Mechanistic insight into the fluorescence activity of forensic fingerprinting reagents.

Fingerprint detection is still the primary investigative technique for deciphering criminal inquiries and identifying individuals. The main forensic fingerprinting reagents (FFRs) currently in use can require multiple treatment steps to produce fingerprints of sufficient quality. Therefore, the development of new, more effective FFRs that require minimal chemical treatment is of great interest in forensic chemistry. In this work, prudently crafted density functional theory and time-dependent density functional theory calculations are utilized to derive mechanistic insight into the optical activity of the non-fluorescent product of ninhydrin, diketohydrindylidenediketohydrindamine (DYDA), and fluorescent product of DFO (1,8-diazafluoren-9-one). We investigate various protonation sites to gain an understanding of isomeric preference in the solid-state material. A relaxed scan of a single torsion angle rotation in the S1 minimized geometry of the O-protonated DYDA isomer suggests a conical intersection upon ∼10° rotation. We show that the absence of a rigid hydrogen-bonded network in the crystal structure of DYDA supports the hypothesis of torsion rotation, which leads de-excitation to occur readily. Conversely, for the fluorescent DFO product, our calculations support an avoided crossing suggestive of a non-radiative mechanism when the torsion angle is rotated by about ∼100°. This mechanistic insight concurs with experimental observations of fluorescence activity in DFO and may aid the photophysical understanding of poorly visualized fingerprints due to weak fluorescence. We show that identifying suggestive avoided crossings via the method described here can be used to initialize thoughts toward the computational design of FFRs.

A comparative evaluation of the disulfur dinitride process for the visualisation of fingermarks on metal surfaces.

The disulfur dinitride process for fingermark visualisation was first reported a decade ago, with promising results obtained for a range of materials including metals. However, the friction sensitive nature of the material and difficulty of synthesis made routine use difficult. Many of these issues have since been addressed, making equipment and chemicals available to build an understanding of how the effectiveness of disulfur dinitride compares to other fingermark visualisation processes currently used on metal surfaces. This enables more informed advice to be given on selection of processes for treatment of metal items, an area of operational interest that encompasses weapons used in violent crime and the increasing incidence in metal theft. This paper reports a comparative study into the effectiveness of disulfur dinitride, cyanoacrylate fuming, vacuum metal deposition, gun blueing and wet powder suspensions on brass, bronze, copper and stainless steel. Experiments were conducted with the surfaces exposed to a range of environments including long term ageing, water/detergent washing, acetone washing and high temperature exposure. The results indicate that disulfur dinitride is an effective process for fingermark visualisation on metal surfaces, including those exposed to adverse environments, and may offer potential improvements over existing processes for those surfaces. Further work, including pseudo-operational trials, is recommended.

The synthesis, full characterisation and utilisation of template-free silica sodalite, a novel polymorph of silica.

Empty glass: Subjecting ethylene glycol silica sodalite to heat (680 degrees C) under a nitrogen atmosphere (i) successfully removes the templating agent to give cubic silica sodalite, which, upon consequent heating under an oxygen atmosphere (ii), transforms into a rhombohedral form of the empty sodalite, in effect a novel polymorph of silica.

Functional correction of T cells derived from patients with the Wiskott-Aldrich syndrome (WAS) by transduction with an oncoretroviral vector encoding the WAS protein.

T-cell dysfunction is thought to be central to the immunodeficiency state seen in patients with the Wiskott-Aldrich syndrome (WAS). Aspects of the WAS phenotype have been corrected in other cell types on introduction of the normal WAS protein (WASP), but the potential for correction of the T-cell defects has not been evaluated. Here we demonstrate that an oncoretroviral vector encoding WASP and green fluorescent protein (GFP), and pseudotyped with the RD114 envelope protein, efficiently transduces primary human T cells derived from WAS patients. Transcription initiated at the oncoretroviral long terminal repeat (LTR) results in levels of WASP that, while lower than those seen in normal control T cells, resulted in correction of the deficient proliferative response to T-cell receptor (TCR) stimulation characteristic of WAS. IL2 secretion after TCR stimulation was partially corrected. Control primary T cells transduced with the same vector responded normally to TCR stimulation, and showed no increase in WASP expression. The demonstration that correction of T cell defects can be achieved by gene transfer supports continued efforts to develop gene therapy for WAS.

Pre-clinical evaluation of an in vitro selection protocol for the enrichment of transduced CD34+ cell-derived human dendritic cells.

The efficient genetic modification of CD34+ cell-derived dendritic cells (DC) will provide a significant advancement towards the development of immunotherapy protocols for cancer, autoimmune disorders and infectious diseases. Recent reports have described the transduction of CD34+ cells via retrovirus- and lentivirus-based gene transfer vectors and subsequent differentiation into functional DC. Since there is significant apprehension regarding the clinical uses of HIV-based vectors, in this report, we compare a murine leukemia virus (MLV)- and a human immunodeficiency virus (HIV)-based bicistronic vector for gene transfer into human CD34+ cells and subsequent differentiation into mature DC. Each vector expressed both EGFP and the dominant selectable marker DHFR(L22Y) allowing for the enrichment of marked cells in the presence of the antifolate drug trimetrexate (TMTX). Both MLV-based and HIV-based vectors efficiently transduced cytokine mobilized human peripheral blood CD34+ cells. However, in vitro expansion and differentiation in the presence of GM-CSF, TNF-alpha, Flt-3L, SCF and IL-4 resulted in a reduction in the percentage of DC expressing the transgene. Selection with TMTX during differentiation increased the percentage of marked DC, resulting in up to 79% (MLV vector) and up to 94% (lentivirus-vector) transduced cells expressing EGFP without loss of DC phenotype. Thus, MLV-based vectors and in vitro selection of transduced human DC show great promise for immunotherapy protocols.

Engraftment of NOD/SCID mice with human CD34(+) cells transduced by concentrated oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein.

Oncoretrovirus vectors pseudotyped with the feline endogenous retrovirus (RD114) envelope protein produced by the FLYRD18 packaging cell line have previously been shown to transduce human hematopoietic progenitor cells with a greater efficiency than similar amphotropic envelope-pseudotyped vectors. In this report, we describe the production and efficient concentration of RD114-pseudotyped murine leukemia virus (MLV)-based vectors. Following a single round of centrifugation, vector supernatants were concentrated approximately 200-fold with a 50 to 70% yield. Concentrated vector stocks transduced prestimulated human CD34(+) (hCD34(+)) cells with approximately 69% efficiency (n = 7, standard deviation = 4.4%) using a single addition of vector at a low multiplicity of infection (MOI = 5). Introduction of transduced hCD34(+) cells into irradiated NOD/SCID recipients resulted in multilineage engraftment with long-term transgene expression. These data demonstrate that RD114-pseudotyped MLV-based vectors can be efficiently concentrated to high titers and that hCD34(+) cells transduced with concentrated vector stocks retain in vivo repopulating potential. These results highlight the potential of RD114-pseudotyped oncoretrovirus vectors for future clinical implementation in hematopoietic stem cell gene transfer.

RD114-pseudotyped oncoretroviral vectors. Biological and physical properties.

Limited functional expression of the viral envelope receptor is a recognized barrier to efficient oncoretroviral mediated gene transfer. To circumvent this barrier we evaluated a number of envelope proteins with respect to gene transfer efficiency into primitive human hematopoietic stem cell populations. We observed that oncoretroviral vectors pseudotyped with the envelope protein of feline endogenous virus (RD114) could efficiently transduce human repopulating cells capable of establishing multilineage hematopoiesis in immunodeficient mice after a single exposure to RD114-pseudotyped vector. Comparable rates of gene transfer with amphotropic and GALV-pseudotyped vectors have been reported, but only after multiple exposures to the viral supernatant. Oncoretroviral vectors pseudotyped with the RD114 or the amphotropic envelopes had similar stability in vitro, indicating that the increased efficiency in gene transfer is at the receptor level likely due to increased receptor expression or an increased receptor affinity for the RD114 envelope. We also found that RD114-pseudotype vectors can be efficiently concentrated, thereby removing any adverse effects of the conditioned media to the long-term repopulating potential of the target human hematopoietic stem cell. These studies demonstrate the potential of RD114-pseudotyped vectors for clinical use.

Long-term engraftment of nonobese diabetic/severe combined immunodeficient mice with human CD34+ cells transduced by a self-inactivating human immunodeficiency virus type 1 vector.

Human hematopoietic cells with in vivo repopulating potential hold much promise as a target for corrective gene transfer for numerous inherited or acquired hematopoietic disorders. Here we demonstrate long-term hematopoietic reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice with human CD34(+) cells transduced by an HIV-1-based self-inactivating (SIN) vector encoding the enhanced green fluorescent protein (EGFP). Human umbilical cord CD34(+) cells were transduced (up to 76%) at a low multiplicity of infection (MOI of 5) in the absence of cytokine prestimulation. Introduction of transduced hCD34(+) cells into irradiated recipients resulted in multilineage engraftment and stable transgene expression for 18 weeks posttransplantation. Bone marrow from transplanted mice contained up to 50% hCD45(+) cells and up to 63% hCD45(+)/EGFP(+) cells. Analysis of extramedullar splenic reconstitution showed up to 13% hCD45(+) cells and up to 41% hCD45(+)/EGFP(+) cells. Analysis of human progenitor cells isolated from bone marrow of recipient animals showed equivalent percentages of EGFP(+) colony-forming cells (CFCs) by fluorescence microscopy and by PCR analysis of provirus sequences, indicating minimal transgene silencing in vivo. These findings demonstrate the utility of lentivirus-based SIN vectors for hematopoietic stem cell gene transfer and provide strong support for their future clinical evaluation.

Self-selection by genetically modified committed lymphocyte precursors reverses the phenotype of JAK3-deficient mice without myeloablation.

Janus kinase 3 (JAK3) is an essential component of cytokine receptor signal transduction pathways required for normal lymphocyte development and function. JAK3 deficiency in both mice and humans results in severe combined immunodeficiency (SCID) and increased susceptibility to opportunistic infections. We have previously shown that JAK3 gene transfer into irradiated recipients could restore immune function. However, since this toxic conditioning would be undesirable for infants in a clinical application, we have tested whether immune function could be restored in nonmyeloablated JAK3-deficient (-/-) mice. Murine JAK3 retroviral vectors were transduced into hematopoietic stem cells from the livers of newborn JAK3(-/-) mice. These cells were then injected intraperitoneally into nonirradiated JAK3(-/-) neonates. Transduced cells were detectable in these mice at time points 4 to 6 months after injection and resulted in significant correction of T and B lymphocyte numbers and circulating immunoglobulin (Ig) levels. After immune challenge with a dose of influenza A virus that was lethal to nonmanipulated JAK3(-/-) mice, mice injected with transduced cells showed development of circulating virus-specific IgG and enhanced survival. This work shows that the large selective advantage for JAK3-corrected lymphoid cells may be sufficient to overcome the need for myeloablative conditioning in JAK3 gene therapy protocols.

Highly efficient gene transfer into cord blood nonobese diabetic/severe combined immunodeficiency repopulating cells by oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein.

Limited expression of the amphotropic envelope receptor is a recognized barrier to efficient oncoretroviral vector-mediated gene transfer. Human hematopoietic cell lines and cord blood-derived CD34(+) and CD34(+), CD38(-) cell populations and the progenitors contained therein were transduced far more efficiently with oncoretroviral particles pseudotyped with the envelope protein of feline endogenous virus (RD114) than with conventional amphotropic vector particles. Similarly, human repopulating cells from umbilical cord blood capable of establishing hematopoiesis in immunodeficient mice were efficiently transduced with RD114-pseudotyped particles, whereas amphotropic particles were ineffective at introducing the proviral genome. After only a single exposure of CD34(+) cord blood cells to RD114-pseudotyped particles, all engrafted nonobese diabetic/severe combined immunodeficiency mice (15 of 15) contained genetically modified human bone marrow cells. Human cells that were positive for enhanced green fluorescent protein represented as much as 90% of the graft. The use of RD114-pseudotyped vectors may be advantageous for therapeutic gene transfer into hematopoietic stem cells. (Blood. 2000;96:1206-1214)

Purification and characterization of an extracellular feruloyl esterase from the thermophilic anaerobe Clostridium stercorarium.

Feruloyl esterases act as accessory enzymes for the complete saccharification of plant cell wall hemicelluloses. Although many fungal feruloyl esterases have been purified and characterized, few bacterial phenolic acid esterases have been characterized. This study shows the extracellular production of a feruloyl esterase by the thermophilic anaerobe Clostridium stercorarium when grown on birchwood xylan. The feruloyl esterase was purified 500-fold in successive steps involving ultrafiltration, preparative isoelectric focusing and column chromatography by anion exchange, gel filtration and hydrophobic interaction. The purified enzyme released ferulic, rho-coumaric, caffeic and sinapinic acid from the respective methyl esters. The purified enzyme also released ferulic acid from a de-starched wheat bran preparation. At pH 8.0 and 65 degrees C, the Km and Vmax values for the hydrolysis of methyl ferulate were 0.04 mmol l-l and 131 micromol min-1 mg-1, respectively; the respective values for methyl coumarate were 0.86 mmol l-l and 18 micromol min-1 mg-1. The purified feruloyl esterase had an apparent mass of 33 kDa under denaturing conditions and showed optimum activity at pH 8.0 and 65 degrees C. At a concentration of 5 mmol l-l, the ions Ca2+, Cu2+, Co2+ and Mn2+ reduced the activity by 70-80%.

Human cord blood CD34+CD38- cell transduction via lentivirus-based gene transfer vectors.

The efficient transfer and sustained expression of a transgene in human hematopoietic cells with in vivo repopulating potential would provide a significant advancement in the development of protocols for the treatment of hematopoietic diseases. Recent advances in the ability to purify and culture hematopoietic cells with the CD34+CD38- phenotype and with in vivo repopulating potential from human umbilical cord blood provide a direct means of testing the ability of transfer vectors to transduce these cells. Here we demonstrate the efficient transduction and expression of enhanced green fluorescent protein (EGFP) in human umbilical cord-derived CD34+CD38- cells, without prestimulation, using a lentivirus-based gene transfer system. Transduced CD34+CD38- cells cultured in serum-free medium supplemented with SCF, Flt-3, IL-3, and IL-6 maintained their surface phenotype for 5 days and expressed readily detectable levels of the transgene. The average transduction efficiency of the CD34+CD38- cells was 59 +/- 7% as determined by flow cytometry. Erythroid and myeloid colonies derived from transduced CD34+CD38- cells were EGFP positive at a high frequency (66 +/- 9%). In contrast, a murine leukemia virus-based vector transduced the CD34+CD38- cells at a low frequency (<4%). These results demonstrate the utility of lentiviral-based gene transfer vectors in the transduction of primitive human hematopoietic CD34+CD38- cells.

High-efficiency transduction and long-term gene expression with a murine stem cell retroviral vector encoding the green fluorescent protein in human marrow stromal cells.

Bone marrow stromal cells (MSCs) are unique mesenchymal cells that have been utilized as vehicles for the delivery of therapeutic proteins in gene therapy protocols. However, there are several unresolved issues regarding their potential therapeutic applications. These include low transduction efficiency, attenuation of transgene expression, and the technical problems associated with drug-based selection markers. To address these issues, we have developed a transduction protocol that yields high-level gene transfer into human MSCs, employing a murine stem cell virus-based bicistronic vector containing the green fluorescent protein (GFP) gene as a selectable marker. Transduction of MSCs plated at low density for 6 hr per day for 3 days with high-titer viral supernatant resulted in a gene transfer efficiency of 80+/-6% (n = 10) as measured by GFP fluorescence. Neither centrifugation nor phosphate depletion increased transduction efficiency. Assessment of amphotropic receptor (Pit-2) expression by RT-PCR demonstrated that all MSCs expressing the receptor were successfully transduced. Cell cycle distribution profiles measured by propidium iodide staining showed no correlation with the susceptibility of MSCs to transduction by the retroviral vector. Human MSCs sequentially transduced with an adenoviral vector encoding the ecotropic receptor and ecotropic retroviral vector encoding GFP demonstrated that all MSCs are susceptible to retroviral transduction. We further showed that both genes of bicistronic vector are expressed for at least 6 months in vitro and that transgene expression did not affect the growth or osteogenic differentiation potential of MSCs. Future studies will be directed toward the development of gene therapy protocols employing this strategy.

Detection of ferulic acid esterase production by Bacillus spp. and lactobacilli.

The production of feruloyl esterase activity by Bacillus spp. and lactobacilli can be detected in an agarplate assay. The assay involves the substitution of the main carbon source in specific agar with ethyl ferulate. A number of Bacillus spp., predominantly B. subtilis strains, were found to exhibit feruloyl esterase activity by this method. Of the examined lactobacilli, Lb. fermentum (NCFB 1751) showed the highest level of ferulic acid esterase activity. The enzyme was released from harvested cells by sonication and showed pH and temperature optima of 6.5 and 30 degrees C respectively.

Development of donor-derived prostate cancer in a recipient following orthotopic heart transplantation.

OBJECTIVE: To report the development of metastatic prostate cancer in a heart transplant recipient without a primary focus in the recipient prostate gland; to present genetic evidence suggesting transplantation of the malignancy from the donor. DESIGN: Histological analysis of donor prostate and recipient prostate and rib. Molecular genetic analysis of prostate and kidney tissue from the donor and peripheral blood leukocytes and rib tissue from the transplant recipient. SETTING: University of Pennsylvania Medical Center. RESULTS: Multiple biopsies of recipient prostate were negative for malignancy but recipient rib contained prostatic adenocarcinoma with osteoblastic bone response. Molecular genetic analysis of recipient rib specimen, which contained both histologically normal and neoplastic cells, was shown to contain a combination of alleles from the donor and recipient at 4 loci. CONCLUSION: Although this is a single case report of an uncommon event, genotyping of polymorphic dinucleotide repeat elements from 4 different chromosomal regions provides strong evidence that the tumor cells arose from donor tissue and were transplanted along with the cardiac allograft.

Isoform-specific regulation of myocardial Na,K-ATPase alpha-subunit in congestive heart failure. Role of norepinephrine.

BACKGROUND: Myocardial ouabain-binding sites and Na,K-ATPase activity are reduced in congestive heart failure (CHF), but the mechanisms by which CHF reduces the Na,K-ATPase remain unknown. We proposed to investigate whether the changes are accompanied by isoform-specific reductions of the Na,K-ATPase alpha-subunit proteins in CHF and whether similar changes could be produced by exogenous norepinephrine administration. METHODS AND RESULTS: CHF was induced in dogs by rapid ventricular pacing at a rate of 225 beats per minute for 8 weeks (protocol 1). A second group of dogs were paced at 100 beats per minute and served as controls. In protocol 2, norepinephrine was infused in normal dogs using a subcutaneous osmotic minipump for 8 weeks. The control dogs received normal saline through the pump. Animals were studied after 8 weeks of pacing or norepinephrine infusion. After the baseline hemodynamics and interstitial norepinephrine concentration had been obtained, the hearts were removed for measuring [3H]ouabain-binding sites and Na,K-ATPase alpha-subunit proteins using isoform-specific monoclonal antibodies. RESULTS: Myocardial [3H]ouabain-binding sites were reduced in dogs with CHF and chronic norepinephrine infusion. The Western blot analysis showed that adult canine hearts possess both alpha 1 and alpha 3 isoforms of the Na,K-ATPase alpha-subunit but not the alpha 2 isoform protein. CHF and NE infusion had no effect on the Na,K-ATPase alpha 1-subunit protein but did reduce the alpha 3 isoform protein significantly. In addition, there was a significant inverse correlation between the amount of myocardial alpha 3 isoform protein and interstitial norepinephrine content in the dogs. In contrast, the specific activity of the sarcolemmal marker 5'-nucleotidase did not differ among the groups of animals. CONCLUSIONS: The reduction of myocardial Na,K-ATPase in CHF is limited to the alpha 3 isoform. Furthermore, because similar changes in myocardial ouabain-binding sites and Na,K-ATPase alpha 3 isoform were produced by chronic norepinephrine infusion, the decrease in the Na,K-ATPase in CHF is most likely mediated via excess sympathetic stimulation.

Burring technique in modern podiatric palliative care.

A review of the literature reveals sparse references regarding the application of burs in the conservative treatment of soft tissues and nails. The most recent publications were in 1946. The authors present an update on the technology and practical application of burs in podiatric medicine, their metallurgy, types, and recommended bur selection for reduction, remodeling, and burnishing of hyperkeratotic tissue and thickened nails.

Reproducibility of quantitative myocardial contrast echocardiography.

To determine whether myocardial contrast echocardiography is quantitatively reproducible, repeated intracoronary injections of sonicated albumin (5%) were performed in eight open chest dogs. Paired injections were performed at baseline, during ischemia produced by ligation of a coronary artery, and during hyperemia induced by intravenous infusion of 0.75 mg/kg body weight of dipyridamole. Contrast washout curves were generated for the left anterior descending coronary artery territory (ischemic area) and left circumflex coronary artery territory (nonischemic area) by beat per beat analysis of frozen end-diastolic frames of left ventricular short-axis views. Peak contrast intensity, contrast washout half-time and area under the curve were derived from these curves. A total of 75 contrast washout curves were analyzed for the study of interinjection, intraobserver and interobserver reproducibility. The correlation coefficients between measurements obtained from paired injections of the echocardiographic contrast agent (interinjection reproducibility) ranged from 0.78 for peak contrast intensity to 0.87 for area under the curve. Percent error varied between 14.7% and 24.7%. The intraobserver variability in measurements was less than the interinjection variability, with a cumulative mean percent error of 17.8% and correlation coefficients of 0.72 (peak contrast intensity), 0.95 (area under the curve) and 0.96 (washout half-time). Interobserver correlation for all indexes was high (r = 0.92 to 0.96). It is concluded that peak contrast intensity, contrast washout half-time and the area under the curve derived from myocardial contrast washout curves can be measured reproducibly from videotapes. In addition, the variability between two injections attempted under identical conditions is greater than reader variability from videotapes.