Search

According to historical records of transatlantic slavery, traders forcibly deported an estimated 12.5 million people from ports along the Atlantic coastline of Africa between the 16th and 19th centuries, with global impacts reaching to the present day, more than a century and a half after slavery's abolition. Such records have fueled a broad understanding of the forced migration from Africa to the Americas yet remain underexplored in concert with genetic data. Here, we analyzed genotype array data from 50,281 research participants, which-combined with historical shipping documents-illustrate that the current genetic landscape of the Americas is largely concordant with expectations derived from documentation of slave voyages. For instance, genetic connections between people in slave trading regions of Africa and disembarkation regions of the Americas generally mirror the proportion of individuals forcibly moved between those regions. While some discordances can be explained by additional records of deportations within the Americas, other discordances yield insights into variable survival rates and timing of arrival of enslaved people from specific regions of Africa. Furthermore, the greater contribution of African women to the gene pool compared to African men varies across the Americas, consistent with literature documenting regional differences in slavery practices. This investigation of the transatlantic slave trade, which is broad in scope in terms of both datasets and analyses, establishes genetic links between individuals in the Americas and populations across Atlantic Africa, yielding a more comprehensive understanding of the African roots of peoples of the Americas.

Short tandem repeats (STRs) are hyper-mutable sequences in the human genome. They are often used in forensics and population genetics and are also the underlying cause of many genetic diseases. There are challenges associated with accurately determining the length polymorphism of STR loci in the genome by next-generation sequencing (NGS). In particular, accurate detection of pathological STR expansion is limited by the sequence read length during whole-genome analysis. We developed TREDPARSE, a software package that incorporates various cues from read alignment and paired-end distance distribution, as well as a sequence stutter model, in a probabilistic framework to infer repeat sizes for genetic loci, and we used this software to infer repeat sizes for 30 known disease loci. Using simulated data, we show that TREDPARSE outperforms other available software. We sampled the full genome sequences of 12,632 individuals to an average read depth of approximately 30× to 40× with Illumina HiSeq X. We identified 138 individuals with risk alleles at 15 STR disease loci. We validated a representative subset of the samples (n = 19) by Sanger and by Oxford Nanopore sequencing. Additionally, we validated the STR calls against known allele sizes in a set of GeT-RM reference cell-line materials (n = 6). Several STR loci that are entirely guanine or cytosines (G or C) have insufficient read evidence for inference and therefore could not be assayed precisely by TREDPARSE. TREDPARSE extends the limit of STR size detection beyond the physical sequence read length. This extension is critical because many of the disease risk cutoffs are close to or beyond the short sequence read length of 100 to 150 bases.

Meiotic nondisjunction and resulting aneuploidy can lead to severe health consequences in humans. Aneuploidy rescue can restore euploidy but may result in uniparental disomy (UPD), the inheritance of both homologs of a chromosome from one parent with no representative copy from the other. Current understanding of UPD is limited to ∼3,300 case subjects for which UPD was associated with clinical presentation due to imprinting disorders or recessive diseases. Thus, the prevalence of UPD and its phenotypic consequences in the general population are unknown. We searched for instances of UPD across 4,400,363 consented research participants from the personal genetics company 23andMe, Inc., and 431,094 UK Biobank participants. Using computationally detected DNA segments identical-by-descent (IBD) and runs of homozygosity (ROH), we identified 675 instances of UPD across both databases. We estimate that UPD is twice as common as previously thought, and we present a machine-learning framework to detect UPD using ROH. While we find a nominally significant association between UPD of chromosome 22 and autism risk, we do not find significant associations between UPD and deleterious traits in the 23andMe database.

Tiger chert was used extensively by Native American people of the Clovis lithic industry as the source material for stone tools. Although tiger chert occurs naturally only within the Bridger Formation of Southwest Wyoming, tiger chert artifacts have been found throughout the American west. This paper explores the previously unreported near-infrared fluorescence emitted from tiger chert upon visible excitation. The two objectives of this study were to demonstrate the utility of near-infrared fluorescence imaging (NIRFI) to tiger chert artifacts and to identify the chemical origin of the observed fluorescence. Tiger chert was analyzed by visible and NIRFI, SEM-EDX, ICP-OES, and fluorescence spectroscopy. NIRFI can be easily used to discriminate between tiger chert artifacts and the environment, since most materials such as soil and most rocks appear black in the resultant image. This suggests NIRFI should be utilized by archeologists at any site where tiger chert has been previously identified. Additionally, NIRFI was used to differentiate between tiger chert artifacts and an artificially weathered, recently-knapped tiger chert point, suggesting that NIRFI could be used to distinguish genuine tiger chert artifacts from counterfeits. Tiger chert fluorescence was confined to the manganese-and-iron-rich surface, suggesting the presence of desert varnish; however, red agate from the same site did not exhibit fluorescence, instead implicating tiger-chert-specific weathering. Thus, the chemical origin of fluorescence was not conclusively identified.

Förster resonance energy transfer (FRET) is the basis for many techniques used in biomedical research. Due to its wide use in molecular sensing, FRET is commonly introduced in many biology, chemistry, and physics courses. While FRET is of great importance in the biophysical sciences, the complexity and difficulty of constructing FRET experiments has resulted in limited usage in undergraduate laboratory settings. Here, we present a practical undergraduate laboratory experiment for teaching FRET using a diverse set of green-emitting fluorescent proteins (FPs) as donors for a cross-linked Yukon orange FP. This laboratory experiment enables students to make the connection of basic lab procedures to real world applications and can be applied to molecular biology, biochemistry, physical chemistry, and biophysical laboratory courses. Published 2018. This article is a U.S. Government work and is in the public domain in the USA., 46(5):516-522, 2018.

Prediction of human physical traits and demographic information from genomic data challenges privacy and data deidentification in personalized medicine. To explore the current capabilities of phenotype-based genomic identification, we applied whole-genome sequencing, detailed phenotyping, and statistical modeling to predict biometric traits in a cohort of 1,061 participants of diverse ancestry. Individually, for a large fraction of the traits, their predictive accuracy beyond ancestry and demographic information is limited. However, we have developed a maximum entropy algorithm that integrates multiple predictions to determine which genomic samples and phenotype measurements originate from the same person. Using this algorithm, we have reidentified an average of >8 of 10 held-out individuals in an ethnically mixed cohort and an average of 5 of either 10 African Americans or 10 Europeans. This work challenges current conceptions of personal privacy and may have far-reaching ethical and legal implications.

Recombinant peptides and proteins have a wide range of applications, especially in medicine and biomedical research. Methods to rapidly and inexpensively manufacture recombinant proteins are needed to realize the full potential of proteins in medicine and to broaden their applications. A one-semester-length laboratory exercise is reported here demonstrating the use of the insect bioreactor system to create recombinant proteins. These laboratory exercises are suitable for students majoring in biochemistry or biotechnology and can be used to teach students basic biochemistry laboratory techniques including bacterial transformation, plasmid DNA isolation, insect cell culture, production of recombinant virus, use of recombinant viruses to deliver transgenes to a living organism, protein isolation by precipitation and chromatography, and protein quantification and protein characterization by electrophoresis and western blotting. The techniques acquired can also be used as the basis for related independent research projects in the second semester.

Ensuring domestic safety from terrorist attack is a daunting challenge because of the wide array of chemical agents that must be screened. A panel of purified fluorescent protein isoforms (FPs) was screened for the ability to detect various explosives, explosive simulants, and toxic agents. In addition to their commonly used visible excitation wavelengths, essentially all FPs can be excited by UV light at 280 nm. Ultraviolet illumination excites electrons in endogenous tryptophan (W) residues, which then relax by Forster Resonance Energy Transfer (FRET) to the chromophore of the FP, and thus the FPs emit with their typical visible spectra. Taking advantage of the fact that tryptophan excitation can be quenched by numerous agents, including nitroaromatics like TNT and nitramines like RDX, it is demonstrated that quenching of visible fluorescence from UV illumination of FPs can be used as the basis for detecting these explosives and explosive degradation products. This work provides the foundation for production of an array of genetically-modified FPs for in vitro biosensors capable of rapid, simultaneous, sensitive and selective detection of a wide range of explosive or toxic agents.

Both phosphointermediate- and vacuolar-type (P- and V-type, respectively) ATPase activities found in cholinergic synaptic vesicles isolated from electric organ are immunoprecipitated by a monoclonal antibody to the SV2 epitope characteristic of synaptic vesicles. The two activities can be distinguished by assay in the absence and presence of vanadate, an inhibitor of the P-type ATPase. Each ATPase has two overlapping activity maxima between pH 5.5 and 9.5 and is inhibited by fluoride and fluorescein isothiocyanate. The P-type ATPase hydrolyzes ATP and dATP best among common nucleotides, and activity is supported well by Mg2+, Mn2+, or Co2+ but not by Ca2+, Cd2+, or Zn2+. It is stimulated by hyposmotic lysis, detergent solubilization, and some mitochondrial uncouplers. Kinetic analysis revealed two Michaelis constants for MgATP of 28 microM and 3.1 mM, and the native enzyme is proposed to be a dimer of 110-kDa subunits. The V-type ATPase hydrolyzes all common nucleoside triphosphates, and Mg2+, Ca2+, Cd2+, Mn2+, and Zn2+ all support activity effectively. Active transport of acetylcholine (ACh) also is supported by various nucleoside triphosphates in the presence of Ca2+ or Mg2+, and the Km for MgATP is 170 microM. The V-type ATPase is stimulated by mitochondrial uncouplers, but only at concentrations significantly above those required to inhibit ACh active uptake. Kinetic analysis of the V-type ATPase revealed two Michaelis constants for MgATP of approximately 26 microM and 2.0 mM. The V-type ATPase and ACh active transport were inhibited by 84 and 160 pmol of bafilomycin A1/mg of vesicle protein, respectively, from which it is estimated that only one or two V-type ATPase proton pumps are present per synaptic vesicle. The presence of presumably contaminating Na+,K(+)-ATPase in the synaptic vesicle preparation is demonstrated.

A protein that binds vesamicol has been purified from a soluble fraction of the Torpedo electric organ homogenate that does not contain synaptic vesicles. The purified vesamicol-binding protein (VBP) has a molecular mass of 470 kDa composed of 30- and 24-kDa subunits. Chemical deglycosylation yielded a single, heterogeneous protein of 24 kDa. The 30-kDa subunit is also sensitive to endo-beta-galactosidase. The dissociation constant of the VBP.vesamicol complex is 0.9 microM, and the Bmax is 5,500 pmol/mg. Antiserum raised to the 30-kDa subunit cross-reacts with the 24-kDa subunit, but not with synaptic vesicles. Drug binding studies and Western blot analysis show that VBP is present in other Torpedo tissues as well as mammalian brain. Immunofluorescence microscopy demonstrates that VBP-like immunoreactivity is not localized exclusively to the nerve terminal regions of the electric organ. Thermal stability, the pH dependence of vesamicol binding, and pharmacological comparisons demonstrate that the VBP is not the cholinergic synaptic vesicle receptor for vesamicol. The implications of this finding for current efforts to develop in vivo diagnostics of cholinergic nerve terminal status based on vesamicol are discussed.

The clustering of type A gamma-aminobutyric acid receptors (GABA(A)R) at discrete and functionally significant domains on the nerve cell surface is an important determinant in the integration of synaptic inputs. To discern the role that the subunits of the GABA(A)R play in determining the receptor's cell surface topography and mobility, the alpha1, beta1, beta3, and gamma2s subunits were transfected into COS7, HEK293, and PC12 cells and the distribution and cell surface mobility of these recombinant receptors were examined. Our results show that alpha1 subunits are retained in the endoplasmic reticulum while beta1 and beta3 subunits are sorted to the plasma membrane where they form clusters. Co-expression and co-assembly of alpha1 and beta3 subunits result in the rescue of intracellular alpha1 subunits, which are transported as alphabeta subunit complexes to the cell surface where they formed clusters. Fluorescence photobleach recovery and single particle tracking of recombinant receptors show that, despite clustering, beta3 subunit homooligomers are mobile within a cell surface domain. Inclusion of alpha1 in beta3 or beta3gamma2s complexes, however, dramatically reduces the receptor's lateral mobility in COS 7 and PC12 cells and anchors GABA(A)Rs on the cell surface, suggesting the formation of a direct link to a component of the cytoskeleton. The mobility of recombinant receptors that include the alpha1 subunit mirrors the mobility of GABA(A)Rs on cell bodies and dendrites of cortical and spinal cord neurons. The results suggest that incorporation of alpha1 subunits give rise to a population of GABA(A)Rs that are immobilized on the cell surface.

The lateral diffusion of 100 nm fluorescent latex microspheres (FS) bound to either N-biotinyl-phosphatidyl-ethanolamine or the glycosylphosphatidylinositol-linked protein Thy1 were monitored in the plasmalemma of primary rat fibroblasts by single particle tracking of FS centroids from digital fluorescence micrographs. A silicon intensified target camera was found to be superior to slow scan cooled CCD and intensified interline transfer CCD cameras for monitoring lateral diffusion of rapidly moving FS with nanometer level precision. To estimate the maximum tracking precision, a 4 sec-sequence comprising 120 images of FS fixed to a cover glass was obtained. The mean distance of the centroids from the origin was 7.5 +/- 0.4 nm, and no centroids were beyond 16 nm from the origin. The SIT camera was then used to track FS attached to lipids and Thy1 molecules on the surface of fibroblasts. The lateral diffusion of lipid-bound FS was unconstrained, and the ensemble averaged diffusion coefficient was 0.80 x 10(-9) cm2/sec. Thy1-bound FS existed in two mobility populations, both of which demonstrated constrained mobility. The rapidly moving population, comprising 61% of the total, had an ensemble diffusion coefficient of 6.1 x 10(-10) cm2/sec, and appeared to be restricted to domains with a mean length of about 700 nm. The slowly moving population, comprising about 39% of the total, had a diffusion coefficient of 5.7 x 10(-12) cm2/sec. These results demonstrate that nanovid can be extended to the realm of fluorescence microscopy and support previous studies indicating that while the lateral mobilities of at least some lipids are not constrained to small domains by barriers to lateral diffusion in the fibroblast plasmalemma, a peripheral membrane protein which is bound only by a lipid anchor can be prevented from diffusing freely.

Publisher Summary This chapter discusses imaging ion channel dynamics in living neurons by fluorescence microscopy. The distribution of ion channels and receptors over the neuronal surface is important for the receipt of incoming synaptic inputs and for the integration of these inputs. Most voltage-gated and ligand-gated ion channels have nonhomogeneous distributions in the neuronal membrane; many are restricted to dendritic, axonal, or somatic domains and are further localized within these domains to regions such as dendritic spines, nodes of Ranvier, or synaptic junctions. Determining where and how ion channels are distributed and maintained is important for a variety of reasons. Ion channels in growth cones have a role in neurite outgrowth mechanisms; they are obligatory for synaptic transmission and are required for amplification of neurotransmitter signals in the postsynaptic membrane. Changes in ion-channel distributions are an important aspect in development and plasticity. Several neurological disorders, including multiple sclerosis, are characterized by a change in ion-channel distribution, which is partially responsible for the clinical manifestations of the disease. Fluorescence techniques are invaluable in the study of ion-channel and receptor distributions and dynamics. Fluorescence microscopy has extremely high sensitivity, the lower limit being the ability to detect a single fluorophore under optimal conditions. Fluorescence measurements are specified by the wavelengths of emission and excitation.

Publisher Summary Acetylcholine (ACh) is stored by synaptic vesicles at about 100-fold higher concentration than present in the cytoplasm. As vesicular ACh is free in solution, energy input is required to establish the gradient of concentration. This is accomplished in a process having at least two macromolecular components that reside in the vesicular membrane. The first component is a proton-pumping ATPase known as a V-type ATPase. It pumps protons into the synaptic vesicle, thus acidifying the vesicle core to about pH 5.4. The enzyme is found in the vacuolar-type intracellular organelles, and it is evolutionally related to the mitochondria1 ATPase. The second component is the ACh transporter (AChT). It exchanges one or more vesicular protons for cytoplasmic ACh, thus accomplishing secondary active transport of ACh. The ACh exhibits a transport Michaelis dissociation constant (KM) of about 0.3 mM, which is less than the 4 mM that can be estimated for the concentration of ACh in the cytoplasm. Proton-exchanging transporters appear to account for vesicular storage of all of the classical neurotransmitters. As a class, these transporters are poorly characterized. A structure-activity study has demonstrated that the potency of vesamicol analogues, as assessed by inhibition of active transport, is highly dependent upon the structure of the analogue. This chapter reports the results of the recent ligand-interaction experiments that were directed toward making a choice between models 1 and 2, and toward the identification of the macromolecular structures of the AChT and VR.

The relationship of the acetylcholine transporter-vesamicol receptor (AcChT-VR) to proteoglycan in Torpedo electric organ synaptic vesicles was investigated. The cholate-solubilized VR was immunoprecipitated by a monoclonal antibody directed against the SV1 epitope located in the glycosaminoglycan portion of the proteoglycan. AcChT that was photoaffinity-labeled with a tritiated high-affinity analogue of AcCh [cyclohexylmethyl cis-N-(4-azidophenacyl)-N-methylisonipecotate] and then denatured in sodium dodecyl sulfate also immunoprecipitated. The labeled AcChT exhibited a M(r) range of 100,000-200,000. Proteoglycan did not engage in detectable nonspecific reversible aggregation that might mask the presence of another subunit during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In vesicles permeabilized with cholate, the enzymes keratanase and testicular hyaluronidase inactivated binding of vesamicol and destroyed the SV1 epitope without detectable proteolysis. Other glycosaminoglycan-degrading enzymes were without effect. The results demonstrate that the AcChT-VR and proteoglycan are very strongly linked and that glycosaminoglycan-like polysaccharide controls the conformation of the VR. The unexpected linkage to proteoglycan suggests that AcChT-VR in intact terminals might communicate with extracellular matrix and participate in stabilization and operation of the synapse.

The distribution of protein and lipid in the plasmalemma of some, and perhaps all, cells is non-homogeneous. Microdomains with compositions that are not representative of the bulk membrane exist in many cell types. This phenomenon is obligatory for spatially segregating certain functions performed by the plasma membrane. Obvious examples are concentration of neurotransmitter receptors at the synaptic gap, and voltage-dependent sodium ion channels at the nodes of Ranvier in myelinated nerve. Evidence has also emerged suggesting that lipids are capable of segregating into non homogeneous domains. A potential consequence of microdomain formation would be altered lateral mobility of components within the bilayer.Recently, nanovid microscopy was used to demonstrate that N-(fluoresceinthiocarbamoyl)- phosphatidyl ethanolamine (FITC-PE) was not constrained by diffusion barriers in fibroblasts. Colloidal gold probes conjugated to antiFITC-PE antibody were observed using DIC optics to undergo Brownian motion on the membrane surface. This study concentrated on the lamellar and super-nuclear regions of the cell membrane.

The distribution of ion channels and receptors over the neuronal surface is important for the receipt of incoming synaptic inputs and for the integration of these inputs. Most voltage-gated and ligand-gated ion channels have non-homogeneous distributions in the neuronal membrane, many being restricted to either dendritic, axonal or somatic domains and further localized within these domains to regions such as dendritic spines, nodes of Ranvier or synaptic junctions (1-3). For example voltage-dependent calcium channels are localized and immobilized on dendrites (4), while voltage-dependent sodium channel are localized on axon hillocks (5) and nodes of Ranvier. Determining where and how ion channels are distributed and maintained is important for a variety of reasons. Ion channels in growth cones have a role in neurite outgrowth mechanisms (6), they are obligatory for synaptic transmission and they are required for amplification of neurotransmitter signals in the post synaptic membrane (7). Changes in ion channel distributions are an important aspect in development and plasticity (8,9).

Using common techniques of molecular biology, students transfect Escherichia coli with cloned DNA (cDNA) plasmids encoding for various isoforms of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The different GFP isoforms can then be purified and characterized.