Miracle-dz/UniFAR-FTUs huggingface dataset:
A Golden Gate-based Protocol for Assembly of Multiplexed gRNA Expression Arrays for CRISPR/Cas9

[Abstract] The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) has become the most broadly used and powerful tool for genome editing. Many applications of CRISPR-Cas9 require the delivery of multiple small guide RNAs (gRNAs) into the same cell in order to achieve multiplexed gene editing or regulation. Using traditional co-transfection of single gRNA expression vectors, the likelihood of delivering several gRNAs into the same cell decreases in accordance with the number of gRNAs. Thus, we have developed a method to efficiently assemble gRNA expression cassettes (2-30 gRNAs) into one single vector using a Golden-Gate assembly method (Vad-Nielsen et al., 2016). In this protocol, we describe the detailed step-by-step instructions for assembly of the multiplexed gRNA expression array. The gRNA scaffold used in our expression array is the gRNA 1.0 system for the Cas9 protein from Streptococcus pyogenes driven by the human U6 promoter.
Inoculación con consorcios nativos de hongos de micorriza arbuscular en Agave angustifolia Haw.

El objetivo de éste trabajo fue determinar la compatibilidad funcional de inóculos nativos de hongos de micorriza arbuscular (HMA) en Agave angustifolia, utilizando parámetros de crecimiento y nutrición como indicadores. En el invernadero del Laboratorio de Suelos del CIIDIR- Unidad Oaxaca, en 2009, se realizó un experimento bifactorial con arreglo combinatorio con cuatro repeticiones y distribución de tratamientos en bloques aleatorizados completos. Los factores experimentales y sus niveles fueron: factor A: plántulas de maguey propagadas por hijuelos vegetativos (HI) y por bulbilos florales (BU); factor B: seis inóculos de consorcios nativos de HMA provenientes del distrito de Tlacolula, Oaxaca (CN1 a CN6), control positivo Glomus intraradices (GI) y control absoluto sin inocular (CON). Las variables de respuesta fueron: altura de la planta, área foliar por planta, longitud y distribución de raíces y biomasa a la cosecha (pesos fresco y seco de hojas, tallo y raíces), concentración y contenido de N y P en hojas y colonización micorrízica. En HI se registró el mayor grado de compatibilidad con el inóculo CN2. En BU se registró compatibilidad con los inóculos CN4, CN6 y GI. Las plantas que fueron inoculadas con CN2 registraron los más altos valores en las variables indicadoras de compatibilidad, seguidas cercanamente por las que recibieron el inóculo CN3. Independientemente de su origen, las plántulas de A. angustifolia mostraron mejor compatibilidad funcional con inóculos nativos que con el inóculo de la especie introducida.
Microboring organisms in living stylasterid corals (Cnidaria, Hydrozoa)

ABSTRACT Microboring or euendolithic microorganisms, which colonize and penetrate various carbonate substrates, are abundant in coral reef ecosystems and play a major role in reef carbonate dissolution. A few studies reported the presence of euendoliths in stylasterid coral skeletons but the biological identity, distribution and abundance of these microorganisms remain largely unknown. Observations of over 100 stylasterid colonies, collected in the Indo-Pacific area, revealed for the first time that the association between these corals and euendolith organisms appears to be quite common in shallow tropical waters. The most abundant euendolith was identified as a cryptic stage in the development of the rhodophyte Porphyra (Conchocelis stage). The euendoliths were observed in the skeletons of seven species of three genera (four Stylaster, two Distichopora and one Lepidotheca). The presence of euendoliths inside skeletons conferred a particular colour to the studied stylasterid corals. Distribution and abundance of microborings varied significantly among stylasterid species and among branches of a single colony and so did the colour of their skeletons. Colonization of skeletons and the associated colour distribution were almost uniform in some stylasterids, forming an upward gradually diminishing or sharply limited gradient. This study shows that patterns of euendolith colonization and growth in stylasterid skeletons may depend on the stage of the euendolith development as well as on their environmental requirements such as light exposure.
Applying Corrections in Single-Molecule FRET

Single-molecule Förster resonance energy transfer (smFRET) experiments can detect the distance between a donor and an acceptor fluorophore on the 3-10nm scale. In ratiometric smFRET experiments, the FRET efficiency is estimated from the ratio of acceptor and total signal (donor + acceptor). An excitation scheme involving two alternating lasers (ALEX) is often employed to discriminate between singly– and doubly-labeled populations thanks to a second ratiometric parameter, the stoichiometry S. Accurate FRET and S estimations requires applying three well-known correction factors: donor emission leakage into the acceptor channel, acceptor direct excitation by the donor excitation laser and the “gamma factor” (i.e. correction for the imbalance between donor and acceptor signals due to different fluorophore’s quantum yields and photon detection effciencies). Expressions to directly correct both raw FRET and S values have been reported in [1] in the context of freely-diffusing smFRET. Here we extend Lee et al. work providing several expressions for the direct excitation coeffcient and highlighting a clear interpretation in terms of physical parameters and experimental quantities. Moreover, we derive a more complete set of analytic expressions for correcting FRET and S. We aim to provide a clear and concise reference for different definitions of correction coeffcients and correction formulas valid for any smFRET experiment both in immobilized and freely-diffusing form.
Role of the His-Asp Catalytic Dyad of Ribonuclease A

Hydrogen-bonded histidine and aspartate residues are found in the active sites of many proteases, lipases, and ribonucleases. We have used a variety of methods to probe the role of the catalytic dyad of bovine pancreatic ribonuclease A (RNase A). In RNase A, His119 and Asp121 form this dyad. Our work relied largely on the analysis of mutant enzymes in which Asp 121 was replaced by an asparagine or alanine residue, or in which His 119 was replaced by an alanine residue. Steady-state kinetic analyses revealed that Aspl21 has a nominal role in catalysis. Our data also showed that the two conformations occupied by His119-one in which it can interact with Asp121 and another. brought about through rotation about the Ca-Cf3 bond, in which it cannot-have similar catalytic activities. The pH-dependence of kcat and Km for the hydrolysis of uridine 2' ,3'-cyclic phosphate revealed an alternative pathway that depends on a group with a pKa of 5.0. Evidence was also found for a residue with a pKa of 3.8, which must be unprotonated to bind substrate. We postulate that this pKa arises from Asp83. IH-NMR titration curves showed that Asp121 has a minimal effect on the pKa of His I 19. In addition. titrations of the unliganded enzymes and the enzymes in the presence of two concentrations of the inhibitor uridine 3'-phosphate revealed a second molecule of inhibitor binds near the catalytic dyad. Surprisingly. the contribution of Asp121 to protein stability is more significant than its contribution to enzymatic catalysis. pH-Dependences of the unfolding transitions indicated that the pKa's of Asp 121 in RNase A in the native and the denatured states were 2.7 and 3.6, respectively, and that His 1 19 is largely responsible for this decrease in the pKa of Asp121. Compensating enthalpy-entropy changes. possibly due to the loss of a hydrogen bond between Ala121 and Lys66, resulted in equivalence in the stability ofDl21N and D121A m RNase A. The two conformations of His 1 19 were calculated to have Keq = 2.5 (PH 1.2) in favor of the interaction with Asp121. I would like to thank all the members of the Raines' lab who helped to make this dissertation possible. Of these, lowe much thanks to Martha Laboissiere for convincing me not to give up. to Brad Kelemen and June Messmore for their constructive criticism of this dissertation, and to Jed Thompson for synthesizing the substrate UpA used …
Squalene: A Trove of Metabolic Actions

Squalene is present in high concentration in the liver of certain sharks and in small concentrations in olive oil. Previous studies showed that its administration decreases hepatic steatosis in male Apoe-knockout mice, but these changes might be complex. Transcriptomics, using DNA microarrays, and proteomics from mitochondrial and microsomal fractions, analyzed by 2D-DIGE and mass spectrometry, were used in these mice that received 1 g/kg/day squalene for 10 weeks. Squalene administration significantly modified the expression of genes such as lipin 1 (Lpin1) and thyroid hormone responsive (Thrsp). Changes in methionine adenosyltransferase 1 alpha (Mat1α), short-chain specific acyl-CoA dehydrogenase (Acads), and thioredoxin domain–containing protein 5 (Txndc5) expressions were consistent with their protein levels. Their mRNA levels were associated with hepatic fat content. These results suggest that squalene action involves changes in hepatic gene expression associated with its anti-steatotic properties. This approach shows new connections between nutrition and gene expression since Txndc5, a gene with unknown biological function, was upregulated by squalene administration. Overall, this nutrigenomic approach illustrates the effects of squalene and provides further support to the idea that not all monounsaturated fatty acid–containing oils behave similarly. Therefore, selection of cultivars producing olive oils enriched in this compound will be a plus.
Dietary composition of endangered seahorses determined by stable isotope analysis

The lack of integrated measures for assessing the feeding ecology of seahorses may restrict the effectiveness of conservation actions on wild populations of worldwide threatened seahorse species. Identifying dietary sources will allow researchers to determine their degree of vulnerability to environmental changes, redefine their conservation status and apply appropriate management strategies. The resource use of the seahorse Hippocampus guttulatus inhabiting coastal waters of Galicia (north-western Iberian Peninsula) was assessed for three populations and 2 years using stable isotope mixing models. The Bayesian mixing model (MixSIAR) estimated the relative contributions of the dietary sources to the seahorse diet and revealed that Caprellidea were the primary source, followed by Gammaridea and Caridea. Mysidae and Annelida represented the less dominant prey. This prey preference can be explained by the foraging behaviour of seahorses. Different contributions of Gammaridea and Caridea to the diet were found among sites, indicating different habitat characteristics and hence different habitat use by seahorses within each site. In addition, differences were encountered among sexes. Caprellidea was the dominant prey for females, whereas Gammaridea was the dominant prey for males. The findings of the present study will contribute to the knowledge of feeding patterns of H. guttulatus, providing relevant data for conservation of this endangered species.
Intracellular Hydrogen Peroxide Detection with Functionalised Nanoelectrodes

Hydrogen peroxide (H2O2) is one of the most important reactive oxygen species and it is involved in a number of cellular processes ranging from signal transduction to immune-defence and oxidative stress. It is of great interest to intracellularly quantify H2O2 to improve the understanding of its role in disease processes. In this study, we present an amperometric nanosensor for the quantification of H2O2 at the single-cell level. Deposition of the electrocatalyst Prussian Blue on carbon nanoelectrodes enables the selective H2O2 reduction at mild potentials. Because of their small size and needle-type shape, these nanoelectrodes can penetrate the membrane of single living cells causing only minimal perturbation. The nanosen¬sors allow the monitoring of penetration-induced oxidative outbursts as well as the uptake of H2O2 from the extracellular environment in single murine macrophages.
Glutathione S-transferase P1, a novel downstream regulator of LRRK2 (G2019S)-induced cytotoxicity in neural cells

The enhanced neurotoxicity of the Parkinson’s disease-associated LRRK2 mutant, G2019S, than its wild-type counter-part has recently been reported. Overexpression of LRRK2 (G2019S) in cultured neural cells results in caspase-3-dependent apoptosis via a yet undefined signaling pathway. Elucidation of the mechanism underlying LRRK2 (G2019S) neurotoxicity may offer new insights into the pathogenesis of Parkinson’s disease. In this study, utilizing two-dimensional gel electrophoresis coupled with mass spectrometry, we have identified glutathione s-transferase P1 (GSTP1) as a selective target whose expression is negatively regulated at the transcriptional levels by LRRK2 (G2019S). Overexpression of LRRK2 (G2019S) in the human neuronal cell line SH-SY5Y markedly suppressed the expression of GSTP1 prior to any manifestation of cell death. This suppression of GSTP1 expression was due to LRRK2 (G2019S) – elicited production of peroxides and subsequent hyper-methylation on the promoter of GSTP1. Moreover, shRNA-mediated knockdown of endogenous GSTP1 expression exacerbated LRRK2 (G2019S) neurotoxicity, whereas overexpression of GSTP1 protected against LRRK2 (G2019S)-induced caspase-3 activation and neuronal apoptosis. In conclusion, the results suggest a previously undefined signaling mechanism underlying the neurotoxic effect of LRRK2 (G2019S), in which LRRK2 (G2019S) triggers oxidative stress in cells and, in turn, results in caspase-dependent apoptosis at least in part by suppressing the expression of GSTP1.
Incomputability at the foundations of physics

In this article, we show that once we assume that there is some uniformity in Nature, empirical laws and theories involving the concepts of Physics can be learned by the scientists. We develop a mathematical framework to explain learnability of empirical relations. Then we classify the degree of learnability according to the degree of predictability by means of timescales. We also use the same mathematical framework to make formal the claim that learnability evolves according not only with the number of observations done, but also with the precision of measurements.
Identification of Growth Hormone Releasing Hormone Gene in Local Buffalo (Bubalus bubalis) Using PCR-RFLP

GHRH is a hypothalamic hormone which stimulates growth hormone secretion in the pituitary gland. The objective of this study was to identify polymorphisms Growth Hormone Releasing Hormone (GHRH) gene of Indonesian buffalo’s. A total of 320 blood samples from Indonesian buffaloes were used to determine polymorphism using PCR-RFLP method. The polymorphism of GHRH gene that spanned within exon 2 and exon 3 was amplified, and their mutation was detected using endonuclease HaeIII. In this study, there were three GHRH/HaeIII genotype (AA genotype 0%, AB genotype 36% and BB genotype 64%) determined by two alleles, A (18%) and B (82%). The frequency of A allele was found 15% for Semarang population, 19% for Mataram population, 2% for Medan population and 40% for Banten population. The frequency of B allele was found 85% for Semarang population, 81% for Mataram population, 98% for Medan population and 60% for Banten population. The observed heterozygosis values were different among populations. The highest heterozygosis (ĥ) 0,485 for Banten population and the lowest was 0.037 for Medan population and the average heterozygosis for all populations (Ĥ) detected was 0.270. Index fixation value of GHRH gene showed there was not fixed into one gene type (Fski ≠ 0). The smallest genetic distance value of GHRH gene was found between Semarang and Mataram population (0.001) and the highest between Medan and Banten population (0.202).
Changes of the colour, total sugar content and respiration rate of broccoli heads during short-term storage

The objective of this study was to investigate the effect of short-term storage (24–96 h) at different temperatures (3°C, 16°C and 21°C) on the colour, respiration rate and total soluble sugar content in broccoli heads. The respiration rate of broccoli heads was also measured at 21°C during 24 h. The results of the study revealed that the colour change (hue angle and L) was the highest in the broccoli heads kept at 3°C after one-day storage. On the fourth day of storage the hue value decreased at 21°C and brightness decreased at 3°C. The respiration rate, measured at 21°C during 24 h, was the highest during the first three hours, later it decreased, and between 10 and 24 h it was almost at the same level. The respiration rate measured on the day of harvest at 16°C and 21°C were almost five times higher than at 3°C. After the 24 and 96 h of storage, the respiration rate was similar at each of the three storage temperatures. There was a significant decrease in the total soluble sugar content only after 96 h of storage at the temperature of 21°C.
PERUVIAN ECONOMIC ASSOCIATION Empowering women through multifaceted interventions: Long-term evidence from a double matching design

Empowering women is a policy goal that has received a lot of interest by policy-makers in the developing world in recent years, yet little is known about effective ways to promote it sustainably. Most existing interventions fail to address the multidimensional nature of empowerment. Using a double matching design to construct the sampling frame and to estimate causal effects, I evaluate the long-term impact of a multifaceted policy intervention designed to improve women’s empowerment in the Atlantic region in Colombia. This intervention provided information about women’s rights, soft-skills and vocational training, seed capital, and mentoring simultaneously. I find that this intervention has mixed results: improvements in incomes and other economic dimensions along with large political and social capital effects, but limited or null impacts on women’s rights knowledge and control over one’s body. Using a list experiment, I even find an increase in the likelihood of intra-household violence. The results highlight the importance of addressing women’s empowerment multidimensional nature in policy innovations designed to foster it, incorporating men in these efforts.
FOREGROUND SELECTION FOR IDENTIFICATION OF PLANTS CARRYING BACTERIAL BLIGHT AND BLAST RESISTANCE GENES IN F2 POPULATION OF COTTONDORA SANNALU

Rice (Oryza sativa L.) is the most important food crop in the world and feeds more than half of the global population. The productivity of rice is being affected by a number of biotic and abiotic factors. Among biotic factors bacterial, fungal and viral diseases covers a large portion causing significant loss in yield. The development of host-plant resistance is considered as the most economical, effective means to control the important diseases. In order to achieve durable resistance, gene pyramiding or gene stacking or both together is advisable to develop new rice varieties with multiple pest and disease resistance or to improve the existing high yielding varieties (HYVs) and hybrids by introgressing the genes for diseases resistance. Marker assisted selection allows the identification of plants carrying multiple resistance genes through foreground selection using gene linked molecular markers. The present investigation entitled “Foreground Selection for identification of plants carrying Bacterial Blight and Blast Resistance genes in F2 Population of Cottondora Sannalu” was undertaken with the objective to develop rice genotype(s) possessing the positive characteristics of a high yielding variety, Cottondora Sannalu (MTU1010) along with bacterial
Novel Partitivirus Infection of Bat White-nose Syndrome (WNS) Fungal Pathogen Pseudogymnoascus destructans Links Eurasian and North American Isolates

Bat White-nose Syndrome (WNS) fungus Pseudogymnoascus destructans had caused mass mortality in the North American bats. A single clone of the pathogen (Hap_1) was likely introduced in the United States while Eurasian population comprised of several haplotypes. The origin and spread of P. destructans remain enigmatic due in part to a lack of precise population markers. We searched for P. destructans mycoviruses as they are highly host-specific, and their spread could provide a window on the origin of the host fungus. We discovered a P. destructans bipartite virus PdPV-1 with two double-stranded RNA (dsRNA) segments - LS (1,683 bp) and SS (1,524 bp) with motifs similar to viral RNA-dependent RNA polymerase (RdRp) and putative capsid proteins (CPs), respectively. Both LS and SS ORFs were embedded only in the positive strand of each dsRNA segment. Sequence alignments and phylogenetic analysis suggested that both segments constitute the genome of a new virus similar to the mycoviruses in the family Partitiviridae genus Gammapartitivirus. Purified viral particles appeared as isometric virions with approximately 33 nm diameters typical of partitiviruses. A newly developed RT-PCR assay revealed that all US isolates and only a few Eurasian isolates were infected with PdPV-1. PdPV-1 was P. destructans - specific as closely related non-pathogenic fungi P. appendiculatus and P. roseus tested negative. Thus, PdPV-1 establishes a link between the Eurasian and North American P. destructans. PdPV-1 could be used as an experimental tool to further investigate fungal biogeography, and the host - pathogen interactions.
Partial Characterization of an Anti- Candida albicans Bacteriocin Produced by a Marine Strain of Bacillus sp., Sh10

The bacteriocin-producing strain Bacillus sp. , Sh10, isolated from the marine environment, exhibited a broad spectrum of antimicrobial activity against different food spoilage and human pathogens, with a maximum inhibitory activity against Candida albicans. The inhibitory compound was sensitive to trypsin but resistant to proteinase K, lysozyme, lipase and α-amylase. It was heat-stable and remained its activity after autoclaving. In addition, the antimicrobial substance demonstrated striking stability at low temperatures (4 and -20°C) for up to one year and retained its activity in a wide pH range from 2 to 11. It was also stable and active in the presence of different surfactants, solvents and heavy metals. Analysis of the partially purified bacteriocin by SDS-PAGE showed an apparent molecular weight of ~11 KDa. This study reveals a remarkable potential of this bacteriocin to be used as a food preservative.
RAPD Variation and Phenetic Relationships for Six Populations of Equisetum pratense in Korea

The phenetic relationships among six natural populations of Equisetum pratense in Korea were investigated at the population level by constructing a tree based on Random Amplified Polymorphic DNA (RAPD) markers. RAPD analysis was also conducted to estimate genetic diversity and the population structure of E. pratense. A mean of 26.7% at the six population levels indicated polymorphism. E. pretense was found to have fewer alleles per locus (1.267) and fewer effective alleles per locus (1.176). Genetic diversity (0.102) in E. pratense is lower than the average for species with similar life history traits. Total genetic diversity values (HT) varied between 0.112 (OPD-07) and 0.445 (OPD-16), for an average overall polymorphic locus of 0.141. Inter-locus variation in the within-population genetic diversity ( HS) was low (0.102). Asexual reproduction, small population size, and the colonization process are proposed as possible factors contributing to the observed low genetic diversity in E. pratense. On a per-locus basis, the proportion of total genetic variation due to differences among populations ( GST) ranged from 0.129 for OPD-07 to 0.455 for OPD-09, with a mean of 0.277. This indicated that about 27.7% of the total variation was among populations. Thus, genetic variation (72.3%) resided within populations. This study contributes new information for research on the taxonomy and population genetics of E. pratense.