Towards the biodegradation pathway of fosfomycin.

Three functionalised propylphosphonic acids were synthesised to study C-P bond cleavage in R. huakuii PMY1. (R)-1-Hydroxy-2-oxopropylphosphonic acid [(R)-5] was prepared by chiral resolution of (±)-dimethyl 1-hydroxy-2-methylallyllphosphonate [(±)-12], followed by ozonolysis and deprotection. The N-(l-alanyl)-substituted (1R,2R)-2-amino-1-hydroxypropylphosphonic acid 10, a potential precursor for 2-oxopropylphosphonic acid (5) in cells, was obtained by coupling the aminophosphonic acid with benzotriazole-activated Z-l-alanine and hydrogenolytic deprotection. (1R*,2R*)-1,2-Dihydroxy-3,3,3-trifluoropropylphosphonic acid, a potential inhibitor of C-P bond cleavage after conversion into its 2-oxo derivative in the cell, was accessed from trifluoroacetaldehyde hydrate via hydroxypropanenitrile 21, which was silylated and reduced to the aldehyde (±)-23. Diastereoselective addition of diethyl trimethylsilyl phosphite furnished diastereomeric α-siloxyphosphonates. The less polar one was converted to the desired racemic phosphonic acid (±)-(1R*,2R*)-9 as its ammonium salt.

Improved reaction conditions for the synthesis of new NKP-1339 derivatives and preliminary investigations on their anticancer potential.

The very promising results of Na-trans-[RuCl4(1H-indazole)2] (NKP-1339) in clinical studies have fuelled renewed interest in the research and development of ruthenium(III) coordination compounds for cancer therapy. By applying an improved synthetic approach to this class of coordination compounds, six new examples of the general formula (cation)-trans-[RuCl4(azole)2], where (cation) = tetrabutylammonium (Bu4N)(+) (1, 2), sodium (3, 4), azolium (5, 6), and azole = 1-methyl-indazole (1, 3, 5), 1-ethyl-indazole (2, 4, 6), have been prepared. All compounds have been characterized by elemental analysis, electrospray ionization (ESI) mass spectrometry, UV-vis-, and NMR-spectroscopy and, if possible, X-ray diffraction analysis. Furthermore, the influence of the alkyl substituent at the position N1 of the indazole backbone on the stability in aqueous media as well as on the biological activity in three human cancer cell lines (CH1, A549, and SW480) and on the cellular accumulation in SW480 cells is discussed.

Monitoring of residual disease by next-generation deep-sequencing of RUNX1 mutations can identify acute myeloid leukemia patients with resistant disease.

We studied the utility and clinical relevance of RUNX1 (runt-related transcription factor 1) mutations and their application as residual disease detection markers using next-generation deep-sequencing. Mutation screening was prospectively performed in 814 acute myeloid leukemia patients. At diagnosis, 211/814 (25.9%) patients harbored mutations with a median clone size of 39% (range: 2-96%). Furthermore, in 57 patients paired samples from diagnosis and relapse were analyzed. In 47/57 (82.5%) cases the same alterations detected at diagnosis were present at relapse, whereas in 1/57 (1.8%) cases the mutation from the diagnostic sample was no longer detectable. Discrepancies were observed in 9/57 (15.8%) cases, also including the occurrence of novel RUNX1 mutations not restricted to those regions affected at diagnosis. Moreover, in 103 patients the prognostic impact of residual levels of RUNX1 mutations during complete remission was studied. Separation of patients according to median residual mutation burden into 'good responders' and 'poor responders' (median: 3.61%; range: 0.03-48.0%) resulted in significant differences of both event-free (median 21.0 vs. 5.7 months, P<0.001) and overall survival (OS; median 56.9 vs. 32.0 months, P=0.002). In conclusion, deep-sequencing revealed that RUNX1 mutations qualify as patient-specific markers for individualized disease monitoring. The measurement of mutation load may refine the assignment into distinct risk categories and treatment strategies.

SF3B1 mutations correlated to cytogenetics and mutations in NOTCH1, FBXW7, MYD88, XPO1 and TP53 in 1160 untreated CLL patients.

We analyzed a large cohort of 1160 untreated CLL patients for novel genetic markers (SF3B1, NOTCH1, FBXW7, MYD88, XPO1) in the context of molecular, immunophenotypic and cytogenetic data. NOTCH1 mutations (mut) (12.3%), SF3B1mut (9.0%) and TP53mut (7.1%) were more frequent than XPO1mut (3.4%), FBXW7mut (2.5%) and MYD88mut (1.5%). SF3B1mut, NOTCH1mut, TP53mut and XPO1mut were highly correlated to unmutated, whereas MYD88mut were associated with mutated IGHV status. Associations of diverse cytogenetic aberrations and mutations emerged: (1) SF3B1mut with del(11q), (2) NOTCH1mut and FBXW7mut with trisomy 12 and nearly exclusiveness of SF3B1mut, (3) MYD88mut with del(13q) sole and low frequencies of SF3B1mut, NOTCH1mut and FBXW7mut. In patients with normal karyotype only SF3B1mut were frequent, whereas NOTCH1mut rarely occurred. An adverse prognostic impact on time to treatment (TTT) and overall survival (OS) was observed for SF3B1mut, NOTCH1mut and TP53 disruption. In multivariate analyses SF3B1mut, IGHV mutational status and del(11q) were the only independent genetic markers for TTT, whereas for OS SF3B1mut, IGHV mutational status and TP53 disruption presented with significant impact. Finally, our data suggest that analysis of gene mutations refines the risk stratification of cytogenetic prognostic subgroups and confirms data of a recently proposed model integrating molecular and cytogenetic data.

Landscape of genetic lesions in 944 patients with myelodysplastic syndromes.

High-throughput DNA sequencing significantly contributed to diagnosis and prognostication in patients with myelodysplastic syndromes (MDS). We determined the biological and prognostic significance of genetic aberrations in MDS. In total, 944 patients with various MDS subtypes were screened for known/putative mutations/deletions in 104 genes using targeted deep sequencing and array-based genomic hybridization. In total, 845/944 patients (89.5%) harbored at least one mutation (median, 3 per patient; range, 0-12). Forty-seven genes were significantly mutated with TET2, SF3B1, ASXL1, SRSF2, DNMT3A, and RUNX1 mutated in >10% of cases. Many mutations were associated with higher risk groups and/or blast elevation. Survival was investigated in 875 patients. By univariate analysis, 25/48 genes (resulting from 47 genes tested significantly plus PRPF8) affected survival (P<0.05). The status of 14 genes combined with conventional factors revealed a novel prognostic model ('Model-1') separating patients into four risk groups ('low', 'intermediate', 'high', 'very high risk') with 3-year survival of 95.2, 69.3, 32.8, and 5.3% (P<0.001). Subsequently, a 'gene-only model' ('Model-2') was constructed based on 14 genes also yielding four significant risk groups (P<0.001). Both models were reproducible in the validation cohort (n=175 patients; P<0.001 each). Thus, large-scale genetic and molecular profiling of multiple target genes is invaluable for subclassification and prognostication in MDS patients.

Combination therapy for neovascular age-related macular degeneration refractory to anti-vascular endothelial growth factor agents.

OBJECTIVE: To examine the outcomes of combination anti-vascular endothelial growth factor (VEGF) and photodynamic therapy (PDT) for the treatment of neovascular age-related macular degeneration (AMD) refractory to anti-VEGF monotherapy. DESIGN: Retrospective, interventional case series. PARTICIPANTS: Twenty-six eyes of 26 patients treated with anti-VEGF monotherapy for neovascular AMD with persistent subretinal or intraretinal fluid after at least 3 anti-VEGF injections in the 7 months before combination treatment. INTERVENTION: Combination anti-VEGF treatment and PDT. MAIN OUTCOME MEASURES: Visual acuity at 1 or 2, 3, and 6 months and central retinal thickness at 1 or 2, 3, and 6 months. Secondary outcome measures were change in number of fluid-free visits and interval between treatments in the 7 months before and 6 months after combination therapy. RESULTS: Statistically significant improvements in logarithm of the minimum angle of resolution visual acuities were present at 1 month (P = 0.01) and 3 months (P = 0.01). Significant decreases in central subfield retinal thickness on optic coherence tomography (OCT) were seen at 1 month (P = 4×10(-5)), 3 months (P = 3×10(-4)), and 6 months (P = 4×10(-5)) as compared with precombination treatment OCT scans. The percentage of patient visits with no subretinal fluid increased from 0.5% to 41% after the initiation of combination therapy (P = 1×10(-5)). The interval between treatments increased from once every 1.6 months in the 7 months before combination treatment to once every 2.7 months in the 6 months after combination treatment (P = 0.002). No ocular complications attributable to PDT were seen. CONCLUSIONS: Rescue therapy with the combination of anti-VEGF and PDT in eyes that have failed anti-VEGF monotherapy resulted in a mean improvement in vision, a decreased central subfield retinal thickness, and an increase in fluid-free intervals. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Intravitreal bevacizumab for treatment of proliferative and nonproliferative type 2 idiopathic macular telangiectasia.

PURPOSE: To determine the effect of treatment with intravitreal bevacizumab on retinal thickness and visual acuity in the nonproliferative and proliferative forms of Type 2 idiopathic macular telangiectasia. METHODS: Retrospective chart review of clinic patients treated with bevacizumab for macular telangiectasia Type 2. Treatment was performed until no further changes were seen after repeated bevacizumab injections. All patients had Snellen visual acuity testing, fundus fluorescein angiography, and measurement of central macular thickness by optical coherence tomography at baseline. Visual acuity and central macular thickness were recorded at follow-up visits. RESULTS: Fourteen eyes of 10 patients were included. In 5 eyes with nonproliferative macular telangiectasia Type 2, average follow-up was 17 months (± 7 months), and no eye demonstrated improvement in visual acuity or decrease in central macular thickness at final follow-up compared with baseline. In 9 eyes with proliferative disease, follow-up averaged 17 months (± 9 months). At 6 weeks, central macular thickness decreased 63 µm (± 58 µm), and acuity improved 1.7 lines (± 2 lines). At final follow-up, central macular thickness decreased 48 µm (± 89 µm) and acuity improved 1.1 lines (± 3 lines). Subretinal neovascularization resolved in eight of nine eyes with proliferative disease after treatment. CONCLUSION: Bevacizumab did not improve acuity or reduce retinal thickness in nonproliferative macular telangiectasia Type 2 at final follow-up. In proliferative macular telangiectasia Type 2, bevacizumab caused involution of neovascularization and improved visual acuity.

Effects of vitrectomy on age-related macular degeneration.

PURPOSE: To determine whether vitrectomy alters the long-term progression of age-related macular degeneration (AMD). DESIGN: Retrospective case-control study. PARTICIPANTS: Forty-four eyes of 22 patients with AMD who underwent vitrectomy in 1 eye were included in the study. The progression of AMD at follow-up in the 22 eyes that underwent vitrectomy was compared with the 22 fellow, nonvitrectomized eyes. METHODS: The charts and photographs of subjects with Age-Related Eye Disease Study category 3 AMD in both eyes who previously underwent vitrectomy surgery for an epiretinal membrane or macular hole were reviewed. Subjects were excluded if they had had a vitrectomy in both eyes, had <2 years of follow-up, had previous choroidal neovascularization (CNV), retinal detachment, diabetic retinopathy, angioid streaks, high myopia, vascular occlusions, or extensive macular scarring in either eye, or insufficient hospital records or photographs to determine the extent of AMD. Clinical notes throughout the follow-up interval were reviewed. Two vitreoretinal specialists independently graded pre- and postvitrectomy fundus photographs of all eyes in a masked fashion. MAIN OUTCOME MEASURES: The development or progression of geographic atrophy of the retinal pigment epithelium and the development of CNV. RESULTS: Twenty-two patients were included. The average follow up interval was 5.5 years (range, 2-15). Choroidal neovascularization developed in 5 control eyes and in 2 vitrectomized eyes, and atrophy developed in 7 control and 4 vitrectomized eyes. The difference between vitrectomized eyes and fellow eyes for the combined end points of RPE geographic atrophy or CNV was significant (P = 0.02). CONCLUSIONS: In this pilot study, we did not detect that vitrectomy increased the progression of AMD. In fact, it was associated with a reduced progression to geographic atrophy or CNV. Additional studies are needed to confirm or refute this association. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any of the materials discussed in this article.

[Rupture of the pectoralis major muscle: classification of injuries and results of operative treatment]. / Musculus-pectoralis-major-Rupturen: Verletzungsklassifikation und Ergebnisse der operativen Versorgung.

AIM: Classification of pectoralis major muscle injuries and results of operative treatment in the Sportsklinik Stuttgart between 1998 and 2004 are analysed. METHODS: 10 sportsmen (2 judo, 8 body-building; 9 male, 1 female) with pectoralis major ruptures received operative treatment in this time period. After clinical examination we used ultrasound, in some cases MRI, for further diagnostics. The follow-up (1-5 years) included a clinical examination, ultrasound, sports level, cosmetics and an isokinetic strength assessment. RESULTS: In 4 cases we found a tear of the musculotendinous junction, 4 cases showed a tear at the humeral insertion and 2 other cases had tears of the muscle belly. There was no sports-specific injury. 6 ruptures underwent immediate (1 week) operative therapy and 4 ruptures had delayed (6 weeks to 4 years) repair of the injury. In 9 cases an anatomic repair was possible, in 1 delayed rupture only an extra-anatomic repair was possible. We had 1 complication with a post-operative wound infection. Based on injury localisation and operative treatment, we classified 3 types of pectoralis major ruptures. The follow-up evaluation showed in 7 cases very good and good results, 2 delayed cases still had a cosmetic defect with reduction of strength. CONCLUSION: From our results on pectoralis major muscle injuries there are 3 types of rupture: type 1: rupture at humeral insertion, type 2: rupture of musculotendinous junction, type 3: rupture of muscle belly. This classification is essential for planning the operative technique and the incision. We recommend, after classification of the rupture, primary operative reconstruction of the pectoralis major muscle.

In the yeast potassium channel, Tok1p, the external ring of aspartate residues modulates both gating and conductance.

The yeast plasma-membrane potassium channel, Tok1p, is a voltage-dependent outward rectifier, the gating and steady-state conductance of which are conspicuously modulated by extracellular [K(+)] ([K(+)](o)). Activation is slow at high [K(+)](o), showing time constants (tau(a)) of approximately 90 ms when [K(+)](o) is 150 mM (depolarizing step to +100 mV), and inactivation is weak (<30%) during sustained depolarization. Lowering [K(+)](o) accelerates activation, increases peak current, and enhances inactivation, so that at 15 mM [K(+)](o) tau(a) is less than 50 ms and inactivation suppresses approximately 60% of peak current. Two negative residues, Asp292 and Asp426, near the mouth of the assembled channel, modulate both kinetics and conductance of the channel. Charge neutralization in the mutant Asp292Asn allows fast activation (tau(a) approximately 20 ms) at high [K(+)](o), peak currents diminishing with decreasing [K(+)](o), and fast, nearly complete, inactivation. The voltage dependence of tau(a) persists in the mutant, but the [K(+)](o) dependence almost disappears. Similar but smaller changes are seen in the Asp426Asn mutant, implying that pore geometry in the functional channel has twofold, not fourfold, symmetry.

AtTPK4, an Arabidopsis tandem-pore K+ channel, poised to control the pollen membrane voltage in a pH- and Ca2+-dependent manner.

The Arabidopsis tandem-pore K(+) (TPK) channels displaying four transmembrane domains and two pore regions share structural homologies with their animal counterparts of the KCNK family. In contrast to the Shaker-like Arabidopsis channels (six transmembrane domains/one pore region), the functional properties and the biological role of plant TPK channels have not been elucidated yet. Here, we show that AtTPK4 (KCO4) localizes to the plasma membrane and is predominantly expressed in pollen. AtTPK4 (KCO4) resembles the electrical properties of a voltage-independent K(+) channel after expression in Xenopus oocytes and yeast. Hyperpolarizing as well as depolarizing membrane voltages elicited instantaneous K(+) currents, which were blocked by extracellular calcium and cytoplasmic protons. Functional complementation assays using a K(+) transport-deficient yeast confirmed the biophysical and pharmacological properties of the AtTPK4 channel. The features of AtTPK4 point toward a role in potassium homeostasis and membrane voltage control of the growing pollen tube. Thus, AtTPK4 represents a member of plant tandem-pore-K(+) channels, resembling the characteristics of its animal counterparts as well as plant-specific features with respect to modulation of channel activity by acidosis and calcium.

Analysis of the role of Caenorhabditis elegans GC-AG introns in regulated splicing.

GC-AG introns represent 0.7% of total human pre-mRNA introns. To study the function of GC-AG introns in splicing regulation, 196 cDNA-confirmed GC-AG introns were identified in Caenorhabditis elegans. These represent 0.6% of the cDNA- confirmed intron data set for this organism. Eleven of these GC-AG introns are involved in alternative splicing. In a comparison of the genomic sequences of homologous genes between C.elegans and Caenorhabditis briggsae for 26 GC-AG introns, the C at the +2 position is conserved in only five of these introns. A system to experimentally test the function of GC-AG introns in alternative splicing was developed. Results from these experiments indicate that the conserved C at the +2 position of the tenth intron of the let-2 gene is essential for developmentally regulated alternative splicing. This C allows the splice donor to function as a very weak splice site that works in balance with an alternative GT splice donor. A weak GT splice donor can functionally replace the GC splice donor and allow for splicing regulation. These results indicate that while the majority of GC-AG introns appear to be constitutively spliced and have no evolutionary constraints to prevent them from being GT-AG introns, a subset of GC-AG introns is involved in alternative splicing and the C at the +2 position of these introns can have an important role in splicing regulation.

The allele-specific suppressor sup-39 alters use of cryptic splice sites in Caenorhabditis elegans.

Mutations in the Caenorhabditis elegans sup-39 gene cause allele-specific suppression of the uncoordination defect of unc-73(e936). e936 is a point mutation that changes the canonical G at the 5' end of intron 16 to a U. This mutation activates three splice donors, two of which define introns beginning with the canonical GU. Use of these two cryptic splice sites causes loss of reading frame; interestingly these messages are not substrates for nonsense-mediated decay. The third splice donor, used in 10% of steady-state e936 messages, is the mutated splice donor at the wild-type position, which defines an intron beginning with UU. In the presence of a sup-39 mutation, these same three splice donors are used, but the ratio of messages produced by splicing at these sites changes. The percentage of unc-73(e936) messages containing the wild-type splice junction is increased to 33% with a corresponding increase in the level of UNC-73 protein. This sup-39-induced change was also observed when the e936 mutant intron region was inserted into a heterologous splicing reporter construct transfected into worms. Experiments with splicing reporter constructs showed that the degree of 5' splice site match to the splicing consensus sequence can strongly influence cryptic splice site choice. We propose that mutant SUP-39 is a new type of informational suppressor that alters the use of weak splice donors.

Selective potentiation of drug cytotoxicity by NSAID in human glioma cells: the role of COX-1 and MRP.

Here, we report that nonsteroidal anti-inflammatory drugs (NSAID) enhance the cytotoxic effects of doxorubicin and vincristine in T98G human malignant glioma cells. The cytotoxicity of BCNU, cisplatin, VM26, camptothecin, and cytarabine is unaffected by NSAID. No free radical formation is induced by doxorubicin or vincristine in the absence or presence of NSAID. Doxorubicin and vincristine cytotoxicity in the absence or presence of NSAID are unaffected by free radical scavengers. Functional inhibitors of phospholipase A2 (PLA2), such as dexamethasone and quinacrine, do not mimick the effects of NSAID. T98G cells, but not LN-18, LN-229, LN-308, or A172 glioma cells, express cyclooxygenase (COX-1) and NSAID do not modulate drug cytotoxicity in the other cell lines, except T98G. Thus, augmentation of doxorubicin and vincristine cytotoxicity by NSAID correlates with COX-1 expression. However, ectopic expression of COX-1 in LN-229 cells does not induce the phenotype of T98G cells, indicating that COX-1 inhibition does not mediate the effects of NSAID on drug cytotoxicity. In contrast, a multidrug resistance (MDR) phenotype due to expression of the multidrug resistance-associated protein (MRP) is most prominent in T98G cells and is amenable to modulation by indomethacin, suggesting that inhibition of MRP is at least in partly responsible for the potentiation of doxorubicin and vincristine cytotoxicity by NSAID.

Antioxidants specifically inhibit cisplatin cytotoxicity of human malignant glioma cells.

BACKGROUND: Human malignant gliomas are resistant to chemotherapy. Here, we examine the modulation of drug-induced cytotoxicity and clonogenic cell death of glioma cells by antioxidants. MATERIALS AND METHODS: We studied the effects on drug toxicity of three structurally unrelated antioxidants, N-acetylcysteine, superoxide dismutase or phenyl-N-tert-butyl-alpha-phenylnitrone, in acute cytotoxicity and clonogenic cell death assays in LN-18, LN-229 and T98G cells. Two fluorescent dyes, 2',7'-dichlorodihydroJluorescein diacetate (DCF-Hr2) and dihydro-rhodamine-123, were used to monitor free radical formation after drug exposure. RESULTS: The antioxidants inhibited acute cytotoxicity and clonogenic cell death induced by cisplatin in all cell lines but had little effect on the toxicity of BCNU, doxorubicin, VM26, vincristine, cytarabine or camptothecin. Cisplatin toxicity was not associated with free radical formation and was not potentiated by L-buthionine-[S,R]-sulfoximine-induced glutathione depletion. CONCLUSION: Antioxidants specifically inhibit cisplatin cytotoxicity of human malignant glioma cells in the absence of drug-induced free radical formation.

Coping with the late effects: differences between depressed and nondepressed polio survivors.

This study examined differences between depressed and nondepressed individuals with a history of paralytic poliomyelitis in terms of demographics, health status and coping strategies. The prevalence of distress and depression in this group of 116 polio survivors was determined. Subjects completed the Brief Symptom Inventory, the Coping with Disability Inventory and a questionnaire concerning their polio histories and self-perceptions of health. Medical assessments were performed by physicians. Only 15.8% of the sample had scores indicating depression and elevated distress. Depressed/distressed polio survivors were more likely to: be living alone, be experiencing further health status deterioration, seek professional help, view their health as poor, report greater pain, be less satisfied with their occupational status and their lives in general and exhibit poorer coping outcome behaviors in relation to their disability. Three factors in coping with the late effects of polio were identified through a factor analysis of the Coping with Disability Inventory: positive self-acceptance, information seeking/sharing about the disability and social activism. Differences between depressed/distressed and other polio survivors were found across these three factors, with depressed/distressed subjects having significantly lower coping scores. These and other results are discussed.