Primate skeletal muscle contains cells capable of sustaining in vitro hematopoiesis

OBJECTIVE: Several investigators recently reported that adult murine skeletal muscle cells possess a remarkable capacity to differentiate into hematopoietic cells. We further examined this biologic process by studying the phenotype and in vitro functional behavior of primate skeletal muscle cells. MATERIALS AND METHODS: Muscles from human abortuses as well as fetal and adult baboons were digested enzymatically and mononuclear cell fractions were isolated. Muscle tissue-derived mononuclear cells (mu-TDMNC) were phenotypically characterized. Both short-term and long-term hematopoietic progenitors were assayed from mu-TDMNC using standard techniques. Gene expression patterns characteristic of hematopoietic and endothelial cells were examined in primary and cultured muscle cells. RESULTS: Primate muscle cells were shown to express the CD34 antigen. Such CD34(+) cells were shown to be CD45(-) and desmin(+), indicating they were not of hematopoietic origin. Fetal but not adult muscle cells contained assayable hematopoietic progenitors. In addition, muscles contained an additional class of progenitors that formed colonies composed of blast cells after prolonged incubation (3-4 weeks). A two-step culture system was established that permitted muscle cells to continue to proliferate when exposed to a hematopoietic environment for 8 months. During this prolonged period of time, the generation of CD34(+), CD56(+), CD11b(+), and CD31(+) as well as von Willebrand factor (vWF)(+) cells were observed. CONCLUSIONS: Our studies indicate that although primate muscle cells contain a significant number of CD34(+) cells, they are likely not of hematopoietic origin. Important ontogenic differences in the hematopoietic potential of primate muscle cells were documented. When exposed to appropriate microenvironmental stimuli, mu-TDMNC displayed an extensive proliferative capacity and contained primitive progenitors with the capacity to generate cells in vitro with phenotypic and genetic properties of hematopoietic and endothelial cells for sustained periods of time. Whether this observation can be accounted for by true transdifferentiation of muscle cells or proliferation of reservoirs of hematopoietic and endothelial progenitor cells residing within skeletal muscle remains unresolved.     

Resolving the HONO formation mechanism in the ionosphere via ab initio molecular dynamic simulations

Solar emission produces copious nitrosonium ions (NO⁺) in the D layer of the ionosphere, 60 to 90 km above the Earth’s surface. NO⁺ is believed to transfer its charge to water clusters in that region, leading to the formation of gaseous nitrous acid (HONO) and protonated water cluster. The dynamics of this reaction at the ionospheric temperature (200–220 K) and the associated mechanistic details are largely unknown. Using ab initio molecular dynamics (AIMD) simulations and transition-state search, key structures of the water hydrates—tetrahydrate NO⁺(H₂O)₄ and pentahydrate NO⁺(H₂O)₅—are identified and shown to be responsible for HONO formation in the ionosphere. The critical tetrahydrate NO⁺(H₂O)₄ exhibits a chain-like structure through which all of the lowest-energy isomers must go. However, most lowest-energy isomers of pentahydrate NO⁺(H₂O)₅ can be converted to the HONO-containing product, encountering very low barriers, via a chain-like or a three-armed, star-like structure. Although these structures are not the global minima, at 220 K, most lowest-energy NO⁺(H₂O)₄ and NO⁺(H₂O)₅ isomers tend to channel through these highly populated isomers toward HONO formation.The ionosphere is the largest layer in the Earth''s atmosphere, ranging in altitude from ∼60 to 1,000 km and includes the thermosphere and parts of the mesosphere and exosphere. The ionosphere contains a high concentration of electrons and ions because of the ionization of gases in that region by short wavelength radiation from the Sun. Therefore, these species play an important role in atmospheric electricity, influencing radio propagation to different regions on the Earth’s surface and space-based navigational systems (1). The D layer is the innermost layer of the ionosphere, ranging from 60 to 90 km in altitude, where Lyman series-α hydrogen radiation from the Sun gives rise to abundant nitrosonium ions (NO⁺). In addition to the ionospheric reaction between NO⁺ and water, explorations of the chemical reactivity of NO⁺ and water clusters (2–4) have implications for understanding the mechanisms of atmospherically relevant reactions in water clusters (5–9).Over the past two decades, several experimental and theoretical studies (10–13) have focused on understanding the chemical and physical properties of the small-sized hydrated nitrosonium ion NO⁺(H₂O)_n, where n = 1–5. Two key processes have been proposed for HONO formation:NO⁺(H₂O)_n + H₂O → {(HONO)H⁺(H₂O)_n}? → H⁺(H₂O)_n + HONO?.[1]Lee and coworkers (14) used vibrational spectroscopy to obtain clear evidence of the rearrangement of the NO⁺(H₂O)_n cluster by observing the appearance of new hydrogen (H)-bonded OH stretching lines. Using quantum molecular dynamics, Ye and Cheng (15) suggested possible structures and corresponding IR spectra for NO⁺(H₂O)_n (n = 1–3) clusters. In a major experimental breakthrough, Relph et al. (16) showed that the extent to which reaction 1 produces HONO and H⁺(H₂O)_n depends on the size and shape of the water clusters. Another key finding was that the reactions for HONO production start with the n = 4 water cluster. Later, the importance of the tetrahydrate isomer NO⁺(H₂O)₄ to its conversion to proton hydrate and HONO at temperature beyond 150 K was further demonstrated experimentally by Eyet et al. (11). Indeed, before Eyet’s study, Siefermann and Abel (17) had already noted that the configurations of the trihydrate and tetrahydrate isomers examined in Relph et al.’s experiment were frozen because of the very low temperature used (5 K). At this low temperature, the most abundant water cluster structures are those found at the global minima of the potential energy surface. At temperatures that are relevant to the ionosphere (200–220 K), these lowest-lying isomers may not directly contribute to the interconversion processes involving the hydrated NO⁺(H₂O)_n ion.An early study suggested that the low rate of reaction 1 can be attributed to the fact that the reactive species responsible for HONO formation include a higher-energy isomer of NO⁺(H₂O)_n that is responsible to the release of a proton (12). Asada et al. (18) reported high-level ab initio molecular-orbital calculations and identified tens of low-energy isomers of NO⁺(H₂O)₄ and NO⁺(H₂O)₅. They also pointed out that relatively higher-energy reactant, transition-state, and product isomers are involved in the formation of HONO from NO⁺(H₂O)_n (n = 4 and 5) clusters. But, the nature of the mechanism by which these relatively higher-energy isomers (in the frozen state at 0 K) can directly contribute to the interconversion processes at temperatures relevant to the ionosphere is little studied.In light of the lack of experimental studies of the dynamics of isomer transformation, we performed Born–Oppenheimer ab initio molecular dynamics (AIMD) simulations to explore the dynamic behaviors of trihydrate NO⁺(H₂O)₃, tetrahydrate NO⁺(H₂O)₄, and pentahydrate NO⁺(H₂O)₅ clusters at 220 K. Our results suggest that 220 K is adequate to drive the isomer interconversion from the lowest-lying isomers to the critical chain-like isomers. Furthermore, based on the climbing image nudged-elastic-band method, a more realistic transition state for the formation of hydrated protons and HONO (coexisting in the tetrahydrate) was identified, with the reactant being the critical tetrahydrate NO⁺(H₂O)₄ isomer. For pentahydrate, two reaction pathways are revealed by the AIMD simulations. Furthermore, the distribution of low-lying isomers at 220 K, including both highly populated critical and less-populated local-minimum isomers, is obtained. In agreement with the results of Relph et al. (16), there is no observed charge transfer between the NO⁺ and the water clusters in the n = 1 or n = 2 NO⁺(H₂O)_n clusters, indicating that these small clusters are inert. The charge transfer observed for clusters with n ≥ 3 suggests the possible formation of HONO from the NO⁺(H₂O)_n clusters. However, the population of water clusters decreases rapidly with increasing n because of the scarcity of the water molecules in the D layer of the ionosphere (17). Therefore, the trihydrate, tetrahydrate, and pentahydrate clusters are most likely the prevailing reactive species for HONO formation in the ionosphere and thus constitute the focus of our AIMD simulations. All the AIMD simulations in this work are performed in the form of the Beche–Lee–Yang–Parr functional (19, 20) with Grimme’s dispersion correction (21) (denoted as BLYP-D method) which can well describe the trend of the charge variation of the NO⁺ hydrates systems (see Fig. S1). Note that our study here is mainly focused on the formation of the HONO species in the hydrates, corresponding to the first step in reaction 1. The detachment of the HONO species from the hydrated proton, which is an endothermic process, is not considered here.Open in a separate windowFig. S1.Charge on the NO varies with the isomers for tetrahydrates on the basis of the Mullikan charge analysis at the BLYP-D and the CCSD(T)/aug-cc-pVTZ//MP2/aug-cc-pVTZ levels of theory [here, CCSD(T) and MP2 refer to the coupled-cluster method with singlet, doublet, and triplet excitations and the second-order Møller–Plesset perturbation theory, respectively]. The CCSD(T)//MP2 denotes that the structure is optimized at the MP2/aug-cc-pVTZ level, whereas the charge analysis is performed at the CCSD(T)/aug-cc-pVTZ level.For the trihydrate cluster, the three experimentally detected low-lying isomers (18) are called 3-α, 3-β, and 3-γ (Fig. 1). Notably, our AIMD simulation shows the isomer interconversion from the two lowest-lying isomers, 3-α and 3-β, to 3-γ (Fig. S2). In the course of AIMD simulation, the N–O₁ distance decreases from ∼2.20 to ∼1.90 Å, concurrent with a slight elongation of the O₁–H₁ and O₁–H₂ bonds, implying the conversion of isomer 3-α or 3-β to the 3-γ isomer (Movies S1A and S1B). No isomerization events are observed in the AIMD simulation with the initial 3-γ configuration, indicating the high thermal stability of 3-γ at 220 K. Hence, the 3-γ isomer is expected to exhibit the highest abundance among trihydrate clusters in the D layer of the ionosphere. Moreover, for the 200-ps AIMD simulation of 3-γ, no evidence of dissociation of the O–H bonds in water molecules was observed, consistent with the previous experiment-based conclusion (16) that trihydrate clusters do not play a major role in reaction 1.Open in a separate windowFig. 1.Illustration of the dynamic-driven isomer interconversion observed in AIMD simulations of the trihydrates and tetrahydrates of NO⁺. The highlighted structures in brackets represent the most likely reaction pathway from the critical (highly populated) isomer 4E to the product isomer 4ii. White, blue, and red spheres represent hydrogen, nitrogen, and oxygen atoms, respectively.Open in a separate windowFig. S2.N–O₁ distance change with time for isomers 3-α (black), 3-β (red), and 3-γ (blue). Time required for the structural transformation from 3-α and 3-β to 3-γ. The atomic label is given. Note that both H₁ and H₂ bind with two different water molecules via H-bond arrangement, which is the so-called “symmetrical H bond.”For the tetrahydrate cluster, the four lowest-lying isomers 4A, 4B, 4C, and 4E (Fig. 1) (18) were selected to investigate their dynamic behaviors at 220 K. As shown in Fig. S3, the O–H bonds of the water molecule directly bound to the NO⁺ are <1.20 Å during the 200-ps AIMD simulations, indicating the absence of HONO-forming reactions in that time period. In contrast, sudden changes in the N–O distance (e.g., at ∼26 ps in Fig. S3A; the definition of N–O distance is given in the caption of Fig. S3) are observed in all four independent AIMD simulations (black lines in Fig. S3), indicating isomer interconversion. To characterize the degree of isomer interconversion, the bond-orientational order parameter (Ψ) given byΨ=1n|∑j=1neinφij|is computed, where n is the total number of atoms j within a given radius cutoff and φ_i?j is the angle between the vector connecting the target atom i with the neighboring atom j and the reference vector connecting the target atom i and the system’s center of mass (marked by the black solid circle in the Inset of Fig. 2A). Two different target atoms with their corresponding neighboring (source) atom j were selected to characterize the structural variation: (i) the N atom in the NO⁺ motif with the O atoms in the water molecules as the source of atom j, and (ii) the O atom (shown by the green sphere in Fig. 2A, Inset) in the water molecule located at the longer end of the chain structure with the O atoms in the other water molecules that forms a hydrogen bond with the target O atom as the source of atom j. In chain-like structures, such as isomer 4E, only one O atom exists within 2.80 Å of the N atom, whereas the target O atom only forms one hydrogen bond with the neighboring water molecule. Hence, the logarithms of Ψ_N?O and Ψ_O?O take values of zero for isomer 4E. For other isomers, either Ψ_N?O or Ψ_O?O is nonzero. Fig. 2A shows the time-dependent order parameters given by the logarithms of Ψ_N?O (red line) and Ψ_O?O (black line), with isomer 4A as the initial structure. The disappearance of the red peaks at ∼26 ps clearly results from the isomer transformation from 4C to 4E (Figs. S3A and ​andS4S4 and Movie S2A). The cyclization of 4E leads to the formation of an additional hydrogen bond with the target O atom, resulting in nonzero values of the logarithm of Ψ_O?O for the ∼50–70-ps time period (see c-4E isomer in Fig. S4). The proximity of the N and O atoms of two nearest water molecules (the corresponding isomer is denoted as d-4E in Fig. S4) results in a small red peak at ∼120 ps. Similar isomer interconversion is also observed in other AIMD simulations with 4B, 4C, or 4E as the initial structure. The corresponding time-evolution data for Ψ_N?O or Ψ_O?O and associate isomer structures are shown in Figs. S5 and ​andS4,S4, respectively.Open in a separate windowFig. 2.(A) Time evolution of the logarithms of the bond-orientational order parameters Ψ_N?O (red line) and Ψ_O?O (black line). (Inset) Illustration of the angle φ_i?j used to calculate Ψ_N?O and Ψ_O?O, where white and red spheres represent oxygen and hydrogen atoms, respectively, and blue and green spheres represent the target nitrogen and oxygen atoms, respectively, used to calculate the bond-orientational order parameter. The pie charts in B–E denote the populations of various isomers observed in four independent AIMD simulations with initial structures of 4A, 4B, 4C, and 4E, respectively. The geometric structures of the isomers are given in Fig. S4.Open in a separate windowFig. S3.Time evolution of the N–O (black line) (where O is the O atom of the nearest H₂O molecule next to the N atom of the NO⁺ ion) and the O–H (red line) (where O–H refers to the averaged O–H bond length for the water molecule nearest to the NO⁺ ion) distances for the AIMD simulations of NO⁺(H₂O)₄ clusters with different initial isomer: (A) isomer 4A, (B) isomer 4B, (C) isomer 4C, and (D) isomer 4E. (Inset) Images represent the initial structures used in each AIMD simulation.Open in a separate windowFig. S4.Geometrical structures of the observed isomers in the AIMD simulation with isomer 4A as the initial structure. The white, blue, and red spheres represent the hydrogen, nitrogen, and oxygen atoms. The arrows and three pairs of circled numbers indicate several possible shifting directions of molecules and corresponding interconversion of isomers.Open in a separate windowFig. S5.Time evolution of the logarithm of the bond-orientational order parameterΨ_N?Ο (red lines) and Ψ_O?O (black lines) for the AIMD simulations starting from isomer 4B (Top), 4C (Middle), and 4E (Bottom), respectively.The chain-like water structure is the critical structure bridging two different isomers during isomer interconversion. As shown in Fig. 2A and Fig. S5, the zero-value interval between two peaks indicates the appearance of isomer 4E during the isomer conversion. More importantly, the population analysis of each isomer over the entire 200-ps AIMD simulation suggests that the chain-like water structure of 4E is much more abundant than the other isomers (Fig. 2 B–E). Specifically, in the four independent AIMD simulations with initial structures of 4A, 4B, 4C, and 4E, the obtained population values are highest for 4E: ∼62.25%, 77.66%, 61.49%, and 86.89%, respectively. Note that at 0 K, BLYP-D functional (19, 20; see Supporting Information, Computational Details) predicts that 4E is lower in energy than 4A (i.e., BLYP-D introduces some biases toward 4E over 4A, see Fig. S6 for MP2 results). But, 4E is still about 1 kcal/mol higher in energy than 4B or 4C at the BLYP-D level. At 220 K, 4E becomes the most thermodynamically favorable isomer at the BLYP-D level. Hence, the 4E isomer can be viewed as the critical isomer among the tetrahydrate clusters and plays a critical role in the D layer of the ionosphere.Open in a separate windowFig. S6.Electronic energies (ΔE), zero-point-energy corrected electronic energies (ΔE+ZPE), and the Gibbs free energies (ΔG) relative to isomer 4A for tetrahydrate isomers at the MP2 level of theory.The HONO-containing tetrahydrate isomer detected experimentally at 5 K (named 4-ii in ref. 16) has nearly the same structure as 4G (Fig. S7). The interconversion between isomer 4-ii and 4G through HONO rotation and flipping of the H₃O⁺ groups was frequently observed in the AIMD simulations (Movie S2B). More importantly, no breaking down of the N–O bonds in HONO was observed in the course of the 200-ps AIMD simulation, suggesting that both isomers are highly stable at 220 K. Therefore, the 4-ii isomer can be viewed as the final product of HONO formation, consistent with the experimental detection of 4-ii at 5 K. To confirm this interpretation, we used climbing image nudge-elastic-band calculations (22) to search for the transition state that bridges the 4E and 4-ii isomers. As shown in Fig. 1, the movement of the water molecule at the short end of the chain-like structure toward the long end and the subsequent formation of two H bonds with two neighboring water molecules led to proton transfer between the two neighboring water molecules, giving rise to the transition state (TS in Fig. 1). Upon passing over the TS, the original water molecule near the short end breaks one hydrogen bond while retaining the other hydrogen bond with the protonated species, concurrent with the formation of HONO species. In this cooperative process, the movement of the water molecule at the short end leads to the formation of a cyclic structure, where the two water molecules and the HONO species act as hydrogen-bond acceptors and form a complete solvation shell around the H₃O⁺ ion. The formation of such a solvation shell can effectively stabilize the central H₃O⁺ ion, a well-established fact in the gas-phase reaction involving ionic clusters (23–26). Due to the stabilization effect, the formation of HONO-containing isomer 4-ii, from the highly populated isomer 4E, entails a low-energy barrier of ∼2.1 kcal/mol. Thus, at the ionospheric temperatures (200–220 K), a chemical equilibrium between 4E and the HONO-containing isomers 4-ii is expected to be an important dynamic channel for HONO formation. Note that in our AIMD simulations, the nuclear quantum effect and the hydrogen tunneling effect are not included. In general, the nuclear quantum effect is equivalent to the lowering of density-functional theory (e.g., BLYP-D) temperature of water by certain degrees (27), whereas the hydrogen tunneling would speed up the proton transfer process in our system not included in the AIMD simulations. Nevertheless, the two effects seem to somewhat offset each other and, as a result, may not affect the qualitative reaction mechanism concluded from the AIMD simulations.Open in a separate windowFig. S7.Geometrical structures of the isomer 4-ii and 4G.Another channel for HONO formation can occur through the pentahydrate NO⁺(H₂O)₅, although the population of pentahydrate clusters is expected to be much lower than that of the tetrahydrate clusters. Here, the lowest-lying four isomers, 5A, 5B, 5D, and 5M (23), are selected as the initial structures in four independent AIMD simulations. In the simulation starting with isomer 5A, no HONO formation or appreciable changes in the N–O and O–H distances were observed within the 200-ps simulation, suggesting that 5A is a highly stable isomer. In contrast, the formation of HONO species is directly observed in the AIMD simulations with initial structures of 5B, 5D, and 5M, indicating that in this case, the HONO-forming reaction has a very low energy barrier. Moreover, as shown in Fig. 3, the final HONO-containing products obtained in the three independent AIMD simulations exhibit the same structure, named 5Γ, which contains a chain-like water structure similar to that found in 4E. To the best of our knowledge, the pentahydrate isomer 5Γ has not been reported in the literature, likely because pentahydrate isomers were previously modeled without considering dynamic effects at 220 K.Open in a separate windowFig. 3.Snapshots of AIMD simulations at different time stages (unit, ps) with initial structures of (A) 5B, (B) 5D, and (C) 5M. White, blue, and red spheres represent hydrogen, nitrogen, and oxygen atoms, respectively. As shown in Fig. 4A, 5C indicates a group of isomers with similar structures. Isomers 5Λ and isomer 5Y are highly populated isomers before the final product 5Γ.As shown by the time evolution of the N–O and O–H distances (Fig. S8), the N–O distance exhibited two sudden decreases, accompanied by sudden increases in the O–H distance. This result suggests that two reaction steps are likely involved in HONO formation in pentahydrate clusters. As shown in Fig. 3A, at ∼13.8 ps, 5B evolves into a group of four intermediate isomers (named isomers 5C-i to 5C-iv) whose structures can all be viewed as derivative from the tetrahydrate isomer 4C (with the addition of one water molecule to different sites of 4C). The added water molecule can move around, thus leading to interconversion of the four isomers as shown in Fig. 4A. The isomer 5C can evolve to a chain-like structure, named 5Λ (e.g., at ∼97.16 ps in Fig. 3A), corresponding to a sudden change in the N–O and O–H distances (Movie S3A). Detailed population analysis of isomers before the formation of HONO (<207.56 ps) indicates high population of both isomer groups 5C (38.63%) and 5Λ (53.19%) (Fig. 4B). Such a population distribution is akin to that for tetrahydrates where the chain-like structure 4E entails the highest population, followed by the isomer 4C (Fig. 2 B and D). The addition of one water molecule can effectively promote the movement of one tail water molecule toward the other end of the chain structure. Meanwhile, proton transfer is observed between the two neighboring water molecules, as shown in Fig. 3A, generating the final product 5Γ (Movie S3B), while the water molecules persist in a chain-like structure similar to that of 4E.Open in a separate windowFig. 4.(A) Schematic illustration of the relocation of one water molecule (with green sphere) around the 4C structure in four different sites, resulting in a group of four different isomers (5C-i to 5C-iv). (B and C) Population of major intermediate isomer observed before the formation of HONO in the AIMD simulation, starting with isomer 5B and 5D, respectively. The isomer structures of 5σ, 5Σ, and 5M′ are illustrated in Fig. S9.Open in a separate windowFig. S8.Time evolution of N–O (black line) and O–H (red line) distance during the AIMD simulation with isomer 5B (Top), 5D (Middle),and 5M (Bottom) as the initial structure.Open in a separate windowFig. S9.Geometry structures for the isomer 5σ, 5Σ, and 5M′ of pentahydrates.In the AIMD simulation starting with the 5D isomer, isomer 5C-i has the highest population before the formation of HONO (<22. 71 ps, Figs. 3B and ​and4C).4C). Here, 5C-i evolves to a three-armed, star-like structure (named 5Y) with the H₃O⁺ at the center, while the N–O distance shortened to ∼1.60 Å and the O–H distance increased to 1.51 Å; The isomer 5Y entails HONO formation (Movie S3C). The HONO-containing 5Y lasts >130 ps during the AIMD simulations, and at ∼155.99 ps the water molecule next to HONO approaches the chain-like structure, further increasing the O–H distance (Fig. S8), and then forms the final product 5Γ (Movie S3D). In the AIMD simulation starting with isomer 5M, which entails a planar cyclic structure, the HONO formation proceeds with a similar path (Fig. 3C) as 5D (Movies S3E and S3F). The same reaction is also observed when a much shorter AIMD time step (0.1 fs) was used in an independent AIMD simulation (Movie S3G). Notably, it converts to the HONO-containing isomer 5Y within 5 ps. Such fast conversion is probably attributed to the initial cyclic water structure which is also observed in the other two cases (Fig. 3). Again, 5Y changes to 5Γ after a relatively longer period of AIMD run.In conclusion, we have shown that the tetrahydrate and pentahydrate structures located at the global minima of potential-energy surface cannot be converted directly to HONO species at the 220-K ionospheric temperature. To achieve HONO formation, the lowest-lying isomers of tetrahydrates must first be converted to the highly populated critical isomer 4E in a dynamic fashion at 220 K. Subsequently, the critical isomer 4E can be converted to the HONO-containing product with encountering very low barriers at 220 K, consistent with previous experiment (11, 17). We also confirmed another experimental finding (18) that the 3γ trihydrate cluster is a highly stable nonreactive cluster, even at 220 K (Fig. 1). However, the addition of one water molecule to 3γ can directly lead to the critical 4E isomer. Thus, the chemical equilibrium between 4E and the product 4ii coupled with the thermodynamically favorable conversion process from the three lowest-lying isomers at 0 K—4A, 4B, and 4C—to the 4E isomer at 220 K represents an important dynamic channel for HONO formation in the ionosphere.Another dynamic channel for HONO formation involves pentahydrate isomers. Upon the addition of one extra water molecule, the formation of HONO can be significantly much faster (11), for example, via a pathway similar to that proposed in the tetrahydrates, namely, via the isomer 5C which contains the motif 4C, followed by the formation of a chain-like water structure akin to 4E and by the bending of the chain to form the product 5Γ. In comparison with the tetrahydrates, the extra water molecule promotes the movement of water molecules, thus leading to the much faster formation of HONO in AIMD simulations. Another possible channel for the formation of HONO could be through the three-armed, star-like precursor isomer 5Y, followed by the combination of a single water molecule with the chain-like water structure to form the product 5Γ. The chemical equilibrium between highly populated 5Λ or 5Y and 5Γ corresponds to the second dynamic channel for HONO formation in the ionosphere. The discovery of these two dynamic channels brings previously unidentified insights into the HONO formation in the 200–220-K temperature range, a key reaction in the D layer of the ionosphere (17).     

Exogenous wild-type p53 gene improved survival of nude mice injected with murine erythroleukemia cell line through amelioration of hemorheological changes

OBJECTIVES: Previous investigations have shown that human wild-type p53 gene (WTp53) inhibits the growth of leukemia and tumor cells in vitro. In the present study, the authors used nude mice and examined the therapeutic role of p53 gene for erythroleukemia in vivo in the absence of MHC effects. METHODS: The nude mice were injected with murine erythroleukemia cells (MEL), MEL cells transfected with wild-type p53 gene (MEL-W), and MEL cells transfected with mutated p53 gene (MEL-M). Abnormalities were found in the hemorheological and biophysical properties of red blood cells in all 3 groups of animals, but the abnormalities were lesser in degree and later in appearance in MEL-W group than in MEL and MEL-M groups. Furthermore, the nude mice in MEL-W group lived longer than those in MEL and MEL-M groups. RESULTS: The results showed that WTp53 restrained the growth of erythroleukemia cells in vivo and reduced the erythroleukemia tumorigenesis in the microcirculation by improving the hemorheological and biophysical properties of MEL cells, which helped to prolong the life span of nude mice suffering from erythroleukemia. CONCLUSION: These results contribute to our knowledge on the use of wild-type p53 gene for the treatment of erythroleukemia disease.     

奋斗呐对白纹伊蚊血淋巴元素与氨基酸含量的影响

目的 :对奋斗呐灭蚊作用机理进行探讨。方法 :以 3种不同浓度 ( 2 .79,1.39与 0 .70μmol/ L )奋斗呐 (顺式氯氰菊酯 )处理白纹伊蚊 4龄幼虫 ,并收集初羽化雌蚊血淋巴 ,分别检测奋斗呐组和对照组的蚊血淋巴元素和氨基酸含量。结果 :比较 3种不同浓度用药组与对照组 ( 0 .2 %丙酮 )蚊血淋巴中 11种元素的含量。表明 2 .79μmol/ L浓度用药组与对照组相比 ,除锌、铬、钙外 ,其他 8种元素含量均显著增高 ;而 1.39μmol/ L和 0 .70μmol/ L组与对照组相比 ,铁、锰等 6- 7种元素含量较对照组高。比较 1.39μmol/ L和 0 .70 μmol/ L奋斗呐组与对照组 (去氯水 )蚊血淋巴中 15种氨基酸含量 ,2个用药组中的 12种氨基酸含量均降低。结论 :奋斗呐对白纹伊蚊成蚊血淋巴中的元素和氨基酸的含量均有一定影响。     

MSCs derived from iPSCs with a modified protocol are tumor-tropic but have much less potential to promote tumors than bone marrow MSCs

Mesenchymal stem or stromal cells (MSCs) have many potential therapeutic applications including therapies for cancers and tissue damages caused by cancers or radical cancer treatments. However, tissue-derived MSCs such as bone marrow MSCs (BM-MSCs) may promote cancer progression and have considerable donor variations and limited expandability. These issues hinder the potential applications of MSCs, especially those in cancer patients. To circumvent these issues, we derived MSCs from transgene-free human induced pluripotent stem cells (iPSCs) efficiently with a modified protocol that eliminated the need of flow cytometric sorting. Our iPSC-derived MSCs were readily expandable, but still underwent senescence after prolonged culture and did not form teratomas. These iPSC-derived MSCs homed to cancers with efficiencies similar to BM-MSCs but were much less prone than BM-MSCs to promote the epithelial–mesenchymal transition, invasion, stemness, and growth of cancer cells. The observations were probably explained by the much lower expression of receptors for interleukin-1 and TGFβ, downstream protumor factors, and hyaluronan and its cofactor TSG6, which all contribute to the protumor effects of BM-MSCs. The data suggest that iPSC-derived MSCs prepared with the modified protocol are a safer and better alternative to BM-MSCs for therapeutic applications in cancer patients. The protocol is scalable and can be used to prepare the large number of cells required for “off-the-shelf” therapies and bioengineering applications.The use of mesenchymal stromal or stem cells (MSCs) in cancer patients or cancer survivors is a promising strategy to improve treatment of advanced cancer (1) and to repair tissues damaged by cancers or by radical cancer therapies (2). Based on the unique homing capability of tissue-derived MSCs to stroma of various primary and metastatic cancers (3–6), MSCs have the potential to treat or even eliminate various cancers by delivering various anticancer agents (7–9). Because of their potential for differentiation (10, 11) and production of immunomodulatory, angiogenic, anti-apoptotic, anti-scarring, and prosurvival factors (12), MSCs have shown promising regeneration potential after radical cancer treatment in animal models, such as soft tissue reconstruction after disfiguring surgeries for head, neck, or breast cancers (13) and salivary gland regeneration for head and neck cancer patients treated with radiotherapy (14, 15). As one example, the combination of osteogenic potential and targeted delivery of anticancer agents make MSCs a promising option to treat tumor-induced osteolysis (16, 17). However, exogenous tissue-derived MSCs, including those from bone marrow, adipose tissues, and umbilical cord, have all shown a tendency to promote rather than inhibit cancers in many circumstances (18–23). Also, endogenous MSCs are a major source of reactive stromal cells that promote growth and metastasis of cancers (4, 24).Moreover, MSCs have a limited proliferation potential and lose some of their important biological functions as they are expanded (25). Therefore, it is difficult to prepare large banks of the cells with uniform biological activities and/or transgene expression required for experiments in large animals and for potential clinical therapies. Another problem is that MSCs are being prepared with a variety of protocols in different laboratories from different donors. As a result, standardization of the cells has been extremely difficult and the data presented in different publications are difficult to compare. Hence large banks of reference cells are needed to advance the MSC research (26).To address the limitations of expandability and standardization, we derived MSCs from induced pluripotent stem cells (iPSCs) with a modified protocol that can be expanded to provide large cell banks from a single cell clone. The protocol produces highly enriched MSC-like cells from iPSCs with high efficiency. The iPSC-derived MSCs (iPSC-MSCs) express the classical surface markers of MSCs, are capable of multilineage mesodermal differentiation and cancer homing, and can be expanded extensively, but do not preserve the pluoripotency of iPSCs. Surprisingly, iPSC-MSCs do not promote epithelial–mesenchymal transition (EMT), invasion, and stemness of cancer cells as is seen with bone marrow-derived MSCs (BM-MSCs). Consistent with these observations, the iPSC-MSCs express much lower levels than BM-MSCs of protumor factors including interleukine-6, prostaglandin E2, SDF1, and hyaluronan before and after exposure to tumor microenvironment. Our data indicated that iPSC-MSCs are a safe alternative to BM-MSCs for cancer therapy and other applications with better expandability and potential for genetic engineering.     

Cardiovascular disease risk factor levels and their relations to CVD rates in China--results of Sino-MONICA project.

BACKGROUND: The Sino-MONICA project is a 7-year study monitoring trends and determinants of cardiovascular disease (CVD) in geographically defined populations in different parts of China. This report focuses on risk factor levels and their relations to CVD rates. DESIGN: Successive surveys on smoking habits, blood pressure (BP), serum total cholesterol (TC), weight and height were conducted in independent random samples of the same populations early (1987-1988) and late (1992-1993) in the study period, by the methodology and criteria of the WHO MONICA project. Associations between risk levels and CVD rates were also assessed by correlation analysis. RESULTS: In general, the mean level of BP in the populations studied was high by international standards. Serum TC and body mass index (BMI) were low compared with the world average. There were significant geographic variations in CVD risk levels, being higher in the north and lower in the south, which correlated with the north-south difference of CVD event rates. CONCLUSIONS: The Sino-MONICA study has established the feasibility of long-term monitoring of CVD events and risk factors with international standardized methods in Chinese communities. The results will have significance in curbing the CVD epidemic not only in China, but also internationally.     

YMDD variants of HBV DNA polymerase gene： Rapid detection and clinicopathological analysis with long-term lamivudine therapy after liver transplantation

AIM: To look for a rapid low-cost technique for the detection of HBV variants. METHODS: Two patients who underwent orthotopic liver transplantation (OLT) for HBV infection were treated with lamivudine (100 mg daily) and HBV infection recurred in the grafted livers. The patients were monitored intensively for liver enzymes, hepatitis B surface antigen (HBsAg) and HBV DNA in serum. Liver biopsy was performed regularly. HBV DNA in a conserved polymerase domain (the YMDD locus) was amplified from serum of each patient by PCR and sequenced. HBV genotypes were analyzed by restriction fragment length polymorphism (RFLP) of the PCR products generated from a fragment of the polymerase gene. RESULTS: YMDD wild-type HBV was detected in one patient by PCR-RFLP and DNA sequencing 19 mo after OLT, and YIDD mutant-type HBV in the other patient, 16 mo after OLT. CONCLUSION: PCR-RFLP assay is an accurate and simple method for genotyping lamivudine-resistant HBV variants.