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Geminiviral minichromosomes were purified to explore epigenetic modifications. The levels of methylation in their covalently closed circular DNA were examined with the help of methylation-dependent restriction (MdR). DNA with 12 superhelical turns was preferentially modified, indicating minichromosomes with 12 nucleosomes leaving an open gap. MdR digestion yielded a specific product of genomic length, which was cloned and Sanger-sequenced, or amplified following ligation-mediated rolling circle amplification and deep-sequenced (circomics). The conventional approach revealed a single cleavage product indicating specific methylations at the borders of the common region. The circomics approach identified considerably more MdR sites in a preferential distance to each other of ~200 nts, which is the DNA length in a nucleosome. They accumulated in regions of nucleosome-free gaps, but scattered also along the genomic components. These results may hint at a function in specific gene regulation, as well as in virus resistance., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background and Purpose: Low 25-hydroxyvitamin D [25(OH)D] levels correlate with higher disease activity in patients with multiple sclerosis (MS). However, it is not clear whether low 25(OH)D levels directly contribute to increased disease activity or merely represent a consequence of reduced endogenous vitamin D synthesis in more disabled MS patients. Furthermore, recent data suggest that bioavailable vitamin D, which also integrates the levels of vitamin D binding proteins and albumin, could be a biologically more relevant parameter than 25(OH)D., Methods: Measured de-seasonalized 25(OH)D3 and vitamin D binding protein and calculated bioavailable and free vitamin D were compared in the baseline serum samples of 76 patients with clinically isolated syndrome enrolled in a longitudinal observational study and in 76 age- and sex-matched healthy controls (HC)., Results: 25(OH)D3 levels were lower in patients with clinically isolated syndrome (P = 0.002) than in HC, and more patients (8/76, 10.5%) than HC (1/76, 1.3%) had 25(OH)D3 levels <25 nmol/l (P = 0.03). In contrast, levels of 25(OH)D2, vitamin D binding protein and calculated levels of free and bioavailable vitamin D did not differ between the two groups., Conclusions: Lower 25(OH)D3 levels already in the earliest phase of disease and in clinically hardly affected patients suggest that low 25(OH)D3 levels are rather a risk factor for than a consequence of MS. Nevertheless, because bioavailable vitamin D levels did not differ between the two groups, the mechanism underlying the association of 25(OH)D3 and MS does not appear to be related to reduced bioavailability of vitamin D., (© 2015 EAN.)

Unlabelled: The C2/AC2 genes of monopartite/bipartite geminiviruses of the genera Begomovirus and Curtovirus encode important pathogenicity factors with multiple functions described so far. A novel function of Abutilon mosaic virus (AbMV) AC2 as a replication brake is described, utilizing transgenic plants with dimeric inserts of DNA B or with a reporter construct to express green fluorescent protein (GFP). Their replicational release upon AbMV superinfection or the individual and combined expression of epitope-tagged AbMV AC1, AC2, and AC3 was studied. In addition, the effects were compared in the presence and in the absence of an unrelated tombusvirus suppressor of silencing (P19). The results show that AC2 suppresses replication reproducibly in all assays and that AC3 counteracts this effect. Examination of the topoisomer distribution of supercoiled DNA, which indicates changes in the viral minichromosome structure, did not support any influence of AC2 on transcriptional gene silencing and DNA methylation. The geminiviral AC2 protein has been detected here for the first time in plants. The experiments revealed an extremely low level of AC2, which was slightly increased if constructs with an intron and a hemagglutinin (HA) tag in addition to P19 expression were used. AbMV AC2 properties are discussed with reference to those of other geminiviruses with respect to charge, modification, and size in order to delimit possible reasons for the different behaviors., Importance: The (A)C2 genes encode a key pathogenicity factor of begomoviruses and curtoviruses in the plant virus family Geminiviridae. This factor has been implicated in the resistance breaking observed in agricultural cotton production. AC2 is a multifunctional protein involved in transcriptional control, gene silencing, and regulation of basal biosynthesis. Here, a new function of Abutilon mosaic virus AC2 in replication control is added as a feature of this protein in viral multiplication, providing a novel finding on geminiviral molecular biology., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

A comprehensive survey on the viral minichromosomes of the begomoviruses Abutilon mosaic virus, tomato yellow leaf curl Sardinia virus, African cassava mosaic virus, Indian cassava mosaic virus (family Geminiviridae) during the course of infections in Nicotiana benthamiana is summarized. Using optimized one-dimensional and two-dimensional gel systems combined with blot hybridization and a standardized evaluation, discrete and heterogeneous virus-specific signals with different DNA forms were compared to trace functions of viral multiplication with inactive/active replication and/or transcription. A quantitative approach to compare the distantly related viruses during the course of infection with the aim to generalize the conclusions for geminiviruses has been developed. Focussing on the distribution of topoisomers of viral supercoiled DNA, which reflect minichromosomal stages, predominant minichromosomes with 12 nucleosomes, less with 13 nucleosomes and no with 11 nucleosomes were found. These results indicate that chromatin with only one open gap to bind transcription factors is the favourite form. The dynamics during infections in dependence on the experimental conditions is discussed with reference to the design of experiments for resistance breeding and molecular analyses., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Importance: Some patients with multiple sclerosis (MS) can either present with or develop severe cognitive impairment during the course of their disease. However, the mechanisms underlying severe cognitive dysfunction in MS are not well understood., Observations: We report on a woman who was diagnosed as having MS at age 33 years and who after giving birth at age 37 years developed cognitive impairment with severe memory dysfunction as the leading symptom. Treatment with different immunotherapies, including cyclophosphamide and natalizumab, did not improve her cognitive deficits, necessitating admission to a nursing home at age 39 years. During a thorough reevaluation at age 43 years, analysis of current and stored cerebrospinal fluid and serum samples demonstrated an intrathecal synthesis of IgG antibodies to the NR1 subunit of the N-methyl-D-aspartate receptor, that is, the characteristic laboratory finding of anti-N-methyl-D-aspartate receptor encephalitis. Although the patient initially stabilized under therapy with corticosteroids, plasma exchange, and mitoxantrone, severe cognitive impairment persisted and she eventually died from the sequelae of her disease., Conclusions and Relevance: This report suggests that the occasional occurrence of severe cognitive impairment in patients with MS may, in some cases, be related to a superimposed antibody-mediated autoimmune encephalitis.

Background: Axonal and neuronal damage are widely accepted as key events in the disease course of multiple sclerosis. However, it has been unclear to date at which stage in disease evolution neurodegeneration begins and whether neuronal damage can occur even in the absence of acute inflammatory attacks., Objective: To characterize inner retinal layer changes in patients with clinically isolated syndrome (CIS)., Method: 45 patients with CIS and age- and sex-matched healthy controls were investigated using spectral domain optical coherence tomography. Patients' eyes were stratified into the following categories according to history of optic neuritis (ON): eyes with clinically-diagnosed ON (CIS-ON), eyes with suspected subclinical ON (CIS-SON) as indicated by a visual evoked potential latency of >115 ms and eyes unaffected by ON (CIS-NON)., Results: CIS-NON eyes showed significant reduction of ganglion cell- and inner plexiform layer and a topography similar to that of CIS-ON eyes. Seven eyes were characterized as CIS-SON and likewise showed significant retinal layer thinning. The most pronounced thinning was present in CIS-ON eyes., Conclusion: Our findings indicate that retinal pathology does occur already in CIS. Intraretinal layer segmentation may be an easily applicable, non-invasive method for early detection of retinal pathology in patients unaffected by ON.

Interferon-β is an established treatment for patients with multiple sclerosis (MS) but its mechanisms of action are not well understood. Viral infections are a known trigger of MS relapses. Toll-like receptors (TLRs) are key components of the innate immune system, which sense conserved structures of viruses and other pathogens. Effects of interferon-β on mRNA levels of all known human TLRs (TLR1-10) and the TLR adaptor molecule MyD88 were analyzed in peripheral blood mononuclear cells (PBMCs) of healthy donors by quantitative real-time PCR and by transcriptome analysis in PBMCs of 25 interferon-β-treated patients with relapsing-remitting MS. Regulation of TLR protein expression by interferon-β was investigated by flow cytometry of leukocyte subsets of healthy subjects and of untreated, interferon-β-, or glatiramer acetate-treated patients with MS. Interferon-β specifically upregulated mRNA expression of TLR3, TLR7, and MyD88 and downregulated TLR9 mRNA in PBMCs of healthy donors as well as in PBMCs of patients with MS. Plasmacytoid dendritic cells (pDCs) were identified as the major cell type responding to interferon-β with increased expression of TLR7 and MyD88 protein. In line with this, expression of TLR7 protein was increased in pDCs of interferon-β-treated, but not untreated or glatiramer acetate-treated patients with MS. Interferon-β-induced upregulation of TLR7 in pDCs is of functional relevance since pre-treatment of PBMCs with interferon-β resulted in a strongly increased production of interferon-α upon stimulation with the TLR7 agonist loxoribine. Flow cytometry confirmed pDCs as the cellular source of interferon-α production induced by activation of TLR7. Thus, upregulation of TLR7 in pDCs and a consequently increased activation of pDCs by TLR7 ligands represents a novel immunoregulatory mechanism of interferon-β. We hypothesize that this mechanism could contribute to a reduction of virus-triggered relapses in patients with MS.

Objective: To assess patients with SSc who present without circulating ANAs or RP., Methods: Five thousand three hundred and ninety patients who fulfilled the ACR criteria for SSc and were enrolled in the EULAR Scleroderma Trials and Research (EUSTAR) database were screened for the absence of both RP and circulating ANA. To differentiate SSc from its mimics, additional information was gathered using a standardized questionnaire., Results: Five thousand three hundred and seventy-eight (99.8%) of the 5390 SSc patients in the EUSTAR database had either detectable ANA or a history of RP. Twelve (0.2%) patients lacked both circulating ANA and RP. Details of the medical history could be obtained for seven patients. Three cases were compatible with ANA-negative and RP-negative SSc and were not typical of any known SSc mimic. Four patients had a malignancy: two had breast cancer, one had multiple myeloma with possible scleromyxoedema and one had bladder carcinoma. There was no temporal relationship between the onset of skin fibrosis and that of the tumour. Although no patient with confirmed nephrogenic systemic fibrosis was identified among the cases of ANA-negative and RP-negative SSc, the presentation of one patient could be compatible with that of nephrogenic systemic fibrosis other than for the absence of chronic kidney disease or of known prior gadolinium exposure., Conclusion: We have identified a very small subgroup of SSc patients who lack both circulating ANA and RP, none of whom fulfils the diagnostic criteria for any known SSc mimic. Prospective studies are needed to elucidate the clinical presentation, evolution and outcome of such patients.

Objectives: The role of the six-minute walk distance (6MWD), measured by a six-minute walk test (6MWT), in the assessment of systemic sclerosis (SSc) patients remains to be evaluated. Here, we have analysed whether 6MWD is associated with clinical parameters obtained by an extended standardised assessment of SSc patients., Methods: In 101 consecutive SSc patients, 6MWD was correlated with disease activity, Scleroderma Health Assessment Questionnaire (SHAQ) score, nutrition status, age, ESR, haemoglobin values, and several lung function parameters., Results: Of the 101 SSc patients, 6 patients were excluded because of diseases that could influence the result of the 6MWT, such as asthma, COPD or peripheral vascular disease. In the remaining 95 patients the median 6MWD was 491.0 m (range 86.0-664.5 m). 6MWD weakly-to-moderately correlated with predicted FVC, FEV1, TLC, DLCO and nutrition status. Moderate negative correlations were found for the SHAQ score and disease activity, weaker correlations for age and BMI. Exclusion of patients with musculoskeletal limitations revealed similar results. Training status of the patients did not affect 6MWD. Multivariate analyses revealed SHAQ score and predicted DLCO values as the best parameters predicting 6MWD. Optimal 6MWD cut-off values for the presence of PAH, predicted FVC values <80%, and dyspnea NYHA III/IV were between 465 m and 480 m., Conclusions: 6MWD is a surrogate marker for disability and complaints in SSc patients. Therefore, 6MWT could provide a valuable outcome parameter although it lacks organ specificity.

320-row CT enables dynamic CT angiography (4D CTA) of the entire intracranial circulation and whole-brain perfusion imaging (CTP). Sixty acute patients with neurological symptoms underwent various 320-row CT-specific protocols, including combined 4D CTA and CTP. Clinical and neuroradiological records were assessed for presumptive diagnoses, final diagnoses, supplementary and follow-up imaging studies. Additional diagnostic benefits delivered by 320-row CT were noted. Out of 60 procedures, 59 were accomplished successfully. Ischemia (n = 19), intracerebral hemorrhage (n = 7) and transient ischemic attacks (n = 10) were the major final diagnoses. Except one small cortical and two small subcortical infarctions all ischemias were diagnosed. All hemorrhages were diagnosed together with their underlying vascular pathology in five atypical cases. In conclusion, 320-row CT is a technically robust procedure being suitable for comprehensive neuroimaging of acute patients. It can provide dynamic angiographic and perfusion data of the whole brain and can deliver additional diagnostic information not available by standard CT.

A monomeric variant of the red fluorescent protein eqFP611, mRuby, is described. With excitation and emission maxima at 558 nm and 605 nm, respectively, and a large Stokes shift of 47 nm, mRuby appears particularly useful for imaging applications. The protein shows an exceptional resistance to denaturation at pH extremes. Moreover, mRuby is about ten-fold brighter compared to EGFP when being targeted to the endoplasmic reticulum. The engineering process of eqFP611 revealed that the C-terminal tail of the protein acts as a natural peroxisomal targeting signal (PTS). Using an mRuby variant carrying the eqFP611-PTS, we discovered that ordered inheritance of peroxisomes is widespread during mitosis of different mammalian cell types. The ordered partitioning is realized by the formation of peroxisome clusters around the poles of the mitotic spindle and ensures that equal numbers of the organelle are inherited by the daughter cells. The unique spectral properties make mRuby the marker of choice for a multitude of cell biological applications. Moreover, the use of mRuby has allowed novel insights in the biology of organelles responsible for severe human diseases.

Confocal fluorescence microscopy and two-photon microscopy have become important techniques for the three-dimensional imaging of intact cells. Their lateral resolution is about 200-300 nm for visible light, whereas their axial resolution is significantly worse. By superimposing the spherical wave fronts from two opposing objective lenses in a coherent fashion in 4Pi microscopy, the axial resolution is greatly improved to approximately 100 nm. In combination with specific tagging of proteins or other cellular structures, 4Pi microscopy enables a multitude of molecular interactions in cell biology to be studied. Here, we discuss the choice of appropriate fluorescent tags for dual-color 4Pi microscopy and present applications of this technique in cellular biophysics. We employ two-color fluorescence detection of actin and tubulin networks stained with fluorescent organic dyes; mitochondrial networks are imaged using the photoactivatable fluorescent protein EosFP. A further example concerns the interaction of nanoparticles with mammalian cells.

Genetically encoded glucose nanosensors have been used to measure steady state glucose levels in mammalian cytosol, nuclei, and endoplasmic reticulum. Unfortunately, the same nanosensors in Arabidopsis thaliana transformants manifested transgene silencing and undetectable fluorescence resonance energy transfer changes. Expressing nanosensors in sgs3 and rdr6 transgene silencing mutants eliminated silencing and resulted in high fluorescence levels. To measure glucose changes over a wide range (nanomolar to millimolar), nanosensors with higher signal-to-noise ratios were expressed in these mutants. Perfusion of leaf epidermis with glucose led to concentration-dependent ratio changes for nanosensors with in vitro K(d) values of 600 microM (FLIPglu-600 microDelta13) and 3.2 mM (FLIPglu-3.2 mDelta13), but one with 170 nM K(d) (FLIPglu-170 nDelta13) showed no response. In intact roots, FLIPglu-3.2 mDelta13 gave no response, whereas FLIPglu-600 microDelta13, FLIPglu-2 microDelta13, and FLIPglu-170 nDelta13 all responded to glucose. These results demonstrate that cytosolic steady state glucose levels depend on external supply in both leaves and roots, but under the conditions tested they are lower in root versus epidermal and guard cells. Without photosynthesis and external supply, cytosolic glucose can decrease to <90 nM in root cells. Thus, observed gradients are steeper than expected, and steady state levels do not appear subject to tight homeostatic control. Nanosensor-expressing plants can be used to assess glucose flux differences between cells, invertase-mediated sucrose hydrolysis in vivo, delivery of assimilates to roots, and glucose flux in mutants affected in sugar transport, metabolism, and signaling.

Proline accumulation and catabolism play significant roles in adaptation to a variety of plant stresses including osmotic stress, drought, temperature, freezing, UV irradiation, heavy metals and pathogen infection. In this study, the gene Delta1 -pyrroline-5-carboxylate dehydrogenase (P5CDH), which catalyzes the second step in the conversion of proline to glutamate, is characterized in a number of cereal species. P5CDH genes from hexaploid wheat, Triticum turgidum (durum wheat), Aegilops tauschii, Triticum monococcum, barley, maize and rice were shown to be conserved in terms of gene structure and sequence, present as a single copy per haploid, non-polyploid genome and located in evolutionarily conserved linkage groups. A wheat cDNA sequence was shown by yeast complementation to encode a functional P5CDH activity. A divergently-transcribed rab7 gene was identified immediately 5' of P5CDH in all grasses examined, except rice. The rab7/P5CDH intergenic region in these species, which presumably encompasses 5' regulatory elements of both genes, showed a distinct pattern of sequence evolution with sequences in juxtaposition to each ORF conserved between barley, wheat, A. tauschii and T. monococcum. More distal 5' sequence in this intergenic region showed a higher rate of divergence, with no homology observed between these regions in the wheat and barley genomes. Maize and rice showed no similarity in regions 5' of P5CDH when compared with wheat, barley, and each other, apart from a 22 bp region of conserved non-coding sequence (CNS) that is similar to a proline response element identified in the promoter of the Arabidopsis proline dehydrogenase gene. A palindromic motif similar to this cereal CNS was also identified 5' of the Arabidopsis AtP5CDH gene showing conservation of this sequence in monocot and dicot lineages.

A family of genetically-encoded metabolite sensors has been constructed using bacterial periplasmic binding proteins (PBPs) linearly fused to protein fluorophores. The ligand-induced conformational change in a PBP allosterically regulates the relative distance and orientation of a fluorescence resonance energy transfer (FRET)-compatible protein pair. Ligand binding is transduced into a macroscopic FRET observable, providing a reagent for in vitro and in vivo ligand-measurement and visualization. Sensors with a higher FRET signal change are required to expand the dynamic range and allow visualization of subtle analyte changes under high noise conditions. Various observations suggest that factors other than inter-fluorophore separation contribute to FRET transfer efficiency and the resulting ligand-dependent spectral changes. Empirical and rational protein engineering leads to enhanced allosteric linkage between ligand binding and chromophore rearrangement; modifications predicted to decrease chromophore rotational averaging enhance the signal change, emphasizing the importance of the rotational freedom parameter kappa2 to FRET efficiency. Tighter allosteric linkage of the PBP and the fluorophores by linker truncation or by insertion of chromophores into the binding protein at rationally designed sites gave rise to sensors with improved signal change. High-response sensors were obtained with fluorescent proteins attached to the same binding PBP lobe, suggesting that indirect allosteric regulation during the hinge-bending motion is sufficient to give rise to a FRET response. The optimization of sensors for glucose and glutamate, ligands of great clinical interest, provides a general framework for the manipulation of ligand-dependent allosteric signal transduction mechanisms.

Background: Metabolomics, i.e., the multiparallel analysis of metabolite changes occurring in a cell or an organism, has become feasible with the development of highly efficient mass spectroscopic technologies. Functional genomics as a standard tool helped to identify the function of many of the genes that encode important transporters and metabolic enzymes over the past few years. Advanced expression systems and analysis technologies made it possible to study the biochemical properties of the corresponding proteins in great detail. We begin to understand the biological functions of the gene products by systematic analysis of mutants using systematic PTGS/RNAi, knockout and TILLING approaches. However, one crucial set of data especially relevant in the case of multicellular organisms is lacking: the knowledge of the spatial and temporal profiles of metabolite levels at cellular and subcellular levels., Methods: We therefore developed genetically encoded nanosensors for several metabolites to provide a basic set of tools for the determination of cytosolic and subcellular metabolite levels in real time by using fluorescence microscopy., Results: Prototypes of these sensors were successfully used in vitro and also in vivo, i.e., to measure sugar levels in fungal and animal cells., Conclusions: One of the future goals will be to expand the set of sensors to a wider spectrum of substrates by using the natural spectrum of periplasmic binding proteins from bacteria and by computational design of proteins with altered binding pockets in conjunction with mutagenesis. This toolbox can then be applied for four-dimensional imaging of cells and tissues to elucidate the spatial and temporal distribution of metabolites as a discovery tool in functional genomics, as a tool for high-throughput, high-content screening for drugs, to test metabolic models, and to analyze the interplay of cells in a tissue or organ., (Copyright (c) 2005 Wiley-Liss, Inc.)

In response to stress, plants accumulate Pro, requiring degradation after release from adverse conditions. Delta1-Pyrroline-5-carboxylate dehydrogenase (P5CDH), the second enzyme for Pro degradation, is encoded by a single gene expressed ubiquitously. To study the physiological function of P5CDH, T-DNA insertion mutants in AtP5CDH were isolated and characterized. Although Pro degradation was undetectable in p5cdh mutants, neither increased Pro levels nor an altered growth phenotype were observed under normal conditions. Thus AtP5CDH is essential for Pro degradation but not required for vegetative plant growth. External Pro application caused programmed cell death, with callose deposition, reactive oxygen species production, and DNA laddering, involving a salicylic acid signal transduction pathway. p5cdh mutants were hypersensitive toward Pro and other molecules producing P5C, such as Arg and Orn. Pro levels were the same in the wild type and mutants, but P5C was detectable only in p5cdh mutants, indicating that P5C accumulation may be the cause for Pro hypersensitivity. Accordingly, overexpression of AtP5CDH resulted in decreased sensitivity to externally supplied Pro. Thus, Pro and P5C/Glu semialdehyde may serve as a link between stress responses and cell death.

Delta1-pyrroline-5-carboxylate (P5C), an intermediate in biosynthesis and degradation of proline (Pro), is assumed to play a role in cell death in plants and animals. Toxicity of external Pro and P5C supply to Arabidopsis suggested that P5C dehydrogenase (P5CDH; EC 1.2.1.12) plays a crucial role in this process by degrading the toxic Pro catabolism intermediate P5C. Also in a Deltaput2 yeast mutant, lacking P5CDH, Pro led to growth inhibition and formation of reactive oxygen species (ROS). Complementation of the Deltaput2 mutant allowed identification of the Arabidopsis P5CDH gene. AtP5CDH is a single-copy gene and the encoded protein was localized to the mitochondria. High homology of AtP5CDH to LuFIS1, an mRNA up-regulated during susceptible pathogen attack in flax, suggested a role for P5CDH in inhibition of hypersensitive reactions. An Arabidopsis mutant (cpr5) displaying a constitutive pathogen response was found to be hypersensitive to external Pro. In agreement with a role in prevention of cell death, AtP5CDH was expressed at a basal level in all tissues analysed. The highest expression was found in flowers that are known to contain the highest Pro levels under normal conditions. External supply of Pro induced AtP5CDH expression, but much more slowly than Pro dehydrogenase (AtProDH) expression. Uncoupled induction of the AtProDH and AtP5CDH genes further supports the hypothesis that P5C levels have to be tightly controlled. These results indicate that, in addition to the well-studied functions of Pro, for example in osmoregulation, the Pro metabolism intermediate P5C also serves as a regulator of cellular stress responses.

The authors examine for relevance and current application what was learned in the building and progress of community medicine at the Mount Sinai School of Medicine, from 1968 to 1993. They look back on their twenty-five years' approach to service development, research and education in this field, as they worked in an urban-based academic medical center in New York and cooperated with universities and their schools of health sciences abroad. A claim is made that this approach, whether in the United States or abroad, while fostering community development produces a diversity of desirable population-oriented new role models for the medical profession.

Through a documented case study the authors identify the critical factors that impede the introduction of prepaid medical care as part of education and practice within a prestigious and well established academic medical center. The inherent conflicts between individual fee-for-service practice and population-based prepaid practice and the resistance to innovations in medical care organization as they surfaced in that center, are presented. The need for a clear understanding of the complexities of HMO development and of an appreciation for the importance of a planning process in which all interested parties are involved, is emphasized. A clear commitment by policy makers, administrators and providers is highlighted as fundamental for the implementation of a system where practitioners are motivated to assume responsibility for the comprehensive care of a defined population that prepays for their services. The rewards as well as the difficulties for institutionalizing commitment to this form of health care delivery and impacting on medical education are discussed.

This paper describes the development of consultancy groups in behavioral science, epidemiology and biostatistics, and information science in a required community medicine rotation with a twenty year history. The addition of consultants to individual student tutors and field preceptors has led to a structure which promotes student-project flexibility, development of critical assessment skills and independent learning while maximizing faculty expertise and effectiveness.

This paper describes the philosophy, process, and outcome of the integration of independent study and classroom training with existing clinical and research experiences in The Mount Sinai Medical Center Department of Community Medicine's general preventive medicine and occupational medicine residencies leading to the creation of the Master of Science in Community Medicine.