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Single-cell technologies have transformed our understanding of human tissues. Yet, studies typically capture only a limited number of donors and disagree on cell type definitions. Integrating many single-cell datasets can address these limitations of individual studies and capture the variability present in the population. Here we present the integrated Human Lung Cell Atlas (HLCA), combining 49 datasets of the human respiratory system into a single atlas spanning over 2.4 million cells from 486 individuals. The HLCA presents a consensus cell type re-annotation with matching marker genes, including annotations of rare and previously undescribed cell types. Leveraging the number and diversity of individuals in the HLCA, we identify gene modules that are associated with demographic covariates such as age, sex and body mass index, as well as gene modules changing expression along the proximal-to-distal axis of the bronchial tree. Mapping new data to the HLCA enables rapid data annotation and interpretation. Using the HLCA as a reference for the study of disease, we identify shared cell states across multiple lung diseases, including SPP1 + profibrotic monocyte-derived macrophages in COVID-19, pulmonary fibrosis and lung carcinoma. Overall, the HLCA serves as an example for the development and use of large-scale, cross-dataset organ atlases within the Human Cell Atlas., (© 2023. The Author(s).)

Purpose: To assess the effect of mask wear on corneal topography and tomography readings., Setting: Tertiary ophthalmic center., Design: Prospective, observational, comparative study., Methods: Subjects underwent imaging with the Tomey TMS-4a topographer and the Oculus Pentacam HR tomographer. Imaging was performed without a mask, and then testing was repeated while wearing 3 different masks: Halyard Level 2, Halyard Level 3, and KN95. Measurements during mask wear were compared with measurements without a mask. The Kruskal-Wallis analysis of variance grouped test was used to compare mean differences without vs with mask wear on measurements. The Chi-square test was used to compare frequency of differences between different masks and against-the-rule vs with-the-rule astigmatism. The frequencies of clinically significant changes in axis of astigmatism, magnitude of astigmatism, and mean keratometry (K) values were calculated., Results: 52 eyes were included in the study with a mean age of 34.71. Mask wear did not show statistically significant differences in mean topography and tomography measurements compared with without mask wear for all parameters. However, the majority, 53% (83/156) had a >10 degrees and 41% (64/156) had >15 degrees change in axis of astigmatism on topography when wearing a mask compared with no mask wear., Conclusions: Mask wear did not result in statistically significant mean changes in K readings on topography and tomography. However, the axis of astigmatism varied >10 degrees in most of the patients. Axis determination should be interpreted with caution regarding refractive surgery and/or toric intraocular lens planning in individuals whose measurements were obtained while wearing a mask., (Copyright © 2022 Published by Wolters Kluwer on behalf of ASCRS and ESCRS.)

Background: Puerperal genital hematoma is an infrequent but potentially life-threatening complication of childbirth. There are three approaches to care: expectant management, surgical evacuation, or uterine artery embolization., Cases: This retrospective case series compares the clinical courses of three patients who developed puerperal genital hematoma and were managed differently. We report the length of time to complete resolution of the hematomas and the associated morbidities for each patient., Conclusion: All three management approaches of puerperal genital hematoma can be effective. Among our three patients, surgical intervention of the puerperal genital hematoma provided the most prompt and definitive management with resolution of all symptoms in 9 days, compared with 3 weeks for expectant management and 20 weeks for treatment with uterine artery embolization. Intervention should be individualized based on the patient's symptoms, stability, and desires with consideration of the hematoma size and location as well as available institutional resources., Competing Interests: Financial Disclosure The authors did not report any potential conflicts of interest., (Copyright © 2022 by the American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. All rights reserved.)

Loss of secretory IgA (SIgA) is common in chronic obstructive pulmonary disease (COPD) small airways and likely contributes to disease progression. We hypothesized that loss of SIgA results from reduced expression of pIgR (polymeric immunoglobulin receptor), a chaperone protein needed for SIgA transcytosis, in the COPD small airway epithelium. pIgR-expressing cells were defined and quantified at single-cell resolution in human airways using RNA in situ hybridization, immunostaining, and single-cell RNA sequencing. Complementary studies in mice used immunostaining, primary murine tracheal epithelial cell culture, and transgenic mice with secretory or ciliated cell-specific knockout of pIgR. SIgA degradation by human neutrophil elastase or secreted bacterial proteases from nontypeable Haemophilus influenzae was evaluated in vitro . We found that secretory cells are the predominant cell type responsible for pIgR expression in human and murine airways. Loss of SIgA in small airways was not associated with a reduction in secretory cells but rather a reduction in pIgR protein expression despite intact PIGR mRNA expression. Neutrophil elastase and nontypeable H. influenzae -secreted proteases are both capable of degrading SIgA in vitro and may also contribute to a deficient SIgA immunobarrier in COPD. Loss of the SIgA immunobarrier in small airways of patients with severe COPD is complex and likely results from both pIgR-dependent defects in IgA transcytosis and SIgA degradation.

Background: Cellular diversity of the lung endothelium has not been systematically characterized in humans. We provide a reference atlas of human lung endothelial cells (ECs) to facilitate a better understanding of the phenotypic diversity and composition of cells comprising the lung endothelium., Methods: We reprocessed human control single-cell RNA sequencing (scRNAseq) data from 6 datasets. EC populations were characterized through iterative clustering with subsequent differential expression analysis. Marker genes were validated by fluorescent microscopy and in situ hybridization. scRNAseq of primary lung ECs cultured in vitro was performed. The signaling network between different lung cell types was studied. For cross-species analysis or disease relevance, we applied the same methods to scRNAseq data obtained from mouse lungs or from human lungs with pulmonary hypertension., Results: Six lung scRNAseq datasets were reanalyzed and annotated to identify >15 000 vascular EC cells from 73 individuals. Differential expression analysis of EC revealed signatures corresponding to endothelial lineage, including panendothelial, panvascular, and subpopulation-specific marker gene sets. Beyond the broad cellular categories of lymphatic, capillary, arterial, and venous ECs, we found previously indistinguishable subpopulations; among venous EC, we identified 2 previously indistinguishable populations: pulmonary-venous ECs (COL15A1 neg ) localized to the lung parenchyma and systemic-venous ECs (COL15A1 pos ) localized to the airways and the visceral pleura; among capillary ECs, we confirmed their subclassification into recently discovered aerocytes characterized by EDNRB , SOSTDC1 , and TBX2 and general capillary EC. We confirmed that all 6 endothelial cell types, including the systemic-venous ECs and aerocytes, are present in mice and identified endothelial marker genes conserved in humans and mice. Ligand-receptor connectome analysis revealed important homeostatic crosstalk of EC with other lung resident cell types. scRNAseq of commercially available primary lung ECs demonstrated a loss of their native lung phenotype in culture. scRNAseq revealed that endothelial diversity is maintained in pulmonary hypertension. Our article is accompanied by an online data mining tool (www.LungEndothelialCellAtlas.com)., Conclusions: Our integrated analysis provides a comprehensive and well-crafted reference atlas of ECs in the normal lung and confirms and describes in detail previously unrecognized endothelial populations across a large number of humans and mice.

Patients with chronic lung disease (CLD) have an increased risk for severe coronavirus disease-19 (COVID-19) and poor outcomes. Here, we analyze the transcriptomes of 611,398 single cells isolated from healthy and CLD lungs to identify molecular characteristics of lung cells that may account for worse COVID-19 outcomes in patients with chronic lung diseases. We observe a similar cellular distribution and relative expression of SARS-CoV-2 entry factors in control and CLD lungs. CLD AT2 cells express higher levels of genes linked directly to the efficiency of viral replication and the innate immune response. Additionally, we identify basal differences in inflammatory gene expression programs that highlight how CLD alters the inflammatory microenvironment encountered upon viral exposure to the peripheral lung. Our study indicates that CLD is accompanied by changes in cell-type-specific gene expression programs that prime the lung epithelium for and influence the innate and adaptive immune responses to SARS-CoV-2 infection., (© 2021. The Author(s).)

Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2 + TMPRSS2 + cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention.

Pulmonary fibrosis (PF) is a form of chronic lung disease characterized by pathologic epithelial remodeling and accumulation of extracellular matrix (ECM). To comprehensively define the cell types, mechanisms, and mediators driving fibrotic remodeling in lungs with PF, we performed single-cell RNA sequencing of single-cell suspensions from 10 nonfibrotic control and 20 PF lungs. Analysis of 114,396 cells identified 31 distinct cell subsets/states. We report that a remarkable shift in epithelial cell phenotypes occurs in the peripheral lung in PF and identify several previously unrecognized epithelial cell phenotypes, including a KRT5 - / KRT17 + pathologic, ECM-producing epithelial cell population that was highly enriched in PF lungs. Multiple fibroblast subtypes were observed to contribute to ECM expansion in a spatially discrete manner. Together, these data provide high-resolution insights into the complexity and plasticity of the distal lung epithelium in human disease and indicate a diversity of epithelial and mesenchymal cells contribute to pathologic lung fibrosis., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Medicinal plants like Aloe barbadensis, Ginkgo biloba, Panax ginseng, and Cassia angustifolia are being increasingly used but self-medication is a major public health challenge because of their numerous interactions. Furthermore, dietary exposure to metals through the consumption of these commercial preparations should be evaluated. Some of these metals may have a nutritional interest but others are metals of toxicological relevance. The content of essential and toxic metals has been analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES) in 80 samples of both organically and conventionally grown aloe, senna, ginseng, and ginkgo pharmaceutical formats to establish their metallic profile, to evaluate their nutritional value and to assess the toxic metal risk resulting from their consumption. Ca (9181 mg/kg ww) in senna, Fe (30.0 mg/kg ww) in ginseng, and Al (15.7 mg/kg ww) in ginseng are noteworthy. The consumption of 9 g/day of ginkgo represents 6.44% of the RDI set for Ca. The consumption of 25 mL/day of aloe syrup is 3.06% (men) and 1.52% (women) of the RDI of Fe. Al exposure from 25 mL/day of aloe consumption is 2.71% of its TDI. Considering the dosages recommended by the manufacturers, the intake of toxic metals does not pose a health risk but the intake of essential metals is worth mentioning.

Matrix-assisted Laser Desorption Ionization-Time of Flight Mass Spectroscopy (MALDI-TOF MS) has been used routinely over the past decade in clinical microbiology laboratories to rapidly characterize diverse microorganisms of medical importance both at the genus and species levels. Currently, there is keen interest in applying MALDI-TOF MS at taxonomic levels beyond species and to characterize environmental isolates. We constructed a model system consisting of 19 isolates of Deinococcus aquaticus obtained from biofilm communities indigenous to diverse substrates (concrete, leaf tissue, metal, and wood) in the Fox River - Lake Winnebago system of Wisconsin to: (1) develop rapid sample preparation methods that produce high quality, reproducible MALDI-TOF spectra and (2) compare the performance of MALDI-TOF MS-based profiling to common DNA-based approaches including 16S rRNA sequencing and genomic diversity by BOX-A1R fingerprinting. Our results suggest that MALDI-TOF MS can be used to rapidly and reproducibly characterize environmental isolates of D. aquaticus at the subpopulation level. MALDI-TOF MS provided higher taxonomic resolution than either 16S rRNA gene sequence analysis or BOX-A1R fingerprinting. Spectra contained features that appeared to permit characterization of isolates into two co-occurring subpopulations. However, reliable strain-level performance required rigorous and systematic standardization of culture conditions and sample preparation. Our work suggests that MALDI-TOF MS offers promise as a rapid, reproducible, and high-resolution approach to characterize environmental isolates of members of the genus Deinococcus . Future work will focus upon application of methods described here to additional members of this ecologically diverse and ubiquitous genus.

New neurons continue to be generated in the subgranular zone of the dentate gyrus of the adult mammalian hippocampus. This process has been linked to learning and memory, stress and exercise, and is thought to be altered in neurological disease. In humans, some studies have suggested that hundreds of new neurons are added to the adult dentate gyrus every day, whereas other studies find many fewer putative new neurons. Despite these discrepancies, it is generally believed that the adult human hippocampus continues to generate new neurons. Here we show that a defined population of progenitor cells does not coalesce in the subgranular zone during human fetal or postnatal development. We also find that the number of proliferating progenitors and young neurons in the dentate gyrus declines sharply during the first year of life and only a few isolated young neurons are observed by 7 and 13 years of age. In adult patients with epilepsy and healthy adults (18-77 years; n = 17 post-mortem samples from controls; n = 12 surgical resection samples from patients with epilepsy), young neurons were not detected in the dentate gyrus. In the monkey (Macaca mulatta) hippocampus, proliferation of neurons in the subgranular zone was found in early postnatal life, but this diminished during juvenile development as neurogenesis decreased. We conclude that recruitment of young neurons to the primate hippocampus decreases rapidly during the first years of life, and that neurogenesis in the dentate gyrus does not continue, or is extremely rare, in adult humans. The early decline in hippocampal neurogenesis raises questions about how the function of the dentate gyrus differs between humans and other species in which adult hippocampal neurogenesis is preserved.

Background: Clinical trials in lung cancer increasingly require patients to provide fresh tumor tissue as a prerequisite to enrollment. The effects of this requirement on enrollment rates, enrollment durations, and patient selection have not been fully elucidated., Methods: The authors retrospectively reviewed data generated by patients who consented to 1 or more interventional lung cancer clinical trials at the University of California-Los Angeles Jonsson Comprehensive Cancer Center between January 2013 and December 2014. Trials were considered to require a biopsy when enrollment was conditional on the procurement of tissue without intervening therapy between procurement and enrollment., Results: In total, 311 patients underwent 368 screening incidents for 1 or more of 19 trials. Trials that required a new biopsy had a longer median screening duration (34 vs 14 days) than trials that did not require a biopsy (P < .001). Trials that required a biopsy had a greater screen failure rate (49.1% vs 26.5%; P < .001), which was largely driven by patients who did not undergo the required biopsy or lacked the required biomarker. Worsening performance status led to the majority of screen failures (56.5%) among biomarker-eligible patients., Conclusions: Although the scientific benefits of obtaining a new biopsy and requiring specific results for trial enrollment are clear, these requirements lead to a lengthening of the screening period, which, in some patients, is associated with clinical decline before enrollment. Implications for the interpretation of data from studies of this design should be explored. Cancer 2017;123:4800-7. © 2017 American Cancer Society., (© 2017 American Cancer Society.)

Thymoma is the most common primary tumor of the anterior mediastinum and often recurs after initial surgical resection. In this case series, percutaneous cryoablation, a locally ablative technique, was used to treat 25 mediastinal and pleural recurrent thymoma lesions in five patients. Safety and short-term efficacy data were collected. In 23 percutaneous cryoablations (92%), there were no or minimal complications. One serious complication, myasthenia gravis flare, occurred. Over the duration of follow-up (median, 331 d), 18 of 20 ablated lesions (90%) showed no evidence of local recurrence. Percutaneous cryoablation shows promise as a safe and effective treatment modality for recurrent thymoma., (Copyright © 2015 SIR. Published by Elsevier Inc. All rights reserved.)

Therapies that target the programmed death-1 (PD-1) receptor have shown unprecedented rates of durable clinical responses in patients with various cancer types. One mechanism by which cancer tissues limit the host immune response is via upregulation of PD-1 ligand (PD-L1) and its ligation to PD-1 on antigen-specific CD8(+) T cells (termed adaptive immune resistance). Here we show that pre-existing CD8(+) T cells distinctly located at the invasive tumour margin are associated with expression of the PD-1/PD-L1 immune inhibitory axis and may predict response to therapy. We analysed samples from 46 patients with metastatic melanoma obtained before and during anti-PD-1 therapy (pembrolizumab) using quantitative immunohistochemistry, quantitative multiplex immunofluorescence, and next-generation sequencing for T-cell antigen receptors (TCRs). In serially sampled tumours, patients responding to treatment showed proliferation of intratumoral CD8(+) T cells that directly correlated with radiographic reduction in tumour size. Pre-treatment samples obtained from responding patients showed higher numbers of CD8-, PD-1- and PD-L1-expressing cells at the invasive tumour margin and inside tumours, with close proximity between PD-1 and PD-L1, and a more clonal TCR repertoire. Using multivariate analysis, we established a predictive model based on CD8 expression at the invasive margin and validated the model in an independent cohort of 15 patients. Our findings indicate that tumour regression after therapeutic PD-1 blockade requires pre-existing CD8(+) T cells that are negatively regulated by PD-1/PD-L1-mediated adaptive immune resistance.

Pulmonary hypertension (PH) may be idiopathic or related to a variety of diseases. The diagnosis, accurate assessment of etiology and severity, prognosis, treatment response, and follow-up of PH can be achieved using a diverse set of diagnostic examinations. In this review, the role of imaging in the evaluation of PH as suggested by the American College of Radiology Appropriateness Criteria Expert Panel on Thoracic Imaging has been discussed. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed every 2 years by a multidisciplinary expert panel. The development and review of the guidelines include an extensive analysis of current medical literature from peer-reviewed journals and the application of a well-established consensus methodology (modified Delphi) to rate the appropriateness of imaging and treatment procedures by the panel. In those instances in which evidence is lacking or not definitive, expert opinion may be used to recommend imaging or treatment.

Radiofrequency ablation (RFA) is used to treat pulmonary malignancies. Although preliminary results are suggestive of a survival benefit, local progression rates are appreciable. Because a patient can undergo repeat treatment if recurrence is detected early, reliable post-RFA imaging follow-up is critical. The purpose of this article is to describe (a) an algorithm for post-RFA imaging surveillance; (b) the computed tomographic (CT) appearance, size, enhancement, and positron emission tomographic (PET) metabolic activity of the ablation zone; and (c) CT, PET, and dual-modality imaging with PET and CT (PET/CT) features suggestive of partial ablation or tumor recurrence and progression. CT is routinely used for post-RFA follow-up. PET and PET/CT have emerged as auxiliary follow-up techniques. CT with nodule densitometry may be used to supplement standard CT. Post-RFA follow-up was divided into three phases: early (immediately after to 1 week after RFA), intermediate (>1 week to 2 months), and late (>2 months). CT and PET imaging features suggestive of residual or recurrent disease include (a) increasing contrast material uptake in the ablation zone (>180 seconds on dynamic images), nodular enhancement measuring more than 10 mm, any central enhancement greater than 15 HU, and enhancement greater than baseline anytime after ablation; (b) growth of the RFA zone after 3 months (compared with baseline) and definitely after 6 months, peripheral nodular growth and change from ground-glass opacity to solid opacity, regional or distant lymph node enlargement, and new intrathoracic or extrathoracic disease; and (c) increased metabolic activity beyond 2 months, residual activity centrally or at the ablated tumor, and development of nodular activity.

Glycerol dehydratase (GD) catalyses glycerol reductive conversion to 3-hydroxypropanaldehyde (3-HPA), this being the first step required for the microbial conversion of glycerol to 1, 3 -propanodiol. GD has been functionally characterised to date and two main groups have been determined, one of them being vitamin B(12)-dependent and the other B(12)-independent. GD evolutionary history has been described and an exhaustive analysis made for detecting the functional residues responsible for type I divergence. GD phylogenetic tree topology was seen to be statistically robust and the data indicated strong purifying selection operating on the GD proteins within it. Two clades were indentified, one for vitamin B(12)-dependent and the other for B(12)- independent classes. The ancient hot-pot residues responsible for protein divergency for each clade were also identified. The basic evolutionary biology for GD proteins has been described, thereby opening the way forward for developing rational mutagenesis studies.

Apicomplexa are an extremely diverse group of unicellular organisms that infect humans and other animals. Despite the great advances in combating infectious diseases over the past century, these parasites still have a tremendous social and economic burden on human societies, particularly in tropical and subtropical regions of the world. Proteases from apicomplexa have been characterized at the molecular and cellular levels, and central roles have been proposed for proteases in diverse processes. In this work, 16 new genes encoding for trypsin proteases are identified in 8 apicomplexan genomes by a genome-wide survey. Phylogenetic analysis suggests that these genes were gained through both intracellular gene transfer and vertical gene transfer. Identification, characterization and understanding of the evolutionary origin of protease-mediated processes are crucial to increase the knowledge and improve the strategies for the development of novel chemotherapeutic agents and vaccines., (Copyright 2010 Beijing Genomics Institute. Published by Elsevier Ltd. All rights reserved.)

The histone-like proteins (HU) belong to a family of DNA architectural proteins that stabilize nucleoprotein complexes. We found a putative HU protein (TgGlmHMM&#95;3045) in Toxoplasma gondii genome that was homologous to the bacterial HU protein. This putative sequence was located in the scaffold TGG&#95;995361 of the chromosome 10. The sequence included the prokaryotic bacterial histone-like domain, KFGSLGlRRRGERVARNPRT (ID number PS00045). HU protein sequences were also found in Plasmodium falciparum, Neospora caninum, Theileria parva and Theileria annulata. We found that the homology of the putative HU protein in Apicomplexa was greater with bacterial histone-like proteins than with eukaryotic histone proteins. The phylogenetic tree indicated that the putative HU protein genes were acquired in Apicomplexa by means of a secondary endosymbiotic event from red algae and later they were transferred from the apicoplast organelle to the nuclear genome.

Rationale and Objectives: The introduction of picture archiving and communication system (PACS) has decreased the time needed to interpret radiology examinations resulting in an increased workflow. Because of concerns that the increase in exam throughput and the use of voice recognition may have a negative impact upon radiology resident education, a survey was conducted to assess the impact of PACS and voice recognition., Materials and Methods: Residents at four diagnostic radiology training programs were surveyed. Survey topics included resident demographics, didactic and technical issues, and areas for improvement., Results: One hundred thirty-four residents were polled with 42 respondents (42/134, 31.3%). The majority have been using PACS for more than 1 year (29/41, 70.7%) to interpret 75-100% of cases (33/39, 84.6%). A majority believed PACS is a superior teaching tool to printed film (28/38, 73.7%). However, only a minority (9/40, 22.5%) indicated that PACS was always used to contain teaching files and to conduct departmental conferences (5/40, 12.5%). The majority of respondents believed PACS have decreased the time needed to interpret diagnostic examinations (33/41, 80.5%). A majority (80.6%, 25/31) indicated that voice recognition takes more time than the traditional dictation and transcription process, where 51.3% (20/39, 51.3%) felt that voice recognition works well less than 50% of the time., Conclusions: Residents believe that PACS has positively affected their learning experience but indicate that it can be better utilized for resident education. Residents believe that voice recognition is less reliable and more time consuming than the traditional dictation system.

The intramolecular Diels-Alder reaction of N-3,5-hexadienoyl ethyl acrylimidates provides an efficient method for the synthesis of cis-fused hexahydroisoquinolones. As a demonstration of the stereochemical control offered by this cycloaddition, two approaches to the construction of the DE rings of reserpine are reported. In the second entry, N-((4-(trimethylsilyl)ethoxymethoxy)methyl-6-benzyloxy-3Z,5E-hexadienoyl)-1-aza-2-ethoxy-1,3-butadiene (40) undergoes cycloaddition to produce as the major product (4aS,7R,8aS)-7-benzyloxy-5-((2-trimethylsilyl)ethoxymethoxy)methyl-3,4,4a,7,8,8a-hexahydroisoquinol-3-one (41). Cycloadduct 41 is then stereospecifically elaborated to (4aS,5S,6R,7R,8aR)-6-methoxy-5-methoxycarbonyl-7-(3,4,5-trimethoxy)benzoyldecahydroisoquinoline-2-carboxylic acid methyl ester (3), a key intermediate previously transformed to reserpine.

Chlorotrimethylsilane completely dealkylates phosphonate esters at elevated temperature in a sealed reaction vessel. These conditions are tolerated by a variety of functional groups and lead to high conversions of dimethyl, diethyl and diisopropyl phosphonates to their corresponding phosphonic acids.

A model was developed that simulated photosynthesis, growth and allocation in tree seedlings. The model was parameterized with data from experiments on seedlings of sycamore (Acer pseudoplatanus L.), Sitka spruce (Picea sitchensis (Bong) Carr.) and young birch trees (Betula pendula Roth.). In these experiments, CO2 concentration ([CO2]) and nutrient addition rate were varied. Parameters quantifying nutrient uptake, translocation and starch synthesis were fitted, based on data from control treatments. Elevated [CO2] and low-nutrient treatments were then used to test the predicted response of growth and allocation against observations. The model accurately predicted total seedling growth in the elevated [CO2] treatments. A response of growth to elevated [CO2] was seen in the birch and sycamore experiments, but not in the Sitka spruce, because of photosynthetic down-regulation. Predictions of allocation were reasonably accurate in the birch and Sitka spruce experiments, but were notably poorer in the sycamore experiments, possibly because of differences in sink strength between root and shoot. In the birch and sycamore experiments, little change in allocation with elevated [CO2] was observed or predicted. This was ascribed to the relative values of K(Tc) and K(Tn), the translocation coefficients that determine the sensitivity of allocation to carbon and nitrogen uptake rates, respectively. Growth and allocation in the low-nutrient treatments were poorly predicted by the model. In Sitka spruce, it was suspected that the photosynthetic parameters measured in August 1994 had been higher earlier in the season, before nutrients became depleted. In sycamore, the discrepancies were thought to relate to differences in sink strength between root and shoot that could not be described by the model.

A deterministic and dynamic model of carbon allocation in walnut seedlings is described. Two experimental data sets were used to calibrate and validate the model. These data included: time course of the carbon content, chemical and isotope composition (12C and 13C) of the kernel and growing organs (roots, stem, leaves), and gas exchange rates during the first 55 days of the life of the plant with continuous 13CO2 feeding. The plant is modelled as a network with nodal organs acting as sources or sinks for carbohydrates. In a sink organ the demand for carbon is the sum of four elementary demands: maintenance respiration, structural growth, growth-associated respiration and carbon storage. The organs of the plant are assumed to be in exponential growth phase. The supply of carbon readily accessible for the organs is the store of soluble sugars present in a local reservoir. Carbon flow in the network is determined by the source/sink activities of the organs and local levels of demand and supply. Two carbon sources are considered: soluble sugars from the kernel, and gross photosynthesis. The rate of synthesis of soluble sugars in the kernel, and measured photosynthesis in the leaves are inputs for the plant model. The outputs are the predicted fluxes of carbon within the seedling; 13C composition,carbohydrate allocation to the growing organs, starch and soluble sugars accumulation, and respiration. The mathematical equations were translated into PSPICE software instructions. After optimisation of the parameter values, the model provided an accurate description of experimental observations in the seed-plant system during the critical transition from heterotrophy to autotrophy, especially C allocation to organs and C partitioning between storage, structural growth and respiration in each organ. The growth of the young plant is supply-limited, except at the earliest stages. A sensitivity analysis suggests that intense competition for carbohydrates dominates the relations among and within organs. Copyright 1998 Academic Press Limited

Antisense oligodeoxynucleotides (ODNs) are capable of inhibiting gene expression via a RNase H mechanism in which the complementary RNA is degraded by RNase H. C-5 propyne dU phosphorothioate ODNs bind selectively and with high affinity to RNA within cells leading to potent antisense inhibition of RNA translation. The effect that increasing steric bulk of C-5-substituted deoxyuridine analogs has on affinity for RNA and ability to inhibit gene expression is discussed. The relative binding affinity was measured by thermal denaturation (Tm) analysis, and antisense activity was determined by inhibition of SV40 T-antigen (TAg) expression in CV1 cells. The results show that antisense activity is not directly correlated to Tm measurements. In vitro analysis (RNase H cleavage, on-rates, and off-rates) and pre-formed ODN/RNA experiments indicate that RNase H activity and intracellular dissociation appear to be major determinants of the antisense potency of the various substituted ODNs. The results of our analysis point to the unique ability of C-5 propyne dU ODNs to selectively bind to RNA within cells and activate cleavage of RNA by RNase H leading to potent inhibition of gene expression.

Phosphorothioate oligodeoxynucleotides containing the C-5 propyne analogs of uridine and cytidine bind RNA with high affinity and are potent antisense inhibitors of gene expression. In a cellular assay, gene-specific antisense inhibition occurred at nanomolar concentrations of oligonucleotide, was dose-dependent and exquisitely sensitive to sequence mismatches, and was correlated with the melting temperature and length of oligonucleotide. Activity was independent of RNA target site and cell type but was detectable only when the oligonucleotides were microinjected or delivered with cell-permeabilizing agents. These oligonucleotides may have important applications in therapy and in studies of gene function.