Polymorphic microsatellite loci for studies of the Ozark hellbender (Cryptobranchus alleganiensis bishopi)

The hellbender is the only North American member of the aquatic salamander family Cryptobranchidae and is a species of conservation concern across its range. We developed eight polymorphic microsatellite loci for hellbenders using a magnetic bead enrichment protocol and a PCR-based detection technique. Allelic diversity averaged 4.0 (±1.8 SD) per locus and heterozygosity averaged 0.56 (±0.30 SD). The hellbender is rare and difficult to study due to its cryptic life history. These loci will provide a valuable resource for population studies, which could inform future conservation and management decisions.     

Genetic relationships of hellbenders in the Ozark highlands of Missouri and conservation implications for the Ozark subspecies (<Emphasis Type="Italic">Cryptobranchus alleganiensis bishopi</Emphasis>)

The hellbender (Cryptobranchus alleganiensis) is an obligately aquatic salamander that is in decline due to habitat loss and disease. Two subspecies of hellbender have been described based on morphological characteristics: C. a. alleganiensis (eastern subspecies) and C. a. bishopi (Ozark hellbender). Current conservation strategies include captive propagation for restorative releases even though information regarding the current levels of genetic variability and structure within populations is not sufficient to effectively plan for conservation of the genetic diversity of the species. To investigate patterns of population structure in the hellbender, we genotyped 276 hellbenders from eight Missouri River drainages, representing both subspecies. Our results showed low levels of within-drainage diversity but strong population structure among rivers, and three distinct genetic clusters. F _ST values ranged from 0.00 to 0.61 and averaged 0.40. Our results confirmed previous reports that C. a. bishopi and C. a. alleganiensis are genetically distinct, but also revealed an equidistant relationship between two groups within C. a. bishopi and all populations of C. a. alleganiensis. Current subspecies delineations do not accurately incorporate genetic structure, and for conservation purposes, these three groups should be considered evolutionarily significant units.     

Rapid high efficiency sensitization of CD8+ T cells to tumor antigens by dendritic cells leads to enhanced functional avidity and direct tumor recognition through an IL-12-dependent mechanism

Myeloid-origin dendritic cells (DCs) can develop into IL-12-secreting DC1 or non-IL-12-secreting DC2 depending on signals received during maturation. Through rapid culture techniques that prepared either mature, CD83+ DC1 or DC2 from CD14+ monocytes in only 2 days followed by a single 6-7 day DC-T cell coculture, we sensitized normal donor CD8+ T cells to tumor Ags (HER-2/neu, MART-1, and gp100) such that peptide Ag-specific lymphocytes constituted up to 16% of the total CD8+ population. Both DC1 and DC2 could sensitize CD8+ T cells that recognized peptide-pulsed target cells. However, with DC2, a general decoupling was observed between recognition of peptide-pulsed T2 target cells and recognition of Ag-expressing tumor cells, with peptide-sensitized T cells responding to tumor only about 15% of the time. In contrast, direct recognition of tumor by T cells was dramatically increased (to 85%) when DC1 were used for sensitization. Enhanced tumor recognition was accompanied by 10- to 100-fold increases in peptide sensitivity and elevated expression of CD8beta, characteristic of high functional avidity T cells. Both of these properties were IL-12-dependent. These results demonstrate the utility of rapid DC culture methods for high efficiency in vitro T cell sensitization that achieves robust priming and expansion of Ag-specific populations in 6 days. They also demonstrate a novel function of IL-12, which is enhancement of CD8+ T cell functional avidity. A new approach to DC-based vaccines that emphasizes IL-12 secretion to enhance functional avidity and concomitant tumor recognition by CD8+ T cells is indicated.     

Isolation and characterization of polymorphic microsatellite loci in the raccoon (Procyon lotor)

We report the isolation and characterization of 17 polymorphic microsatellite loci in the North American raccoon (Procyon lotor). These loci exhibit high levels of allelic diversity, with between four and 13 alleles per locus, and heterozygosity, with observed values of 0.500-1.000 in a sample of 20 individuals. All genotypes conformed to Hardy-Weinberg expectations and there were no instances of linkage disequilibrium detected.     

A comparison of olanzapine and risperidone on the risk of psychiatric hospitalization in the naturalistic treatment of patients with schizophrenia

BACKGROUND: Decreasing hospital admissions is important for improving outcomes for people with schizophrenia and for reducing cost of hospitalization, the largest expenditure in treating this persistent and severe mental illness. This prospective observational study compared olanzapine and risperidone on one-year psychiatric hospitalization rate, duration, and time to hospitalization in the treatment of patients with schizophrenia in usual care. METHODS: We examined data of patients newly initiated on olanzapine (N = 159) or risperidone (N = 112) who continued on the index antipsychotic for at least one year following initiation. Patients were participants in a 3-year prospective, observational study of schizophrenia patients in the US. Outcome measures were percent of hospitalized patients, total days hospitalized per patient, and time to first hospitalization during the one-year post initiation. Analyses employed a generalized linear model with adjustments for demographic and clinical variables. A two-part model was used to confirm the findings. Time to hospitalization was measured by the Kaplan-Meier survival formula. RESULTS: Compared to risperidone, olanzapine-treated patients had significantly lower hospitalization rates, (24.1% vs. 14.4%, respectively, p = 0.040) and significantly fewer hospitalization days (14.5 days vs. 9.9 days, respectively, p = 0.035). The mean difference of 4.6 days translated to $2,502 in annual psychiatric hospitalization cost savings per olanzapine-treated patient, on average. CONCLUSIONS: Consistent with prior clinical trial research, treatment-adherent schizophrenia patients who were treated in usual care with olanzapine had a lower risk of psychiatric hospitalization than risperidone-treated patients. Lower hospitalization costs appear to more than offset the higher medication acquisition cost of olanzapine.     

Engineering of B800 bacteriochlorophyll binding site specificity in the Rhodobacter sphaeroides LH2 antenna

The light-harvesting 2 complex (LH2) of the purple phototrophic bacterium Rhodobacter sphaeroides is a highly efficient, light-harvesting antenna that allows growth under a wide-range of light intensities. In order to expand the spectral range of this antenna complex, we first used a series of competition assays to measure the capacity of the non-native pigments 3-acetyl chlorophyll (Chl) a, Chl?d, Chl?f or bacteriochlorophyll (BChl) b to replace native BChl?a in the B800 binding site of LH2. We then adjusted the B800 site and systematically assessed the binding of non-native pigments. We find that Arg_?10 of the LH2 β polypeptide plays a crucial role in binding specificity, by providing a hydrogen-bond to the 3-acetyl group of native and non-native pigments. Reconstituted LH2 complexes harbouring the series of (B)Chls were examined by transient absorption and steady-state fluorescence spectroscopies. Although slowed 10-fold to ~6?ps, energy transfer from Chl?a to B850 BChl?a remained highly efficient. We measured faster energy-transfer time constants for Chl?d (3.5?ps) and Chl?f (2.7?ps), which have red-shifted absorption maxima compared to Chl?a. BChl?b, red-shifted from the native BChl?a, gave extremely rapid (≤0.1?ps) transfer. These results show that modified LH2 complexes, combined with engineered (B)Chl biosynthesis pathways in vivo, have potential for retaining high efficiency whilst acquiring increased spectral range.     

Consequences of saturation mutagenesis of the protein ligand to the B-side monomeric bacteriochlorophyll in reaction centers from Rhodobacter capsulatus

In bacterial reaction centers (RCs), photon-induced initial charge separation uses an A-side bacteriochlorophyll (BChl, B_A) and bacteriopheophytin (BPh, H_A), while the near-mirror image B-side B_B and H_B cofactors are inactive. Two new sets of Rhodobacter capsulatus RC mutants were designed, both bearing substitution of all amino acids for the native histidine M180 (M-polypeptide residue 180) ligand to the core Mg ion of B_B. Residues are identified that largely result in retention of a BChl in the B_B site (Asp, Ser, Pro, Gln, Asn, Gly, Cys, Lys, and Thr), ones that largely harbor the Mg-free BPh in the B_B site (Leu and Ile), and ones for which isolated RCs are comprised of a substantial mixture of these two RC types (Ala, Glu, Val, Met and, in one set, Arg). No protein was isolated when M180 is Trp, Tyr, Phe, or (in one set) Arg. These findings are corroborated by ground state spectra, pigment extractions, ultrafast transient absorption studies, and the yields of B-side transmembrane charge separation. The changes in coordination chemistries did not reveal an RC with sufficiently precise poising of the redox properties of the B_B-site cofactor to result in a high yield of B-side electron transfer to H_B. Insights are gleaned into the amino acid properties that support BChl in the B_B site and into the widely observed multi-exponential decay of the excited state of the primary electron donor. The results also have direct implications for tuning free energies of the charge-separated intermediates in RCs and mimetic systems.

     

High yield of secondary B-side electron transfer in mutant Rhodobacter capsulatus reaction centers

From the crystal structures of reaction centers (RCs) from purple photosynthetic bacteria, two pathways for electron transfer (ET) are apparent but only one pathway (the A side) operates in the native protein-cofactor complex. Partial activation of the B-side pathway has unveiled the true inefficiencies of ET processes on that side in comparison to analogous reactions on the A side. Of significance are the relative rate constants for forward ET and the competing charge recombination reactions. On the B side, these rate constants are nearly equal for the secondary charge-separation step (ET from bacteriopheophytin to quinone), relegating the yield of this process to < 50%. Herein we report efforts to optimize this step. In surveying all possible residues at position 131 in the M subunit, we discovered that when glutamic acid replaces the native valine the efficiency of the secondary ET is nearly two-fold higher than in the wild-type RC. The positive effect of M131 Glu is likely due to formation of a hydrogen bond with the ring V keto group of the B-side bacteriopheophytin leading to stabilization of the charge-separated state involving this cofactor. This change slows charge recombination by roughly a factor of two and affords the improved yield of the desired forward ET to the B-side quinone terminal acceptor.