Mutations in glucocerebrosidase cause the lysosomal storage disorder Gaucher's disease and are the most common risk factor for Parkinson's disease. Therapies to restore the enzyme's function in the brain hold great promise for treating the neurological implications. Thus, we developed blood-brain barrier penetrant therapeutic molecules by fusing transferrin receptor-binding moieties to β-glucocerebrosidase (referred to as GCase-BS). We demonstrate that these fusion proteins show significantly increased uptake and lysosomal efficiency compared to the enzyme alone. In a cellular disease model, GCase-BS rapidly rescues the lysosomal proteome and lipid accumulations beyond known substrates. In a mouse disease model, intravenous injection of GCase-BS leads to a sustained reduction of glucosylsphingosine and can lower neurofilament-light chain plasma levels. Collectively, these findings demonstrate the potential of GCase-BS for treating GBA1-associated lysosomal dysfunction, provide insight into candidate biomarkers, and may ultimately open a promising treatment paradigm for lysosomal storage diseases extending beyond the central nervous system.

( TAR DNA-binding protein 43 (TDP-43) is a key player in neurodegenerative diseases including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Accumulation of TDP-43 is associated with neuronal death in the brain. How increased and disease-causing mutant forms of TDP-43 induce cell death remains unclear. Here we addressed the role of TDP-43 during neural development and show that reduced TDP-43 causes defects in neural stem/progenitor cell proliferation but not cell death. However, overexpression of wild type and TDP-43A315T proteins induce p53-dependent apoptosis of neural stem/progenitors and human induced pluripotent cell (iPS)-derived immature cortical neurons. We show that TDP-43 induces expression of the proapoptotic BH3-only genes Bbc3 and Bax, and that p53 inhibition rescues TDP-43 induced cell death of embryonic mouse, and human cortical neurons, including those derived from TDP-43(G298S) ALS patient iPS cells. Hence, an increase in wild type and mutant TDP-43 induces p53-dependent cell death in neural progenitors developing neurons and this can be rescued. These findings may have important implications for accumulated or mutant TDP-43 induced neurodegenerative diseases.

Genome editing in eukaryotes became easier in the last years with the development of nucleases that induce double strand breaks in DNA at user-defined sites. CRISPR/Cas9-based genome editing is currently one of the most powerful strategies. In the easiest case, a nuclease (e.g. Cas9) and a target defining guide RNA (gRNA) are transferred into a target cell. Non-homologous end joining (NHEJ) repair of the DNA break following Cas9 cleavage can lead to inactivation of the target gene. Specific repair or insertion of DNA with Homology Directed Repair (HDR) needs the simultaneous delivery of a repair template. Recombinant Lentivirus or Adenovirus genomes have enough capacity for a nuclease coding sequence and the gRNA but are usually too small to also carry large targeting constructs. We recently showed that a baculovirus-based multigene expression system (MultiPrime) can be used for genome editing in primary cells since it possesses the necessary capacity to carry the nuclease and gRNA expression constructs and the HDR targeting sequences. Here we present new Acceptor plasmids for MultiPrime that allow simplified cloning of baculoviruses for genome editing and we show their functionality in primary cells with limited life span and induced pluripotent stem cells (iPS).

Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including, synthetic and structural biology, cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity, thus impeding unrestricted multigene expression. We developed MultiPrime, a modular, non-cytotoxic, non-integrating, baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters. MultiPrime viruses efficiently transduce a wide range of cell types, including non-dividing primary neurons and induced-pluripotent stem cells (iPS). We show that MultiPrime can be used for reprogramming, and for genome editing and engineering by CRISPR/Cas9. Moreover, we implemented dual-host-specific cassettes enabling multiprotein expression in insect and mammalian cells using a single reagent. Our experiments establish MultiPrime as a powerful and highly efficient tool, to deliver multiple genes for a wide range of applications in primary and established mammalian cells.

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of children diarrhea in the world. Adhesion of ETEC to small intestine is an important virulence trait. One of the most prevalent colonization factors (CFs) in human is CFA/I fimbriae and CfaE which is the required binding factor for adhesion of ETEC to intestinal mucosa. We optimized cfaE gene codons according to codon bias of E. coli to achieve a high level of recombinant protein expression. The optimized gene was expressed in E. coli and rCFaE protein was used for mice immunization. Blocking activity of the obtained antibody was examined by microplate agglutination inhibition test. SDS-PAGE analysis indicated that the optimized sequence of cfaE produces a suitable amount of rCFaE in comparison with native gene sequence. This optimized rCFaE protein could induces strong humoral response in mice and the antibody obtained against rCFaE inhibited the adhesion of ETEC to human group A erythrocytes. It is concluded that codon optimization is a useful approach for obtaining large quantities of recombinant rCFaE protein. With regard to the results of hemagglutination inhibition test, codon optimization and increased production of recombinant protein expressed in E. coli did not affect the immunogenicity potential of CFaE.

Enterotoxigenic Escherichia coli (ETEC) is the most significant agent leading to childhood diarrhea and death in developing countries. This bacterium is the cause of 380 thousand deaths in children under five years of age. Due to its prevalence as well as difficulties in its treatment, designing effective vaccines against ETEC is a goal of the World Health Organization (WHO). The Colonization factor B (CfaB) as major subunit of fimberiae has a critical role in bacterial attachment to small intestine epithelium. Hence, the molecule alone or together with other candidate molecules has been considered in vaccine design. In this work, we produced recombinant CfaB in E. coli with the aim of studying its immunogenicity as a component of vaccine. The cfaB gene was cloned into pET28a and regarding the presence of rare codons in cfaB gene, it was not expressed. Therefore, an optimized gene with codon preferences was synthesized and cloned into pET28a vector and subsequently expressed. The recombinant protein was purified with Ni-NTA column and used as an antigen for mice immunization and in ELISA test. Microplate agglutination inhibition test was utilized to show antibody blocking activity. In conclusion, codon optimization is a useful approach for obtaining large quantities of a desired protein. Relying on agglutination inhibition experiment, anti-CfaB serum was able to block the binding of colonization factor antigen I (CFA/I) fimbriated ETEC to erythrocytes. Key words: Enterotoxigenic Escherichia coli (ETEC), colonization factor antigen I, expression, agglutination inhibition

Background: Enterotoxigenic Escherichia coli bacterium is the most important bacterial agent causing diarrhea. Specific virulence factors, such as enterotoxins and colonization factors, distinguish ETEC from other classes of diarrheagenic E.coli. In this study, heat-labile toxin was purified which could be utilized for anti-toxin assay in GM1 gangelioside receptor-ELISA based method and for identification of ETEC producing toxin. Materials and Methods: In this experimental study, bacterial strain producing heat-labile toxin was first cultivated for production and purification of toxin. Then supernatant soluble proteins were precipitated with ammonium sulfate and purified using biochemical methods. Finally, purified protein was dialyzed against Tris 0.02 mM pH 8 and analyzed on gel electrophoresis. GM1 gangelioside receptor-ELISA based method was used for detection and assessment of the purified toxin. Through this method, the effect of anti-recombinant heat-labile toxin B subunit neutralization on heat-labile toxin was investigated. Results: Toxin purification was revealed by the presence of 12 and 28 KD protein bands. This study demonstrated that anti-recombinant heat-labile toxin B subunit antibody can detect the purified toxin and can inhibit its binding to GM1 receptor up to 80%. Conclusion: Purification of heat-labile toxin and gangelioside receptor-ELISA assay can be used for accurate detection and epidemiological study of clinical isolates.

Aims: It has been estimated that gastroenteritis is caused by bacteria in 30-70% of cases. Enterotoxigenic Escherichia coli or ETEC is one of the most common agents causing diarrhea. Protective immunity may be induced against this disease. Designing and producing a vaccine against this disease is one of the purposes of World Health Organization. Vaccine candidate molecules have to induce protective immunity against a broad spectrum of ETEC bacteria. Most ETEC strains can produce labile toxin; therefore labile toxin may be a proper candidate for being used as a vaccine molecule. The aim of this study was to optimize Heat-labile Toxin B Subunit expression in order to investigate its immunological properties. Materials & Methods: Optimizing of 3 parameters (IPTG concentration, time and temperature of promoter induction) was performed. Recombinant protein was purified with Ni-NTA column. Purified Heat-labile Toxin B Subunit was injected to mice subcutaneously in 4 sessions. Blood samples were taken during the interval between the injections and after last injection. Then, ELISA was performed. Results: The optimum expression occurred at 1mM IPTG concentration, after 3 hours and at 37oC. Recombinant protein was highly purified (>95%) with Ni-NTA column. Also, ELISA showed high titer of antibody production in mice. Conclusion: Expressed Heat-labile Toxin B Subunit is an immunogenic protein and can be one of the important

The human brain is a highly organized structure and the cerebral cortex in particular has expanded massively in size during evolution. The cerebral cortex is arranged into layers of specialized neuron subtypes formed during development by orchestrated stem cell maintenance, expansion, fate commitment and differentiation. The cortical neural stem cells generate billions of neurons in a systematic fashion. The mechanisms and their interplay that control most aspects of human brain development are unclear. This is partially due to the ethical and practical challenges associated with analyzing fetal human development. Recent progress into understanding the formation of the human brain has taken advantage of in vitro modeling of corticogenesis using pluripotent cells. Human pluripotent stem cells and procedures developed for their differentiation provided previously unavailable opportunities to study the mechanisms involved in development of the cerebral cortex. These human cell culture models can be applied to address specific biological questions and have been successfully utilized to investigate mechanisms associated, not only with normal brain development, but also neuropsychiatric disorders. Here, we review the recent literature that uses these cell culture models to study human corticogenesis. Then, we discuss the challenges and limitations of the current models.