Evaluation of a Continuous Quantification Method of Apoptosis and Necrosis in Tissue Cultures

In tissue-engineering and other life sciences, there is a growing need for real-time, non-destructive information on apoptosis and necrosis in 2D and 3D tissue cultures. Previously, propidium iodide was applied as a fluorescent marker for monitoring necrosis. In the current study this technique was extended with a fluorescent apoptosis marker, YO-PRO-1, to discriminate between both stages of cell death. The main goal was to evaluate the performance of YO-PRO-1 and propidium iodide during monitoring periods of up to 3 days. Apoptosis was induced in C2C12 cultures and the numbers of YP-positive and PI-positive nuclei were counted in time. The performance of the dual staining was evaluated with a metabolic measure and a probe intensity study. Cell metabolism was unaffected during the first 24 h of testing. In conclusion, the YP/PI dual staining method was found to be a powerful tool in obtaining real-time spatial information on viability in cell and tissue culture without culture disruption.     

Epitope specific immunity elicited by a synthetic streptococcal antigen without carrier or adjuvant

A polypeptide fragment of type 24 streptococcal M protein (pep M24) has been shown to raise protective anti-streptococcal antibodies in rabbits and humans when administered with adjuvants. More recently, such protective antibodies were shown to be evoked by a synthesized 35-residue sub-peptide fragment (S-CB7 synthetic cyanogen bromide fragment 7) of pep M24. We now show that the weak pep M24 immunogen induces high titers of long lasting antibodies when associated with murabutide, a synthetic derivative of MDP (NAcMur-L-Ala-D-Glnn-butyl-ester) which is currently undergoing clinical trials. We demonstrate also that the polymerized synthetic S-CB7 administered without adjuvant or carrier evokes a strong epitope specific, protective immune response in mice primed with the parent pep M24. A booster dose of polymerized S-CB7 induced antibodies directed specifically against the S-CB7 structure whereas a booster dose of pep M24 evoked antibodies recognizing additional determinants of the whole pep M24 molecule.     

A supravital staining technique for honey bee spermatozoa

ABSTRACT A new supravital staining technique is described for honey bee, Apis mellifera L., spermatozoa using the fluorochromes, propidium iodide and Hoechst 33342 (H342), a bis-benzimidazole derivative. Propidium iodide binds to the DNA of sperm which lack membrane integrity and H342 binds to the DNA of all sperm. This assay is a simple and rapid method for determining the percentage nonviabiiity of a male honey bee's sperm. The recommended staining procedure is to incubate sperm in a solution of 5 μ.g/ml H342 and 10 μ.g/ml propidium iodide in modified Kiev solution for 15–20 min. After incubation, wet mounts of the sperm-stain suspension are examined using fluorescence microscopy. Percentage nonviabiiity is determined by the ratio of propidium iodide stained sperm to H342 stained sperm.     

A Decade of Progress in Targeted Therapy for Advanced Thyroid Cancer: An Overview

A better understanding of the molecular aberrations prevalent in thyroid cancers had led to significant advances in the management of advanced thyroid cancer. The landscape of thyroid cancer treatment has grown rapidly. Molecular profiling is the key to identify actionable targets for treatment of advanced disease. In the past decade, there have been regulatory approvals of 9 kinase inhibitors or kinase inhibitor combinations. There are now drugs approved for all of the different types of thyroid cancers, including anaplastic thyroid cancer. However, these drugs are not curative and therefore new strategies and treatments continue to be sought.     

Reduction in accumulation of [3H]triphenylmethylphosphonium cation in neuroblastoma cells caused by optical probes of membrane potential: Evidence for interactions between carbocyanine dyes and lipophilic anions

The accumulation of [³H]triphenylmethylphosphonium cation in neuroblastoma N1E 115 cells in the presence of tetraphenylboron is reduced by 3,3′-diethylthiadicarbocyanine iodide and by 3,3′-dipropylthiadicarbocyanine iodide. This reduction in uptake of the lipophilic cation is not due to the carbocyanine dyes depolarizing the plasma membrane of these cells but due to an interaction between the carbocyanine dyes and tetraphenylboron leaving less of the lipophilic anion free in solution to assist uptake of the lipophilic cation. This interaction is shown to have a 1:1 stoicheiometry.     

Structure-function study of cathelicidin-derived bovine antimicrobial peptide BMAP-28: Design of its cell-selective analogs by amino acid substitutions in the heptad repeat sequences

Although BMAP-28 is a potent cathelicidin-derived bovine antimicrobial peptide, its cytotoxic activity against the human and other mammalian cells is of concern for converting it into a novel antimicrobial drug. We have identified a short leucine and isoleucine zipper sequences at the N- and C-terminals of BMAP-28, respectively. To understand the possible role of these structural elements in BMAP-28, a number of alanine-substituted analogs were designed, synthesized and characterized along with the wild-type peptide. The substitution of amino acids at single or multiple ‘a’ position(s) of these structural motifs by alanine showed significant effects on the cytotoxic activity of the molecule on the human red blood cells (hRBCs) and 3T3 cells without showing much effects on their MIC values against the selected bacteria. BMAP-28 and all its analogs depolarized the Escherichia coli cells with almost equal efficacy. In contrast, the alanine-substituted analogs of BMAP-28 depolarized hRBCs much less efficiently than the parent molecule. Results further showed that BMAP-28 assembled appreciably onto the live E. coli and hRBC. However, the selected less toxic analogs of BMAP-28 although assembled as good as the parent molecule onto the live E. coli cells, their assembly onto the live mammalian hRBCs was much weaker as compared to that of the wild-type molecule. Looking at the remarkable similarity with the data presented in our previous work on melittin, it appears that probably the heptad repeat sequence possesses a general role in maintaining the cytotoxicity of the antimicrobial peptides against the mammalian cells and assembly therein.     

Mycobacterium tuberculosis ketol-acid reductoisomerase down-regulation affects its ability to persist,and its survival in macrophages and in mice

Branched-chain amino acids (BCAAs) leucine, isoleucine and valine biosynthetic pathways have been reported from plants, fungi and bacteria including Mycobacterium tuberculosis (Mtb) but are absent in animals. This makes interventions with BCAAs biosynthesis an attractive proposition for antimycobacterial drug discovery. In the present study, Mycobacterium tuberculosis H37Ra (Mtb-Ra) ketol-acid reductoisomerase encoding ORF MRA_3031 was studied to establish its role in Mtb-Ra growth and survival. Recombinant knockdown (KD) and complemented (KDC) strains along with wild-type (WT) Mtb-Ra were studied under in-vitro and ex-vivo conditions. KD was defective for survival inside macrophages and showed time dependent decrease in its colony forming unit (CFU) counts, while, WT and KDC showed time dependent increase in CFUs, after macrophage infection. Also, KD showed reduced ability to form persister cells, had altered membrane permeability against ethidium bromide and nile red dyes, and had reduced biofilm maturation, compared to WT and KDC. The in-vivo studies showed that KD infected mice had lower CFU counts in lungs, compared to WT. In summary Mtb shows survival deficit in macrophages and in mice after ketol-acid reductoisomerase down-regulation.     

Tyrosine nitration of a humanized anti-cocaine mAb differentially affects ligand binding of cocaine and its metabolites

Tetranitromethane was used to selectively modify tyrosine residues of a humanized anti-cocaine mAb (h2E2), under development for the treatment of cocaine use disorders. The effect of mild tyrosine nitration on the affinity of cocaine and two high affinity cocaine metabolites, cocaethylene and benzoylecgonine, was assessed using differential scanning fluorimetry to measure ligand affinities via ligand-induced thermal stabilization of the mAb antigen binding region. Nitrated tyrosine residues were identified by mass spectral analysis of thermolysin peptides. One objective was to understand the binding affinity differences observed for these three ligands, which are not explained by the published crystal structure of the h2E2 mAb Fab fragment co-crystalized with benzoylecgonine, since the carboxylic acid of benzoylecgonine that is esterified to form cocaine and cocaethylene is not in contact with the mAb. Importantly, the binding affinity of the cocaine metabolite benzoylecgonine was not decreased by mild nitration, whereas the binding affinities of cocaine and cocaethylene were decreased about two-fold. These ligands differ only in the substituent attached to the carboxylate moiety of the compound, with benzoylecgonine having an unesterified carboxylate, and cocaine and cocaethylene having methyl and ethyl esters, respectively, at this position. The results are consistent with nitration of light chain tyrosine residue 34, resulting in a less favorable interaction with cocaine and cocaethylene carboxylate esters, while not affecting binding of benzoylecgonine. Thus, light chain Tyr34 residue may have molecular interactions with cocaine and cocaethylene not present for benzoylecgonine, leading to the observed affinity differences for these three ligands.