In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

Enzymes of the AlkB and CYP153 families catalyze the first step in the catabolism of medium-chain-length alkanes, selective oxidation of the alkane to the 1-alkanol, and enable their host organisms to utilize alkanes as carbon sources. Small, gaseous alkanes, however, are converted to alkanols by evolutionarily unrelated methane monooxygenases. Propane and butane can be oxidized by CYP enzymes engineered in the laboratory, but these produce predominantly the 2-alkanols. Here we report the in vivo-directed evolution of two medium-chain-length terminal alkane hydroxylases, the integral membrane di-iron enzyme AlkB from Pseudomonas putida GPo1 and the class II-type soluble CYP153A6 from Mycobacterium sp. strain HXN-1500, to enhance their activity on small alkanes. We established a P. putida evolution system that enables selection for terminal alkane hydroxylase activity and used it to select propane- and butane-oxidizing enzymes based on enhanced growth complementation of an adapted P. putida GPo12(pGEc47{Delta}B) strain. The resulting enzymes exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation. This in vivo evolution system could be useful for directed evolution of enzymes that function efficiently to hydroxylate small alkanes in engineered hosts

The Highly Unusual Chemical Composition of the Hercules Dwarf Spheroidal Galaxy

We report on the abundance analysis of two red giants in the faint Hercules dwarf spheroidal (dSph) galaxy. These stars show a remarkable deficiency in the neutron-capture elements, while the hydrostatic alpha-elements (O, Mg) are strongly enhanced. Our data indicate [Ba/Fe] and [Mg/Fe] abundance ratios of <-2 dex and ~+0.8 dex, respectively, with essentially no detection of other n-capture elements. In contrast to the only other dSph star with similar abundance patterns, Dra 119, which has a very low metallicity at [Fe/H]=-2.95 dex, our objects, at [Fe/H]~-2.0 dex, are only moderately metal poor. The measured ratio of hydrostatic/explosive alpha-elements indicates that high-mass (~35 M_sun) Type II supernovae progenitors are the main, if not only, contributors to the enrichment of this galaxy. This suggests that star formation and chemical enrichment in the ultrafaint dSphs proceeds stochastically and inhomogeneously on small scales, or that the IMF was strongly skewed to high mass stars. The neutron capture deficiencies and the [Co/Fe] and [Cr/Fe] abundance ratios in our stars are similar to those in the extremely low metallicity Galactic halo. This suggests that either our stars are composed mainly of the ejecta from the first, massive, population III stars (but at moderately high [Fe/H]), or that SN ejecta in the Hercules galaxy were diluted with ~30 times less hydrogen than typical for extreme metal-poor stars.Comment: 5 pages, 3 figures, accepted by Astrophysical Journal Letter

Tug-of-war in motility assay experiments

The dynamics of two groups of molecular motors pulling in opposite directions on a rigid filament is studied theoretically. To this end we first consider the behavior of one set of motors pulling in a single direction against an external force using a new mean-field approach. Based on these results we analyze a similar setup with two sets of motors pulling in opposite directions in a tug-of-war in the presence of an external force. In both cases we find that the interplay of fluid friction and protein friction leads to a complex phase diagram where the force-velocity relations can exhibit regions of bistability and spontaneous symmetry breaking. Finally, motivated by recent work, we turn to the case of motility assay experiments where motors bound to a surface push on a bundle of filaments. We find that, depending on the absence or the presence of a bistability in the force-velocity curve at zero force, the bundle exhibits anomalous or biased diffusion on long-time and large-length scales

A novel universal algorithm for filament network tracing and cytoskeleton analysis

The rapid development of advanced microscopy techniques over recent decades has significantly increased the quality of imaging and our understanding of subcellular structures, such as the organization of the filaments of the cytoskeleton using fluorescence and electron microscopy. However, these recent improvements in imaging techniques have not been matched by similar development of techniques for computational analysis of the images of filament networks that can now be obtained. Hence, for a wide range of applications, reliable computational analysis of such two-dimensional methods remains challenging. Here, we present a new algorithm for tracing of filament networks. This software can extract many important parameters from grayscale images of filament networks, including the mesh hole size, and filament length and connectivity (also known as Coordination Number). In addition, the method allows sub-networks to be distinguished in two-dimensional images using intensity thresholding. We show that the algorithm can be used to analyze images of cytoskeleton networks obtained using different advanced microscopy methods. We have thus developed a new improved method for computational analysis of two-dimensional images of filamentous networks that has wide applications for existing imaging techniques. The algorithm is available as open-source software

Heel impact forces during barefoot versus minimally shod walking among Tarahumara subsistence farmers and urban Americans

Despite substantial recent interest in walking barefoot and in minimal footwear, little is known about potential differences in walking biomechanics when unshod versus minimally shod. To test the hypothesis that heel impact forces are similar during barefoot and minimally shod walking, we analysed ground reaction forces recorded in both conditions with a pedography platform among indigenous subsistence farmers, the Tarahumara of Mexico, who habitually wear minimal sandals, as well as among urban Americans wearing commercially available minimal sandals. Among both the Tarahumara (n = 35) and Americans (n = 30), impact peaks generated in sandals had significantly (p < 0.05) higher force magnitudes, slower loading rates and larger vertical impulses than during barefoot walking. These kinetic differences were partly due to individuals' significantly greater effective mass when walking in sandals. Our results indicate that, in general, people tread more lightly when walking barefoot than in minimal footwear. Further research is needed to test if the variations in impact peaks generated by walking barefoot or in minimal shoes have consequences for musculoskeletal health

Five Kepler target stars that show multiple transiting exoplanet candidates

We present and discuss five candidate exoplanetary systems identified with the Kepler spacecraft. These five systems show transits from multiple exoplanet candidates. Should these objects prove to be planetary in nature, then these five systems open new opportunities for the field of exoplanets and provide new insights into the formation and dynamical evolution of planetary systems. We discuss the methods used to identify multiple transiting objects from the Kepler photometry as well as the false-positive rejection methods that have been applied to these data. One system shows transits from three distinct objects while the remaining four systems show transits from two objects. Three systems have planet candidates that are near mean motion commensurabilities---two near 2:1 and one just outside 5:2. We discuss the implications that multitransiting systems have on the distribution of orbital inclinations in planetary systems, and hence their dynamical histories; as well as their likely masses and chemical compositions. A Monte Carlo study indicates that, with additional data, most of these systems should exhibit detectable transit timing variations (TTV) due to gravitational interactions---though none are apparent in these data. We also discuss new challenges that arise in TTV analyses due to the presence of more than two planets in a system.Comment: Accepted to Ap

The SPLASH Survey: A Spectroscopic Analysis of the Metal-Poor, Low-Luminosity M31 dSph Satellite Andromeda X

Andromeda X (And X) is a newly discovered low-luminosity M31 dwarf spheroidal galaxy (dSph) found by Zucker et al. (2007) in the Sloan Digital Sky Survey (SDSS - York et al. 2000). In this paper, we present the first spectroscopic study of individual red giant branch stars in And X, as a part of the SPLASH Survey (Spectroscopic and Photometric Landscape of Andromeda's Stellar Halo). Using the Keck II telescope and multiobject DEIMOS spectrograph, we target two spectroscopic masks over the face of the galaxy and measure radial velocities for ~100 stars with a median accuracy of sigma_v ~ 3 km/s. The velocity histogram for this field confirms three populations of stars along the sight line: foreground Milky Way dwarfs at small negative velocities, M31 halo red giants over a broad range of velocities, and a very cold velocity ``spike'' consisting of 22 stars belonging to And X with v_rad = -163.8 +/- 1.2 km/s. By carefully considering both the random and systematic velocity errors of these stars (e.g., through duplicate star measurements), we derive an intrinsic velocity dispersion of just sigma_v = 3.9 +/- 1.2 km/s for And X, which for its size, implies a minimum mass-to-light ratio of M/L =37^{+26}_{-19} assuming the mass traces the light. Based on the clean sample of member stars, we measure the median metallicity of And X to be [Fe/H] = -1.93 +/- 0.11, with a slight radial metallicity gradient. The dispersion in metallicity is large, sigma([Fe/H]) = 0.48, possibly hinting that the galaxy retained much of its chemical enrichment products. We discuss the potential for better understanding the formation and evolution mechanisms for M31's system of dSphs through (current) kinematic and chemical abundance studies, especially in relation to the Milky Way sample. (abridged version)Comment: Accepted for Publication in Astrophys. J. 14 pages including 7 figures and 2 tables (journal format

Validation of the SNACOR clinical scoring system after transarterial chemoembolisation in patients with hepatocellular carcinoma

Background Transarterial chemoembolisation is the standard of care for intermediate stage (BCLC B) hepatocellular carcinoma, but it is challenging to decide when to repeat or stop treatment. Here we performed the first external validation of the SNACOR (tumour Size and Number, baseline Alpha-fetoprotein, Child-Pugh and Objective radiological Response) risk prediction model. Methods A total of 1030 patients with hepatocellular carcinoma underwent transarterial chemoembolisation at our tertiary referral centre from January 2000 to December 2016. We determined the following variables that were needed to calculate the SNACOR at baseline: tumour size and number, alpha-fetoprotein level, Child-Pugh class, and objective radiological response after the first transarterial chemoembolisation. Overall survival, time-dependent area under receiver-operating characteristic curves, Harrell’s C-index, and the integrated Brier score were calculated to assess predictive ability. Finally, multivariate analysis was performed to identify independent predictors of survival. Results The study included 268 patients. Low, intermediate, and high SNACOR scores predicted a median survival of 31.5, 19.9, and 9.2 months, respectively. The areas under the receiver-operating characteristic curve for overall survival were 0.641, 0.633, and 0.609 at 1, 3, and 6 years, respectively. Harrell’s C-index was 0.59, and the integrated Brier Score was 0.175. Independent predictors of survival included tumour size (P < 0.001), baseline alpha-fetoprotein level (P < 0.001) and Child-Pugh class (P < 0.004). Objective radiological response (P = 0.821) and tumour number (P = 0.127) were not additional independent predictors of survival. Conclusions The SNACOR risk prediction model can be used to identify patients with a dismal prognosis after the first transarterial chemoembolisation who are unlikely to benefit from further transarterial chemoembolisation. However, Harrell’s C-index showed only moderate performance. Accordingly, this risk prediction model can only serve as one of several components used to make the decision about whether to repeat treatment

Impact evaluation report: Egypt’s Takaful Cash Transfer Program: Second round report

Egypt introduced the Takaful and Karama Program (TKP), a pair of targeted cash transfer schemes in 2015. In 2018, the International Food Policy Research Institute (IFPRI) completed the first round of impact evaluation of TKP, based on household survey data collected after the first 15 months of the program. In the period between the first-round evaluation and the data collection for this report in January 2022, Egypt faced an enormous economic shock in the COVID-19 pandemic with a complete loss of tourism, which before the crisis was responsible for 12% of GDP and 10% of employment (IMF, 2021). A follow-up evaluation was designed to assess whether impacts estimated from the first round have been sustained and whether longer duration of treatment has led to impacts on additional outcome variables. This follow-up impact evaluation was conducted using a regression discontinuity (RD) design similar to the first round but using a different sample of households much more tightly concentrated around the 4500 thresholds to address concerns about the smaller discontinuity. We find that households invested in assets, particularly productive assets and Takaful households reduced their debt burdens. There were no measurable impacts of the Takaful program on household consumption or poverty, which may be partially explained by changes in household demographics. We also find that Takaful improved school enrollment and attendance and contributed to household’s ability to cope with shocks