The Design of Pooling Experiments for Screening a Clone Map

We consider nonadaptive pooling designs for unique-sequence screening of a 1530-clone map ofAspergillus nidulans.The map has the properties that the clones are, with possibly a few exceptions, ordered and no more than 2 of them cover any point on the genome. We propose two subdesigns of the Steiner systemS(3, 5, 65), one with 65 pools and approximately 118 clones per pool, the other with 54 pools and about 142 clones per pool. Each design allows 1 or 2 positive clones to be detected, even in the presence of substantial experimental error rates. More efficient designs are possible if the overlap information in the map is exploited, if there is no constraint on the number of clones in a pool, and if no error tolerance is required. An information theory lower bound requires at least 12 pools to satisfy these minimal criteria, and an “interleaved binary” design can be constructed on 20 pools, with about 380 clones per pool. However, the designs with more pools have important properties of robustness to various possible errors and general applicability to a wider class of pooling experiments.     

Model-assisted Design of Experiments as a concept for knowledge-based bioprocess development

Bioprocess and Biosystems Engineering - Design of Experiments methods offer systematic tools for bioprocess development in Quality by Design, but their major drawback is the user-defined choice of...     

Design and Evaluation of a Novel Matrix Type Multiple Units as Biphasic Gastroretentive Drug Delivery Systems

A biphasic gastroretentive floating drug delivery system with multiple-unit mini-tablets based on gas formation technique was developed to maintain constant plasma level of a drug concentration within the therapeutic window. The system consists of loading dose as uncoated core units, and prolonged-release core units are prepared by direct compression process; the latter were coated with three successive layers, one of which is seal coat, an effervescent (sodium bicarbonate) layer, and an outer polymeric layer of polymethacrylates. The formulations were evaluated for quality control tests, and all the parameters evaluated were within the acceptable limits. The system using Eudragit RL30D and combination of them as polymeric layer could float within acceptable time. The drug release was linear with the square root of time. The rapid floating and the controlled release properties were achieved in this present study. When compared with the theoretical release profile, the similarity factor of formulation with coating of RS:RL (1:3)–7.5%, was observed to be 74, which is well fitted into zero-order kinetics confirming that the release from formulation is close to desired release profile. The stability samples showed no significant change in dissolution profiles (p > 0.05). In vivo gastric residence time was examined by radiograms, and it was observed that the units remained in the stomach for about 5 h.     

MORE: Mixed Optimization for Reverse Engineering-An Application to Modeling Biological Networks Response via Sparse Systems of Nonlinear Differential Equations

Reverse engineering is the problem of inferring the structure of a network of interactions between biological variables from a set of observations. In this paper, we propose an optimization algorithm, called MORE, for the reverse engineering of biological networks from time series data. The model inferred by MORE is a sparse system of nonlinear differential equations, complex enough to realistically describe the dynamics of a biological system. MORE tackles separately the discrete component of the problem, the determination of the biological network topology, and the continuous component of the problem, the strength of the interactions. This approach allows us both to enforce system sparsity, by globally constraining the number of edges, and to integrate a priori information about the structure of the underlying interaction network. Experimental results on simulated and real-world networks show that the mixed discrete/continuous optimization approach of MORE significantly outperforms standard continuous optimization and that MORE is competitive with the state of the art in terms of accuracy of the inferred networks.     

Use of Experimental Factorial Design for Optimization of Hexavalent Chromium Removal by a Bacterial Consortium: Soil Microcosm Bioremediation

Hexavalent chromium Cr(VI) is regularly introduced into the environment through diverse anthropogenic activities. It is highly toxic, mutagenic and carcinogenic, and because of its solubility in water, chromate contamination can be difficult to contain. Bacteria can reduce chromate to insoluble and less toxic trivalent chromium Cr(III), and thus increasing attention is paid to chromate bioremediation to reduce its ecotoxicological impacts. In this study, the factorial design 2³ was employed to optimize critical parameters responsible for higher Cr(VI) removal by a bacterial consortium. The factors considered were pH, temperature, and inoculum size at two markedly different levels. All three dependent variables have significant effect on Cr(VI) reduction. Optimal Cr(VI) removal by the bacterial consortium occurred at pH 9, temperature 37°C, and inoculum size OD = 3. Analysis of variance (ANOVA) showed a high coefficient of determination (R²) value of 0.984, thus ensuring a satisfactory adjustment of the second-order regression model with the experimental data. In addition, the effect of bioaugmentation of Cr(VI)-polluted soil microcosms with the bacterial consortium was investigated using the best factor levels. Contaminated soil by 20 and 60 mg/Kg of Cr(VI) showed reductions of 83% and 65% of initial Cr(VI) by the bacterial consortium, suggesting that this bacterial consortium might diminish phytoavailable Cr(VI) in soil and be useful for cleaning up chromium-contaminated sites.     

Optimization of Process Parameters for a Quasi-Continuous Tablet Coating System Using Design of Experiments

The aim of this study was to identify and optimize the critical process parameters of the newly developed Supercell quasi-continuous coater for optimal tablet coat quality. Design of experiments, aided by multivariate analysis techniques, was used to quantify the effects of various coating process conditions and their interactions on the quality of film-coated tablets. The process parameters varied included batch size, inlet temperature, atomizing pressure, plenum pressure, spray rate and coating level. An initial screening stage was carried out using a 2^6−1(IV) fractional factorial design. Following these preliminary experiments, optimization study was carried out using the Box–Behnken design. Main response variables measured included drug-loading efficiency, coat thickness variation, and the extent of tablet damage. Apparent optimum conditions were determined by using response surface plots. The process parameters exerted various effects on the different response variables. Hence, trade-offs between individual optima were necessary to obtain the best compromised set of conditions. The adequacy of the optimized process conditions in meeting the combined goals for all responses was indicated by the composite desirability value. By using response surface methodology and optimization, coating conditions which produced coated tablets of high drug-loading efficiency, low incidences of tablet damage and low coat thickness variation were defined. Optimal conditions were found to vary over a large spectrum when different responses were considered. Changes in processing parameters across the design space did not result in drastic changes to coat quality, thereby demonstrating robustness in the Supercell coating process.     

A Practical Approach for the Selection, Pilot Testing, Design, and Monitoring of In Situ Air Sparging/Biosparging Systems

The use of in situ air sparging (IAS) has increased rapidly since the early 1990s, and it is now likely to be the most practiced engineered in situ remediation option when targeting the treatment of hydrocarbon-impacted aquifers. To date, IAS system design has remained largely empirical, with significant variability in approaches and results. Here, the valuable knowledge gained from IAS studies and applications over the past decade has been integrated into a new paradigm for feasibility assessment, pilot testing, design, and operation. The basis for this Design Paradigm, the initial feasibility assessment, monitoring, and the overall design approach are discussed in detail here; other referenced documents contain the details of specific recommended activities. The proposed design approach is unique in that it contains two design routes; the first is a non-site-specific approach requiring minimal site characterization and testing (Standard Design Approach), while the second is a more site-specific approach (Site-Specific Design Approach).     

CRISPys: Optimal sgRNA Design for Editing Multiple Members of a Gene Family Using the CRISPR System

The development of the CRISPR–Cas9 system in recent years has made eukaryotic genome editing, and specifically gene knockout for reverse genetics, a simple and effective task. The system is directed to a genomic target site by a programmed single-guide RNA (sgRNA) that base-pairs with it, subsequently leading to site-specific modifications. However, many gene families in eukaryotic genomes exhibit partially overlapping functions, and thus, the knockout of one gene might be concealed by the function of the other. In such cases, the reduced specificity of the CRISPR–Cas9 system, which may lead to the modification of genomic sites that are not identical to the sgRNA, can be harnessed for the simultaneous knockout of multiple homologous genes. We introduce CRISPys, an algorithm for the optimal design of sgRNAs that would potentially target multiple members of a given gene family. CRISPys first clusters all the potential targets in the input sequences into a hierarchical tree structure that specifies the similarity among them. Then, sgRNAs are proposed in the internal nodes of the tree by embedding mismatches where needed, such that the efficiency to edit the induced targets is maximized. We suggest several approaches for designing the optimal individual sgRNA and an approach to compute the optimal set of sgRNAs for cases when the experimental platform allows for more than one. The latter may optionally account for the homologous relationships among gene-family members. We further show that CRISPys outperforms simpler alignment-based techniques by in silico examination over all gene families in the Solanum lycopersicum genome.     

Design and Screening of a Glial Cell-Specific,Cell Penetrating Peptide for Therapeutic Applications in Multiple Sclerosis

Multiple Sclerosis (MS) is an autoimmune, neurodegenerative disease of the central nervous system (CNS) characterized by demyelination through glial cell loss. Current and proposed therapeutic strategies to arrest demyelination and/or promote further remyelination include: (i) modulation of the host immune system; and/or (ii) transplantation of myelinating/stem or progenitor cells to the circulation or sites of injury. However, significant drawbacks are inherent with both approaches. Cell penetrating peptides (CPP) are short amino acid sequences with an intrinsic ability to translocate across plasma membranes, and theoretically represent an attractive vector for delivery of therapeutic peptides or nanoparticles to glia to promote cell survival or remyelination. The CPPs described to date are commonly non-selective in the cell types they transduce, limiting their therapeutic application in vivo. Here, we describe a theoretical framework for design of a novel CPP sequence that selectively transduces human glial cells (excluding non-glial cell types), and conduct preliminary screens of purified, recombinant CPPs with immature and matured human oligodendrocytes and astrocytes, and two non-glial cell types. A candidate peptide, termed TD2.2, consistently transduced glial cells, was significantly more effective at transducing immature oligodendrocytes than matured progeny, and was virtually incapable of transducing two non-glial cell types: (i) human neural cells and (ii) human dermal fibroblasts. Time-lapse confocal microscopy confirms trafficking of TD2.2 (fused to EGFP) to mature oligodendrocytes 3–6 hours after protein application in vitro. We propose selectivity of TD2.2 for glial cells represents a new therapeutic strategy for the treatment of glial-related disease, such as MS.     

Upregulation of Myelin Gene Expression by a Physically-Modified Saline via Phosphatidylinositol 3-Kinase-Mediated Activation of CREB: Implications for Multiple Sclerosis

An increase in central nervous system (CNS) remyelination and a decrease in CNS inflammation are important steps to halt the progression of multiple sclerosis (MS). RNS60 is a bioactive aqueous solution generated by subjecting normal saline to Taylor–Couette–Poiseuille flow under elevated oxygen pressure. Recently we have demonstrated that RNS60 exhibits anti-inflammatory properties. Here, we describe promyelinating property of RNS60. RNS60, but not normal saline (NS), RNS10.3 (TCP-modified saline without excess oxygen) or PNS60 (saline containing excess oxygen without TCP modification), stimulated the expression of myelin-specific genes and proteins (myelin basic protein, MBP; myelin oligodendrocyte glycoprotein, MOG and proteolipid protein, PLP) in primary mouse oligodendroglia and mixed glial cells. While investigating the mechanisms, we found that RNS60 treatment induced the activation of cAMP response element binding protein (CREB) in oligodendrocytes, ultimately leading to the recruitment of CREB to the promoters of myelin-specific genes. Furthermore, activation of type 1A p110β/α, but not type 1B p110γ, phosphatidylinositol-3 (PI-3) kinase by RNS60 together with abrogation of RNS60-mediated activation of CREB and upregulation of myelin genes by LY294002 (a specific inhibitor of PI-3 kinase) suggest that RNS60 upregulates the activation of CREB and the expression of myelin-specific molecules in oligodendrocytes via activation of PI3 kinase. These results highlight a novel promyelinating property of RNS60, which may be of benefit for MS and other demyelinating disorders.     

Design of a Fluorescent Electrophoretic Mobility Shift Assay Improved for the Quantitative and Multiple Analysis of Protein-DNA Complexes

We describe a protocol for the fluorescent electrophoretic mobility shift assay improved for the quantitative analysis of protein-DNA complexes. Fluorescent-labeled oligonucleotide probes incubated with nuclear proteins were followed by electrophoresis. The signals for protein-DNA complexes were measured and normalized with fluorescent-labeled marker using fragment analysis software. This assay proved reliable measurement and multiple detection of DNA binding proteins.     

Optimization Design of a Multi-slot Nanoantenna Based on Genetic Algorithm for Energy Harvesting

A new genetic algorithm (GA)-based multi-slot nanoantenna is proposed for energy harvesting, which consists of two element nanoantennas with rectangular shape and with double bowtie double ring (DBDR) slot. The DBDR slot structure can enhance the electric field to increase the absorptivity of nanoantennas; however, the larger parameter space of multi-slot is more hardly controlled. Therefore, we use GA to optimize the parameters of the DBDR slot nanoantenna and the Finite-Difference Time-Domain method to calculate the absorptivity. It is found that absorptivity of the optimized nanoantenna is over 77% in 400–1800 nm waveband. We attribute the better absorbing property of the nanoantenna to the synergistic effect of the localized surface plasmon resonance enhancement and coupling between slots.

     

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson''s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic α-SYN mouse models develop gradual α-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson''s disease.     

In Silico Design of a Poly-epitope Vaccine for Urinary Tract Infection Based on Conserved Antigens by Modern Vaccinology

International Journal of Peptide Research and Therapeutics - Immunoinformatics or vaccine informatics focuses on applying computational approaches to advance vaccine research and development...     

Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction

Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning. DNA chemical synthesis has limits on both its length and yield, thus full-length genes have to be hierarchically constructed from synthesized DNA fragments. Gibson Assembly and its derivatives are the simplest methods to assemble multiple double-stranded DNA fragments. Currently, up to 12 dsDNA fragments can be assembled at once with Gibson Assembly according to its vendor. In practice, the number of dsDNA fragments that can be assembled in a single reaction are much lower. We have developed a rational design method for gene construction that allows high-number dsDNA fragments to be assembled into full-length genes in a single reaction. Using this new design method and a modified version of the Gibson Assembly protocol, we have assembled 3 different genes from up to 45 dsDNA fragments at once. Our design method uses the thermodynamic analysis software Picky that identifies all unique junctions in a gene where consecutive DNA fragments are specifically made to connect to each other. Our novel method is generally applicable to most gene sequences, and can improve both the efficiency and cost of gene assembly.     

Challenges of Diagnosing Acute HIV-1 Subtype C Infection in African Women: Performance of a Clinical Algorithm and the Need for Point-of-Care Nucleic-Acid Based Testing

Background

Prompt diagnosis of acute HIV infection (AHI) benefits the individual and provides opportunities for public health intervention. The aim of this study was to describe most common signs and symptoms of AHI, correlate these with early disease progression and develop a clinical algorithm to identify acute HIV cases in resource limited setting.

Methods

245 South African women at high-risk of HIV-1 were assessed for AHI and received monthly HIV-1 antibody and RNA testing. Signs and symptoms at first HIV-positive visit were compared to HIV-negative visits. Logistic regression identified clinical predictors of AHI. A model-based score was assigned to each predictor to create a risk score for every woman.

Results

Twenty-eight women seroconverted after a total of 390 person-years of follow-up with an HIV incidence of 7.2/100 person-years (95%CI 4.5–9.8). Fifty-seven percent reported ≥1 sign or symptom at the AHI visit. Factors predictive of AHI included age <25 years (OR = 3.2; 1.4–7.1), rash (OR = 6.1; 2.4–15.4), sore throat (OR = 2.7; 1.0–7.6), weight loss (OR = 4.4; 1.5–13.4), genital ulcers (OR = 8.0; 1.6–39.5) and vaginal discharge (OR = 5.4; 1.6–18.4). A risk score of 2 correctly predicted AHI in 50.0% of cases. The number of signs and symptoms correlated with higher HIV-1 RNA at diagnosis (r = 0.63; p<0.001).

Conclusions

Accurate recognition of signs and symptoms of AHI is critical for early diagnosis of HIV infection. Our algorithm may assist in risk-stratifying individuals for AHI, especially in resource-limited settings where there is no routine testing for AHI. Independent validation of the algorithm on another cohort is needed to assess its utility further. Point-of-care antigen or viral load technology is required, however, to detect asymptomatic, antibody negative cases enabling early interventions and prevention of transmission.     

Design and Simulation of a Novel Tunable Terahertz Biosensor Based on Metamaterials for Simultaneous Monitoring of Blood and Urine Components

In this essay, a tunable metamaterial-based biosensor is proposed for simultaneous monitoring of blood components including cells, plasma, water, thrombus, and urine components as well as glucose, albumin, and urea. The proposed biosensor is based on optical sensors, and it provides real-time, label-free, and direct detection, small size, and cost-effectiveness that can be an alternative tool to other conventional methods. The influence of operating frequency, sample thickness, temperature, and radiation angle on the performance of the sensor is investigated by the finite element method (FEM). Numerical results show that the maximum sensitivity and figure of merit (FoM) in the high frequency are 500 (nm/RIU) and 2000, and for low frequency are 136 (µm/RIU) and 155, respectively. The footprint of the structure is 0.34 µm2, which is remarkably smaller than the other reported biosensing structures. The proposed biosensor has the potential to provide high sensitivity, high FoM, and a wide operating range for biomedical applications.