No overall hyposialylation in hereditary inclusion body myopathy myoblasts carrying the homozygous M712T GNE mutation

Hereditary inclusion body myopathy (HIBM) is a unique group of neuromuscular disorders characterized by adult-onset, slowly progressive distal and proximal muscle weakness, which is caused by mutations in UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme in the biosynthetic pathway of sialic acid. In order to investigate the consequences of the mutated GNE enzyme in muscle cells, we have established cell cultures from muscle biopsies carrying either kinase or epimerase mutations. While all myoblasts carrying a mutated GNE gene show a reduction in their epimerase activity, only the cells derived from the patient carrying a homozygous epimerase mutation present also a significant reduction in the overall membrane bound sialic acid. These results indicate that although mutations in each of the two GNE domains result in an impaired enzymatic activity and the same HIBM phenotype, they do not equally affect the overall sialylation of muscle cells. This lack of correlation suggests that the pathological mechanism of the disease may not be linked solely to the well-characterized sialic acid pathway.     

Death-associated protein 3 localizes to the mitochondria and is involved in the process of mitochondrial fragmentation during cell death

Death-associated protein 3 (DAP3) was previously isolated in our laboratory as a positive mediator of cell death. It is a 46-kDa protein containing a GTP binding domain that was shown to be essential for the induction of cell death. DAP3 functions downstream of the receptor signaling complex, and its death-promoting effects depend on caspase activity. Recent reports have suggested that DAP3 is localized to the mitochondria, but no functional significance of this localization has been reported so far. Here, we study the sub-cellular localization and cellular function of human DAP3 (hDAP3). We found that hDAP3 is localized to the mitochondria and, in contrast to cytochrome c, is not released to the cytoplasm following several cell death signals. Overexpression of hDAP3 induced dramatic changes in the mitochondrial structure involving increased fragmentation of the mitochondria. Both the mitochondrial localization of hDAP3 and its GTP-binding activity were essential for the fragmentation. The punctiform mitochondrial morphology was similar to that observed upon treatment of HeLa cells with staurosporine. In fact, reduction of endogenous hDAP3 protein by RNA interference partially attenuated staurosporine-induced mitochondrial fission. Thus, hDAP3 is a necessary component in the molecular pathway that culminates in fragmented mitochondria, probably reflecting its involvement in the fission process. These results, for the first time, provide a specific functional role for hDAP3 in mitochondrial maintenance.     

Targeted overexpression of IL-18 binding protein at the central nervous system overrides flexibility in functional polarization of antigen-specific Th2 cells

The current study shows that functional polarization of Ag-specific CD4(+) Th2 cells entering the CNS during the accelerating phase of experimental autoimmune encephalomyelitis is flexible and dependent on the cytokine milieu there. Thus, targeted cell/gene therapy by Ag-specific T cells overexpressing IL-18 binding protein overrides this flexibility and induces infectious spread of T cell tolerance. Using a congenic system, we demonstrated that at this time, Ag-specific Th2 cells accumulate at the CNS but then arrest of IL-4 production. A manipulation of targeted cell/gene delivery was then used to detect whether this function is dependent on the cytokine milieu there. Targeted overexpression of IL-18 binding protein, a natural inhibitor of IL-18, restored the ability of these Ag-specific Th2 cells to produce IL-4 and subsequently induce protective spread of Th2 polarization. These findings not only suggest a novel way of therapy, but also explain why shifting the balance of Ag-specific T cells toward Th2 suppresses ongoing experimental autoimmune encephalomyelitis, whereas a direct transfer of these cells is ineffective.     

High fertigation frequency and phosphorus level: Effects on summer-grown bell pepper growth and blossom-end rot incidence

The objective was to examine the effects of fertigation frequency and P application rate on bell pepper growth and blossom-end rot (BER) incidence, under hot conditions. The experiment comprised six treatments: two concentrations of phosphorus (3 and 30 mg L^–1) combined with three fertigation frequencies (two and eight events per day, and for 1.5 min every 25 min throughout the day). Increasing the fertigation frequency significantly increased the plants acquisition of nutrients, especially phosphorus and manganese. A significant linear regression was obtained between aboveground biomass, and leaf P concentration in the early vegetative stage. Based on the linear regression, 96% of the dry weight variations could be explained by differences in leaf P concentration, indicating that the main effect of fertigation frequency was related to improved P mobilization and uptake. Increasing the daily fertigation frequency from two to eight and to 30 applications reduced the number of BER fruits from 7 to 3 and to 2 per plant, respectively, and accordingly, increased the yield of export-quality fruits from 6.5 to 10 and to 10.5 per plant, respectively. The Mn concentration in plants exposed to low fertigation frequency were low, probably in the deficiency range, but they increased with increasing fertigation frequency. A negative correlation was found between the accumulated number of BER-affected fruits throughout the experiment and fruit-Mn concentrations. In light of recent findings that BER effects in the fruit tissue include the production of oxygen free-radicals and diminution of anti-oxidative compounds and enzymatic activities, and the known crucial role of manganese in enzyme activities and in detoxification of oxygen free-radicals, the relationships between BER incidence and fruit-Mn concentration may indicate that BER is related to Mn deficiency. Future researches are needed to validate this hypothesis.     

Macroalgae Biorefinery from <Emphasis Type="Italic">Kappaphycus alvarezii:</Emphasis> Conversion Modeling and Performance Prediction for India and Philippines as Examples

Marine macroalgae are potential sustainable feedstock for biorefinery. However, this use of macroalgae is limited today mostly because macroalgae farming takes place in rural areas in medium- and low-income countries, where technologies to convert this biomass to chemicals and biofuels are not available. The goal of this work is to develop models to enable optimization of material and exergy flows in macroalgal biorefineries. We developed models for the currently widely cultivated red macroalgae Kappaphycus alvarezii being biorefined for the production of bioethanol, carrageenan, fertilizer, and biogas. Using flux balance analysis, we developed a computational model that allows the prediction of various fermentation scenarios and the identification of the most efficient conversion of K. alvarezii to bioethanol. Furthermore, we propose the potential implementation of these models in rural farms that currently cultivate Kappaphycus in Philippines and in India.     

miRNA normalization enables joint analysis of several datasets to increase sensitivity and to reveal novel miRNAs differentially expressed in breast cancer

Different miRNA profiling protocols and technologies introduce differences in the resulting quantitative expression profiles. These include differences in the presence (and measurability) of certain miRNAs. We present and examine a method based on quantile normalization, Adjusted Quantile Normalization (AQuN), to combine miRNA expression data from multiple studies in breast cancer into a single joint dataset for integrative analysis. By pooling multiple datasets, we obtain increased statistical power, surfacing patterns that do not emerge as statistically significant when separately analyzing these datasets. To merge several datasets, as we do here, one needs to overcome both technical and batch differences between these datasets. We compare several approaches for merging and jointly analyzing miRNA datasets. We investigate the statistical confidence for known results and highlight potential new findings that resulted from the joint analysis using AQuN. In particular, we detect several miRNAs to be differentially expressed in estrogen receptor (ER) positive versus ER negative samples. In addition, we identify new potential biomarkers and therapeutic targets for both clinical groups. As a specific example, using the AQuN-derived dataset we detect hsa-miR-193b-5p to have a statistically significant over-expression in the ER positive group, a phenomenon that was not previously reported. Furthermore, as demonstrated by functional assays in breast cancer cell lines, overexpression of hsa-miR-193b-5p in breast cancer cell lines resulted in decreased cell viability in addition to inducing apoptosis. Together, these observations suggest a novel functional role for this miRNA in breast cancer. Packages implementing AQuN are provided for Python and Matlab: https://github.com/YakhiniGroup/PyAQN.     

The Effect of Correlated Neuronal Firing and Neuronal Heterogeneity on Population Coding Accuracy in Guinea Pig Inferior Colliculus

It has been suggested that the considerable noise in single-cell responses to a stimulus can be overcome by pooling information from a large population. Theoretical studies indicated that correlations in trial-to-trial fluctuations in the responses of different neurons may limit the improvement due to pooling. Subsequent theoretical studies have suggested that inherent neuronal diversity, i.e., the heterogeneity of tuning curves and other response properties of neurons preferentially tuned to the same stimulus, can provide a means to overcome this limit. Here we study the effect of spike-count correlations and the inherent neuronal heterogeneity on the ability to extract information from large neural populations. We use electrophysiological data from the guinea pig Inferior-Colliculus to capture inherent neuronal heterogeneity and single cell statistics, and introduce response correlations artificially. To this end, we generate pseudo-population responses, based on single-cell recording of neurons responding to auditory stimuli with varying binaural correlations. Typically, when pseudo-populations are generated from single cell data, the responses within the population are statistically independent. As a result, the information content of the population will increase indefinitely with its size. In contrast, here we apply a simple algorithm that enables us to generate pseudo-population responses with variable spike-count correlations. This enables us to study the effect of neuronal correlations on the accuracy of conventional rate codes. We show that in a homogenous population, in the presence of even low-level correlations, information content is bounded. In contrast, utilizing a simple linear readout, that takes into account the natural heterogeneity, even of neurons preferentially tuned to the same stimulus, within the neural population, one can overcome the correlated noise and obtain a readout whose accuracy grows linearly with the size of the population.     

Levator veli palatini muscle and eustachian tube function

Thirty previously unoperated patients with submucous cleft palate, occult submucous cleft palate, and unilateral congenital paralysis of the levator veli palatini muscle were examined. All patients were subjected to a comprehensive otoscopic, endoscopic, audiologic, and tympanometric evaluation. A correlation was made between levator veli palatini muscle anomalies, eustachian tube orifice anomalies, and middle ear ventilation and disorders. Normal middle ear ventilation was found in 23 patients. Negative middle ear pressure that consequently normalized following treatment of coexisting sinusitis was found in 3 patients. Only in 4 patients was chronic middle ear disease found. In one of them, middle ear effusion disappeared following successful treatment of sinusitis. Our conclusion is that the levator veli palatini muscle has no significant function in the opening mechanism of the eustachian tube and must be considered as a velopharyngeal valve muscle only.     

Cloning Rosa hybrid phenylacetaldehyde synthase for the production of 2-phenylethanol in a whole cell Escherichia coli system

2-Phenylethanol (2-PE) is a desirable compound in the food and perfumery industries with a characteristic rose fragrance. Until now, most of the studied biotechnological processes to produce 2-PE were conducted using natural 2-PE-producing yeasts. Only several researches were conducted in other genetically engineered microorganisms that simulated the Ehrlich pathway for the conversion of amino acids to fusel alcohols. Here, a novel metabolic pathway has been designed in Escherichia coli to produce 2-PE, using the Rosa hybrid phenylacetaldehyde synthase (PAAS), a pyridoxal 5′-phosphate (PLP)-dependent enzyme capable of transforming l-phenylalanine (l-phe) into phenylacetaldehyde by decarboxylation and oxidation. To overcome the enzyme insolubility in E. coli, several plasmids and host strains were tested for their expression ability. The desired results were obtained by using the pTYB21 plasmid containing the intein tag from the Saccharomyces cerevisiae VMA1. It was discovered that the intein PAAS activity is temperature-dependent, working well in the range of 25 to 30 °C but losing most of its activity at 37 °C. When external PLP cofactor was added, the cells produced 0.39 g l^-1 2-PE directly from l-phe. In addition, a biotransformation that was based only on internal de novo PLP synthesis produced 0.34 g l^-1 2-PE, thus creating for the first time an E. coli strain that can produce 2-PE from l-phe without the need for exterior cofactor additions.