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Merkel cell carcinoma (MCC) is a rare malignant cutaneous neuroendocrine tumour affecting mainly elderly patients and is more common in the West than in Asia. It is associated with Merkel cell polyomavirus (MCPyV), immunosuppression, and ultraviolet light. In this study, we retrospectively investigated the first series of MCC from Taiwan and identified 19 cases from three tertiary centres. All patients were males with a median age of 67.5. Twelve (63%) cases occurred in the extremities, with one unique case presenting initially as nodal metastasis of unknown primary. Immunohistochemically, the great majority of tumours expressed CK20 (89%), synaptophysin (89%), and INSM1 (84%), with none positive for TTF1. Eleven (58%) cases were positive for MCPyV by immunohistochemistry (clone CM2B4). All patients were treated with excision, including four with additional radiotherapy and one with radiotherapy and chemotherapy. Nodal status and treatment modalities significantly affected survival. The median survival time of MCPyV-positive cases was much longer than the negative cases (median 40 vs. 10 months). In summary, we presented the first report on the clinicopathological features of MCC in Taiwan, with 58% cases associated with MCPyV. The prognosis of patients with MCPyV-positive tumours was better than those negative for MCPyV.

Follicular lymphoma (FL) is usually a nodal lymphoma expressing CD10, rarely with leukaemic presentation (FL-LP).We searched for FL-LP in our institution from 2000 to 2018 and characterised the neoplastic cells by flow cytometry, immunohistochemistry and fluorescence in situ hybridization. Thirteen (6.1%) of 212 FL cases were FL-LP, all de novo neoplasms. The leukaemic cells were small in 12 cases and large in one. All had concurrent FL, mostly (92%; 12/13) low-grade. The single case with large leukaemic cells had a concurrent primary splenic low-grade FL and a double-hit large B-cell lymphoma in the marrow.CD10 was expressed in the leukaemic cells in 38% (5/13) cases by flow cytometry and in 77% (10/13) cases in tumours (p= 0.0471). IGH/BCL2 reciprocal translocation was identified in 85% (11/13) cases. Most patients were treated with chemotherapy. In a median follow-up time of 36 months, nine patients were in complete remission. The 2- and 5-year survival rates were at 100% and 83%, respectively. In this study, we characterised a series of de novo FL-LP in Taiwan. All patients had concurrent nodal and/or tissue tumours, which might suggest that these patients seek medical help too late.The lower CD10 expression rate by flow cytometry than by immunohistochemistry might be due to different epitopes for these assays. Alternatively, loss of CD10 expression might play a role in the pathogenesis of leukaemic change. The clinical course of FL-LP could be aggressive, but a significant proportion of the patients obtained complete remission with chemotherapy.

Sensitivity and resolution of molecular analysis are often compromised by the use of techniques that measure the ensemble average of large cell populations. Having a research tool to nondestructively identify the KSHV replication stage in an infected cell would not only allow us to effectively isolate cells of interest from cell populations but also enable more precise sample selection for advanced single-cell analysis. We prepared a recombinant KSHV that can report on its replication stage in host cells by differential fluorescence emission. Consistent with previous host gene expression studies, our experiments reveal the highly heterogenic nature of KSHV replication/gene expression at individual cell levels. The utilization of a newly developed reporter-KSHV and initial characterization of KSHV replication in single cells are presented., Molecular mechanisms of Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation have been studied primarily by measuring the total or average activity of an infected cell population, which often consists of a mixture of both nonresponding and reactivating cells that in turn contain KSHVs at various stages of replication. Studies on KSHV gene regulation at the individual cell level would allow us to better understand the basis for this heterogeneity, and new preventive measures could be developed based on findings from nonresponding cells exposed to reactivation stimuli. Here, we generated a recombinant reporter virus, which we named “Rainbow-KSHV,” that encodes three fluorescence-tagged KSHV proteins (mBFP2-ORF6, mCardinal-ORF52, and mCherry-LANA). Rainbow-KSHV replicated similarly to a prototype reporter-KSHV, KSHVr.219, and wild-type BAC16 virus. Live imaging revealed unsynchronized initiation of reactivation and KSHV replication with diverse kinetics between individual cells. Cell fractionation revealed temporal gene regulation, in which early lytic gene expression was terminated in late protein-expressing cells. Finally, isolation of fluorescence-positive cells from nonresponders increased dynamic ranges of downstream experiments 10-fold. Thus, this study demonstrates a tool to examine heterogenic responses of KSHV reactivation for a deeper understanding of KSHV replication. IMPORTANCE Sensitivity and resolution of molecular analysis are often compromised by the use of techniques that measure the ensemble average of large cell populations. Having a research tool to nondestructively identify the KSHV replication stage in an infected cell would not only allow us to effectively isolate cells of interest from cell populations but also enable more precise sample selection for advanced single-cell analysis. We prepared a recombinant KSHV that can report on its replication stage in host cells by differential fluorescence emission. Consistent with previous host gene expression studies, our experiments reveal the highly heterogenic nature of KSHV replication/gene expression at individual cell levels. The utilization of a newly developed reporter-KSHV and initial characterization of KSHV replication in single cells are presented.

Small-conductance Ca2+-activated K+ (SK) channels regulate the excitability of cardiomyocytes by integrating intracellular Ca2+ and membrane potentials on a beat-to-beat basis. The inextricable interplay between activation of SK channels and Ca2+ dynamics suggests the pathology of one begets another. Yet, the exact mechanistic underpinning for the activation of cardiac SK channels remains unaddressed. Here, we investigated the intracellular Ca2+ microdomains necessary for SK channel activation. SK currents coupled with Ca2+ influx via L-type Ca2+ channels (LTCCs) continued to be elicited after application of caffeine, ryanodine or thapsigargin to deplete SR Ca2+ store, suggesting that LTCCs provide the immediate Ca2+ microdomain for the activation of SK channels in cardiomyocytes. Super-resolution imaging of SK2, Cav1.2 Ca2+ channel, and ryanodine receptor 2 (RyR2) was performed to quantify the nearest neighbor distances (NND) and localized the three molecules within hundreds of nanometers. The distribution of NND between SK2 and RyR2 as well as SK2 and Cav1.2 was bimodal, suggesting a spatial relationship between the channels. The activation mechanism revealed by our study paved the way for the understanding of the roles of SK channels on the feedback mechanism to regulate the activities of LTCCs and RyR2 to influence local and global Ca2+ signaling.

Background: In stage I/II natural killer (NK)/T-cell lymphoma, concurrent chemoradiotherapy (CCRT) had previously been shown to result in superior outcome compared with anthracycline-containing regimens, which have since been considered ineffective. The role of CCRT in comparison with approaches employing nonanthracycline-containing chemotherapy (CT) and sequential radiotherapy (RT) in such patients remains to be defined. Patients and methods: Three hundred and three untreated patients (207 men, 96 women; median age: 51, 18-86 years) with stage I/II NK/T-cell lymphoma who had received nonanthracycline-containing regimens were collected from an international consortium and retrospectively analyzed. Treatment included single modality (CT and RT), sequential modalities (CT+RT; RT+CT) and concurrent modalities (CCRT; CCRT+CT). The impact of clinicopathologic parameters and types of treatment on complete response (CR) rate, progression-free-survival (PFS) and overall-survival (OS) was evaluated. Results: For CR, stage (P=0.027), prognostic index for NK/T-cell lymphoma (PINK) (P=0.026) and types of initial treatment (P=0.011) were significant prognostic factors on multivariate analysis. On Cox regression analysis, ECOG performance score (P=0.021) and PINK-EBV DNA (PINK-E) (P=0.002) significantly impacted on PFS; whereas ECOG performance score (P=0.008) and stage (P

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

High concentrations of sub-micron nanoparticles have been shown to be released during laser hair removal (LHR) procedures. These emissions pose a potential biohazard to healthcare workers that have prolonged exposure to LHR plume.We sought to demonstrate that cold sapphire skin cooling done in contact mode might suppress plume dispersion during LHR.A total of 11 patients were recruited for laser hair removal. They were treated on the legs and axilla with a 755 or 1064 nm millisecond-domain laser equipped with either (i) cryogen spray (CSC); (ii) refrigerated air (RA); or (iii) contact cooling with sapphire (CC). Concentration of ultrafine nanoparticles1 μm were measured just before and during LHR with the three respective cooling methods.For contact cooling (CC), counts remained at baseline levels, below 3,500 parts per cubic centimeter (ppc) for all treatments. In contrast, the CSC system produced large levels of plume, peaking at times to over 400,000 ppc. The CA cooled system produced intermediate levels of plume, about 35,000 ppc (or about 10× baseline).Cold Sapphire Skin cooling with gel suppresses plume during laser hair removal, potentially eliminating the need for smoke evacuators, custom ventilation systems, and respirators during LHR. Lasers Surg. Med. 50:280-283, 2018. © 2017 Wiley Periodicals, Inc.

Background Occupational exposures to ultrafine particles in the plume generated during laser hair removal procedures, the most commonly performed light based cosmetic procedure, have not been thoroughly characterized. Acute and chronic exposures to ambient ultrafine particles have been associated with a number of negative respiratory and cardiovascular health effects. Thus, the aim of this study was to measure airborne concentrations of particles in a diameter size range of 10 nm to 1 μm in procedure rooms during laser hair removal procedures. Methods TSI Model 3007 Condensation Particle Counters were used to quantify the particle count concentrations in the waiting and procedure rooms of a dermatology office. Particle concentrations were sampled before, during, and after laser hair removal procedures, and characteristics of each procedure were noted by the performing dermatologist. Results Twelve procedures were sampled over 4 days. Mean ultrafine particle concentrations in the waiting and procedure rooms were 14,957.4 particles/cm3 and 22,916.8 particles/cm3 (p

The MAPK phosphatase MKP1 (DUSP1) is overexpressed in many human cancers, including chemoresistant and radioresistant breast cancer cells, but its functional contributions in these settings are unclear. Here, we report that after cell irradiation, MKP1 translocates into mitochondria, where it prevents apoptotic induction by limiting accumulation of phosphorylated active forms of the stress kinase JNK. Increased levels of mitochondrial MKP1 after irradiation occurred in the mitochondrial inner membrane space. Notably, cell survival regulated by mitochondrial MKP1 was responsible for conferring radioresistance in HER2-overexpressing breast cancer cells, due to the fact that MKP1 serves as a major downstream effector in the HER2-activated RAF–MEK–ERK pathway. Clinically, we documented MKP1 expression exclusively in HER2-positive breast tumors, relative to normal adjacent tissue from the same patients. MKP1 overexpression was also detected in irradiated HER2-positive breast cancer stem-like cells (HER2+/CD44+/CD24−/low) isolated from a radioresistant breast cancer cell population after long-term radiation treatment. MKP1 silencing reduced clonogenic survival and enhanced radiosensitivity in these stem-like cells. Combined inhibition of MKP1 and HER2 enhanced cell killing in breast cancer. Together, our findings identify a new mechanism of resistance in breast tumors and reveal MKP1 as a novel therapeutic target for radiosensitization. Cancer Res; 74(24); 7498–509. ©2014 AACR.

Locally concentrated nuclear factors ensure efficient binding to DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable preinitiation complexes. We have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around Kaposi's sarcoma-associated herpesvirus (KSHV) genomes in the host cell nucleus. Using immunofluorescence labeling of latent nuclear antigen (LANA) protein, together with fluorescence in situ RNA hybridization (RNA-FISH) of the intron region of immediate early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At the single-cell level, we found that not all episomes were uniformly transcribed following reactivation stimuli. However, those episomes that were being transcribed would spontaneously aggregate to form transcriptional “factories,” which recruited a significant fraction of cellular RNA polymerase II. Focal assembly of “viral transcriptional factories” decreased the pool of cellular RNA polymerase II available for cellular gene transcription, which consequently impaired cellular gene expression globally, with the exception of selected ones. The viral transcriptional factories localized with replicating viral genomic DNAs. The observed colocalization of viral transcriptional factories with replicating viral genomic DNA suggests that KSHV assembles an “all-in-one” factory for both gene transcription and DNA replication. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be a previously unexplored aspect of KSHV gene regulation by confiscation of a limited supply of RNA polymerase II in infected cells. IMPORTANCE B cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) harbor multiple copies of the KSHV genome in the form of episomes. Three-dimensional imaging of viral gene expression in the nucleus allows us to study interactions and changes in the physical distribution of these episomes following stimulation. The results showed heterogeneity in the responses of individual KSHV episomes to stimuli within a single reactivating cell; those episomes that did respond to stimulation, aggregated within large domains that appear to function as viral transcription factories. A significant portion of cellular RNA polymerase II was trapped in these factories and served to transcribe viral genomes, which coincided with an overall decrease in cellular gene expression. Our findings uncover a strategy of KSHV gene regulation through focal assembly of KSHV episomes and a molecular mechanism of late gene expression.

Cutaneous biopsies often heal with little or no scarring. Prior studies have shown an alarming percentage of patients who incorrectly identify biopsy sites at the time of surgery.To investigate the safety and utility of an ultraviolet (UV)-fluorescent tattoo for biopsy site identification.A preclinical proof of concept was established with skin culture. An UV-fluorescent tattoo was applied to discarded neonatal foreskin in culture medium. The stability of the tattooed skin was examined clinically and histologically. One patient with a recurrent basal cell carcinoma in a difficult-to-identify location underwent tattoo application at the time of biopsy to demarcate the site. The patient was monitored for tattoo reaction and referred for surgical excision.The cultured tissue exhibited stable UV fluorescence with daily washing. Tissue histology demonstrated tattoo particles lining the skin edge under fluorescent microscopy. The patient was reluctant to undergo another surgical procedure and instead returned to our clinic at 3 months and 17 months after the biopsy for management of other tumors. The patient had no symptoms of allergic reaction to the tattoo dye. The fluorescent tattoo remains invisible under visible light and visible only under Wood's light.The present study documents the utility of an UV-fluorescent tattoo to locate a biopsy site.

Background: Rituximab has been associated with hepatitis B virus reactivation (HBV-R). However, the characteristics and scope of this association remain largely undefined. Methods: We completed a comprehensive literature search of all published rituximab-associated HBV-R cases and from the Food and Drug Administration (FDA) Adverse Event Reporting System (AERS) MedWatch database. Literature and FDA cases were compared for completeness, and a meta-analysis was completed. Results: One hundred and eighty-three unique cases of rituximab-associated HBV-R were identified from the literature (n = 27 case reports, n = 156 case series). The time from last rituximab to reactivation was 3 months (range 0–12), although 29% occurred >6 months after last rituximab. Within FDA data (n = 118 cases), there was a strong signal for rituximab-associated HBV-R [proportional reporting ratio = 28.5, 95% confidence interval (CI) 23.9–34.1; Empiric Bayes Geometric Mean = 26.4, 95% CI 21.4–31.1]. However, the completeness of data in FDA reports was significantly inferior compared with literature cases (P < 0.0001). Among HBV core antibody (HBcAb(+)) series, the pooled effect of rituximab-based therapy showed a significantly increased risk of HBV-R compared with nonrituximab-treated patients (odds ratio 5.73, 95% CI 2.01–16.33; Z = 3.33, P = 0.0009) without heterogeneity (χ2 = 2.12, P = 0.5473). Conclusions: The FDA AERS provided strong HBV-R safety signals; however, literature-based cases provided a significantly more complete description. Furthermore, meta-analysis of HBcAb(+) series identified a more than fivefold increased rate of rituximab-associated HBV-R.

BACKGROUND Dermatologists have championed Mohs micrographic surgery (MMS) for its unsurpassed treatment success for skin cancers, safety profile, cost-effectiveness, and tissue-sparing quality. It is unclear whether patients undergoing MMS also value these characteristics. OBJECTIVE To evaluate patients' preoperative expectations of MMS and identify the factors that may influence such expectations METHODS The study prospectively recruited participants who were newly diagnosed with skin cancer and referred for MMS. A questionnaire listing the characteristics of MMS was given to the participants asking them to score the importance of each characteristic on a 10-point scale. The participants were also asked to provide information regarding their gender, age, subjective health status, education level, family annual income, and their referral source RESULTS On average, participants placed the highest value, in descending order, on a treatment that yielded the highest cure rate, reconstruction initiation only after complete tumor removal, and the surgeon being a skin cancer specialist. Overall, participants placed high values on characteristics of MMS that dermatologists have long esteemed. CONCLUSION Our data corroborate that MMS is a valuable procedure that meets the expectations not just of physicians, but also of patients. The authors have indicated no significant interest with commercial supporters.

Contamination of groundwater with vinyl chloride (VC), a known human carcinogen, is a common environmental problem at plastics manufacturing, dry cleaning, and military sites. At many sites, there is the potential to cleanup VC groundwater plumes with aerobic VC-oxidizing microorganisms (e.g., methanotrophs, etheneotrophs, and VC-assimilating bacteria). Environmental biotechnologies that reveal the presence and activity of VC-oxidizing bacteria in contaminated groundwater samples would provide valuable lines of evidence that bioremediation of VC is occurring at a site. We applied targeted shotgun mass spectrometry-based proteomic methods to ethene-enriched groundwater microcosms from a VC-contaminated site. Polypeptides from the enzymes alkene monooxygenase (EtnC) and epoxyalkane:CoM transferase (EtnE), both of which are expressed by aerobic etheneotrophs and VC-assimilating bacteria, were identified in 7 of the 14 samples analyzed. Bioinformatic analysis revealed that 2 EtnC and 5 EtnE peptides were unique to deduced EtnC and EtnE sequences from two different cultivated strains. In addition, several partial EtnE genes sequenced from microcosms matched with observed EtnE peptides. Our results have revealed broader etheneotroph functional gene diversity and demonstrate the feasibility, speed, and accuracy of applying a targeted metaproteomics approach to identifying protein biomarkers from etheneotrophs in complex environmental samples.

Arginine deprivation as an anticancer therapy has historically been met with limited success. The development of pegylated arginine deiminase (ADI-PEG20) has renewed interest in arginine deprivation for the treatment of some cancers. The efficacy of ADI-PEG20 is directly correlated with argininosuccinate synthetase (ASS) deficiency. CWR22Rv1 prostate cancer cells do not express ASS, the rate-limiting enzyme in arginine synthesis, and are susceptible to ADI-PEG20 in vitro. Interestingly, apoptosis by 0.3 μg/mL ADI-PEG20 occurs 96 hours posttreatment and is caspase independent. The effect of ADI-PEG20 in vivo reveals reduced tumor activity by micropositron emission tomography as well as reduced tumor growth as a monotherapy and in combination with docetaxel against CWR22Rv1 mouse xenografts. In addition, we show autophagy is induced by single amino acid depletion by ADI-PEG20. Here, autophagy is an early event that is detected within 1 to 4 hours of 0.3 μg/mL ADI-PEG20 treatment and is an initial protective response to ADI-PEG20 in CWR22Rv1 cells. Significantly, the inhibition of autophagy by chloroquine and Beclin1 siRNA knockdown enhances and accelerates ADI-PEG20–induced cell death. PC3 cells, which express reduced ASS, also undergo autophagy and are responsive to autophagy inhibition and ADI-PEG20 treatment. In contrast, LNCaP cells highly express ASS and are therefore resistant to both ADI-PEG20 and autophagic inhibition. These data point to an interrelationship among ASS deficiency, autophagy, and cell death by ADI-PEG20. Finally, a tissue microarray of 88 prostate tumor samples lacked expression of ASS, indicating ADI-PEG20 is a potential novel therapy for the treatment of prostate cancer. [Cancer Res 2009;69(2):700–8]

Distinguishing angioimmunoblastic T-cell lymphoma from peripheral T-cell lymphoma, unspecified, using morphology, immunophenotype and molecular genetics Aims: To identify distinguishing histological, immunophenotypic and molecular genetic features between angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma (PTL). Methods: Nodal T-cell lymphomas examined (n =137), included AITL (n = 89), PTL (n = 22), anaplastic large cell lymphoma (n = 16) and 'AITL/PTL indeterminate' (n = 10) with overlapping features between AITL and PTL, showing morphology typical of AITL but lacking follicular dendritic cell expansion. Immunohistochemistry for CD3, CD20, CD21 and CD10, in situ hybridization for Epstein-Barr virus encoded RNA (EBER) and polymerase chain reaction for T-cell and B-cell clonality analysis were performed. Results: Of the AITLs, 74/89 showed typical morphology, whereas 15/89 showed hyperplastic follicles. AITL and 'AITL/PTL indeterminate' showed a polymorphous infiltrate and prominent vascularity in all cases. In both groups, CD10 was present in the majority and clear cells and EBER positivity were specific (but not universal) features lacking in PTL. Detection of T-cell clonality was significantly higher in AITL (90%) compared with PTLu (59%). Conclusions: Clear cells and EBV infection (when present) are useful distinguishing features and CD10 a sensitive and specific marker of AITL. Hyperplastic follicles are present in a significant minority of AITL. AITL/PTL indeterminate probably falls within the spectrum of AITL rather than PTL.

1Alpha,25-dihydroxyvitamin D3 (1,25D3) displays potent antiproliferative activity in a variety of tumor model systems and is currently under investigation in clinical trials in cancer. Studies were initiated to explore its potential in nonsmall cell lung cancer (NSCLC), as effective approaches to the treatment of that disease are needed. In evaluating factors that may affect activity in NSCLC, the authors found that CYP24 (25-hydroxyvitamin D3-24-hydroxylase), the enzyme that catabolizes 1,25D3, is frequently expressed in NSCLC cell lines but not in the nontumorigenic bronchial epithelial cell line, Beas2B. CYP24 expression by RT-PCR was also detected in 10/18 primary lung tumors but in only 1/11 normal lung tissue specimens. Tumor-specific CYP24 upregulation was confirmed at the protein level via immunoblot analysis of patient-matched normal lung tissue and lung tumor extracts. Enzymatically active CYP24 is expected to desensitize NSCLC cells to 1,25D3. The authors therefore implemented a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for 1,25D3 and its CYP24-generated metabolites to determine whether NSCLC cells express active enzyme. Analysis of NSCLC cell cultures revealed time-dependent loss of 1,25D3 coincident with the appearance of CYP24-generated metabolites. MK-24(S)-S(O)(NH)-Ph-1, a specific inhibitor of CYP24, slowed the loss of 1,25D3 and increased 1,25D3 half-life. Furthermore, combination of 1,25D3 with MK-24(S)-S(O)(NH)-Ph-1 resulted in a significant decrease in the concentration of 1,25D3 required to achieve maximum growth inhibition in NSCLC cells. These data suggest that increased CYP24 expression in lung tumors restricts 1,25D3 activity and support the preclinical evaluation of CYP24 inhibitors for lung cancer treatment.

4 páginas, 1 figura., Multiple cutaneous and uterine leiomyomata (MCL) is an autosomal dominant disorder characterized by the development of benign smooth muscle tumours (leiomyomas) in the skin and uterus of affected women, and in the skin of affected men. In rare cases, MCL has been associated with a predisposition to the rare type II papillary renal cell cancer, also known as hereditary leiomyomatosis and renal cell cancer. The genetic locus for MCL has been mapped to chromosome 1q42.3–43 and subsequently, germline mutations in the fumarate hydratase (FH) gene have been identified. In addition, analysis of FH in some tumours of MCL patients revealed a second mutation inactivating the wild-type allele, suggesting that FH may function as a tumour suppressor gene. Here, we report two cases of MCL patients with FH mutations, designated as T287P and R190L. T287P represents a novel mutation of a highly conserved amino acid of the FH protein. In addition, a patient with an unusual clinical presentation of MCL was found to have the recurrent mutation, R190L, raising the possibility of incorporating FH sequencing as a diagnostic tool. Our findings extend the allelic series of mutations in FH and support its status as the underlying cause of MCL., This work was supported in part by the Skin Disease Research Center, Department of Dermatology, Columbia University (P30 AR44535) and the Authority for Research and Development, Hebrew University of Jerusalem (to A. Zlotogorski).

The technical difficulty of laparoscopic partial nephrectomy (LPN) lies mainly in the steps required during warm ischemia time (WIT), which includes tumor excision and renal reconstruction. We present a renal-suspension traction system to place the tumor in stable optimal view during the critical steps of LPN.Thirty-three patients underwent LPN from October 2002 through December 2003. Eight had a renal sling placed intraoperatively because of difficult access to the tumor. Perioperative parameters were assessed. The renal hilum was dissected and the tumor exposed. To keep the tumor oriented perfectly toward the camera and the working ports, a 2-0 braided polyglactin (Vicryl) traction suture was passed through Gerota's fascia, catching the renal capsule; brought out through the abdominal wall; and secured to the skin with a clamp.All surgical margins were negative. For the traction and nontraction groups, the mean sizes of the tumor were 2.5 cm and 2.2 cm, respectively (P = 0.426). The estimated blood loss was 125 mL and 246 mL respectively (P = 0.041). The WIT average 27.4 minutes and 30.12 minutes (P = 0.470). The surgical time was 192 minutes and 235 minutes respectively, (P = 0.062). Based on our findings, we have devised a renal suspension traction algorithm for specific tumor locations during LPN.The renal suspension traction system allows precision in tumor excision and renal reconstruction during the critical steps of LPN. With the renal suspension system we devised, we are able to simplify LPN for tumors located away from optimal port access.

The new 24-phenylsulfone 4a, a low-calcemic analog of the natural hormone 1alpha,25-dihydroxyvitamin D(3), is a potent (IC(50) = 28nM) and highly selective inhibitor of the human 24-hydroxylase enzyme CYP24.

Summary 2B4 (CD244) is a member of the CD2 subset of the immunoglobulin superfamily and functions as a triggering molecule on natural killer (NK) cells. Previously, we have found that 2B4-mediated activation of NK cells involves complex interactions involving LAT, Ras, Raf, ERK and p38 and that cytolytic function and cytokine production may be regulated by distinct pathways. Here we assessed the role of protein kinase C (PKC) in 2B4-mediated cytotoxicity of YT cells, a human NK cell line. Our data indicate that PKC-δ is activated upon stimulation with monoclonal antibody against 2B4. Treatment with the PKC inhibitor, bisindolylmaleimide I (Go6850), of YT cells or YT cells depleted of Ca2+-dependent isoforms of PKC prior to 2B4 stimulation, resulted in inhibition of natural cytotoxicity and redirected antibody-dependent cellular cytotoxicity. However, inhibition of PKC failed to block 2B4 stimulation of interferon-γ secretion as opposed to pretreatment with LY294002, a phosphoinositide 3-kinase inhibitor. We also examined the effect of phorbol 12-myristate 13-acetate (PMA) induction on 2B4 gene transcription. PMA induction resulted in a more than two-fold increase of 2B4 transcription. However, when we introduced a three-base substitution mutation to disrupt the activator protein-1 binding site at (−106 to −100) in the 2B4 promoter, we found complete loss of transcriptional activity, including the two-fold increase due to PMA induction of PKC. The present study indicated that PKC may play an important role in 2B4 signalling and activator protein-1 activation.

Researchers have shown that the five major dimensions of personality (extraversion, agreeableness, conscientiousness, neuroticism, and openness to experience) and two additional factors (irritability and positive activity) are evident from adolescence. This study attempted to replicate and extend these results in a longitudinal study of 102 Swedish children, followed from 2.3 to 15.2 years of age. Item analyses revealed consistently reliable irritability, conscientiousness, and positive activity factors, whereas the internal reliability of the extraversion, agreeableness, neuroticism, and openness to experience factors increased over time. Irritability and positive activity were not independent of the other factors. Scores on most of the personality factors were fairly stable over time. Over time, children became less extraverted, more agreeable, and more conscientious. Neuroticism and openness to experience increased in Phase III, although openness then decreased in Phase V. Validity of the original factors was demonstrated by correlations with independent assessments of the children's cognitive performance and adjustment to school.

2B4 (CD244), a member of the CD2 subset of the Ig superfamily receptors, is expressed on all human NK cells, a subpopulation of T cells, basophils and monocytes. 2B4 activates NK cell mediated cytotoxicity, induces secretion of IFN-γ and matrix metalloproteinases, and NK cell invasiveness. Although there has been several molecules shown to interact with 2B4, the signaling mechanism of 2B4-mediated activation of NK cells is still unknown. In this study, we found cross-linking of 2B4 on YT cells, a human NK cell line, results in the increased DNA binding activity of activator protein-1 (AP-1), an important regulator of nuclear gene expression in leukocytes. We investigated the possible role of various signaling molecules that may be involved in the activation of lytic function of YT cells via 2B4. Treatment of YT cells with various specific inhibitors indicate that 2B4-stimulation of YT cells in spontaneous and Ab-dependent cytotoxicity is Ras/Raf dependent and involves multiple MAPK signaling pathways (ERK1/2 and p38). However, only inhibitors of transcription and p38 inhibited 2B4-mediated IFN-γ release indicating distinct pathways are involved in cytotoxicity and cytokine release. In this study we also show that 2B4 constitutively associates with the linker for activation of T cells (LAT) and that 2B4 may mediate NK cell activation via a LAT-dependent signaling pathway. These results indicate that 2B4-mediated activation of NK cells involves complex interactions involving LAT, Ras, Raf, ERK and p38 and that cytolytic function and cytokine production may be regulated by distinct pathways.

Hepatic cell-specific expression of the human apolipoprotein B (apoB) gene is controlled by at least four cis-acting elements located between positions −128 and +122 [Chuang, S. S., & Das, H. K. (1996) Biochem. Biophys. Res. Commun.220, 553–562]. A negative cis-acting element (+20 to +40) is located in the first nontranslated exon of the human apoB gene, and apoB regulatory factor-3 (BRF-3) interacts with this. In this paper, we report the purification and characterization of BRF-3 from rat liver nuclear extracts. BRF-3 has been purified to apparent homogeneity by DEAE–cellulose, heparin–agarose, and DNA-specific affinity chromatography. Purified BRF-3 produced two polypeptide bands with apparent molecular masses of 70 kDa and 67 kDa in SDS/PAGE as detected by silver staining. Both 70-kDa and 67-kDa proteins have been found to hybridize specifically with labeled double-stranded oligonucleotide containing BRF-3 binding site in a South-Western blot. Double-stranded oligonucleotide containing mutations in the BRF-3 binding site was found to abolish DNA binding by these two proteins. Amino acid sequences of tryptic peptides derived from affinity purified 70-kDa and 67-kDa rat BRF-3 proteins were found to have 100% sequence homologies with DNA topoisomerase I. These data suggest that the 70-kDa and 67-kDa forms of BRF-3 are derived by proteolytic cleavage of topoisomerase I, and therefore, topoisomerase I may play an important role in transcriptional regulation of apoB.

Since 1999 we have made 2 modifications in the nerve sparing approach to radical retropubic prostatectomy (RRP), namely early release of the neurovascular bundles (NVBs) before division of the posterior membranous urethra and the use of 2.5x optical loupe magnification during NVB preservation. We retrospectively reviewed our results.Between January 1998 and August 2003, 507 men underwent RRP for prostate cancer. All surgeries were performed by a single surgeon (CBB). Bilateral nerve sparing procedures were performed in 313 men. Modifications were introduced sequentially to the surgical technique and potency rates were compared to those of patients operated on before these modifications. Patients were divided into groups based on the technique of nerve sparing as group 1 (standard release of the NVB), group 2 (early release of the NVB) and group 3 (early release with loupe magnification). All patients were followed for a minimum of 5 months. Postoperative potency rates were compared among the 3 groups.Mean followup was 15.9 months. Mean patient age was 56.2 years. The overall potency rate among groups 1, 2 and 3 was 40.5%, 54.8% and 66.1%, respectively. Mean time to potency was 10.7, 8.5 and 2.0 months, respectively. Significant differences were found in the overall potency rate among all groups (p0.05). Mean time to potency was significantly improved between groups 1 and 3 (p0.05) and between groups 2 and 3 (p0.05).Minor modifications in nerve sparing technique lead to improved postoperative potency rates and decreased time to potency in men undergoing RRP.

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine(1). This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)(1,10). Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)(1,2,3). Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation(4,5). Although the essential components of this pathway are well-characterized(6,7,8,9), many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy(11,12). Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Potentially harmful chemicals are released when tissues are vaporized. Laser hair removal (LHR) causes heating and often vaporization of hairs, producing both a signature malodorous plume and visible particulates.To characterize the chemical composition and quantify the ultrafine particle content of the plume generated during LHR.In the laser center of a large academic hospital, discarded terminal hairs from the trunk and extremities were collected from 2 adult volunteers. The hair samples were sealed in glass gas chromatography chambers and treated with a laser. The laser plume was analyzed by gas chromatography-mass spectrometry (GC-MS). During LHR treatment, two 6-L negative pressure canisters were used to capture 30 seconds of laser plume, and a portable condensation particle counter was used to measure ultrafine particulates (1 µm). Ultrafine particle concentrations were measured within the treatment room, within the waiting room, and outside the building.The chemical content of the laser plume was analyzed with GC-MS and screened for aerosolized toxins using Environmental Protection Agency-certified methods. The ambient concentration of ultrafine particles during LHR was measured by condensation particle counters.Analysis with GC-MS identified 377 chemical compounds. Sixty-two of these compounds, of which 13 are known or suspected carcinogens and more than 20 are known environmental toxins, exhibited strong absorption peaks. During LHR, the portable condensation particle counters documented an 8-fold increase compared with the ambient room baseline level of ultrafine particle concentrations (ambient room baseline, 15 300 particles per cubic centimeter [ppc]; LHR with smoke evacuator, 129 376 ppc), even when a smoke evacuator was in close proximity (5.0 cm) to the procedure site. When the smoke evacuator was turned off for 30 seconds, there was a more than 26-fold increase in particulate count compared with ambient baseline levels (ambient baseline, 15 300 ppc; LHR without smoke evacuator for 30 seconds, 435 888 ppc).These findings establish the concern that the burning-hair plume often present during LHR should be considered a biohazard, warranting the use of smoke evacuators, good ventilation, and respiratory protection, especially for health care workers with prolonged exposure to LHR plume.

Apolipoprotein B-100, produced primarily in the human liver, is the sole protein component of low-density lipoprotein and serves as a ligand for the LDL receptor. Two cis-acting positive elements between −128 and −70 control hepatic cell-specific expression of the human apoB gene (H. K. Das, T. Leff, and J. L. Breslow, J. Biol. Chem. 263: 11452-11458, 1988). In this study, two apoB cis-acting elements (+20 to +40; +43 to +53) have been identified using DNase I footprint analysis. Through in vitro mutagenesis and transient transfection experiments in Hep G2 and HeLa cells, the element (+20 to +40) was observed to have a negative effect on transcription of the apoB gene. The element (+43 to +53) was found to have a strong positive effect on apoB gene transcription in Hep G2 cells and mild positive effect in HeLa cells. Therefore these two cis-acting elements mediate hepatic-cell specific expression of the apolipoprotein gene by interacting with trans-acting protein factors.

An estimated 18,000 individuals are trafficked into the United States each year from all over the world, and are forced into hard labor or commercial sex work. Despite their invisibility, some victims are known to have received medical care while under traffickers' control. Our project aimed to characterize trafficking victims' encounters in US health care settings.The study consisted of semi-structured interviews with six Key Informants who work closely with trafficking victims (Phase I) and 12 female trafficking survivors (Phase II). All survivors were recruited through the Coalition to Abolish Slavery and Trafficking, an NGO in Los Angeles, and all were trafficked into Los Angeles. Interviews were conducted in English and six other languages, with the assistance of professional interpreters. Using a framework analysis approach that focused on victims' encounters in health care settings, we assessed interview transcript content and coded for themes. We used an exploratory pile-sorting technique to aggregate similar ideas and identify overarching domains.The survivors came from 10 countries. Eight had experienced domestic servitude, three had survived sex trafficking, and one had experienced both. Half the survivors reported that they had visited a physician while in their traffickers' control, and another worked in a health care facility. All Key Informants described other victims who had received medical care. For domestic servants, medical visits were triggered by injury and respiratory or systemic illness, while sex trafficking victims were seen by health professionals for sexually transmitted infections and abortion. Trafficking victims were prevented from disclosing their status to health care providers by fear, shame, language barriers, and limited interaction with medical personnel, among other obstacles.This exploration of survivors' experiences in health care settings supports anecdotal reports that US health care providers may unwittingly encounter human trafficking victims. Increasing awareness of human trafficking, and modifying practice to facilitate disclosure, could improve victim identification.

Background Squamous cell carcinoma (SCC) in situ (SCCIS) is often treated without any pathologic confirmation of tumor clearance. It is unclear how often an invasive SCC is harbored within a lesion in which the initial biopsy demonstrated SCCIS because of inadequate sampling. This study examines the final histologic diagnosis of cases in which the initial biopsies were diagnosed as SCCIS and evaluates factors that may correlate with a histologic upstaging of the diagnosis. Methods We prospectively recruited 29 consecutive patients with biopsy-proven SCCIS sent for Mohs micrographic surgery (MMS). Each tumor underwent MMS, and the central blocks of the Mohs debulking specimens were horizontally sectioned at 30-μm intervals until exhausted. A fellowship-trained Mohs surgeon and a board-certified dermatopathologist processed and examined these sections to determine the final histologic diagnosis of the tumor. Results Of the 29 subjects with biopsy-proven SCCIS, nine were found to harbor invasive SCC on final histology. Of the remaining lesions, seven had residual SCCIS, whereas the rest exhibited only actinic keratoses or scars. Approximately 31% of lesions showed evidence of invasive SCC. Correlating the clinical characteristics of the lesions with their corresponding final histologic diagnoses, the lesions harboring invasive SCC were more likely to demonstrate clinical signs of residual tumor (scales and papular changes) and be larger than 1.4 cm in diameter. Limitations Our experience at a single institution in the northeastern United States may not be reflective of a wider population. There is also a possible referral bias, because only lesions with high clinical suspicion for invasive SCC were referred for MMS. Conclusion Although biopsy-proven SCCIS is often treated with modalities that are best suited for superficial disease and do not involve a final pathologic confirmation of clearance (e.g., cryotherapy, electrodesiccation and curettage), this study demonstrated that up to 31% of biopsy-proven SCCIS lesions may harbor invasive SCC. Clinical signs of residual tumor and a diameter larger than 1.4 cm are statistically significant predictors of underlying invasive SCC. These data suggest that treatment modalities that include histologic control of tumor removal should also be strongly considered for the treatment of select biopsy-proven SCCIS meeting the above criteria.

Chronic hepatitis C virus (HCV) infection ultimately leads to chronic hepatitis, hepatic cirrhosis and hepatocellular carcinoma (HCC). As the standard treatment is not completely efficacious, a safer and more effective agent against HCV infection needs to be developed. In this report, we demonstrated that 3-hydroxy caruilignan C (3-HCL-C) isolated from Swietenia macrophylla stems exhibited high anti-HCV activity at both protein and RNA levels at nontoxic concentrations, with an EC(50) value of 10.5 ± 1.2 μm. Combinations of 3-HCL-C and interferon-α (IFN-α), an HCV NS5B polymerase inhibitor (2'-C-methylcytidine; NM-107) or an HCV NS3/4A protease inhibitor (Telaprevir; VX-950) increased the suppression of HCV RNA replication. The results suggested that 3-HCL-C may be a potential anti-viral agent. We then demonstrated that 3-HCL-C interfered with HCV replication by inducing IFN-stimulated response element transcription and IFN-dependent anti-viral gene expression.

Background Toxic epidermal necrolysis syndrome (TEN) is a rare, life-threatening, drug-related skin reaction with a high mortality rate. To date, only a few studies with insufficient sample sizes have been conducted to analyse SCORTEN in Asian populations with TEN. Objective To analyse SCORTEN and other related factors that affect TEN patients in Taiwan. Methods A retrospective review of medical records of 101 patients with TEN from 1992 to 2009. Results There were 62 cases of adverse reactions to a single medication and 39 cases of idiopathic reaction, from multiple medications, or infectious pathogens, of 101 TEN patients. Of the seven individual SCORTEN parameters, only associate malignancy, detached or compromised body surface area >10%, serum urea and bicarbonate were statistically significant in the multivariate analysis. Factors such as 1.5 times baseline serum creatinine levels, urine output of less than 0.5 mL/kg for 6 h and acute renal failure were connected with subsequent mortality. Conclusion The SCORTEN score is effective in predicting the outcome in Taiwanese TEN patients. A number of factors are predictors of mortality. In our study, we determine renal insufficiency and failure to be a marker for predicting a poor outcome.

T cell and natural killer (NK)/T-cell neoplasms are rare and may occasionally present as leukaemia. We retrospectively searched T cell and NK/T-cell tumours in a single institution in Taiwan from January 2000 to December 2009 and identified 137 (19.1%) patients with T cell and NK/T-cell tumours among 718 patients with lymphoid neoplasms. Among these 137 patients, 18 (13.1%) presented with leukaemia including T-lymphoblastic lymphoma/leukaemia (T-LBL), T-cell prolymphocytic leukaemia, aggressive NK-cell leukaemia, adult T-cell lymphoma/leukaemia (ATLL), T-cell large granular lymphocytic (T-LGL) leukaemia and unspecified peripheral T-cell lymphoma. Cases with concurrent lymphoma, higher absolute leukaemic cell counts and elevated lactate dehydrogenase level carried a poorer prognosis. The survival was dichotomous, with a very poor prognosis for patients with T-LBL, T-cell prolymphocytic leukaemia, aggressive NK-cell leukaemia, ATLL in acute phase and unspecified peripheral T-cell lymphoma, while those with T-LGL leukaemia and ATLL in chronic phase had a favourable outcome.

The controversy over human gene patents was reignited in March 2010 when a US Federal District Court decided that isolated human gene sequences are not patentable.1 An appeal is pending, and the US Department of Justice has just filed a friend-of-the-court brief in the case arguing that such gene sequences should not be patentable.2 Because this case may eventually find its way to the US Supreme Court, the ruling could have significant implications for gene-based medical therapies and for the biotechnology industry overall. It is therefore important to assess both the past and present context of this controversy, taking into account scientific research, health care access, and ethical concerns. Patents are government grants that give their owners the right to exclude anyone else from making, using, or selling the inventions they describe.3 To be eligible for a patent, the invention must be new, useful, and not obvious to someone working in the same field as the inventor.4 Laws of nature, physical phenomena, and abstract ideas are not patentable, but, according to the US Supreme Court, “anything under the sun that is made by man” is potentially patentable.5 For example, a microorganism as it exists in nature is not patentable, but a version of that microorganism that has been genetically modified by man is. The US Patent and Trademark Office (USPTO) relies on this reasoning when issuing patents on isolated human gene sequences, because human intervention is required to extract and purify them.6 The European Union takes a similarly open stance toward gene patents and has issued a directive permitting isolated human gene sequences to be patented.7 Not surprisingly, gene patents have flourished. It is estimated that nearly 20% of human genes are associated with at least one US patent and that the number of DNA-related patents exceeds 40,000.8 The current controversy over gene patents involves Myriad Genetics, Inc., a biotechnology company that owns patents on isolated forms of 2 human genes, BRCA1 and BRCA2, which have been linked to hereditary breast and ovarian cancer. As the owner of the patent, Myriad can prevent others from using BRCA1 and BRCA2 in research, diagnostic, and therapeutic applications. Myriad also has the ability to charge high prices to companies wishing to use these genes. Thus, the American Civil Liberties Union and the Public Patent Foundation filed a lawsuit claiming that Myriad’s patents were invalid, stifled biomedical research and diagnostic testing, and limited patients’ access to medical care. The court agreed with the plaintiffs and found that isolated gene sequences are not patentable because they are not “markedly different” from the gene sequences as they exist in nature.1 This was a surprising result, given that the matter of the patentability of isolated human gene sequences in the United States had been considered settled. While this case could be dismissed as an aberration, certain to be overturned or curtailed on appeal, other judges also have questioned the patentability of isolated gene sequences.9 As noted, the US Department of Justice has taken the position that such sequences should not be patentable.2 Accordingly, there is a real possibility that US courts will bar the patenting of human genes. Such a decision would place the United States in opposition to the European Union, and because the USPTO and its European counterpart, the European Patent Office (EPO), are among the largest patent offices in the world,10 the resulting discordance could have profound implications for international policy. Although the Myriad case is concerned with genetic diagnostic testing, the District Court ruling calls into question the validity of numerous patents that have been issued for other gene-based inventions, including gene-based biologic therapeutics. Many existing patented biologic drugs are biotech blockbusters, such as human insulin and insulin analogues, the rheumatoid arthritis drug etanercept, and the anemia drug erythropoietin. While the patents for such blockbuster agents generally cover more than the isolated and purified gene sequences on which they are based, the uncertainty over the scope of patent protection for biologic drugs could deter potential investment, retarding the development of new gene-based therapies and adversely affecting the biotechnology industry. Given the impact of a potential ban on gene patenting, we need to further consider the arguments for and against such patents. Some opponents claim that human gene patents hinder scientific research, because investigators hoping to work with patented genes need to seek permission from the patent owners, thereby increasing costs and inefficiencies. These costs, they suggest, may become so prohibitive that researchers will stop working with patented genes altogether. However, these claims have not yet been substantiated. In fact, there is little evidence that gene patents are actually having a deterrent effect on biotechnology research,11,12 suggesting that various stakeholders may be using licensing agreements or other workarounds to facilitate research. From the standpoint of health care access, some critics of gene patenting argue that if a particular patented gene therapy or diagnostic test is deemed medically necessary, a patient will have little choice but to obtain the treatment or test from the patent owner or the owner’s licensee. Indeed, there is evidence that patenting of diagnostic tests does restrict patients’ options.8 Although such restrictions could increase patient burden and costs, this effect is not unique to human gene patents; any patents granted to health care products can have such effects. Thus, the high cost of patented therapies is a drawback of the patent system as a whole and is not specific to gene patents. After all, our patent system is based on the premise that patent exclusivity is needed to incentivize invention and innovation. Pharmaceutical companies have long contended that patent and market exclusivity allows them to recoup the costs of drug development and finance new research.13 While gene patents may play a crucial role in promoting biotechnology innovation, there is insufficient evidence to determine whether the patent system is the optimal way of encouraging such innovation. Furthermore, some posit that the human genome is qualitatively different from other naturally occurring entities and is even distinct from the DNA of plants and other animals. Therefore, the argument goes, human dignity should preclude anyone from owning patents on human genes.14 Moreover, if the human genome is part of man’s common heritage and if individuals have an inalienable right to ownership of their bodies, including their genes, what right does any single individual have to own part of the genome?15,16 The difficulty with these arguments is that the patent owner merely owns the isolated gene and does not control the genetic information encoded by that gene. In fact, these moral objections were made with respect to Myriad’s gene patents in opposition proceedings before the EPO, although they were ultimately rejected.17,18 Perhaps the underlying thrust of these arguments is that by allowing patent owners to control isolated gene sequences, we may unwittingly be enabling them to control the encoded genetic information common to us all. A final decision in the Myriad case may be a few years off, depending on whether the US Supreme Court decides to weigh in. But are we, as a society, prepared for this decision when important questions remain unanswered? For example, more evidence is still needed to determine whether gene patents actually impede or encourage the development, commercialization, and/or utilization of important therapies and diagnostic tools. We need to critically consider whether controlling an isolated gene sequence translates into de facto control of the genetic information encoded by that sequence. How different is an isolated gene sequence from the gene sequence as it resides in the human body? The scientific community has a responsibility to enrich the debate on this controversial topic by providing empirically based, thoroughly deliberated answers to these and other questions. The answers will ultimately help inform the important decision society must make regarding the propriety of patenting human genes.

Apolipoprotein B100 (apoB), the only protein of low-density lipoprotein, is produced primarily in the liver and serves as a ligand for the low-density lipoprotein receptor. Hepatic cell-specific expression of the human apoB gene is controlled by at least two cis-acting positive elements located between positions-128 and -70 (H. K. Das, T. Leff, and J.L. Breslow, J. Biol. Chem. 263:11452-11458, 1988). The distal element (-128 to -85) appears to be liver specific since it shows positive activity in HepG2 cells and negative activity in HeLa cells. The proximal element (-84 to -70) acts as a positive element in both these cell lines, and two rat liver nuclear proteins, BRF-1 and C/EBP, bind to two overlapping sites (-84 to -60 and -70 to -50, respectively). By gel mobility shift assay, we have identified a rat liver nuclear protein (BRF-2) which binds to the distal element (-128 to -85) of the apoB gene. This putative trans-acting factor has been purified to apparent homogeneity by DEAE-cellulose, heparin-agarose, and DNA-specific affinity chromatography. The purified BRF-2 has an apparent molecular mass of 120 kDa and was found to specifically recognize sequence -128 to -85; BRF-2 also produced a strong hypersensitive site at nucleotide position -95 with copper-orthophenanthroline reagent. A double-stranded oligonucleotide (-128 to -85) containing a 3-nucleotide (TTC) insertion between position -95 and -94 was found to abolish DNA binding by BRF-2. This result suggests that the region surrounding the hypersensitive site -95 is important for protein-DNA interaction. By using apoB promoter fragments containing various internal deletions as templates for gel mobility shift assay, the region between -104 and -85 was identified to be crucial for binding by BRF-2. We propose that BRF-2 may play an important role in the tissue-specific regulation of apoB gene transcription.

This study explores maternal and paternal parenting practices (authoritative or authoritarian) and parental values and goals for toddlers among Chinese mothers and fathers in Canada and China. The participants included 126 families of 1-year-old toddlers (67 Chinese Canadians and 59 mainland Chinese). The findings revealed that Chinese Canadian parents were more supportive of authoritative practices, and Chinese parents were more likely to support authoritarian practices. Between mothers and fathers, gender differences were found within countries. Interparental agreement for parenting beliefs varied by infant gender and country. For parental values, parents generally endorsed self-confidence as the most important trait for their toddlers. Endorsement of other traits (collectivistic and individualistic) varied in importance. Links among parenting beliefs and desired personality traits for their children were also explored.

The spread of HIV between immune cells is greatly enhanced by cell-cell adhesions called virological synapses, although the underlying mechanisms have been unclear. With use of an infectious, fluorescent clone of HIV, we tracked the movement of Gag in live CD4 T cells and captured the direct translocation of HIV across the virological synapse. Quantitative, high-speed three-dimensional (3D) video microscopy revealed the rapid formation of micrometer-sized “buttons” containing oligomerized viral Gag protein. Electron microscopy showed that these buttons were packed with budding viral crescents. Viral transfer events were observed to form virus-laden internal compartments within target cells. Continuous time-lapse monitoring showed preferential infection through synapses. Thus, HIV dissemination may be enhanced by virological synapse-mediated cell adhesion coupled to viral endocytosis.

Although heat shock protein 70 (HSP70) has been suggested to be a stress marker or to play a protective role in brain injury, the relevance of its pathological expression in epilepsy is unclear. We investigated the expression of HSP70 in brain tissue from human temporal lobe epilepsy (TLE) patients and from kainic acid (KA)-induced seizure-related neuronal damage in vivo and in vitro. The human TLE tissue showed severe neuronal loss and gliosis in hippocampal CA3 area. The KA-induced neuronal damage was similar to pathological changes of the TLE hippocampus. An increased number of TUNEL-positive cells were observed at day 5 when compared with day 2 after seizure induction. Intense HSP70 immunofluorescence was observed in hippocampal CA3 pyramidal neurons of rat, 2 days following KA administration, which then declined in labeling by day 5. No HSP70 expression was found in Fluoro-Jade B positive dying neurons by double staining. Western blot analysis showed an increased level of p53 and Bax expression following KA treatment. In vitro, there was no apparent difference in the degree of apoptosis between HSP70 siRNA- and control empty vector-transfected primary neurons following KA treatment. Our results revealed that HSP70 was a useful indicator of stressed neurons in acute phase of epilepsy, but not associated with neuronal death, thereby suggesting that HSP70 played no role in neuroprotection during an epileptogenic state.

Our previous report has confirmed that visually presented words can elicit neuromagnetic activities from the visual cortex, angular gyrus and Broca's area. It is not clear how the words are visuospatially transformed and recognized by the human brain. Mirror reading is characterized by reading which runs in the opposite direction to normal reading, with reversals of letters. It would be very interesting to find out if there are any neuromagnetic differences between the mirror reading and normal reading. Four right-handed healthy adults have been studied with a whole cortex Magnetoencephalography (MEG) system. The stimuli consisted of eight normal oriented words and eight inverted exclamation mark section "reversed words" (mirror-image of the words). All stimuli were randomly presented on the screen in front of the subjects using DirectX. MEG data were analyzed using both single dipole modeling and synthetic aperture magnetometry (SAM). Four responses to the reversed words were identified in all four subjects. In comparison to the normally oriented words, the reversed words elicited a stronger response at a latency of 248+/-6 ms. SAM results indicated that the reversed words invoked strong activations in the left and right parietal cortices but the normally oriented words did not. The mirror reading elicited one magnetic response which is different from that of the normal reading. The difference between the mirror reading and the normal reading in terms of neuromagnetic activation may reflect the development of novel representations for reversed words.

Our previous studies have demonstrated that there is a motion-related magnetic response evoked by moving sound but not stationary sound. The motion-related response reflects the dynamic activities of brain processing of auditory motion. This study focuses on the effect of attention on the response. Six normal hearing subjects were studied using a 151-channel whole-cortex magnetoencephalography (MEG) system. Four sound stimuli were designed: two sounds moved from left to right (rightward) and the other two sounds moved from right to left (leftward). Two sounds moving in the same direction were separated with an attention task. The attention task was a mathematical calculation. The results showed that when subjects listened to the moving sound, there was a clearly auditory motion related response at a latency of 649 +/- 14.2 ms. However, when subjects were doing the calculation while listening to the moving sounds, the motion related response completely disappeared. Strikingly, there was a dent at a latency of 600 ms. To our knowledge, this is probably the first study showing a neuromagnetic response which can be clearly elicited or inhibited by an attention task. We consider that the motion related response has the potential to be an objective index for the study of attention.

Conventional visual identification of epileptic spike is a challenging problem in the clinical application of magnetoencephalography (MEG). More importantly, the conventional method has problems of detecting other abnormalities such as high frequency oscillation in the human epileptic brain. The objective of this study was to develop a new approach using magnetic spectral analysis and spatial filtering. Twelve patients with seizure have been studied with a whole cortex MEG system. Fifteen epochs were recorded for each patient; each epoch was 120 seconds. Neuromagnetic spectrum was analyzed using a new method called accumulated spectrogram. Focal increases of spectral power were localized using synthetic aperture magnetometry (SAM). The MEG results were then compared with clinical findings. Focal increases of spectral power have been identified in all patients (12/12, 100%). The locations of the focal increases of spectral power were in agreement with dipole locations of spikes in 9 patients (9/12, 75%). A comparison between MEG results and clinical findings indicated that SAM revealed focal epileptic activities in two patients when dipole fitting failed. The results suggest that epileptic regions could be quantitatively identified and accurately localized using accumulated spectrogram and SAM. In comparison to visual identification of spike, the new approach is objective and sensitive, and provides the possibility of analyzing much wider frequency bands.

It is difficult to localize the somatosensory cortex in children under 6 years using magnetoencephalography (MEG) with conventional median nerve stimulation. One main reason is that MEG data recorded from small children have poor signal-to-noise ratio due to their small heads and short hands. To find a better approach, this study investigated neuromagnetic activities following finger stimulation using spatially filtered MEG. Four healthy children have been studied with a whole cortex MEG system. Electric stimulation was applied to the thumb and the middle fingers with two Digital Rings. Two trials were recorded for each hand. Focal increases of spectral power were localized using spectrogram and synthetic aperture magnetometry (SAM). A clear response at a latency of 21 ms was identified in 3 children (3/4). Dipole modeling localized the somatosensory cortex in 2 children (2/4). SAM successfully localized the somatosensory cortex in 4 children (4/4). Interestingly, the functional region estimated for the thumb was significantly larger than that of the middle finger (p0.01). However, it is possible that this effect is due to a larger source amplitude rather than larger source volume. Our results demonstrated that the developed approach could map the somatosensory cortex in children ranging in age from 3 to 6. To our knowledge, this is the first report using non-invasive methods to provide quantitative data indicating that the functional area of the thumb is larger than that of the middle finger in small children.

23 páginas, 3 figuras, 1 tabla., Multiple cutaneous and uterine leiomyomata syndrome (MCL) is an autosomal dominant disease characterized by the presence of concurrent benign tumors of smooth muscle origin (leiomyoma) in the skin and uterus of affected females, and in the skin of affected males. MCL can also be associated with type II papillary renal cell cancer (HLRCC). The genetic locus for MCL and HLRCC was recently mapped to chromosome 1q42.3-43 and subsequently, dominantly inherited mutations in the fumarate hydratase gene (FH) were identified. Importantly, analysis of the FH gene in tumors of MCL patients revealed a second mutation inactivating the wild-type allele in some tumors. Based on these findings, it has been suggested that FH may function as a tumor suppressor gene in MCL. Here, we report the analysis of the FH gene in a group of 11 MCL families, with the identification of 8 different mutations accounting for the disease in all families. One of the mutations, 905-1G>A, has been identified in 4 families of Iranian origin. The analysis of highly polymorphic markers in the vicinity of the FH gene showed a shared haplotype in these 4 families, suggesting that 905-1G>A represents a founder mutation. Collectively, identification of 5 novel and 3 recurrent mutations further supports the role of FH in the pathogenesis of MCL., Supported in part by the Skin Disease Research Center, Department of Dermatology, Columbia University (P30 AR44535), a National Institutes of Health research grant (K01-HG0005501), and a research grant from the Women's Health Program, supported by HWZOA. G. S. C. is a fellow of Howard Hughes Medical Institute-Medical Student Training Fellowship.

Malignant mesothelioma of the tunica vaginalis is rare, and is usually not diagnosed until surgery is undertaken. Reports on the ultrasound features of this tumour are limited. We present an unusual case with ultrasound features mimicking an adenomatoid tumour.