Chemical profiling of the street cocktail drug 'nyaope' in South Africa using GC-MS II: Stability studies of the cannabinoid, opiate and antiretroviral components during sample storage.

Nyaope is a mixture of low grade heroin, cannabis products, antiretroviral drugs and other materials added as bulking agents. It is a highly physically additive mixture which is smoked by users. As part of the development of a method for the analysis and profiling of nyaope this study evaluates the stability of the cannabinoid, opiate and antiretroviral components of nyaope during storage following seizure. Conditions used were those typically used for storage of drug seizures: in a desiccator in a refrigerator, in a desiccator in the dark at room temperature, in a desiccator in daylight at room temperature and ambient room temperature in the dark in a cabinet used for storage of drug seizures. Street samples of cannabis (Δ9-tetrahydrocannabinol) and heroin were mixed with efavirenz and nevirapine tablets to mimic a nyaope sample. The samples were homogenized and transferred into glass bottles and extracted with tertiary butyl alcohol (tBuOH) and analysed by gas chromatography - mass spectrometry (GC-MS) after the powdered drugs had been stored for intervals of 0 and 24 h under each storage condition. The data obtained indicates that the target drug components in nyaope samples decompose and that for comparison purposes the drug extracts should be prepared in tBuOH immediately after seizure because of the decomposition of the drug components during storage prior to extraction and analysis. The implications of this work are that law enforcement agencies dealing with nyaope and wanting to compare drug samples may need to change their practice around how the drug is handled after seizure but prior to analysis.

Chemical profiling of the street cocktail drug 'nyaope' in South Africa using GC-MS I: Stability studies of components of 'nyaope' in organic solvents.

Nyaope, a street drug commonly found in South Africa, is a mixture of low grade heroin, cannabis products, antiretroviral drugs and other materials added as cutting agents. It is a highly physiologically addictive substance which is smoked by users. Little work has been published on the chemical analysis and profiling of nyaope. Sample preparation prior to chromatographic or spectrometric analysis normally involves dissolution of the sample in an organic solvent. This study determined the most suitable organic solvent in which the common components of nyaope, namely Δ9-tetrahydrocannabinol, diamorphine, caffeine, dextromethorphan, phenacetin and the antiretrovirals efavirenz and nevirapine, which have different chemical characteristics, are stable during extraction and prior to analysis of nyaope samples i.e. autosampler stability. Street samples of cannabis (Δ9-tetrahydrocannabinol), heroin (diamorphine) and antiretrovirals were mixed to mimic a nyaope sample and dissolved in the organic solvents dichloromethane, ethanol, ethyl acetate, hexane, isopropanol and tertiary butyl alcohol. Analysis was performed after intervals of 0, 1, 6, 8, 24, 48 and 72h, prior to analysis by gas chromatography-mass spectrometry. Tertiary butyl alcohol resulted in the most stable extracts of the main nyaope components after 72h of storage. The analysis was also repeated on actual street samples of nyaope. These results show that tertiary butyl alcohol is a suitable solvent for sample preparation for the identification, comparison and profiling of nyaope samples.

Burkitt's lymphoma-associated c-Myc mutations converge on a dramatically altered target gene response and implicate Nol5a/Nop56 in oncogenesis.

Burkitt's lymphomas (BLs) acquire consistent point mutations in a conserved domain of Myc, Myc Box I. We report that the enhanced transforming activity of BL-associated Myc mutants can be uncoupled from loss of phosphorylation and increased protein stability. Furthermore, two different BL-associated Myc mutations induced similar gene expression profiles independently of T58 phosphorylation, and these profiles are dramatically different from MycWT. Nol5a/Nop56, which is required for ribosomal RNA methylation, was identified as a gene hyperactivated by the BL-associated Myc mutants. We show that Nol5a is necessary for Myc-induced cell transformation, enhances MycWT-induced cell transformation and increases the size of MycWT-induced tumors. Thus, Nol5a expands the link between Myc-induced regulation of nucleolar target genes, which are rate limiting for cell transformation and tumor growth.

Forensic Analysis of Cathinones.

In the past decade there has been a significant increase in the popularity of synthetic cathinones in the illegal drug market. They have been easily available from Internet-based vendors as well as at "head shops" and "smart shops". The recent prominence of synthetic cathinones can be attributed to their stimulatory properties similar to those of amphetamines. This paper provides a review on the current popular cathinone derivatives, their history and prevalence in the illegal drug market, legislation of these drugs in various countries, pharmacology, toxicology, and metabolism studies, analysis of toxicology samples (blood, urine, and hair) and criminalistic samples (seized, purchased via the Internet, and synthesized). From the reviewed literature, it is concluded that the products sold as "legal highs" do not only contain cathinone but also cathinone derivatives, and adulterants such as caffeine, lidocaine, and inorganic materials. Full toxicity data is currently unavailable for this drug class and hence more research is required with regard to their analysis and metabolism. Moreover, clandestine chemists are constantly synthesizing new derivatives and hence forensic chemists often need to synthesize and characterize these drugs to confirm the identity of the seized samples. This is expensive as well as time-consuming. Therefore, there is a need for national and international collaboration among forensic chemists to overcome this difficulty.

Specific regulation of mRNA cap methylation by the c-Myc and E2F1 transcription factors.

Methylation of the mRNA 5' guanosine cap is essential for efficient gene expression. The 5' methyl cap binds to eIF4E, which is the first step in the recruitment of mRNA to the 40S ribosomal subunit. To investigate whether mRNA cap methylation is regulated in a gene-specific manner, we established a method to detect the relative level of cap methylation on specific mRNAs. We found that two transcription factors, c-Myc and E2F1, induce cap methylation of their transcriptional target genes, and therefore, c-Myc and E2F1 upregulate gene expression by simultaneously inducing transcription and promoting translation. c-Myc-induced cap methylation is greater than transcriptional induction for the majority of its target genes, indicating that this is a major mechanism by which Myc regulates gene expression.

An N-Myc truncation analogous to c-Myc-S induces cell proliferation independently of transactivation but dependent on Myc homology box II.

Myc promotes both normal cell proliferation and oncogenic transformation through the activation and repression of target genes. The c-Myc-S protein is a truncated form of c-Myc that is produced in some cells from translation initiation at an internal AUG codon. We report that c-Myc-S and a similar truncated form of N-MycWT can fully rescue the proliferation defect in myc-null fibroblasts, but rescue is dependent on the highly conserved Myc homology box II (MBII). Global gene expression studies show that the N-Myc equivalent of c-Myc-S is defective for virtually all transcriptional activation of Myc target genes but remains active for the majority of transcriptional repression. Repression by Myc-S is dependent on MBII, but it does not bind to several known nuclear cofactors. These data suggest that repression by Myc involves recruitment of a novel MBII-dependent cofactor.

Displacement of nuclear fragments into the vitreous complicating phacoemulsification surgery in the UK: clinical features, outcomes and management.

AIMS: To study the clinical features, management and outcomes of displacement of nuclear fragments into the vitreous (DNFV) complicating phacoemulsification in the UK. METHODS: Cases were collected prospectively between March 2003 and March 2004 inclusive by active surveillance through the British Ophthalmological Surveillance Unit. Details were obtained using incidence questionnaires and follow-up questionnaires after 6 months. The data used in this paper were obtained from the follow-up questionnaires. RESULTS: 610 cases were confirmed during the reporting period, for which 387 follow-up questionnaires were received. In 67% of cases, a best-corrected visual acuity of 6/12 or better was reported at final follow-up. The most common immediate sequelae of DNFV were intraocular inflammation (85%), corneal oedema (55%) and an intraocular pressure >30 mm Hg (34%). Pars plana vitreolensectomy was used in 97% of cases, and fragmatome ultrasound lensectomy was used in over half of these procedures. The median time from cataract surgery to pars plana vitrectomy for the removal of DNFV was 3 days, and most patients (68%) had vitrectomy within 1 week of the first procedure. An intraocular lens had been inserted at the time of the complicated cataract surgery (defined as a "primary IOL") in 40% of cases, and over three-quarters of these primary IOLs were subsequently removed (with or without a replacement IOL). Only 67% of eyes that had a primary IOL inserted after DNFV were pseudophakic at final follow-up, in contrast with 79% of eyes that were left aphakic after DNFV (p = 0.008). A best-corrected visual acuity of 6/60 or worse was reported in 14% of cases at final follow-up and was most commonly associated with persistent uveitis, corneal oedema, cystoid macular oedema, optic atrophy or retinal detachment. CONCLUSIONS: DNFV complicating cataract surgery was followed by a secondary procedure in 97% of cases. About three-quarters (77%) of "primary IOLs" inserted at the time of DNFV were subsequently removed or replaced, and eyes that had received a primary IOL had significantly less chance of being pseudophakic at final follow-up than eyes that had been left primarily aphakic at the time of the complicated cataract surgery. The delay before secondary intervention was shorter, fragmatome ultrasound lensectomy use was higher, and the retinal detachment rate was lower than in previous studies. Affected eyes still had a worse outcome in terms of visual acuity compared with eyes after uncomplicated cataract surgery.

Displacement of nuclear fragments into the vitreous complicating phacoemulsification surgery in the UK: incidence and risk factors.

AIMS: To study the epidemiology and risk factors contributing to displacement of nuclear fragments into the vitreous (DNFV) complicating phacoemulsification in the UK. METHODS: Cases were collected prospectively between March 2003 and March 2004 by active surveillance through the British Ophthalmological Surveillance Unit (BOSU). Case-control analysis of risk factors was performed by visiting 10 randomly selected centres using a total of 521 cases of uncomplicated phacoemulsification. Validation analysis to assess under-reporting was performed in a total of 13 randomly selected units. RESULTS: 610 cases of DNFV were confirmed during the reporting period. The estimated incidence of DNFV was 0.19-0.28%. The group with complications was significantly older than the control group (mean 76.8 vs 74.3 years: p<0.001). Significant preoperative risk factors were posterior synechiae (5.1% vs 2.2%), incomplete pupil dilation (59.5% vs 8.8%), pseudoexfoliation (5.6% vs 1.4%) and previous vitrectomy (7.8% vs 2.2%). Significant operative variables related to surgical experience, topical (14.3% vs 3.1%) and sub-Tenon's (51.4% vs 37.2%) anaesthesia, and requirement for vision blue (trypan blue ophthalmic solution) (13.7% vs 2.4%). CONCLUSIONS: The estimated incidence of DNFV during phacoemulsification surgery in the UK is two or three per 1000 operations. Risk factors have been identified that should help to guide case selection for phacoemulsification surgery and modify techniques.

Presumed infectious endophthalmitis following cataract surgery in the UK: a case-control study of risk factors.

AIMS: To study risk factors for presumed infectious endophthalmitis complicating cataract surgery in the United Kingdom. METHODS: Two hundred and fourteen clinically diagnosed patients with presumed infectious endophthalmitis were compared with 445 control patients throughout the United Kingdom in a prospective case-control study. The cases were identified through the British Ophthalmological Surveillance Unit reporting card system. Control patients undergoing cataract surgery from 13 'control centres' throughout the United Kingdom were selected randomly. Risk factors were identified by univariate and multivariate logistic regression analyses. Pertinent variables relating to the cataract extraction procedure, antimicrobial prophylaxis, ophthalmic and medical history were analysed with regard to postoperative infection. RESULTS: Statistically significant risk factors in the multivariate model included inpatient cataract surgery (P=0.001), surgery in dedicated eye theatres (P<0.001), consultant grade surgeon (compared to registrar) (P=0.001), posterior capsule tear during cataract surgery (P=0.001). The use of face masks by the scrub nurse and surgeon during cataract surgery (P<0.001) and the administration of subconjunctival antibiotics at the end of surgery (P<0.001) were protective against postoperative infection. CONCLUSIONS: In order to minimise the risk of postoperative endophthalmitis we would recommend the wearing of face masks by the surgeon and scrub nurse during cataract surgery and subconjunctival antibiotics at the end of surgery.

E-cadherin repression contributes to c-Myc-induced epithelial cell transformation.

c-Myc oncoprotein is overexpressed in a significant proportion of human epithelial cancers, and experimental overexpression of c-Myc in epithelial cells promotes tumour formation. However, it is not known how c-Myc promotes epithelial cell tumour formation. We report that c-Myc expression in human mammary epithelial cells induces a dramatic change in cell morphology, with some characteristics of an 'epithelial to mesenchymal transition'. E-cadherin expression is repressed by a post-transcriptional mechanism in cells expressing c-Myc. Furthermore, E-cadherin repression is necessary for c-Myc-induced cell transformation.

Transcriptional activation by the Myc oncoprotein.

The Myc transcription factor functions as a downstream effector of most mitogenic signals. Myc is synthesized rapidly in response to extracellular mitogenic signals, and blocking Myc induction abolishes or at least severely attenuates any mitogenic response. Furthermore, ectopic Myc expression can often bypass the requirement for extracellular signals for entry into S phase. Thus, the Myc transcription factor is both necessary and in many ways sufficient to promote the growth of diverse cell types. Given this potent biological activity, it is not surprising that mutations in the myc gene are among the most frequent in human and animal cancers. Understanding the molecular basis of Myc function has been a central issue in the fields of cancer biology and signal transduction for 20 years.

The use of vapour phase ultra-violet spectroscopy for the analysis of arson accelerants in fire scene debris.

A method has been developed for the analysis of arson accelerants in fire scene debris by vapour phase ultra-violet (UV) spectroscopy. The method is rapid, inexpensive, simple to use and is sufficiently sensitive and discriminating to be of use for the analysis of crime scene samples. Application to casework samples is described. On occasion, the method offers additional information to that which can be obtained by gas chromatography-flame ionisation detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS) and represents a useful adjunct to these techniques. In addition, the method offers advantages where the use of GC-MS analysis of arson accelerants in fire scene debris is not a practical proposition.

The ATM-related domain of TRRAP is required for histone acetyltransferase recruitment and Myc-dependent oncogenesis.

The ATM-related TRRAP protein is a component of several different histone acetyltransferase (HAT) complexes but lacks the kinase activity characteristic of other ATM family members. We identified a novel function for this evolutionarily conserved domain in its requirement for the assembly of a functional HAT complex. Ectopic expression of TRRAP protein with a mutation in the ATM-related domain inhibits Myc-mediated oncogenic transformation. The Myc-binding region of TRRAP maps to a separable domain, and ectopic expression of this domain inhibits cell growth. These findings demonstrate that the ATM-related domain of TRRAP forms a structural core for the assembly and recruitment of HAT complexes by transcriptional activators.

E2F transcriptional activation requires TRRAP and GCN5 cofactors.

The E2F family of transcription factors regulates the temporal transcription of genes involved in cell cycle progression and DNA synthesis. E2F transactivation is antagonized by retinoblastoma protein (pRb), which recruits chromatin-remodeling proteins such as histone deacetylases and SWI.SNF complexes to the promoter to repress transcription. We hypothesized that E2F proteins must reverse the pRb-imposed chromatin structure to stimulate transcription. If this is true, E2F proteins should recruit proteins capable of histone acetylation. Here we map the E2F-4 transactivation domain and show that E2F-1 and E2F-4 transactivation domains bind the acetyltransferase GCN5 and cofactor TRRAP in vivo. TRRAP and GCN5 co-expression stimulated E2F-mediated transactivation, and c-Myc repressed E2F transactivation dependent on an intact TRRAP/GCN5 binding motif. The transactivation domain of E2F-4 recruited proteins with significant histone acetyltransferase activity in vivo, and this activity required catalytically active GCN5. E2F-4 proteins with subtle mutations in the transactivation domain exhibited a positive correlation among transcriptional activation and GCN5 and TRRAP binding capacity and associated acetyltransferase activity. We conclude that E2F stimulates transcription by recruiting acetyltransferase activity and the essential cofactors GCN5 and TRRAP. These results provide a mechanism for E2F transcription factors to overcome pRb-mediated dominant repression of transcription.

Systemic non-Hodgkin's lymphoma presenting as a serpiginous choroidopathy: report of a case and review of the literature.

Intraocular lymphoma is a rare tumour, the two main types being primary central nervous system non-Hodgkin's lymphoma (CNS-NHL) and, less commonly, systemic lymphoma that has spread to involve the eye. We present a case of a systemic NHL resulting in a serpiginous choroidopathy. To our knowledge, this presentation has not previously been reported. The diagnosis of this case was made based on the temporal relationship between the clinical activity of the choroidopathy and its response to chemotherapy. Diagnosis of intraocular lymphoma on vitreous biopsy is notoriously difficult, and this case is presented along with a review of the literature regarding vitreous biopsy and diagnostic techniques.

Complementation of Myc-dependent cell proliferation by cDNA expression library screening.

The targeted knockout of the c-myc gene from rat fibroblasts leads to a stable defect in cell proliferation. We used complex cDNA libraries expressed from retroviral vectors and an efficient sorting procedure to rapidly select for cDNAs that can restore the growth rate of c-myc deficient cells. All of the biologically active cDNAs contained either c-myc or N-myc, suggesting that no other cellular genes can effectively bypass the requirement for c-myc in fibroblast proliferation. This approach provides a powerful screening method for cell cycle changes in genetically defined systems.

An ATPase/helicase complex is an essential cofactor for oncogenic transformation by c-Myc.

The c-Myc transactivation domain was used to affinity purify tightly associated nuclear proteins. Two of these proteins were identified as TIP49 and a novel related protein called TIP48, both of which are highly conserved in evolution and contain ATPase/helicase motifs. TIP49 and TIP48 are complexed with c-Myc in vivo, and binding is dependent on a c-Myc domain essential for oncogenic activity. A missense mutation in the TIP49 ATPase motif acts as a dominant inhibitor of c-Myc oncogenic activity but does not inhibit normal cell growth, indicating that functional TIP49 protein is an essential mediator of c-Myc oncogenic transformation. The TIP49 and TIP48 ATPase/helicase proteins represent a novel class of cofactors recruited by transcriptional activation domains that function in diverse pathways.

The c-Myc transactivation domain is a direct modulator of apoptotic versus proliferative signals.

We have assayed the oncogenic, proliferative, and apoptotic activities of the frequent mutations that occur in the c-myc gene in Burkitt's lymphomas. Some alleles have a modest (50 to 60%) increase in transforming activity; however, the most frequent Burkitt's lymphoma allele (T58I) had an unexpected substantial decrease in transforming activity (85%). All alleles restored the proliferation function of c-Myc in cells that grow slowly due to a c-myc knockout. There was discordance for some alleles between apoptotic and oncogenic activities, but only the T58A allele had elevated transforming activity with a concomitant reduced apoptotic potential. We discovered a novel missense mutation, MycS71F, that had a very low apoptotic activity compared to wild-type Myc, yet this mutation has never been found in lymphomas, suggesting that there is no strong selection for antiapoptotic c-Myc alleles. MycS71F also induced very low levels of cytochrome c release from mitochondria, suggesting a mechanism of action for this mutation. Phosphopeptide mapping provided a biochemical basis for the dramatically different biological activities of the transformation-defective T58I and transformation-enhanced T58A c-Myc alleles. Furthermore, the antiapoptotic survival factor insulin-like growth factor 1 was found to suppress phosphorylation of T58, suggesting that the c-Myc transactivation domain is a direct target of survival signals.

The essential cofactor TRRAP recruits the histone acetyltransferase hGCN5 to c-Myc.

The c-Myc protein functions as a transcription factor to facilitate oncogenic transformation; however, the biochemical and genetic pathways leading to transformation remain undefined. We demonstrate here that the recently described c-Myc cofactor TRRAP recruits histone acetylase activity, which is catalyzed by the human GCN5 protein. Since c-Myc function is inhibited by recruitment of histone deacetylase activity through Mad family proteins, these opposing biochemical activities are likely to be responsible for the antagonistic biological effects of c-Myc and Mad on target genes and ultimately on cellular transformation.