Protozoan parasites of the genus Leishmania cause leishmaniasis, a disease with variable clinical manifestations that affects millions of people worldwide. Infection with L. donovani can result in ...fatal visceral disease. In Panama, Colombia, and Costa Rica, L. panamensis is responsible for most of the reported cases of cutaneous and mucocutaneous leishmaniasis. Studying a large number of drug candidates with the methodologies available to date is quite difficult, given that they are very laborious for evaluating the activity of compounds against intracellular forms of the parasite or for performing in vivo assays. In this work, we describe the generation of L. panamensis and L. donovani strains with constitutive expression of the gene that encodes for an enhanced green fluorescent protein (eGFP) integrated into the locus that encodes for 18S rRNA (ssu). The gene encoding eGFP was obtained from a commercial vector and amplified by polymerase chain reaction (PCR) to enrich it and add restriction sites for the BglII and KpnI enzymes. The eGFP amplicon was isolated by agarose gel purification, digested with the enzymes BglII and KpnI, and ligated into the Leishmania expression vector pLEXSY-sat2.1 previously digested with the same set of enzymes. The expression vector with the cloned gene was propagated in E. coli, purified, and the presence of the insert was verified by colony PCR. The purified plasmid was linearized and used to transfect L. donovani and L. panamensis parasites. The integration of the gene was verified by PCR. The expression of the eGFP gene was evaluated by flow cytometry. Fluorescent parasites were cloned by limiting dilution, and clones with the highest fluorescence intensity were selected using flow cytometry.
Axenic Euglena cultures were grown under different LED conditions, medium compositions, and durations of cultivation to identify the factors involved in tuning Euglena biomass quality, especially ...protein and paramylon contents. Compositional analysis suggests that paramylon and protein concentrations in biomass can be tuned to different extents depending on medium and process conditions, including medium compositions, glucose concentrations, and illuminations of various LED spectra. Through the optimization of these parameters, the biomass productivities under specified conditions reached ~50–68 g/L. The protein-rich (>50 % dry basis) Euglena cultures can be produced with higher biomass productivities (38 g/L) under mixotrophic conditions with the presence of white and red LED lights and the modified medium composition. The cell morphology studies of Euglena cultures under various cultivation conditions indicate the correlation of slender cell-shape to the cell feature of high-protein contents in the biomass. The fresh cellular volumes and dry weights of paramylon-rich round-shaped cells were also estimated to be 5–9 times higher than those of protein-rich slender-shaped cells. The present study demonstrates an effective method to produce high-protein Euglena biomass with higher productivity in a shorter time.
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•The relationship of paramylon and protein contents in Euglena is tightly inverse to each other.•Light property, higher ratios of CM/ammonium-to-glucose, and enriched trace elements could enhance protein accumulation.•Mixotrophic cultivation could produce Euglena biomass up to 68 g/L.•Protein-rich (>50 %) Euglena biomass could be produced up to 38 g/L.•Paramylon-rich Euglena cells have 10 times the dry cellular weight of protein-rich cells.
A one-pot electrochemical nickel-catalyzed decarboxylative sp2–sp3 cross-coupling reaction has been developed using redox-active esters prepared in situ from alkyl carboxylates ...and N-hydroxyphthalimide tetramethyluronium hexafluorophosphate (PITU). This undivided cell one-pot method enables C–C bond formation using inexpensive, benchtop-stable reagents with isolated yields up to 95% with good functional group tolerance, which includes nitrile, ketone, ester, alkene and selectivity over other aromatic halogens.
The purpose of this study was to define baseline variability of apparent diffusion coefficient (ADC) on diffusion-weighted MR imaging (DWI) in patients with head and neck squamous cell carcinoma ...(HNSCC) and to compare it with early treatment-induced ADC change.
Patients with American Joint Committee on Cancer stages III and IV HNSCC were imaged with two baseline DWI examinations 1 week apart and a third DWI examination during the 2nd week of curative-intent chemoradiation therapy. Mean ADC was measured in the primary tumor and largest lymph node for each patient on the three DWI scans. Mean baseline percentage differences (%∆ADC) were compared with intratreatment change. The repeatability coefficient for baseline %∆ADC was calculated and compared with intratreatment %∆ADC. Repeatability was also assessed with Bland-Altman plots and the intraclass correlation coefficient (ICC).
Sixteen patients underwent double baseline imaging, with 14 also undergoing intratreatment imaging. Baseline nodal disease ADC could be measured in 16 patients, but ADC in primary tumors could only be measured in five patients. The nodal mean (SD) baseline %∆ADC was 8% (± 7%), which was significantly different compared with intratreatment changes of 32% (± 31%) (p = 0.01). Baseline ICC was 0.86 for nodal disease and 0.99 for primary tumor (excellent correlation). The calculated repeatability coefficient for baseline nodal ADC was 15%. No patients had decreases in intratreatment ADC of more than 15%.
Baseline ADC variability for HNSCC is less than intratreatment ADC change for nodal disease. Assessment of response should consider intrinsic baseline variability.
Due to the absence of transcriptional regulation of gene expression in
parasites, it is now well accepted that several forms of genomic variations modulate the levels of critical proteins through ...changes in gene dosage. We previously observed many of these variations in our reference laboratory strain of
(PSC-1 strain), including chromosomes with an increased somy and the presence of a putative linear minichromosome derived from chromosome 34. Here, we compared the previously described genomic variations with those occurring after exposure of this strain to increasing concentrations of trivalent antimony (Sb
), as well as those present in two geographically unrelated clinical isolates of
. We observed changes in the somy of several chromosomes, amplifications of several chromosomal regions, and copy number variations in gene arrays after exposure to Sb
. Occurrence of amplifications potentially beneficial for the Sb-resistant phenotype appears to be associated with the loss of other forms of amplification, such as the linear minichromosome. In contrast, we found no evidence of changes in somy or amplification of relatively large chromosomal regions in the clinical isolates. In these isolates, the predominant amplifications appear to be those that generate genes arrays; however, in many cases, the amplified arrays have a notably higher number of copies than those from the untreated and Sb-treated laboratory samples.
High-throughput protein expression platforms are increasingly used to produce proteins for many applications: to support studies in structure/function, regulation and proteomics, as well as for ...direct use as potential biotherapeutic agents for medical applications. Here we describe a device that we refer to as the flask density reader (FDR) consisting of a through-beam laser and sensor, and a customized culture flask-receiving nest. The FDR has been integrated onto GNF System™'s automated protein expression platform to enable rapid, noninvasive, fully automated spectrophotometric determination of cell densities in suspension mammalian cell cultures. The FDR reduces the risk of culture contamination from frequent flask sampling and greatly reduces the time and effort needed to count cells using off-line methods.
Rapid quantitative methods for characterizing small molecules, peptides, proteins, or RNAs in a broad array of cellular assays would allow one to discover new biological activities associated with ...these molecules and also provide a more comprehensive profile of drug candidates early in the drug development process. Here we describe a robotic system, termed the automated compound profiler, capable of both propagating a large number of cell lines in parallel and assaying large collections of molecules simultaneously against a matrix of cellular assays in a highly reproducible manner. To illustrate its utility, we have characterized a set of 1,400 kinase inhibitors in a panel of 35 activated tyrosine-kinase-dependent cellular assays in dose-response format in a single experiment. Analysis of the resulting multidimensional dataset revealed subclusters of both inhibitors and kinases with closely correlated activities. The approach also identified activities for the p38 inhibitor BIRB796 and the dual src/abl inhibitor BMS-354825 and exposed the expected side activities for Glivec/STI571, including cellular inhibition of c-kit and platelet-derived growth factor receptor. This methodology provides a powerful tool for unraveling the cellular biology and molecular pharmacology of both naturally occurring and synthetic chemical diversity.
Herein are reported initial efforts to develop a generally accessible flow process, applying a heterogenous nanocatalyst to aqueous micelle-enabled Suzuki-Miyaura coupling reactions. Also disclosed ...is a new engineering solution (
i.e.
, a self-draining back pressure regulator), which, when applied, enabled 1.5 hours of continuous operation leading to the production of 20 grams of a pharmaceutical intermediate.
Plugged flow chemistry in water has been developed using a heterogeneous catalyst and applied to Suzuki-Miyaura cross-couplings.
To investigate the feasibility of the log-demons deformable image registration (DIR) method to correct eddy current and field inhomogeneity distortions while preserving diffusion tensor information. ...Diffusion-weighted images (DWIs) are susceptible to distortions caused by eddy current and echo-planar imaging (EPI) gradients. We propose a post-acquisition correction algorithm using the log-demons DIR technique for eddy current and field inhomogeneity distortions of DWI. The new correction technique was applied to DWI acquired using a diffusion phantom and the multiple acquisitions for standardization of structural imaging validation and evaluation (MASSIVE) brain database. This method is compared to previous methods using cross-correlation, mutual information (MI). In the phantom study, the log-demons algorithm reduced eddy current and field inhomogeneity distortions while preserving diffusion tensor information when compared to affine and demon's registration techniques. Analysis of the tensor metrics using percent difference and the root mean square of the apparent diffusion coefficient and fractional anisotropy found that the log-demons algorithm outperforms the other algorithms in terms of preserving diffusion information. In the MASSIVE study, the average MI of all slices increased for both eddy current and field inhomogeneity distortion correction. The average absolute differences of all slices between corrected images with opposing gradients were also on average decreased. This work indicates that the log-demons DIR algorithm is feasible to reduce eddy current and field inhomogeneity distortions while preserving quantitative diffusion information.
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