Protein kinase C delta null mice exhibit structural alterations in articular surface,intra-articular and subchondral compartments

IntroductionStructural alterations in intra-articular and subchondral compartments are hallmarks of osteoarthritis, a degenerative disease that causes pain and disability in the aging population. Protein kinase C delta (PKC-δ) plays versatile functions in cell growth and differentiation, but its role in the articular cartilage and subchondral bone is not known.MethodsHistological analysis including alcian blue, safranin O staining and fluorochrome labeling were used to reveal structural alterations at the articular cartilage surface and bone–cartilage interface in PKC-δ knockout (KO) mice. The morphology and organization of chondrocytes were studied using confocal microscopy. Glycosaminoglycan content was studied by micromass culture of chondrocytes of PKC-δ KO mice.ResultsWe uncovered atypical structural demarcation between articular cartilage and subchondral bone of PKC-δ KO mice. Histology analyses revealed a thickening of the articular cartilage and calcified bone–cartilage interface, and decreased safranin O staining accompanied by an increase in the number of hypertrophic chondrocytes in the articular cartilage of PKC-δ KO mice. Interestingly, loss of demarcation between articular cartilage and bone was concomitant with irregular chondrocyte morphology and arrangement. Consistently, in vivo calcein labeling assay showed an increased intensity of calcein labeling in the interface of the growth plate and metaphysis in PKC-δ KO mice. Furthermore, in vitro culture of chondrocyte micromass showed a decreased alcian blue staining of chondrocyte micromass in the PKC-δ KO mice, indicative of a reduced level of glycosaminoglycan production.ConclusionsOur data imply a role for PKC-δ in the osteochondral plasticity of the interface between articular cartilage and the osteochondral junction.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0720-4) contains supplementary material, which is available to authorized users.     

Optimizing clinical dosing of combination broadly neutralizing antibodies for HIV prevention

Broadly neutralizing antibodies (bNAbs) are promising agents to prevent HIV infection and achieve HIV remission without antiretroviral therapy (ART). As with ART, bNAb combinations are likely needed to cover HIV’s extensive diversity. Not all bNAbs are identical in terms of their breadth, potency, and in vivo longevity (half-life). Given these differences, it is important to optimally select the composition, or dose ratio, of combination bNAb therapies for future clinical studies. We developed a model that synthesizes 1) pharmacokinetics, 2) potency against a wide HIV diversity, 3) interaction models for how drugs work together, and 4) correlates that translate in vitro potency to clinical protection. We found optimization requires drug-specific balances between potency, longevity, and interaction type. As an example, tradeoffs between longevity and potency are shown by comparing a combination therapy to a bi-specific antibody (a single protein merging both bNAbs) that takes the better potency but the worse longevity of the two components. Then, we illustrate a realistic dose ratio optimization of a triple combination of VRC07, 3BNC117, and 10–1074 bNAbs. We apply protection estimates derived from both a non-human primate (NHP) challenge study meta-analysis and the human antibody mediated prevention (AMP) trials. In both cases, we find a 2:1:1 dose emphasizing VRC07 is nearly optimal. Our approach can be immediately applied to optimize the next generation of combination antibody prevention and cure studies.     

Grassland birds and rotational-grazing in the northeast: Breeding ecology,survival and management opportunities

We studied long-term (2002–2009) demographics of bobolinks (Dolichonyx oryzivorus) and savannah sparrows (Passerculus sandwichensis) breeding in rotationally grazed systems in the northeastern United States. Both nest success (6–44% bobolink; 7–48% savannah sparrow) and annual productivity (0.43–2.83 bobolink; 0.70–2.35 savannah sparrow) varied annually. Predation (48%) and trampling by cows (32%) accounted for most failed nests. Annual adult apparent survival ranged from 23% to 85% and showed substantial variation by species and sex. Although grazing resulted in substantial loss of vegetative cover, savannah sparrows responded similarly to predation and grazing-induced nest failure (time to renest, distance moved between nests) and generally remained in the same paddock to renest. Minimum paddock size to accommodate one female within this rotation was 70 m × 70 m (0.49 ha), allowing a female to stay within a paddock and move (approx. 50 m) between nesting attempts. To balance pasturing objectives and birds' needs in paddocks first grazed before 25 June, managers in the Northeast should rest that paddock 42–50 days before the second grazing. © 2011 The Wildlife Society.     

Expression of the human atypical kinase ADCK3 rescues coenzyme Q biosynthesis and phosphorylation of Coq polypeptides in yeast coq8 mutants

Coenzyme Q (ubiquinone or Q) is a lipid electron and proton carrier in the electron transport chain. In yeast Saccharomyces cerevisiae eleven genes, designated COQ1 through COQ9, YAH1 and ARH1, have been identified as being required for Q biosynthesis. One of these genes, COQ8 (ABC1), encodes an atypical protein kinase, containing six (I, II, III, VIB, VII, and VIII) of the twelve motifs characteristically present in canonical protein kinases. Here we characterize seven distinct Q-less coq8 yeast mutants and show that unlike the coq8 null mutant, each maintained normal steady-state levels of the Coq8 polypeptide. The phosphorylation states of Coq polypeptides were determined with two-dimensional gel analyses. Coq3p, Coq5p, and Coq7p were phosphorylated in a Coq8p-dependent manner. Expression of a human homolog of Coq8p, ADCK3(CABC1) bearing an amino-terminal yeast mitochondrial leader sequence, rescued growth of yeast coq8 mutants on medium containing a nonfermentable carbon source and partially restored biosynthesis of Q(6). The phosphorylation state of several of the yeast Coq polypeptides was also rescued, indicating a profound conservation of yeast Coq8p and human ADCK3 protein kinase function in Q biosynthesis.     

Expression of recombinant Pseudomonas stutzeri di-heme cytochrome c4 by high-cell-density fed-batch cultivation of Pseudomonas putida

The gene of the di-heme protein cytochrome c(4) from Pseudomonas stutzeri was expressed in Pseudomonas putida. High-yield expression of the protein was achieved by high-cell-density fed-batch cultivation using an exponential glucose feeding strategy. The recombinant cytochrome c(4) protein was purified to apparent homogeneity and analyzed by electronic absorption spectroscopy, nanoflow electrospray ionization time-of-flight mass spectrometry, and electrochemistry. Cyclic voltammograms and UV-vis electronic absorption spectra were indistinguishable from the equivalent data of native P. stutzeri cytochrome c(4). Furthermore, the calculated and experimentally determined molecular masses of recombinant cytochrome c(4) were identical. Biochemical characterization of both wild-type and mutant derivatives of the protein will be greatly enhanced and facilitated by the described high-yield fermentation and rapid isolation procedure.     

Molecular identification of bacteria in tracheal aspirate fluid from mechanically ventilated preterm infants

Background

Despite strong evidence linking infections to the pathogenesis of bronchopulmonary dysplasia (BPD), limitations of bacterial culture methods have precluded systematic studies of airway organisms relative to disease outcomes. Application of molecular bacterial identification strategies may provide new insight into the role of bacterial acquisition in the airways of preterm infants at risk for BPD.

Methods

Serial (within 72 hours, 7, 14, and 21 days of life) tracheal aspirate samples were collected from 10 preterm infants with gestational age ≤34 weeks at birth, and birth weight of 500–1250 g who required mechanical ventilation for at least 21 days. Samples were analyzed by quantitative real time PCR assays for total bacterial load and by pyrosequencing for bacterial identification.

Results

Subjects were diagnosed with mild (1), moderate (3), or severe (5) BPD. One patient died prior to determination of disease severity. 107,487 sequences were analyzed, with mean of 3,359 (range 1,724–4,915) per sample. 2 of 10 samples collected <72 hours of life contained adequate bacterial DNA for successful sequence analysis, one of which was from a subject exposed to chorioamnionitis. All other samples exhibited bacterial loads >70copies/reaction. 72 organisms were observed in total. Seven organisms represented the dominant organism (>50% of total sequences) in 31/32 samples with positive sequences. A dominant organism represented>90% of total sequences in 13 samples. Staphylococcus, Ureaplasmaparvum, and Ureaplasmaurealyticum were the most frequently identified dominant organisms, but Pseudomonas, Enterococcus, and Escherichia were also identified.

Conclusions

Early bacterial colonization with diverse species occursafter the first 3 days of life in the airways of intubated preterm infants, and can be characterized by bacterial load and marked species diversity. Molecular identification of bacteria in the lower airways of preterm infants has the potential to yield further insight into the pathogenesis of BPD.     

Expression of the Cameleon calcium biosensor in fungi reveals distinct Ca2+ signatures associated with polarized growth,development, and pathogenesis

Calcium is a universal messenger that translates diverse environmental stimuli and developmental cues into specific cellular and developmental responses. While individual fungal species have evolved complex and often unique biochemical and structural mechanisms to exploit specific ecological niches and to adjust growth and development in response to external stimuli, one universal feature to all is that Ca²⁺-mediated signaling is involved. The lack of a robust method for imaging spatial and temporal dynamics of subcellular Ca²⁺ (i.e., “Ca²⁺ signature”), readily available in the plant and animal systems, has severely limited studies on how this signaling pathway controls fungal growth, development, and pathogenesis. Here, we report the first successful expression of a FRET (Förster Resonance Energy Transfer)-based Ca²⁺ biosensor in fungi. Time-lapse imaging of Magnaporthe oryzae, Fusarium oxysporum, and Fusarium graminearum expressing this sensor showed that instead of a continuous gradient, the cytoplasmic Ca²⁺ ([Ca²⁺]_c) change occurred in a pulsatile manner with no discernable gradient between pulses, and each species exhibited a distinct Ca²⁺ signature. Furthermore, occurrence of pulsatile Ca²⁺ signatures was age and development dependent, and major [Ca²⁺]_c transients were observed during hyphal branching, septum formation, differentiation into specialized plant infection structures, cell–cell contact and in planta growth. In combination with the sequenced genomes and ease of targeted gene manipulation of these and many other fungal species, the data, materials and methods developed here will help understand the mechanism underpinning Ca²⁺-mediated control of cellular and developmental changes, its role in polarized growth forms and the evolution of Ca²⁺ signaling across eukaryotic kingdoms.