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Research over the past decade has identified several of the key limiting features of multidrug resistance (MDR) in cancer therapy applications, such as evolving glycoprotein receptors at the surface of the cell that limit therapeutic uptake, metabolic changes that lead to protection from multidrug resistant mediators which enhance degradation or efflux of therapeutics, and difficulty ensuring retention of intact and functional drugs once endocytosed. Nanoparticles have been demonstrated to be effective delivery vehicles for a plethora of therapeutic agents, and in the case of nucleic acid based agents, they provide protective advantages. Functionalizing cell penetrating peptides, also known as protein transduction domains, onto the surface of fluorescent quantum dots creates a labeled delivery package to investigate the nuances and difficulties of drug transport in MDR cancer cells for potential future clinical applications of diverse nanoparticle-based therapeutic delivery strategies. In this study, eight distinct cell penetrating peptides were used (CAAKA, HSV1-VP22, HIV-TAT, HIV-gp41, Ku-70, hCT(9-32), integrin-β3, and K-FGF) to examine the different cellular uptake profiles in cancer versus drug resistant melanoma (A375 & A375-R), mesothelioma (MSTO & MSTO-R), and glioma (rat 9L and 9L-R, and human U87 & LN18) cell lines. The results of this study demonstrate that cell penetrating peptide uptake varies with drug resistance status and cell type, likely due to changes in cell surface markers. This study provides insight into developing functional nanoplatform delivery systems in drug resistant cancer models.

Use of tobacco products is injurious to health in men and women. However, tobacco use by pregnant women receives greater scrutiny because it can also compromise the health of future generations. More men smoke cigarettes than women. Yet the impact of nicotine use by men upon their descendants has not been as widely scrutinized. We exposed male C57BL/6 mice to nicotine (200 μg/mL in drinking water) for 12 wk and bred the mice with drug-naïve females to produce the F1 generation. Male and female F1 mice were bred with drug-naïve partners to produce the F2 generation. We analyzed spontaneous locomotor activity, working memory, attention, and reversal learning in male and female F1 and F2 mice. Both male and female F1 mice derived from the nicotine-exposed males showed significant increases in spontaneous locomotor activity and significant deficits in reversal learning. The male F1 mice also showed significant deficits in attention, brain monoamine content, and dopamine receptor mRNA expression. Examination of the F2 generation showed that male F2 mice derived from paternally nicotine-exposed female F1 mice had significant deficits in reversal learning. Analysis of epigenetic changes in the spermatozoa of the nicotine-exposed male founders (F0) showed significant changes in global DNA methylation and DNA methylation at promoter regions of the dopamine D2 receptor gene. Our findings show that nicotine exposure of male mice produces behavioral changes in multiple generations of descendants. Nicotine-induced changes in spermatozoal DNA methylation are a plausible mechanism for the transgenerational transmission of the phenotypes. These findings underscore the need to enlarge the current focus of research and public policy targeting nicotine exposure of pregnant mothers by a more equitable focus on nicotine exposure of the mother and the father., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following potential competing interests. Pradeep Bhide: Dr. Bhide is a co-founder and consultant to Avekshan LLC, Tallahassee, FL, a pharmaceutical enterprise engaged in the development of novel therapies for attention deficit hyperactivity disorder (ADHD). Dr. Bhide is an inventor in following patents or patent applications relevant to ADHD therapy: US Patent, “Class of non-stimulant treatment and ADHD and related disorders” (#US9623023 B2), and US patent application, “Methods and compositions to prevent addiction (#US20130289061 A1). Deirdre McCarthy: Ms. McCarthy is a co-founder and consultant to Avekshan LLC, Tallahassee, FL, a pharmaceutical enterprise engaged in the development of novel therapies for attention deficit hyperactivity disorder (ADHD). Thomas Spencer: Dr. Spencer received research support or was a consultant from the following sources: Alcobra, Avekshan, Ironshore, Lundbeck, Shire Laboratories Inc, Sunovion, the FDA, and the Department of Defense. Consultant fees are paid to the Clinical Trials Network at the Massachusetts General Hospital (MGH) and not directly to Dr. Spencer. Dr. Spencer has been on an advisory board for the following pharmaceutical companies: Alcobra. Dr. Spencer received research support from Royalties and Licensing fees on copyrighted ADHD scales through MGH Corporate Sponsored Research and Licensing. Through MGH corporate licensing, Dr. Spencer is an inventor on a US Patent, “Class of non-stimulant treatment and ADHD and related disorders” (#US9623023 B2), and US patent application, “Methods and compositions to prevent addiction" (#US20130289061 A1). Joseph Biederman: Dr. Biederman is currently receiving research support from the following sources: AACAP, The Department of Defense, Food & Drug Administration, Headspace, Lundbeck, Neurocentria Inc., NIDA, PamLab, Pfizer, Shire Pharmaceuticals Inc., Sunovion, and NIH. Dr.  Biederman has a financial interest in Avekshan LLC, a company that develops treatments for attention deficit hyperactivity disorder (ADHD). His interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. Dr. Biederman’s program has received departmental royalties from a copyrighted rating scale used for ADHD diagnoses, paid by Ingenix, Prophase, Shire, Bracket Global, Sunovion, and Theravance; these royalties were paid to the Department of Psychiatry at MGH.  In 2017, Dr. Biederman is a consultant for Aevi Genomics, Akili, Guidepoint, Ironshore, Medgenics, and Piper Jaffray. He is on the scientific advisory board for Alcobra and Shire. He received honoraria from the MGH Psychiatry Academy for tuition-funded CME courses. Through MGH corporate licensing, he is an inventor on US Patent, “Class of non-stimulant treatment and ADHD and related disorders” (#US9623023 B2), and US patent application, “Methods and compositions to prevent addiction (#US20130289061 A1). In 2016, Dr. Biederman received honoraria from the MGH Psychiatry Academy for tuition-funded CME courses, and from Alcobra and APSARD. He was on the scientific advisory board for Arbor Pharmaceuticals. He was a consultant for Akili and Medgenics. He received research support from Merck and SPRITES. Thomas Morgan, Sara Lowe, and Matthew Williamson have no competing interests to declare.

Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent increases in the pro-apoptotic mitochondrial protein BAX leading to a loss of mitochondrial membrane potential as demonstrated by a 25% decrease in JC-1 red:green fluorescence ratio. Disruption of mitochondrial membrane integrity was accompanied by efflux of the apoptosis inducing factor (AIF) from the mitochondria and translocation to the nucleus with a significant increase in reactive oxygen species (ROS) after 24h of zinc deficiency. Measurement of caspase cleavage, mRNA, and treatment with caspase inhibitors revealed the involvement of caspases 2, 3, 6, and 7 in zinc deficiency-mediated apoptosis. Down-stream targets of caspase activation, including the nuclear structure protein lamin and polyADP ribose polymerase (PARP), which participates in DNA repair, were also cleaved. Transfection with a dominant-negative p53 construct and use of the p53 inhibitor, pifithrin-μ, established that these alterations were largely dependent on p53. Together these data identify a cascade of events involving mitochondrial p53 as well as p53-dependent caspase-mediated mechanisms leading to apoptosis during zinc deficiency., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Mesenchymal stem cells (MSC) have been identified as having great potential as autologous cell therapeutics to treat traumatic brain injury and spinal injury as well as neuronal and cardiac ischemic events. All future clinical applications of MSC cell therapies must allow the MSC to be harvested, transfected, and induced to express a desired protein or selection of proteins to have medical benefit. For the full potential of MSC cell therapy to be realized, it is desirable to systematically alter the protein expression of therapeutically beneficial biomolecules in harvested MSC cells with high fidelity in a single transfection event. We have developed a delivery platform on the basis of the use of a solid gold nanoparticle that has been surface modified to produce a fusion containing a zwitterionic, pentapeptide designed from Bax inhibiting peptide (Ku70) to enhance cellular uptake and a linearized expression vector to induce enhanced expression of brain-derived neurotrophic factor (BDNF) in rat-derived MSCs. Ku70 is observed to effect >80% transfection following a single treatment of femur bone marrow isolated rat MSCs with efficiencies for the delivery of a 6.6 kbp gene on either a Au nanoparticle (NP) or CdSe/ZnS quantum dot (QD). Gene expression is observed within 4 d by optical measurements, and secretion is observed within 10 d by Western Blot analysis. The combination of being able to selectively engineer the NP, to colocalize biological agents, and to enhance the stability of those agents has provided the strong impetus to utilize this novel class of materials to engineer primary MSCs.

Objectives: Proliferating adult stem cells in the subgranular zone of the dentate gyrus have the capacity not only to divide, but also to differentiate into neurons and integrate into the hippocampal circuitry. The present study identifies several hippocampal genes putatively regulated by zinc and tests the hypothesis that zinc deficiency impairs neuronal stem cell differentiation., Methods: Genes that regulate neurogenic processes were identified using microarray analysis of hippocampal mRNA isolated from adult rats fed zinc-adequate or zinc-deficient (ZD) diets. We directly tested our hypothesis with cultured human neuronal precursor cells (NT2), stimulated to differentiate into post-mitotic neurons by retinoic acid (RA), along with immunocytochemistry and western analysis., Results: Microarray analysis revealed the regulation of genes involved in cellular proliferation. This analysis also identified a number of genes known to be involved in neuronal differentiation, including the nuclear RA receptor, retinoid X receptor (RXR), doublecortin, and a transforming growth factor-beta (TGF-β) binding protein (P < 0.05). Zinc deficiency significantly reduced RA-induced expression of the neuronal marker proteins doublecortin and β-tubulin type III (TuJ1) to 40% of control levels (P < 0.01). This impairment of differentiation may be partially mediated by alterations in TGF-β signaling. The TGF-β type II receptor, responsible for binding TGF-β during neuronal differentiation, was increased 14-fold in NT2 cells treated with RA (P < 0.001). However, this increase was decreased by 60% in ZD RA-treated cells (P < 0.001)., Discussion: This research identifies target genes that are involved in governing neurogenesis under ZD conditions and suggests an important role for TGF-β and the trace metal zinc in regulating neuronal differentiation.

A fluorescent heteroditopic indicator for the zinc(II) ion possesses two different zinc(II) binding sites. The sequential coordination of zinc(II) at the two sites can be transmitted into distinct fluorescence changes. In the heteroditopic ligand system that our group developed, the formations of mono- and dizinc(II) complexes along an increasing gradient of zinc(II) concentration lead to fluorescence enhancement and an emission bathochromic shift, respectively. The extents of these two changes determine the sensitivity and, ultimately, the effectiveness of the heteroditopic indicator in quantifying zinc(II) ion over a large concentration range. In this work, a strategy to increase the degree of fluorescence enhancement upon the formation of the monozinc(II) complex of a heteroditopic ligand under simulated physiological conditions is demonstrated. Fluorination of the pyridyl groups in the pentadentate N,N,N'-tris(pyridylmethyl)ethyleneamino group reduces the apparent pK(a) value of the high-affinity site, which increases the degree of fluorescence enhancement as the monozinc(II) complex is forming. However, fluorination impairs the coordination strength of the high-affinity zinc(II) binding site, which in the triply fluorinated ligand reduces the binding strength to the level of the low-affinity 2,2'-bipyridyl. The potential of the reported ligands in imaging zinc(II) ion in living cells was evaluated. The subcellular localization properties of two ligands in five organelles were characterized. Both benefits and deficiencies of these ligands were revealed, which provides directions for the near future in this line of research.

Chicken lines were classified into six distinct groups based on expression of B-G molecules by peripheral red blood cells (RBC). In addition to the previously reported 48 kD subunits of homodimeric B-G molecules, subunits of 60, 40, and possibly 20 kD were detected in certain of the chicken lines. Several of the chicken lines express the previously reported 40 and 44 kD subunits of heterodimeric B-G molecules; however, B21B21 chickens expressed 44 kD subunits only and B5B5 and B13B13 chickens did not express detectable levels of any heterodimeric-related molecules. These studies provide further evidence for the polymorphic nature of the B-G antigens.

Monoclonal antibody R7-3 recognized an erythroid specific cell surface molecule with a m.w. of approximately 98 kilodaltons (Kd) under nonreducing conditions and molecules of 40 and 44 Kd under reducing conditions on both embryonic- and adult-derived peripheral RBC. Immunochemical characterization, including limited peptide map analyses of these molecules, provided evidence that mAb R7-3 was recognizing the MHC coded B-G heterodimer. This is the first report of a monoclonal antibody that recognizes the B-G heterodimer. Affinity binding studies suggested that mAb R7-3 preferentially recognized the 44 Kd molecule. Immune depletion analyses demonstrated the presence of a single population of B-G heterodimers. Endoglycosidase-F and neuraminidase digestions suggested that the 44 and 40 Kd molecules contained very little, if any, carbohydrate. B-G heterodimer expression was examined on primitive and definitive RBC during embryonic development. B-G heterodimer expression was not detected on RBC of other avians.

Antiserum prepared against sucrose gradient purified reticuloendotheliosis virus (REV) recognized the chicken transferrin receptor. Molecules immunoprecipitated from red blood cells (RBC) obtained from embryonic chickens with either the anti-REV reagent or a chicken transferrin immunomatrix were demonstrated to be identical by co-migration in both reducing and nonreducing SDS-polyacrylamide gels and in two-dimensional isoelectric focusing analyses, reciprocal immunodepletion analyses and by peptide mapping. The chicken transferrin receptor was shown to be a 190,000 dalton cell surface membrane molecule consisting of two similar disulfide-bonded subunits of approximately 95,000 daltons. The chicken transferrin receptor was expressed on erythroid cell surface membranes as 95,000 dalton monomers as well as 190,000 dalton dimers. The chicken transferrin receptor was expressed on all differentiation/maturation stages, including mature RBC, of both the primitive and definitive type I erythroid cell series. In adult chickens, the transferrin receptor was expressed by immature erythroid cells in the bone marrow, but not by mature circulating RBC. REV-transformed immature lymphoid cells and avian erythroblastosis virus (AEV)-transformed erythroid cells expressed dimers composed of 95,000 and 110,000 dalton subunits. Comparisons among V8 protease derived peptides from 95,000 dalton transferrin receptors obtained from RBC and REV-transformed lymphoid cells revealed a high degree of homology; however, the 95,000 dalton molecules isolated from REV-transformed lymphoid cells exhibited a 56,000 dalton peptide that was unique. Cloned AEV-transformed erythroleukemia cells induced to differentiate by supplementation of the media with 1 mM butyric acid expressed elevated transferrin receptor levels. Both serological and peptide mapping studies demonstrated the human transferrin receptor on K562 cells and the chicken transferrin receptor to be distinct. However, chicken transferrin was shown to be capable of reacting with the human transferrin receptors on K562 cells.