Fluorescence labeling of anchor-modified Mart-1 peptide for increasing its affinity for HLA-A*0201: Hit two targets with one arrow

One of the most successful strategies in designing peptide-based cancer vaccines is modifying natural epitope peptides to increase their binding strength to human leukocyte antigens (HLAs). Anchor-modified Mart-1 peptide (ELAGIGILTV) is among the artificial epitope peptides with the highest binding affinity for HLA-A*0201. In this study, by fluorescence labeling of its either C- or N-terminus with N^ε-(5-carboxyfluorescein)-l -lysine, we not only made it traceable but also drastically increased its binding strength to HLA-A*0201. HLA streptamer, for the first time, is introduced for measuring the binding constants (K_a) of the labeled peptides. The affinity of the labeled peptides for the HLA-A*201 of the MCF-7 cells was extraordinarily high and co-incubating them with the highest possible amount of the unlabeled peptide, as a competitor, did not significantly prohibit them from binding to the HLA. The reproducibility of the obtained results was confirmed by using the T2 cell line. The HLA-deficient K562 cell line was used as the negative control. With in silico simulations, we found two hydrophobic pockets on both sides of HLA-A*0201 for anchoring the C- or N-terminal 5-carboxyfluorescein probe, which can explain the extraordinary affinity of the labeled peptides for the HLA-A*0201.     

Climate tracking by freshwater fishes suggests that fish diversity in temperate lakes may be increasingly threatened by climate warming

Aim

Many freshwater fishes are migrating poleward to more thermally suitable habitats in response to warming climates. In this study, we aimed to identify which freshwater fishes are most sensitive to climatic changes and asked: (i) how fast are lakes warming? (ii) how fast are fishes moving? and (iii) are freshwater fishes tracking climate?

Location

Ontario, Canada.

Methods

We assembled a database containing time series data on climate and species occurrence data from 10,732 lakes between 1986 and 2017. We calculated the rate of lake warming and climate velocity for these lakes. Climate velocities were compared with biotic velocities, specifically the rate at which the northernmost extent of each species shifted north.

Results

Lakes in Ontario warmed by 0.2°C decade⁻¹ on average, at a climate velocity of 9.4 km decade⁻¹ between 1986 and 2017. In response, some freshwater fishes have shifted their northern range boundaries with considerable interspecific variation ranging from species moving southwards at a rate of −58.9 km decade⁻¹ to species ranges moving northwards at a rate of 83.6 km decade⁻¹ over the same time period. More freshwater fish species are moving into northern lakes in Ontario than those being lost. Generally, predators are moving their range edges northwards, whereas prey fishes are being lost from northern lakes.

Main Conclusions

The concurrent loss of cooler refugia, combined with antagonistic competitive and predatory interactions with the range expanding species, has resulted in many commercially important predators moving their range edges northwards, whereas prey species have contracted their northern range edge boundaries. Trophic partitioning of range shifts highlights a previously undocumented observation of the loss of freshwater fishes from lower trophic levels in response to climate-driven migrations.     

Pro-haemostatic effect of DDAVP is partially derived through non-classical (CD14dim/CD16++) monocytes residing the spleen

The splenic endothelial Weibel-palade bodies are one of the most important candidate organelles to release von Willebrand factor upon stimulation with desmopressin. However, the presence of functional desmopressin-specific receptor has not yet been demonstrated on endothelial cells. Experimental evidences are in favour of an indirect pro-haemostatic effect of desmopressin, but the exact mediator and its cellular origin are largely elusive. Here, we report partially hampered desmopressin response in a splenectomised severe haemophilia A/Beta Thalassemia patient without any genetic variant relevant to his incomplete desmopressin response. To further investigate the role of the spleen in this phenomenon, the release of VWF from desmopressin-treated human splenic endothelial cells was assessed in vitro. As a result, desmopressin induced the release of VWF from endothelial cells when the cells were co-cultured with non-classical (CD14^dim/CD16⁺⁺), but not other subtypes of monocytes or PBMCs. This in vitro study which resembles close proximity of endothelial cells of sinusoids to monocyte reservoir reside in parenchyma of subcapsular red pulp of the spleen sheds a light upon the role of this highly vascularized VWF-producing organ in driving indirect effect of desmopressin.     

2-Aminothiazole-Oxadiazole Bearing N-Arylated Butanamides: Convergent Synthesis,Tyrosinase Inhibition,Kinetics, Structure-Activity Relationship,and Binding Conformations

A multi-step synthesis of novel bi-heterocyclic N-arylated butanamides was consummated through a convergent strategy and the structures of these medicinal scaffolds, 7a–h , were corroborated using spectral techniques. The in vitro analysis of these hybrid molecules revealed their potent tyrosinase inhibition as compared to the standard used. The kinetics mechanism was investigated through Lineweaver-Burk plots which exposed that, 7f , inhibited tyrosinase enzyme non-competitively by forming the enzyme-inhibitor complex. The inhibition constants K_i calculated from Dixon plots for this compound was 0.025 μM. Their binding conformations were ascertained by in silico computational studies whereby these molecules disclosed good binding energy values (kcal/mol). So, it was anticipated from the current research that these bi-heterocyclic butanamides might be probed as imperative therapeutic agents for melanogenesis.     

The influence of in vitro procedures which diminish NADPH cytochrome c reductase activity on oxidative drug metabolism in lung and liver microsomes

Rabbit lung and liver microsomes were subjected to three procedures which decreased NADPH cytochrome c reductase activity; flavoprotein antibody, trypsin and subtilisin digestion. The effects on benzphetamine and p-nitroanisode demethylation and amine metabolic-intermediate complex formation were investigated. In general, the proteolytic digestion had a greater inhibitory effect on oxidation reactions for a given loss of NADPH cytochrome c reductase activity than did flavoprotein antibody; and of the two proteases, subtilisin, which also diminises the cytochrome b₅ reduction pathway, had a greater inhibitory effect than trypsin. Subtilisin digestion had similar effects in both liver and lung microsomes; a loss of flavoprotein without a loss of cytochrome P-450; but whereas all three oxidative reactions decreased in unison as the flavoprotein was lost in the liver, benzphetamine demethylation was less susceptible to flavoprotein depletion than the other two reactions in lung microsomes. With trypsin digestion flavoprotein was removed without loss of cytochrome P-450 only in lung microsomes; in liver microsomes the cytochrome P-450 was susceptible to tryptic degradation. In lung microsomes, benzphetamine and p-nitroanisole demethylations were less susceptible to flavoprotein loss than metabolic-intermediate complex formation.     

Choriocarcinoma metastatic to the breast diagnosed by fine needle aspiration.

Fine needle aspiration (FNA) was used to study nodules in the left breast of a patient with a previous history of uterine choriocarcinoma. The FNA smears contained numerous malignant mononucleated cells and multinucleated giant cells. The cytologic diagnosis was metastatic choriocarcinoma, which was confirmed by histologic study of excised tissue. That diagnosis would have been difficult to make cytologically if the previous history had not been known; the differential diagnosis of multinucleated giant cells in an aspirate from a breast mass is discussed.     

Forensic STR profiling based smart barcode,a highly efficient and cost effective human identification system

In forensic science, the human identification is the major goal in criminal cases and in paternity dispute, based on DNA profiling of individual. Analysis of short tandem repeat (STRs) markers used as a reliable technique for this purpose. In this study the main objective was to develop a human identification based on DNA fingerprinting using Human Identification Barcode System (HIBS). HIBS may be used to provide a unique molecular signature of human in the form of a barcode. DNA was isolated from blood by using PCR technique to detect bands to design human barcode using self-designed system. HIBS is a web based application that can be accessed via www.hibs.com.pk. HIBS can be accessed with internet access and may be introduced on security checkpoints to identify an individual based on his own DNA instead of conventional procedures of identification. The barcode generated through DNA fingerprinting will be stored in a HIBS, and may also be a part of CNIC. It may be successfully used against suicide bombers, target killers, etc., as even a single blood spot, a few skin cells, root of the hair etc., to identify such culprits. It may also be effectively used to relieve the individuals with false accusations.