The dual epigenetic role of PRMT5 in acute myeloid leukemia: gene activation and repression via histone arginine methylation.

Changes in the enzymatic activity of protein arginine methyltransferase (PRMT) 5 have been associated with cancer; however, the protein's role in acute myeloid leukemia (AML) has not been fully evaluated. Here, we show that increased PRMT5 activity enhanced AML growth in vitro and in vivo while PRMT5 downregulation reduced it. In AML cells, PRMT5 interacted with Sp1 in a transcription repressor complex and silenced miR-29b preferentially via dimethylation of histone 4 arginine residue H4R3. As Sp1 is also a bona fide target of miR-29b, the miR silencing resulted in increased Sp1. This event in turn led to transcription activation of FLT3, a gene that encodes a receptor tyrosine kinase. Inhibition of PRMT5 via sh/siRNA or a first-in-class small-molecule inhibitor (HLCL-61) resulted in significantly increased expression of miR-29b and consequent suppression of Sp1 and FLT3 in AML cells. As a result, significant antileukemic activity was achieved. Collectively, our data support a novel leukemogenic mechanism in AML where PRMT5 mediates both silencing and transcription of genes that participate in a 'yin-yang' functional network supporting leukemia growth. As FLT3 is often mutated in AML and pharmacologic inhibition of PRMT5 appears feasible, the PRMT5-miR-29b-FLT3 network should be further explored as a novel therapeutic target for AML.

A Retrospective Analysis of the Management of Postoperative Discitis: A Single Institutional Experience.

STUDY DESIGN: Retrospective study. PURPOSE: The aim of the study was to study the impact and outcome of conservative management and surgical intervention in cases of postoperative discitis. OVERVIEW OF LITERATURE: Postoperative discitis is a rare but often misdiagnosed cause of failed back syndrome. There is paucity of literature regarding management guidelines of postoperative discitis. METHODS: The study was carried out over a period of 6 years. Eighteen patients with postoperative discitis were included in the study. RESULTS: Conservative management with antibiotics, analgesics and bed rest were started in all the study cases. Posterior transpedicular fixation after re-exploration debridement and curettage of disc space granulation tissue was conducted in five patients in whom conservative management failed. CONCLUSIONS: Early diagnosis and appropriate management is the key to effective treatment of postoperative discitis. Conservative management leads to excellent results in majority of cases. Surgical intervention with posterior interbody fusion and debridement is helpful when conservative treatment fails.

Pharmacological targeting of miR-155 via the NEDD8-activating enzyme inhibitor MLN4924 (Pevonedistat) in FLT3-ITD acute myeloid leukemia.

High levels of microRNA-155 (miR-155) are associated with poor outcome in acute myeloid leukemia (AML). In AML, miR-155 is regulated by NF-κB, the activity of which is, in part, controlled by the NEDD8-dependent ubiquitin ligases. We demonstrate that MLN4924, an inhibitor of NEDD8-activating enzyme presently being evaluated in clinical trials, decreases binding of NF-κB to the miR-155 promoter and downregulates miR-155 in AML cells. This results in the upregulation of the miR-155 targets SHIP1, an inhibitor of the PI3K/Akt pathway, and PU.1, a transcription factor important for myeloid differentiation, leading to monocytic differentiation and apoptosis. Consistent with these results, overexpression of miR-155 diminishes MLN4924-induced antileukemic effects. In vivo, MLN4924 reduces miR-155 expression and prolongs the survival of mice engrafted with leukemic cells. Our study demonstrates the potential of miR-155 as a novel therapeutic target in AML via pharmacologic interference with NF-κB-dependent regulatory mechanisms. We show the targeting of this oncogenic microRNA with MLN4924, a compound presently being evaluated in clinical trials in AML. As high miR-155 levels have been consistently associated with aggressive clinical phenotypes, our work opens new avenues for microRNA-targeting therapeutic approaches to leukemia and cancer patients.

Comprehensive quantum chemical and spectroscopic (FTIR, FT-Raman, 1H, 13C NMR) investigations of O-desmethyltramadol hydrochloride an active metabolite in tramadol--an analgesic drug.

O-desmethyltramadol is one of the main metabolites of tramadol widely used clinically and has analgesic activity. The FTIR and FT-Raman spectra of O-desmethyl tramadol hydrochloride are recorded in the solid phase in the regions 4000-400 cm(-1) and 4000-100 cm(-1), respectively. The observed fundamentals are assigned to different normal modes of vibration. Theoretical studies have been performed as its hydrochloride salt. The structure of the compound has been optimised with B3LYP method using 6-31G(**) and cc-pVDZ basis sets. The optimised bond length and bond angles are correlated with the X-ray data. The experimental wavenumbers were compared with the scaled vibrational frequencies determined by DFT methods. The IR and Raman intensities are determined with B3LYP method using cc-pVDZ and 6-31G(d,p) basic sets. The total electron density and molecular electrostatic potential surfaces of the molecule are constructed by using B3LYP/cc-pVDZ method to display electrostatic potential (electron+nuclei) distribution. The electronic properties HOMO and LUMO energies were measured. Natural bond orbital analysis of O-desmethyltramadol hydrochloride has been performed to indicate the presence of intramolecular charge transfer. The (1)H and (13)C NMR chemical shifts of the molecule have been anlysed.

Primidone--an antiepileptic drug--characterisation by quantum chemical and spectroscopic (FTIR, FT-Raman, 1H, 13C NMR and UV-Visible) investigations.

The solid phase FTIR and FT-Raman spectra of primidone were recorded in the regions 4000-400 cm(-1) and 4000-100 cm(-1), respectively. The vibrational spectra were analysed and the observed fundamentals were assigned and analysed. The experimental wavenumbers were compared with the theoretical scaled vibrational wavenumbers determined by DFT methods. The Raman intensities were also determined with B3LYP/6-31G(d,p) method. The total electron density and molecular electrostatic potential surface of the molecule were constructed by using B3LYP/6-311++G(d,p) method to display electrostatic potential (electron+nuclei) distribution. The HOMO and LUMO energies were measured. Natural bond orbital analysis of primidone has been performed to indicate the presence of intramolecular charge transfer. The (1)H and (13)C NMR spectra were recorded and the chemical shifts of the molecule were calculated.

Conformational, vibrational, NMR and DFT studies of N-methylacetanilide.

A detailed conformational, vibrational, NMR and DFT studies of N-methylacetanilide have been carried out. In DFT, B3LYP method have been used with 6-31G(**), 6-311++G(**) and cc-pVTZ basis sets. The vibrational frequencies were calculated resulting in IR and Raman frequencies together with intensities and Raman depolarisation ratios. The dipole moment derivatives were computed analytically. Owing to the complexity of the molecule, the potential energy distributions of the vibrational modes of the compound are also calculated. Isoelectronic molecular electrostatic potential surface (MEP) and electron density surface were examined. (1)H and (13)C NMR isotropic chemical shifts were calculated and the assignments made are compared with the experimental values. The energies of important MO's of the compound were also determined from TD-DFT method.

Structural, vibrational, electronic investigations and quantum chemical studies of 2-amino-4-methoxybenzothiazole.

Extensive vibrational investigations of 2-amino-4-methoxybenzothiazole have been carried out with FTIR and FT-Raman spectral techniques. The electronic structure of the molecule has been analysed by UV-Visible and NMR spectroscopies. The DFT studies were carried out with B3LYP and HF methods utilising 6-31G(d,p), 6-311++G(d,p) and cc-pVDZ basis sets to determine the structural, thermodynamical, vibrational, electronic characteristics of the compound and also to understand the electronic and steric influence of the methoxy amino groups on the skeletal frequencies. The mixing of the fundamental modes was determined with the help of total energy distribution (TED). The energies of the frontier molecular orbitals have also been determined. The kinetic and thermodynamic stability and chemical hardness of the molecule have been determined. Complete NBO analysis was also carried out to find out the intramolecular electronic interactions and their stabilisation energy. (1)H and (13)C NMR chemical shifts and the electronic transitions of the molecule are also discussed.

Increased anti-leukemic activity of decitabine via AR-42-induced upregulation of miR-29b: a novel epigenetic-targeting approach in acute myeloid leukemia.

Histone deacetylase (HDAC) inhibitors either alone or in combination with hypomethylating agents have limited clinical effect in acute myeloid leukemia (AML). Previously, we demonstrated that AML patients with higher miR (microRNA)-29b expression had better response to the hypomethylating agent decitabine. Therefore, an increase in miR-29b expression preceding decitabine treatment may provide a therapeutic advantage. We previously showed that miR-29b expression is suppressed by a repressor complex that includes HDACs. Thus, HDAC inhibition may increase miR-29b expression. We hypothesized that priming AML cells with the novel HDAC inhibitor (HDACI) AR-42 would result in increased response to decitabine treatment via upregulation of miR-29b. Here, we show that AR-42 is a potent HDACI in AML, increasing miR-29b levels and leading to downregulation of known miR-29b targets (that is, SP1, DNMT1, DNMT3A and DNMT3B). We then demonstrated that the sequential administration of AR-42 followed by decitabine resulted in a stronger anti-leukemic activity in vitro and in vivo than decitabine followed by AR-42 or either drug alone. These preclinical results with AR-42 priming before decitabine administration represent a promising, novel treatment approach and a paradigm shift with regard to the combination of epigenetic-targeting compounds in AML, where decitabine has been traditionally given before HDACIs.

A comparative study on vibrational, conformational and electronic structure of 1,2-dimethyl-5-nitroimidazole and 2-methyl-5-nitroimidazole.

The FTIR and FT-Raman spectra of 1,2-dimethyl-5-nitroimidazole and 2-methyl-5-nitroimidazole have been recorded in the regions 4000-400 and 4000-100 cm(-1), respectively. The conformational analyses were performed and the energies of the different possible conformers were determined. The geometry of different conformers of the compounds were optimised with B3LYP and B3PW91 methods using 6-311++G and aug-cc-pVTZ basis sets to characterise all stationary points as minima. The optimised structural parameters of the most stable conformer were used in the vibrational frequency calculations. The Raman activities were also determined with B3LYP method using 6-311++G basis sets. The force constants obtained from the B3LYP/6-311++G method have been utilised in the normal coordinate analysis. The temperature dependence of the thermodynamic properties heat capacity at constant pressure (C(p)), entropy (S) and enthalpy change (ΔH(0→T)) for both the compounds were also determined by B3LYP/6-311++G method. The total electron density and MESP surfaces of the molecules were constructed by NBO analysis using B3LYP/6-311++G method to display electrostatic potential (electron+nuclei) distribution, molecular shape, size, and dipole moments of the molecule. The electronic properties HOMO and LUMO energies were measured. The influences of nitro and methyl groups on the skeletal modes have been investigated.

Structural characteristics and harmonic vibrational analysis of the stable conformer of 2,3-epoxypropanol by quantum chemical methods.

The FT-IR and FT-Raman spectra of H bond inner conformer of 2,3-epoxypropanol have been recorded in the regions 3700-400 and 3700-100 cm(-1), respectively. The spectra were interpreted in terms of fundamentals modes, combination and overtone bands. The normal coordinate analysis was carried out to confirm the precision of the assignments. The structure of the conformers H bond inner and H bond outer1 were optimised and the structural characteristics were determined by density functional theory (DFT) using B3LYP and MP2 methods with 6-31G** and 6-311++G** basis sets. The vibrational frequencies were calculated in all these methods and were compared with the experimental frequencies which yield good agreement between observed and calculated frequencies. The electronic properties HOMO and LUMO energies were measured by time-dependent TD-DFT approach.

Fatal intratumoral hemorrhage in posterior fossa tumors following ventriculoperitoneal shunt.

We report on two patients with rare major intratumoral hemorrhage following ventriculoperitoneal shunt in posterior fossa tumors. A 28-year-old woman with a midline posterior fossa lesion, whose imaging features suggested a fourth ventricular ependymoma with obstructive hydrocephalus, was subjected to a right ventriculoperitoneal shunt. Her consciousness deteriorated, and she experienced massive intratumoral hemorrhage and later died. An eight-year-old girl presented with raised intracranial pressure and ataxia caused by vermian astrocytoma with obstructive hydrocephalus. She also developed a massive tumor bleed following a ventriculoperitoneal shunt and was subjected to emergency decompression of the tumor with the bleeding. She remained vegetative at discharge and died 18 months later. Intratumoral hemorrhage is a rare but important cause of morbidity and mortality in patients with posterior fossa tumors who undergo ventriculoperitoneal shunt.

Intensive care management of head injury patients without routine intracranial pressure monitoring.

BACKGROUND: Head injury contributes significantly to mortality and morbidity in India. Evaluation of the available trauma care facilities may help improve outcome. AIM: To evaluate the factors influencing the mortality of patients with head injury who had intensive care management and evolve strategies to improve outcome. SETTING AND DESIGN: Retrospective study in a tertiary hospital where intracranial pressure monitoring (ICPM) is not routinely practiced. MATERIALS AND METHODS: All patients with head injury managed in the intensive care unit in a two-year period were included. The factors evaluated were age, vital signs, Glasgow Coma scale score (GCS) at admission, pupillary light reflex (PR), oculocephalic reflex (OCR), hemodynamic stability, computerized tomography (CT) findings, diabetes mellitus, anemia, infections and abnormalities of serum sodium. RESULTS: We analyzed 208 patients (202 without ICPM). In-hospital mortality was 64 (31%). Only 24 (11.5%) patients were admitted within one hour of injury, while one-third arrived after six hours. The clinical factors (at admission) that influenced mortality included age, GCS, PR, OCR and diastolic blood pressure (DBP). Effacement of the basal cisterns in the initial and repeat CT scans, hyperglycemia, hemodynamic instability and serum sodium imbalances were associated with higher mortality. The independent predictors of mortality by logistic regression were initial GCS, DBP, hemodynamic instability and effacement of cisterns on repeat CT. CONCLUSIONS: Mortality following head injury is high. Pre-hospital emergency medical services are disorganized. The key to reducing mortality within the limitations of our current trauma system is maintenance of DBP>70 mmHg and SBP>90 mmHg from the time of first contact.

Genetic modification of cerebral arterial wall: implications for prevention and treatment of cerebral vasospasm.

Genetic modification of cerebral vessels represents a promising and novel approach for prevention and/or treatment of various cerebral vascular disorders, including cerebral vasospasm. In this review, we focus on the current understanding of the use of gene transfer to the cerebral arteries for prevention and/or treatment of cerebral vasospasm following subarachnoid hemorrhage (SAH). We also discuss the recent developments in vascular therapeutics, involving the autologous use of progenitor cells for repair of damaged vessels, as well as a cell-based gene delivery approach for the prevention and treatment of cerebral vasospasm.

Evaluation of the immunocontraceptive potential of Escherichia coli-expressed recombinant dog ZP2 and ZP3 in a homologous animal model.

Dog zona pellucida glycoprotein 2 (dZP2), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was cloned and expressed as a polyhistidine fusion protein in Escherichia coli to evaluate the immunocontraceptive efficacy of ZP glycoproteins. The recombinant dZP2 (rec-dZP2) revealed a 70 kDa band corresponding to the full length transcript, as well as several low molecular mass fragments in western blot analysis. In addition to rec-dZP2, E. coli expressed recombinant dog ZP glycoprotein 3 (rec-dZP3), which has also been evaluated for its efficacy to block fertility in a homologous system. Three groups of female dogs (n = 4 per group) were immunized with rec-dZP2 conjugated to diphtheria toxoid (rec-dZP2-DT), rec-dZP3 conjugated to DT (rec-dZP3-DT) and DT alone. Immunization of female dogs with rec-dZP2-DT and rec-dZP3-DT led to generation of antibodies against the respective ZP proteins as well as to DT. Subsequent to mating, the four female dogs immunized with rec-dZP2-DT all conceived, which is indicative of failure of the anti-rec-dZP2 antibodies to block fertility. In the group of dogs immunized with rec-dZP3-DT, three of four animals did not conceive when mated with males of proven fertility. The block in fertility was associated with anti-dZP3 antibody titres. Ovarian histopathology revealed that the block in fertility in the group immunized with rec-dZP3-DT is probably manifested by inhibition in the development of follicles and is due to atretic changes in the zona pellucida. These results, although preliminary, indicate that immunization with dZP3 may be a feasible proposition to control dog populations provided that adequate antibody titres are achieved.

Novel human testis-specific cDNA: molecular cloning, expression and immunobiological effects of the recombinant protein.

A differential display-polymerase chain reaction was employed to obtain a testis-specific cDNA fragment. On screening the human testis-(lambda)gt10-cDNA library with testis-specific cDNA fragment, a novel cDNA encoding for a sperm antigen, designated TSA-1, was obtained. It has a novel open reading frame (ORF) of 471 base pairs encoding for 156 amino acids. The computer generated translated protein has a calculated molecular mass of 17.4 kDa and contains a potential N-glycosylation site at amino acids 122-124. The hydrophilicity analysis of the amino acid sequence suggested that this protein is a membrane-anchored peptide. Extensive analysis for tissue-specificity by Northern blots and RT-PCR-Southern blot procedures using various human tissues indicated that TSA-1 was specifically expressed only in the human testis. Based on the results of in vitro transcription and translation experiments, the TSA-1 (ORF) was subcloned into pGEX-6P-3 vector and expressed using the glutathione S-transferase gene fusion system. Antibodies (Ab) against the purified recombinant protein specifically recognized the approximately 17 kDa recombinant TSA-1, and a approximately 24 kDa band in human sperm extract in the Western blot procedure. The recombinant TSA-1 Ab recognized the acrosomal, equatorial, mid-piece, and tail regions of human sperm cell in indirect immunofluorescence, bound to live human sperm in the immunobeads binding technique (IBT) and caused a significant concentration-dependent inhibition of human sperm acrosome reaction. These findings indicate that the novel sperm-specific recombinant TSA-1 has a role in sperm function and may have applications in the development of a contraceptive vaccine, and in the specific diagnosis and treatment of male infertility.

Artefactual skin disease in children and adolescents.

As in adults, artefactual skin disease in children and adolescents has heterogenous presentations with multifactorial aetiology. We report a series of 32 young patients aged 8-16 years. There were 24 females and 8 males. In over half of the cases the lesions were on the head and neck. The types of lesions encountered included physical injury producing grazing, erosions and deep ulcers, chemical and thermal burns, hair cutting and shaving and skin painting. We discuss the approach that we believe should be taken with these patients, emphasizing the role of dermatology-psychiatry liaison in their management. Of great importance is the avoidance of confrontation of the patient but the clear exposition of the nature of the problem to the parents. The major aim should be to have the family accept the need for expert psychiatric assistance.

Dog zona pellucida glycoprotein-3 (ZP3): expression in Escherichia coli and immunological characterization.

An internal fragment (978 bp) corresponding to the dog zona pellucida glycoprotein-3 (DZP3), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was amplified by polymerase chain reaction from a full-length cDNA clone. The amplified SacI and PstI restricted fragment was cloned in-frame downstream of the T5 promoter under lac operator control for expression in the pQE-30 vector. Recombinant DZP3 (rec-DZP3) was expressed as a polyhistidine fusion protein in Escherichia coli. Optimum expression of rec-DZP3 was observed at 1.0 mM isopropyl-beta-D-thiogalactopyronoside. Immunoblots with a murine monoclonal antibody, MA-451 (raised against porcine ZP3beta-a homologue of DZP3 and cross-reactive with dog zona pellucida), revealed a major band of 42 kDa. Localization studies revealed that the recombinant protein was present only in an insoluble intracellular fraction. Further optimization studies revealed that the level of expression of rec-DZP3 was significantly higher in Luria broth medium containing glycerol rather than glucose and maximum expression was observed when cultures were induced during the mid-log phase of growth. Batch fermentation with glycerol as the carbon source yielded 30 mg/L of rec-DZP3 compared to 4 mg/L from a shake flask culture. Immunization of two male rabbits with Ni-NTA-purified rec-DZP3 and two female dogs with the rec-DZP3 conjugated to diphtheria toxoid generated high antibody titers against rec-DZP3 as determined by enzyme-linked immunosorbent assay. Rabbit immune serum reacted with porcine ZP3beta but failed to react with porcine ZP3alpha in a Western blot. Moreover, antisera when tested by indirect immunofluorescence on dog ovarian sections showed positive fluorescence with zona pellucida. The availability of rec-DZP3 will help in evaluating its efficacy for fertility regulation in stray dogs.