Growth dynamics of nanocolumnar thin films deposited by magnetron sputtering at oblique angles.

The morphology of numerous nanocolumnar thin films deposited by the magnetron sputtering technique at oblique geometries and at relatively low temperatures has been analyzed for materials as different as Au, Pt, Ti, Cr, TiO2, Al, HfN, Mo, V, WO3and W. Despite similar deposition conditions, two characteristic nanostructures have been identified depending on the material: a first one defined by highly tilted and symmetric nanocolumnar structures with a relatively high film density, and a second one characterized by rather vertical and asymmetric nanocolumns, with a much lower film density. With the help of a model, the two characteristic nanostructures have been linked to different growth dynamics and, specifically, to different surface relaxation mechanisms upon the incorporation of gaseous species with kinetic energies above the surface binding energy. Moreover, in the case of Ti, a smooth structural transition between the two types of growths has been found when varying the value of the power used to maintain the plasma discharge. Based on these results, the existence of different surface relaxation mechanisms is proposed, which quantitatively explains numerous experimental results under the same conceptual framework.

Low temperature nucleation of thermochromic VO2crystal domains in nanocolumnar porous thin films.

The low temperature formation of monoclinic VO2crystal domains in nanocolumnar vanadium/oxygen thin films prepared by magnetron sputtering at oblique angles is analyzed. The synthesis procedure involved the deposition of amorphous nanocolumnar VO1.9thin films at room temperature and its subsequent annealing at temperatures between 250 °C and 330 °C in an oxygen atmosphere. The thermochromic transition of these films was found at a temperature of 47 °C when the annealing temperature was 270 °C and 58 °C when the annealing temperature was 280 °C and 290 °C, presenting a clear drop of the optical transmittance in the infrared region of the spectrum. The significant downshift in the temperature window to obtain VO2in comparison with compact films and other strategies in literature is explained by the particular morphology of the nanocolumnar structures, which contains numerous defects along with open and embedded porosity.

Experience with the use of siltuximab in patients with SARS-CoV-2 infection.

OBJECTIVE: The study aims to describe characteristics and clinical outcome of patients with SARS-CoV-2 infection that received siltuximab according to a protocol that aimed to early block the activity of IL-6 to avoid the progression of the inflammatory flare. METHODS: Retrospective review of the first 31 patients with SARS-CoV-2 treated with siltuximab, in Hospital Clinic of Barcelona or Hospital Universitario Salamanca, from March to April 2020 with positive polymerase-chain reaction (PCR) from a nasopharyngeal swab. RESULTS: The cohort included 31 cases that received siltuximab with a median (IQR) age of 62 (56-71) and 71% were males. The most frequent comorbidity was hypertension (48%). The median dose of siltuximab was 800 mg ranging between 785 and 900 mg. 7 patients received siltuximab as a salvage therapy after one dose of tocilizumab. At the end of the study, a total of 26 (83.9) patients had been discharged alive and the mortality rate was 16.1% but only 1 out of 24 that received siltuximab as a first line option (4%). CONCLUSIONS: Siltuximab is a well-tolerated alternative to tocilizumab when administered as a first line option in patients with COVID-19 pneumonia within the first 10 days from symptoms onset and high C-reactive protein.

Real-life use of remdesivir in hospitalized patients with COVID-19.

OBJECTIVE: Controversial results on remdesivir efficacy have been reported. We aimed to report our real-life experience with the use of remdesivir from its availability in Spain. METHODS: We performed a descriptive study of all patients admitted for ≥48 hours with confirmed COVID-19 who received remdesivir between the 1st of July and the 30th of September 2020. RESULTS: A total of 123 patients out of 242 admitted with COVID-19 at our hospital (50.8%) received remdesivir. Median age was 58 years, 61% were males and 56.9 % received at least one anti-inflammatory treatment. No adverse events requiring remdesivir discontinuation were reported. The need of intensive care unit admission, mechanical ventilation and 30-days mortality were 19.5%, 7.3% and 4.1%, respectively. CONCLUSIONS: In our real-life experience, the use of remdesivir in hospitalized patients with COVID-19 was associated with a low mortality rate and good safety profile.

Mycoplasma penetrans bacteremia in an immunocompromised patient detected by metagenomic sequencing: a case report.

BACKGROUND: Mycoplasma sp. are well recognized as etiological agents of respiratory and sexually transmitted disease. Mycoplasma penetrans, a species of Mycoplasma sp., has been frequently detected in HIV-positive patients and associated with the progression of HIV-associated disease. To date, there is only a single case report describing M. penetrans as the causative agent of a severe respiratory tract infection in a HIV-negative patient. CASE PRESENTATION: In this report, we describe the case of M. penetrans bacteremia in a HIV-negative, 38-year-old, female, immunocompromised, solid organ transplant patient (combined kidney and pancreas transplantation in 2016), who was admitted to our hospital with anemic uterine bleeding and fever of 38.3 °C. Several hours before her admission at our university hospital, a latex bladder catheter was inserted into her uterus and she complained about fatigue, dizziness and ongoing vaginal bleeding. Laboratory examination showed severe anemia, but microbiological examination was inconspicuous (culture negative vaginal and cervical smears, negative urine culture). Bacterial blood cultures showed a growth signal after 4 h, but microscopic examination with Gram staining and subcultures on different agar media did not identify bacterial pathogens. To identify the bacterial cause of malignancy in the patient, metagenomic sequencing of the blood culture was performed that identified M. penetrans. CONCLUSION: Metagenomic sequencing identified M. penetrans in an immunosuppressed patient with culture-negative bacteremia. Clinicians should be aware of the opportunistic potential of M. penetrans that may cause severe infections in certain vulnerable patient populations and the limitations of culture and Gram staining for confirming the presence of fastidious bacterial pathogens like Mycoplasma spp.

Evaluating methodologies for species delimitation: the mismatch between phenotypes and genotypes in lichenized fungi (Bryoria sect. Implexae, Parmeliaceae).

In many lichen-forming fungi, molecular phylogenetic analyses lead to the discovery of cryptic species within traditional morphospecies. However, in some cases, molecular sequence data also questions the separation of phenotypically characterised species. Here we apply an integrative taxonomy approach - including morphological, chemical, molecular, and distributional characters - to re-assess species boundaries in a traditionally speciose group of hair lichens, Bryoria sect. Implexae. We sampled multilocus sequence and microsatellite data from 142 specimens from a broad intercontinental distribution. Molecular data included DNA sequences of the standard fungal markers ITS, IGS, GAPDH, two newly tested loci (FRBi15 and FRBi16), and SSR frequencies from 18 microsatellite markers. Datasets were analysed with Bayesian and maximum likelihood phylogenetic reconstruction, phenogram reconstruction, STRUCTURE Bayesian clustering, principal coordinate analysis, haplotype network, and several different species delimitation analyses (ABGD, PTP, GMYC, and DISSECT). Additionally, past population demography and divergence times are estimated. The different approaches to species recognition do not support the monophyly of the 11 currently accepted morphospecies, and rather suggest the reduction of these to four phylogenetic species. Moreover, three of these are relatively recent in origin and cryptic, including phenotypically and chemically variable specimens. Issues regarding the integration of an evolutionary perspective into taxonomic conclusions in species complexes, which have undergone recent diversification, are discussed. The four accepted species, all epitypified by sequenced material, are Bryoria fuscescens, B. glabra, B. kockiana, and B. pseudofuscescens. Ten species rank names are reduced to synonymy. In the absence of molecular data, they can be recorded as the B. fuscescens complex. Intraspecific phenotype plasticity and factors affecting the speciation of different morphospecies in this group of Bryoria are outlined.

Antibiotic selection in the treatment of acute invasive infections by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is characterized by an important intrinsic resistance to antibiotics and it possess an extraordinary ability to develop resistance to nearly all available antimicrobials through selection of mutations. We review some of the pharmacodynamic principles of antibiotics predicting efficacy, clinical experience with monotherapy and combination therapy, and principles for antibiotic treatment for empirical and directed treatment of P. aeruginosa invasive infections.

Kinetic energy-induced growth regimes of nanocolumnar Ti thin films deposited by evaporation and magnetron sputtering.

We experimentally analyze different growth regimes of Ti thin films associated to the existence of kinetic energy-induced relaxation mechanisms in the material's network when operating at oblique geometries. For this purpose, we have deposited different films by evaporation and magnetron sputtering under similar geometrical arrangements and at low temperatures. With the help of a well-established growth model we have found three different growth regimes: (i) low energy deposition, exemplified by the evaporation technique, carried out by species with typical energies in the thermal range, where the morphology and density of the film can be explained by solely considering surface shadowing processes, (ii) magnetron sputtering under weak plasma conditions, where the film growth is mediated by surface shadowing mechanisms and kinetic-energy-induced relaxation processes, and (iii) magnetron sputtering under intense plasma conditions, where the film growth is highly influenced by the plasma, and whose morphology is defined by nanocolumns with similar tilt than evaporated films, but with much higher density. The existence of these three regimes explains the variety of morphologies of nanocolumnar Ti thin films grown at oblique angles under similar conditions in the literature.

Ochratoxin a in human breast milk, maternal and placental blood from Cochabamba-Bolivia / Presencia de ocratoxina a en leche, placenta y sangre materna en Cochabamba-Bolivia

The nephrotoxic and carcinogenic mycotoxin Ochratoxin A (OTA) is a contaminant in a wide variety of foods. Mothers who ingest food contaminated with OTA during the gestation and lactation period can transfer this mycotoxin to the fetus as to the infant, respectively. To quantify the transmission of Ochratoxin A from mother to newborn and to infant, samples were taken from maternal blood and umbilical cord of women at the moment of delivery and breast milk of ladies in lactation period in the city of Cochabamba-Bolivia. From a total of 31 samples of maternal plasma and its respective cord, 4 were positive in both sample types (incidence 13%, 12% allowable relative error to the population at 95% confidence). Maternal plasma samples had a range of 0.3-10.2 ng of Ochratoxin A/ml with a mean of 3.0 ng/ml (+/- SD=3,0). Similarly, in the umbilical cord plasma Ochratoxin A was also detected with values of 0.3 to 1.7 ng/ml and with a mean of 0.7+/- 0.65 ng/ml. In two samples the concentrations of ochratoxin A were higher in umbilical cord blood (0,5 and 1.7 ng/mL) than their corresponding pairs of maternal blood (0.3 and 1.3 ng /ml, respectively) with an average concentration ratio of 1.5 ng/ml. Two other samples presented positive in ochratoxin A in umbilical cord blood and negative in their corresponding pairs of maternal blood confirming the transfer of the mycotoxin through human placental barrier. Out of 29 samples the breast milk samples from the Bolivian mothers collected during the summer period, 13 (44.8%) were found positive in a ranged from 20.2 to 146.1 ng/l. Concerning the samples collected in the winter season, only one (2.4%) out of 42 had a measurable ochratoxin A concentration of 28.3 ng/1, and seven samples showed traces of the toxin, below the detection limit

La micotoxina nefrotóxica y cancerígena Ocratoxina A (OTA) es un contaminante en una amplia variedad de alimentos. Las madres que ingieren alimentos contaminados con OTA durante el período de gestación y lactancia pueden transferir esta micotoxina al feto y al bebé, respectivamente. Para cuantificar la transmisión de la ocratoxina A de la madre al feto y al niño, se tomaron muestras de sangre materna y cordón umbilical de mujeres en el momento del parto y de leche materna de madres en el período de lactancia en la ciudad de Cochabamba-Bolivia. De un total de 31 muestras de plasma materno y su respectivo cordón umbilical, 4 resultaron positivas en ambos tipos de muestra, (incidencia 13%, error admisible de 12% respecto a la población con 95% de confianza). Las muestras de plasma materno se encontraron en un rango de 0,3 a 10,2 ng/ml con una media de 3,0 ng/ml (+/- SD = 3,0). De igual manera, en el plasma de cordón umbilical se detectó también OTA en cuatro muestras, con valores de 0,3 a 1,7 ng/ml y con una media de 0,7 ng/ml (+/- SD=0.65). En dos muestras las concentraciones de ocratoxina A eran mayores en la sangre del cordón umbilical (0,5 y 1,7 ng/ml) que sus correspondientes pares de la sangre materna (0,3 y 1,3 ng/ml, respectivamente) con una relación de concentraciones promedio de 1,5. Otras dos muestras presentaron positivo en OTA en la sangre del cordón umbilical y negativo en sus correspondientes pares de sangre materna. Confirmándose así el traspaso de esta micotoxina a través de la barrera placentaria humana. De otra parte, de un total de 29 muestras de leche materna humana colectadas de madres bolivianas durante la estación de verano, 13 (44.8 %) se encontraron positivas en un rango de 20,2 a 146,1 ng/l. Respecto a las muestras de leche colectadas durante la estación de invierno solo 1 (2,4%) de 42 fueron positivas en ocratoxina a una concentración de 28,3 ng/l y siete muestras mostraron trazas de la toxina por debajo del límite de detección

Structural control in porous/compact multilayer systems grown by magnetron sputtering.

In this work we analyze a phenomenon that takes place when growing magnetron sputtered porous/compact multilayer systems by alternating the oblique angle and the classical configuration geometries. We show that the compact layers develop numerous fissures rooted in the porous structures of the film below, in a phenomenon that amplifies when increasing the number of stacked layers. We demonstrate that these fissures emerge during growth due to the high roughness of the porous layers and the coarsening of a discontinuous interfacial region. To minimize this phenomenon, we have grown thin interlayers between porous and compact films under the impingement of energetic plasma ions, responsible for smoothing out the interfaces and inhibiting the formation of structural fissures. This method has been tested in practical situations for compact TiO2/porous SiO2 multilayer systems, although it can be extrapolated to other materials and conditions.

Nanocolumnar association and domain formation in porous thin films grown by evaporation at oblique angles.

Porous thin films grown at oblique angles by evaporation techniques are formed by tilted nanocolumnar structures which, depending on the material type and growth conditions, associate along certain preferential directions, giving rise to large domains. This arrangement, commonly denoted as bundling association, is investigated in the present work by performing fundamental experiments and growth simulations. It is proved that trapping processes of vapor species at the film surface, together with the shadowing mechanism, mediate the anisotropic widening of the nanocolumns and promote their preferential coalescence along certain directions, giving rise to domains with different shape and size. The role of these two processes is thoroughly studied in connection with the formation of these domains in materials as different as SiO2 and TiO2.

Laser Treatment of Nanoparticulated Metal Thin Films for Ceramic Tile Decoration.

This paper presents a new method for the fabrication of metal-like decorative layers on glazed ceramic tiles. It consists of the laser treatment of Cu thin films prepared by electron-beam evaporation at glancing angles. A thin film of discontinuous Cu nanoparticles was electron-beam-evaporated in an oblique angle configuration onto ceramic tiles and an ample palette of colors obtained by laser treatment both in air and in vacuum. Scanning electron microscopy along with UV-vis-near-IR spectroscopy and time-of-flight secondary ion mass spectrometry analysis were used to characterize the differently colored layers. On the basis of these analyses, color development has been accounted for by a simple model considering surface melting phenomena and different microstructural and chemical transformations of the outmost surface layers of the samples.

Growth regimes of porous gold thin films deposited by magnetron sputtering at oblique incidence: from compact to columnar microstructures.

Growth regimes of gold thin films deposited by magnetron sputtering at oblique angles and low temperatures are studied from both theoretical and experimental points of view. Thin films were deposited in a broad range of experimental conditions by varying the substrate tilt angle and background pressure, and were analyzed by field emission scanning electron microscopy and grazing-incidence small-angle x-ray scattering techniques. Results indicate that the morphological features of the films strongly depend on the experimental conditions, but can be categorized within four generic microstructures, each of them defined by a different bulk geometrical pattern, pore percolation depth and connectivity. With the help of a growth model, a microstructure phase diagram has been constructed where the main features of the films are depicted as a function of experimentally controllable quantities, finding a good agreement with the experimental results in all the studied cases.

Efectos inmediatos de la manipulación occipito-atlo-axoidea en el apoyo podal: estudio baropodométrico / Immediate effects of occipito-atloaxoid manipulation on foot support: baropodometric study

Introducción: El equilibrio postural es mantenido gracias a un sistema de aferencias y eferencias en continua adaptación. El objetivo de estas adaptaciones es el mantenimiento de la horizontalidad de la mirada y los centros laberínticos. Para esto el raquis cervical superior tiene un rol importante asegurando la orientación de la cabeza en el espacio. Este estudio trata de evaluar el efecto sobre el apoyo de la normalización propioceptiva del raquis suboccipital. Objetivos: Evaluar cambios inmediatos en la distribución de presiones en el apoyo plantar tras la técnica de thrust occipitoatlo-axoidea (TOAA). Material y Métodos: Se realizó un estudio experimental controlado aleatorizado (ECCA) de carácter explicativo, simple ciego con estrategia de evaluador cegado. Se evaluó a cada sujeto antes y después de la intervención o placebo mediante plataforma de presión. Los sujetos fueron evaluados sin calzado y recibiendo órdenes protocolizadas. La muestra fue de 46 sujetos (25 hombres / 22 mujeres) con una edad media de 24,98±3,04 años. Se utilizó para la comparación intergrupal de la varianza el estadístico ANOVA para las variables paramétricas y la prueba U de Mann Whitney para las no paramétricas. Resultados: Se observó aumento de la «presión máxima» (p=0,044) y del «Porcentaje de carga sobre el pie izquierdo» (p=0,048) acercándolo al reparto equitativo bilateral. Conclusiones: La manipulación occipito-atlo-axoidea aumenta la presión máxima de apoyo y aproxima el porcentaje de carga al reparto equitativo bilateral en sujetos sanos. Los resultados inducen a pensar de cambios en el apoyo tras la técnica, lo cual debe ser comprobado en estudios posteriores con muestras más amplias (AU)

Introduction: Postural balance is maintained thanks to a continually changing system of inputs and outputs. The goal of these changes is to maintain the sight and labyrinthine centres horizontal. To do this, the upper cervical spine has an important role, ensuring the head’s direction within the area. This study is about assessing the effect on the support of proprioceptive normalisation of the suboccipital spine. Objectives: To assess immediate changes in pressure distribution on the arch support after occipito-atlo-axoid thrust (OAAT). Material and methods: A single blind randomised controlled trial (RCT) of an experimental explanatory nature was carried out using the strategy of a blind (no connection between the assessor and inspector) assessor. Each subject was assessed before and after the procedure or placebo using a pressure platform. The subjects were assessed without footwear receiving standardised orders. The sample had 46 subjects (25 men and 22 women) with an average age of 24.98±3.04. For comparison between groups of the variance for parametric variables, the ANOVA statistic was used and for the non-parametric variables the Mann Whitney U test was used. Results: An increase was seen in the «maximum pressure» (p=0.044) and in the «load percentage on the left foot» (p=0.048) coming close to equitable bilateral distribution. Conclusions: Occipito-atlo-axoid manipulation increases the maximum support pressure and approaches the percentage for equitable bilateral load distribution in healthy subjects. The results could lead to considering changes in support after the technique, which must be verified in later studies with larger samples (AU)

Transplantation of hepatocytes in nonhuman primates: a preclinical model for the treatment of hepatic metabolic diseases.

BACKGROUND: The transplantation of isolated hepatocytes in large animals, including nonhuman primates, must be evaluated before clinical trials are performed. However, in the absence of large transgenic animals and large-animal (as opposed to small-animal) models of genetic deficiencies, it is difficult to evaluate the fate of transplanted hepatocytes, their localization, survival, and function within the parenchyma of the host liver. In this work, we aimed to develop a technique for delivering hepatocytes to the liver of a nonhuman primate and to evaluate their localization and functionality in the short term. METHODS: A 20% hepatectomy was performed in 34 cynomolgus monkeys (Macaca fascicularis) and hepatocytes were isolated. Hepatocytes were labeled in vitro with a recombinant retrovirus expressing the beta-galactosidase gene and returned to the liver by infusion through a portal catheter left in place. Liver biopsies were performed 4 and 7 d after transplantation. RESULTS: Twenty-four monkeys underwent surgery to define the necessary technical adjustments and to optimize conditions. Six monkeys died. The whole protocol, including the transplantation of genetically marked hepatocytes and procurement of liver biopsies, was performed in the remaining 10 monkeys. In eight monkeys, transplanted hepatocytes expressing the beta-galactosidase gene were widely distributed in the portal tracts, sinusoids, and hepatocyte plates of the host liver 4 and 7 d after transplantation. CONCLUSIONS: We have developed an experimental nonhuman primate model for the evaluation of hepatocyte transplantation. We demonstrated the engraftment and functioning of transplanted hepatocytes in the host liver 4 and 7 d after transplantation.

[Allotransplantation in utero and immortalization of primate fetal hepatocytes]. / Allotransplantation in utero et immortalisation d'hépatocytes foetaux de primates.

We are developing cell therapy approaches on non-human primates as a preclinical model for the treatment of hepatic metabolic diseases. In foetuses, the tissues, including liver, are in expansion, which should facilitate hepatocytes engraftment, and the immune system becomes fully mature only after birth. We have set out conditions for isolation of fetal hepatocytes from macaca mulatta at the end of the 2nd trimester of gestation (90-100 days), their cryopreservation and retroviral transduction. Two different routes of administration of hepatocytes were evaluated: the umbilical vein which was deleterious for the foetuses, and the intraparenchymatous injection which was well tolerated by the animals. Administration of hepatocytes into the hepatic parenchyma resulted in microchimerism and allogenic cells were visualized 9 days after transplantation. Another approach has been to immortalize simian foetal hepatocytes using a retroviral vector expressing SV40 Large T flanked by lox sites. A cell line has been established for 2 years, which is not tumorigenic when injected subcutaneously into nude mice and display characteristics of bipotent hepatoblasts, precursors of hepatocytes and biliary cells. After orthotopic transplantation into nude mice via the portal vein, these cells expressed albumin until the sacrifice of the animals (17 days). The next steps will be to define conditions for transplantation of retrovirally transduced fetal primary and/or immortalized hepatocytes into young foetuses (60 days of gestation) and post-natally.

Engraftment of autologous retrovirally transduced hepatocytes after intraportal transplantation into nonhuman primates: implication for ex vivo gene therapy.

The main impediment to effective ex vivo liver gene therapy of metabolic diseases is the lack of experimental work on large animals to resolve such important issues as effective gene delivery, cell-processing techniques, and the development of appropriate vectors. We have used a nonhuman primate, as a preclinical model, to analyze the limiting steps of this approach using recombinant retroviruses. Seven monkeys (Macaca fascicularis) underwent the complete protocol: their left liver lobe was resected, a catheter was placed in the inferior mesenteric vein and connected to an infusion chamber, and the hepatocytes were isolated, cultured, and transduced with a retroviral vector containing the beta-galactosidase gene. The hepatocytes were harvested and returned to the host via the infusion chamber. Biopsies were taken 4-40 days later. No animal was killed in the course of the experiments. They all tolerated the procedure well. We have developed and defined conditions that permit the proliferation and transduction of up to 90% of the plated hepatocytes. A significant proportion of genetically modified cells, representing up to 3% of the liver mass, were safely delivered to the liver via the chamber. Polymerase chain reaction analysis detected integrated viral DNA sequences and quantitative analysis of the in situ beta-Gal-expressing hepatocytes indicated that a significant amount of transduced hepatocytes, up to 2%, had become integrated into the liver and were functional. These results represent substantial advances in the development of the ex vivo approach and suggest that this approach is of clinical relevance for liver-directed gene therapy.

[Effect of portal branch ligation on liver regeneration in the rat]. / Effet de la ligature portale partielle sur la régénération hépatique chez le rat.

GOAL: The aim of this study was to assess liver regeneration after partial portal ligation. METHODS: 70% partial portal occlusion was obtained by ligation of the left portal vein branch. Total liver weight ratio were measured 96 hours after partial portal occlusion and in sham operated animals. The kinetics of hepatocytes division was evaluated by measuring the incorporation of 5-bromo-21-deoxyuridine into replicating cells at various time points by immunohistochemistry. RESULTS: Partial portal occlusion did not alter the total liver weight 96 hours after surgery. It resulted in atrophy of the ligated lobes and hypertrophy of the lobes with preserved portal flow. Hypertrophy was associated to an increase of the percentage of replicating hepatocytes. The replication rate was maximum at 28 hours with a peak at 12.5% and was prolonged beyond the 48th hour. CONCLUSIONS: Partial portal occlusion results in major and prolonged regeneration process in the liver lobes with preserved portal flow.

A novel heptameric sequence (TTAGTAA) is the binding site for a protein required for high level expression of pcbAB, the first gene of the penicillin biosynthesis in Penicillium chrysogenum.

The first two genes pcbAB and pcbC of the penicillin biosynthesis pathway are expressed from a 1.01-kilobase bidirectional promoter region. A series of sequential deletions were made in the pcbAB promoter region, and the constructions with the modified promoters coupled to the lacZ reporter gene were introduced as single copies at the pyrG locus in Penicillium chrysogenum npe10. Three regions, boxes A, B, and C, produced a significant decrease in expression of the reporter gene when deleted. Protein-DNA complexes were observed by using the electrophoretic mobility shift assay with boxes A and B (complexes AG1, BG1, BG2, and BL1) but not with box C. Uracil interference assay showed that a protein in P. chrysogenum cell extracts interacts with the thymines in a palindromic heptanucleotide TTAGTAA. Point mutations and deletion of the entire TTAGTAA sequence supported the involvement of this sequence in the binding of a transcriptional activator named penicillin transcriptional activator 1 (PTA1). In vivo studies using constructions carrying point mutations in the TTAGTAA sequence (or a deletion of the complete heptanucleotide) confirmed that this intact sequence is required for high level expression of the pcbAB gene. The TTAGTAA sequence resembles the target sequence of BAS2 (PHO2), a factor required for expression of several genes in yeasts.

In utero allotransplantation of retrovirally transduced fetal hepatocytes in primates: feasibility and short-term follow-up.

In utero allotransplantation of fetal hepatocytes into a preimmune fetus could be used in early treatment of many inherited hepatic metabolic diseases. This study was designed to assess the tolerance to hepatocyte transplantation and to test the feasability and toxicity of such an injection in a primate model. Fetal hepatocytes were obtained from two 120-day-old Macaca mulatta fetuses and cryopreserved. They were thawed, cultured in vitro, and transduced with a recombinant retrovirus expressing beta-galactosidase. Transduction efficiency was 75-85%. Three unrelated fetuses (90, 100, and 104 days old) were each given 1-2 x 10(7) transduced cells via the umbilical vein. This caused vasospasm and severe bradycardia. Two fetuses died in the 48 hours after transplantation; the third survived and was killed at the end of gestation. No evidence of the infused cells was found. Three fetuses (90 days old) were, therefore, given 3-4 10(7) hepatocytes by direct intrahepatic injection. All the fetuses survived without side effect. Donor cells were not apparent from histochemical staining and PCR reactions. There was no evidence of inflammatory reaction. These findings indicate that the protocole could be improved by increasing the number of transplanted cells and using specific hepatic promoters in the retroviral vectors to achieve an effective postnatal chimerism.