Risk factors for major complications after surgical treatment of primary ileocecal Crohn's disease. A multicentric Latin American experience.

INTRODUCTION: Complications after ileocecal resection for Crohn's disease (CD) are frequent. The aim of this study was to analyze risk factors for postoperative complications after these procedures. MATERIALS AND METHODS: We conducted a retrospective analysis of patients treated surgically for Crohn's disease limited to the ileocecal region during an 8-year period at 10 medical centers specialized in inflammatory bowel disease (IBD) in Latin America. Patients were allocated into 2 groups: those who presented major postoperative complications (Clavien-Dindo > II), the "postoperative complication" (POC) group; and those who did not, the "no postoperative complication" (NPOC) group. Preoperative characteristics and intraoperative variables were analyzed to identify possible factors for POC. RESULTS: In total, 337 patients were included, with 51 (15.13%) in the POC cohort. Smoking was more prevalent among the POC patients (31.37 vs. 17.83; P = .026), who presented more preoperative anemia (33.33 vs. 17.48%; P = .009), required more urgent care (37.25 vs. 22.38; P = .023), and had lower albumin levels. Complicated disease was associated with higher postoperative morbidity. POC patients had a longer operative time (188.77 vs. 143.86 min; P = .005), more intraoperative complications (17.65 vs. 4.55%; P < .001), and lower rates of primary anastomosis. In the multivariate analysis, both smoking and intraoperative complications were independently associated with the occurrence of major postoperative complications. CONCLUSION: This study shows that risk factors for complications after primary ileocecal resections for Crohn's disease in Latin America are similar to those reported elsewhere. Future efforts in the region should be aimed at improving these outcomes by controlling some of the identified factors.

Comprehensive analysis (aerobic/anaerobic, molecular recognitions, band-position and degradation-mechanism) of undoped and Co-doped anatase-brookite - An experimental/theoretical evaluation of the less-studied TiO2 mixed phase.

The molecular recognition (MRec) effect is required in the initial phase of organic reactions. The second stage involves molecular-orientations and molecular-orbitals energy-levels (MOrbE). The components of a reaction must be compatible in terms MRec and MOrbE. Therefore, the comprehension of photocatalytic systems applied in wastewater treatment will be improved if the MRec effect is also considered as an important factor. The purpose of this study is to provide a comprehensive understanding of the less studied anatase-brookite mixed-phase (doped and undoped). Anatase/brookite photocatalytic systems were evaluated utilizing experimental/theoretical approaches in H2O (aerobic/anaerobic) environments with Vis-light and the organic pollutant (OrPo) methyl orange (MO). The compatibility of MRec and MOrbE of anatase-brookite mixed-phase (with the different reactive system components) confirmed this is the optimal combination for photocatalytic application. Using the sol-gel method, AM-TiO2NP (amorphous), TiO2NP (crystalline), and TiO2NP-Co0.1 at% (crystalline Co-doped) anatase-brookite mixed-phase photocatalysts were obtained. The morphology and surface were characterized using XRD, BET, SEM, HR-TEM, FT-IR and XPS. Employing UV-vis DRS and PL, photo-response and electron-hole recombination were studied. LVS and Mott-Schottky plot were employed to determine photo-electrochemical activity. The results of TiO2NP photocatalytic degradation in both aerobic and anaerobic environments are remarkable. The results of molecular dynamics (MD) simulation and Fukui Function (FF) based on density functional theory (DFT) validate the remarkable photocatalytic MO degradation.

Oral antiseptics against coronavirus: in-vitro and clinical evidence.

Angiotensin converting enzyme 2 (ACE2) is the cellular receptor for SARS-CoV-2, so ACE2-expressing cells can act as target cells and are susceptible to infection. ACE2 receptors are highly expressed in the oral cavity, so this may be a potential high-risk route for SARS-CoV-2 infection. Furthermore, the virus can be detected in saliva, even before COVID-19 symptoms appear, with the consequent high risk of virus transmission in asymptomatic/presymptomatic patients. Reducing oral viral load could lead to a lower risk of transmission via salivary droplets or aerosols and therefore contribute to the control of the pandemic. Our aim was to evaluate the available evidence testing the in-vitro and in-vivo effects of oral antiseptics to inactivate or eradicate coronaviruses. The criteria used were those described in the PRISMA declaration for performing systematic reviews. An electronic search was conducted in Medline (via PubMed) and in Web of Sciences, using the MeSH terms: 'mouthwash' OR 'oral rinse' OR 'mouth rinse' OR 'povidone iodine' OR 'hydrogen peroxide' OR 'cetylpyridinium chloride' AND 'COVID-19' OR 'SARS-CoV-2' OR 'coronavirus' OR 'SARS' OR 'MERS'. The initial search strategy identified 619 articles on two electronic databases. Seventeen articles were included assessing the virucidal efficacy of oral antiseptics against coronaviruses. In conclusion, there is sufficient in-vitro evidence to support the use of antiseptics to potentially reduce the viral load of SARS-CoV-2 and other coronaviruses. However, in-vivo evidence for most oral antiseptics is limited. Randomized clinical trials with a control group are needed to demonstrate its clinical efficacy.

Teleconsulta en la pandemia por Coronavirus: desafíos para la telemedicina pos-COVID-19 / Teleconsultation in a pandemic due to Coronavirus: challenges for telemedicine in the post-COVID-19 era

Con el objetivo de detener la transmisión del coronavirus (declarado como pandemia por la Organización Mundial de la Salud [OMS]), y prevenir la propagación de la enfermedad COVID-19, el Gobierno de Colombia decretó un período de aislamiento social y confinamiento obligatorio en el territorio nacional a partir del 24 de marzo de 2020. El Instituto de Coloproctología (ICO) diseñó un programa de teleconsulta, junto con la entidad prestadora de los servicios de salud (EPS) SURA, apoyado en las tecnologías de la información disponibles. Se buscó mantener las actividades propias de la coloproctología y la fisioterapia del piso pélvico para garan-tizar a los pacientes la atención y el acceso ininterrumpido. Luego de 25 días, se programaron 626 consultas (coloproctología 62 % y fisioterapia del piso pélvico 38 %) con un porcentaje de ejecución del 94 % y una resolutividad del 78 %. Estas cifras demuestran que la telemedicina, al igual que otras actividades basadas en el teletrabajo, llegó para quedarse y trae consigo altos niveles de satisfacción para médicos, pacientes y entidades prestadoras de los servicios de salud. En este campo, también, el mundo nunca será el mismo.(AU)

After being decreed, as of March 24, 2020, social isolation and mandatory confinement began in the national territory with the aim of stopping the transmission and preventing the spread of the coronavirus as it was declared a pandemic by the World Organization of Health (WHO). The Instituto de Coloproctología (ICO) designed and developed, together with its largest insurance company EPS SURA and through the available technologies of information, a teleconsultation program, with the aim of maintaining the activities of colorectal surgery and pelvic floor biofeedback and thus guaranteeing uninterrupted medical care and access for our patients. After twenty-five days, 626 consultations were scheduled (colorectal surgery 62 % and pelvic floor biofeedback 38 %) with a percentage of execution of 94 % and a resolution of 78%. This shows that teleme-dicine, like all teleworking activities, came to stay with high levels of satisfaction, in a world that will never be the same again.(AU)

Low loss optical waveguides fabricated in LiTaO3 by swift heavy ion irradiation.

Optical waveguides are fabricated by irradiation of LiTaO3 with a variety of swift heavy ions that provide increasing levels of both nuclear and electronic damage rates, including C, F and Si ions, in the energy range of 15-40 MeV. A systematic study of the role of the ion fluence has been carried out in the broad range of 1e13-2e15 at/cm2. The kinetics of damage is initially of nuclear origin for the lowest fluences and stopping powers and, then, is enhanced by the electronic excitation (for F and Si ions) in synergy with the nuclear damage. Applying suitable annealing treatments, optical propagation losses values as low as 0.1 dB have been achieved. The damage rates found in LiTaO3 have been compared with those known for the reference LiNbO3 and discussed in the context of the thermal spike model.

Zika y casos de microcefalia en el estado Zulia: aspectos clínicos y epidemiológicos del brote, año 2016 / Zika and cases of microcephaly in the state of Zulia: clinical and epidemiological aspects of the sprout, year 2016

El Zika es una enfermedad emergente en Venezuela y en el estado Zulia, evoluciona generalmente de manera benigna y puede producir complicaciones como síndrome Guillain Barré y microcefalia en recién nacidos. Se plantea describir los aspectos clínicos, epidemiológicos del brote de Zika y los casos de microcefalia. La investigación es descriptiva, transversal, realizada de mayo 2016 a marzo 2017. La población fue de 1.155 casos sospechosos, la muestra 228 pacientes, resultando 108 positivos por PCR en tiempo real. El mayor número de casos se presentó en la edad de 17 a 33 años (59,3%), predomina el sexo femenino (81,5%), en los municipios Maracaibo y San Francisco (75%). En relación a la sintomatología, la frecuencia fue erupción (84,3%), cefalea y fiebre (76,9%), menos común la artralgia (65,7%) y mialgias (47,2%). Durante el brote, se presentaron dos muertes del sexo masculino, representando el 1,9%; 146 casos con el síndrome de Guillain Barré y 7 casos con microcefalia correspondiente al sexo masculino (85%), con una edad gestacional del recién nacido de 37,4 semanas y sobrevida de 85%. La principal recomendación, debe estar orientada al control del vector transmisor de la enfermedad y hacer el diagnóstico de los casos sospechosos para su confirmación.

Zika is an emerging disease in Venezuela and in the state of Zulia, it generally evolves in a benign manner and can produce complications such as Guillain-Barré syndrome and microcephaly in newborns. Itis proposed to describe the clinical and epidemiological aspects of the Zika outbreak and the cases of microcephaly. The research is descriptive, cross-sectional, conducted from May 2016 to March 2017. The population consisted of 1,155 suspected cases, with a sample of 228 patients, resulting in 108 positive PCR in real time. The largest number of cases occurred at the age of 17 to 33 years (59.3%), with a predominance of females (81.5%), in the municipalities of Maracaibo and San Francisco (75%). In relation to the symptomatology, the frequent are rash (84.3%), headache and fever (76.9%), while arthralgia (65.7%) and myalgia (47.2%) are less common. During the outbreak, there were two deaths of the male sex, representing 1.9%; 146 cases with Guillain-Barré syndrome and 7 cases with microcephaly, corresponding to the male sex (85%), with a newborn gestational age of 37.4 weeks and 85% survival. The main recommendation should be directed to the control of the transmitting vector of the disease and to make the diagnosis of the suspicious cases for their confirmation.

Pharmacokinetics and toxicity of the novel oral demethylating agent zebularine in laboratory and tumor bearing dogs.

The purpose of this study was to determine the plasma pharmacokinetics (PK) and toxicity of zebularine, an oral cytidine analog with demethylating activity, in dogs. Plasma zebularine concentrations were determined by HPLC-MS/MS following an oral zebularine dose of 8 or 4 mg kg-1 . Plasma zebularine clearance was constant. Mean maximum concentration (Cmax ) was 23 ± 4.8 and 8.6 ± 1.4 µM following 8 and 4 mg kg-1 , respectively. Mean half-life was 5.7 ± 0.84 and 7.1 ± 2.1 following 8 and 4 mg kg-1 , respectively. A single 8 mg kg-1 dose was well tolerated. Daily 4 mg kg-1 treatment in three laboratory dogs resulted in grade 4 neutropenia (n = 3), grade 1 anorexia (n = 2) and grade 1 or 2 dermatologic changes (n = 2). All adverse events resolved with supportive care. A 4 mg kg-1 dose every 21 days was well tolerated. A follow-up dose escalation study is in progress with a lower starting dose.

¿Es realmente la azitromicina una opción terapéutica en la salmonelosis intestinal? / Is azithromycin really a therapeutic option in intestinal salmonellosis?

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Batch and Pulsed Fed-Batch Cultures of Aspergillus flavipes FP-500 Growing on Lemon Peel at Stirred Tank Reactor.

Aspergillus flavipes FP-500 grew up on submerged cultures using lemon peel as the only carbon source, developing several batch and pulsed fed-batch trials on a stirred tank reactor. The effect of carbon source concentration, reducing sugar presence and initial pH on exopectinase and endopectinase production, was analyzed on batch cultures. From this, we observed that the highest substrate concentration favored biomass (X max) but had not influence on the corresponding specific production (q p) of both pectinases; the most acid condition provoked higher endopectinase-specific productions but had not a significant effect on those corresponding to exopectinases; and reducing sugar concentrations higher than 1.5 g/L retarded pectinase production. On the other hand, by employing the pulsed fed-batch operation mode, we observed a prolonged growth phase, and an increase of about twofold on endopectinase production without a significant raise on biomass concentration. So, pulsed fed-batch seems to be a good alternative for obtaining higher endopectinase titers by using high lemon peel quantities without having mixing and repression problems to the system. The usefulness of unstructured kinetic models for explaining, under a theoretic level, the behavior of the fungus along the batch culture with regard to pectinase production was evident.

Relación médico-paciente y mercado de la salud en Colombia / The doctor-patient relationship and the healthcare market in Colombia / A relação médico-paciente e o mercado da saúde na Colômbia

Este artículo versa sobre el deterioro de la relación médico-paciente por el sistema de salud colombiano basado en principios neoliberales del mercado de la salud. Son tan numerosos los datos y las evidencias del deterioro de la atención en salud en Colombia durante las dos últimas décadas, que se hace innecesario emplear una metodología cuantitativa de administración de pruebas para denunciarlo. Aquí se muestra la precarización de la relación médico-paciente en Colombia, mediante testimonios de expertos y observación propia como usuario del sistema...

This paper addresses the deterioration of the doctor-patient relationship within the Colombian healthcare system based on neoliberal principles of the healthcare market. The data on and evidence of the deterioration of healthcare services in Colombia over the past two decades are so numerous that it is unnecessary to use quantitative test management methodology to denounce this. Here, the way in which the doctor-patient relationship in Colombia has become precarious is shown through expert testimony and personal observation as a system user...

Este artigo trata da deterioração da relação médico-paciente pelo sistema de saúde colombiano, baseado nos princípios neoliberais do mercado da saúde. São tão numerosos os dados e as evidências da deterioração dos serviços de saúde na Colômbia durante as últimas duas décadas, que não é necessário utilizar uma metodologia quantitativa de gerenciamento de teste para denunciá-lo. Aqui se mostra a precarização da relação médico-paciente na Colômbia mediante testemunhas de especialistas e observações próprias como usuário do sistema...

Isolation of Actinobacillus pleuropneumoniae from layer hens showing clinical signs of infectious coryza.

Actinobacillus pleuropneumoniae is the causal agent of porcine pleuropneumonia, which is a highly contagious respiratory disease that affects swine nearly exclusively. An isolate with characteristics of some Pasteurellaceae family members (Gram-negative bacterium, pleomorphic, and NAD-dependent) was isolated from layer hens showing clinical signs of infectious coryza. This bacterium presented hemolysis on rabbit red blood cell agar plates, and PCR amplification and sequencing of its 16S rDNA gene indicated 99% identity with A. pleuropneumoniae serotypes 3 and 7. The presence of a putative apxIIA gene was also determined by PCR. A single, smooth colony of this bacterium inoculated in five, 7-day-old chicken embryos via the yolk sac route induced 100% mortality. However, inoculation into 10-wk-old, specific-pathogen-free chickens induced only light facial swelling, and reisolation of the inoculated bacterium was negative.

The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx).

The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.

Disruption of the MYC-miRNA-EZH2 loop to suppress aggressive B-cell lymphoma survival and clonogenicity.

c-MYC (hereafter MYC) overexpression has been recognized in aggressive B-cell lymphomas and linked to adverse prognosis. MYC activation results in widespread repression of micro-RNA (miRNA) expression and associated with lymphoma aggressive progression. Our recent study identified a MYC-miRNA-EZH2 feed-forward loop linking overexpression of MYC, EZH2 and miRNA repression. Here, using a novel small-molecule BET bromodomain inhibitor, JQ1, and the EZH2 inhibitor, DZNep, we demonstrated that combined treatment of JQ1 and DZNep cooperatively disrupted MYC activation, resulting in a greater restoration of miR-26a expression and synergistically suppressed lymphoma growth and clonogenicity in aggressive lymphoma cells. Furthermore, CHIP assay demonstrated that MYC recruited EZH2 to miR-26a promoter and cooperatively repressed miR-26a expression in aggressive lymphoma cell lines, as well as primary lymphoma cells. Loss- or gain-of-function approaches revealed that miR-26a functioned as a tumor suppressor miRNA and mediated the combinatorial effects of JQ1 and DZNep. These findings represent a novel promising approach for silencing MYC-miRNA-EZH2 amplification loop for combinatorial therapy of aggressive B-cell lymphomas.

Silencing of Kruppel-like factor 2 by the histone methyltransferase EZH2 in human cancer.

The Kruppel-like factor (KLF) proteins are multitasked transcriptional regulators with an expanding tumor suppressor function. KLF2 is one of the prominent members of the family because of its diminished expression in malignancies and its growth-inhibitory, pro-apoptotic and anti-angiogenic roles. In this study, we show that epigenetic silencing of KLF2 occurs in cancer cells through direct transcriptional repression mediated by the Polycomb group protein Enhancer of Zeste Homolog 2 (EZH2). Binding of EZH2 to the 5'-end of KLF2 is also associated with a gain of trimethylated lysine 27 histone H3 and a depletion of phosphorylated serine 2 of RNA polymerase. Upon depletion of EZH2 by RNA interference, short hairpin RNA or use of the small molecule 3-Deazaneplanocin A, the expression of KLF2 was restored. The transfection of KLF2 in cells with EZH2-associated silencing showed a significant anti-tumoral effect, both in culture and in xenografted nude mice. In this last setting, KLF2 transfection was also associated with decreased dissemination and lower mortality rate. In EZH2-depleted cells, which characteristically have lower tumorigenicity, the induction of KLF2 depletion 'rescued' partially the oncogenic phenotype, suggesting that KLF2 repression has an important role in EZH2 oncogenesis. Most importantly, the translation of the described results to human primary samples demonstrated that patients with prostate or breast tumors with low levels of KLF2 and high expression of EZH2 had a shorter overall survival.

Hypermethylation of specific microRNA genes in MLL-rearranged infant acute lymphoblastic leukemia: major matters at a micro scale.

MLL-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year) is the most aggressive type of childhood leukemia. To develop more suitable treatment strategies, a firm understanding of the biology underlying this disease is of utmost importance. MLL-rearranged ALL displays a unique gene expression profile, partly explained by erroneous histone modifications. We recently showed that t(4;11)-positive infant ALL is also characterized by pronounced promoter CpG hypermethylation. In this study, we investigated whether this widespread hypermethylation also affected microRNA (miRNA) expression. We identified 11 miRNAs that were downregulated in t(4;11)-positive infant ALL as a consequence of CpG hypermethylation. Seven of these miRNAs were re-activated after exposure to the de-methylating agent Zebularine. Interestingly, five of these miRNAs are associated either with MLL or MLL fusions, and for miR-152 we found both MLL and DNA methyltransferase 1 (DNMT1) as potential targeted genes. Finally, a high degree of methylation of the miR-152 CpG island was strongly correlated with a poor clinical outcome. Our data suggests that inhibitors of methylation have a potential beyond re-expression of hypermethylated protein-coding genes in t(4;11)-positive infant ALL. In this study, we provide additional evidence that they should be tested for their efficacy in MLL-rearranged infant ALL in in vivo models.

Wealth of opportunity - the C1 domain as a target for drug development.

The diacylglycerol-responsive C1 domains of protein kinase C and of the related classes of signaling proteins represent highly attractive targets for drug development. The signaling functions that are regulated by C1 domains are central to cellular control, thereby impacting many pathological conditions. Our understanding of the diacylglycerol signaling pathways provides great confidence in the utility of intervention in these pathways for treatment of cancer and other conditions. Multiple compounds directed at these signaling proteins, including compounds directed at the C1 domains, are currently in clinical trials, providing strong validation for these targets. Extensive understanding of the structure and function of C1 domains, coupled with detailed insights into the molecular details of ligand - C1 domain interactions, provides a solid basis for rational and semi-rational drug design. Finally, the complexity of the factors contributing to ligand - C1 domain interactions affords abundant opportunities for manipulation of selectivity; indeed, substantially selective compounds have already been identified.

Adolescents as victims of familial violence: a hospital based surveillance.

Adolescent abuse is an important and understudied issue in society. The objective of this study was to examine the epidemiology of physical injuries due to maltreatment among adolescents aged 10-19 years. Subjects came from seven hospitals/trauma centres in Washington DC that were involved in the Washington DC Initiative to Reduce Infant Mortality and Prevention of Childhood Injuries Study. From 1996-1998, information was gathered about all injuries to adolescents aged 10-19 years that resulted in a visit to a participating emergency department. This paper focuses on the subset 178 adolescents aged 10-19 years who presented with physical injuries due to maltreatment. It was found that 55% of victims of abuse were female. Abuse victims were more likely to be female than those with unintentional injury. The most common injuries were contusions to the extremities (29%). Mothers were the most common perpetrators (48%). A total of 64% of victims were assaulted with an object/weapon and the most common object used was a belt. There are some similarities and some important differences between patterns of maltreatment in adolescents vs. younger children. Increased awareness of maltreatment among older children is a critical step in increasing and improving screening and prevention practices among health-care professionals.

Gallibacterium anatis-secreted metalloproteases degrade chicken IgG.

Gallibacterium anatis (previously named Pasteurella haemolytica-like) is considered a normal inhabitant of genital and upper respiratory tracts of healthy chickens, but it is also associated with different pathological conditions. Secreted metalloproteases from field and reference G. anatis cultures were obtained by methanol precipitation and were characterized. Proteins of molecular mass higher than 100 kDa showing proteolytic activity were observed in 10% polyacrylamide gels copolymerized with 1% bovine casein. They were active at alkaline pH, and inhibited by ethylenediamine tetraacetic acid. Their activity was stable at 50 degrees C, but partially inhibited at 60 degrees C, and totally inhibited at higher temperatures. Secreted proteins were able to degrade chicken IgG after 24 h of incubation, and cross-reacted with a polyclonal antibody against purified protease from Actinobacillus pleuropneumoniae. Secreted metalloproteases could play a role in infections caused by G. anatis.

Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua.

An important group of antimalarial drugs consists of the endoperoxide sesquiterpene lactone artemisinin and its derivatives. Only little is known about the biosynthesis of artemisinin in Artemisia annua L., particularly about the early enzymatic steps between amorpha-4,11-diene and dihydroartemisinic acid. Analyses of the terpenoids from A. annua leaves and gland secretory cells revealed the presence of the oxygenated amorpha-4,11-diene derivatives artemisinic alcohol, dihydroartemisinic alcohol, artemisinic aldehyde, dihydroartemisinic aldehyde and dihydroartemisinic acid. We also demonstrated the presence of a number of biosynthetic enzymes such as the amorpha-4,11-diene synthase and the--so far unknown--amorpha-4,11-diene hydroxylase as well as artemisinic alcohol and dihydroartemisinic aldehyde dehydrogenase activities in both leaves and glandular trichomes. From these results, we hypothesise that the early steps in artemisinin biosynthesis involve amorpha-4,11-diene hydroxylation to artemisinic alcohol, followed by oxidation to artemisinic aldehyde, reduction of the C11-C13 double bond to dihydroartemisinic aldehyde and oxidation to dihydroartemisinic acid.