An improved pre-clinical patient-derived liquid xenograft mouse model for acute myeloid leukemia

Background

Xenotransplantation of patient-derived AML (acute myeloid leukemia) cells in NOD-scid Il2rγ ^null (NSG) mice is the method of choice for evaluating this human hematologic malignancy. However, existing models constructed using intravenous injection in adult or newborn NSG mice have inferior engraftment efficiency, poor peripheral blood engraftment, or are difficult to construct.

Methods

Here, we describe an improved AML xenograft model where primary human AML cells were injected into NSG newborn pups intrahepatically.

Results

Introduction of primary cells from AML patients resulted in high levels of engraftment in peripheral blood, spleen, and bone marrow (BM) of recipient mice. The phenotype of engrafted AML cells remained unaltered during serial transplantation. The mice developed features that are consistent with human AML including spleen enlargement and infiltration of AML cells into multiple organs. Importantly, we demonstrated that although leukemic stem cell activity is enriched and mediated by CD34⁺CD117⁺ subpopulation, CD34⁺CD117^? subpopulation can acquire CD34⁺CD117⁺ phenotype through de-differentiation. Lastly, we evaluated the therapeutic potential of Sorafenib and Regorafenib in this AML model and found that periphery and spleen AML cells are sensitive to these treatments, whereas BM provides a protective environment to AML.

Conclusions

Collectively, our improved model is robust, easy-to-construct, and reliable for pre-clinical AML studies.

     

Crystal structure of human seminal diferric lactoferrin at 3.4 Angstrom resolution

Lactoferrin was purified from human seminal fluid obtained from the semen bank. The purified samples were saturated with Fe3+ and crystallized by microdialysis method. The crystals belong to orthorhombic space group P21212, with a = 55.9 Angstrom. b = 97.2 Angstrom, c = 156.1 Angstrom and Z = 4. The structure was determined with molecular replacement method and refined to an R factor of 18.7% for all the data to 3.4 Angstrom resolution. The overall structure of seminal lactoferrin is similar to human colostrum lactoferrin. The amino acid sequence of seminal lactoferrin shows that it has one amino acid less than human colostrum lactoferrin and the structure of its N-terminal region is far more ordered than other lactoferrins. The structure of the iron-binding site and its immediate surroundings indicate well defined features.     

Transformation and Transport of Nitrogen Forms in a Sandy Entisol Following a Heavy Loading of Ammonium Nitrate Solution: Field Measurements and Model Simulations

Understanding the factors influencing water and nutrient transport through soil profile is important for the efficient management of nutrient and irrigation to minimize nutrient leaching below the rootzone. Transport of NO₃-N and NH₄-N was studied in a Candler fine sand following a heavy loading of a liquid fertilizer containing ammonium nitrate. Both NO₃-N and NH₄-N transported quite rapidly (within 3?d) and accumulated above the clay layer at about depth of 2.7?m. The concentrations of NH₄-N and NO₃-N approached background levels throughout the soil profile by 184?d. More than 50% of ammonium and nitrate contained in the spilled solution leached from the entire depth of soil profile sampled during the first 95?d. The cumulative amount of rainfall during this period was 329?mm, which accounted for 65% of the total rainfall for the entire study period. The concentrations of NH₄-N and NO₃-N at various depths within the entire soil profile reasonably predicted by the Leaching Estimation and Chemistry Model (LEACHM) and compared favorably with the measured concentrations, however, there are few places with high concentrations. The cumulative amount of leachate at the bottom of the soil profile predicted by LEACHM represented 90% of total rainfall that occurred during the study period. This demonstrated a substantial potential for leaching of soluble nutrients through the sandy soil profile.     

Short stature and an interesting association

Untreated hypothyroidism in children usually results in delayed puberty, but juvenile hypothyroidism causes isosexual precocious puberty in a rare syndrome called Van Wyk Grumbach syndrome, with a complete reversal to the pre pubertal state following thyroid hormone replacement therapy. We report here, a 7-year-old girl who presented with short stature, constipation and isosexual precocious puberty due to the long standing untreated severe hypothyroidism with this syndrome.     

Denitrification in the vadose zone and in surficial groundwater of a sandy entisol with citrus production

A portion of nitrate (NO ₃ ⁻ ), a final breakdown product of nitrogen (N) fertilizers, applied to soils and/or that produced upon decomposition of organic residues in soils may leach into groundwater. Nitrate levels in water excess of 10 mg L⁻¹ (NO₃–N) are undesirable as per drinking water quality standards. Nitrate concentrations in surficial groundwater can vary substantially within an area of citrus grove which receives uniform N rate and irrigation management practice. Therefore, differences in localized conditions which can contribute to variations in gaseous loss of NO ₃ ⁻ in the vadose zone and in the surficial aquifer can affect differential concentrations of NO₃–N in the groundwater at different points of sampling. The denitrification capacity and potential in a shallow vadose zone soil and in surficial groundwater were studied in two large blocks of a citrus grove of ‘Valencia’ orange trees (Citrus sinensis (L.) Obs.) on Rough lemon rootstock ( Citrus jambhiri (L.)) under a uniform N rate and irrigation program. The NO₃–N concentration in the surficial groundwater sampled from four monitoring wells (MW) within each block varied from 5.5- to 6.6-fold. Soil samples were collected from 0 to 30, 30 to 90, or 90 to 150 cm depths, and from the soil/groundwater interface (SGWI). Groundwater samples from the monitoring wells (MW) were collected prior to purging (stagnant water) and after purging five well volumes. Without the addition of either C or N, the denitrification capacity ranged from 0.5 to 1.53, and from 0.0 to 2.25 mg N₂O–N kg⁻¹ soil at the surface soil and at the soil/groundwater interface, respectively. The denitrification potential increased by 100-fold with the addition of 200 mg kg⁻¹ each of N and C. The denitrification potential in the groundwater also followed a pattern similar to that for the soil samples. Denitrification potential in the soil or in the groundwater was greatest near the monitor well with shallow depth of vadose zone (MW3). Cumulative N₂O–N emission (denitrification capacity) from the SGWI soil samples and from stagnant water samples strongly correlated to microbial most probable number (MPN) counts (r² = 0.84 – 0.89), and dissolved organic C (DOC) (r² = 0.96 – 0.97). Denitrification capacity of the SGWI samples moderately correlated to water-filled pore space (WFPS) (r² = 0.52). However, extractable NO₃-N content of the SGWI soil samples poorly (negative) correlated to denitrification capacity (r² = 0.35). However, addition C, N or both to the soil or water samples resulted in significant increase in cumulative N₂O emission. This study demonstrated that variation in denitrification capacity, as a result of differences in denitrifier population, and the amount of readily available carbon source significantly (at 95% probability level) influenced the variation in NO₃–N concentrations in the surficial groundwater samples collected from different monitoring wells within an area with uniform N management. This revised version was published online in June 2006 with corrections to the Cover Date.     

Culture independent characterization of bacteria associated with the mucus of the coral <Emphasis Type="Italic">Acropora digitifera</Emphasis> from the Gulf of Mannar

Corals are sessile eukaryotic hosts which provide a unique surface for microbial colonization. Culture independent studies show that the coral mucus and tissue harbour diverse and abundant prokaryotic communities. However, little is known about the diversity of bacteria associated with the corals of Gulf of Mannar. The present study characterised the bacterial diversity associated with the mucus of the coral Acropora digitifera from the Gulf of Mannar by 16S rRNA gene clone library construction. The bacterial communities of the mucus of A. digitifera were diverse, with representatives within the Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes and several unclassified bacteria. The culture independent bacterial population was totally different from our previous culture dependent study of the mucus and tissue of the same coral. 36% of the bacteria in the clone library of A. digitifera were found to be novel after full length sequencing of the 16S rRNA gene wherein several clones were found to be novel at the Genus and species level. The current study further supports the findings that Actinobacteria amount to a certain proportion among bacterial communities associated with corals.     

Crystal structure of a novel regulatory 40-kDa mammary gland protein (MGP-40) secreted during involution

We have determined the crystal structure of a novel regulatory protein (MGP-40) from the mammary gland. This protein is implicated as a protective signaling factor that determines which cells are to survive the drastic tissue remodeling that occurs during involution. It has been indicated that certain cancers could surreptitiously utilize the proposed normal protective signaling by proteins of this family to extend their own survival and thereby allow them to invade the organ and metastasize. In view of this, MGP-40 could form an important target for rational structure-based drug design against breast cancer. It is a single chain, glycosylated protein with a molecular mass of 40 kDa. It was isolated from goat dry secretions and has been cloned and sequenced. It was crystallized by microdialysis from 20 mg ml(-1) solution in 0.1 m Tris-HCl, pH 8.0, and equilibrated against the same solution containing 19% ethanol. Its x-ray structure has been determined by molecular replacement and refined to a 2.9 A resolution. The protein adopts a beta/alpha domain structure with a triose-phosphate isomerase barrel conformation in the core and a small alpha+beta folding domain. A single glycosylation site containing two N-acetylglucosamine units has been observed in the structure. Compared with chitinases and chitinase-like proteins the most important mutation in this protein pertains to a change from Glu to Leu at position 119, which is part of the so-called active site sequence in the form of Asp(115), Leu(119), and Asp(186) and in this case resulting in the loss of chitinase activity. The orientations of two Trp residues Trp(78) and Trp(331) in the beta barrel reduces the free space, drastically impairing the binding of saccharides/polysaccharides. However, the site and mode of binding of this protein to cell surface receptors are not yet known.     

Linking phospholipid flippases to vesicle-mediated protein transport

Type IV P-type ATPases (P4-ATPases) are a large family of putative phospholipid translocases (flippases) implicated in the generation of phospholipid asymmetry in biological membranes. P4-ATPases are typically the largest P-type ATPase subgroup found in eukaryotic cells, with five members in Saccharomyces cerevisiae, six members in Caenorhabditis elegans, 12 members in Arabidopsis thaliana and 14 members in humans. In addition, many of the P4-ATPases require interaction with a noncatalytic subunit from the CDC50 gene family for their transport out of the endoplasmic reticulum (ER). Deficiency of a P4-ATPase (Atp8b1) causes liver disease in humans, and studies in a variety of model systems indicate that P4-ATPases play diverse and essential roles in membrane biogenesis. In addition to their proposed role in establishing and maintaining plasma membrane asymmetry, P4-ATPases are linked to vesicle-mediated protein transport in the exocytic and endocytic pathways. Recent studies have also suggested a role for P4-ATPases in the nonvesicular intracellular trafficking of sterols. Here, we discuss the physiological requirements for yeast P4-ATPases in phospholipid translocase activity, transport vesicle budding and ergosterol metabolism, with an emphasis on Drs2p and its noncatalytic subunit, Cdc50p.     

Evidence for mouse sulfhydryl oxidase‐assisted cross‐linking of major seminal vesicle proteins

Copulatory plug formation in animals is a general phenomenon by which competition is reduced among rival males. In mouse, the copulatory plug formation results from the coagulation of highly viscous seminal vesicle secretion (SVS) that is rich in proteins, such as dimers of SVS I, SVS I + II + III, and SVS II. These high‐molecular‐weight complexes (HMWCs) are also reported to be the bulk of proteins in the copulatory plug of the female mouse following copulation. In addition, mouse SVS contributes to the existence of sulfhydryl oxidase (Sox), which mediates the disulfide bond formation between cysteine residues. In this study, flavin adenine dinucleotide (FAD)‐dependent Sox was purified from mouse SVS using ion exchange and high‐performance liquid chromatography. The purified enzyme was identified to be Sox, based on western blot analysis with Sox antiserum and its capability of oxidizing dithiothreitol as substrate. The pH optima and thermal stability of the enzyme were determined. Among the metal ions tested, zinc showed an inhibitory effect on Sox activity. A prosthetic group of the enzyme was identified as FAD. The K_m and V_max of the enzyme was also determined. In addition to purification and biochemical characterization of seminal vesicle Sox, the major breakthrough of this study was proving its cross‐linking activity among SVS I–III monomers to form HMWCs in SVS.