Assigning alleles to different loci in amplifications of duplicated loci

Duplicated loci, for example those associated with major histocompatibility complex (MHC) genes, often have similar DNA sequences that can be coamplified with a pair of primers. This results in genotyping difficulties and inaccurate analyses. Here, we present a method to assign alleles to different loci in amplifications of duplicated loci. This method simultaneously considers several factors that may each affect correct allele assignment. These are the sharing of identical alleles among loci, null alleles, copy number variation, negative amplification, heterozygote excess or heterozygote deficiency, and linkage disequilibrium. The possible multilocus genotypes are extracted from the alleles for each individual and weighted to estimate the allele frequencies. The likelihood of an allele configuration is calculated and is optimized with a heuristic algorithm. Monte‐Carlo simulations and three empirical MHC data sets are used as examples to evaluate the efficacy of our method under different conditions. Our new software, mhc‐typer V1.1, is freely available at https://github.com/huangkang1987/mhc-typer .     

Invasion by activated macrophages requires delivery of nascent membrane‐type‐1 matrix metalloproteinase through late endosomes/lysosomes to the cell surface

Macrophage migration into injured or infected tissue is a key aspect in the pathophysiology of many diseases where inflammation is a driving factor. Membrane‐type‐1 matrix metalloproteinase (MT1‐MMP) cleaves extracellular matrix components to facilitate invasion. Here we show that, unlike the constitutive MT1‐MMP surface recycling seen in cancer cells, unactivated macrophages express low levels of MT1‐MMP. Upon lipopolysaccharide (LPS) activation, MT1‐MMP synthesis dramatically increases 10‐fold at the surface by 15 hours. MT1‐MMP is trafficked from the Golgi complex to the surface via late endosomes/lysosomes in a pathway regulated by the late endosome/lysosome R‐SNAREs VAMP7 and VAMP8. These form two separate complexes with the surface Q‐SNARE complex Stx4/SNAP23 to regulate MT1‐MMP delivery to the plasma membrane. Loss of either one of these SNAREs leads to a reduction in surface MT1‐MMP, gelatinase activity and reduced invasion. Thus, inhibiting MT1‐MMP transport through this pathway could reduce macrophage migration and the resulting inflammation.     

Vertical mixing in a shallow tropical reservoir

Limnology - This paper presents observations of diurnal cycles of stratification and vertical mixing in Kranji Reservoir, a shallow tropical reservoir with an average depth of 6.7 m...     

Do intraspecific life history patterns follow interspecific predictions? A test using latitudinal variation in ringed seals

Mammals adapted to unpredictable and low-energy environments often evolve a “bet-hedging” life history strategy characterized by less costly reproductive outputs over a longer and slower-growing life. In contrast, species adapted to more predictable (i.e., low variation) and higher energy environments may evolve greater fecundity over a shorter and faster-growing life. We tested whether this known interspecific pattern also occurs within a species. We compared life history traits of the ringed seal (Pusa hispida) in the Canadian High Arctic to those closer to the southern limit of the species' circumpolar distribution. We found that northern seals grew slower than southern seals (Brody growth coefficient), achieved a greater asymptotic body weight (82 and 69 kg vs. 74 and 54 kg female and male, respectively), reached sexual maturity later (6.1 years vs. 4.5 years), had lower fecundity (1.8 years vs. 1.3 years interbirth interval), longer average lifespan (5 years vs. 3 years median age), and greater movements (1,269 vs. 681 km). Mating systems also likely differed with northern seals showing morphological evidence of a promiscuous mating system with potential sperm competition as indicated by greater relative testes size. The northern region was also characterized by more unpredictable environmental timing of seasonal events, such as spring sea ice breakup. Life history variation between the intraspecific groups of seals appears to agree with interspecific patterns and provides a better understanding of how species' life history parameters shift in concert with environmental conditions.     

Angular range,sampling and noise considerations for inverse light scattering analysis of nuclear morphology

In recent years, significant work has been devoted to the use of angle‐resolved elastic scattering for the extraction of nuclear morphology in tissue. By treating the nucleus as a Mie scattering object, techniques such as angle‐resolved low‐coherence interferometry (a/LCI) have demonstrated substantial success in identifying nuclear alterations associated with dysplasia. Because optical biopsies are inherently noninvasive, only a small, discretized portion of the 4π scattering field can be collected from tissue, limiting the amount of information available for diagnostic purposes. In this work, we comprehensively characterize the diagnostic impact of variations in angular sampling, range and noise for inverse light scattering analysis of nuclear morphology, using a previously reported dataset from 40 patients undergoing a/LCI optical biopsy for cervical dysplasia. The results from this analysis are applied to a benchtop scanning a/LCI system which compromises angular range for wide‐area scanning capability. This work will inform the design of next‐generation optical biopsy probes by directing optical design towards parameters which offer the most diagnostic utility.      

Effect of siRNA nuclease stability on the in vitro and in vivo kinetics of siRNA-mediated gene silencing

Small interfering RNA (siRNA) molecules achieve sequence-specific gene silencing through the RNA interference (RNAi) mechanism. Here, live-cell and live-animal bioluminescent imaging (BLI) is used to directly compare luciferase knockdown by unmodified and nuclease-stabilized siRNAs in rapidly (HeLa) and slowly (CCD-1074Sk) dividing cells to reveal the impact of cell division and siRNA nuclease stability on the kinetics of siRNA-mediated gene silencing. Luciferase knockdown using unmodified siRNAs lasts approximately 1 week in HeLa cells and up to 1 month in CCD-1074Sk cells. There is a slight increase in the duration of luciferase knockdown by nuclease-stabilized siRNAs relative to unmodified siRNAs after cationic lipid transfection, but this difference is not observed after electroporation. In BALB/cJ mice, a fourfold increase in maximum luciferase knockdown is observed after hydrodynamic injection (HDI) of nuclease-stabilized siRNAs relative to unmodified siRNAs, yet the overall kinetics of the recovery after knockdown are nearly identical. By using a mathematical model of siRNA-mediated gene silencing, the trends observed in the experimental data can be duplicated by changing model parameters that affect the stability of the siRNAs before they reach the cytosolic compartment. Based on these findings, we hypothesize that the stabilization advantages of nuclease-stabilized siRNAs originate primarily from effects prior to and during internalization before the siRNAs can interact with the intracellular RNAi machinery.     

Synthesis and reactivity of nonstabilized diazo sugars

1,6-Anhydro-4-deoxy-4-diazo-2,3-O-isopropylidene-beta-D-lyxo-hexopyranose (4) is a stable crystalline compound readily accessible by an improved synthetic procedure. It has been used as a model for evaluating the reactivity of the diazo group, when not stabilized by an adjacent carbonyl function, in a rigid chiral matrix. A range of carbene-type, electrophile-promoted, and 1,3-dipolar reactions were evaluated, leading to 4,4'-alkene dimers, 4-deoxy-3-enose and related derivatives, 4,4-dihalo compounds, 4-spirocyclopropane derivatives, 4-spiropyrazole structures, and by skeletal rearrangement, branched-chain anhydropentose structures having a bicyclo[2.2.2] skeleton.