulating glucose and lipid metabolism. Recently, AMPK has been implicated in the control of adipose tissue content. Yet, the nature of this action is controversial. We examined the effect on F442a adipocytes of the AMPK activator-AICAR. Activation of AMPK induced dose-dependent apoptotic cell death, inhibition of lipolysis, and downregulatation key adipogenic genes, such as peroxisome proliferator-activated receptor (PPARgamma) and CCAAT/enhancer-binding protein alpha (C/EBPalpha). We have identified the alpha-subunit of the eukaryotic initiation factor-2 (eIF2alpha) as a target gene which is phosphorylated following AICAR treatment. Such phosphorylation is one of the best-characterized mechanisms for downregulating protein synthesis. 2-Aminopurine (2-AP), an inhibitor of eIF2alpha kinases, could overcome the apoptotic effect of AICAR, abolishing the reduction of PPARgamma and C/EBPalpha and the lipolytic properties of AMPK. Thus, AMPK may diminish adiposity via reduction of fat cell number through eIF2alpha-dependent translation shutdown.     

Toll-like receptor deficiency worsens inflammation and lymphedema after lymphatic injury

Mechanisms regulating lymphedema pathogenesis remain unknown. Recently, we have shown that lymphatic fluid stasis increases endogenous danger signal expression, and these molecules influence lymphatic repair (Zampbell JC, et al. Am J Physiol Cell Physiol 300: C1107-C1121, 2011). Endogenous danger signals activate Toll-like receptors (TLR) 2, 4, and 9 and induce homeostatic or harmful responses, depending on physiological context. The purpose of this study was to determine the role of TLRs in regulating tissue responses to lymphatic fluid stasis. A surgical model of lymphedema was used in which wild-type or TLR2, 4, or 9 knockout (KO) mice underwent tail lymphatic excision. Six weeks postoperatively, TLR KOs demonstrated markedly increased tail edema compared with wild-type animals (50-200% increase; P < 0.01), and this effect was most pronounced in TLR4 KOs (P < 0.01). TLR deficiency resulted in decreased interstitial and lymphatic transport, abnormal lymphatic architecture, and fewer capillary lymphatics (40-50% decrease; P < 0.001). Lymphedematous tissues of TLR KOs demonstrated increased leukocyte infiltration (P < 0.001 for TLR4 KOs), including higher numbers of infiltrating CD3+ cells (P < 0.05, TLR4 and TLR9 KO), yet decreased infiltrating F4/80+ macrophages (P < 0.05, all groups). Furthermore, analysis of isolated macrophages revealed twofold reductions in VEGF-C (P < 0.01) and LYVE-1 (P < 0.05) mRNA from TLR2-deficient animals. Finally, TLR deficiency was associated with increased collagen type I deposition and increased transforming growth factor-β1 expression (P < 0.01, TLR4 and TLR9 KO), contributing to dermal fibrosis. In conclusion, TLR deficiency worsens tissue responses to lymphatic fluid stasis and is associated with decreased lymphangiogenesis, increased fibrosis, and reduced macrophage infiltration. These findings suggest a role for innate immune responses, including TLR signaling, in lymphatic repair and lymphedema pathogenesis.     

Lymphatic function is regulated by a coordinated expression of lymphangiogenic and anti-lymphangiogenic cytokines

Lymphangiogenic cytokines such as vascular endothelial growth factor-C (VEGF-C) are critically required for lymphatic regeneration; however, in some circumstances, lymphatic function is impaired despite normal or elevated levels of these cytokines. The recent identification of anti-lymphangiogenic molecules such as interferon-γ (IFN-γ), transforming growth factor-β1, and endostatin has led us to hypothesize that impaired lymphatic function may represent a dysregulated balance in the expression of pro/anti-lymphangiogenic stimuli. We observed that nude mice have significantly improved lymphatic function compared with wild-type mice in a tail model of lymphedema. We show that gradients of lymphatic fluid stasis regulate the expression of lymphangiogenic cytokines (VEGF-A, VEGF-C, and hepatocyte growth factor) and that paradoxically the expression of these molecules is increased in wild-type mice. More importantly, we show that as a consequence of T-cell-mediated inflammation, these same gradients also regulate expression patterns of anti-lymphangiogenic molecules corresponding temporally and spatially with impaired lymphatic function in wild-type mice. We show that neutralization of IFN-γ significantly increases inflammatory lymph node lymphangiogenesis independently of changes in VEGF-A or VEGF-C expression, suggesting that alterations in the balance of pro- and anti-lymphangiogenic cytokine expression can regulate lymphatic vessel formation. In conclusion, we show that gradients of lymphatic fluid stasis regulate not only the expression of pro-lymphangiogenic cytokines but also potent suppressors of lymphangiogenesis as a consequence of T-cell inflammation and that modulation of the balance between these stimuli can regulate lymphatic function.     

Vascular endothelial growth factor-D is a key molecule that enhances lymphatic metastasis of soft tissue sarcomas

Studies on lymph node metastasis of soft tissue sarcomas are insufficient because of its rarity. In this study, we examined the expressions of vascular endothelial growth factor (VEGF)-C and VEGF-D in soft tissue sarcomas metastasized to lymph nodes. In addition, the effects of the two molecules on the barrier function of a lymphatic endothelial cell monolayer against sarcoma cells were analyzed. We examined 7 patients who had soft tissue sarcomas with lymph node metastases and who had undergone neither chemotherapy nor radiotherapy before lymphadenectomy. Immunohistochemistry revealed that 2 of 7 sarcomas that metastasized to lymph nodes expressed VEGF-C both in primary and metastatic lesions. On the other hand, VEGF-D expression was detected in 4 of 7 primary and 7 of 7 metastatic lesions, respectively. Interestingly, 3 cases that showed no VEGF-D expression at primary sites expressed VEGF-D in metastatic lesions. Recombinant VEGF-C at 10(-8) and VEGF-D at 10(-7)and 10(-8)g/ml significantly increased the random motility of lymphatic endothelial cells compared with controls. VEGF-D significantly increased the migration of sarcoma cells through lymphatic endothelial monolayers. The fact that VEGF-D induced the migration of fibrosarcomas through the lymphatic endothelial monolayer is the probable reason for the strong relationship between VEGF-D expression and lymph node metastasis in soft tissue sarcomas. The important propensities of this molecule for the increase of lymph node metastases are not only lymphangiogenesis but also down-regulation of the barrier function of lymphatic endothelial monolayers, which facilitates sarcoma cells entering the lymphatic circulation.     

Genome Evolution Due to Allopolyploidization in Wheat

The wheat group has evolved through allopolyploidization, namely, through hybridization among species from the plant genera Aegilops and Triticum followed by genome doubling. This speciation process has been associated with ecogeographical expansion and with domestication. In the past few decades, we have searched for explanations for this impressive success. Our studies attempted to probe the bases for the wide genetic variation characterizing these species, which accounts for their great adaptability and colonizing ability. Central to our work was the investigation of how allopolyploidization alters genome structure and expression. We found in wheat that allopolyploidy accelerated genome evolution in two ways: (1) it triggered rapid genome alterations through the instantaneous generation of a variety of cardinal genetic and epigenetic changes (which we termed “revolutionary” changes), and (2) it facilitated sporadic genomic changes throughout the species’ evolution (i.e., evolutionary changes), which are not attainable at the diploid level. Our major findings in natural and synthetic allopolyploid wheat indicate that these alterations have led to the cytological and genetic diploidization of the allopolyploids. These genetic and epigenetic changes reflect the dynamic structural and functional plasticity of the allopolyploid wheat genome. The significance of this plasticity for the successful establishment of wheat allopolyploids, in nature and under domestication, is discussed.     

Infantile cerebellar-retinal degeneration associated with a mutation in mitochondrial aconitase, ACO2

Degeneration of the cerebrum, cerebellum, and retina in infancy is part of the clinical spectrum of lysosomal storage disorders, mitochondrial respiratory chain defects, carbohydrate glycosylation defects, and infantile neuroaxonal dystrophy. We studied eight individuals from two unrelated families who presented at 2-6 months of age with truncal hypotonia and athetosis, seizure disorder, and ophthalmologic abnormalities. Their course was characterized by failure to acquire developmental milestones and culminated in profound psychomotor retardation and progressive visual loss, including optic nerve and retinal atrophy. Despite their debilitating state, the disease was compatible with survival of up to 18 years. Laboratory investigations were normal, but the oxidation of glutamate by muscle mitochondria was slightly reduced. Serial brain MRI displayed progressive, prominent cerebellar atrophy accompanied by thinning of the corpus callosum, dysmyelination, and frontal and temporal cortical atrophy. Homozygosity mapping followed by whole-exome sequencing disclosed a Ser112Arg mutation in ACO2, encoding mitochondrial aconitase, a component of the Krebs cycle. Specific aconitase activity in the individuals' lymphoblasts was severely reduced. Under restrictive conditions, the mutant human ACO2 failed to complement a yeast ACO1 deletion strain, whereas the wild-type human ACO2 succeeded, indicating that this mutation is pathogenic. Thus, a defect in mitochondrial aconitase is associated with an infantile neurodegenerative disorder affecting mainly the cerebellum and retina. In the absence of noninvasive biomarkers, determination of the ACO2 sequence or of aconitase activity in lymphoblasts are warranted in similarly affected individuals, based on clinical and neuroradiologic grounds.