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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.05.012 Byline: J.F. Evers (a)(b), D. Muench (b), C. Duch (b) Keywords: Synaptogenesis; Dendritic growth; Motoneuron; Filopodia; Postembryonic development; 3D reconstruction Abstract: Dendritic filopodia are long thin protrusions occurring predominantly on developing neurons. Data from different systems suggest a range of crucial functions for filopodia in central circuit formation, including steering of dendritic growth, branch formation, synaptogenesis, and spinogenesis. Are the same filopodia competent to mediate all these processes, do filopodia acquire different functions through development, or do different filopodial types with distinct functions exist? In this study, 3-dimensional reconstructions from confocal image stacks demonstrate the existence of two morphologically and functionally distinct types of filopodia located on the dendritic tips versus the dendritic shafts of the same developing motoneuron. During dendritic growth, both filopodial types undergo a process of stage-specific morphogenesis. Using novel quantification strategies of 3-dimensional co-localization analysis for immunocytochemically labeled presynaptic specializations along postsynaptic filopodia, we find that presynaptic terminals accumulate along filopodia towards the dendrites at both stable dendritic shafts and on growing dendritic tips. On tips, this is likely to reflect synaptotrophic growth of the dendrite. At stable shafts, however, presynaptic sites become relocated along filopodia towards dendritic branches. This indicates the interactive growth of both pre- and postsynaptic partner towards one another during synaptogenesis, using filopodia as guides. Author Affiliation: (a) University of Cambridge, Department of Zoology, Downingstreet, Cambridge CB2 3EJ, UK (b) Freie Universitat Berlin, FB Biologie/Chemie/Pharmazie, Institut fur Biologie-Neurobiologie, Konigin-Luise-Str. 28/30, 14195 Berlin, Germany Article History: Received 8 February 2006; Revised 3 May 2006; Accepted 10 May 2006

Abstract Background Puf proteins have important roles in controlling gene expression at the post-transcriptional level by promoting RNA decay and repressing translation. The Pumilio homology domain (PUM-HD) is a conserved region within Puf proteins that binds to RNA with sequence specificity. Although Puf proteins have been well characterized in animal and fungal systems, little is known about the structural and functional characteristics of Puf-like proteins in plants. Results The Arabidopsis and rice genomes code for 26 and 19 Puf-like proteins, respectively, each possessing eight or fewer Puf repeats in their PUM-HD. Key amino acids in the PUM-HD of several of these proteins are conserved with those of animal and fungal homologs, whereas other plant Puf proteins demonstrate extensive variability in these amino acids. Three-dimensional modeling revealed that the predicted structure of this domain in plant Puf proteins provides a suitable surface for binding RNA. Electrophoretic gel mobility shift experiments showed that the Arabidopsis AtPum2 PUM-HD binds with high affinity to BoxB of the Drosophila Nanos Response Element I (NRE1) RNA, whereas a point mutation in the core of the NRE1 resulted in a significant reduction in binding affinity. Transient expression of several of the Arabidopsis Puf proteins as fluorescent protein fusions revealed a dynamic, punctate cytoplasmic pattern of localization for most of these proteins. The presence of predicted nuclear export signals and accumulation of AtPuf proteins in the nucleus after treatment of cells with leptomycin B demonstrated that shuttling of these proteins between the cytosol and nucleus is common among these proteins. In addition to the cytoplasmically enriched AtPum proteins, two AtPum proteins showed nuclear targeting with enrichment in the nucleolus. Conclusions The Puf family of RNA-binding proteins in plants consists of a greater number of members than any other model species studied to date. This, along with the amino acid variability observed within their PUM-HDs, suggests that these proteins may be involved in a wide range of post-transcriptional regulatory events that are important in providing plants with the ability to respond rapidly to changes in environmental conditions and throughout development.

Abstract Background Starch accumulation and degradation in chloroplasts is accomplished by a suite of over 30 enzymes. Recent work has emphasized the importance of multi-protein complexes amongst the metabolic enzymes, and the action of associated non-enzymatic regulatory proteins. Arabidopsis At5g39790 encodes a protein of unknown function whose sequence was previously demonstrated to contain a putative carbohydrate-binding domain. Results We here show that At5g39790 is chloroplast-localized, and binds starch, with a preference for amylose. The protein persists in starch binding under conditions of pH, redox and Mg+2 concentrations characteristic of both the day and night chloroplast cycles. Bioinformatic analysis demonstrates a diurnal pattern of gene expression, with an accumulation of transcript during the light cycle and decline during the dark cycle. A corresponding diurnal pattern of change in protein levels in leaves is also observed. Sequence analysis shows that At5g39790 has a strongly-predicted coiled-coil domain. Similar analysis of the set of starch metabolic enzymes shows that several have strong to moderate coiled-coil potential. Gene expression analysis shows strongly correlated patterns of co-expression between At5g39790 and several starch metabolic enzymes. Conclusion We propose that At5g39790 is a regulatory scaffold protein, persistently binding the starch granule, where it is positioned to interact by its coiled-coil domain with several potential starch metabolic enzyme binding-partners.

Abstract Background The plant peroxisomal multifunctional protein (MFP) possesses up to four enzymatic activities that are involved in catalyzing different reactions of fatty acid β-oxidation in the peroxisome matrix. In addition to these peroxisomal activities, in vitro assays revealed that rice MFP possesses microtubule- and RNA-binding activities suggesting that this protein also has important functions in the cytosol. Results We demonstrate that MFP is an authentic microtubule-binding protein, as it localized to the cortical microtubule array in vivo, in addition to its expected targeting to the peroxisome matrix. MFP does not, however, interact with the three mitotic microtubule arrays. Microtubule co-sedimentation assays of truncated versions of MFP revealed that multiple microtubule-binding domains are present on the MFP polypeptide. This indicates that these regions function together to achieve high-affinity binding of the full-length protein. Real-time imaging of a transiently expressed green fluorescent protein-MFP chimera in living plant cells illustrated that a dynamic, spatial interaction exits between peroxisomes and cortical microtubules as peroxisomes move along actin filaments or oscillate at fixed locations. Conclusion Plant MFP is associated with the cortical microtubule array, in addition to its expected localization in the peroxisome. This observation, coupled with apparent interactions that frequently occur between microtubules and peroxisomes in the cell cortex, supports the hypothesis that MFP is concentrated on microtubules in order to facilitate the regulated import of MFP into peroxisomes.

Purpose: The financial sustainability of the US healthcare system is a growing concern in an environment of declining reimbursement and rising costs. Variable Centers for Medicare and Medicaid (CMS) reimbursement and denial rates for specific imaging examinations exist across sites of service, adding complexity to financial planning for healthcare organizations. Understanding the financial implications of site of service in existing CMS reimbursement for imaging may be of strategic importance for organizations going forward., Materials and Methods: Current Procedural Terminology (CPT) codes were obtained for common cross-sectional imaging examinations using the 2022 CMS Medicare Physician Fee Schedule. Using reimbursement rates with historical volumes and denial rates, a simulation was created to estimate the overall reimbursement of paired hospital outpatient departments (HOPD) and free-standing office (FSO) sites. A baseline simulation was performed with random allocation of imaging examinations between sites of service, and an optimized simulation was performed to estimate the maximum financial impact of targeted allocation between sites. These simulations were performed for paired CT and MR scanners separately., Results: For CT, the baseline simulation estimated annual average reimbursement for combined HOPD and FSO was $3.25M. Reimbursement increased to $3.51M after optimized reallocation of studies between sites of service, resulting in an expected gain of $260,162 for a set of paired HOPD and FSO scanners. For MR, the same approach resulted in baseline reimbursement of $2.51M, increasing to $2.60M upon reallocation between sites for an expected gain of $87,532. Assuming a stable cost of service delivery, this approach would result in improved margins of 8% for CT and 3.5% for MR. There were 28 CT and 19 MRI daily patient imaging appointments at each respective HOPD and FSO scanners, unchanged between baseline and optimized cases. Differences in reimbursement rates between sites were the dominant driver of increased margins at low denial rates, although denial rates became dominant at values greater than 50%., Conclusion: Given CMS payment and denial rate variability, optimally allocating imaging studies between sites of service may improve reimbursement for the same services delivered. Although financial incentives exist for site allocation, such decisions should require physician input to assess safety and appropriate level of care. This work contributes to an understanding of financial incentives of existing reimbursement policy and may guide future policy design towards high value care., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Purpose of Review: The purpose of this review is to describe the Southeastern Kidney Transplant Coalition's mission, vision, goals, and Early Transplant Access registry as an example of a community/academic collaboration dedicated to improving access to transplantation and reducing inequities in transplant access., Recent Findings: The barriers and facilitators to referral and evaluation for kidney transplantation are not necessarily the same as for waitlisting and transplantation. Recent findings suggest that inequities in transplant access are multilevel and multifactorial and require continued community engagement to improve access to kidney transplantation across patients, health systems, and populations., Summary: Community-engaged approaches are critical to ensuring that inequities in transplant access - which may vary across regions -- are not only described but are addressed in practice in a sustainable manner.

Disruption of epigenetic regulation is a hallmark of acute myeloid leukemia (AML), but epigenetic therapy is complicated by the complexity of the epigenome. Herein, we developed a long-term primary AML ex vivo platform to determine whether targeting different epigenetic layers with 5-azacytidine and LSD1 inhibitors would yield improved efficacy. This combination was most effective in TET2 mut AML, where it extinguished leukemia stem cells and particularly induced genes with both LSD1-bound enhancers and cytosine-methylated promoters. Functional studies indicated that derepression of genes such as GATA2 contributes to drug efficacy. Mechanistically, combination therapy increased enhancer-promoter looping and chromatin-activating marks at the GATA2 locus. CRISPRi of the LSD1-bound enhancer in patient-derived TET2 mut AML was associated with dampening of therapeutic GATA2 induction. TET2 knockdown in human hematopoietic stem/progenitor cells induced loss of enhancer 5-hydroxymethylation and facilitated LSD1-mediated enhancer inactivation. Our data provide a basis for rational targeting of cooperating aberrant promoter and enhancer epigenetic marks driven by mutant epigenetic modifiers. SIGNIFICANCE: Somatic mutations of genes encoding epigenetic modifiers are a hallmark of AML and potentially disrupt many components of the epigenome. Our study targets two different epigenetic layers at promoters and enhancers that cooperate to aberrant gene silencing, downstream of the actions of a mutant epigenetic regulator. This article is highlighted in the In This Issue feature, p. 813 ., (©2019 American Association for Cancer Research.)

Background: Vascular endothelial growth factor (VEGF) is commonly overexpressed in a variety of tumor types including lung cancer. As a key regulator of angiogenesis, it promotes tumor survival, growth, and metastasis through the activation of the downstream protein kinase B (AKT) and extracellular signal-regulated kinase (ERK 1/2) activation. The VEGF promoter contains a 36 bp guanine-rich sequence (VEGFq) which is capable of forming quadruplex (four-stranded) DNA. This sequence has been implicated in the down-regulation of both basal and inducible VEGF expression and represents an ideal target for inhibition of VEGF expression., Results: Our experiments demonstrate sequence-specific interaction between a G-rich quadruplex-forming oligonucleotide encoding a portion of the VEGFq sequence and its double stranded target sequence, suggesting that this G-rich oligonucleotide binds specifically to its complementary C-rich sequence in the genomic VEGF promoter by strand invasion. We show that treatment of A549 non-small lung cancer cells (NSCLC) with this oligonucleotide results in decreased VEGF expression and growth inhibition. The VEGFq oligonucleotide inhibits proliferation and invasion by decreasing VEGF mRNA/protein expression and subsequent ERK 1/2 and AKT activation. Furthermore, the VEGFq oligonucleotide is abundantly taken into cells, localized in the cytoplasm/nucleus, inherently stable in serum and intracellularly, and has no effect on non-transformed cells. Suppression of VEGF expression induces cytoplasmic accumulation of autophagic vacuoles and increased expression of LC3B, suggesting that VEGFq may induce autophagic cell death., Conclusion: Our data strongly suggest that the G-rich VEGFq oligonucleotide binds specifically to the C-rich strand of the genomic VEGF promoter, via strand invasion, stabilizing the quadruplex structure formed by the genomic G-rich sequence, resulting in transcriptional inhibition. Strand invading oligonucleotides represent a new approach to specifically inhibit VEGF expression that avoids many of the problems which have plagued the therapeutic use of oligonucleotides. This is a novel approach to specific inhibition of gene expression., Competing Interests: The authors have declared that no competing interests exist.

We herein report the design and synthesis of the first nanomolar binding inhibitor of STAT5 protein. Lead compound 13a , possessing a phosphotyrosyl-mimicking salicylic acid group, potently and selectively binds to STAT5 over STAT3, inhibits STAT5-SH2 domain complexation events in vitro , silences activated STAT5 in leukemic cells, as well as STAT5's downstream transcriptional targets, including MYC and MCL1 , and, as a result, leads to apoptosis. We believe 13a represents a useful probe for interrogating STAT5 function in cells as well as being a potential candidate for advanced preclinical trials.

Objective: We tested the effects of structured health care for the diabetic foot in one region in Germany aiming to reduce the number of major amputations., Research Design and Methods: In a prospective study we investigated patients with diabetic foot in a structured system of outpatient, in-patient and rehabilitative treatment. Subjects were recruited between January 1st, 2000 and December 31, 2007. All participants underwent a two-year follow-up. The modified University of Texas Wound Classification System (UT) was the basis for documentation and data analysis. We evaluated numbers of major amputations, rates of ulcer healing and mortality. In order to compare the effect of the structured health care program with usual care in patients with diabetic foot we evaluated the same parameters at another regional hospital without interdisciplinary care of diabetic foot (controls)., Results: 684 patients with diabetic foot and 508 controls were investigated. At discharge from hospital 28.3% (structured health care program, SHC) vs. 23.0% (controls) of all ulcers had healed completely. 51.5% (SHC) vs. 49.8% (controls) were in UT grade 1.Major amputations were performed in 32 subjects of the structured health care program group (4.7%) vs. 110 (21.7%) in controls (p<0.0001). Mortality during hospitalization was 2.5% (SHC) vs. 9.4% in controls (p<0.001)., Conclusions: With the structured health care program we achieved a significant reduction of major amputation rates by more than 75% as compared to standard care.

The processes of cellular growth regulation and cellular metabolism are closely interrelated. The c-Myc oncogene is a "master regulator" which controls many aspects of both of these processes. The metabolic changes which occur in transformed cells, many of which are driven by c-Myc overexpression, are necessary to support the increased need for nucleic acids, proteins, and lipids necessary for rapid cellular proliferation. At the same time, c-Myc overexpression results in coordinated changes in level of expression of gene families which result in increased cellular proliferation. This interesting duality of c-Myc effects places it in the mainstream of transformational changes and gives it a very important role in regulating the "transformed phenotype." The effects induced by c-Myc can occur either as a "primary oncogene" which is activated by amplification or translocation or as a downstream effect of other activated oncogenes. In either case, it appears that c-Myc plays a central role in sustaining the changes which occur with transformation. Although efforts to use c-Myc as a therapeutic target have been quite frustrating, it appears that this may change in the next few years., (©2012 AACR)