Targeting oncogenic interleukin‐7 receptor signalling with N‐acetylcysteine in T cell acute lymphoblastic leukaemia

Activating mutations of the interleukin‐7 receptor (IL7R) occur in approximately 10% of patients with T cell acute lymphoblastic leukaemia (T‐ALL). Most mutations generate a cysteine at the transmembrane domain leading to receptor homodimerization through disulfide bond formation and ligand‐independent activation of STAT5. We hypothesized that the reducing agent N‐acetylcysteine (NAC), a well‐tolerated drug used widely in clinical practice to treat acetaminophen overdose, would reduce disulfide bond formation, and inhibit mutant IL7R‐mediated oncogenic signalling. We found that treatment with NAC disrupted IL7R homodimerization in IL7R‐mutant DND‐41 cells as assessed by non‐reducing Western blot, as well as in a luciferase complementation assay. NAC led to STAT5 dephosphorylation and cell apoptosis at clinically achievable concentrations in DND‐41 cells, and Ba/F3 cells transformed by an IL7R‐mutant construct containing a cysteine insertion. The apoptotic effects of NAC could be rescued in part by a constitutively active allele of STAT5. Despite using doses lower than those tolerated in humans, NAC treatment significantly inhibited the progression of human DND‐41 cells engrafted in immunodeficient mice. Thus, targeting leukaemogenic IL7R homodimerization with NAC offers a potentially effective and feasible therapeutic strategy that warrants testing in patients with T‐ALL.     

海绵质骨螺旋钉拔出试验之生物力学分析

评估螺旋钉设计优劣的简便方法之一为拔出试验。虽然比较用之海绵质骨试件可取自相同部位，自同一方向植入螺旋钉，并具有相同基准。然而因各处海绵质骨骼结构不尽相同，至今尚未有人探讨如使用不同之海绵质骨结构，例如近似等向性之杆状结构，或是板与杆组合之有强列方向排列者，对于螺旋钉拔出之最大力量有多大影响？采用牛股骨远端部位及牛颈椎Ｃ２～Ｃ６作为不同结构特性之试体。并各由两相互垂直之方向旋入螺旋钉，来做拔出试验。最后并建立一三维有限元素模型，用以研究螺纹周围之变形模式及其预测拔出最大力量的准确性。模型分析中以应变能密度准则作为破坏之参考标准。     

Hard superconducting nitrides

Detailed study of the equation of state, elasticity, and hardness of selected superconducting transition-metal nitrides reveals interesting correlations among their physical properties. Both the bulk modulus and Vickers hardness are found to decrease with increasing zero-pressure volume in NbN, HfN, and ZrN. The computed elastic constants from first principles satisfy c11 > c12 > c44 for NbN, but c11 > c44 > c12 for HfN and ZrN, which are in good agreement with the neutron scattering data. The cubic delta-NbN superconducting phase possesses a bulk modulus of 348 GPa, comparable to that of cubic boron nitride, and a Vickers hardness of 20 GPa, which is close to sapphire. Theoretical calculations for NbN show that all elastic moduli increase monotonically with increasing pressure. These results suggest technological applications of such materials in extreme environments.     

Fine-needle aspiration of cystic nephroma (multilocular cyst of the kidney).

Cystic nephromas are rare tumors of the kidney most commonly affecting boys or adult females. The fine-needle aspiration cytomorphology has not yet been described. A renal cystic mass in a 56 year old female was aspirated under ultrasound guidance. Papanicolaou stained smears of the cyst fluid revealed markedly atypical cells forming papillary clusters. Subsequent nephrectomy showed a typical cystic nephroma with lining epithelium resembling that seen in the aspirate. The cytomorphology of cystic nephroma has been misdiagnosed as renal cell carcinoma in the literature. Low cellularity, absence of necrosis, and paucity of single cells are features that should raise the possibility of cystic nephroma in a cystic renal mass.     

HIPDM single photon emission computed tomography brain imaging in partial onset secondarily generalized tonic-clonic seizures

HIPDM-Single photon emission computed tomography brain imaging was performed during interictal and ictal stages in three patients with complex partial seizures and secondarily generalized tonic-clonic seizures. In all three patients, interictal studies demonstrated decreased regional cerebral perfusion (rCP) and ictal studies showed increased rCP in the epileptogenic region. The demonstration of focal hyperperfusion by SPECT performed during secondarily generalized tonic-clonic seizures suggests that rCP in the epileptic focus remains higher than in other cerebral regions during immediate postictal stages, even in secondarily generalized seizures.     

Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus

We sought to determine whether intramuscular injection of a recombinant adeno-associated virus (rAAV) vector expressing human factor IX (hF.IX) could direct expression of therapeutic levels of the transgene in experimental animals. High titer (10¹²–10¹³ vector genomes/ml) rAAV expressing hF.IX was prepared, purified, and injected into hindlimb muscles of C57BL/6 mice and Rag 1 mice. In the immunocompetent C57BL/6 mice, immunofluorescence staining of muscle harvested 3 months after injection demonstrated the presence of hF.IX protein, and PCR analysis of muscle DNA was positive for AAV DNA, but no hF.IX was detected in mouse plasma. Further studies showed that these mice had developed circulating antibodies to hF.IX. In follow-up experiments in Rag 1 mice, which carry a mutation in the recombinase activating gene-1 and thus lack functional B and T cells, similar results were seen on DNA analysis of muscle, but these mice also demonstrated therapeutic levels (200–350 ng/ml) of F.IX in the plasma. The time course of F.IX expression demonstrates that levels gradually increase over a period of several weeks before reaching a plateau that is stable 6 months after injection. In other experiments we demonstrate colocalization of hF.IX and collagen IV in intersitial spaces between muscle fibers. Collagen IV has recently been identified as a F.IX-binding protein; this finding explains the unusual pattern of immunofluorescent staining for F.IX shown in these experiments. Thus rAAV can be used to direct stable expression of therapeutic levels of F.IX after intramuscular injection and is a feasible strategy for treatment of patients with hemophilia B.     

Nicotinic Acid Metabolism, V. A Cobamide Coenzyme-Dependent Conversion of α-Methyleneglutaric Acid to Dimethylmaleic Acid

A new B(12)-coenzyme-dependent isomerization, catalyzed by extracts of a nicotinate-fermenting clostridium, results in the conversion of alpha-methyleneglutaric acid to dimethylmaleic acid. These two acids are intermediates in the multistep anaerobic process wherein nicotinate is converted, ultimately, to one mole each of propionate, acetate, carbon dioxide, and ammonia.Dimethylmaleic acid reacts in its anhydride form with 2,4-dinitrophenylhydrazine to form N-2',4'-dinitrophenyl-anilino-3,4-dimethylmaleimide. The characteristic reddish color exhibited by the latter derivative in alkaline solution serves as a convenient quantitative assay for dimethylmaleic acid. Comparison of the 2,4-dinitrophenylhydrazine derivatives of the product of the enzymic reaction and of synthetic dimethylmaleic anhydride showed them to be identical in every respect.     

Incongruity of Imaging Using Fluorescent 2-DG Conjugates Compared to 18F-FDG in Preclinical Cancer Models

Purpose

We compared the use of near-infrared conjugates of 2-deoxyglucose (NIR 2-DG) to 2-deoxy-2-[¹⁸F]fluoro-d-glucose (¹⁸F-FDG) for the purposes of imaging tumors, as well as response to therapy.

Procedures

Uptake of both ¹⁸F-FDG and NIR 2-DG within gastrointestinal stromal tumor xenografts were imaged before and after nilotinib treatment. Confocal microscopy was performed to determine NIR 2-DG distribution in tumors.

Results

Treatment with nilotinib resulted in a rapid reduction in ¹⁸F-FDG uptake and reduced tumor cell viability which was predictive of long-term antitumor efficacy. In contrast, optical imaging with NIR 2-DG probes was unable to differentiate control from niltonib-treated animals, and microscopic analysis revealed no change in probe distribution as a result of treatment.

Conclusions

These results suggest that conjugation of large bulky fluorophores to 2-DG disrupts the facilitated transport and retention of these probes in cells. Therefore, optical imaging of NIR 2-DG probes cannot substitute for ¹⁸F-FDG positron emission tomography imaging as a biomarker of tumor cell viability and metabolism.     

6]-gingerol induces Ca2+ mobilization in Madin-Darby canine kidney cells

[6]-gingerol, a major phenolic compound derived from ginger (Zingiber officinale), is a potential chemopreventive compound that can induce stress in cancer cells and cause apoptotic cell death. This study examines the early signaling effects of [6]-gingerol on renal cells. It was found that [6]-gingerol caused a slow and sustained rise of [Ca2+]i in a concentration-dependent manner. [6]-gingerol also induced a [Ca2+]i rise when extracellular Ca2+ was removed, but the magnitude was reduced by 80%. Depletion of intracellular Ca2+ stores with CCCP, a mitochondrial uncoupler, did not affect the action of [6]-gingerol. In a Ca2+-free medium, the [6]-gingerol-induced [Ca2+]i rise was partially abolished by depleting stored Ca2+ with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). The elevation of [6]-gingerol-caused [Ca2+]i in a Ca2+-containing medium was not affected by modulation of protein kinase C activity. The [6]-gingerol-induced Ca2+ influx was blocked by nicardipine. U73122, an inhibitor of phospholipase C, abolished ATP (but not [6]-gingerol)-induced [Ca2+]i rise. These findings suggest that [6]-gingerol induces a significant rise in [Ca2+]i in MDCK renal tubular cells by stimulating both extracellular Ca2+ influx and thapsigargin-sensitive intracellular Ca2+ release via as yet unidentified mechanisms.