Spatial structure and genetic diversity of three tropical tree species with different habitat preferences within a natural forest

Analyses of the spatial distribution pattern, spatial genetic structure and genetic diversity were carried out using a 33-ha plot in a hill dipterocarp forest for three dipterocarps with different habitat preferences, i.e. Shorea curtisii on the ridges, Shorea leprosula in the valleys and Shorea macroptera both on the ridges and in the valleys. The significant spatial aggregation in small-diameter trees of all the three species was explained by limited seed dispersal. At the large-diameter trees, only S. macroptera showed random distribution and this might further prove that S. macroptera is habitat generalist, whilst S. curtisii and S. leprosula are habitat specific. The levels of genetic diversity estimated based on five microsatellite loci were high and comparable in all the three studied species. As the three studied species reproduced mainly through outcrossing, the observed high levels of genetic diversity might support the fact that the plant mating system can be used as guideline to infer the levels of genetic diversity, regardless of whether the species is habitat specific or habitat generalist. The lack of spatial genetic structure but significant aggregation in the small-diameter trees of all the three species might indicate limited seed dispersal but extensive pollen flow. Hence, if seed dispersal is restricted but pollen flow is extensive, significant spatial aggregation but no spatial genetic structure will be observed at the small-diameter trees, regardless of whether the species is habitat specific or habitat generalist. The inferred extensive pollen flow might indicate that energetic pollinators are involved in the pollination of Shorea species in the hill dipterocarp forests.     

Cloning of the rainbow trout (Oncorhynchus mykiss) Mx2 and Mx3 cDNAs and characterization of trout Mx protein expression in salmon cells.

Two rainbow trout (Oncorhynchus mykiss) Mx cDNAs were cloned by using RACE (rapid amplification of cDNA ends) PCR and were designated RBTMx2 and RBTMx3. The deduced RBTMx2 and RBTMx3 proteins were 636 and 623 amino acids in length with molecular masses of 72 and 70.8 kDa, respectively. These proteins, along with the previously described RBTMx1 protein (G. D. Trobridge and J. A. Leong, J. Interferon Cytokine Res. 15:691-702, 1995), have between 88.7 and 96.6% identity at the amino acid level. All three proteins contain the tripartite GTP binding domain and leucine zipper motif common to Mx proteins. A monospecific polyclonal antiserum to an Escherichia coli-expressed fragment of RBTMx3 was generated, and that reagent was found to react with all three rainbow trout Mx proteins. Subsequently, endogenous Mx production in RTG-2 cells induced with poly(IC) double-stranded RNA was detected by immunoblot analysis. The cellular localization of the rainbow trout proteins was determined by transient expression of the RBTMx cDNAs in CHSE-214 (chinook salmon embryo) cells. A single-cell transient-transfection assay was used to examine the ability of each Mx cDNA clone to inhibit replication of the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV). No significant inhibition in the accumulation of the IHNV nucleoprotein was observed in cells expressing either trout Mx1, Mx2, or Mx3 in transiently transfected cells.     

Prediction of non-linear pharmacokinetics in humans of an antibody-drug conjugate (ADC) when evaluation of higher doses in animals is limited by tolerability: Case study with an anti-CD33 ADC

For antibody-drug conjugates (ADCs) that carry a cytotoxic drug, doses that can be administered in preclinical studies are typically limited by tolerability, leading to a narrow dose range that can be tested. For molecules with non-linear pharmacokinetics (PK), this limited dose range may be insufficient to fully characterize the PK of the ADC and limits translation to humans. Mathematical PK models are frequently used for molecule selection during preclinical drug development and for translational predictions to guide clinical study design. Here, we present a practical approach that uses limited PK and receptor occupancy (RO) data of the corresponding unconjugated antibody to predict ADC PK when conjugation does not alter the non-specific clearance or the antibody-target interaction. We used a 2-compartment model incorporating non-specific and specific (target mediated) clearances, where the latter is a function of RO, to describe the PK of anti-CD33 ADC with dose-limiting neutropenia in cynomolgus monkeys. We tested our model by comparing PK predictions based on the unconjugated antibody to observed ADC PK data that was not utilized for model development. Prospective prediction of human PK was performed by incorporating in vitro binding affinity differences between species for varying levels of CD33 target expression. Additionally, this approach was used to predict human PK of other previously tested anti-CD33 molecules with published clinical data. The findings showed that, for a cytotoxic ADC with non-linear PK and limited preclinical PK data, incorporating RO in the PK model and using data from the corresponding unconjugated antibody at higher doses allowed the identification of parameters to characterize monkey PK and enabled human PK predictions.     

Selfing and inbreeding depression in seeds and seedlings of Neobalanocarpus heimii (Dipterocarpaceae)

We evaluated the degree of selfing and inbreeding depression at the seed and seedling stages of a threatened tropical canopy tree, Neobalanocarpus heimii, using microsatellite markers. Selection resulted in an overall decrease in the level of surviving selfed progeny from seeds to established seedlings, indicating inbreeding depression during seedling establishment. Mean seed mass of selfed progeny was lower than that of outcrossed progeny. Since the smaller seeds suffered a fitness disadvantage at germination in N. heimii, the reduced seed mass of selfed progeny would be one of the determinants of the observed inbreeding depression during seedling establishment. High selfing rates in some mother trees could be attributed to low local densities of reproductive individuals, thus maintenance of a sufficiently high density of mature N. heimii should facilitate regeneration and conservation of the species.     

Coupling spatial segregation with synthetic circuits to control bacterial survival

Engineered bacteria have great potential for medical and environmental applications. Fulfilling this potential requires controllability over engineered behaviors and scalability of the engineered systems. Here, we present a platform technology, microbial swarmbot, which employs spatial arrangement to control the growth dynamics of engineered bacteria. As a proof of principle, we demonstrated a safeguard strategy to prevent unintended bacterial proliferation. In particular, we adopted several synthetic gene circuits to program collective survival in Escherichia coli: the engineered bacteria could only survive when present at sufficiently high population densities. When encapsulated by permeable membranes, these bacteria can sense the local environment and respond accordingly. The cells inside the microbial swarmbot capsules will survive due to their high densities. Those escaping from a capsule, however, will be killed due to a decrease in their densities. We demonstrate that this design concept is modular and readily generalizable. Our work lays the foundation for engineering integrated and programmable control of hybrid biological–material systems for diverse applications.     

A neutralizing monoclonal antibody specific for the dimer interface region of IL-8

We have generated two mAbs, 6G4.2.5 and A5.12.14, that are similarly capable of neutralizing the biologic activity of wild-type IL-8. To characterize these antibodies further, their reactivity against a series of engineered IL-8 monomer and dimer variants was examined using a neutrophil degranulation assay. While 6G4.2.5 was found to block effectively the biologic activity of all variants regardless of their dimerization status, the results for A5.12.14 differed dramatically. A5.12.14 fully inhibited the agonist activity of one of the monomer variants, partially blocked the activity of another, and had no effect on the activity of two other variants. These results suggested that the binding epitope of A5.12.14 was being affected by the particular amino acid substitutions introduced into the dimer interface region of the variants to disfavor dimerization. If A5.12.14 indeed binds to the dimer interface region of IL-8, it could be predicted that this mAb would be unable to inhibit the activity of dimeric IL-8. This was confirmed in studies which showed that A5.12.14 had no demonstrable effect on the activity of a constitutively dimeric IL-8 variant. These studies represent the first example of a mAb specific for the dimerization status of IL-8.     

The sleeper Bostrichthys sinensis (family Eleotridae) stores glutamine and reduces ammonia production during aerial exposure

Bostrichthys sinensis inhabits brackish water, living in the crevices of the river mouths of Shang Xi and Guangdong, China. In its natural habitat, it may encounter aerial exposure frequently during low tides, and it usually remains quiescent in the absence of water. Upon aerial exposure in the laboratory, the ammonia excretion rate decreased to one-fourth that of the submerged control. Although all the enzymes of the ornithine-urea cycle were detected in the liver of this fish, the activity of hepatic carbamoyl phosphate synthetase was too low for the cycle to be functioning. Indeed, ammonia accumulated in the tissues and was not converted to urea. Results indicate that ammonia produced through amino acid catabolism was detoxified to glutamine during the first 24 h of aerial exposure. The excess amount of glutamine stored in the muscle during this period couldaccount approximately for the reduction in ammonia equivalent excreted. There was indeed a significant increase in the activity of glutamine synthetase from the liver of specimens exposed to terrestrial conditions. In contrast to the production of alanine, formation of glutamine is energetically expensive. Since B. sinensis remained relatively inactive on land, the reduction in energy demand for muscular activity might provide it with the opportunity to exploit glutamine formation as a means to detoxify ammonia. After 72 h of aerial exposure, B. sinensis reduced internal ammonia production, possibly through reductions in proteolysis and amino acid catabolism, to avoid excessive accumulation of ammonia.     

Surface Antigens of Smooth Brucellae

Surface antigens of smooth brucellae were extracted by ether-water, phenol-water, trichloroacetic acid, and saline and examined by immunoelectrophoresis and gel diffusion with antisera from infected and immunized rabbits. Ether-water extracts of Brucella melitensis contained a lipopolysaccharide protein component, which was specific for the surface of smooth brucellae and was correlated with the M agglutinogen of Wilson and Miles, a polysaccharide protein component devoid of lipid which was not restricted to the surface of smooth brucellae and was not correlated with the smooth agglutinogen (component 1), and several protein components which were associated with internal antigens of rough and smooth brucellae. Immunoelectrophoretic analysis of ether-water extracts of B. abortus revealed only two components, a lipopolysaccharide protein component, which was correlated with the A agglutinogen, and component 1. Component 1 from B. melitensis and B. abortus showed identity in gel diffusion tests, whereas component M from B. melitensis and component A from B. abortus showed partial identity with unabsorbed antisera and no cross-reactions with monospecific sera. Attempts to prepare monospecific sera directly by immunization of rabbits with cell walls or ether-water extracts were unsuccessful. Absorption of antisera with heavy fraction of ether-water extracts did not always result in monospecific sera. It was concluded (as has been described before) that the A and M antigens are present on a single antigenic complex, in different proportions depending upon the species and biotype, and that this component is a lipopolysaccharide protein complex of high molecular weight that diffuses poorly through agar gel. Components 1, A, and M were also demonstrated in trichloroacetic acid and phenol-water extracts. With all extracts, B. melitensis antigen showed greater diffusibility in agar than B. abortus antigens. After mild acid hydrolysis, B. abortus ether-water extract was able to diffuse more readily.     

The Notch pathway in prostate development and cancer

Abstract The Notch family of transmembrane receptors are important mediators of cell fate determination. Accordingly, Notch signaling is intimately involved in the development of numerous tissues. Recent findings have highlighted a critical role for Notch signaling in normal prostate development. Notch signaling is required for embryonic and postnatal prostatic growth and development, for proper cell lineage specification within the prostate, as well as for adult prostate maintenance and regeneration following castration and hormone replacement. Evidence for Notch as a regulator of prostate cancer development, progression, and metastasis has also emerged. This review summarizes our current understanding of the role of Notch pathway elements, including members of the Jagged, Delta-like, hairy/enhancer-of-split, and hairy/enhancer-of-split related with YRPW motif families, in prostate development and tumorigenesis. Data supporting Notch pathway elements as oncogenes and tumor suppressors in prostate tumors, as well as data implicating Notch receptors and ligands as potential markers of normal prostate stem/progenitor cells and prostate cancer stem/initiating cells, are also presented.