Concurrent Whole-Genome Haplotyping and Copy-Number Profiling of Single Cells

Methods for haplotyping and DNA copy-number typing of single cells are paramount for studying genomic heterogeneity and enabling genetic diagnosis. Before analyzing the DNA of a single cell by microarray or next-generation sequencing, a whole-genome amplification (WGA) process is required, but it substantially distorts the frequency and composition of the cell’s alleles. As a consequence, haplotyping methods suffer from error-prone discrete SNP genotypes (AA, AB, BB) and DNA copy-number profiling remains difficult because true DNA copy-number aberrations have to be discriminated from WGA artifacts. Here, we developed a single-cell genome analysis method that reconstructs genome-wide haplotype architectures as well as the copy-number and segregational origin of those haplotypes by employing phased parental genotypes and deciphering WGA-distorted SNP B-allele fractions via a process we coin haplarithmisis. We demonstrate that the method can be applied as a generic method for preimplantation genetic diagnosis on single cells biopsied from human embryos, enabling diagnosis of disease alleles genome wide as well as numerical and structural chromosomal anomalies. Moreover, meiotic segregation errors can be distinguished from mitotic ones.     

Carry‐over Effects of Size at Metamorphosis in Red‐eyed Treefrogs: Higher Survival but Slower Growth of Larger Metamorphs

Most animals have complex life histories, composed of a series of ecologically distinct stages, and the transitions between stages are often plastic. Anurans are models for research on complex life cycles. Many species exhibit plastic timing of and size at metamorphosis, due to both environmental constraints on larval growth and development and adaptive plastic responses to environmental variation. Models predicting optimal timing of metamorphosis balance cost/benefit ratios across stages, assuming that size affects growth and mortality rates in each stage. Much research has documented such effects in the larval period, but we lack an equal understanding of juvenile growth and mortality. Here, we examine how variation in size at metamorphosis in the Neotropical red‐eyed treefrog, Agalychnis callidryas, affects post‐metamorphic growth, foraging, and behavior in the lab as well as growth and survival in the field. Surprisingly, many individuals lost mass for weeks after metamorphosis. In the lab, larger metamorphs lost more mass following metamorphosis, ate similar amounts, had lower food conversion efficiencies, and grew more slowly after mass loss ceased than did smaller ones. In field cages larger metamorphs were more likely to survive than smaller ones; just one froglet died in the lab. Our data suggest that size‐specific differences in physiology and behavior influence these trends. Comparing across species and studies, large size at metamorphosis generally confers higher survival; size effects on growth rates vary substantially among species, in both magnitude and direction, but may be stronger in the tropics.     

Systematic Mapping of WNT-FZD Protein Interactions Reveals Functional Selectivity by Distinct WNT-FZD Pairs

The seven-transmembrane-spanning receptors of the FZD_1–10 class are bound and activated by the WNT family of lipoglycoproteins, thereby inducing a complex network of signaling pathways. However, the specificity of the interaction between mammalian WNT and FZD proteins and the subsequent signaling cascade downstream of the different WNT-FZD pairs have not been systematically addressed to date. In this study, we determined the binding affinities of various WNTs for different members of the FZD family by using bio-layer interferometry and characterized their functional selectivity in a cell system. Using purified WNTs, we show that different FZD cysteine-rich domains prefer to bind to distinct WNTs with fast on-rates and slow off-rates. In a 32D cell-based system engineered to overexpress FZD₂, FZD₄, or FZD₅, we found that WNT-3A (but not WNT-4, -5A, or -9B) activated the WNT-β-catenin pathway through FZD_2/4/5 as measured by phosphorylation of LRP6 and β-catenin stabilization. Surprisingly, different WNT-FZD pairs showed differential effects on phosphorylation of DVL2 and DVL3, revealing a previously unappreciated DVL isoform selectivity by different WNT-FZD pairs in 32D cells. In summary, we present extensive mapping of WNT-FZD cysteine-rich domain interactions complemented by analysis of WNT-FZD pair functionality in a unique cell system expressing individual FZD isoforms. Differential WNT-FZD binding and selective functional readouts suggest that endogenous WNT ligands evolved with an intrinsic natural bias toward different downstream signaling pathways, a phenomenon that could be of great importance in the design of FZD-targeting drugs.     

Increased palmitate intake: higher acylcarnitine concentrations without impaired progression of β-oxidation

Palmitic acid (PA) is associated with higher blood concentrations of medium-chain acylcarnitines (MCACs), and we hypothesized that PA may inhibit progression of FA β-oxidation. Using a cross-over design, 17 adults were fed high PA (HPA) and low PA/high oleic acid (HOA) diets, each for 3 weeks. The [1-¹³C]PA and [13-¹³C]PA tracers were administered with food in random order with each diet, and we assessed PA oxidation (PA OX) and serum AC concentration to determine whether a higher PA intake promoted incomplete PA OX. Dietary PA was completely oxidized during the HOA diet, but only about 40% was oxidized during the HPA diet. The [13-¹³C]PA/[1-¹³C]PA ratio of PA OX had an approximate value of 1.0 for either diet, but the ratio of the serum concentrations of MCACs to long-chain ACs (LCACs) was significantly higher during the HPA diet. Thus, direct measurement of PA OX did not confirm that the HPA diet caused incomplete PA OX, despite the modest, but statistically significant, increase in the ratio of MCACs to LCACs in blood.     

KSRP is critical in governing hepatic lipid metabolism through controlling Per2 expression

Hepatic lipid metabolism is controlled by integrated metabolic pathways. Excess accumulation of hepatic TG is a hallmark of nonalcoholic fatty liver disease, which is associated with obesity and insulin resistance. Here, we show that KH-type splicing regulatory protein (KSRP) ablation reduces hepatic TG levels and diet-induced hepatosteatosis. Expression of period 2 (Per2) is increased during the dark period, and circadian oscillations of several core clock genes are altered with a delayed phase in Ksrp^−/− livers. Diurnal expression of some lipid metabolism genes is also disturbed with reduced expression of genes involved in de novo lipogenesis. Using primary hepatocytes, we demonstrate that KSRP promotes decay of Per2 mRNA through an RNA-protein interaction and show that increased Per2 expression is responsible for the phase delay in cycling of several clock genes in the absence of KSRP. Similar to Ksrp^−/− livers, both expression of lipogenic genes and intracellular TG levels are also reduced in Ksrp^−/− hepatocytes due to increased Per2 expression. Using heterologous mRNA reporters, we show that the AU-rich element-containing 3′ untranslated region of Per2 is responsible for KSRP-dependent mRNA decay. These findings implicate that KSRP is an important regulator of circadian expression of lipid metabolism genes in the liver likely through controlling Per2 mRNA stability.     

Bird movements at rotor heights measured continuously with vertical radar at a Dutch offshore wind farm

Assessing the impacts of avian collisions with wind turbines requires reliable estimates of avian flight intensities and altitudes, to enable accurate estimation of collision rates, avoidance rates and related effects on populations. At sea, obtaining such estimates visually is limited not only by weather conditions but, more importantly, because a high proportion of birds fly at night and at heights above the range of visual observation. We used vertical radar with automated bird‐tracking software to overcome these limitations and obtain data on the magnitude, timing and altitude of local bird movements and seasonal migration measured continuously at a Dutch offshore wind farm. An estimated 1.6 million radar echoes representing individual birds or flocks were recorded crossing the wind farm annually at altitudes between 25 and 115 m (the rotor‐swept zone). The majority of these fluxes consisted of gull species during the day and migrating passerines at night. We demonstrate daily, monthly and seasonal patterns in fluxes at rotor heights and the influence of wind direction on flight intensity. These data are among the first to show the magnitude and variation of low‐altitude flight activity across the North Sea, and are valuable for assessing the consequences of developments such as offshore wind farms for birds.     

Using miniaturized radiotelemetry to discover the breeding grounds of the endangered New Zealand Storm Petrel Fregetta maoriana

Identification of breeding sites remains a critical step in species conservation, particularly in procellariiform seabirds whose threat status is of global concern. We designed and conducted an integrative radiotelemetry approach to uncover the breeding grounds of the critically endangered New Zealand Storm Petrel Fregetta maoriana (NZSP), a species considered extinct before its rediscovery in 2003. Solar‐powered automated radio receivers and hand‐held telemetry were used to detect the presence of birds on three island groups in the Hauraki Gulf near Auckland, New Zealand. At least 11 NZSP captured and radiotagged at sea were detected at night near Te Hauturu‐o‐Toi/Little Barrier Island with the detection of an incubating bird leading to the discovery of the first known breeding site for this species. In total, four NZSP breeding burrows were detected under mature forest canopy and three adult NZSP and two NZSP chicks were ringed. Telemetry data indicated NZSP showed strong moonlight avoidance behaviour over the breeding site, had incubation shifts of approximately 5 days and had a breeding season extending from February to June/July, a different season from other Procellariiformes in the region. Radiotelemetry, in combination with rigorously collected field data on species distribution, offers a valuable technique for locating breeding grounds of procellariiform seabirds and gaining insights into breeding biology while minimizing disturbance to sensitive species or damage to fragile habitat. Our study suggests an avenue for other breeding ground searches in one of the most threatened avian Orders, and highlights the general need for information on the location of breeding sites and understanding the breeding biology in data‐deficient birds.     

Selective Sensitization of Zinc Finger Protein Oxidation by Reactive Oxygen Species through Arsenic Binding

Cysteine oxidation induced by reactive oxygen species (ROS) on redox-sensitive targets such as zinc finger proteins plays a critical role in redox signaling and subsequent biological outcomes. We found that arsenic exposure led to oxidation of certain zinc finger proteins based on arsenic interaction with zinc finger motifs. Analysis of zinc finger proteins isolated from arsenic-exposed cells and zinc finger peptides by mass spectrometry demonstrated preferential oxidation of C3H1 and C4 zinc finger configurations. C2H2 zinc finger proteins that do not bind arsenic were not oxidized by arsenic-generated ROS in the cellular environment. The findings suggest that selectivity in arsenic binding to zinc fingers with three or more cysteines defines the target proteins for oxidation by ROS. This represents a novel mechanism of selective protein oxidation and demonstrates how an environmental factor may sensitize certain target proteins for oxidation, thus altering the oxidation profile and redox regulation.     

On the serial homology of the pectoral and pelvic girdles of tetrapods

While fore‐ and hindlimbs are commonly assumed to be serially homologous, the serial homology of the pectoral and pelvic girdles is more ambiguous. We investigate the degree to which a common history, developmental program, and gene network are shared between the girdles relative to the rest of the appendicular skeleton. Paleontological data indicate that pectoral appendages arose millions of years before pelvic appendages. Recent embryological and genetic data suggest that the anatomical similarity between the fore‐ and hindlimbs arose through the sequential, derived deployment of similar developmental programs and gene networks, and is therefore not due to ancestral serial homology. Much less developmental work has however been published about the girdles. Here, we provide the first detailed review of the developmental programs and gene networks of the pectoral and pelvic girdles. Our review shows that, with respect to these programs and networks, there are fewer similarities between pelvic and pectoral girdles than there are between the limbs. The available data therefore support recent hypotheses that the anatomical similarities between the fore‐ and hindlimbs arose during the fin‐to‐limb transition through the derived co‐option of similar developmental mechanisms, while the phylogenetically older pectoral and pelvic girdles have remained more distinct since their evolutionary origin.