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Recovering the patterns of glacial erosion over time is key to understanding feedbacks between climate and tectonic processes. Glacial erosion rates have been shown to systematically increase worldwide toward the present since the late Cenozoic, a behavior interpreted as the response of glaciers to a cooling and increasingly variable climate. However, the validity of this signal has been questioned, and suggested to be affected by the incompleteness of the sedimentary record, which can introduce a time dependent bias in the time averaged rates. In this study, we present new glacial erosion rates estimated from sediment accumulations in Lago Argentino, Patagonia, a proglacial basin with a nearly complete preserved sedimentary record. The erosion rates are estimated through the past 20,000 years and averaged over time intervals ranging from sub‐decadal to millennial, allowing us to explore erosion rate variability through time and within a glacial cycle. The data show that erosion rates have varied substantially, from 0.43 ±$\pm $0.12 to 82.38 ±$\pm $17.58 mm/yr, with no systematic increase (or decrease) through time. Rather, erosion occurs during discrete, intense events separated by times of quiescence. In addition, we find that glacial erosion rates have comparable magnitudes when averaged over similar time intervals. Our data show a power‐law increase in glacial erosion rates with decreasing averaging time interval, consistent with other observations globally. Given our observed intermittent character of glacial erosion, we attribute this increase to a time averaging bias, rather than to an escalation in magnitude of erosional pulses toward the present. Glaciers are powerful agents of erosion and sensitive to climate. Their action competes with tectonic forces in limiting the heights of mountains. Understanding how fast glaciers carve into the landscape over time helps us understand the relationship between climate and tectonics. Glacial erosion rates have been shown to increase over the past 10 million years, interpreted as an effect of a cooling climate worldwide. The same signal has been interpreted to result from how rates are estimated, particularly if rates are averaged over time intervals that contain periods of low or no erosion. In this study, we present glacial erosion rates calculated from data imaging glacial sediments preserved in a lake in Patagonia, Argentina. To understand the effect of the time interval length, we calculate glacial erosion rates for the past 20,000 years over different time intervals. We find that rates show no straightforward increase toward the present. Rather, erosion occurs during discrete events separated by times of low erosion. Rates positively correlate with the length of the averaging time interval. We attribute this behavior to a bias resulting from the length of time over which erosion rates are averaged, rather than a genuine signal in erosion rate trends. Glacial erosion rates estimated over decadal to millennial time scales do not systematically increase toward the presentArgentino and Upsala Glacier erosion rates are timescale dependent, but age independentEstimated glacial erosion rates are affected by a time bias due to the intermittent character of glacial erosion Glacial erosion rates estimated over decadal to millennial time scales do not systematically increase toward the present Argentino and Upsala Glacier erosion rates are timescale dependent, but age independent Estimated glacial erosion rates are affected by a time bias due to the intermittent character of glacial erosion

The Korteweg–de Vries equation that describes surface gravity water wave dynamics in shallow water is well known to admit cnoidal wave solutions, i.e. periodic travelling waves with stationary wave shape. Such type of periodic wave patterns can be also found in the deep-water waves with the envelopes that follow the dynamics of the nonlinear Schrödinger equation (NLS). A particular class of NLS periodic and stationary solutions are cnoidal (CN) and dnoidal (DN) envelopes. This one parameter family of solutions has as limiting cases either the envelope soliton or a constant background. In this experimental study, we discuss the physical features of such waves and emphasize the particular effect of dissipation, as observed in several hydrodynamic experiments that have been conducted in several water wave facilities with different dimensions. Experiments on such type of periodic wave envelopes in a large facility have been already reported in the early 1990s. These studies demonstrated significant deviation of the DN-type envelopes from theory. Here, we show that these deviations are due to the effect of dissipation that can be qualitatively considered by adapting the NLS framework accordingly. Reducing the amplitude of the carrier wave makes the wave field susceptible to dissipation effects. Our experiments prove that the dissipation is indeed responsible for phase-shift pulsations for DN-type envelopes.

We evaluated the impact of genomic polymorphisms in folate-metabolizing, DNA synthesis and DNA repair enzymes on the clinical outcome of 108 patients with myelodysplastic syndromes (MDS) receiving best supportive care (BSC) or azacitidine. A statistically significant association between methylenetetrahydrofolate reductase (MTHFR) 677T/T, thymidylate synthase (TS) 5′-untranslated region (UTR) 3RG, TS 3′-UTR −6 bp/−6 bp, XRCC1 399G/G genotypes and short survival was found in patients receiving BSC by multivariate analysis (P<0.001; P=0.026; P=0.058; P=0.024). MTHFR 677T/T, TS 3′-UTR −6 bp/−6 bp and XRCC1 399G/G genotypes were associated with short survival in patients receiving azacitidine by multivariate analysis (P<0.001; P=0.004; P=0.002). We then performed an exploratory analysis to evaluate the effect of the simultaneous presence of multiple adverse variant genotypes. Interestingly, patients with ⩾1 adverse genetic variants had a short survival, independently from their International Prognostic Scoring System (IPSS) and therapy received. To our knowledge, this is the first study showing that polymorphisms in folate-metabolizing pathway, DNA synthesis and DNA repair genes could influence survival of MDS patients.

On 7 May 2015, a Mw4.0 earthquake occurred near Venus, northeast Johnson County, Texas, in an area of the Bend Arch‐Fort Worth Basin that reports long‐term, high‐volume wastewater disposal and that has hosted felt earthquakes since 2009. In the weeks following the Mw4.0 earthquake, we deployed a local seismic network and purchased nearby active‐source seismic reflection data to capture additional events, characterize the causative fault, and explore potential links between ongoing industry activity and seismicity. Hypocenter relocations of the resulting local earthquake catalog span ~4–6 km depth and indicate a fault striking ~230°, dipping to the west, consistent with a nodal plane of the Mw4.0 regional moment tensor. Fault plane solutions indicate normal faulting, with Baxes striking parallel to maximum horizontal compressive stress. Seismic reflection data image the reactivated basement fault penetrating the Ordovician disposal layer and Mississippian production layer, but not displacing post‐Lower Pennsylvanian units. Template matching at regional seismic stations indicates that low‐magnitude earthquakes with similar waveforms began in April 2008, with increasing magnitude over time. Pressure data from five saltwater disposal wells within 5 km of the active fault indicate a disposal formation that is 0.9–4.8 MPa above hydrostatic. We suggest that the injection of 28,000,000 m3of wastewater between 2006 and 2015 at these wells led to an increase in subsurface pore fluid pressure that contributed to inducing this long‐lived earthquake sequence. The 2015 Mw4.0 event represents the largest event in the continuing evolution of slip on the causative fault. Seismicity spans nearly 10 years with maximum magnitude increasing with timeWastewater disposal at five wells has led to an overpressured formation that is hydraulically connected with the seismogenic basementInduced earthquakes occur only where the host fault is optimally oriented for failure within the regional stress field

Production of lactate even in the presence of sufficient levels of oxygen (aerobic glycolysis) seems the prevalent energy metabolism pathway in cancer cells. The analysis of altered expression of effectors causing redirection of glucose metabolism would help to characterize this phenomenon with possible therapeutic implications. We analyzed mRNA expression of the key enzymes involved in aerobic glycolysis in normal mucosa (NM), primary tumor (PT) and liver metastasis (LM) of colorectal cancer (CRC) patients (pts) who underwent primary tumor surgery and liver metastasectomy. Tissues of 48 CRC pts were analyzed by RT-qPCR for mRNA expression of the following genes: hexokinase-1 (HK-1) and 2 (HK-2), embryonic pyruvate kinase (PKM-2), lactate dehydrogenase-A (LDH-A), glucose transporter-1 (GLUT-1), voltage-dependent anion-selective channel protein-1 (VDAC-1). Differences in the expression of the candidate genes between tissues and associations with clinical/pathologic features were studied. GLUT-1, LDH-A, HK-1, PKM-2and VDAC-1mRNA expression levels were significantly higher in PT/LM tissues compared with NM. There was a trend for higher expression of these genes in LM compared with PT tissues, but differences were statistically significant for LDH-Aexpression only. RASmutation-positive disease was associated with high GLUT-1mRNA expression levels only. Right-sided colon tumors showed significantly higher GLUT-1, PKM-2and LDH-AmRNA expression levels. High glycolytic profile was significantly associated with poor prognosis in 20 metastatic, RAS-mutated pts treated with first-line chemotherapy plus Bevacizumab. Altered expression of effectors associated with upregulated glucose uptake and aerobic glycolysis occurs in CRC tissues. Additional analyses are warranted for addressing the role of these changes in anti-angiogenic resistance and for developing novel therapeutics.

Detailed models of crustal structure at volcanic passive margins offer insight into the role of magmatism and the distribution of igneous addition during continental rifting. The Eastern North American Margin (ENAM) is a volcanic passive margin that formed during the breakup of Pangea ∼200 Myr ago. The offshore, margin‐parallel East Coast Magnetic Anomaly (ECMA) is thought to mark the locus of syn‐rift magmatism. Previous widely spaced margin‐perpendicular studies seismically imaged igneous addition as seaward dipping reflectors (SDRs) and high velocity lower crust (HVLC; >7.2 km/s) beneath the ECMA. Along‐strike imaging is necessary to more accurately determine the distribution and volume of igneous addition during continental breakup. We use wide‐angle, marine active‐source seismic data from the 2014–2015 ENAM Community Seismic Experiment to determine crustal structure beneath a ∼370‐km‐long section of the ECMA. P‐wave velocity models based on data from short‐period ocean bottom seismometers reveal a ∼21‐km‐thick crust with laterally variable lower crust velocities ranging from 6.9 to 7.5 km/s. Sections with HVLC (>7.2 km/s) alternate with two ∼30‐km‐wide areas where the average velocities are less than 7.0 km/s. This variable structure indicates that HVLC is discontinuous along the margin, reflecting variable amounts of intrusion along‐strike. Our results suggest that magmatism during rifting was segmented. The HVLC discontinuities roughly align with locations of Mid‐Atlantic Ridge fracture zones, which may suggest that rift segmentation influenced later segmentation of the Mid‐Atlantic Ridge. The East Coast of the United States is a passive margin that formed during continental breakup of Pangea, the most recent supercontinent. Although passive margins are generally not locales of active faulting and magmatism, by investigating their current structure, we gain insight into processes during past rifting. We know that extensive volcanism and magmatic addition to the Earth's crust occurred during the breakup of Pangea, but we do not fully understand how the distribution changes from north to south along the margin. To study these rocks, which are buried 10–20 km beneath the seafloor, we use ocean bottom seismometers to determine the sound speed of rocks beneath the surface, which can tell us about composition and rock type. We found that the thickness and extent of magma‐derived rocks are variable along the margin, with gaps up to 30 km wide. This variability is likely related to processes happening in the continental rift which may have influenced the structure of the Mid‐Atlantic Ridge that formed after rifting ended. High velocity lower crust beneath the East Coast Magnetic Anomaly is variable along‐strike and discontinuous in some placesThe lower crust variability reflects variable intrusion into lower continental crust and represents magmatic segmentation during riftingThe high velocity lower crust discontinuities are coincident to Mid‐Atlantic Ridge fracture zones, suggesting a rift‐to‐ridge connection High velocity lower crust beneath the East Coast Magnetic Anomaly is variable along‐strike and discontinuous in some places The lower crust variability reflects variable intrusion into lower continental crust and represents magmatic segmentation during rifting The high velocity lower crust discontinuities are coincident to Mid‐Atlantic Ridge fracture zones, suggesting a rift‐to‐ridge connection

The discipline of seismology is based on observations of ground motion that are inherently undersampled in space and time. Our basic understanding of earthquake processes and our ability to resolve 4D Earth structure are fundamentally limited by data volume. Today, Big Data Seismology is an emergent revolution involving the use of large, data‐dense inquiries that is providing new opportunities to make fundamental advances in these areas. This article reviews recent scientific advances enabled by Big Data Seismology through the context of three major drivers: the development of new data‐dense sensor systems, improvements in computing, and the development of new types of techniques and algorithms. Each driver is explored in the context of both global and exploration seismology, alongside collaborative opportunities that combine the features of long‐duration data collections (common to global seismology) with dense networks of sensors (common to exploration seismology). The review explores some of the unique challenges and opportunities that Big Data Seismology presents, drawing on parallels from other fields facing similar issues. Finally, recent scientific findings enabled by dense seismic data sets are discussed, and we assess the opportunities for significant advances made possible with Big Data Seismology. This review is designed to be a primer for seismologists who are interested in getting up‐to‐speed with how the Big Data revolution is advancing the field of seismology. Seismology is the scientific discipline by which vibrational waves that travel through the Earth are used to study a range of natural processes, from the structure and layering of the subsurface to the dynamics of earthquake rupture. Long propelled by advances in instrumentation and the availability of waveform observations, seismology has entered an era of Big Data where new opportunities come hand in hand with new challenges. This review article gives an overview of recent scientific advances powered by Big Data Seismology, along with emerging trends, potential challenges, and future opportunities. The review encompasses a range of subjects from seismic imaging and earthquake source physics to education and outreach, and is designed to be a jumping off point for readers interested in how big data is changing the face of seismology research. Big Data Seismology is an emergent subdiscipline that uses “big data” inquiries to explore fundamental science questions in seismologyThree drivers of Big Data Seismology are the growth of large data volumes, the development of new algorithms, and advances in computingBig Data Seismology is being applied to study earthquakes, to better resolve Earth structure, and to open new frontiers in seismology Big Data Seismology is an emergent subdiscipline that uses “big data” inquiries to explore fundamental science questions in seismology Three drivers of Big Data Seismology are the growth of large data volumes, the development of new algorithms, and advances in computing Big Data Seismology is being applied to study earthquakes, to better resolve Earth structure, and to open new frontiers in seismology

Recent insights into the molecular pathogenesis of colorectal cancer (CRC) have given rise to specific targetdirected therapies, including monoclonal antibodies (mAb) against epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF). These drugs have been approved as first, second and third line therapies for metastatic CRC (mCRC) and the advent of target-specific cancer therapeutics has remarkably improved the outcomes of patients with CRC. The molecular mechanisms underlying the clinical response to these drugs are not fully understood, although recent studies have elucidated the effect of intracellular signaling pathways involving in particular RAS/RAF/MAPK signaling on the safety and efficacy of target-specific drugs. Activating mutations of KRAS and BRAF genes are genetic events in tumorigenesis and these mutations are implicated as predictive factors in determining response, in particular to anti-EGFR drugs, and additional data suggest that other EGFR downstream pathways such as PI3K/PTEN/Akt or JAK/STAT are also important when considering mechanisms of EGFR antibody resistance. Recently the European Medical Agency (EMEA) approved the use of the mAb Panitumumab (December 2007) and approved a license extension for the use of the mAb Cetuximab in combination with chemotherapy as first-line treatment (October 2008) in mCRC patients with no mutations in the codon 12 and 13 of KRAS gene. The predictive value of KRAS mutations in the treatment of CRC has been very useful to clinicians and patients in terms of decision making, avoiding toxicities, and decreasing financial burden. This success also encourages researchers to find new markers with the same strong predictive value. Future studies also need to identify patterns of multiple mutations to further increase the power of patient selection for anti-EGFR therapy. These advances allow us to truly enter a new and exciting era of individualized therapy in oncology. Here we review the molecular basis of EGFR-targeted therapies and the resistance to treatments conferred by KRAS and other gene mutations as well as the laboratory methods used to detect all these genetic variations.

The native HIV-1 Tat protein was chosen as vaccine candidate for phase I clinical trials in both uninfected (ClinicalTrials.gov identifier: NCT00529698) and infected volunteers (ClinicalTrials.gov identifier: NCT00505401). The rationale was based on the role of Tat in the natural infection and AIDS pathogenesis, on the association of Tat-specific immune responses with the asymptomatic stage and slow-progression rate as well as on its sequence conservation among HIV clades (http://www.hiv1tat-vaccines.info/). The parallel conduction in the same clinical centers of randomized, double blind, placebo-controlled phase I studies both in healthy, immunologically competent adults and in HIV-infected, clinically asymptomatic, individuals represents a unique occasion to compare the vaccine-induced immune response in both the preventive and therapeutic setting. In both studies, the same lot of the native Tat protein was administered 5 times, every four weeks, subcute (SC) with alum adjuvant or intradermic (ID), in the absence of adjuvant, at 7.5 ig, 15 ig or 30 ig doses, respectively. The primary and secondary endpoints of these studies were the safety and immunogenicity of the vaccine candidate, respectively. The study lasted 52 weeks and monitoring was conducted for on additional 3 years. The results of both studies indicated that the Tat vaccine is safe and well tolerated both locally and systemically and it is highly immunogenic at all the dosages and by both routes of administration. Vaccination with Tat induced a balanced immune response in uninfected and infected individuals. In particular, therapeutic immunization induced functional antibodies and partially reverted the marked Th1 polarization of anti-Tat immunity seen in natural infection, and elicited a more balanced Th1/Th2 immune response. Further, the number of CD4 T cells correlated positively with anti-Tat antibody titers. Based on these results, a phase II study is ongoing in infected drug-treated individuals (http://www.hiv1tat-vaccines.info/).

We report the results of the treatment of nine children with an aneurysmal bone cyst of the distal fibula (seven cysts were juxtaphyseal, and two metaphyseal). The mean age of the children was 10 years and 3 months (7 years and 4 months to 12 years and 9 months). All had open physes. All cysts were active and in seven cases substituted and expanded the entire width of the bone (type-2 lesions). The mean longitudinal extension was 5.7 cm (3 to 10). The presenting symptoms were pain, swelling and pathological fracture. Moderate fibular shortening was evident in one patient.In six patients curettage was performed, using phenol as adjuvant in three. Three with juxtaphyseal lesions underwent resection. A graft from the contralateral fibula (one case) and allografts (two cases) were positioned at the edge of the physis for reconstruction. The mean follow-up was 11.6 years (3.1 to 27.5). There was no recurrence.At the final follow-up there was no significant difference in the American Orthopaedic Foot and Ankle Society scores (excellent/good in all cases) and in growth disturbance, alignment, stability and bone reconstitution, but in the resection group the number of operations, including removal of hardware, complications (two minor) and time of immobilisation/orthosis, were increased. Movement of the ankle was restricted in one patient.The potential risks in the management of these lesions include recurrence, physeal injury, instability of the ankle and hardware and graft complications. Although resection is effective it should be reserved for aggressive or recurrent juxtaphyseal lesions.

Newly acquired, deep-penetration Broadband Onshore-Offshore Lithospheric Investigation of Venezuela and the Antilles Arc Region seismic reflection data from offshore western Venezuela (Bonaire Basin) and around the Leeward Antilles are combined with existing geologic and geophysical data sets to examine the chronology of Late Cretaceous-Cenozoic tectonic events in this part of the Caribbean-South American plate boundary zone. These tectonic events have controlled the maturation and structural trapping of known hydrocarbons in the offshore Bonaire Basin and the adjacent onland Falcón Basin. We infer three tectonic phases that are constrained using these combined data sets. (1) The late Eocene-early Oligocene, north-south opening of the 3-6-km (1.8-3.7-mi)-thick Falcón-Bonaire Basin occurred along east-west-striking normal fault systems that have locally been inverted by later tectonic phases. These Paleogene normal faults rifted the Upper Cretaceous arc crust and Paleogene marine depositional sequences within the offshore Bonaire Basin. (2) Northwest-striking normal faults crosscut the older normal faults of the Bonaire Basin and Leeward Antilles and form deep, submarine rifts that contain up to 4 km (2.5 mi) of sedimentary fill and form deep-water channels between islands of the Leeward Antilles. Offshore well data and age of onshore sediments in the Falcón Basin indicate that this second phase of rifting occurred mainly during the late Oligocene to early Miocene and remains active to the present. (3) Inversion of the subaerial Falcón Basin commenced during the middle Miocene. This inversion phase is reflected in the present-day pattern of an east-northeast-trending fold-thrust belt that can be traced over 200 km (124 mi) along strike in the Falcón Basin. A second offshore fold-thrust belt (La Vela) can be traced over a distance of 175 km (108 mi) along strike and parallel to the northeast-trending Falcón Basin coast. Restoration of imbricate thrusts seen on seismic lines perpendicular to the La Vela fold-thrust belt indicates a minimum of 7 km (4.3 mi) of northeast-southwest--directed, thin-skinned shortening. Geochemical work indicates that source rocks for scattered occurrences of hydrocarbons in the Falcón Basin and its coastal zone are Paleogene and Miocene marine shale. Reservoir rocks are Tertiary marine sandstone and shale deposited in Paleogene rifts formed during the first tectonic phase in the late Eocene to early Oligocene. Structural traps were formed by thrusting during the second tectonic phase in the late Oligocene to early Miocene. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease (IBD) present in childhood in 15% to 25% of cases. The aim of therapy in children is not only to guarantee normal growth but also to prevent relapse and to maintain remission. Steroids are effective to induce remission; however, resistance, dependency, and irreversible side effects can develop. The aim of this study was to determine whether treatment with repeated infusions of autologous red blood cells (RBCs) loaded with dexamethasone 21-phosphate (Dex 21-P) is safe and allows maintenance of long-term remission in children with steroid-dependent Crohn disease (CD).

Inflammatory bowel disease (IBD) present in childhood in 15% to 25% of cases. The aim of therapy in children is not only to guarantee normal growth but also to prevent relapse and to maintain remission. Steroids are effective to induce remission; however, resistance, dependency, and irreversible side effects can develop. The aim of this study was to determine whether treatment with repeated infusions of autologous red blood cells (RBCs) loaded with dexamethasone 21-phosphate (Dex 21-P) is safe and allows maintenance of long-term remission in children with steroid-dependent Crohn disease (CD). Eighteen consecutive pediatric patients who met the inclusion criteria were admitted to the study. Infusions of autologous RBCs loaded with Dex 21-P were performed every 4 weeks; the mean duration of treatment was 24 months. At the beginning of treatment and after 6, 12, and 24 months, we performed clinical evaluation according to the Pediatric Crohn Disease Activity Index (pCDAI). Assessment of body mass indexamethasone and bone mineral density by means of computerized bone mineralometry-dual energy x-ray absorptiometry, endoscopic evaluation, and hematic morning cortisol determination were also performed. During treatment, the mean pCDAI significantly decreased (P< 0.05); 78% of patients discontinued steroids. Determination of morning cortisol showed suppression only on the first day after infusion, followed by normalization of values. Endoscopic findings showed remission in 44% of patients. None of the patients experienced serious side effects. These data suggest that repeated infusions of RBCs loaded with Dex 21-P can be safe and useful to maintain long-term remission in pediatric patients with moderately active CD.

Evaluating the presence of leakage on fluorescein angiography (FA) in patients with age-related macular degeneration (AMD) retreated with photodynamic therapy (PDT) can be difficult. New diagnostic tools such as optical coherence tomography (OCT) might help to optimize PDT management.Thirty AMD patients scheduled for regular follow-up FA in conjunction with PDT treatment were also scanned with OCT. Follow-up data at 9 months were retrieved from the patients’ medical records. Inter-observer agreement [kappa (?) coefficient] for the presence of leakage on FA, for OCT parameters for leakage, and agreement between FA and OCT evaluations were calculated. The indication for retreatment was evaluated using the leakage analysis based on FA alone, OCT alone, and both examinations combined, and compared to the actual follow-up of the patients at 9 months.Agreement between the two observers for the presence of leakage on FA was moderate (?=0.51). OCT agreement between the two observers for the presence of leakage was good (?=0.85). Agreement between FA and OCT for the presence of leakage was poor (?=0.16). Follow-up data at 9 months on all patients were analyzed. Seven out of 30 patients were not retreated at the time of examination, and four of these patients (57%) remained stable without further treatment. Twenty-three patients did receive a PDT treatment at the time of examination; and eight of these patients did not show leakage on OCT, and five of these patients (62%) remained stable without additional treatment. In contrast, only three out of 15 patients (20%) with leakage on both FA and OCT remained stable during this 9 month follow-up period.Inter-observer agreement for the presence of leakage was moderate for FA and good for OCT. There was considerable disagreement between leakage as judged by OCT and by FA. OCT could be of help in the decision regarding PDT retreatment. Assuming that 57% of the patients without leakage either on FA or OCT would remain stable without retreatment, the rate of probable ineffective retreatment could be reduced from 35% to 20%.

Reduced glutathione (GSH) is present in millimolar concentrations in mammalian cells. It is involved in many cellular functions such as detoxification, amino acid transport, production of coenzymes, and the recycling of vitamins E and C. GSH acts as a redox buffer to preserve the reduced intracellular environment. Decreased glutathione levels have been found in numerous diseases such as cancer, viral infections, and immune dysfunctions. Many antioxidant molecules, such as GSH and N-acetylcysteine (NAC), have been demonstrated to inhibit in vitro and in vivo viral replication through different mechanisms of action. Accumulating evidence suggests that intracellular GSH levels in antigen-presenting cells such as macrophages, influence the Th1/Th2 cytokine response pattern, and more precisely, GSH depletion inhibits Th1-associated cytokine production and/or favours Th2 associated responses. It is known that GSH is not transported to most cells and tissues in a free form. Therefore, a number of different approaches have been developed in the last years to circumvent this problem. This review discusses the capacity of some new molecules with potent pro- GSH effects either to exert significant antiviral activity or to augment GSH intracellular content in macrophages to generate and maintain the appropriate Th1/Th2 balance. The observations reported herein show that pro-GSH molecules represent new therapeutic agents to treat antiviral infections and Th2-mediated diseases such as allergic disorders and AIDS.

Double-stranded oligodeoxynucleotides (ODN) containing the consensus binding sequence of a transcription factor are valuable tools for the manipulation of gene expression at the transcriptional level by means of the decoy strategy. The approach involves flooding the cells with enough ODN decoy to compete for binding of the transcription factor with its consensus sequence in target genes. The technique has been proven effective in vitro and in vivo, suggesting its use in therapy. Therefore, great attention has been recently focused on chemical modifications enhancing biostability of the oligonucleotides so as to improve their pharmacological properties. Unfortunately, benefits in terms of nuclease resistance have been often negated by a decrease in the affinity and / or specificity of transcription factor binding. To circumvent these problems, circular dumbbell and chimeric decoy oligonucleotides, the latter comprising a central stretch of transcription factor recruiting deoxynucleotides flanked by modified nucleotides, have been introduced. Nevertheless, these approaches have the limitation of leaving the ODNs still sensitive to endonuclease cleavage. To address these concerns, we have recently investigated the use of a new class of nucleotide analogs termed locked nucleic acids (LNA) in the designing of decoy molecules for the transcription factor κB (NF-κB). In this article, we review the advantages of LNA in the design of ODN decoys pointing to the feasibility of introducing modifications in the transcription factor binding sequence in order to obtain molecules with increased stability compared to end-capped ODNs, while remaining efficiently recognized by NF-κB.

AbstractLoss of E-cadherin expression has been related with an adverse outcome in patients with resected gastric cancer. More recently, experimental models with cancer cell lines showed that chemosensitivity may be affected by the E-cadherin expression status. We investigated whether E-cadherin expression is correlated with the response to chemotherapy and the survival of patients with advanced gastric cancer. Consecutive patients with advanced gastric cancer who underwent palliative chemotherapy were considered eligible for study entry. Measurable disease, complete follow-up information and availability of tumor specimens for immunohistochemistry were mandatory inclusion criteria. In 70 assessable patients, 30 patients had locoregional disease and 40 patients had visceral metastases. Chemotherapy consisted of cisplatin/fluorouracil/folinic acid in 33 patients and cisplatin/fluorouracil/epirubicin/folinic acid in 37 patients. There were 13 patients with complete response, 20 with partial response, 20 with stable disease and 17 patients progressed. Thirty-eight patients had >80% E-cadherin-positive cancer cells (positive E-cadherin expression); 15 cases showed 25–70% (reduced E-cadherin expression), and in the remaining 17 cases E-cadherin expression was &lt;10% (negative E-cadherin expression). The response to chemotherapy was unrelated to the E-cadherin expression status. Conversely, survival in the 32 patients with reduced/negative E-cadherin expression (25 weeks) was significantly worse than that observed in the 38 patients with preserved E-cadherin expression (36 weeks) (p &lt; 0.01). E-cadherin expression retained its independent prognostic role in the multivariate analysis. E-cadherin expression may give prognostic information in patients with advanced gastric cancer, but it does not seem to possess a predictive role in vivo. Some of the mechanisms inducing E-cadherin downregulation, like hypermethylation, may be potentially reversible, and they deserve further investigation as the target of novel therapeutic strategies.Copyright © 2004 S. Karger AG, Basel

Human red blood cells, once in circulation, survive for 120 days and are then removed through an immunomediated mechanism by resident macrophages. We have taken advantage of these unique properties to develop a new drug delivery system. In fact, a number of conventional and new drugs can be encapsulated into human erythrocytes by several procedures. Once re-infused in the same donor the circulating red cells behave as a slow drug delivery system. Alternatively, drug-loaded red cells can be modified to increase their phagocytosis by inducing band 3 clustering and opsonization by autologous IgGs and complement (up to C3b). Macrophages recognize and phagocytose such drug-loaded red cells through the Fc and C3b receptors. This drug targeting system permits the administration of membrane-impermeable molecules to macrophages and allows to optimize treatment of human infections by a number of pathogens with a tropism for macrophages. Furthermore, as reviewed in this paper, modified red cells can be conveniently used to deliver peptides, modified proteins, antisense nucleotides, or small organic molecules. The red cell-based drug targeting system has been proved to be effective and safe in preclinical studies. The administration of drugs by autologous red cells has been proved to be effective in humans with more than 600 administrations.

SummaryHuman red blood cells, once in circulation, survive for 120 days and are then removed through an immunomediated mechanism by resident macrophages. We have taken advantage of these unique properties to develop a new drug delivery system. In fact, a number of conventional and new drugs can be encapsulated into human erythrocytes by several procedures. Once re-infused in the same donor the circulating red cells behave as a slow drug delivery system. Alternatively, drug-loaded red cells can be modified to increase their phagocytosis by inducing band 3 clustering and opsonization by autologous IgGs and complement (up to C3b). Macrophages recognize and phagocytose such drug-loaded red cells through the Fc and C3b receptors. This drug targeting system permits the administration of membrane-impermeable molecules to macrophages and allows to optimize treatment of human infections by a number of pathogens with a tropism for macrophages. Furthermore, as reviewed in this paper, modified red cells can be conveniently used to deliver peptides, modified proteins, antisense nucleotides, or small organic molecules. The red cell-based drug targeting system has been proved to be effective and safe in preclinical studies. The administration of drugs by autologous red cells has been proved to be effective in humans with more than 600 administrations. Copyright © 2004 S. Karger GmbH, Freiburg

Among the antiviral agents developed for the treatment of human viral infections, nucleoside analogs represent the largest group. However, much remains to do to improve their pharmacokinetic properties, to increase their efficacy, to reduce the selection of drug-resistent strains and to reduce their toxic side effects. Towards this end many nucleotide dimers have been synthesized in the last years in several laboratories. Such compounds have several advantages compared to the administration of nucleoside analogs as single drugs: 1) can act as prodrugs for a slow delivery of monomers in circulation; 2) can be encapsulated into autologous erythrocytes to perform as bioreactors converting a non diffusible dimer into a diffusible nucleoside analog to be released in circulation; 3) can be targeted to macrophages by proper drug targeting systems; 4) can overcome the limiting phosphorylating activities of several infectable cell types; 5) can have the advantage of a combination therapy with the administration of a single compound. In this review, dimers developed in our laboratory will be reported. In particular, the heterodinucleotide AZTpPMPA and the homodinucleotide Bis-PMEA are shown to be able to act as prodrugs when administered to mice releasing the single monomer in circulation. The homodinucleotide AZTp2AZT and the dimer AZTp2EMB once encapsulated in human erythrocytes are converted by erythrocyte enzymes into diffusible nucleosides and slowly released from the carrier cells. The dimers AZTp2AZT, AZTp2ACV, ACVpPMPA, AZTpPMPA and Bis-PMEA were targeted to macrophages where a very effective protection against virus replications was obtained. Thus, nucleotide dimers could be used as effective prodrugs for drug delivery in the treatment of viral infections improving the pharmacokinetic of single moieties and can be efficiently targeted to selected cell types with intracellular release of a phosphorylated (active) nucleoside.

AbstractCurrent studies are investigating new E-cadherin gene (CDH1) mutations that may be responsible for diffuse gastric cancer susceptibility. Recently, a novel CDH1 germline variant presenting a G/A nucleotide change at cDNA position 2494 has been found in Japanese patients with familial diffuse gastric cancer. The consequent amino acid variation (Val/Met) may alter the binding activity to beta-catenin and the adhesive function of the E-cadherin protein. We have investigated its frequency in Italian cases of sporadic diffuse gastric cancer a well as in healthy controls. Peripheral blood samples were collected from consecutive patients with sporadic, diffuse gastric cancer and from healthy controls in the District of Urbino, Marche Region, Central Italy. After DNA extraction, standard techniques for molecular analyses were used to investigate the 2494 G/A germline nucleotide change in CDH1 cDNA. None of the 48 patients and 48 controls showed the G/A 2494 nucleotide change. Assuming a binomial distribution of the mutation among individuals and the absence of mutations in the 48 patients, the 95% upper bound for the underlying mutation frequency was 7.4%. The novel CDH1 nucleotide change is uncommon in Italian patients with sporadic diffuse gastric cancer. Given these results, further analyses in large population-based studies are not advisable.Copyright © 2003 S. Karger AG, Basel

The 26S proteasome is a multicatalytic complex that acts as primary protease of the ubiquitin-mediated proteolytic pathway in eukaryotes. We provide here the first evidence that the proteasome plays a key role in regulating pollen tube growth. Immunoblotting experiments revealed the presence of high levels of free ubiquitin and ubiquitin conjugates in rehydrated and germinating pollen of kiwifruit [Actinidia deliciosa var. deliciosa (A. Chev) C. F. Liang et A. R. Ferguson]. Proteasome activity, assayed fluorometrically, accompanied the progression of germination. Specific inhibitors of proteasome function such as benzyloxycarbonyl-leucinyl-leucinyl-leucinal (MG-132), clasto-lactacystin beta-lactone, and epoxomicin significantly decreased tube growth or altered tube morphology. High-molecular mass, ubiquitinated proteins accumulated in MG-132- and beta-lactone-treated pollen, indicating that proteasome function was effectively impaired. The inhibitors were also able to decrease in vitro proteasome activity in pollen extracts. Because MG-132 can inhibit calpains, as well as the proteasome, trans-epoxy succinyl-L-leucylamido-(4-guanidino) butane (E-64), an inhibitor of cysteine proteases, was investigated. Some reduction in tube growth rate was observed, but only at 80 microM E-64, and no abnormal tubes were produced. Furthermore, no inhibition of tube growth was observed when another inhibitor of cysteine proteases, leupeptin, or inhibitors of serine and aspartic proteases (phenylmethylsulfonyl fluoride and pepstatin) were used. Our results indicate that protein turnover during tube organization and elongation in kiwifruit pollen is important, and our results also implicate the ubiquitin/26S proteasome as the major proteolytic pathway involved.

Monocyte-derived macrophages (MDMs) play a central role in the pathogenesis of infection by human immunodeficiency virus (HIV-1) and represent one of the main reservoirs of the virus in the body. In addition, MDMs can easily be infected by various herpes viruses, including herpes simplex virus type 1 (HSV-1). We have synthesized a new antiviral agent (Bis-PMEA) that consists of two 9-(2-phosphonylmethoxyethyl)adenine (PMEA) molecules bound by a phosphate bridge. This nucleotide analogue, like the parent compound PMEA, has strong and selective activity against HIV-1 and HSV-1. A drug-targeting system previously developed in our laboratory was used for the selective delivery of these drugs to macrophages. Bis-PMEA and PMEA were encapsulated into autologous erythrocytes by a procedure of hypotonic dialysis and isotonic resealing. Loaded erythrocytes were modified to increase their recognition and phagocytosis by human macrophages. By administering Bis-PMEA-loaded erythrocytes to macrophages, 47% of Bis-PMEA and 28% of PMEA was still present 10 days after phagocytosis; in contrast, only 12% of PMEA was found in macrophages receiving PMEA-loaded erythrocytes. Bis-PMEA-loaded erythrocytes were then added to macrophages infected with HIV-1 and HSV-1 and their antiviral activity evaluated. Remarkable protection was obtained against HIV-1 and HSV-1 infection (95 and 85%, respectively). Therefore, Bis-PMEA acts as an efficient antiviral prodrug that, following selective targeting to macrophages by means of loaded erythrocytes, can protect a refractory cell compartment.

Monocyte-derived macrophages (M/M) are considered important in vivoreservoirs for different kinds of viruses, including HIV. Hence, therapeutic strategies are urgently needed to protect these cells from virus infection or to control viral replication. In this paper, we report the synthesis, target delivery and in vitroefficacy of a new heterodinucleotide (AZTpPMPA), able to inhibit HIV-1 production in human macrophages. AZTpPMPA consists of two established anti-HIV drugs [zidovudine (AZT) and tenofovir (PMPA)] chemically coupled together by a phosphate bridge. This drug is not able to prevent p24 production when administered for 18 h to M/M experimentally infected with HIV-1 Bal (inhibition 27%), but can almost completely suppress virus production when given encapsulated into autologous erythrocytes (inhibition of p24 production 97%). AZTpPMPA is slowly converted to PMPA, AZT monophosphate and AZT (36 h half-life at 37°C) by cell-resident enzymes. Thus AZTpPMPA should be considered a new prodrug of AZT and PMPA that is able to provide stechiometric amounts of both nucleoside analogues to macrophage cells and to overcome the low phosphorylating activity of M/M for AZT and the modest permeability of PMPA.

IRMA (the acronym stands for InfraRed Microscope Analysis) is a hyperspectral imaging spectrometer which is capable, in its present design, to achieve a spatial resolution of 38 μm in the 0.8–5 μm infrared spectral range. IRMA has the goal to first ever quantitatively characterize the mineralogy and the microphysical structure of the materials of the Martian soils and rocks down to the depth available to a lander sampling mechanism. The experiment has been selected by the Italian Space Agency (ASI), in the framework of the Italian participation to the NASA's Mars Surveyor Programme, to be part of the Italian Package for Scientific Experiments (IPSE). IPSE was to fly on-board the ill-fated MARS2003 MARS SAMPLE RETURN Mission, postponed during the early months of 2000 to a later date (possibly 2007). IRMA present state of development is compatible with a flight readiness in 2007.

This paper presents the results of a small-angle neutron scattering (SANS) study of He-bubble growth in martensitic steel F82H-mod. The investigated samples had been homogeneously implanted with 400 appm He at 250°C, then submitted to 2 h annealings up to 975°C, together with a non-implanted sample of the same material (one for each temperature) serving as a reference to isolate the SANS signal arising from the He-bubbles. An as-implanted sample was also investigated together with its thermal reference. The He-bubble volume distributions, obtained from the SANS data and discussed with reference to uncertainties arising from background subtraction, show that the uniform distribution of the as-implanted sample, with bubbles approximately 15 Å in size, evolves during high temperature annealing into a bimodal one with a secondary population of bubbles as large as 100 Å approximately.

Nuclear Factor-kB (NF-kB), is a transcription factor composed of dimeric complexes of p50 (NF-kB1) or p52 (NF-kB2) usually associated with members of the Rel family (p65, c-Rel, Rel B) which have potent transactivation domains. Different combinations of NF-kB/Rel proteins bind distinct kB sites to regulate the transcription of different genes. In resting cells NF-kB resides in the cytoplasm in inactive form, complexed to members of a family of inhibitory proteins referred to as IkB. The bound IkB masks the NF-kB nuclear localization signal and thereby inhibits its nuclear transport. NF-kB can be activated by a variety of signals relevant to pathophysiology including inflammatory cytokines and bacterial lipopolysaccharides (LPS) as well as oxidative and fluid mechanical stress. Upon activation by these stimuli, IkB is phosphorylated and subsequently degraded. Phosphorylation targets IkB for ubiquitination and degradation by the 26S proteasome thus leading to NF-kB nuclear translocation. The same proteolytic pathway is involved in the processing of the p105 and p100 precursors to generate mature p50 and p52 subunits, respectively. Once in the nucleus, NF-kB is able to regulate the expression of many genes involved in the immune and inflammatory responses (i.e. inflammatory cytokines and adhesion molecules). Thus, new approaches to modulating NF-kB activation, and as a consequence inflammatory or metastatic processes, may take advantage of the selectivity of the ubiquitination and ATP-dependent proteolytic processes leading to IkB turnover. This review will analyze the current strategies aimed at interfering with NF-kB activation and will consider the ubiquitination system as a new selective target for the development of new anti-inflammatory therapies.

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The behavioral performance of young and aged rats was studied in a repeated-trials test. Young animals reacted to both spatial displacement and novelty, whereas most aged rats lost the ability to react to novelty although maintaining spatial memory. The cluster analysis procedure performed on all the tested subjects enabled the recognition of a consistent group of the aged sample (35%) with a mild degree of spatial and non-spatial memory impairment. Spatial memory impairment of some of the aged animals was also evaluated in the Morris water maze test. On the fifth day of the task, we observed a very low percentage of impaired aged animals, which partially corresponded to the impaired group identified by the object recognition test. In contrast, the subgroup of mildly impaired rats performed similarly to the young animals. We advance that the Morris water maze might represent a stressful experimental condition for aged rats, enhancing the motivational level of animals subjected to this procedure. This condition may alter the cognitive responses. As a consequence, the “mildly impaired” rats, which may be considered an interesting group for investigating memory-enhancing drugs, will infrequently be recognized with the Morris water maze test. Cognitive impairment in aged rats should be studied utilizing a sensitive test in which motivation does not substantially influence the results of the test.

The Patagonian slab window has been proposed to enhance the solid Earth response to ice mass load changes in the overlying Northern and Southern Patagonian Icefields (NPI and SPI, respectively). Here, we present the first regional seismic velocity model covering the entire north‐south extent of the slab window. A slow velocity anomaly in the uppermost mantle indicates warm mantle temperature, low viscosity, and possibly partial melt. Low velocities just below the Moho suggest that the lithospheric mantle has been thermally eroded over the youngest part of the slab window. The slowest part of the anomaly is north of 49°S, implying that the NPI and the northern SPI overlie lower viscosity mantle than the southern SPI. This comprehensive seismic mapping of the slab window provides key evidence supporting the previously hypothesized connection between post‐Little Ice Age anthropogenic ice mass loss and rapid geodetically observed glacial isostatic uplift (≥4 cm/yr). A gap in the subducting plate beneath Patagonia has enabled hotter, less viscous mantle material to flow underneath South America. Icefields in the Austral Andes above the gap in the plate have recently been shrinking, removing weight that had caused the continent to flex downward. We use seismic data to image the subsurface structure and find very low seismic velocity within and around the gap, as well as thinning of the rigid South American lithosphere overlying the gap. The low mantle velocity implies that mantle viscosity is also low beneath the shrinking icefields, and low viscosity enables the region to rebound upwards. We observe anomalously low upper mantle shear wave velocities (<4.1 km/s) in the Patagonian slab windowThe lithospheric mantle has been thermally eroded over the youngest part of the slab windowLow viscosities in the slab window link observed rapid geodetic uplift to geologically recent ice mass loss in Patagonia We observe anomalously low upper mantle shear wave velocities (<4.1 km/s) in the Patagonian slab window The lithospheric mantle has been thermally eroded over the youngest part of the slab window Low viscosities in the slab window link observed rapid geodetic uplift to geologically recent ice mass loss in Patagonia

Four different hexokinase (HK) isoenzymes are distributed in different proportions in human tissues. Fibroblasts contain HK type I as the predominant glucose phosphorylating activity, the same isoenzyme that predominates in red blood cells (RBC). We have established cell lines from two patients homozygous for RBC HK deficiency but carrying different mutations. In one case (HK-Melzo) the residual RBC enzyme shows a marked heat instability but possesses normal kinetic and regulatory properties; in the other (HK-Napoli), the enzyme is characterized by an increased Kifor glucose-1,6-diphosphate. These properties are also retained by the fibroblasts' hexokinase. Glucose utilization by cultured fibroblasts from these patients was markedly reduced in the cell lines where HK deficiency was more pronounced. However, cells with only 30% HK activity retained their full ability to utilize glucose in the hexose monophosphate pathway. This was shown to be true not only under basal conditions but also in the presence of oxidative agents such as methylene blue. Significant reduction of the ATP level was also found in HK-Melzo fibroblasts. Thus, HK deficiency is associated with reduced glucose utilization and normal hexose monophosphate shunt rates. Results previously obtained on RBC support similar conclusions.

The LP-BM5 murine retrovirus-induced immunodeficiency model was used to evaluate the efficacy and toxicity of long-term 2′,3′-dideoxycytidine (DDC) therapy. A mean plasma drug concentration of 0.2 + 0.02 μm of DDC for 3 months was found to reduce splenomegaly, lymphoadenopathy and hypergammaglobulinemia in infected mice. However, DDC also reduced spleen weight in control mice and spleen haemopoiesis in both infected and uninfected animals. In the bone marrow the most prominent feature of DDC treatment was a marked reduction of megakariocytes, while in the liver an hepatocellular vacuolation was evident in uninfected animals. DDC reduced, but did not prevent, LP-BM5 integration in lymph node DNA and Pr 60gagexpression in spleen lymphocytes and bone marrow cells. Furthermore, DDC reduced the mitochondrial DNA content and restored the mitogen proliferation of T cells but not that of B cells in infected mice. Thus, DDC appears to be of some, but limited, efficacy in murine AIDS, with a toxicity profile involving more cell types than previously thought.

Among glycolytic enzyme defects, hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1; HK) deficiency is a very rare disease where the predominant clinical effect is nonspherocytic hemolytic anemia. Here we report the characterization at molecular level of the HK type I cDNA from a patient with hemolytic anemia due to hexokinase deficiency. PCR amplification and sequence of the cDNA revealed the presence of a deletion and of a single nucleotide substitution, both in heterozygous form. In particular, the deletion, 96 bp long, concerns nucleotides 577 to 672 in the HK cDNA sequence and was never found in the cDNAs of 14 unrelated normal subjects. The sequence of the HK allele without deletion showed a single nucleotide substitution from T to C at position 1667 which causes the amino acid change from Leu⁵²⁹ to Ser. This heterozygous mutation at nt 1667 was confirmed by direct sequencing of the patient genomic DNA, but when DNAs from 10 normal controls were examined by this technique the substitution at nt 1667 was never found. From these results we concluded that the patient is carrying a point mutation at nt 1667 of one HK allele and a 96 nt deletion in the other allele. In normal subjects two differences from the published cDNA sequence were documented.

One possible explanation for the failure of human immunodeficiency virus type 1 (HIV-1) antiretroviral inhibitors to block the clinical progression of the infection may be a failure to maintain adequate drug levels at the site of viral replication. We have previously found that exposure of human monoblastoid cells (U937) for several months to a therapeutically relevant concentration (0.1 μM) of 2′,3′-dideoxycytidine (zalcitabine, ddC) allowed the isolation of a drug-resistant cell line characterized by a normal drug transport but a reduced ability to accumulate 2′,3′-dideoxycytidine 5′-triphosphate (the active antiretroviral form of the drug). In this paper we show that the drug-resistant cells were indistinguishable from normal cells in terms of surface CD4 receptors. The susceptibility of parental and ddC-resistant U937 cells to infection by HIV-1 was similar, as measured by proviral DNA formation. However, HIV-1 p24 production and the number of infectious virus particles produced were significantly lower in the drug-resistant compared to control cells. Addition of 0.1 μM ddC inhibited viral production by up to 92% in the control cells but had no effect on ddC-resistant cells. Thus, human cells exposed to therapeutically relevant ddC concentrations for several months show a reduced ddC anabolism and allow ddC-sensitive HIV-1 to replicate in the presence of inhibitory ddC concentrations.

Red blood cell glucose metabolism was investigated in a male patient with de novo trisomy 10p. According to previous evidence, when assigning hexokinase gene locus in the 10p11 leads to pter region, a triplex dosage effect of hexokinase activity (HK) was found, while all the other erythrocyte glycolytic enzymes were in the normal values range. Red blood cell glucose utilization was 2.87 mumole/hr/ml RBC as compared to 1.43 in normal controls; the rate of glucose metabolized through the hexose monophosphate shunt (HMPS) was unchanged. Glucose-6- phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, and dihydroxyacetone phosphate increased with respect to normal controls, while normal levels of 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and ATP were found. The HK activity increased in all the red blood cell fractions obtained by density gradient ultracentrifugation. However, a small difference in the distribution of cells through the gradient was evident. The experiments reported in this article show that in the red blood cells of patients with trisomy 10p, an increased level of HK leads to higher concentrations of glucose- 6-phosphate and to a faster glucose utilization in the Embden-Meyerhof pathway, while the HMPS rate is unchanged.

The antiviral agent 2′,3′-dideoxycytidine (ddC) has been shown to be active against HIV-1 infectivity. However, conflicting results have been reported concerning its efficacy in macrophages. Because macrophages possess low levels of the kinase(s) responsible for ddC phosphorylation, we investigated the ability of ddC and 2′,3′-dideoxycytidine 5′-trisphosphate (ddCTP) to suppress HIV-1 and LP-BM5 replication in these cells. Retrovirus replication was only partially inhibited in the two systems investigated by a high (1 μM) ddC concentration. The direct administration of ddCTP, using autologous red blood cells as a delivery system, was found to inhibit HIV-1 and LP-BM5 replication by more than 90% in macrophages without affecting major cell functions. These data, together with those already reported for FIV [Magnani et al.(1994) AIDS Res Hum Retroviruses10: 1179-1186], suggest that the anabolic phosphorylation of ddC is an important determinant of its anti-HIV activity and that pharmacological interventions that modulate ddC metabolism may be useful for improving its antiretroviral activity in macrophages.

Summary The addition of ingestible particles (opsonized erythrocytes or latex beads) or a phorbol ester activates monocytes — derived human macrophages (MDHM) cultured in vitro, and markedly reduces virion release from HIV-infected MDHM as well as their ability to transmit the infection to cocultured lymphoid CD 4-positive CEM cells.

Four different hexokinase (HK) isoenzymes are distributed in different proportions in human tissues. Fibroblasts contain HK type I as the predominant glucose phosphorylating activity, the same isoenzyme that predominates in red blood cells (RBC). We have established cell lines from two patients homozygous for RBC HK deficiency but carrying different mutations. In one case (HK-Melzo) the residual RBC enzyme shows a marked heat instability but possesses normal kinetic and regulatory properties; in the other (HK-Napoli), the enzyme is characterized by an increased Kifor glucose-1,6-diphosphate. These properties are also retained by the fibroblasts' hexokinase. Glucose utilization by cultured fibroblasts from these patients was markedly reduced in the cell lines where HK deficiency was more pronounced. However, cells with only 30% HK activity retained their full ability to utilize glucose in the hexose monophosphate pathway. This was shown to be true not only under basal conditions but also in the presence of oxidative agents such as methylene blue. Significant reduction of the ATP level was also found in HK-Melzo fibroblasts. Thus, HK deficiency is associated with reduced glucose utilization and normal hexose monophosphate shunt rates. Results previously obtained on RBC support similar conclusions.

The adverse effect of light or moderate maternal drinking during pregnancy on the well being of the newborn has been investigated. The study group included 2145 live births in the obstetric units of 11 Italian cities between February 1989 and July 1990. A detailed life style questionnaire was administered to the mothers. Information on the newborn was collected from clinical records as well as from a clinical examination. Both univariate and multivariate analyses were suggestive of a decrease in mean birth weight associated with maternal drinking during pregnancy, especially in women who also smoked during pregnancy. This effect was higher in male newborns. The occurrence of low birth weight (< 2500 g.) was more frequent in women drinking during pregnancy in both smokers and non-smokers (for this latter group an effect is suggested only for a daily consumption of more than 10 grams of absolute alcohol). Maternal alcohol drinking of more than 20 grams of absolute alcohol per day also increased the risk of preterm delivery (OR = 2.35; 95% CI: .98 – 5.59). Finally, an increase in the rate of early jaundice was found, also associated with maternal drinking (OR = 3.30; 95% CI: 1.03 – 10.54).

Conjugate ubiquitin was previously found in the nucleus, cytoplasm, and membranes of eukaryotic cells while the enzymes of the ubiquitin-conjugating system appear to be cytoplasmic. We have prepared the mitochondrial fraction from rabbit brain by discontinuous density gradient ultracentrifugation and by Western blotting, using a specific antibody against conjugate ubiquitin, showing that it contains ubiquitin conjugates in a very wide molecular weight range. Electron microscopy and measurement of specific enzyme markers show that this fraction not only contains mitochondria but also some endoplasmic reticulum vesicles. Immunostaining with anti-ubiquitin IgG followed by immunodecoration with colloidal gold particles provides evidence for the presence of conjugate ubiquitin both in mitochondria and in the endoplasmic reticulum. Furthermore, this “mitochondrial fraction” shows a pronounced ATP-dependent ability to conjugate 125I-ubiquitin into a number of endogenous proteins as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Addition of E1, E2, and E3, the enzymes of the ubiquitin conjugating system purified from rabbit reticulocytes, does not further increase this ubiquitination nor incorporate 125I-ubiquitin into additional protein bands. The same mitochondrial fraction is not able to carry out any ATP-dependent degradation of 125I-albumin; however, it contains an isopeptidase activity able to release the covalently incorporated 125I-ubiquitin and is also able to conjugate 125I-ubiquitin to exogenous proteins as oxidized RNase. By affinity chromatography on ubiquitin-agarose of fraction II of a crude Triton X-100 extract of the mitochondrial fraction, several proteins corresponding in Mr to the E1 and E2s enzymes were obtained. These proteins were also able to form specific ubiquitin-thiol ester bounds on sodium dodecyl sulfate-polyacrylamide gels and to support 125I-ubiquitin conjugation to oxidized RNase. Detergent fractionation of the mitochondrial fraction provided evidence for a possible localization of the ubiquitin conjugating activity in the mitochondrial external membrane and endoplasmic reticulum. The presence of an active ubiquitin protein conjugating system in mitochondria and endoplasmic reticulum may be related to the turnover of organelle proteins as well as to specific cell functions such as import of proteins into mitochondria and ubiquitination of externally oriented membrane-bound proteins.