Dissecting diversity:clonal analysis of normal and malignant hematopoiesis using cellular barcodes

Leukemia is clonally heterogenous, consisting of multiple groups of cells with distinct (epi-)genetic alterations, which may alter essential cell functions. Clonal heterogeneity is thought to drive leukemia evolution, therapy resistance and relapse. To improve treatment of leukemia patients and prevent relapse, we need to extend our knowledge of clonal heterogeneity in leukemia. Here, we used cellular barcoding of patient-derived leukemia cells to trace their clonal behavior in murine xenografts. Cellular barcoding relies on the viral integration of synthetic DNA sequences of fixed length into the genome of the leukemia cells. Upon cell division, barcodes are inherited by a cell’s progeny. As a result, the barcode composition in a given cell population reflects the number of clonogenic cells and their relative fitness. First, to optimize reliability of barcode detection, we compared different data-analysis pipelines. We showed that the number of counted barcode clones can differ several-fold depending on the selected data-analysis strategy, and provide a method for optimization. Second, applying this method to murine xenografts transplanted with pediatric leukemia samples, we demonstrate that these mice harbor tens to hundreds of leukemia clones, which are asymmetrically distributed across the murine body. As a consequence, approximately half of the leukemia clones remained undetected when only one anatomical location is analyzed. Moreover, barcodes allowed for the identification of chemotherapy-resistant leukemia clones in mice. Combining cellular barcoding with other clone tracing methods will further improve our understanding of clonal heterogeneity in leukemia and potentially result in the development of new and improved treatment strategies of leukemia patients

Clonal analysis of patient-derived samples using cellular barcodes

Cellular barcoding is a relatively simple method that allows quantitative assessment of the clonal dynamics of normal, nonmalignant hematopoietic stem cells and of leukemia. Cellular barcodes are (semi-)random synthetic DNA sequences of a fixed length, which are used to uniquely mark and track cells over time. A successful barcoding experiment consists of several essential steps, including library production, transfection, transduction, barcode retrieval, and barcode data analysis. Key challenges are to obtain sufficient number of barcoded cells to conduct experiments and reliable barcode data analysis. This is especially relevant for experiments using primary leukemia cells (which are of limited availability and difficult to transduce), when studying low levels of chimerism, or when the barcoded cell population is sorted in different smaller subpopulations (e.g., lineage contribution of normal hematopoietic stem cells in murine xenografts). In these settings, retrieving accurate barcode data from low input material using standard PCR amplification techniques might be challenging and more sophisticated approaches are required. In this chapter we describe the procedures to transfect and transduce patient-derived leukemia cells, to retrieve barcoded data from both high and low input material, and to filter barcode data from sequencing noise prior to quantitative clonal analysis

Influence of Slip on the Plateau-Rayleigh Instability on a Fibre

The Plateau-Rayleigh instability of a liquid column underlies a variety of fascinating phenomena that can be observed in everyday life. In contrast to the case of a free liquid cylinder, describing the evolution of a liquid layer on a solid fibre requires consideration of the solid-liquid interface. In this article, we revisit the Plateau-Rayleigh Instability of a liquid coating a fibre by varying the hydrodynamic boundary condition at the fibre-liquid interface, from no-slip to slip. While the wavelength is not sensitive to the solid-liquid interface, we find that the growth rate of the undulations strongly depends on the hydrodynamic boundary condition. The experiments are in excellent agreement with a new thin film theory incorporating slip, thus providing an original, quantitative and robust tool to measure slip lengths

Signatures of slip in dewetting polymer films

Thin liquid polymer films on hydrophobic substrates are susceptable to rupture and formation of holes, which in turn initiate a complex dewetting process that eventually evolves into characteristic stationary droplet patterns. Experimental and theoretical studies suggest that the specific type of droplet pattern largely depends on the nature of the polymer-substrate boundary condition. To follow the morphological evolution numerically over long time scales and for the multiple length scales involved has so far been a major challenge. In this study a highly adaptive finite-element based numerical scheme is presented that allows for large-scale simulations to follow the evolution of the dewetting process deep into the nonlinear regime of the model equations, capturing the complex dynamics including shedding of droplets. In addition, the numerical results predict the previouly unknown shedding of satellite droplets during the destabilisation of liquid ridges, that form during the late stages of the dewetting process. While the formation of satellite droplets is well-known in the context of elongating fluid filaments and jets, we show here that for dewetting liquid ridges this property can be dramatically altered by the interfacial condition between polymer and substrate, namely slip

Signatures of slip in dewetting polymer films

Thin liquid polymer films on hydrophobic substrates are susceptable to rupture and formation of holes, which in turn initiate a complex dewetting process that eventually evolves into characteristic stationary droplet patterns. Experimental and theoretical studies suggest that the specific type of droplet pattern largely depends on the nature of the polymer-substrate boundary condition. To follow the morphological evolution numerically over long time scales and for the multiple length scales involved has so far been a major challenge. In this study a highly adaptive finite-element based numerical scheme is presented that allows for large-scale simulations to follow the evolution of the dewetting process deep into the nonlinear regime of the model equations, capturing the complex dynamics including shedding of droplets. In addition, the numerical results predict the previouly unknown shedding of satellite droplets during the destabilisation of liquid ridges, that form during the late stages of the dewetting process. While the formation of satellite droplets is well-known in the context of elongating fluid filaments and jets, we show here that for dewetting liquid ridges this property can be dramatically altered by the interfacial condition between polymer and substrate, namely slip

Coconut coir as a sustainable nursery growing media for seedling production of the ecologically diverse quercus species

Peat, a non-sustainable resource, is still predominately used in forest nurseries. Coconut coir might provide an alternative, renewable, and reliable growing media but few studies have evaluated this media type in forest nurseries. We assessed the influence of pure coir, in combination with various fertilization regimes, on the growth and physiology of three ecologically diverse Quercus species seedlings (Q. robur, Q. pubescens, and Q. ilex) during nursery cultivation. Seedlings were grown using peat and pure coir in combination with three fertilization treatments (standard, K-enriched, and P-enriched). Data were collected for: (1) growth and physiological traits; (2) detailed above- and below-ground morphological traits by destructive analysis; and (3) NPK content in leaves, shoot and roots, and in the growing media, following cultivation. Peat and coir in combination with the various fertilization treatments affected above- and below-ground morphology and, to a lesser extent, the physiological traits of Quercus seedlings. Large effects of the substrate occurred for most morphological variables, with peat being more effective than coir in all studied species. Fertilization also produced significant differences. The effect of K-enriched fertilization on plant growth was clear across the three species and the two growing media. P-enriched fertilization in peat was the only combination that promoted a higher amount of this element in the tissues at the end of cultivation. Despite their smaller size, seedlings produced in coir were compatible with standard Quercus forest stocktype size, and showed a proportionally higher root system development and fibrosity. Our results suggest that coir can be used as an alternative substrate to grow Quercus species seedlings, and that fertilization can offset coir deficiencies in chemical properties. As several functional traits drive planting performance under varying environmental conditions. according to the Target Plant Concept, coir might thus serve as an acceptable material for seedling cultivation in some cases

Detection of chemotherapy-resistant patient-derived acute lymphoblastic leukemia clones in murine xenografts using cellular barcodes

Clonal heterogeneity fuels leukemia evolution, therapeutic resistance, and relapse. Upfront detection of therapy-res istant leukemia clones at diagnosis may allow adaptation of treatment and prevention of relapse, but this is hampered by a paucity of methods to identify and trace single leukemia-propagating cells and their clonal offspring. Here, we tested methods of cellular barcoding analysis, to trace the in vivo competitive dynamics of hundreds of patient-derived leukemia clones upon chemotherapy-mediated selective pressure. We transplanted Nod/Scid/Il2Rg(-/-) (NSG) mice with barcoded patient-derived or SupB15 acute lymphoblastic leukemia (ALL) cells and assessed clonal responses to dexamethasone, methotrexate, and vincristine, longitudinally and across nine anatomic locations. We illustrate that chemotherapy reduces clonal diversity in a drug-dependent manner. At end-stage disease, methotrexate-treated patientderived xenografts had significantly fewer clones compared with placebo-treated mice (100 +/- 10 vs. 160 +/- 15 clones, p = 0.0005), while clonal complexity in vincristineand dexamethasone-treated xenografts was unaffected (115 +/- 33 and 150 +/- 7 clones, p = NS). Using tools developed to assess differential gene expression, we determined whether these clonal patterns resulted from random clonal drift or selection. We identified 5 clones that were reproducibly enriched in methotrexate-treated patient-derived xenografts, suggestive of pre-existent resistance. Finally, we found that chemotherapymediated selection resulted in a more asymmetric distribution of leukemia clones across anatomic sites. We found that cellular barcoding is a powerful method to trace the clonal dynamics of human patient-derived leukemia cells in response to chemotherapy. In the future, integration of cellular barcoding with single-cell sequencing technology may allow in-depth characterization of therapy-resistant leukemia clones and identify novel targets to prevent relapse. (C) 2020 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc

Donor-to-Donor Heterogeneity in the Clonal Dynamics of Transplanted Human Cord Blood Stem Cells in Murine Xenografts

Umbilical cord blood (UCB) provides an alternative source of hematopoietic stem cells (HSCs) for allogeneic transplantation. Administration of sufficient donor HSCs is critical to restore recipient hematopoiesis and to maintain long-term polyclonal blood formation. However, due to lack of unique markers, the frequency of HSCs among UCB CD34(+) cells is the subject of ongoing debate, urging for reproducible strategies for their counting. Here, we used cellular barcoding to determine the frequency and clonal dynamics of human UCB HSCs and to determine how data analysis methods affect these parameters. We transplanted lentivirally barcoded CD34(+) cells from 20 UCB donors into Nod/Scid/IL2Ry(-/-) (NSG) mice (n = 30). Twelve recipients (of 8 UCB donors) engrafted with >1% GFP(+) cells, allowing for clonal analysis by multiplexed barcode deep sequencing. Using multiple definitions of clonal diversity and strategies for data filtering, we demonstrate that differences in data analysis can change clonal counts by several orders of magnitude and propose methods to improve their consistency. Using these methods, we show that the frequency of NSG-repopulating cells was low (median similar to 1 HSC/10(4) CD34(+) UCB cells) and could vary up to 10-fold between donors. Clonal patterns in blood became increasingly consistent over time, likely reflecting initial output of transient progenitors, followed by long-term HSCs with stable hierarchies. The majority of long-term clones displayed multilineage output, yet clones with lymphoid- or myeloid-biased output were also observed. Altogether, this study uncovers substantial interdonor and analysis-induced variability in the frequency of UCB CD34(+) clones that contribute to post-transplant hematopoiesis. As clone tracing is increasingly relevant, we urge for universal and transparent methods to count HSC clones during normal aging and upon transplantation. (C) 2019 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc

Tarantulas (Araneae : Theraphosidae) in the pet trade in South Africa

CITATION: Shivambu, T.C. et al. 2020. Tarantulas (Araneae: Theraphosidae) in the pet trade in South Africa. African Zoology 55(4):323-336. doi:10.1080/15627020.2020.1823879The original publication is available at https://www.tandfonline.com/toc/tafz20/currentMany alien species have been introduced around the world as part of the pet trade, and some have escaped captivity and become invasive. In South Africa, many species of tarantula (Theraphosidae) are kept as pets. It is not known which species are traded, which are most popular, and whether their names are correctly applied. Online traders and physical pet stores were investigated between 2015 and 2016 to determine the extent or size of trade, species composition, most popular species, and their invasion history elsewhere. In total, 36 specimens, three individuals from 12 putative species, were also purchased for DNA barcoding targeting the COI gene region to quantify the accuracy of tarantula identification by traders. In total, 195 tarantula species were advertised for sale, and the most popular species were Brachypelma albopilosum Valerio, 1980 (n = 199), B. vagans Ausserer, 1875 (n = 132), and Grammostola rosea Walckenaer, 1837 (n = 120). The composition of shared species differed between the sources and most of the species were advertised online. Only one of the popular species, B. vagans, has been recorded as being invasive elsewhere. Only 36% of the barcoded specimens matched existing barcodes in online repositories that had the same species name. The three individuals from 12 putative species were not in the same terminal clade as those of conspecifics in the Barcode of Life Data System (BOLD) and the NCBI GenBank reference sequences. A large proportion of the known tarantula species are traded in South Africa and must be included in management and risk assessments to avoid potential invasions