Biochemical interactions between methotrexate and 1-β-d-arabinofuranosylcytosine in hematopoietic cells of children: a Pediatric Oncology Group study

Summary Children with acute lymphocytic leukemia (ALL) in remission were treated with overlapping sequential infusions of methotrexate (MTX) and 1--d-arabinofuranosylcytosine (araC) as part of continuation therapy. The doses and the sequence were chosen to mimic conditions that produced greater than additive antineoplastic activity with these two drugs in preclinical studies. To assess the potential for the drug combination to exhibit greater than additive effect in vivo, we investigated several biochemical parameters that had been associated with synergism in vitro. Because the patients were in remission, the intracellular parameters could only be measured in cytologically normal hematopoietic cells. We observed that (1) the mean plasma concentrations of MTX and araC were above those required to obtain a greater than additive cytotoxicity with the two drugs in tissue culture; (2) MTX did not have a significant antipurine effect in bone marrow mononuclear cells; (3) the mean intracellular concentration of deoxycytidine triphosphate (dCTP) was significantly lower after treatment with the drug combination than after therapy with araC alone; and (4) the ratio of araC triphosphate (araCTP) to dCTP was 2.6 times higher after treatment with the combination than after araC alone. These results indicate that it is possible to achieve in patients the biochemical conditions associated with the greater than additive antineoplastic activity of MTX and araC in vitro.Abbreviations ALL acute lymphocytic leukemia - araC 1--d-arabinofuranosyluracil - araCTP araC triphosphate - araU 1--d-arabinofuranosyluracil - dNTPs deoxyribonucleoside triphosphates - MTX methotrexate - TCA trichloroacetic acid Supported in part by grants from the National Cancer Institute, National Institutes of Health (CA-38 053; CA-33572, CA-32278, CA-38 859, CA-29 691, and CA-30 969). Preliminary reports on the biochemical data were published by E. M. N., A. M. T., and D. P. inProc Am Assoc Cancer Res 24: 133 (1983) and those on the clinical data, by R. A K., E. M. N., D. P. R. B. R., M. B. H., Y. R., and A. I. F. inProc Am Soc Clin Oncol 3: 201 (1984)     

Expanding allelic and phenotypic spectrum of ZC4H2-related disorder: A novel hypomorphic variant and high prevalence of tethered cord

ZC4H2 (MIM# 300897) is a nuclear factor involved in various cellular processes including proliferation and differentiation of neural stem cells, ventral spinal patterning and osteogenic and myogenic processes. Pathogenic variants in ZC4H2 have been associated with Wieacker-Wolff syndrome (MIM# 314580), an X-linked neurodevelopmental disorder characterized by arthrogryposis, development delay, hypotonia, feeding difficulties, poor growth, skeletal abnormalities, and dysmorphic features. Zebrafish zc4h2 null mutants recapitulated the human phenotype, showed complete loss of vsx2 expression in brain, and exhibited abnormal swimming and balance problems. Here we report 7 new patients (four males and three females) with ZC4H2-related disorder from six unrelated families. Four of the 6 ZC4H2 variants are novel: three missense variants, designated as c.142T>A (p.Tyr48Asn), c.558G>A (p.Met186Ile) and c.602C>T (p.Pro201Leu), and a nonsense variant, c.618C>A (p.Cys206*). Two variants were previously reported : a nonsense variant c.199C>T (p.Arg67*) and a splice site variant (c.225+5G>A). Five patients were on the severe spectrum of clinical findings, two of whom had early death. The male patient harboring the p.Met186Ile variant and the female patient that carries the p.Pro201Leu variant have a relatively mild phenotype. Of note, 4/7 patients had a tethered cord that required a surgical repair. We also demonstrate and discuss previously under-recognized phenotypic features including sleep apnea, arrhythmia, hypoglycemia, and unexpected early death. To study the effect of the missense variants, we performed microinjection of human ZC4H2 wild-type or variant mRNAs into zc4h2 null mutant zebrafish embryos. The p.Met186Ile mRNA variant was able to partially rescue vsx2 expression while p.Tyr48Asn and p.Pro201Leu mRNA variants were not. However, swimming and balance problems could not be rescued by any of these variants. These results suggest that the p.Met186Ile is a hypomorphic allele. Our work expands the genotypes and phenotypes associated with ZC4H2-related disorder and demonstrates that the zebrafish system is a reliable method to determine the pathogenicity of ZC4H2 variants.     

Immunological evidence of an early seroconversion to oncogenic Merkel cell polyomavirus in healthy children and young adults

Oncogenic Merkel cell polyomavirus (MCPyV) provokes a widespread and asymptomatic infection in humans. Herein, sera from healthy children and young adults (HC, n = 344) aged 0–20 years old were evaluated for anti-MCPyV immunoglobulin G (IgG) and IgM antibodies employing a recently developed immunoassay. Serum MCPyV IgG data from healthy subjects (HS, n = 510) and elderlies (ES, n = 226), aged 21–65/66–100 years old, from our previous studies, were included. The anti-MCPyV IgG and IgM rates in HC sera were 40.7% and 29.7%, respectively. A lower prevalence of anti-MCPyV IgGs was found in HC aged 0–5 years old (13%) compared to 6–10 (52.3%), 11–15 (60.5%) and 16–20 years old (61.6%) cohorts. Age-stratified HCs exhibited similar anti-MCPyV IgM rates (27.9%–32.9%). Serological profiles indicated that anti-MCPyV IgGs and IgMs had low optical densities (ODs) during the first years of life, while IgM ODs appeared to decrease throughout young adulthood. A lower anti-MCPyV IgGs rate was found in HC (40.7%) than HS (61.8%) and ES (63.7%). Upon the 5-years range age-stratification, a lower anti-MCPyV IgGs rate was found in the younger HC cohort aged 0–5 years old compared to the remaining older HC/HS/ES cohorts (52.3%–72%). The younger HC cohort exhibited the lowest anti-MCPyV IgG ODs than the older cohorts. Low anti-MCPyV IgMs rates and ODs were found in the 21–25 (17.5%) and 26–30 (7.7%) years old cohorts. Our data indicate that, upon an early-in-life seroconversion, the seropositivity for oncogenic MCPyV peaks in late childhood/young adulthood and remains at high prevalence and relatively stable throughout life.     

Mixed Hyperbranched/Triblock Copolymer Micelle Assemblies: Physicochemical Properties and Potential for Drug Encapsulation

Mixed micelles have numerous advantages while requiring little to no effort in preparation. This study aims to produce mixed micelle nanostructures from a linear triblock copolymer and a hyperbranched random copolymer, and is able to be loaded with the weakly water-soluble drugs curcumin and indomethacin. Different preparation techniques are employed to produce mixed micelles comprised of Pluronic F127 block copolymer, and hyperbranched poly[(ethylene glycol) methyl ether methacrylate-co-lauryl methacrylate], H-[P(OEGMA-co-LMA)], copolymer. Few studies have dabbled in these types of coassemblies, which provides insight into how structural differences of each copolymer can affect the formation of micelles. To determine the properties of the emerging nanostructures in aqueous environments, including their size, homogeneity, and surface charge, different physicochemical techniques are used, such as light scattering and spectroscopic methods. The results reveal that the copolymers combine, and spontaneously self-assemble into mixed micelle-like nanostructures in aqueous environments, whereas both systems of neat and drug-loaded nanostructures exhibit desirable properties such as small average micelle hydrodynamic radii and low size polydispersity indices. The nanostructures that result from the effective encapsulation of curcumin exhibit outstanding stability over 169 days. The fluorescent qualities of curcumin persist after encapsulation, making the novel nanostructures excellent candidates for bioimaging applications.     

Hydrophobic Bombyx mori Silk Fibroin: Routes to Functionalization with Alkyl Chains

Bombyx mori silk fibroin (SF) is a very versatile biopolymer due to its biocompatibility and exceptional mechanical properties which make possible its use as a functional material in several applications. SF can be modified with a large variety of chemical approaches which endow the material with tailored chemical–physical properties. Here, a systematic investigation of different routes is reported to graft long alkyl chains on SF based on both liquid- and solid-phase, aiming to modulate its hydrophobic behavior. The liquid phase method involves direct activation of SF tyrosine residues via diazo coupling and cycloaddition reactions, generating hydrophobic materials insoluble in any common solvent. The solid phase approach consists of the chemical modification of drop-casted SF films by esterification of hydroxyl groups of serine, threonine, and tyrosine SF residues with acyl chlorides of fatty acids. For the solid-state functionalization, a new class of hydrophobic pendant groups is synthesized, based on triple esters of gallic acid anhydrides, that are reacted with the biopolymer to further enhance its resulting hydrophobic features.     

Dyadic reciprocal associations between self-efficacy and planning predict sedentary behaviour

Objectives

There are two alternative hypotheses regarding bidirectional associations between self-efficacy and planning in predicting health behaviour change: self-efficacy may establish planning (cultivation hypothesis) or planning may enable the formation of self-efficacy (enabling hypothesis). This study investigates the order in which these two social cognitions are linked in adult–adult dyads in the context of sedentary behaviours (SB).

Design

A longitudinal study with 4 measurement points, spanning 8 months.

Methods

A total of 320 dyads (age: 18–90 years) were enrolled. Dyads included a focus person (who received the recommendation to reduce SB and intended to change their SB), and their partners, who were willing to support the focus persons and intended to reduce their own SB as well. Data were collected at Time 1 (T1), Time 2 (1 week later, T2), Time 3 (T3, 2 months after T1) and Time 4 (T4, 8 months after T1). SB was measured with accelerometers at (T1 and T4). Mediation models with individual and dyadic reciprocal effects were tested with path analyses.

Results

Only one indirect effect was found: A higher level of partners' SB reduction-specific self-efficacy at T2 was related to the focus person's more frequent planning to reduce SB at T3, which, in turn, predicted lower SB time among partners at T4.

Conclusions

The findings provide partial support for the cultivation model (self-efficacy prompting planning) and for dyadic reciprocal associations in the context of SB time reduction among adult dyads.     

Management of fibrous histiocytoma of the corneoscleral limbus: Report of a case and review of the literature

Background: Fibrous histiocytomas of the corneoscleral limbus are rare tumors. We present an additional case and review the treatment approaches in the literature. So far, only light and electron microscopic studies have been performed. We used immunohistochemical stains to further characterize the cellular composition. Methods: The excised lesion was routinely fixed and processed for light microscopy and immunohistochemical studies. For comparison, three dermal fibrous histiocytomas were also examined and processed similarly. Results: The corneolimbal tumor was mainly composed of fibroblasts and histiocytes with a large amount of interstitial collagen, arranged in a storiform pattern. Immunohistochemical stains were positive for histiocytes, fibroblasts, and a marker for mesenchymal cells. The staining pattern of the dermal lesions was similar. Conclusion: Fibrous histiocytomas of the corneolimbal region are morphologically benign, slowly growing and infiltrative tumors. Complete resection, especially of the deep margin, is suggested. The immunohistochemical staining pattern is similar to that of dermal fibrous histiocytomas, which behave in a benign manner.     

Immunohistochemical detection of p140trkA and p75LNGFR neurotrophin receptors in neuroblastoma

In neuroblastoma, high levels of mRNA for p14h ^trkA and p75 ^LNGFR neurotrophin receptors are predictive of favorable outcome. Their evaluation by Northern blot, however, requires substantial amounts of tissue and this prevents their routine evaluation as well as the possibility for multicenter studies to be easily carried out. In an attempt to overcome these limitations, the feasibility and reliability of determining both neurotrophin receptors on cryostat sections by immunohistochemistry were assessed, and these findings were compared to those obtained from Northern blot analysis. Primary tumor samples from 28 untreated patients at all stages were evaluated by using H10 anti-p140 ^trkA and ME20.4 anti-p75 ^LNGFR mAbs. Although weak, positiveimmunostaining was found in 9 of 28 tumors for p140 ^trkA and in 5 of 28 tumors for p75 ^LNGFR . As compared to Northern blot, the concordance rate was 79% (22 of 28 cases) for p140 ^trkA (p < 0.05) and 71% (20 of 28 cases) for p75 ^LNGFR (p < 0.05). No case negative for Northern blot was found to be positive with immunohistochemistry. Since only high mRNA levels for both receptors have been shown to be clinically relevant, their immunohistochemical detection, although less sensitive than Northern blot, can be just as sufficient and reliable as a prognostic tool, and possibly with a better cost-benefit ratio.     

Molecular approach to the nucleo-melanosomal interaction in human melanoma cells

This paper presents evidence that L-tyrosine oxidation products and 5,6-dihydroxyindole, an intermediate of melanin synthesis bind to and modify DNA structure, as tested by extracting cell DNA, using topoisomerase I and denaturation assays. When supercoiled plasmid pCU18 or pBR322 DNAs are treated with 5,6-dihydroxyindole the supercoiled species disappear and are converted to species less mobile in a gel retardation test with respect to relaxed DNA. 5,6-Dihydroxyindole causes an easier acid denaturation of the double helix. The results, that are dose dependent,would point to both intercalation and cross-linking of DNA by 5,6-dihydroxyindole and its oxidation product(s). ³H-L-tyrosine deriving radioactivity, bound to nuclear DNA, is higher at low pH, (5.6) if compared to pH 6.8. The highest radioactivity bound to cell DNA is found during the transition from the amelanotic to the melanotic phenotype in human melanoma cell lines. As a control, the binding of ³H-L-tyrosine radioactivity to human prostate fibroblast DNA was investigated.