197 research outputs found
Semi-automated Magnetic Bead-Based Antibody Selection from Phage Display Libraries
Phage display of combinatorial antibody libraries is a very efficient method for selecting recombinant antibodies against a wide range of molecules. It has been applied very successfully for the generation of therapeutic antibodies for more than a decade. To increase robustness and reproducibility of the selection procedure, we developed a semi-automated selection method for the generation of recombinant antibodies from phage display libraries. In this procedure, the selection targets are specifically immobilised to magnetic particles which can then by automatically handled by a magnetic particle processor. At present up to 96 samples can be handled simultaneously. Applying the processor allows standardisation of panning parameters such as washing conditions, incubation times, or to perform parallel selections on same targets under different buffer conditions. Additionally, the whole protocol has been streamlined to carry out bead loading, phage selection, phage amplification between selection rounds and magnetic particle ELISA for confirmation of binding activity in microtiter plate formats. Until now, this method has been successfully applied to select antibody fragments against different types of target, such as peptides, recombinant or homologous proteins, or chemical compounds
Suppression of p75 Neurotrophin Receptor Surface Expression with Intrabodies Influences Bcl-xL mRNA Expression and Neurite Outgrowth in PC12 Cells
Background: Although p75 neurotrophin receptor (p75NTR) is the first neurotrophin receptor isolated, its diverse physiological functions and signaling have remained elusive for many years. Loss-of-function phenotypic analyses for p75NTR were mainly focused at the genetic level; however these approaches were impacted by off-target effect, insufficient stability, unspecific stress response or alternative active splicing products. In this study, p75NTR surface expression was suppressed for the first time at the protein level by endoplasmic reticulum (ER) retained intrabodies. Results: Three monoclonal recombinant antibody fragments (scFv) with affinities in the low nanomolar range to murine p75NTR were isolated by antibody phage display. To suppress p75NTR cell surface expression, the encoding genes of these scFvs extended by the ER retention peptide KDEL were transiently transfected into the neuron-like rat pheochromocytoma cell line PC12 and the mouse neuroblastoma x mouse spinal cord hybrid cell line NSC19. The ER retained intrabody construct, SH325-G7-KDEL, mediated a downregulation of p75NTR cell surface expression as shown by flow cytometry. This effect was maintained over a period of at least eight days without activating an unfolded protein response (UPR). Moreover, the ER retention of p75NTR resulted in downregulation of mRNA levels of the anti-apoptotic protein Bcl-xL as well as in strong inhibition of NGF-induced neurite outgrowth in PC12 cells. Conclusion: The ER retained intrabody SH325-G7-KDEL not only induces phenotypic knockdown of this p75NTR but als
Isolation and Characterisation of a Human-Like Antibody Fragment (scFv) That Inactivates VEEV In Vitro and In Vivo
Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required
Improvement and efficient display of Bacillus thuringiensis toxins on M13 phages and ribosomes
Infected pancreatic necrosis: outcomes and clinical predictors of mortality. A post hoc analysis of the MANCTRA-1 international study
: The identification of high-risk patients in the early stages of infected pancreatic necrosis (IPN) is critical, because it could help the clinicians to adopt more effective management strategies. We conducted a post hoc analysis of the MANCTRA-1 international study to assess the association between clinical risk factors and mortality among adult patients with IPN. Univariable and multivariable logistic regression models were used to identify prognostic factors of mortality. We identified 247 consecutive patients with IPN hospitalised between January 2019 and December 2020. History of uncontrolled arterial hypertension (p = 0.032; 95% CI 1.135-15.882; aOR 4.245), qSOFA (p = 0.005; 95% CI 1.359-5.879; aOR 2.828), renal failure (p = 0.022; 95% CI 1.138-5.442; aOR 2.489), and haemodynamic failure (p = 0.018; 95% CI 1.184-5.978; aOR 2.661), were identified as independent predictors of mortality in IPN patients. Cholangitis (p = 0.003; 95% CI 1.598-9.930; aOR 3.983), abdominal compartment syndrome (p = 0.032; 95% CI 1.090-6.967; aOR 2.735), and gastrointestinal/intra-abdominal bleeding (p = 0.009; 95% CI 1.286-5.712; aOR 2.710) were independently associated with the risk of mortality. Upfront open surgical necrosectomy was strongly associated with the risk of mortality (p < 0.001; 95% CI 1.912-7.442; aOR 3.772), whereas endoscopic drainage of pancreatic necrosis (p = 0.018; 95% CI 0.138-0.834; aOR 0.339) and enteral nutrition (p = 0.003; 95% CI 0.143-0.716; aOR 0.320) were found as protective factors. Organ failure, acute cholangitis, and upfront open surgical necrosectomy were the most significant predictors of mortality. Our study confirmed that, even in a subgroup of particularly ill patients such as those with IPN, upfront open surgery should be avoided as much as possible. Study protocol registered in ClinicalTrials.Gov (I.D. Number NCT04747990)
Dieta suplementada com prebiótico, probiótico e simbiótico no cultivo de camarões marinhos
Pre-treatment of Malaysian agricultural wastes toward biofuel production
Various renewable energy technologies are under considerable interest due to the projected depletion of our primary sources of energy and global warming associated with their utilizations. One of the alternatives under focus is renewable fuels produced from agricultural wastes. Malaysia, being one of the largest producers of palm oil, generates abundant agricultural wastes such as fibers, shells, fronds, and trunks with the potential to be converted to biofuels. However, prior to conversion of these materials to useful products, pre-treatment of biomass is essential as it influences the energy utilization in the conversion process and feedstock quality. This chapter focuses on pre-treatment technology of palm-based agriculture waste prior to conversion to solid, liquid, and gas fuel. Pre-treatment methods can be classified into physical, thermal, biological, and chemicals or any combination of these methods. Selecting the most suitable pre-treatment method could be very challenging due to complexities of biomass properties. Physical treatment involves grinding and sieving of biomass into various particle sizes whereas thermal treatment consists of pyrolysis and torrefaction processes. Additionally biological and chemical treatment using enzymes and chemicals to derive lignin from biomass are also discussed
Analyst information precision and small earnings surprises
This study proposes and tests an alternative to the extant earnings management explanation for zero and small positive earnings surprises (i.e., analyst forecast errors). We argue that analysts’ ability to strategically induce slight pessimism in earnings forecasts varies with the precision of their information. Accordingly, we predict that the probability that a firm reports a small positive instead of a small negative earnings surprise is negatively related to earnings forecast uncertainty, and we present evidence consistent with this prediction. Our findings have important implications for the earnings management interpretation of the asymmetry around zero in the frequency distribution of earnings surprises. We demonstrate how empirically controlling for earnings forecast uncertainty can materially change inferences in studies that employ the incidence of zero and small positive earnings surprises to categorize firms as suspected of managing earnings
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