Refining siRNA in vivo transfection: silencing SPHK1 reveals its key role in C5a-induced inflammation in vivo

The transfection of siRNA in vivo is essential for the study of gene functions, target validation, and for gene therapy. However, the successful delivery of siRNA in whole organisms is still very difficult to achieve. A high-pressure delivery technique, called the "hydrodynamics" method, has been used for siRNA transfection in mice. However, it is a method based on a high-speed and high-volume of i.v. injection, which makes it very difficult to implement in vivo, due to vascular breakage. Here, we systematically investigated ways to optimize the siRNA delivery, in order to avoid strong side effects, while achieving a high-efficiency siRNA-gene knockdown. We show here that the amount of siRNA delivered is crucial, as using too little or too much siRNA minimizes the knockdown effect. We demonstrate that by carefully identifying an optimal-minimal volume, and an optimal amount of siRNA, we achieve a high knockdown effect, with a 100% survival rate. We have previously shown that SphK1 plays a key role in anaphylatoxin (C5a) signaling in neutrophils and macrophages. Our approach, optimizing the dosage of siRNA, allowed us to successfully silence our target gene-product (SphK1) in vivo, and enabled us to validate SphK1 as a key player in our in vivo model of C5a-induced acute peritonitis and systemic inflammation including multi-organ damage, demonstrating that this improved siRNA-silencing method not only allowed us to identify SphK1 as a key therapeutic target, but brings us a step closer to the usage of siRNA for therapeutic intervention.     

A two-plane tubular photobioreactor for outdoor culture of Spirulina

A photobioreactor in the form of a 245-m-long loop made of plexiglass tubes having an inner diameter of 2.6 cm was designed and constructed for outdoor culture of Spirulina. The loop was arranged in two planes, with 15 8-m-long tubes in each plane. In the upper plane, the tubes were placed in the vacant space between the ones of the lower plane. The culture recycle was performed either with two airlifts, one per plane, or with two peristaltic pumps. The power required for water recycle in the tubular photobioreactor, with a Reynolds number of 4000, was 3.93 x 10(-2) W m(-2). The photobioreactor contained 145 L of culture and covered an overall area of 7.8 m(2). The photobioreactor operation was computer controlled. Viscosity measurements performed on Spirulina cultures having different biomass concentrations showed non-Newtonian behavior displaying decreasing viscosity with an increasing shear rate. The performance of the two-plane photobioreactor was tested under the climatic conditions of central Italy (latitude 43.8 degrees N, longitude 11.3 degrees E). A biomass concentration of 3.5 g L(-1) was found to be adequate for outdoor culture of Spirulina. With a biomass concentration of 6.3 g L(-1), the biomass output rate significantly decreased. The net biomass output rate reached a mean value of 27.8 g m(-2) d(-1) in July; this corresponded to a net photosynthetic efficiency of 6.6% (based on visible irradiance). (c) 1993 John Wiley & Sons, Inc.     

Mutational spectrum of BRAF gene in colorectal cancer patients in Saudi Arabia

Colorectal cancer (CRC) is one of the topmost causes of death in males in Saudi Arabia. In females, it was also within the top five cancer types. CRC is heterogeneous in terms of pathogenicity and molecular genetic pathways. It is very important to determine the genetic causes of CRC in the Saudi population. BRAF is one of the major genes involved in cancers, it participates in transmitting chemical signals from outside the cells into the nucleus of the cells and it is also shown to participate in cell growth. In this study, we mapped the spectrum of BRAF mutations in 100 Saudi patients with CRC. We collected tissue samples from colorectal cancer patients, sequenced the BRAF gene to identify gene alterations, and analyzed the data using different bioinformatics tools. We designed a three-dimensional (3D) homology model of the BRAF protein using the Swiss Model automated homology modeling platform to study the structural impact of these mutations using the Missense3D algorithm. We found six mutations in 14 patients with CRC. Four of these mutations are being reported for the first time. The novel frameshift mutations observed in CRC patients, such as c.1758delA (E586E), c.1826insT (Q609L), c.1860insA and c.1860insA/C (M620I), led to truncated proteins of 589, 610, and 629 amino acids, respectively, and potentially affected the structure and the normal functions of BRAF. These findings provide insights into the molecular etiology of CRC in general and to the Saudi population. BRAF genetic testing may also guide treatment modalities, and the treatment may be optimized based on personalized gene variations.     

Structurally related but genetically unrelated antibody lineages converge on an immunodominant HIV-1 Env neutralizing determinant following trimer immunization

Understanding the molecular mechanisms by which antibodies target and neutralize the HIV-1 envelope glycoprotein (Env) is critical in guiding immunogen design and vaccine development aimed at eliciting cross-reactive neutralizing antibodies (NAbs). Here, we analyzed monoclonal antibodies (mAbs) isolated from non-human primates (NHPs) immunized with variants of a native flexibly linked (NFL) HIV-1 Env stabilized trimer derived from the tier 2 clade C 16055 strain. The antibodies displayed neutralizing activity against the autologous virus with potencies ranging from 0.005 to 3.68 μg/ml (IC₅₀). Structural characterization using negative-stain EM and X-ray crystallography identified the variable region 2 (V2) of the 16055 NFL trimer to be the common epitope for these antibodies. The crystal structures revealed that the V2 segment adopts a β-hairpin motif identical to that observed in the 16055 NFL crystal structure. These results depict how vaccine-induced antibodies derived from different clonal lineages penetrate through the glycan shield to recognize a hypervariable region within V2 (residues 184–186) that is unique to the 16055 strain. They also provide potential explanations for the potent autologous neutralization of these antibodies, confirming the immunodominance of this site and revealing that multiple angles of approach are permissible for affinity/avidity that results in potent neutralizing capacity. The structural analysis reveals that the most negatively charged paratope correlated with the potency of the mAbs. The atomic level information is of interest to both define the means of autologous neutralization elicited by different tier 2-based immunogens and facilitate trimer redesign to better target more conserved regions of V2 to potentially elicit cross-neutralizing HIV-1 antibodies.     

Disease associated cellular machinery in anaphylaxis – And the de novo paradigm shift

Anaphylaxis is a sudden immune reaction against an allergen that can potentially lead to Anaphylactic Shock (AS). This immune reaction is characterized by an increase in Immunoglobulin-E (IgE) type of antibodies that bind with FcεRI receptors on mast cells to release inflammatory mediators. Various intracellular signaling molecules downstream of IgE/ FcεRI axis play a potential role in cytokine, chemokine and eicosanoid secretion as well as degranulation of immune cells causing vasodilation, vascular permeability, and reduction of intravascular volume leading to cardiovascular collapse. Here, we discuss the cellular machinery of anaphylaxis and the de novo paradigm shift in the cellular aspects of AS.     

A polymer nanostructured Fabry-Perot interferometer based biosensor

A polymer nanostructured Fabry–Perot interferometer (FPI) based biosensor is reported. Different from a conventional FPI, the nanostructured FPI has a layer of Au-coated nanopores inside its cavity. The Au-coated nanostructure layer offers significant enhancement of optical transducing signals due to the localized surface plasmon resonance effect and also due to the significantly increased sensing surface area, which is up to at least two orders of magnitude larger than that of a conventional FPI-based biosensor. Using this technical platform, the immobilization of captures proteins (protein A) on the nanostructure layer and their binding with immunoglobulin G (IgG) has been monitored in real time, resulting in the shift of the interference fringes of the optical transducing signals. Current results show that the limit-of-detection of the biosensor should be lower than 10 pg/mL for IgG-protein A binding.     

Deciphering the key molecular and cellular events in neutrophil transmigration during acute inflammation

Recruitment of leukocytes circulating in our blood to the sites of infection or tissue damage is the key phenomenon in the acute inflammatory response(s). Among the leukocytes, neutrophils are primarily recruited into the areas of acute inflammation. When neutrophils interact with activated endothelium of the blood vessels, they become migratory and cross the endothelial layer of the blood vessel wall in a process called as leukocyte extravasation. Identifying and understanding the gene regulation of this extravasation phenomenon is one of the key objective of biomedical research aimed at ameliorating or alleviating the symptoms of various diseases, such as rheumatoid arthritis, asthma, anaphylaxis, atherosclerosis, ulcerative colitis etc., that are exacerbated by inappropriate inflammatory stimuli. Here, we decipher and discuss the key genes implicated in the leukocyte transmigration using the acute inflammation model called as the Dextran Sulphate Sodium (DSS) induced Colitis in mice as a classic paradigm.     

Growth characteristics of Rhodopseudomonas palustris cultured outdoors, in an underwater tubular photobioreactor, and investigation on photosynthetic efficiency

The underwater tubular photobioreactor is a fully controlled outdoor system to study photosynthetic bacteria. Before growing bacteria cells outdoors, two modified van Niel medium (vN-A, vN-B) were tested under artificial light. During exponential growth, the specific growth rates were 0.0416 and 0.0434 h⁻¹, respectively; vN-B was chosen for outdoor experiments. The growth behavior of Rhodopseudomonas palustris was investigated under a natural light–dark cycle (sunrise–sunset, 15L/9D) and a forced light–dark cycle (9:00–19:00, 10L/14D). Relationships between solar radiations, daily growth rates, and biomass output rates were also investigated. After determining the elemental biomass molar composition and its combustion heat, some trends of photosynthetic efficiency (PE) were obtained over daylight. The PE trends were always of the oscillatory type, with the exception of that achieved at low biomass concentration. Under a natural light/dark cycle, the maximum PE (11.2%) was attained at sunset, while under a forced light/dark cycle, the highest PE (8.5%) was achieved in the morning. Three initial biomass concentrations were investigated (0.65, 1.01, and 1.54 g l⁻¹). The stoichiometric equation for bacteria cells indicated that 87.7% of the carbon of acetic acid was converted to biomass and only 12.3% was lost as CO₂.     

A compilation of Bioactive Compounds from Ayurveda

This review deals with the key bioactive compounds and the role of medicinal plants in Ayurvedic systems of medicine in India and their earlier investigation. There has been an increase in demand for the Phytopharmaceutical products of Ayurvĕda in Western countries, because of the fact that the allopathic drugs have more side effects. Many pharmaceutical companies are now concentrating on manufacturing of Ayurvĕdic Phytopharmaceutical products. Ayurvĕda is the Indian traditional system of medicine, which also deals about pharmaceutical science. Different type of plant parts used for the Ayurvedic formulation; overall out line of those herbal scenario and its future prospects for the scientific evaluation of medicinal plants used by traditional healers are also discussed. In India most of them, where Ayurvedic treatment is frequently used, for their ailments and provides instructions to local people how to prepare medicine from the herbs. As much as possible importance is also given for the taxonomic literature.