Single-dose pharmacokinetic and toxicity analysis of pyrrole–imidazole polyamides in mice

Purpose: Pyrrole–imidazole (Py-Im) polyamides are programmable, sequence-specific DNA minor groove–binding ligands. Previous work in cell culture has shown that various polyamides can be used to modulate the transcriptional programs of oncogenic transcription factors. In this study, two hairpin polyamides with demonstrated activity against androgen receptor signaling in cell culture were administered to mice to characterize their pharmacokinetic properties. Methods: Py-Im polyamides were administered intravenously by tail vein injection. Plasma, urine, and fecal samples were collected over a 24-h period. Liver, kidney, and lung samples were collected postmortem. Concentrations of the administered polyamide in the plasma, excretion, and tissue samples were measured using LC/MS/MS. The biodistribution data were analyzed by both non-compartmental and compartmental pharmacokinetic models. Animal toxicity experiments were also performed by monitoring weight loss after a single subcutaneous (SC) injection of either polyamide. Results: The biodistribution profiles of both compounds exhibited rapid localization to the liver, kidneys, and lungs upon injection. Plasma distribution of the two compounds showed distinct differences in the rate of clearance, the volume of distribution, and the AUCs. These two compounds also have markedly different toxicities after SC injection in mice. Conclusions: The variations in pharmacokinetics and toxicity in vivo stem from a minor chemical modification that is also correlated with differing potency in cell culture. The results obtained in this study could provide a structural basis for further improvement of polyamide activity both in cell culture and in animal models

Case Report: Is Surgical Treatment Beneficial for Intracranial Basal Ganglia Cunninghamellamycosis Following Haematopoietic Stem Cell Transplantation?

Cunninghamellamycosis is an unusual but often highly fatal mucormycosis caused by Cunninghamella bertholletiae, which belongs to the basal lineage order Mucorales. It is especially fatal when the central nervous system is involved. So far, there are few reported cases of surgical treatment for intracranial mucormycosis in children after allogeneic haematopoietic stem cell transplantation (HSCT). The surgical management of deep-seated basal ganglia fungal lesions remains controversial, and its clinical benefits are not yet well established. Herein, we present a rare case of disseminated mucormycosis caused by C. bertholletiae involving the lung and intracranial basal ganglia after homologous leucocytic antigen-matched sibling donor HSCT. The patient was successfully treated for intracranial cunninghamellamycosis with neuroendoscopic surgery and systemic wide-spectrum antifungal treatment and achieved pulmonary recovery without recurrent C. bertholletiae infection or neurologic sequelae. Over the follow-up period of 13 months, there were no adverse events associated with the intracranial surgical debridement, and the patient remained in good health.</jats:p

Application of Next-Generation Sequencing in Infections After Allogeneic Haematopoietic Stem Cell Transplantation: A Retrospective Study

This retrospective study aimed to determine the characteristics of infection and diagnostic efficacy of next-generation sequencing (NGS) in patients with fever after allogeneic hematopoietic stem cell transplantation (allo-HSCT). A total of 71 patients with fever after HSCT were enrolled in this study. Compared with conventional microbiological test (CMT), we found that the sensitivity of NGS versus CMT in peripheral blood samples was 91.2% vs. 41.2%, and that NGS required significantly less time to identify the pathogens in both monomicrobial infections (P=0.0185) and polymicrobial infections (P= 0.0027). The diagnostic performance of NGS was not affected by immunosuppressant use. Viruses are the most common pathogens associated with infections. These results indicated that the sensitivity, timeliness, and clinical significance of NGS are superior for the detection of infections. Although NGS has the advantage of identifying a wide range of potential pathogens, the positive rate is related closely to the sample type. Therefore, we recommend that, in the clinical application of NGS to detect pathogens in patients after allo-HSCT, an appropriate sample type and time should be selected and submitted to improve the positive rate and accuracy of NGS. NGS holds promise as a powerful technology for the diagnosis of fever after HSCT.</jats:p

Mechanisms of Senescence-Related NKG2D Ligands Release and Immune Escape Induced by Chemotherapy in Neuroblastoma Cells

Chemotherapy-induced senescence promotes immunocyte aggregation in the tumor microenvironment by upregulating the surface expression of activating ligands in cancer cells. However, these senescent tumor cells cannot be completely cleared and can induce tumor recurrence. Previous studiesshowed that soluble natural killer (NK) group 2D (NKG2D) ligands impair the recognition of multiple immune cells. In this study, we established an in vitro senescence model using neuroblastoma cells subjected to low-dose Chemotherapeutic drug doxorubicin or the Aurora A inhibitor MLN8237. The results showed that different neuroblastoma cell lines showed increased secretion of the NKG2D ligand MHC class I polypeptide-related sequence A/B (MICA/B) following proteolysis after treatment, with MICA/B subsequently recruited to exosomes to downregulate NKG2D expression in NK cells. Interestingly, disintegrin and metalloproteinase domain-containing 10 (ADAM10) was upregulated in senescent tumor cells, and combined treatment with the ADAM10 inhibitor GI254023X and chemotherapeutic drugs inhibited MICA/B secretion and enhanced recognition and killing by NK cells. Additionally, we found that expression of the long noncoding RNA MALAT1 was significantly increased in senescent neuroblastoma cells, and that MALAT1 served as a sponge for microRNA (miR)-92a-3p to counteract miR-92a-3p-mediated repression of ADAM10 levels. Furthermore, administration of a MALAT1 inhibitor or an miR-92a-3p mimic reduced the MICA/B shedding and enhanced recognition and killing by NK cells. These results confirmed that low-dose chemotherapy induces senescence in neuroblastoma cells, and that senescent tumor cells promote the shedding of the NKG2D ligand MICA/B through the MALAT1/miR-92a/ADAM10 axis, thereby contributing to the formation of a suppressive immune microenvironment and promoting immune escape.</jats:p

G-to-T Transversions and Small Tandem Base Deletions Are the Hallmark of Mutations Induced by Ultraviolet A Radiation in Mammalian Cells^†

Ultraviolet A (UVA) radiation received from the sun and from the widespread use of tanning beds by populations residing in areas of northern latitude represents a potential risk factor for human health. The genotoxic and cancer-causing effects of UVA have remained controversial. A mutagenic role for UVA based on DNA damage formation by reactive oxygen species as well as by generation of photoproducts such as cyclobutane pyrimidine dimers (CPDs) has been suggested. Here, we investigated the mutagenicity of UVA in relation to its DNA damaging effects in transgenic Big Blue mouse embryonic fibroblasts. We determined the formation of a typical oxidative DNA lesion, 8-oxo-7,8-dihydro-2‘-deoxyguanosine (8-oxo-dG), and of CPDs, as well as quantified the induction of mutations in the cII transgene in cells irradiated with a 2000 W UVA lamp. UVA irradiation at a dose of 18 J/cm2 produced significant levels of 8-oxo-dG in DNA (P < 0.03) but did not yield detectable CPDs. UVA irradiation also increased the cII mutant frequency almost 5-fold over background (P < 0.01) while showing moderate cytotoxicity (70% cell viability). UVA-induced mutations were characterized by statistically significant increases in G-to-T transversions and small tandem base deletions (P = 0.0075, P = 0.008, respectively) relative to spontaneously derived mutations. This mutational spectrum differs from those previously reported for UVA in other test systems; however, it corresponds well with the known spectrum of mutations established for oxidative base lesions such as 8-oxo-dG. We conclude that UVA has the potential to trigger carcinogenesis owing to its mutagenic effects mediated through oxidative DNA damage

Prevention of early renal disease, dyslipidaemia and lipid peroxidation in STZ-diabetic rats by LR-9 and LR-74, novel AGE inhibitors

Background: Increased formation of advanced glycation/lipoxidation end-products (AGEs/ALEs) has been implicated in the pathogenesis of various diabetic complications. Several compounds have been developed as inhibitors of AGE/ALE formation. We examined the effects of two new AGE/ALE inhibitors, LR-9 and LR-74, on the development of early renal disease and lipid metabolism in streptozotocin (STZ)-induced diabetic rats. Methods: Diabetic Sprague-Dawley rats were treated with either of the LR compounds for 32 weeks. Progression of renal disease was evaluated by measurements of urinary albumin and plasma creatinine concentrations. AGE/ALE and nitrotyrosine levels in kidneys were determined by immunohistochemistry. AGE-induced chemical modification of the tail tendon collagen and levels of Nε-(carboxymethyl) and (carboxyethyl)- lysines (CML and CEL) in skin collagen were measured. Plasma lipids and their lipid hydroperoxide concentrations were also determined. In vitro, both compounds were tested for inhibiting lipid peroxidation reactions. Results: Treatment of either LR compounds significantly inhibited the increase in albuminuria, creatinaemia, hyperlipidaemia and lipid peroxidation in diabetic rats without any effect on hyperglycaemia. Both compounds also reduced CML-AGE and nitrotyrosine accumulation in kidney glomeruli and tubules, AGE-linked fluorescence and cross-linking of tail collagen, and levels of CML and CEL in skin collagen. In vitro, LR compounds inhibited the oxidation of human low-density lipoprotein (LDL). Conclusion: Both compounds can inhibit the progression of renal disease and also prevent dyslipidaemia in type-1 diabetic animals. These compounds may have an additional beneficial effect as an antioxidant against lipid peroxidation, and thus may provide alternative therapeutic options for the treatment of various diabetic macrovascular complications. Copyright © 2005 John Wiley & Sons, Ltd

Table1_Case report: Genetic analysis of a novel intronic inversion variant in the SPTB gene associated with hereditary spherocytosis.pdf

Background: Hereditary spherocytosis (HS) is a congenital haemolytic anaemia attributed to dysregulation or abnormal quantities of erythrocyte membrane proteins. Currently, the most common erythrocytic gene, spectrin β (SPTB), variants are located in exons and give rise to mRNA defects. However, the genetic characteristics and pathogenic mechanisms of SPTB intronic variants are not completely understood. This study aimed to analyse a rare intronic inversion variant in the SPTB gene associated with HS, and explore the impact of the variant on SPTB mRNA splicing.Method: The clinical manifestations of the patient were summarised and analysed for spherocytosis phenotype diagnosis. The pathogenic variant was identified in the proband using targeted next-generation and Sanger sequencing. RNA sequencing was performed to analyse whether SPTB gene splicing and expression were affected.Results: Targeted next-generation sequencing identified a novel disease-associated intronic inversion variant of the SPTB gene in the proband. The inversion variant was located between intron 19 and 20, and contained the entire exon 20 and partial sequences of adjacent introns. Sanger sequencing confirmed that the intronic inversion variant only appeared in the genome of the proband, not in his parents. RNA sequencing revealed that the variant could result in the skipping of exon 20 and reduced expression of SPTB mRNA.Conclusion: This study identifies a rare intronic inversion variant in the SPTB gene associated with hereditary spherocytosis. The pathogenic variant can lead to exon 20 skipping and decreased SPTB gene expression. This finding has not been previously reported in any literature. This study can expand the intronic variant spectrum of the SPTB gene, deepen our understanding of HS pathogenesis, and contribute to the genetic diagnosis and clinical management of patients.</p