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Chirality is determinant for sphingosine biofunctions and pharmacological activity, yet the reasons for the biological chiral selection are not well understood. Here, we characterized the intra- and intermolecular interactions at the headgroup of the cytotoxic anhydrophytosphingosine jaspine B, revealing chirality-dependent correlations between the puckering of the ring core and the formation of amino-alcohol hydrogen bond networks, both in the monomer and the monohydrate. Following the specific synthesis of a shortened 3-carbon side-chain molecule, denoted jaspine B3, six different isomers were observed in a jet expansion using broadband (chirped-pulsed) rotational spectroscopy. Additionally, a single isomer of the jaspine B3 monohydrate was observed, revealing the insertion of water in between the hydroxy and amino groups and the formation of a network of O-H···N-H···Oring hydrogen bonds. The specific jaspine B3 stereochemistry thus creates a double-faced molecule where the exposed lone-pair electrons may easily catalyze the formation of intermolecular aggregates and determine the sphingosine biological properties., Funding support from the Spanish MICINN-FEDER (grant PGC2018-098561-B-C22) is gratefully acknowledged. M.J. and R.T.S. are thankful for predoctoral contracts from the MICINN and UVa, respectively.

Sphingosine [(2S,3R,4E)-2-amino-4-octadecene-1,3-diol] is the most common sphingoid base in mammals. Ceramides are N-acyl sphingosines. Numerous small variations on this canonical structure are known, including the 1-deoxy, the 4,5-dihydro, and many others. However, whenever there is a Δ4 double bond, it adopts the trans (or E) configuration. We synthesized a ceramide containing 4Z-sphingosine and palmitic acid (cis-pCer) and studied its behavior in the form of monolayers extended on an air-water interface. cis-pCer acted very differently from the trans isomer in that, upon lateral compression of the monolayer, a solid-solid transition was clearly observed at a mean molecular area ≤44 Å2·molecule-1, whose characteristics depended on the rate of compression. The solid-solid transition, as well as states of domain coexistence, could be imaged by atomic force microscopy and by Brewster-angle microscopy. Atomistic molecular dynamics simulations provided results compatible with the experimentally observed differences between the cis and trans isomers. The data can help in the exploration of other solid-solid transitions in lipids, both in vitro and in vivo, that have gone up to now undetected because of their less obvious change in surface properties along the transition, as compared to cis-pCer. Fil: Fanani, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Busto, Jon V.. Universidad del País Vasco; España. Consejo Superior de Investigaciones Científicas; España Fil: Sot, Jesús. Consejo Superior de Investigaciones Científicas; España. Universidad del País Vasco; España Fil: Abad, José L.. Consejo Superior de Investigaciones Científicas. Instituto de Química Avanzada de Catalunya; España Fil: Fabrías, Gemma. Consejo Superior de Investigaciones Científicas. Instituto de Química Avanzada de Catalunya; España Fil: Saiz, Leonor. University of California; Estados Unidos. Massachusetts Institute of Technology; Estados Unidos Fil: Vilar, Jose M. G.. Universidad del País Vasco; España. Consejo Superior de Investigaciones Científicas; España Fil: Goñi, Félix M.. Consejo Superior de Investigaciones Científicas; España. Universidad del País Vasco; España Fil: Maggio, Bruno. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Alonso, Alicia. Consejo Superior de Investigaciones Científicas; España. Universidad del País Vasco; España

The combination of daunorubicin (dnr) and cytarabine (Ara-C) is a cornerstone of treatment for acute myelogenous leukemia (AML); resistance to these drugs is a major cause of treatment failure. Ceramide, a sphingolipid (SL), plays a critical role in cancer cell apoptosis in response to chemotherapy. Here, we investigated the effects of chemotherapy selection pressure with Ara-C and dnr on SL composition and enzyme activity in the AML cell line HL-60. Resistant cells, those selected for growth in Ara-C- and dnr-containing medium (HL-60/Ara-C and HL-60/dnr, respectively), demonstrated upregulated expression and activity of glucosylceramide synthase, acid ceramidase (AC), and sphingosine kinase 1 (SPHK1); were more resistant to ceramide than parental cells; and displayed sensitivity to inhibitors of SL metabolism. Lipidomic analysis revealed a general ceramide deficit and a profound upswing in levels of sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) in HL-60/dnr cells versus parental and HL-60/Ara-C cells. Both chemotherapyselected cells also exhibited comprehensive upregulations in mitochondrial biogenesis consistent with heightened reliance on oxidative phosphorylation, a property that was partially reversed by exposure to AC and SPHK1 inhibitors and that supports a role for the phosphorylation system in resistance. In summary, dnr and Ara-C selection pressure induces acute reductions in ceramide levels and large increases in S1P and C1P, concomitant with cell resilience bolstered by enhanced mitochondrial remodeling. Thus, strategic control of ceramide metabolism and further research to define mitochondrial perturbations that accompany the drug-resistant phenotype offer new opportunities for developing therapies that regulate cancer growth., This work was supported by National Institutes of Health Grant P01 CA171983 and by a grant from the Brody Brothers Foundation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors declare that they have no conflicts of interest with the contents of this article.

Producción Científica, Sparteine is a quinolizidine alkaloid used as chiral auxiliary in asymmetric synthesis. We examine whether hydration by a single molecule can flip sparteine from the most stable trans conformation to the bidentate cis arrangement observed in catalytic complexation to a metal center. Sparteine and the dimer sparteine-water were generated in a supersonic jet expansion with H216O and H218O, and characterized by broadband chirped-pulse microwave spectroscopy. Despite the bidentate water dimer was predicted with larger binding energy, a single isomer was observed for the monohydrated cluster, with sparteine retaining the trans conformation observed for the free molecule. The absence of the bidentate dimer is attributed to kinetic control of the cluster formation, favoring the pre-expansion most abundant monomer. The structural properties of the O-H···N hydrogen bond in the dimer are compared with complexes of other secondary and tertiary amines., 2018-07-21, MINECO-FEDER (CTQ2015-68148-C2-2-P)

10 páginas, 8 figuras -- PAGS nros. 2739-2748, Neural stem cells depend on insulin-like growth factor I (IGF-I) for differentiation. We analysed how activation and inhibition of the PI 3-kinase–Akt signalling affects the number and differentiation of mouse olfactory bulb stem cells (OBSCs). Stimulation of the pathway with insulin and/or IGF-I, led to an increase in Akt phosphorylated on residues Ser473 and Thr308 (P-AktSer473 and P-AktThr308, respectively) in proliferating OBSCs, and in differentiating cells. Conversely, P-AktSer473 levels decreased by 50% in the OB of embryonic day 16.5-18.5 IGF-I knockout mouse embryos. Overexpression of PTEN, a negative regulator of the PI 3-kinase pathway, caused a reduction in the basal levels of P-AktSer473 and P-AktThr308 and a minor reduction in IGF-I-stimulated P-AktSer473. Although PTEN overexpression decreased the proportion of neurons and astrocytes in the absence of insulin/IGF-I, it did not alter the proliferation or survival of OBSCs. Accordingly, overexpression of a catalytically inactive PTEN mutant promoted OBSCs differentiation. Inhibition of PI 3-kinase by LY294002 produced strong and moderate reductions in IGF-I-stimulated P-AktSer473 and P-AktThr308, respectively. Consequently, LY294002 reduced the proliferation of OBSCs and the number of neurons and astrocytes, and also augmented cell death. These findings indicate that OBSC differentiation is more sensitive to lower basal levels of P-Akt than proliferation or death. By regulating P-Akt levels in opposite ways, IGF-I and PTEN contribute to the fine control of neurogenesis in the olfactory bulb, This work was funded by the grants from the MEC (Spain) BFU 2004-2352 to F.d.P., SAF2004-05798 to C.V.-A. and BMC 2003-07751 to E.J.d.l.R., and from the Fundación "la Caixa" NE03/72-02 to C.V.-A. G.O., M.J.Y.-B, E.V.-V. and H.R.M.-G. were supported by doctoral fellowships either from the MEC or the "Comunidad Autónoma de Madrid"

Laser in situ keratomileusis (LASIK) was performed in the left eye of a 57-year-old man for residual ametropia after phacoemulsification. The patient was given topical tobramycin and a corticosteroid for 1 week postoperatively. Fifteen days later, he developed 3 corneal infiltrates beneath the flap with a gas bubble, suggesting an anaerobic infection. Tobramycin and ofloxacin were administered every 2 hours, but the condition worsened. Corneal scrapings were taken from beneath the flap for microbiological cultures and a polymerase chain reaction (PCR) test. The PCR amplification was negative for fungi and mycobacteria and positive for bacterial DNA. Sequence analysis showed Propionibacterium granulosum as the causal agent, but cultures were negative. Treatment with vancomycin and cefazolin led to clinical improvement, with resolution of corneal infiltrates. Anaerobic microorganisms can cause keratitis after LASIK. Polymerase chain reaction amplification and DNA typing can help detect microorganisms involved in these ocular infections.

This study aimed to analyze the effect of velocity-based resistance training (RT) with moderate loads and few repetitions per set combined with jumps and sprints on physical performance in young soccer players of different ages. A total of 44 elite youth soccer players belonging to 3 teams participated in this study: an under-16 team (U16, n = 17) and an under-18 team (U18, n = 16) performed maximal velocity RT program for 26 weeks in addition to typical soccer training, whereas an under-21 team (U21, n = 11) did not perform RT. Before and after the training program, all players performed 20-m running sprint (T20), countermovement jump (CMJ), a progressive isoinertial loading test in squat to determine the load that elicited a ∼ 1 m · s(-1) velocity (V1LOAD) and an incremental field test to determine maximal aerobic speed (MAS). U16 showed significantly (p = 0.000) greater gains in V1LOAD than U18 and U21 (100/0/0%). Only U16 showed significantly (p = 0.01) greater gains than U21 (99/1/0%) in CMJ height. U18 obtained a likely better effect on CMJ performance than U21 (89/10/1%). The beneficial effects on T20 between groups were unclear. U16 showed a likely better effect on MAS than U21 (80/17/3%), whereas the rest of comparisons were unclear. The changes in CMJ correlated with the changes in T20 (r = -0.49) and V1LOAD (r = 0.40). In conclusion, velocity-based RT with moderate load and few repetitions per set seems to be an adequate method to improve physical performance in young soccer players.

The goal of this study was to determine whether sequence analysis of internal transcribed spacer/5.8S ribosomal DNA (rDNA) can be used to detect fungal pathogens in patients with ocular infections (endophthalmitis and keratitis). Internal transcribed spacer 1 (ITS1) and ITS2 and 5.8S rDNA were amplified by PCR and seminested PCR to detect fungal DNA. Fifty strains of 12 fungal species (yeasts and molds) were used to test the selected primers and conditions of the PCR. PCR and seminested PCR of this region were carried out to evaluate the sensitivity and specificity of the method. It proved possible to amplify the ITS2/5.8S region of all the fungal strains by this PCR method. All negative controls (human and bacterial DNA) were PCR negative. The sensitivity of the seminested PCR amplification reaction by DNA dilutions was 1 organism per PCR, and the sensitivity by cell dilutions was fewer than 10 organisms per PCR. Intraocular sampling or corneal scraping was undertaken for all patients with suspected infectious endophthalmitis or keratitis (nonherpetic), respectively, between November 1999 and February 2001. PCRs were subsequently performed with 11 ocular samples. The amplified DNA was sequenced, and aligned against sequences in GenBank at the National Institutes of Health. The results were PCR positive for fungal primers for three corneal scrapings, one aqueous sample, and one vitreous sample; one of them was negative by culture. Molecular fungal identification was successful in all cases. Bacterial detection by PCR was positive for three aqueous samples and one vitreous sample; one of these was negative by culture. Amplification of ITS2/5.8S rDNA and molecular typing shows potential as a rapid technique for identifying fungi in ocular samples.

Several infiltrates appeared in the upper midperipheral cornea of a 29-year-old woman who had had uneventful corneal collagen crosslinking (CXL) with riboflavin and ultraviolet-A light (UVA) for the treatment of keratoconus in the right eye. Staphylococcus epidermidis keratitis was confirmed by microbiological studies, which guided treatment with topical fortified antibiotic agents. Before CXL, the best spectacle-corrected visual acuity (BSCVA) in the right eye was 20/25, the manifest refraction was -0.25 -0.25 x 125, and the anterior segment was normal under biomicroscopy. Five months after the procedure, the BSCVA was 20/22, the manifest refraction was +1.00 -2.50 x 40, and slitlamp examination revealed a mild residual haze in the upper midperipheral cornea. Collagen crosslinking with riboflavin-UVA is a minimally invasive method but traditionally requires epithelial removal, which could be a predisposing factor to bacterial keratitis.

The CCR5 chemokine receptor plays a crucial role in the initiation of in vivo HIV infection, acting as a critical coreceptor molecule for primary strains. Individuals with mutations in the CCR5 gene that reduce its level of expression are resistant to HIV-1 infection. Since these mutations are not associated with any known clinical condition, CCR5 may be an ideal target for anti-HIV therapy. We have designed an artificial hammerhead ribozyme, denoted RzR5-76, targeted to exon 2 of the human CCR5 mRNA. When RzR5-76 activity is induced in HEK 293 cells transfected with a CCR5 expression plasmid, the surface levels of this chemokine receptor are reduced up to 60%. The results indicate that this inhibitory effect is mainly due to the catalytic activity of the ribozyme and not to its antisense properties. These preliminary data suggest that intracellular ribozymes could be used in vivo to block HIV-1 entry into human cells.

The first case ofAlternaria infectoriaocular infection is reported. Keratitis and endophthalmitis developed after eye-perforating trauma from a lemon tree branch. Two months after surgery and empirical steroid and antibiotic treatment, diagnosis by molecular methods was performed. PCR amplification was positive for a fungus after 4 h. Antifungal treatment with amphotericin B and fluconazole was initiated immediately. DNA sequence analysis showedAlternaria infectoriato be the causal agent. After topical and systemic administration of antifungal treatment, ocular inflammation disappeared and visual acuity improved. DNA typing was found to be a useful tool to achieve early identification of the causal agent.

Dihydroceramides, the precursors of ceramides in the de novo sphingolipid synthesis, have been recently implicated in active signalling. We previously demonstrated that dihydroceramide accumulation, in response to treatment with the dihydroceramide desaturase inhibitor XM462, induced autophagy with no sign of cell death in the gastric carcinoma HCG27 cell line. Here we show that XM462 treatment induces a transient early increase in dihydroceramides that are successively metabolized into other sphingolipids. Dihydroceramides accumulation is associated with cyclin D1 expression modulation, delayed G1/S transition of cell cycle and increased autophagy. Moreover, XM462 treatment induces ER stress via the activation of the translation inhibitor eIF2α and the pro-survival transcriptional factor Xbp1. Exogenous addition of a short chain dihydroceramide analog reproduces the effects of endogenous accumulation of dihydroceramides, causing cell cycle delay of the G1/S transition, autophagy enhancement, eIF2α activation and Xbp1 splicing. Blocking autophagy with 3-methyladenine abrogates the effect of XM462 on cell cycle and reduces cell survival to XM462 treatment. Furthermore, the XM462-induced survival response is able to reduce etoposide toxicity in HCG27 and HCT116 cancer cells. Our data suggest a role of dihydroceramide in regulating cell proliferation and survival.

PURPOSE To report a patient who presented an infectious keratitis 4 years after laser in situ keratomileusis (LASIK) without any other predisposing risk factor than the LASIK procedure itself. CASE REPORT We report a 32-year-old man operated by LASIK in January 2006 who presented with infectious keratitis in the OD in April 2010. Clinical examination showed a corneal abscess at 10-o'clock position in the interface and fibrin and Tyndall 4+ in the anterior chamber. Microbiological analysis identified Pseudomonas aeruginosa as the cause of infection. The patient was given ofloxacin, sulfate neomycin, polymyxin B, and prednisolone acetate to be used every 2 h. Treatment led to clinical improvement with resolution of corneal infiltrate. Keratitis with intact epithelium by Pseudomonas can occur up to 4 years after LASIK. CONCLUSIONS LASIK treatment is a predisposing factor for bacterial keratitis even years after surgery. This report demonstrates the importance of continued postoperative vigilance by patient and his/her clinician.

The T-box brain 1 (Tbr1) gene encodes a transcription factor necessary for the maintenance and/or differentiation of glutamatergic cells in the olfactory bulb (OB) and cortex, although its precise function in the development of glutamatergic neurons is not known. Furthermore, Tbr1 has not been reported to regulate the formation of glial cells. We show that Tbr1 is expressed during the initial stages in the generation of glutamatergic mitral neurons from dividing progenitors in the E12.5 mouse OB. Retroviral-mediated overexpression of Tbr1 in cultured embryonic and adult OB stem cells (OBSC) produces a marked increase in the number of TuJ1+ neurons (including VGLUT1+ glutamatergic and GABA+ neurons) and O4+ oligodendrocytes. Moreover, transduction of Tbr1 inhibits the production of GFAP+ astrocytes from both cultured OBSC and dividing progenitor cells in vivo. These results show that the expression of Tbr1 in neural stem and progenitor cells prevents them from following an astrocyte fate during OB development. Our findings suggest that the transduction of Tbr1 into neural stem cells could be useful to increase the production of neurons and oligodendrocytes in studies of neuroregeneration. © 2010.

We report 2 cases of severe corneal infections caused by Serratia marcescens after laser in situ keratomileusis (LASIK). Twenty-four hours after LASIK, 2 patients developed infectious keratitis, 1 bilaterally. In each eye, the corneal flap was edematous, ulcerated, and detached from the stromal bed. Treatment included removal of the necrotic flap and aggressive antibiotic therapy. Cultures from corneal exudates were positive for S marcescens. After 1 year, both patients had a loss of best corrected visual acuity (BCVA) ranging from 20/40 to 20/22 because of irregular astigmatism. Overrefraction with a hard contact lens resulted in a BCVA of 20/20 in the 3 affected eyes. Slitlamp examination showed trace subepithelial haze without severe corneal scarring. Videokeratography disclosed areas of paracentral inferior steepening resembling keratoconus. Refraction and videokeratography remained stable after 6 months of follow-up. Ulcerative keratitis caused by S marcescens is a potential complication of LASIK. Bilateral involvement may occur if bilateral simultaneous surgery is performed.

PURPOSE: To contribute toward assessing the effectiveness of polymerase chain reaction as a rapid method in diagnosis of torpid keratitis caused by opportunistic fungi. METHODS: Interventional case report. A 50-year-old man with a corneal abscess in the right eye treated for a period of 6 months with different combinations of broad-spectrum antibiotics and steroids was referred to our center. Corneal scraping was taken for microbiological study, including classic cultures and polymerase chain reaction. Amplified DNA was sequenced to identify the pathogen. RESULTS: Polymerase chain reaction amplification was negative for Acanthamoeba species and positive for fungi. The sequence analysis showed Alternaria alternata as the causal agent in 24 hours. Cultures confirmed the identification in 10 days. CONCLUSION: Polymerase chain reaction amplification with subsequent DNA-typing was revealed to be a useful method for detection of ocular pathogens such as A. alternata involved in cases of torpid keratitis, even in the presence of broad-spectrum antimicrobial therapy.

PURPOSE AND METHODS: Corneal interface central nodules appeared in a patient who underwent uncomplicated laser in situ keratomileusis (LASDX) retreatment for residual myopia. RESULTS/CONCLUSIONS: Nocardia asteroides keratitis was confirmed by microbiologic studies, which guided treatment. Six months after the appearance of the keratitis, the patient's uncorrected visual acuity was 20/45, and spectacle-corrected visual acuity was 20/40. The postoperative refraction was +0.75 -0.75 X 95°, and slit-lamp examination revealed a clear cornea with a mild rounded scar in the central area. Night halos and starbursts were the main complaints in this patient. The immediate management of lifting the corneal flap for stromal bed scraping, fast microbial identification, and proper treatment was the key for the results in this patient. [J Refract Surg 1997;13:314-317]