The dynamic nature of netrin-1 and the structural basis for glycosaminoglycan fragment-induced filament formation.

Netrin-1 is a bifunctional chemotropic guidance cue that plays key roles in diverse cellular processes including axon pathfinding, cell migration, adhesion, differentiation, and survival. Here, we present a molecular understanding of netrin-1 mediated interactions with glycosaminoglycan chains of diverse heparan sulfate proteoglycans (HSPGs) and short heparin oligosaccharides. Whereas interactions with HSPGs act as platform to co-localise netrin-1 close to the cell surface, heparin oligosaccharides have a significant impact on the highly dynamic behaviour of netrin-1. Remarkably, the monomer-dimer equilibrium of netrin-1 in solution is abolished in the presence of heparin oligosaccharides and replaced with highly hierarchical and distinct super assemblies leading to unique, yet unknown netrin-1 filament formation. In our integrated approach we provide a molecular mechanism for the filament assembly which opens fresh paths towards a molecular understanding of netrin-1 functions.

Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor.

The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems. KEY POINTS: • Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1 • Biolayer interferometry allows target protein quantitation in expression media • High production yields in the bioreactor do not impair Netrin-1 proteoglycan quality.

Boron rich nanotube drug carrier system is suited for boron neutron capture therapy.

Boron neutron capture therapy (BNCT) is a two-step therapeutic process that utilizes Boron-10 in combination with low energy neutrons to effectively eliminate targeted cells. This therapy is primarily used for difficult to treat head and neck carcinomas; recent advances have expanded this method to cover a broader range of carcinomas. However, it still remains an unconventional therapy where one of the barriers for widespread adoption is the adequate delivery of Boron-10 to target cells. In an effort to address this issue, we examined a unique nanoparticle drug delivery system based on a highly stable and modular proteinaceous nanotube. Initially, we confirmed and structurally analyzed ortho-carborane binding into the cavities of the nanotube. The high ratio of Boron to proteinaceous mass and excellent thermal stability suggest the nanotube system as a suitable candidate for drug delivery into cancer cells. The full physicochemical characterization of the nanotube then allowed for further mechanistic molecular dynamic studies of the ortho-carborane uptake and calculations of corresponding energy profiles. Visualization of the binding event highlighted the protein dynamics and the importance of the interhelical channel formation to allow movement of the boron cluster into the nanotube. Additionally, cell assays showed that the nanotube can penetrate outer membranes of cancer cells followed by localization around the cells' nuclei. This work uses an integrative approach combining experimental data from structural, molecular dynamics simulations and biological experiments to thoroughly present an alternative drug delivery device for BNCT which offers additional benefits over current delivery methods.

Solution structure of the cytoplasmic domain of NhaP2 a K+/H+ antiporter from Vibrio cholera.

NhaP2 is a K+/H+ antiporter from Vibrio cholerae which consists of a transmembrane domain and a cytoplasmic domain of approximately 200 amino acids, both of which are required for cholera infectivity. Here we present the solution structure for a 165 amino acid minimal cytoplasmic domain (P2MIN) form of the protein. The structure reveals a compact N-terminal domain which resembles a Regulator of Conductance of K+ channels (RCK) domain connected to a more open C-terminal domain via a flexible 20 amino acid linker. NMR titration experiments showed that the protein binds ATP through its N-terminal domain, which was further supported by waterLOGSY and Saturation Transfer Difference NMR experiments. The two-domain organisation of the protein was confirmed by BIOSAXS, which also revealed that there are no detectable-ATP-induced conformational changes in the protein structure. Finally, in contrast to all known RCK domain structures solved to date, the current work shows that the protein is a monomer.

Structure and hydrodynamics of a DNA G-quadruplex with a cytosine bulge.

The identification of four-stranded G-quadruplexes (G4s) has highlighted the fact that DNA has additional spatial organisations at its disposal other than double-stranded helices. Recently, it became clear that the formation of G4s is not limited to the traditional G3+NL1G3+NL2G3+NL3G3+ sequence motif. Instead, the G3 triplets can be interrupted by deoxythymidylate (DNA) or uridylate (RNA) where the base forms a bulge that loops out from the G-quadruplex core. Here, we report the first high-resolution X-ray structure of a unique unimolecular DNA G4 with a cytosine bulge. The G4 forms a dimer that is stacked via its 5'-tetrads. Analytical ultracentrifugation, static light scattering and small angle X-ray scattering confirmed that the G4 adapts a predominantly dimeric structure in solution. We provide a comprehensive comparison of previously published G4 structures containing bulges and report a special γ torsion angle range preferentially populated by the G4 core guanylates adjacent to bulges. Since the penalty for introducing bulges appears to be negligible, it should be possible to functionalize G4s by introducing artificial or modified nucleotides at such positions. The presence of the bulge alters the surface of the DNA, providing an opportunity to develop drugs that can specifically target individual G4s.

Human DDX21 binds and unwinds RNA guanine quadruplexes.

Guanine quadruplexes (G4s) are an important structure of nucleic acids (DNA and RNA) with roles in several cellular processes. RNA G4s require specialized unwinding enzymes, of which only two have been previously identified. We describe the results of a simple and specific mass spectrometry guided method used to screen HEK293T cell lysate for G4 binding proteins. From these results, we validated the RNA helicase protein DDX21. DDX21 is an established RNA helicase, but has not yet been validated as a G4 binding protein. Through biochemical techniques, we confirm that DDX21-quadruplex RNA interactions are direct and mediated via a site of interaction at the C-terminus of the protein. Furthermore, through monitoring changes in nuclease sensitivity we show that DDX21 can unwind RNA G4. Finally, as proof of principle, we demonstrate the ability of DDX21 to suppress the expression of a protein with G4s in the 3΄ UTR of its mRNA.

The lack of OmpF, but not OmpC, contributes to increased antibiotic resistance in Serratia marcescens.

The environmental organism Serratia marcescens is one of the primary causes of numerous nosocomial outbreaks and opportunistic infections. Multi-drug resistance is now a common feature among S. marcescens clinical isolates, complicating the efficacy of treatment. Recent reports have attributed antibiotic resistance to altered porin expression as well as perturbation of the intrinsic AmpC beta-lactamase production pathway. In this study, we aimed to genetically correlate the absence of OmpF and OmpC classical porins with increased antibiotic resistance. In generating isogenic porin mutant strains, we avoided incorporating additional resistance through the use of antibiotic cassettes in gene replacement and adopted an alternative strategy in creating clean unmarked mutant strains. We found that lack of OmpF, but not OmpC, significantly increased antibiotic MIC values to the beta-lactam drugs such as ampicillin and cefoxitin as well as to nitrofurantoin. Furthermore, we found that cefoxitin did not induce intrinsic AmpC beta-lactamase production, indicating that the increased MIC values were a result of reduced permeability of cefoxitin due to the lack of OmpF. Genetic deletion of both ompF and ompC did not compromise the integrity of the bacterial cell envelope in optimal growth conditions, suggesting that other outer-membrane porins may function in a compensatory role to facilitate nutrient uptake and cell envelope integrity. Taken together, to our knowledge this is the first study that genetically correlates increased antibiotic resistance with altered porin expression in S. marcescens.

Purificación del factor IX recombinante expresado en leche de cerdas transgénicas / Purification of recombinant factor IX expressed in milk of transgenic sows

Se purificó a partir de la leche de cerdas transgénicas, factor IX recombinante, y se obtuvieron rendimientos entre 1 a 2 g de esta proteína por litro, lo que resulta una nueva vía para la obtención de este producto con una alta eficiencia, ya que su expresión es 1 000 veces superior a la del factor IX plasmático humano. Mediante la combinación de 2 pasos cromatográficos: intercambio iónico en DEAE-Shephadex A-50 y cromatografía de afinidad con heparina, se realizó la purificación del factor IX, con esta leche como material de partida. Se estudiaron diferentes métodos para la eliminación de las caseínas, contaminante principal del proceso, y se escogió finalmente la ultracentrifugación, por las numerosas ventajas que presenta con respecto a la precipitación isoeléctrica y la precipitación por sales. El factor IX puede ser purificado de la leche transgénica con una alta pureza utilizando métodos cromatográficos que no usan inmunoafinidad y son finalmente escalables en la producción industrial, lo cual proporciona nuevas perspectivas para el tratamiento de la hemofilia B mediante la preparación de posibles formulaciones orales.

Recombinant factor IX was purified from milk of transgenic sows, and yieldings between 1 and 2 g of this protein per liter were obtained. This is a new way to get this product with a high efficiency, since its expression is 1 000 times higher than of the human plasmatic factor IX. By combining 2 chromatographic steps (ion exchange in DEAE-Shephadex A-50 and affinity chromatography with heparin), the factor IX was purified, with this milk as a starting material. Different methods were studied to eliminate caseins, the main pollutant of the process, and ultracentrifugation was selected due to its numerous advantages over the isoelectric precipitation and salt precipitation. Factor IX may be purified from transgenic milk with an elevated purity by chromatographic methods that do not use immunoaffinity and are finally scalable in industrial production, which provides new perspectives for treating hemophilia B by preparing new oral formulations.

Purificación del factor IX recombinante expresado en leche de cerdas transgénicas / Purification of recombinant factor IX expressed in milk of transgenic sows

Se purificó a partir de la leche de cerdas transgénicas, factor IX recombinante, y se obtuvieron rendimientos entre 1 a 2 g de esta proteína por litro, lo que resulta una nueva vía para la obtención de este producto con una alta eficiencia, ya que su expresión es 1 000 veces superior a la del factor IX plasmático humano. Mediante la combinación de 2 pasos cromatográficos: intercambio iónico en DEAE-Shephadex A-50 y cromatografía de afinidad con heparina, se realizó la purificación del factor IX, con esta leche como material de partida. Se estudiaron diferentes métodos para la eliminación de las caseínas, contaminante principal del proceso, y se escogió finalmente la ultracentrifugación, por las numerosas ventajas que presenta con respecto a la precipitación isoeléctrica y la precipitación por sales. El factor IX puede ser purificado de la leche transgénica con una alta pureza utilizando métodos cromatográficos que no usan inmunoafinidad y son finalmente escalables en la producción industrial, lo cual proporciona nuevas perspectivas para el tratamiento de la hemofilia B mediante la preparación de posibles formulaciones orales(AU)

Pasteurización de soluciones de inmunoglobulinas de uso intravenoso. 2. El ph ácido como elemento estabilizante / Pasteurization of intravenous immunoglobulin solutions. 2. pH as a stabilizer element

Una solución de inmunoglobulinas de uso intravenoso (intacglobin©, Cuba), libre de estabilizantes, fue pasteurizada a baja fuerza iónica y pH ácidos. El contenido de polímeros se determinó por cromatografía en gel, y se obtuvo el mejor resultado a pH 3,5 con 3,8 porciento de polímeros y 72,7 porciento de monómeros. La estabilidad de la inmunoglobulina G (IgG), tratada a pH de 3,0 a 7,0, fue evaluada por espectrofotometría. La actividad antígeno-anticuerpo fue medida por un ELISA tipo Sandwich de doble antígeno, y se obtuvo como resultado una disminución del título de anticuerpos a los pH más ácidos. Se demostró que la baja fuerza iónica y el pH ácido son 2 condiciones necesarias para lograr un bajo nivel de polímeros durante la pasteurización de las soluciones de IgG. Se encontraron requisitos para que el proceso transcurra sin la adición de estabilizantes en mejores condiciones que las reportadas por otros autores

Pasteurización de soluciones de inmunoglobulinas de uso intravenoso. 2. El ph ácido como elemento estabilizante / Pasteurization of intravenous immunoglobulin solutions. 2. pH as a stabilizer element

Una solución de inmunoglobulinas de uso intravenoso (intacglobin©, Cuba), libre de estabilizantes, fue pasteurizada a baja fuerza iónica y pH ácidos. El contenido de polímeros se determinó por cromatografía en gel, y se obtuvo el mejor resultado a pH 3,5 con 3,8 porciento de polímeros y 72,7 porciento de monómeros. La estabilidad de la inmunoglobulina G (IgG), tratada a pH de 3,0 a 7,0, fue evaluada por espectrofotometría. La actividad antígeno-anticuerpo fue medida por un ELISA tipo Sandwich de doble antígeno, y se obtuvo como resultado una disminución del título de anticuerpos a los pH más ácidos. Se demostró que la baja fuerza iónica y el pH ácido son 2 condiciones necesarias para lograr un bajo nivel de polímeros durante la pasteurización de las soluciones de IgG. Se encontraron requisitos para que el proceso transcurra sin la adición de estabilizantes en mejores condiciones que las reportadas por otros autores (AU)

Pasteurización de soluciones de inmunoglobulinas de uso intravenoso. Uso de azúcares como estabilizantes / Pasteurization of intravenous immunoglobulin solutions. Use of sugars as stabilizers

Se realizó la pasteurización a partir de la unión de los sobrenadantes II de 4 lotes de plasma normal sometidos a fraccionamiento alcohólico, con la utilización de los estabilizantes siguientes: sorbitol, sacarosa y dextrosa al 30 porciento. El grado de agregación molecular se determinó por cromatografía en gel. Entre los estabilizantes utilizados, el sorbitol presentó los mejores resultados, con formación de polímeros que fluctúan entre el 10 - 15 porciento, valores que se corresponden con los reportados por otros autores. Estos polímeros según los requerimientos de la OMS deben ser eliminados por otros procedimientos para obtener valores inferiores al 10 porciento

Pasteurización de soluciones de inmunoglobulinas de uso intravenoso. 1. Uso de azúcares como estabilizantes / Pasteurization of intravenous immunoglobulin solutions. Use of sugars as stabilizers

Se realizó la pasteurización a partir de la unión de los sobrenadantes II de 4 lotes de plasma normal sometidos a fraccionamiento alcohólico, con la utilización de los estabilizantes siguientes: sorbitol, sacarosa y dextrosa al 30 porciento. El grado de agregación molecular se determinó por cromatografía en gel. Entre los estabilizantes utilizados, el sorbitol presentó los mejores resultados, con formación de polímeros que fluctúan entre el 10 - 15 porciento, valores que se corresponden con los reportados por otros autores. Estos polímeros según los requerimientos de la OMS deben ser eliminados por otros procedimientos para obtener valores inferiores al 10 porciento (AU)