Clinical Outcomes of Open-Wedge Varus Derotation Osteotomy in Legg-Calve-Perthes Disease Among 6-12-Year-Old Children.

Background Legg-Calve-Perthes (LCP) disease during early childhood might lead to impaired blood circulation to the femoral head causing osteoarthritis and avascular necrosis of the femoral head. In most cases of children diagnosed after the age of nine years, the progression of LCP disease is associated with poor outcomes causing disability. This study aimed to evaluate the clinical and radiological outcomes of varus derotation osteotomy in children with LCD disease with a late presentation at 6-12 years of age. Methodology This prospective study was performed among 25 6-12-year-old children who were diagnosed with LCD disease. Children were classified per Herring's Lateral Pillar and Catterall classification. They were treated with open-wedge varus derotation osteotomy, and clinical, radiological, and functional parameters were assessed postoperatively. The postoperative grading was done using the Catterall classification. Results In this study, significant improvement in the postoperative mean Harris Hip Score (82.96 ± 4.3 vs. 85.36 ± 3.75; p = 0.000) was observed. In addition, the mean muscle mass reduced postoperatively compared to the preoperative mass (2.64 ± 3.82 vs. 8.52 ± 3.73 mm; p = 0.000). Based on the Catterall classification, 56% of children showed good outcomes, and 36% showed fair outcomes. Regarding complications, 20% had postoperative infections. Conclusions In our study, open-wedge varus derotation osteotomy showed improved clinical and radiological outcomes in children with late presentation of LCD disease.

Author Correction: Computationally designed antibody-drug conjugates self-assembled via affinity ligands.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Computationally designed antibody-drug conjugates self-assembled via affinity ligands.

Antibody-drug conjugates (ADCs) combine the high specificity of antibodies with cytotoxic payloads. However, the present strategies for the synthesis of ADCs either yield unstable or heterogeneous products or involve complex processes. Here, we report a computational approach that leverages molecular docking and molecular dynamics simulations to design ADCs that self-assemble through the non-covalent binding of the antibody to a payload that we designed to act as an affinity ligand for specific conserved amino acid residues in the antibody. This method does not require modifications to the antibody structure and yields homogenous ADCs that form in less than 8 min. We show that two conjugates, which consist of hydrophilic and hydrophobic payloads conjugated to two different antibodies, retain the structure and binding properties of the antibody and its biological specificity, are stable in plasma and improve anti-tumour efficacy in mice with non-small cell lung tumour xenografts. The relative simplicity of the approach may facilitate the production of ADCs for the targeted delivery of cytotoxic payloads.

A safe and efficacious Pt(ii) anticancer prodrug: design, synthesis, in vitro efficacy, the role of carrier ligands and in vivo tumour growth inhibition.

Diaminocyclohexane-Pt(ii)-phenalenyl complexes (1 and 2) showed an appropriate balance between efficacy and toxicity. Compound 2 showed nearly two-fold higher tumour growth inhibition than oxaliplatin in a murine NSCLC tumour model, when a combined drug development approach was used. The fluorescent properties of phenalenone were utilized to understand the mechanistic details of the drug.

Development of a facile antibody-drug conjugate platform for increased stability and homogeneity.

Despite the advances in the design of antibody-drug conjugates (ADCs), the search is still ongoing for novel approaches that lead to increased stability and homogeneity of the ADCs. We report, for the first time, an ADC platform technology using a platinum(ii)-based linker that can re-bridge the inter-chain cysteines in the antibody, post-reduction. The strong platinum-sulfur interaction improves the stability of the ADC when compared with a standard maleimide-linked ADC thereby reducing the linker-drug exchange with albumin significantly. Moreover, due to the precise conserved locations of cysteines, both homogeneity and site-specificity are simultaneously achieved. Additionally, we demonstrate that our ADCs exhibit increased anticancer efficacy in vitro and in vivo. The Pt-based ADCs can emerge as a simple and exciting proposition to address the limitations of the current ADC linker technologies.

The dual inhibitory effect of thiostrepton on FoxM1 and EWS/FLI1 provides a novel therapeutic option for Ewing's sarcoma.

The poor prognosis of Ewing's sarcoma (EWS), together with its high lethal recurrence rate and the side­effects of current treatments, call for novel targeted therapies with greater curative effectiveness and substantially reduced side­effects. The oncogenic chimeric protein EWS/FLI1 is the key malignancy driver in most EWSs, regulating numerous target genes, many of which influence cell cycle progression. It has often been argued that targeting proteins regulated directly or indirectly by EWS/FLI1 may provide improved therapeutic options for EWS. In this context, our study examined FoxM1, a key cell cycle regulating transcription factor, reported to be expressed in EWS and influenced by EWS/FLI1. Thiostrepton, a naturally occurring small molecule, has been shown to selectively inhibit FoxM1 expression in cancer cells. We demonstrate that in EWS, in addition to inhibiting FoxM1 expression, thiostrepton downregulates the expression of EWS/FLI1, both at the mRNA and protein levels, leading to cell cycle arrest and, ultimately, to apoptotic cell death. We also show that thiostrepton treatment reduces the tumorigenicity of EWS cells, significantly delaying the growth of nude mouse xenograft tumors. Results from this study demonstrate a novel action of thiostrepton as inhibitor of the expression of the EWS/FLI1 oncoprotein in vitro and in vivo, and that it shows greater efficacy against EWS than against other tumor types, as it is active on EWS cells and tumors at concentrations lower than those reported to have effective inhibitory activity on tumor cells derived from other cancers. Owing to the dual action of this small molecule, our findings suggest that thiostrepton may be particularly effective as a novel agent for the treatment of EWS patients.

Targeted disruption of glutathione peroxidase 4 in mouse skin epithelial cells impairs postnatal hair follicle morphogenesis that is partially rescued through inhibition of COX-2.

Selenoproteins are essential molecules for the mammalian antioxidant network. We previously demonstrated that targeted loss of all selenoproteins in mouse epidermis disrupted skin and hair development, and caused premature death. In the current study, we targeted specific selenoproteins for epidermal deletion to determine whether similar phenotypes developed. Keratinocyte-specific knockout mice lacking either the glutathione peroxidase 4 (GPx4) or thioredoxin reductase 1 (TR1) gene were generated by cre-lox technology using K14-cre. TR1 knockout mice had a normal phenotype in resting skin, whereas GPx4 loss in the epidermis caused epidermal hyperplasia, dermal inflammatory infiltrate, dysmorphic hair follicles, and alopecia in perinatal mice. Unlike epidermal ablation of all selenoproteins, mice ablated for GPx4 recovered after 5 weeks and had a normal life span. GPx1 and TR1 were upregulated in the skin and keratinocytes of GPx4-knockout mice. GPx4 deletion reduces keratinocyte adhesion in culture and increases lipid peroxidation and cyclooxygenase-2 (COX-2) levels in cultured keratinocytes and whole skin. Feeding a COX-2 inhibitor to nursing mothers partially prevents development of the abnormal skin phenotype in knockout pups. These data link the activity of cutaneous GPx4 to the regulation of COX-2 and hair follicle morphogenesis, and provide insight into the function of individual selenoprotein activity in maintaining cutaneous homeostasis.

Auto-stimulatory action of secreted caveolin-1 on the proliferation of Ewing's sarcoma cells.

Caveolin-1 (CAV1) is highly expressed in Ewing's sarcoma (EWS). We previously showed that increased cellular CAV1 is associated with the regulation of the tumorigenicity, drug resistance and metastatic ability of EWS cells. Because several studies reported that melanoma and prostate cancer cells, which express relatively high CAV1 levels, secrete CAV1, and that secreted CAV1 is associated with tumor progression, our study explored the possibility that EWS cells also secreted CAV1 and that secreted CAV1 may contribute to EWS pathobiology. Results from experiments involving the ectopic expression of a Myc-tagged CAV1 protein in EWS cells as well as the supplementation of culture media with purified CAV1 protein followed by its intracellular localization using immunofluorescence demonstrated that EWS cells secrete CAV1, that they are able to take up the secreted protein, and that extracellular CAV1 enhances EWS cell proliferation. These findings strongly support the notion that secreted CAV1 may also contribute to the malignant properties of EWS.

Selenoproteins are essential for proper keratinocyte function and skin development.

Dietary selenium is known to protect skin against UV-induced damage and cancer and its topical application improves skin surface parameters in humans, while selenium deficiency compromises protective antioxidant enzymes in skin. Furthermore, skin and hair abnormalities in humans and rodents may be caused by selenium deficiency, which are overcome by dietary selenium supplementation. Most important biological functions of selenium are attributed to selenoproteins, proteins containing selenium in the form of the amino acid, selenocysteine (Sec). Sec insertion into proteins depends on Sec tRNA; thus, knocking out the Sec tRNA gene (Trsp) ablates selenoprotein expression. We generated mice with targeted removal of selenoproteins in keratin 14 (K14) expressing cells and their differentiated descendents. The knockout progeny had a runt phenotype, developed skin abnormalities and experienced premature death. Lack of selenoproteins in epidermal cells led to the development of hyperplastic epidermis and aberrant hair follicle morphogenesis, accompanied by progressive alopecia after birth. Further analyses revealed that selenoproteins are essential antioxidants in skin and unveiled their role in keratinocyte growth and viability. This study links severe selenoprotein deficiency to abnormalities in skin and hair and provides genetic evidence for the role of these proteins in keratinocyte function and cutaneous development.

Selenoprotein T deficiency alters cell adhesion and elevates selenoprotein W expression in murine fibroblast cells.

Mammalian selenoproteins have diverse functions, cellular locations, and evolutionary histories, but all use the amino acid selenocysteine (Sec), often present in the enzyme's active site. Only about half of mammalian selenoproteins have been functionally characterized, with most being oxidoreductases. The cellular role of selenoprotein T (SelT), manifesting a CxxU motif in a thioredoxin-like fold and localized to Golgi and the endoplasmic reticulum, is not known. To examine its biological function, we knocked down SelT expression in mouse fibroblast cells and found that SelT deficiency alters cell adhesion and enhances the expression of several oxidoreductase genes, while decreasing the expression of genes involved in cell structure organization, suggesting the involvement of SelT in redox regulation and cell anchorage. Furthermore, we found that the loss of SelT elevates expression of another selenoprotein, selenoprotein W (SepW1). SelT and SepW1 belong to the same protein family, suggesting that SepW1 may functionally compensate for SelT.

Selenoproteins regulate macrophage invasiveness and extracellular matrix-related gene expression.

BACKGROUND: Selenium, a micronutrient whose deficiency in diet causes immune dysfunction and inflammatory disorders, is thought to exert its physiological effects mostly in the form of selenium-containing proteins (selenoproteins). Incorporation of selenium into the amino acid selenocysteine (Sec), and subsequently into selenoproteins is mediated by Sec tRNA([Ser]Sec). RESULTS: To define macrophage-specific selenoprotein functions, we generated mice with the Sec tRNA([Ser]Sec) gene specifically deleted in myeloid cells. These mutant mice were devoid of the "selenoproteome" in macrophages, yet exhibited largely normal inflammatory responses. However, selenoprotein deficiency led to aberrant expression of extracellular matrix-related genes, and diminished migration of macrophages in a protein gel matrix. CONCLUSION: Selenium status may affect immune defense and tissue homeostasis through its effect on selenoprotein expression and the trafficking of tissue macrophages.

A rare presentation in a case of parathyroid carcinoma.

Parathyroid carcinoma is a very rare endocrine malignancy, which usually presents with features of hypercalcaemia and a neck mass. Here a case of parathyroid carcinoma, whose only presenting feature was multiple pathological fractures, without any palpable neck mass is reported. En masse resection of the parathyroid mass along with ipsilateral hemithyroidectomy was performed, while the fractures were treated conservatively. At three years follow-up the patient does not have any recurrence or any evidence of metastasis.

Spindle cell sarcoma of foot with metastasis--a case report.

A 34-year-old male presented with right sided chest pain, shortness of breath and a swelling over his left foot. Chest radiology revealed a mass lesion in the right middle lobe which on CT guided biopsy showed features of spindle cell sarcoma. Surgical excision of both the lung and foot masses were done, both of which on histopathological examination proved to be spindle cell sarcoma.

A functional link between housekeeping selenoproteins and phase II enzymes.

Sec (selenocysteine) is biosynthesized on its tRNA and incorporated into selenium-containing proteins (selenoproteins) as the 21st amino acid residue. Selenoprotein synthesis is dependent on Sec tRNA and the expression of this class of proteins can be modulated by altering Sec tRNA expression. The gene encoding Sec tRNA (Trsp) is a single-copy gene and its targeted removal in liver demonstrated that selenoproteins are essential for proper function wherein their absence leads to necrosis and hepatocellular degeneration. In the present study, we found that the complete loss of selenoproteins in liver was compensated for by an enhanced expression of several phase II response genes and their corresponding gene products. The replacement of selenoprotein synthesis in mice carrying mutant Trsp transgenes, wherein housekeeping, but not stress-related selenoproteins are expressed, led to normal expression of phase II response genes. Thus the present study provides evidence for a functional link between housekeeping selenoproteins and phase II enzymes.

Loss of housekeeping selenoprotein expression in mouse liver modulates lipoprotein metabolism.

Selenium is incorporated into proteins as selenocysteine (Sec), which is dependent on its specific tRNA, designated tRNA([Ser]Sec). Targeted removal of the tRNA([Ser]Sec) gene (Trsp) in mouse hepatocytes previously demonstrated the importance of selenoproteins in liver function. Herein, analysis of plasma proteins in this Trsp knockout mouse revealed increases in apolipoprotein E (ApoE) that was accompanied by elevated plasma cholesterol levels. The expression of genes involved in cholesterol biosynthesis, metabolism and transport were also altered in knockout mice. Additionally, in two transgenic Trsp mutant mouse lines (wherein only housekeeping selenoprotein synthesis was restored), the expression of ApoE, as well as genes involved in cholesterol biosynthesis, metabolism and transport were similar to those observed in wild type mice. These data correlate with reports that selenium deficiency results in increased levels of ApoE, indicating for the first time that housekeeping selenoproteins have a role in regulating lipoprotein biosynthesis and metabolism.

Evolutionary dynamics of eukaryotic selenoproteomes: large selenoproteomes may associate with aquatic life and small with terrestrial life.

BACKGROUND: Selenocysteine (Sec) is a selenium-containing amino acid that is co-translationally inserted into nascent polypeptides by recoding UGA codons. Selenoproteins occur in both eukaryotes and prokaryotes, but the selenoprotein content of organisms (selenoproteome) is highly variable and some organisms do not utilize Sec at all. RESULTS: We analyzed the selenoproteomes of several model eukaryotes and detected 26 and 29 selenoprotein genes in the green algae Ostreococcus tauri and Ostreococcus lucimarinus, respectively, five in the social amoebae Dictyostelium discoideum, three in the fly Drosophila pseudoobscura, and 16 in the diatom Thalassiosira pseudonana, including several new selenoproteins. Distinct selenoprotein patterns were verified by metabolic labeling of O. tauri and D. discoideum with 75Se. More than half of the selenoprotein families were shared by unicellular eukaryotes and mammals, consistent with their ancient origin. Further analyses identified massive, independent selenoprotein losses in land plants, fungi, nematodes, insects and some protists. Comparative analyses of selenoprotein-rich and -deficient organisms revealed that aquatic organisms generally have large selenoproteomes, whereas several groups of terrestrial organisms reduced their selenoproteomes through loss of selenoprotein genes and replacement of Sec with cysteine. CONCLUSION: Our data suggest many selenoproteins originated at the base of the eukaryotic domain and show that the environment plays an important role in selenoproteome evolution. In particular, aquatic organisms apparently retained and sometimes expanded their selenoproteomes, whereas the selenoproteomes of some terrestrial organisms were reduced or completely lost. These findings suggest a hypothesis that, with the exception of vertebrates, aquatic life supports selenium utilization, whereas terrestrial habitats lead to reduced use of this trace element due to an unknown environmental factor.

Selective restoration of the selenoprotein population in a mouse hepatocyte selenoproteinless background with different mutant selenocysteine tRNAs lacking Um34.

Novel mouse models were developed in which the hepatic selenoprotein population was targeted for removal by disrupting the selenocysteine (Sec) tRNA([Ser]Sec) gene (trsp), and selenoprotein expression was then restored by introducing wild type or mutant trsp transgenes. The selenoprotein population was partially replaced in liver with mutant transgenes encoding mutations at either position 34 (34T-->A) or 37 (37A-->G) in tRNA([Ser]Sec). The A34 transgene product lacked the highly modified 5-methoxycarbonylmethyl-2'-O-methyluridine, and its mutant base A was converted to I34. The G37 transgene product lacked the highly modified N(6)-isopentenyladenosine. Both mutant tRNAs lacked the 2'-methylribose at position 34 (Um34), and both supported expression of housekeeping selenoproteins (e.g. thioredoxin reductase 1) in liver but not stress-related proteins (e.g. glutathione peroxidase 1). Thus, Um34 is responsible for synthesis of a select group of selenoproteins rather than the entire selenoprotein population. The ICA anticodon in the A34 mutant tRNA decoded Cys codons, UGU and UGC, as well as the Sec codon, UGA. However, metabolic labeling of A34 transgenic mice with (75)Se revealed that selenoproteins incorporated the label from the A34 mutant tRNA, whereas other proteins did not. These results suggest that the A34 mutant tRNA did not randomly insert Sec in place of Cys, but specifically targeted selected selenoproteins. High copy numbers of A34 transgene, but not G37 transgene, were not tolerated in the absence of wild type trsp, further suggesting insertion of Sec in place of Cys in selenoproteins.

SelT, SelW, SelH, and Rdx12: genomics and molecular insights into the functions of selenoproteins of a novel thioredoxin-like family.

Selenium is an essential trace element in many life forms due to its occurrence as a selenocysteine (Sec) residue in selenoproteins. The majority of mammalian selenoproteins, however, have no known function. Herein, we performed extensive sequence similarity searches to define and characterize a new protein family, designated Rdx, that includes mammalian selenoproteins SelW, SelV, SelT and SelH, bacterial SelW-like proteins and cysteine-containing proteins of unknown function in all three domains of life. An additional member of this family is a mammalian cysteine-containing protein, designated Rdx12, and its fish selenoprotein orthologue. Rdx proteins are proposed to possess a thioredoxin-like fold and a conserved CxxC or CxxU (U is Sec) motif, suggesting a redox function. We cloned and characterized three mammalian members of this family, which showed distinct expression patterns in mouse tissues and different localization patterns in cells transfected with the corresponding GFP fusion proteins. By analogy to thioredoxin, Rdx proteins can use catalytic cysteine (or Sec) to form transient mixed disulfides with substrate proteins. We employed this property to identify cellular targets of Rdx proteins using affinity columns containing mutant versions of these proteins. Rdx12 was found to interact with glutathione peroxidase 1, whereas 14-3-3 protein was identified as one of the targets of mammalian SelW, suggesting a mechanism for redox regulation of the 14-3-3 family of proteins.

Pregnane and Xenobiotic Receptor (PXR/SXR) resides predominantly in the nuclear compartment of the interphase cell and associates with the condensed chromosomes during mitosis.

Pregnane and Xenobiotic Receptor (PXR) is a transcription factor that is activated by a diverse range of xenobiotics and endogenous metabolites including steroids, bile acids and about 50% of the prescription drugs. In specific cell types (e.g. liver and intestine) it serves as a 'xenosensor' by regulating expression of a network of genes involved in xenobiotic clearance from the body. PXR expression in several cancerous tissues and its regulated expression of multi-drug resistance proteins highlight its significance in prognosis of malignancies. The view that subcellular localization and ligand induced movements of transcription factors is one of the major phenomena in regulating transcriptional activity, we used a green fluorescent protein tagged PXR chimera to study its dynamic behaviour in living cells. Under all experimental conditions, PXR was observed to be a predominantly nuclear protein maintaining a dynamic equilibrium between the nuclear and cytoplasmic compartments of the interphase cells. Interestingly, for the first time, a member of the nuclear receptor superfamily, PXR, has been observed to be associated with condensed chromosomes during all the mitotic stages of cell division. The significance of PXR association with mitotic chromosomes is discussed.

Golgi localization and dynamics of hyaluronan binding protein 1 (HABP1/p32/C1QBP) during the cell cycle.

Hyaluronan binding protein 1 (HABP1) is a negatively charged multifunctional mammalian protein with a unique structural fold. Despite the fact that HABP1 possesses mitochondrial localization signal, it has also been localized to other cellular compartments. Using indirect immunofluorescence, we examined the sub-cellular localization of HABP1 and its dynamics during mitosis. We wanted to determine whether it distributes in any distinctive manner after mitotic nuclear envelope disassembly or is dispersed randomly throughout the cell. Our results reveal the golgi localization of HABP1 and demonstrate its complete dispersion throughout the cell during mitosis. This distinctive distribution pattern of HABP1 during mitosis resembles its ligand hyaluronan, suggesting that in concert with each other the two molecules play critical roles in this dynamic process.