Nuclear immobilization of DsRed1 tagged proteins: A novel tool for studying DNA–protein interactions?

AbstractDsRed1 is a red fluorescent protein that can be used as a fusion partner with other proteins to determine their subcellular localization, similarly to the popular green fluorescent proteins (GFP). Here, we report that fusion of DsRed1 to estrogen receptor α (ERα) renders the transcription factor immobile within the nucleus. Furthermore, we show that the immobilization is dependent on DNA interaction and that the binding to the DNA can be direct as well as indirect for DsRed to immobilize with its fusion partners. This observation could provide a new tool to be used for the identification of target genes containing low affinity binding sites for several transcription factors including ERα. In addition, it could be employed for studies on protein–DNA interactions as well as protein–protein interactions during protein complex formation on chromatin in the event of transcription initiation and regulation

Lumbar spine segmentation in MR images: a dataset and a public benchmark

This paper presents a large publicly available multi-center lumbar spine magnetic resonance imaging (MRI) dataset with reference segmentations of vertebrae, intervertebral discs (IVDs), and spinal canal. The dataset includes 447 sagittal T1 and T2 MRI series from 218 patients with a history of low back pain. It was collected from four different hospitals and was divided into a training (179 patients) and validation (39 patients) set. An iterative data annotation approach was used by training a segmentation algorithm on a small part of the dataset, enabling semi-automatic segmentation of the remaining images. The algorithm provided an initial segmentation, which was subsequently reviewed, manually corrected, and added to the training data. We provide reference performance values for this baseline algorithm and nnU-Net, which performed comparably. We set up a continuous segmentation challenge to allow for a fair comparison of different segmentation algorithms. This study may encourage wider collaboration in the field of spine segmentation, and improve the diagnostic value of lumbar spine MRI

Proteomic Profiling of Plasmodium Sporozoite Maturation Identifies New Proteins Essential for Parasite Development and Infectivity

Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquito—early and late oocysts containing midgut sporozoites, and the mature, infectious salivary gland sporozoites. Despite the morphological similarity between midgut and salivary gland sporozoites, their proteomes are markedly different, in agreement with their increase in hepatocyte infectivity. The different sporozoite proteomes contain a large number of stage specific proteins whose annotation suggest an involvement in sporozoite maturation, motility, infection of the human host and associated metabolic adjustments. Analyses of proteins identified in the P. falciparum sporozoite proteomes by orthologous gene disruption in the rodent malaria parasite, P. berghei, revealed three previously uncharacterized Plasmodium proteins that appear to be essential for sporozoite development at distinct points of maturation in the mosquito. This study sheds light on the development and maturation of the malaria parasite in an Anopheles mosquito and also identifies proteins that may be essential for sporozoite infectivity to humans

A P. falciparum NF54 Reporter Line Expressing mCherry-Luciferase in Gametocytes, Sporozoites, and Liver-Stages

Transgenic malaria parasites expressing fluorescent and bioluminescent proteins are valuable tools to interrogate malaria-parasite biology and to evaluate drugs and vaccines. Using CRISPR/Cas9 methodology a transgenic Plasmodium falciparum (Pf) NF54 line was generated that expresses a fusion of mCherry and luciferase genes under the control of the Pf etramp10.3 gene promoter (line [email protected]). Pf etramp10.3 is related to rodent Plasmodium uis4 and the uis4 promoter has been used to drive high transgene expression in rodent parasite sporozoites and liver-stages. We examined transgene expression throughout the complete life cycle and compared this expression to transgenic lines expressing mCherry-luciferase and GFP-luciferase under control of the constitutive gapdh and eef1a promoters. The [email protected] parasites express mCherry in gametocytes, sporozoites, and liver-stages. While no mCherry signal was detected in asexual blood-stage parasites above background levels, luciferase expression was detected in asexual blood-stages, as well as in gametocytes, sporozoites and liver-stages, with the highest levels of reporter expression detected in stage III-V gametocytes and in sporozoites. The expression of mCherry and luciferase in gametocytes and sporozoites makes this transgenic parasite line suitable to use in in vitro assays that examine the effect of transmission blocking inhibitors and to analyse gametocyte and sporozoite biology

Three Members of the 6-cys Protein Family of Plasmodium Play a Role in Gamete Fertility

The process of fertilization is critically dependent on the mutual recognition of gametes and in Plasmodium, the male gamete surface protein P48/45 is vital to this process. This protein belongs to a family of 10 structurally related proteins, the so called 6-cys family. To identify the role of additional members of this family in Plasmodium fertilisation, we performed genetic and functional analysis on the five members of the 6-cys family that are transcribed during the gametocyte stage of P. berghei. This analysis revealed that in addition to P48/45, two members (P230 and P47) also play an essential role in the process of parasite fertilization. Mating studies between parasites lacking P230, P48/45 or P47 demonstrate that P230, like P48/45, is a male fertility factor, consistent with the previous demonstration of a protein complex containing both P48/45 and P230. In contrast, disruption of P47 results in a strong reduction of female fertility, while males remain unaffected. Further analysis revealed that gametes of mutants lacking expression of p48/45 or p230 or p47 are unable to either recognise or attach to each other. Disruption of the paralog of p230, p230p, also specifically expressed in gametocytes, had no observable effect on fertilization. These results indicate that the P. berghei 6-cys family contains a number of proteins that are either male or female specific ligands that play an important role in gamete recognition and/or attachment. The implications of low levels of fertilisation that exist even in the absence of these proteins, indicating alternative pathways of fertilisation, as well as positive selection acting on these proteins, are discussed in the context of targeting these proteins as transmission blocking vaccine candidates

Rapid Attachment of Adipose Stromal Cells on Resorbable Polymeric Scaffolds Facilitates the One-Step Surgical Procedure for Cartilage and Bone Tissue Engineering Purposes

The stromal vascular fraction (SVF) of adipose tissue provides an abundant source of mesenchymal stem cells. For clinical application, it would be beneficial to establish treatments in which SVF is obtained, seeded onto a scaffold, and returned into the patient within a single surgical procedure. In this study, we evaluated the suitability of both a macroporous poly(L-lactide-co-caprolactone) and a porous collagen type I/III scaffold for this purpose. Surprisingly, cell attachment was rapid (similar to 10 min) and sequestered the majority of adipose stem cells, as deduced from colony-forming unit assays. Proliferation occurred in both polymeric scaffolds. Upon chondrogenic induction, upregulation of chondrogenic genes, production of glycosaminoglycans, and accumulation of collagen type II was observed, indicating differentiation of scaffold-attached SVF cells along the chondrogenic lineage. Osteogenic differentiation was achieved in both scaffold types, as visualized by up-regulation of osteogenic genes, increase of alkaline phosphatase production over time, and accumulation of bone sialoprotein and osteonectin. In conclusion, this study identifies both poly(L-lactide-co-caprolactone) and collagen type I/III as promising scaffold materials for rapid attachment of adipose stem cell-like (stromal) cells, enhancing the development of one-step surgical concepts for cartilage and bone tissue engineering. (C) 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:853-860, 201

Molecular changes in the degenerated goat intervertebral disc

Study Design. Caprine lumbar intervertebral discs (IVD) were collected from previous studies and categorized as normal, mildly, or severely degenerated. The biochemical composition and the RNA profiles present in both the nucleus pulposus (NP) and the anulus fibrosus (AF) were analyzed. Objective. To determine the molecular changes occurring in a disc degeneration model, evaluating the mechanism through which the degeneration develops in this model. Summary of Background Data. Recently we described an IVD degeneration model in the goat by injecting chondroitinase ABC. This results in mild progressive disc degeneration. Methods. One hundred nine caprine IVDs were assigned to 3 classes: no degeneration, mild, or severe degeneration. Collagen content, collagen cross-links (hydroxylysyl pyridinoline) and the ratio between the glycosaminoglycans (GAGs) and hydroxyprolines (Hyp) (GAG/Hyp ratio) in the NP and AF samples were studied. Furthermore, the gene expression of collagen type I, type II, and aggrecan as well as a desintegrin and metalloproteinase with thrombospondin motifs (ADAMTIS)-2, ADAMTS-14, and matrix metalloproteinases-13 were studied. Results. Collagen content was increased in severely degenerated NPs and decreased in severely degenerated AFs. Collagen cross-links were decreased in the severely degenerated NPs indicating de novo deposition of immature, reducible cross-linked collagens. The GAG/Hyp ratio found in none-degenerate goat discs was comparable to human ratios and decreased in degenerated discs, similar as in humans. The ADAMTS genes were increasingly detectable in the degenerated discs. The matrix metalloproteinases-13 gene increased significantly in degenerated discs. The expression of collagen type I increased in degenerated discs while aggrecan decreased. Conclusion. Changes in the GAG/Hyp ratio of chemically induced degeneration in goat IVD resemble the changes seen in humans. Gene expression profiles match the pattern of degeneration, suggesting that the injection of chondroitinase ABC might mimic the onset of human disc degeneratio

Lumbar spine segmentation in MR images: a dataset and a public benchmark

Abstract This paper presents a large publicly available multi-center lumbar spine magnetic resonance imaging (MRI) dataset with reference segmentations of vertebrae, intervertebral discs (IVDs), and spinal canal. The dataset includes 447 sagittal T1 and T2 MRI series from 218 patients with a history of low back pain and was collected from four different hospitals. An iterative data annotation approach was used by training a segmentation algorithm on a small part of the dataset, enabling semi-automatic segmentation of the remaining images. The algorithm provided an initial segmentation, which was subsequently reviewed, manually corrected, and added to the training data. We provide reference performance values for this baseline algorithm and nnU-Net, which performed comparably. Performance values were computed on a sequestered set of 39 studies with 97 series, which were additionally used to set up a continuous segmentation challenge that allows for a fair comparison of different segmentation algorithms. This study may encourage wider collaboration in the field of spine segmentation and improve the diagnostic value of lumbar spine MRI

SPIDER - Lumbar spine segmentation in MR images: a dataset and a public benchmark

<p>This is a large publicly available multi-center lumbar spine magnetic resonance imaging (MRI) dataset with reference segmentations of vertebrae, intervertebral discs (IVDs), and spinal canal. The dataset includes 447 sagittal T1 and T2 MRI series from 218 studies of 218 patients with a history of low back pain. The data was collected from four different hospitals. There is an additional hidden test set, not available here, used in the accompanying SPIDER challenge on spider.grand-challenge.org. We share this data to encourage wider participation and collaboration in the field of spine segmentation, and ultimately improve the diagnostic value of lumbar spine MRI.</p><p>Which MRI studies are assigned to the training and validation sets can be found in the overview file. This file also provides the biological sex for all patients and the age for the patients for which this was available. It also includes a number of scanner and acquisition parameters for each individual MRI study. The dataset also comes with radiological gradings found in a separate file for the following degenerative changes:</p><p>1.    Modic changes (type I, II or III)</p><p>2.    Upper and lower endplate changes / Schmorl nodes (binary)</p><p>3.    Spondylolisthesis (binary)</p><p>4.    Disc herniation (binary)</p><p>5.    Disc narrowing (binary)</p><p>6.    Disc bulging (binary)</p><p>7.    Pfirrman grade (grade 1 to 5). </p><p>All radiological gradings are provided per IVD level.</p><p> </p&gt