Poly(methyl metacrylate) conductive fiber optic transducers as dual biosensor platforms

Herein the development of an alternative optic-conductive fiber configuration applied for the construction of biosensing platforms. This new approach is based on applying the chemical polymerization of pyrrole onto the surface of polymethyl metacrylate (PMMA) fibers to create a polymer—a conductive surface, onto which an additional photoactive polypyrrole-benzophenone (PpyBz) film is electrochemically generated upon the fiber surface. Irradiation of the benzophenone groups embedded in the Ppy films with UV radiation (350 nm) formed active radicals that allowed the covalent attachment of the desired bioreceptors. Characterization of the amperometric biosensing matrix was accomplished by using a model Urease (Urs) through electrochemical impedance spectroscopy (EIS) and amperometry. Both techniques have shown a low charge transfer resistance (340 kΩ) and a high sensitivity (12.3 μA mM⁻¹ cm⁻²). Thereafter, the construction of an optical biosensing matrix based on horseradish peroxidase (HRP) production of photons was carried out. The high signal to noise (S/N) ratio (1600) indicated clearly that this approach can serve as a new platform to replace glass optical fibers based on biosensors.     

Rational confederation of genes and diseases: NGS interpretation via GeneCards,MalaCards and VarElect

Background

A key challenge in the realm of human disease research is next generation sequencing (NGS) interpretation, whereby identified filtered variant-harboring genes are associated with a patient’s disease phenotypes. This necessitates bioinformatics tools linked to comprehensive knowledgebases. The GeneCards suite databases, which include GeneCards (human genes), MalaCards (human diseases) and PathCards (human pathways) together with additional tools, are presented with the focus on MalaCards utility for NGS interpretation as well as for large scale bioinformatic analyses.

Results

VarElect, our NGS interpretation tool, leverages the broad information in the GeneCards suite databases. MalaCards algorithms unify disease-related terms and annotations from 69 sources. Further, MalaCards defines hierarchical relatedness—aliases, disease families, a related diseases network, categories and ontological classifications. GeneCards and MalaCards delineate and share a multi-tiered, scored gene-disease network, with stringency levels, including the definition of elite status—high quality gene-disease pairs, coming from manually curated trustworthy sources, that includes 4500 genes for 8000 diseases. This unique resource is key to NGS interpretation by VarElect. VarElect, a comprehensive search tool that helps infer both direct and indirect links between genes and user-supplied disease/phenotype terms, is robustly strengthened by the information found in MalaCards. The indirect mode benefits from GeneCards’ diverse gene-to-gene relationships, including SuperPaths—integrated biological pathways from 12 information sources. We are currently adding an important information layer in the form of “disease SuperPaths”, generated from the gene-disease matrix by an algorithm similar to that previously employed for biological pathway unification. This allows the discovery of novel gene-disease and disease–disease relationships. The advent of whole genome sequencing necessitates capacities to go beyond protein coding genes. GeneCards is highly useful in this respect, as it also addresses 101,976 non-protein-coding RNA genes. In a more recent development, we are currently adding an inclusive map of regulatory elements and their inferred target genes, generated by integration from 4 resources.

Conclusions

MalaCards provides a rich big-data scaffold for in silico biomedical discovery within the gene-disease universe. VarElect, which depends significantly on both GeneCards and MalaCards power, is a potent tool for supporting the interpretation of wet-lab experiments, notably NGS analyses of disease. The GeneCards suite has thus transcended its 2-decade role in biomedical research, maturing into a key player in clinical investigation.

     

Optically derived metabolic and hemodynamic parameters predict hippocampal neurogenesis in the BTBR mouse model of autism

In this study, we made use of dual‐wavelength laser speckle imaging (DW‐LSI) to assess cerebral blood flow (CBF) in the BTBR‐genetic mouse model of autism spectrum disorder, as well as control (C57Bl/6J) mice. Since the deficits in social behavior demonstrated by BTBR mice are attributed to changes in neural tissue structure and function, we postulated that these changes can be detected optically using DW‐LSI. BTBR mice demonstrated reductions in both CBF and cerebral oxygen metabolism (CMRO₂), as suggested by studies using conventional neuroimaging technologies to reflect impaired neuronal activation and cognitive function. To validate the monitoring of CBF by DW‐LSI, measurements with laser Doppler flowmetry (LDF) were also performed which confirmed the lowered CBF in the autistic‐like group. Furthermore, we found in vivo cortical CBF measurements to predict the rate of hippocampal neurogenesis, measured ex vivo by the number of neurons expressing doublecortin or the cellular proliferation marker Ki‐67 in the dentate gyrus, with a strong positive correlation between CBF and neurogenesis markers (Pearson, r = 0.78; 0.9, respectively). These novel findings identifying cortical CBF as a predictive parameter of hippocampal neurogenesis highlight the power and flexibility of the DW‐LSI and LDF setups for studying neurogenesis trends under normal and pathological conditions.      

Antiviral activity of 3(2H)- and 6-chloro-3(2H)-isoflavenes against highly diverged, neurovirulent vaccine-derived, type2 poliovirus sewage isolates

Background

Substituted flavanoids interfere with uncoating of Enteroviruses including Sabin-2 polio vaccine strains. However flavanoid resistant and dependent, type-2 polio vaccine strains (minimally-diverged), emerged during in vitro infections. Between 1998–2009, highly-diverged (8 to >15%) type-2, aVDPV₂s, from two unrelated persistent infections were periodically isolated from Israeli sewage.

Aim

To determine whether highly evolved aVDPV₂s derived from persistent infections retained sensitivity to isoflavenes.

Methods

Sabin-2 and ten aVDPV₂ isolates from two independent Israeli sources were titered on HEp2C cells in the presence and absence of 3(2H)- Isoflavene and 6-chloro-3(2H)-Isoflavene. Neurovirulence of nine aVDPV₂s was measured in PVR-Tg-21 transgenic mice. Differences were related to unique amino acid substitutions within capsid proteins.

Principal Findings

The presence of either flavanoid inhibited viral titers of Sabin-2 and nine of ten aVDPV₂s by one to two log₁₀. The tenth aVDPV₂, which had unique amino acid substitution distant from the isoflavene-binding pocket but clustered at the three- and five-fold axies of symmetry between capsomeres, was unaffected by both flavanoids. Genotypic neurovirulence attenuation sites in the 5′UTR and VP1 reverted in all aVDPV₂s and all reacquired a full neurovirulent phenotype except one with amino acid substitutions flanking the VP1 site.

Conclusion

Both isoflavenes worked equally well against Sabin 2 and most of the highly-diverged, Israeli, aVDPV₂s isolates. Thus, functionality of the hydrophobic pocket may be unaffected by selective pressures exerted during persistent poliovirus infections. Amino acid substitutions at sites remote from the drug-binding pocket and adjacent to a neurovirulence attenuation site may influence flavanoid antiviral activity, and neurovirulence, respectively.     

Mutation in TECPR2 Reveals a Role for Autophagy in Hereditary Spastic Paraparesis

We studied five individuals from three Jewish Bukharian families affected by an apparently autosomal-recessive form of hereditary spastic paraparesis accompanied by severe intellectual disability, fluctuating central hypoventilation, gastresophageal reflux disease, wake apnea, areflexia, and unique dysmorphic features. Exome sequencing identified one homozygous variant shared among all affected individuals and absent in controls: a 1 bp frameshift TECPR2 deletion leading to a premature stop codon and predicting significant degradation of the protein. TECPR2 has been reported as a positive regulator of autophagy. We thus examined the autophagy-related fate of two key autophagic proteins, SQSTM1 (p62) and MAP1LC3B (LC3), in skin fibroblasts of an affected individual, as compared to a healthy control, and found that both protein levels were decreased and that there was a more pronounced decrease in the lipidated form of LC3 (LC3II). siRNA knockdown of TECPR2 showed similar changes, consistent with aberrant autophagy. Our results are strengthened by the fact that autophagy dysfunction has been implicated in a number of other neurodegenerative diseases. The discovered TECPR2 mutation implicates autophagy, a central intracellular mechanism, in spastic paraparesis.     

Signaling pathways involved in human sperm hyperactivated motility stimulated by Zn2+

To fertilize the egg, sperm cells must reside in the female reproductive tract for several hours during which they undergo chemical and motility changes collectively called capacitation. During capacitation, the sperm develop a unique type of motility known as hyperactivated motility (HAM). The semen contains Zn²⁺ in millimolar concentrations, whereas in the female reproductive tract, the concentration is around 1 µM. In this study, we characterize the role of Zn²⁺ in human sperm capacitation focusing on its effect on HAM. Western blot analysis revealed the presence of GPR39‐type Zn‐receptor localized mainly in the sperm tail. Zn²⁺ at micromolar concentration stimulates HAM, which is mediated by a cascade involving GPR39–adenylyl cyclase (AC)–cyclic AMP (cAMP)–protein kinase A–tyrosine kinase Src (Src)–epidermal growth factor receptor and phospholipase C. Both the transmembrane AC and the soluble‐AC are involved in the stimulation of HAM by Zn²⁺. The development of HAM is precisely regulated by cAMP, in which relatively low concentration (5–10 µM) stimulated HAM, whereas at 30 µM no stimulation occurred. A similar response was seen when different concentrations of Zn²⁺ were added to the cells; low Zn²⁺ stimulated HAM, whereas at relatively high Zn²⁺, no effect was seen. We further demonstrate that the Ca²⁺‐channel CatSper involved in Zn²⁺‐stimulated HAM. These data support a role for extracellular Zn²⁺ acting via GPR39 to regulate signaling pathways in sperm capacitation, leading to HAM induction.     

Differential expression of microRNA in serum fractions and association of Argonaute 1 microRNAs with heart failure

The serum or plasma microRNA (miRNA) molecules have been suggested as diagnostic and prognostic biomarkers, in various pathological conditions. However, these molecules are also found in different serum fractions, such as exosomes and Argonaute (Ago) protein complexes. Ago1 is the predominant Ago protein expressed in heart tissue. The objective of the study was to examine the hypothesis that Ago1‐associated miRNAs may be more relevant to cardiac disease and heart failure compared with the serum. In total, 84 miRNA molecules were screened for their expression in the whole serum, exosomes and Ago1, and Ago2 complexes. Ago1‐bound miR‐222‐3p, miR‐497‐5p and miR‐21‐5p were significantly higher, and let‐7a‐5p was significantly lower in HF patients compared with healthy controls, whereas no such difference was observed for those markers in the serum samples among the groups. A combination of these 4 miRNAs into an Ago1‐HF score provided a ROC curve with an AUC of 1, demonstrating clear discrimination between heart failure patients and healthy individuals. Ago1 fraction might be a better and more specific platform for identifying HF‐related miRNAs compared with the whole serum.