Completely serum-free and chemically defined adipocyte development and maintenance

Background aims

In vitro engineered adipose tissue is in great demand to treat lost or damaged soft tissue or to screen for new drugs, among other applications. However, today most attempts depend on the use of animal-derived sera. To pave the way for the application of adipose tissue–engineered products in clinical trials or as reliable and robust in vitro test systems, sera should be completely excluded from the production process. In this study, we aimed to develop an in vitro adipose tissue model in the absence of sera and maintain its function long-term.

Nutritional studies on species and mutants of Lepista,Cantharellus, Pleurotus and Volvariella

The purpose of this study is to determine the ability of select Agaricales species to utilize various sources of carbon, nitrogen, vitamins, and growth hormones. Fungi selected for the studies include:Cantharellus clavatus Fries,C. cibarius Fries,Lepista nuda (Bull. ex. Fries)Cooke,Pleurotus ostreatus (Jacq. ex. Fries)Kummer, andVolvariella volvacea (Bull. ex. Fries)Singer. Three strains ofC. cibarius and one mutant ofV. volvacea (V135), V134, were employed to determine if nutritional requirement differences occurred. One species,V. volvacea, is grown commercially as a cottage industry in the Orient (Alicbusan &Ela, 1961) while the other species currently have no commercial value. All species studied possess pleasing flavors and have potential use in the mushroom production industry.A literature compilation of the nutritional regulation of basidiocarp formation and vegetative growth of Agaricales was made with specific mention to the named species (Volz &Beneke, 1969). Recent nutritional studies with one or more of the specific species include those byYusef &Allam (1967), andEger (1970).     

A preliminary study of keratinophilic fungi from Abaco Island,the Bahamas

The soil of two Bahamian communities on Abaco Island was examined for keratinophilic fungi. Isolated fungal species in soil identifies possible causal agents of dermatophytoses. Inhabitants of the area may have contaminated the soil, or species present may represent a potential source of new clinical cases. Fungal pathogens have been reported as free living soil saprophytes.     

PsbI affects the stability, function, and phosphorylation patterns of photosystem II assemblies in tobacco

Photosystem II (PSII) core complexes consist of CP47, CP43, D1, D2 proteins and of several low molecular weight integral membrane polypeptides, such as the chloroplast-encoded PsbE, PsbF, and PsbI proteins. To elucidate the function of PsbI in the photosynthetic process as well as in the biogenesis of PSII in higher plants, we generated homoplastomic knock-out plants by replacing most of the tobacco psbI gene with a spectinomycin resistance cartridge. Mutant plants are photoautotrophically viable under green house conditions but sensitive to high light irradiation. Antenna proteins of PSII accumulate to normal amounts, but levels of the PSII core complex are reduced by 50%. Bioenergetic and fluorescence studies uncovered that PsbI is required for the stability but not for the assembly of dimeric PSII and supercomplexes consisting of PSII and the outer antenna (PSII-LHCII). Thermoluminescence emission bands indicate that the presence of PsbI is required for assembly of a fully functional Q(A) binding site. We show that phosphorylation of the reaction center proteins D1 and D2 is light and redox-regulated in the wild type, but phosphorylation is abolished in the mutant, presumably due to structural alterations of PSII when PsbI is deficient. Unlike wild type, phosphorylation of LHCII is strongly increased in the dark due to accumulation of reduced plastoquinone, whereas even upon state II light phosphorylation is decreased in delta psbI. These data attest that phosphorylation of D1/D2, CP43, and LHCII is regulated differently.     

Phylodynamic Inference for Structured Epidemiological Models

Coalescent theory is routinely used to estimate past population dynamics and demographic parameters from genealogies. While early work in coalescent theory only considered simple demographic models, advances in theory have allowed for increasingly complex demographic scenarios to be considered. The success of this approach has lead to coalescent-based inference methods being applied to populations with rapidly changing population dynamics, including pathogens like RNA viruses. However, fitting epidemiological models to genealogies via coalescent models remains a challenging task, because pathogen populations often exhibit complex, nonlinear dynamics and are structured by multiple factors. Moreover, it often becomes necessary to consider stochastic variation in population dynamics when fitting such complex models to real data. Using recently developed structured coalescent models that accommodate complex population dynamics and population structure, we develop a statistical framework for fitting stochastic epidemiological models to genealogies. By combining particle filtering methods with Bayesian Markov chain Monte Carlo methods, we are able to fit a wide class of stochastic, nonlinear epidemiological models with different forms of population structure to genealogies. We demonstrate our framework using two structured epidemiological models: a model with disease progression between multiple stages of infection and a two-population model reflecting spatial structure. We apply the multi-stage model to HIV genealogies and show that the proposed method can be used to estimate the stage-specific transmission rates and prevalence of HIV. Finally, using the two-population model we explore how much information about population structure is contained in genealogies and what sample sizes are necessary to reliably infer parameters like migration rates.     

Refined crystal structures of Escherichia coli and chicken liver dihydrofolate reductase containing bound trimethoprim

Refined crystal structures are reported for complexes of Escherichia coli and chicken dihydrofolate reductase containing the antibiotic trimethoprim (TMP). Structural comparison of these two complexes reveals major geometrical differences in TMP binding that may be important in understanding the stereo-chemical basis of this inhibitor's selectivity for bacterial dihydrofolate reductases. For TMP bound to chicken dihydrofolate reductase we observe an altered binding geometry in which the 2,4-diaminopyrimidine occupies a position in closer proximity (by approximately 1 A) to helix alpha B compared to the pyrimidine position for TMP or methotrexate bound to E. coli dihydrofolate reductase. One important consequence of this deeper insertion of the pyrimidine into the active site of chicken dihydrofolate reductase is the loss of a potential hydrogen bond that would otherwise form between the carbonyl oxygen of Val-115 and the inhibitor's 4-amino group. In addition, for TMP bound to E. coli dihydrofolate reductase, the inhibitor's benzyl side chain is positioned low in the active-site pocket pointing down toward the nicotinamide-binding site, whereas, in chicken dihydrofolate reductase, the benzyl group is accommodated in a side channel running upward and away from the cofactor. As a result, the torsion angles about the C5-C7 and C7-C1' bonds for TMP bound to the bacterial reductase (177 degrees, 76 degrees) differ significantly from the corresponding angles for TMP bound to chicken dihydrofolate reductase (-85 degrees, 102 degrees). Finally, when TMP binds to the chicken holoenzyme, the Tyr-31 side chain undergoes a large conformational change (average movement is 5.4 A for all atoms beyond C beta), rotating down into a new position where it hydrogen bonds via an intervening water molecule to the backbone carbonyl oxygen of Trp-24.     

A chemotactic signaling surface on CheY defined by suppressors of flagellar switch mutations.

CheY is the response regulator protein that interacts with the flagellar switch apparatus to modulate flagellar rotation during chemotactic signaling. CheY can be phosphorylated and dephosphorylated in vitro, and evidence indicates that CheY-P is the activated form that induces clockwise flagellar rotation, resulting in a tumble in the cell's swimming pattern. The flagellar switch apparatus is a complex macromolecular structure composed of at least three gene products, FliG, FliM, and FliN. Genetic analysis of Escherichia coli has identified fliG and fliM as genes in which mutations occur that allele specifically suppress cheY mutations, indicating interactions among these gene products. We have generated a class of cheY mutations selected for dominant suppression of fliG mutations. Interestingly, these cheY mutations dominantly suppressed both fliG and fliM mutations; this is consistent with the idea that the CheY protein interacts with both switch gene products during signaling. Biochemical characterization of wild-type and suppressor CheY proteins did not reveal altered phosphorylation properties or evidence for phosphorylation-dependent CheY multimerization. These data indicate that suppressor CheY proteins are specifically altered in the ability to transduce chemotactic signals to the switch at some point subsequent to phosphorylation. Physical mapping of suppressor amino acid substitutions on the crystal structure of CheY revealed a high degree of spatial clustering, suggesting that this region of CheY is a signaling surface that transduces chemotactic signals to the switch.     

Substance P-immunoreactive neurons in the brainstem of the cat related to cardiovascular centers

Summary The distribution of substance P-immunoreactivity (SP-IR) in the brainstem and spinal cord of normal and colchicine-pretreated cats was analysed using the peroxidase-antiperoxidase (PAP) technique. Numerous SP-IR fibers are present in the nucleus solitarius, nucleus dorsalis nervi vagi and nucleus spinalis nervi trigemini, various parts of the formatio reticularis, substantia grisea centralis mesencephali, locus coeruleus and nucleus parabrachialis. SP-IR perikarya occur in the substantiae gelatinosa and intermedia of the spinal cord, the nucleus spinalis nervi trigemini-pars caudalis, the nucleus dorsalis nervi vagi, and the nucleus solitarius, as well as in the adjacent formatio reticularis and the medullary nuclei of the raphe. In addition, SP-IR cell bodies are located in the nuclei raphe magnus and incertus, ventral and dorsal to the nucleus tegmentalis dorsalis (Gudden), nucleus raphe dorsalis, substantia grisea centralis mensencephali, locus coeruleus, nucleus parabrachialis and colliculus superior.The results indicate that SP-IR neurons may be involved in the regulation of cardiovascular functions both at the central and peripheral level. A peripheral afferent portion seems to terminate in the nucleus solitarius and an efferent part is postulated to originate from the nucleus dorsalis nervi vagi and from the area of the nuclei retroambiguus, ambiguus and retrofacialis.     

Identification of polystyrene nanoparticle penetration across intact skin barrier as rare event at sites of focal particle aggregations

The question whether nanoparticles can cross the skin barrier is highly debated. Even in intact skin rare events of deeper penetration have been reported, but technical limitations and possible artifacts require careful interpretation. In this study, horizontal scanning by 2‐photon microscopy (2 PM) of full‐thickness human skin samples placed in a lateral position yielded highly informative images for skin penetration studies of fluorescently tagged nanoparticles. Scanning of large fields of view allowed for detailed information on interfollicular and follicular penetration in tissue blocks without damaging the sample. Images in histomorphological correlation showed that 2P‐excited fluorescence signals of fluorescently tagged 20 and 200 nm polystyrene nanoparticles preferentially accumulated in the stratum corneum (SC) and in the upper part of vellus hair follicles (HFs). Rare events of deeper penetration in the SC and in the infundibulum of vellus HFs were observed at sites of high focal particle aggregations. Wide‐field 2 PM allows for imaging of nanoparticle penetration in large tissue blocks, whereas total internal reflection microscopy (TIRFM) enables selective detection of individual nanoparticles as well as clusters of nanoparticles in the SC and within the epidermal layer directly beneath the SC, thus confirming barrier crossing with high sensitivity.      

A functional genetic marker for apple red skin coloration across different environments

The red skin color desired by most apple consumers is not easy to achieve in warm climates, as the expression of MYB10, which regulates red pigmentation in apple, is influenced negatively by high temperatures. We describe the development and validation of a genetic marker for red skin coloration that effectively predicts color in a warm summer environment in Spain, as well as more temperate climates in New Zealand and Italy. Following the determination of a major-effect quantitative trait locus (QTL) controlling red skin coloration on linkage group (LG)9, using four segregating populations grown in New Zealand, and screened using the IRSC apple 8-K single-nucleotide polymorphism (SNP) array, the most significant SNP marker (ss475879531) was transformed into a marker suitable for use in a real-time PCR assay. This marker was validated using five apple seedling populations growing in a warm summer environment in Spain, demonstrating that the marker system efficiently predicts red skin coloration and can be used for marker assisted selection, even under conditions considered adverse for skin color development.