The C2 domain of PKCdelta is a phosphotyrosine binding domain

In eukaryotic cells, the SH2 and PTB domains mediate protein-protein interactions by recognizing phosphotyrosine residues on target proteins. Here we make the unexpected finding that the C2 domain of PKCdelta directly binds to phosphotyrosine peptides in a sequence-specific manner. We provide evidence that this domain mediates PKCdelta interaction with a Src binding glycoprotein, CDCP1. The crystal structure of the PKCdelta C2 domain in complex with an optimal phosphopeptide reveals a new mode of phosphotyrosine binding in which the phosphotyrosine moiety forms a ring-stacking interaction with a histidine residue of the C2 domain. This is also the first example of a protein Ser/Thr kinase containing a domain that binds phosphotyrosine.     

Controlled release of nutrients to mammalian cells cultured in shake flasks

Though cell culture-based protein production processes are rarely carried out under batch mode of operation, cell line and initial process development operations are usually carried out in batch mode due to simplicity of operation in widely used scale down platforms like shake flasks. Nutrient feeding, if performed, is achieved by bolus addition of concentrated feed solution at different intervals, which leads to large transient increases in nutrient concentrations. One negative consequence is increased waste metabolite production. We have developed a hydrogel-based nutrient delivery system for continuous feeding of nutrients in scale down models like shake flasks without the need for manual feed additions or any additional infrastructure. Continuous delivery also enables maintaining nutrient concentrations at low levels, if desired. The authors demonstrate the use of these systems for continuous feeding of glucose and protein hydrolysate to a suspension Chinese Hamster Ovary (CHO) culture in a shake flask. Glucose feeding achieved using the glucose-loaded hydrogel resulted in a 23% higher integral viable cell density and an 89% lower lactate concentration at the end of the culture when compared with a bolus-feed of glucose.     

Compositional modification of nascent in vitro dental plaques by human host-defence peptides

Salivary host-defence peptides include defensins, histatins and cathelicidin. We have investigated the effects of these peptides on the microbial composition of dental plaques. Salivary consortia, established within hydroxyapatite disc models, were exposed during development to physiological levels of human neutrophil proteins (HNP) 1 and 2; human β defensins (hβD) 1, 2 and 3; histatins (His) 5 and 8; and cathelicidin (LL37). Effects on aggregation and microbial composition were determined using fluorescence microscopy; and differential culture with PCR-DGGE, respectively. LIVE/DEAD microscopic analysis indicated that HDPs decreased total bacterial viability, whilst β defensins, paired HNPs, His 5, His 8 and the HDPs combined inhibited bacterial aggregation. According to differential culture, all test HDPs (except His 5) significantly decreased the abundance of Gram-negative anaerobes and lactobacilli (except HNP 2, hβD 1, paired HNPs and His 5). Combined HNPs and paired hβD 1 and 3 inhibited streptococci, whereas HNP 1, hβD 1, hβD 3, His 5 and LL37 increased streptococcal numbers. According to cluster analyses of DGGE profiles, HDP-exposed plaques were compositionally distinct from undosed controls. Thus, whilst HDPs reportedly exhibit variable potency against oral bacteria in endpoint susceptibly tests, exposure of nascent plaques can markedly influence bacterial viability, composition and microbial aggregation.     

Microwave-Assisted Synthesis,Antimicrobial Activity,and SAR Study of 3-(2-Chlorobenzyl)-6-(Substituted Phenyl)-7H-[1,2,4]Triazolo[3,4-b][1,3,4]Thiadiazines

Russian Journal of Bioorganic Chemistry - An efficient, simple, and greener method has been described for the synthesis of 3-(2-chlorobenzyl)-6-(substituted...     

Establishment and characterization of lung co-culture spheroids for paclitaxel loaded Eudragit® RL 100 nanoparticle evaluation

3D cell cultures are regarded as a better and more relevant approach for screening drugs and therapeutics, particularly due to their likeness with the in vivo conditions. Spheroids offer an intermediate platform between in vitro and in vivo models, for conducting tumor-based investigations. In this study, a simple setup was developed for consistent generation of lung co-culture spheroids, which were developed using the cancer cell lines A549, NCI H460, and fibroblast cells WI-38. The potential of these spheroids for evaluating the toxicity of Eudragit® RL 100 nanoparticles (ENP) was explored. Monodisperse ENP, having the size range of 140–200 nm was prepared using the nanoprecipitation method. These were loaded with the poorly water-soluble anticancer drug paclitaxel. The evaluation of toxicity and uptake of drug-loaded ENP revealed that 2D monolayers were more sensitive to treatment than 3D spheroids. Within spheroids, co-cultures were more resistant to the treatment than monocultures. Overall, our findings demonstrated that the lung co-culture spheroids were a suitable model for accelerating the efficacy and toxicity-related investigations of novel drug delivery systems.     

Treatment Failure of Isavuconazole in a Patient with Cryptococcosis

Mycopathologia - Isavuconazole is a broad-spectrum azole that is FDA-approved for the treatment of aspergillosis and mucormycosis; data on the use of isavuconazole for the treatment and prevention...     

LocARNA-P: accurate boundary prediction and improved detection of structural RNAs

Current genomic screens for noncoding RNAs (ncRNAs) predict a large number of genomic regions containing potential structural ncRNAs. The analysis of these data requires highly accurate prediction of ncRNA boundaries and discrimination of promising candidate ncRNAs from weak predictions. Existing methods struggle with these goals because they rely on sequence-based multiple sequence alignments, which regularly misalign RNA structure and therefore do not support identification of structural similarities. To overcome this limitation, we compute columnwise and global reliabilities of alignments based on sequence and structure similarity; we refer to these structure-based alignment reliabilities as STARs. The columnwise STARs of alignments, or STAR profiles, provide a versatile tool for the manual and automatic analysis of ncRNAs. In particular, we improve the boundary prediction of the widely used ncRNA gene finder RNAz by a factor of 3 from a median deviation of 47 to 13 nt. Post-processing RNAz predictions, LocARNA-P's STAR score allows much stronger discrimination between true- and false-positive predictions than RNAz's own evaluation. The improved accuracy, in this scenario increased from AUC 0.71 to AUC 0.87, significantly reduces the cost of successive analysis steps. The ready-to-use software tool LocARNA-P produces structure-based multiple RNA alignments with associated columnwise STARs and predicts ncRNA boundaries. We provide additional results, a web server for LocARNA/LocARNA-P, and the software package, including documentation and a pipeline for refining screens for structural ncRNA, at http://www.bioinf.uni-freiburg.de/Supplements/LocARNA-P/.