Molecular polymorphisms for phylogeny, pedigree and population structure studies

A number of types of molecular polymorphisms can be used for studying genetic relationship and evolutionary history. Microsatellites are hypervariable and can be very useful tools to determine population structure, distinguish sibling species, as well as verifying parental relationships and pedigrees. However, while microsatellite polymorphisms are useful for solving relationships between populations within a species, relations among species or genera will probably be obscured due to a high degree of homoplasy —identity arising from evolutionary convergence not by descent. For long range evolutionary history, such as phylogeny from old world monkey to human, mtDNA markers may be better candidates. The aim of this thesis is to assess molecular polymorphisms of different types and their optimal use in different situations. Two widely separated taxa were used for testing –the green monkey Chlorocebus sabaeus, and the sibling dipteran flies Bactrocera tryoni and B. neohumeralis, known collectively as the Queensland fruit fly. In the present study a complete 16,550 bp mtDNA sequence of the green monkey Chlorocebus sabaeus is reported for the fist time and has been annotated (Chapter 2). Knowledge of the mtDNA genome contributes not only to identification of large scale single nucleotide polymorphisms (SNPs) (Chapter 4) or other mtDNA polymorphisms development, but also to primate phylogenetic and evolutionary study (Chapter 3). Microsatellites used for the green monkey paternity and pedigree studies were developed by cross-amplification using human primers (Chapter 5). For studies of population structure and species discrimination in Queensland fruit fly (Chapter 7), microsatellites were isolated from a genomic library of Bactrocera tryoni (Chapter 6) The total length of 16550 bp of complete mtDNA of the green monkey C. sabaeus, which has been sequenced and annotated here, adds a new node to the primate phylogenetic tree, and creates great opportunity for SNP marker development. The heteroplasmic region was cloned and five different sequences from a single individual were obtained; the implication of this are discussed. The phylogenetic tree reconstructed using the complete mtDNA sequence of C. sabaeus and other primates was used to solve controversial taxonomic status of C. sabaeus. Phylogenies of primate evolution using different genes from mtDNA are discussed. Primate evolutionary trees using different substitution types are compared and the phylogenetic trees constructed using transversions for the complete mtDNA were found close to preconceived expectations than those with transversions + transitions. The sequence of C. sabaeus 12SrRNA reported here agrees with the one published by ven der Kuyl et al. (1996), but additional SNPs were identified. SNPs for other regions of mtDNA were explored using dHPLC. Twenty two PCR segments for 96 individuals were tested by dHPLC. Fifty five SNPs were found and 10 haplogroups were established. Microsatellite markers were used to construct a genealogy for a colony of green monkeys (C. sabaeus) in the UCLA Vervet Monkey Research Colony. Sixteen microsatellites cross-amplified from human primers were used to conduct paternity analysis and pedigree construction. Seventy-eight out of 417 offspring were assigned paternity successfully. The low success rate is attributed to a certain proportion of mismatches between mothers and offspring; the fact that not all candidate fathers were sampled, the limitations of microsatellite polymorphisms; and weakness of the exclusion method for paternity assessment. Due to the low success rate, the pedigree is split into a few small ones. In a complicated pedigree composed of 75 animals and up to four generations with multiple links a power male mated with 8 females and contributed 10 offspring to the pedigree. Close inbreeding was avoided. Population structure within two species of Queensland fruit fly Bactrocera tryoni and Bactrocera neohumeralis (Tephritidae: Diptera) is examined using microsatellite polymorphisms. Queensland fruit flies B. tryoni and B. neohumeralis are sympatric sibling species that have similar morphological and ecological features. They even share polymorphism at the molecular level. Mating time difference is the main mechanism by which they maintain separate species. In the present study, 22 polymorphic and scorable microsatellites were isolated from B. tryoni and tested in the two species sampled from sympatric distribution areas. Pairwise genetic distance analysis showed explicit differentiation in allele frequencies between the two species, but very weak differences between conspecific populations. Gene flow is higher within B. tryoni than within B. neohumeralis, and gene exchange between the two species exists. An averaging linkage clustering tree constructed by UPGMA showed two major clusters distinguishing the two species, and it appears that population structure is highly correlated with geographic distance. The relationship between molecular markers, evolution, and selection are discussed using comparative studies within two large taxa: primate and insect. The degree of conservation and polymorphism in microsatellites varies between taxa, over evolutionary time

Molecular polymorphisms for phylogeny, pedigree and population structure studies

A number of types of molecular polymorphisms can be used for studying genetic relationship and evolutionary history. Microsatellites are hypervariable and can be very useful tools to determine population structure, distinguish sibling species, as well as verifying parental relationships and pedigrees. However, while microsatellite polymorphisms are useful for solving relationships between populations within a species, relations among species or genera will probably be obscured due to a high degree of homoplasy —identity arising from evolutionary convergence not by descent. For long range evolutionary history, such as phylogeny from old world monkey to human, mtDNA markers may be better candidates. The aim of this thesis is to assess molecular polymorphisms of different types and their optimal use in different situations. Two widely separated taxa were used for testing –the green monkey Chlorocebus sabaeus, and the sibling dipteran flies Bactrocera tryoni and B. neohumeralis, known collectively as the Queensland fruit fly. In the present study a complete 16,550 bp mtDNA sequence of the green monkey Chlorocebus sabaeus is reported for the fist time and has been annotated (Chapter 2). Knowledge of the mtDNA genome contributes not only to identification of large scale single nucleotide polymorphisms (SNPs) (Chapter 4) or other mtDNA polymorphisms development, but also to primate phylogenetic and evolutionary study (Chapter 3). Microsatellites used for the green monkey paternity and pedigree studies were developed by cross-amplification using human primers (Chapter 5). For studies of population structure and species discrimination in Queensland fruit fly (Chapter 7), microsatellites were isolated from a genomic library of Bactrocera tryoni (Chapter 6) The total length of 16550 bp of complete mtDNA of the green monkey C. sabaeus, which has been sequenced and annotated here, adds a new node to the primate phylogenetic tree, and creates great opportunity for SNP marker development. The heteroplasmic region was cloned and five different sequences from a single individual were obtained; the implication of this are discussed. The phylogenetic tree reconstructed using the complete mtDNA sequence of C. sabaeus and other primates was used to solve controversial taxonomic status of C. sabaeus. Phylogenies of primate evolution using different genes from mtDNA are discussed. Primate evolutionary trees using different substitution types are compared and the phylogenetic trees constructed using transversions for the complete mtDNA were found close to preconceived expectations than those with transversions + transitions. The sequence of C. sabaeus 12SrRNA reported here agrees with the one published by ven der Kuyl et al. (1996), but additional SNPs were identified. SNPs for other regions of mtDNA were explored using dHPLC. Twenty two PCR segments for 96 individuals were tested by dHPLC. Fifty five SNPs were found and 10 haplogroups were established. Microsatellite markers were used to construct a genealogy for a colony of green monkeys (C. sabaeus) in the UCLA Vervet Monkey Research Colony. Sixteen microsatellites cross-amplified from human primers were used to conduct paternity analysis and pedigree construction. Seventy-eight out of 417 offspring were assigned paternity successfully. The low success rate is attributed to a certain proportion of mismatches between mothers and offspring; the fact that not all candidate fathers were sampled, the limitations of microsatellite polymorphisms; and weakness of the exclusion method for paternity assessment. Due to the low success rate, the pedigree is split into a few small ones. In a complicated pedigree composed of 75 animals and up to four generations with multiple links a power male mated with 8 females and contributed 10 offspring to the pedigree. Close inbreeding was avoided. Population structure within two species of Queensland fruit fly Bactrocera tryoni and Bactrocera neohumeralis (Tephritidae: Diptera) is examined using microsatellite polymorphisms. Queensland fruit flies B. tryoni and B. neohumeralis are sympatric sibling species that have similar morphological and ecological features. They even share polymorphism at the molecular level. Mating time difference is the main mechanism by which they maintain separate species. In the present study, 22 polymorphic and scorable microsatellites were isolated from B. tryoni and tested in the two species sampled from sympatric distribution areas. Pairwise genetic distance analysis showed explicit differentiation in allele frequencies between the two species, but very weak differences between conspecific populations. Gene flow is higher within B. tryoni than within B. neohumeralis, and gene exchange between the two species exists. An averaging linkage clustering tree constructed by UPGMA showed two major clusters distinguishing the two species, and it appears that population structure is highly correlated with geographic distance. The relationship between molecular markers, evolution, and selection are discussed using comparative studies within two large taxa: primate and insect. The degree of conservation and polymorphism in microsatellites varies between taxa, over evolutionary time

Time-resolved propagation of polarized light in turbid media: experiment and theory

This paper presents our study of time-resolved propagation of polarized light in scattering media. Monte Carlo simulated time-resolved Stokes vectors of transmitted light were compared with the experimental results. A satisfying match has been obtained

Pain perception of older adults in nursing home and home care settings: evidence from China

Background In the past decade, the number of long-term care (LTC) services for older adults in China has grown annually by an average of 10%. Older adults, their family members, and policymakers in China are concerned about patient outcomes in different care settings because older adults who have a similar functional status and LTC needs may choose either nursing home care or home care. The aim of this study was to compare pain perception in nursing home care and home care settings for physically dependent older adults in China. Methods Multi-stage sampling method was used to recruit respondents aged 65 and older from Yichang City, China, in 2015. The researchers employed a two-step analytical strategy—zero-inflated ordered probit regression followed by propensity score matching method—to model the effect of contrasting residence types on pain perception. Results Zero-inflated ordered probit regression analysis with participants unmatched (n = 484) showed that compared with older adults who received home care, those who received nursing home care did not have more severe pain (β = 0.088, SE = 0.196, p = 0.655). After propensity-score matching, the research found that older adults in the home care group perceived less pain compared with the nursing home group (β = 0.489, SE = 0.169, p = 0.004). Conclusions The older adults who received home care perceived significantly less pain than the nursing home residents. The pain of older adults may differ based on the type of LTC services and therapy intensity they received, and home care might lead to less pain and better comfort than nursing home care

Polarization-gated imaging techniques based on time-resolved Stokes vectors for filament tissues

We report the discovery of using various time-resolved Stokes vector components for more efficient imaging of filament tissues. In particular, the Stokes vector components S2 and S3 were found more useful, compared with the other two components. The time-resolved Stokes vectors of signals transmitting chicken breast tissue, mouse cardiac muscle tissue, and polystyrene micro-sphere solution are compared. A target of chicken bone in chicken breast tissue is used for demonstrating the effectiveness of various Stokes vector components for imaging purpose. Also, Monte Carlo simulations were conducted for comparison

Exploring the Association Between Morgellons Disease and Lyme Disease: Identification of Borrelia Burgdorferi in Morgellons Disease Patients

Morgellons disease (MD) is a complex skin disorder characterized by ulcerating lesions that have protruding or embedded filaments. Many clinicians refer to this condition as delusional parasitosis or delusional infestation and consider the filaments to be introduced textile fibers. In contrast, recent studies indicate that MD is a true somatic illness associated with tickborne infection, that the filaments are keratin and collagen in composition and that they result from proliferation and activation of keratinocytes and fibroblasts in the skin. Previously, spirochetes have been detected in the dermatological specimens from four MD patients, thus providing evidence of an infectious process. Methods & Results Based on culture, histology, immunohistochemistry, electron microscopy and molecular testing, we present corroborating evidence of spirochetal infection in a larger group of 25 MD patients. Irrespective of Lyme serological reactivity, all patients in our study group demonstrated histological evidence of epithelial spirochetal infection. Strength of evidence based on other testing varied among patients. Spirochetes identified as Borrelia strains by polymerase chain reaction (PCR) and/or in-situ DNA hybridization were detected in 24/25 of our study patients. Skin cultures containing Borrelia spirochetes were obtained from four patients, thus demonstrating that the organisms present in dermatological specimens were viable. Spirochetes identified by PCR as Borrelia burgdorferi were cultured from blood in seven patients and from vaginal secretions in three patients, demonstrating systemic infection. Based on these observations, a clinical classification system for MD is proposed. Conclusions Our study using multiple detection methods confirms that MD is a true somatic illness associated with Borrelia spirochetes that cause Lyme disease. Further studies are needed to determine the optimal treatment for this spirochete-associated dermopathy. Keywords: Morgellons disease; Lyme disease; Borrelia burgdorferi ; Spirochetes; Dermopath

Study of polarization evolution in phantom tissues with ultrafast optics techniques: Monte Carlo simulations and experiments

This paper presents our study results of polarized short pulse transmission through phantom tissues made of polystyrene particle solutions with various concentrations and particle sizes. To improve the quality of optical imaging using an ultrafast light source, study is required to fully understand the evolution of the polarization state in the sample, as well as the time- and polarization-dependent distributions of optical intensity exiting from samples. Temporal profiles of the Stokes vectors and the degree of polarization are measured experimentally. The results agree well with those resulting from Monte Carlo simulations. Analyses based on the Stokes-Mueller formalism show that the first scattering event determines the spatial patterns of the transmitted Stokes vectors. When a detected area at the output surface of the sample is symmetric about the incident beam, the temporal profile of transmittance is independent of the incident polarization state. The linear relationship between the average order of scatters and the light propagation time can be used to explain the exponential decay of the degree of polarization and the inversely proportional relationship between the FWHM of the degree of polarization and the scatterer concentration

Retinoid production using metabolically engineered Escherichia coli with a two-phase culture system

<p>Abstract</p> <p>Background</p> <p>Retinoids are lipophilic isoprenoids composed of a cyclic group and a linear chain with a hydrophilic end group. These compounds include retinol, retinal, retinoic acid, retinyl esters, and various derivatives of these structures. Retinoids are used as cosmetic agents and effective pharmaceuticals for skin diseases. Retinal, an immediate precursor of retinoids, is derived by β-carotene 15,15'-mono(di)oxygenase (BCM(D)O) from β-carotene, which is synthesized from the isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Retinoids are chemically unstable and biologically degraded via retinoic acid. Although extensive studies have been performed on the microbial production of carotenoids, retinoid production using microbial metabolic engineering has not been reported. Here, we report retinoid production using engineered <it>Escherichia coli </it>that express exogenous BCM(D)O and the mevalonate (MVA) pathway for the building blocks synthesis in combination with a two-phase culture system using a dodecane overlay.</p> <p>Results</p> <p>Among the BCM(D)O tested in <it>E. coli</it>, the synthetic retinoid synthesis protein (SR), based on bacteriorhodopsin-related protein-like homolog (Blh) of the uncultured marine bacteria 66A03, showed the highest β-carotene cleavage activity with no residual intracellular β-carotene. By introducing the exogenous MVA pathway, 8.7 mg/L of retinal was produced, which is 4-fold higher production than that of augmenting the MEP pathway (<it>dxs </it>overexpression). There was a large gap between retinal production and β-carotene consumption using the exogenous MVA pathway; therefore, the retinal derivatives were analyzed. The derivatives, except for retinoic acid, that formed were identified, and the levels of retinal, retinol, and retinyl acetate were measured. Amounts as high as 95 mg/L retinoids were obtained from engineered <it>E. coli </it>DH5α harboring the synthetic <it>SR </it>gene and the exogenous MVA pathway in addition to <it>dxs </it>overexpression, which were cultured at 29°C for 72 hours with 2YT medium containing 2.0% (w/v) glycerol as the main carbon source. However, a significant level of intracellular degradation of the retinoids was also observed in the culture. To prevent degradation of the intracellular retinoids through <it>in situ </it>extraction from the cells, a two-phase culture system with dodecane was used. The highest level of retinoid production (136 mg/L) was obtained after 72 hours with 5 mL of dodecane overlaid on a 5 mL culture.</p> <p>Conclusions</p> <p>In this study, we successfully produced 136 mg/L retinoids, which were composed of 67 mg/L retinal, 54 mg/L retinol, and 15 mg/L retinyl acetate, using a two-phase culture system with dodecane, which produced 68-fold more retinoids than the initial level of production (2.2 mg/L). Our results demonstrate the potential use of <it>E. coli </it>as a promising microbial cell factory for retinoid production.</p

Potential therapeutic application of gold nanoparticles in B-chronic lymphocytic leukemia (BCLL): enhancing apoptosis

B-Chronic Lymphocytic Leukemia (CLL) is an incurable disease predominantly characterized by apoptosis resistance. We have previously described a VEGF signaling pathway that generates apoptosis resistance in CLL B cells. We found induction of significantly more apoptosis in CLL B cells by co-culture with an anti-VEGF antibody. To increase the efficacy of these agents in CLL therapy we have focused on the use of gold nanoparticles (GNP). Gold nanoparticles were chosen based on their biocompatibility, very high surface area, ease of characterization and surface functionalization. We attached VEGF antibody (AbVF) to the gold nanoparticles and determined their ability to kill CLL B cells. Gold nanoparticles and their nanoconjugates were characterized using UV-Visible spectroscopy (UV-Vis), transmission electron microscopy (TEM), thermogravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS). All the patient samples studied (N = 7) responded to the gold-AbVF treatment with a dose dependent apoptosis of CLL B cells. The induction of apoptosis with gold-AbVF was significantly higher than the CLL cells exposed to only AbVF or GNP. The gold-AbVF treated cells showed significant down regulation of anti-apoptotic proteins and exhibited PARP cleavage. Gold-AbVF treated and GNP treated cells showed internalization of the nanoparticles in early and late endosomes and in multivesicular bodies. Non-coated gold nanoparticles alone were able to induce some levels of apoptosis in CLL B cells. This paper opens up new opportunities in the treatment of CLL-B using gold nanoparticles and integrates nanoscience with therapy in CLL. In future, potential opportunities exist to harness the optoelectronic properties of gold nanoparticles in the treatment of CLL