Associating somatic mutation with clinical outcomes through kernel regression and optimal transport

Somatic mutations in cancer patients are inherently sparse and potentially high dimensional. Cancer patients may share the same set of deregulated biological processes perturbed by different sets of somatically mutated genes. Therefore, when assessing the associations between somatic mutations and clinical outcomes, gene-by-gene analysis is often under-powered because it does not capture the complex disease mechanisms shared across cancer patients. Rather than testing genes one by one, an intuitive approach is to aggregate somatic mutation data of multiple genes to assess their joint association with clinical outcomes. The challenge is how to aggregate such information. Building on the optimal transport method, we propose a principled approach to estimate the similarity of somatic mutation profiles of multiple genes between tumor samples, while accounting for gene–gene similarities defined by gene annotations or empirical mutational patterns. Using such similarities, we can assess the associations between somatic mutations and clinical outcomes by kernel regression. We have applied our method to analyze somatic mutation data of 17 cancer types and identified at least five cancer types, where somatic mutations are associated with overall survival, progression-free interval, or cytolytic activity.     

Surface imprinted bio-nanocomposites for affinity separation of a cellular DNA repair protein

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional DNA repair protein localized in different subcellular compartments. The mechanisms responsible for the highly regulated subcellular localization and “interactomes” of this protein are not fully understood but have been closely correlated to the posttranslational modifications in different biological context. In this work, we attempted to develop a bio-nanocomposite with antibody-like properties that could capture APE1 from cellular matrices to enable the comprehensive study of this protein. By fixing the template APE1 on the avidin-modified surface of silica-coated magnetic nanoparticles, we first added 3-aminophenylboronic acid to react with the glycosyl residues of avidin, followed by addition of 2-acrylamido-2-methylpropane sulfonic acid as the second functional monomer to perform the first step imprinting reaction. To further enhance the affinity and selectivity of the binding sites, we carried out the second step imprinting reaction with dopamine as the functional monomer. After the polymerization, we modified the nonimprinted sites with methoxypoly (ethylene glycol) amine (mPEG-NH₂). The resulting molecularly imprinted polymer-based bio-nanocomposite showed high affinity, specificity, and capacity for template APE1. It allowed for the extraction of APE1 from the cell lysates with high recovery and purity. Moreover, the bound protein could be effectively released from the bio-nanocomposite with high activity. The bio-nanocomposite offers a very useful tool for the separation of APE1 from various complex biological samples.     

High-efficiency and multilocus targeted integration in CHO cells using CRISPR-mediated donor nicking and DNA repair inhibitors

Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all-in-one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site-specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double-cut and double-nick donor formats significantly improved targeting accuracy by 2.3–8.3-fold and 19–22-fold, respectively, compared to standard circular donors. Notably, Cas9-mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low-fidelity outcomes. A screen of 10 molecules that modulate the major mammalian DNA repair pathways identified two inhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%–55% efficiency at two loci simultaneously in selected clones. The CRISPR-based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications.     

MORTALITY OF SEA OTTERS IN PRINCE WILLIAM SOUND FOLLOWING THE EXXON VALDEZ OIL SPILL

Abstract: This paper presents an estimate of the total number of sea otters that died as a direct consequence of the oil spill that occurred when the T/V Exxon Valdez grounded in Prince William Sound, Alaska on 24 March 1989. We compared sea otter counts conducted from small boats throughout the Sound during the summers of 1984 and 1985 to counts made after the spill during the summer of 1989. We used ratio estimators, corrected for sighting probability, to calculate otter densities and population estimates for portions of the Sound affected by the oil spill. We estimated the otter population in the portion of Prince William Sound affected by the oil was 6,546 at the time of the spill and that the post-spill population in the summer of 1989 was 3,898, yielding a loss estimate of approximately 2,650. Bootstrapping techniques were used to approximate confidence limits on the loss estimate of about 500–5,000 otters. The wide confidence limits are a result of the complex scheme required to estimate losses and limitations of the data. Despite the uncertainty of the loss estimate it is clear that a significant fraction of the otters in the spill zone survived. We observed otters persisting in relatively clean embayments throughout the oil spill zone suggesting that the highly convoluted coastline of Prince William Sound produced refuges that allowed some sea otters in the oil spill area to survive.     

SURVIVAL RATES FOR THE HAWAIIAN MONK SEAL (MONACHUS SCHAUINSLANDI)

Abstract: Endangered Hawaiian monk seal ( Monachs schauinslandi ) pups at all the major breeding islands in the Northwestern Hawaiian Islands have been tagged since the early 1980s. Pups were double flipper tagged as soon as possible post-weaning. With few exceptions, an extensive tag resighting effort was conducted annually at the same islands. These resighting data were used to estimate seal survival rates from the time of tagging to age one at all locations using the ratio of seals alive in the second year to number of pups tagged. These survival rates among the islands, from weaning to age one, averaged over the years of the study, ranged from 0.80 to 0.90. For young seals over age one, capture-recapture methods were used to calculate survival pooled through several years, and these rates ranged from 0.85 to 0.98. At French Frigate Shoals and Laysan Island, the higher numbers of tagged pups allowed separate estimates of male and female survival to be calculated. These rates suggested that survival of immature females was better than males. Beginning in 1989, survival of immature seals at French Frigate Shoals declined sharply.     

Phosphorylation states greatly regulate the activity and gating properties of Cav3.1 T-type Ca2+ channels

Ca_v3.1 T-type Ca²⁺ channels play pivotal roles in neuronal low-threshold spikes, visceral pain, and pacemaker activity. Phosphorylation has been reported to potently regulate the activity and gating properties of Ca_v3.1 channels. However, systematic identification of phosphorylation sites (phosphosites) in Ca_v3.1 channel has been poorly investigated. In this work, we analyzed rat Ca_v3.1 protein expressed in HEK-293 cells by mass spectrometry, identified 30 phosphosites located at the cytoplasmic regions, and illustrated them as a Ca_v3.1 phosphorylation map which includes the reported mouse Ca_v3.1 phosphosites. Site-directed mutagenesis of the phosphosites to Ala residues and functional analysis of the phospho-silent Ca_v3.1 mutants expressed in Xenopus oocytes showed that the phospho-silent mutation of the N-terminal Ser18 reduced its current amplitude with accelerated current kinetics and negatively shifted channel availability. Remarkably, the phospho-silent mutations of the C-terminal Ser residues (Ser1924, Ser2001, Ser2163, Ser2166, or Ser2189) greatly reduced their current amplitude without altering the voltage-dependent gating properties. In contrast, the phosphomimetic Asp mutations of Ca_v3.1 on the N- and C-terminal Ser residues reversed the effects of the phospho-silent mutations. Collectively, these findings demonstrate that the multiple phosphosites of Ca_v3.1 at the N- and C-terminal regions play crucial roles in the regulation of the channel activity and voltage-dependent gating properties.     

Investigation of <i>GhFAT</i> Genes Related to Seed Oil Content and Fatty Acid Composition in <i>Gossypium hirsutum</i> L.

Fatty Acyl-ACP thioesterase (FAT) is a key enzyme controlling oil biosynthesis in plant seeds. FATs can be divided into two subfamilies, FATA and FATB according to their amino acid sequences and substrate specificity. The Upland cotton genome contains 20 GhFAT genes, amongst which 6 genes were of the GhFATA subfamily and 14 of the GhFATB subfamily. The 20 GhFAT genes are unevenly distributed on 14 chromosomes. The GhFATA genes have 5 or 7 exons and the GhFATB genes have 6 or 7 exons. All GhFAT proteins have the conserved Acyl-ACP_TE domain and PLN02370 super family, the typical characteristics of plant thioesterases. Analyses of the expression level of GhFATs and the compositions of fatty acid in 5–60 days-post-anthesis seeds showed that the ratio of saturated fatty acids to unsaturated fatty acids was consistent with the expression profile of GhFATB12, GhFATB3, and GhFATB10; the ratio of monounsaturated fatty acid to polyunsaturated fatty acids was consistent with the expression profile of GhFATA3. The oil contents of mature cottonseeds were positively correlated with the contents of palmitic acid and linolenic acid as well as seed vigor. These results provide essential information for further exploring the role(s) of the specific GhFATs in determining oil biosynthesis and cottonseed compositions.     

D-amino acid levels in human physiological fluids

Plasma, urine, cerebrospinal fluid (CSF), and amniotic fluid were examined to determine whether free D-amino acids were present and if so at what levels. It was found that D-amino acids exist in all physiological fluids tested, but that their level varied, considerably. The lowest levels of D-amino acids were usually found in amniotic fluid or CSF (almost always <1% of the corresponding L-amino acid). The highest levels were found in urine (usually tenth percent to low percent levels). Pipecolic acid seemed to be different from the other amino acids tested in that it was excreted primarily as the D-enantiomer (often >90%). Correspondingly high levels of D-pipecolic acid were not found in plasma. Some of the trends found in this work seemed to be analogous to those found in a recent rodent study. © 1993 Wiley-Liss, Inc.     

An ancient enzyme finds a new home: Prevalence and neofunctionalization of trypsin in marine phytoplankton

Trypsin is an ancient protease best known as a digestive enzyme in animals, and traditionally believed to be absent in plants and protists. However, our recent studies have revealed its wide presence and important roles in marine phytoplankton. Here, to gain a better understanding on the importance of trypsin in phytoplankton, we further surveyed the distribution, diversity, evolution and potential ecological roles of trypsin in global ocean phytoplankton. Our analysis indicated that trypsin is widely distributed both taxonomically and geographically in marine phytoplankton. Furthermore, by systematic comparative analyses we found that algal trypsin could be classified into two subfamilies (trypsin I and trypsin II) and exhibited highly duplicated and diversified during evolution. We also observed markedly different domain sequences and organizations between and within the subfamilies, suggesting potential neofunctionalization. Diatoms contain both subfamilies of trypsin, with higher numbers of genes and more environment-responsive expression of trypsin than other lineages. The duplication and subsequent neofunctionalization of the trypsin family may be important in diatoms for adapting to dynamical environmental conditions, contributing to diatoms' dominance in the coastal oceans. This work advances our knowledge on the distribution and neofunctionalization of this ancient enzyme and creates a new window of research on phytoplankton biology.     

Effect of light intensity and ammonium-N on carotenogenesis ofTrentepohlia odorata andDunaliella bardawil

AxenicTrentepohlia odorata was cultured at three different NH₄Cl levels (3.5 × 10^–2, 3.5 × 10^–3, 3.5 × 10^–4 M) and three different light intensities (48, 76, 122 µmol m^–2 s^–1). Chloride had no effect on growth over this range of concentration. High light intensity and high NH₄Cl concentration enhanced the specific growth rate. The carotenoid content increased under a combination of high light intensity and low N concentration. WhenD. bardawil was exposed to the same combination of growth conditions, there was an increase in its carotenoid content. The light saturation and the light inhibition constants (K _s andK _i, respectively) for growth, and the saturation constant (K _m) for NH₄Cl were determined. TheK _s andK _i values were higher inT. odorata (66.7 and> 122 mol m^–2 s^–1, respectively) than inD. bardawil (5.1 and 14.7 µmol m^–2 s^–1, respectively). TheK _m value determined at 122 µmol m^–2 s^–1, however, was lower inT. odorata (0.048 µM) than inD. bardawil (0.062 µM).Author for correspondence