Miracle-dz/UniFAR-FTUs huggingface dataset:
In situ genetic dissection of the boundary element Miscadestral pigmentation in the Bithorax complex of Drosophila melanogaster

In the past, dissection of cis-regulatory elements (enhancers, boundary elements, Polycomb Response Elements, etc.) was mostly done using transgenic assays. The study of such elements at the endogenous genomic location was, in many cases, not possible due to technical restrictions. Nowadays, the CRISPR/Cas9 method allows the precise modification, and hence dissection of cis-regulatory elements in their natural genomic environment, allowing an unbiased observation of their role in the regulation of their target gene. In this thesis I will describe the dissection of a well studied cis-regulatory element at its endogenous genomic location: the Miscadestral pigmentation (Mcp) boundary in the Drosophila melanogaster Bithorax Hox Complex (BX-C). In Drosophila melanogaster the Miscadestral pigmentation boundary element and the adjacent Polycomb Response Element divide the abdominal-A (abd-A) from the Abdominal-B (Abd-B) gene. Deletion of this elements leads to spurious interaction of abdominal-A cis-regulatory elements with the Abdominal-B gene. Such incorrect interactions of enhancers have to be prevented not only in the Hox cluster but throughout the genome to allow precise gene regulation. The dissection of such an element as a model system can give insights on how the genome is organized in independent gene regions. I used the CRISPR/Cas9 method in combination with both Non- Homologous-End-Joining and Homologous-Recombination DNA re- pair mechanisms to induce deletions in the Mcp region, and to establish a phiC31-integrase dependent landing site in the Mcp genomic location. The established landing site can be used to bring back into the Mcp locus modified genetic material, for example Mcp sequences with small deletions or completely unrelated boundary sequences from other genomic loci or species. The endogenous Mcp boundary and the Polycomb Response Element are dissected in great detail, and the obtained results demonstrate that the function of the boundary depends on a single CCCTC-binding factor (CTCF, a known boundary associated factor) site and on four Pleiohomeotic (Pho, part of the Polycomb Protein Group) sites in the Polycomb Response Element. Those results are further refined using classical boundary element assays in transgenic flies. Furthermore, I study the role of the orientation of those regulatory DNA sequences in the endogenous genomic locus. Surprisingly, some deletions in the Mcp region give rise to phenotypes not associated previously with this boundary, suggesting the presence of additional cis-regulatory elements other than the already known boundary element and PRE. This study further demonstrates the versatility of the CRISPR/Cas9 method in studying cis-regulatory elements in their endogenous genomic position, a possibility that will enable more precise and clean investigation of gene regulation in the future.
Influence of Different Auxins on Rooting of Rosemary, Sage and Elderberry

Aims: The aim of research is to examine the influence of auxins IAA, IBA, NAA and commercial rooting product on rooting of cuttings from rosemary ( Rosmarinus officinialis L.), sage ( Salvia officinialis L.) and elderberry ( Sambucus nigra L). Study Design: Completely randomized design. Methodology: Fifty cuttings from each experimental species ( Rosmarinus officinialis L., Salvia officinialis and Sambucus nigra L.), with height of 15 cm, were treated with freshly made solutions of different auxin and planted for rooting in the rooting beds. After 100 days of planting we measure: height of cuttings, number of rooted cuttings, number of roots, root length and rooting percentage. Results: The conducted research shows that growth regulators applied during the experiment, including the commercial rooting product, positively influenced rooting process of vegetative cuttings of rosemary and sage. Application of auxins as IAA, IBA and NAA in concentration of only 5 ppm have stimulating effect on rooting of the rosemary (IBA – 92%), sage (IBA – 54%; NAA – 50%) and elderberry cuttings (K1 and IAA – 14%). Conclusion: This study is first report for application of auxins on rosemary, sage and elderberry cuttings collected from local populations in Republic of Macedonia. Application of auxins in concentrations of 5 ppm positively affected rosemary, sage and elderberry. The results presented in this paper show that the vegetative propagation of medicinal and aromatic species might be stimulated by auxins utilization even in very low concentrations. It should be noted that further research should be extended to more medicinal and aromatic plants with significant economic importance as well as with application of several higher concentration of auxins.
Genome-wide identification, phylogenetic analysis, and expression profiling of the BBX family genes in pear

ABSTRACT B-box containing proteins (BBXs) are a class of zinc finger transcription factor proteins that contain one or two B-box domains and play important roles in plant growth and development. The BBX gene family has been identified in the Arabidopsis and rice genomes. However, no comprehensive identification has been reported in pear. In this study, 37 genes were identified as candidate BBXs in Chinese white pear (Pyrus bretschneideri Rehd) and denominated following the uniform nomenclature in Arabidopsis. We also performed analyses of their phylogenic relationships, structures, chromosomal locations, promoter regions, Gene Ontology (GO) annotations, and expression patterns. The identified genes were classified into five groups based on phylogeny and domain constitution analysis. PbBBXs were unevenly distributed in 12 out of 17 pear chromosomes. The cis-elements in the promoters of PbBBXs were mainly related to light, stress, and hormone response. Gene expression analysis by quantitative real-time PCR showed that PbBBXs had differential expression in different plant organs and could be classified into four major groups. PbBBXs also showed differential responses to low temperature and dehydration treatments. Overall, this study provided useful information for further functional analysis of BBXs in pear.
Effect of soil moisture level and phosphorus rate on phosphorus uptake and growth of corn (Zea mays L)

The effect of soil moisture level and phosphorus fertilization on corn ( Zea mays L.) growth and P uptake was evaluated in a pot experiment under greenhouse conditions. Two soils, Lewisburg silt loam (fine, mixed, mesit, Typic Hapludalf) and Nipe clay (clayey, oxidic, isohyperthermic, Anionic Acrudox) were included in the study. The three moisture levels were M2 = field capacity, M2 = fluctuation between field capacity and 50% available water, and M3 = fluctuation between field capacity and permanent wilting point. The phosphorus treatments were P1 = 0 kg P/ha, P2=112 kg P/ha and P3 = 224 kg P/ha. Fresh and dry matter yield of corn plants grown on Lewisburg soil increased significantly with phosphorus fertilization. The field capacity treatment (M1) significantly outyielded the other two moisture treatments when 11 2 kg P/ha was applied. At 224 kg P/ha there was no significant difference in yield between M1 and M2 treatments, but both these treatments significantly outyielded the M3 treatment. Corn plants grown on Nipe soil did not respond to phosphorus or moisture treatments. Phosphorus applications to Lewisburg soil significantly increased phosphorus uptake by corn plants. Phosphorus uptake was 3.89, 21.60 and 42.73 mg/pot for 0, 112 and 224 kg P treatments, respectively. An increase in moisture stress decreased P uptake with M1, M2 and M3 yielding 27.88, 22.91 and 17.42 mg P/pot, respectively. Corn plants grown on Nipe soil showed a slight increase in P uptake with the application of 224 kg P/ha. but this increase was not significantly different from that of the other two P treatments. Nor did moisture stress affect P uptake. The lack of response of corn plants to P fertilization and moisture level of Nipe soil can be attributed to a high P fixing capacity of the soil.
Distribution pattern, threatened status and conservation measures of fishes with relation to water quality and habitat characteristics of Bhavanisagar Reservoir, Tamil Nadu

This study documents the water quality, habitat characteristics and freshwater fish diversity of Bhavanisagar Reservoir, Tamil Nadu. We recorded 52 species represented by 8 orders, 13 families and 38 genera. The Cyprinid family was found to be dominant in the present study. Various diversity indices have been used to assess the fish diversity. The highest species diversity was found out in Bhavanisagar Dam (S1) and lowest species diversity was recorded in Aapakkudal (S7) sites. Fish diversity was also correlated with physicochemical variables and habitat characteristics. Among 52 fish species collected from 10 study sites of the upstream/ downstream areas of Bhavanisagar reservoir, of which 2 species (4%) were found to be critically endangered, 41 species were least concerned (79%), 5 species were near threatened (9%) and 4 species were in vulnerable category (8%). The threat status of fishes found in the Bhavanisagar reservoir strongly suggests the need for effective conservation measures to conserve the fish species richness.
Molecular identification of Prototheca zopfii genotype 2 mastitis isolates and their influence on the milk somatic cell count

Algae from the genus Prototheca are the only plant-like microorganisms which can cause inflammation and alterations in the mammary gland. Prototheca mastitis is usually recognized as a chronic and symptomless disease with reduced milk production and a very high somatic cell count. Molecular identification of Prototheca spp. is helpful for the differentiation of pathogenic from non-pathogenic strains which are probably milk contaminants. Genotype-specific PCR assays, based on the 18S rDNA gene sequences, have recently been developed to differentiate three genotypes of Prototheca zopfii, of which Prototheca zopfii genotype 3 was reclassified in a new species: Prototheca blaschkeae. P. zopfii genotype 2 is characterized as the main causative agent of Prototheca mastitis that leads to significant economic losses in primary milk production. The purpose of this study was to give a molecular characterization of Prototheca strains isolated in cases of subclinical and clinical mastitis, as well as to determine the influence of these pathogenic algae on the milk somatic cell count. After microbiological examination, algae from the genus Prototheca were isolated in pure cultures from 1.8% of all tested milk samples, and all 13 (100%) isolates were determined as Prototheca zopfii genotype 2 by a genotype-specific PCR. This study has provided the first molecular identification of Prototheca zopfii genotype 2 in the Republic of Serbia. In the case of subclinical Prototheca mastitis, the somatic cell count was 4,175,244 ± 1,233,685/mL of milk. A distinctly higher somatic cell count (P<0.05) was found in the quarters infected by Prototheca zopfii genotype 2 than in the quarters infected by Staphylococcus aureus, which is the most common mastitis causative agent worldwide. The results from this study support previous observations that P. zopfii genotype 2 is the main causative agent of Prototheca mastitis which leads to a significant increase in the somatic cell count in the milk.
ANALYSIS OF VEGETATIVE COMPATIBILITY GROUPS OF ITALIAN AND DUTCH ISOLATES OF FUSARIUM OXYSPORUM f. sp. LACTUCAE

Fusarium oxysporum f.sp. lactucae (FOL), the causal agent of lettuce fusarium wilt, has spread to several countries where lettuce is grown. To date, four races of FOL have been identified, but only race 1 has been detected in Europe; race 2 and race 3 have been identified in Japan and in Taiwan. A new physiological race has been isolated in The Netherlands. The vegetative-compatibility group approach has been used to determine genetic diversity within a group of forty-eight FOL strains of different origin, with special attention to the Italian and Dutch isolates of the pathogen, in order to obtain a better understanding of the recent epidemics that have been observed in Europe. On the basis of the complementation pattern, all the Italian isolates of FOL tested were assigned to VCG 0300. FOL isolates belonging to races 2 and 3 belonged to VCG 0301 and 0302, respectively. The isolates obtained from lettuce in The Netherlands belonged to a new VCG, numbered 0303. The results support the hypothesis that FOL race 4 might have been selected locally in Dutch fields.
Growth Performance of the Monsoon River Prawn Macrobrachium malcolmsonii on Formulated Feeds with Combinations of Pulses and Cereals along with Groundnut Oilcake and Soya Meal

This study was conducted to categorize the growth performance of feeds formulated with combinations of pulses and cereals (horse gram and ragi, green gram and pearl millet, and cow gram and wheat) along with groundnut oilcake and soya meal on the post larvae (PL) of the monsoon river prawn, Macrobrachium malcolmsonii . This is to promote the inland aquaculture of this economically important species. Two types of feeds with different proportions of pulse and cereal (type-A: 25% pulse + 25% cereal; type-B: 40% pulse + 10% cereal) were prepared in each combination along with equal proportion of groundnut oil cake and soya meal (20% each). Tapioca flour (5%) and egg albumin (4%) were used as binding agents. Vitamin B-complex (1%) was also mixed. These feeds were fed to M. malcolmsonii PL for a period of 45 days. The efficacy of these feeds on growth performance and biochemical constituents were assessed. Commercially available standard Scampi feed was served as control. The overall influence of these feeds on growth, and concentrations of total protein, amino acid, carbohydrate and lipid of PL were found to be the best in scampi feed followed by the feed rich in pulse (type-B) and the feed with equal proportion of pulses and cereals (type-A). Among three combinations of pulses and cereals were tested, the feed formulated with cow gram and wheat showed the best overall performance, followed by green gram and pearl millet, and horse gram and ragi. Â All the results were confirmed through paired sample t-test, DMRT, and two-way ANOVA. The survival rate was found to be better in formulated feeds fed PL when compared with control. It is assumed that the additive/ preservative added in the commercially available scampi feed may be the reason for the lower survival rate recorded. Moreover, t he commercially available scampi feed is not always affordable to small farmers. Therefore, the farm made feeds with locally available commodities of pulses and cereals can be prepared and used in a sustainable manner for healthy promotion of Macrobrachium culture.
The Increase in LEAP-2 mRNA Suggests a Synergistic Probiotics-Doxycycline Interaction in Chickens

Background: Extensive interaction between gut microbiota and the host immune system has significant impact on chicken performance. Objective: Therefore, the influence of doxycycline, administered with or without Lactobacillus spp. , on PepT1 and LEAP-2 mRNAs expression in duodenum, jejunum and liver was investigated in Duc chickens. Methods: One-day-old Duc broiler chickens were divided in four groups. Chickens without treatment served as controls. Five days after hatching the second group was treated with Lactobacillus brevis, L.plantarum and L.bulgaricus probiotics via feed for 15 days. The third group was treated with probiotics (as in group 2) and doxycycline (10 mg/kg bw, via drinking water for five days, started 15 days after hatching). The fourth group received antibiotic only as described above. Water and food (broiler starter without drugs) were supplied ad libitum. Samples from liver, duodenum and jejunum were collected at the end of the treatment with doxycycline (21 days after hatching). Expression levels of LEAP-2 and PepT1 mRNAs were determined by qRT-PCR. Results: Doxycycline administered alone or in combination with probiotics, provoked a statistically significant upregulation of LEAP-2 mRNA in the liver and in the duodenum. Abundance of PepT1 mRNA was increased in the duodenum. Administration of doxycycline alone caused a moderate induction of LEAP-2 mRNA and down-regulation of PepT1 mRNA in the liver. Conclusion: Up-regulation of the studied antimicrobial peptides provoked by combination of Lactobacilli and doxycycline might be beneficial in terms of host protection.
BioID indicates the involvement of the TRAPP protein TrappC11 in autophagy

The Transport protein particle (TRAPP) complexes are molecular machines that function in various membrane transport processes including the secretory pathway. Recently, one form of the TRAPP complex, TRAPPIII, has been shown to be involved in autophagy, a cellular recycling process that delivers cytoplasmic components to lysosomes for degradation. Mutations in TrappC11, one subunit in TRAPPIII, have been reported to be associated with muscular dystrophy and intellectual disability. To understand mutated TrappC11 could lead to the phenotypes, I employed BioID to identify TrappC11 interacting proteins. To perform BioID, I constructed TrappC11 fused to BirA*, a biotin ligase that catalyzes the biotinylation of proteins within proximity to the fusion protein. The biotinylated proteins were then isolated and identified by mass spectrometry. The BioID approach revealed the autophagy-related proteins Atg2A, Atg2B, WDR45, p62/SQSTM1, and NBR1 as potential TrappC11 interacting partners, suggesting the involvement of TrappC11 in autophagy. Consistently, transient and stable knockdown of TrappC11 in HeLa cells both resulted in an accumulation of autophagosomes in non-starved cells as well as an increase of cellular lipid droplets. The distribution of lysosomes was also effected by the TrappC11 knockdown. Moreover, expression of eGFP-TrappC11 resulted in the formation of cytoplasmic puncta that partially overlapped with p62/SQSTM1, consistent with the BioID data that suggested an interaction between TrappC11 and this selective autophagy receptor. Taken together, my data indicate that TrappC11 might have an important role in non-starvation induced autophagy.
Drought stress increased survival and development of emerald ash borer larvae on coevolved Manchurian ash and implicates phloem‐based traits in resistance

Emerald ash borer (Agrilus planipennis Fairmaire, EAB) is causing widespread ash (Fraxinus spp.) mortality as it invades North America and Eastern Europe. Resistance of its coevolved hosts, including Manchurian ash (Fraxinus mandshurica Rupr.), is considered to limit EAB outbreaks and ash mortality in its native Asia, although an understanding of resistance mechanisms is still developing. Such knowledge may facilitate breeding for resistance and management of EAB in its invaded ranges. In the present study, controlled egg inoculations were used to investigate resistance mechanisms impacting larval performance, as well as to characterize the effects of water and nutrient availability on inter‐ and intra‐specific variation in resistance phenotypes based on larval outcomes. Larval survival and growth rates were lower on coevolved Manchurian ash than on evolutionarily naïve white ash (Fraxinus americana L.). Water stress decreased tree growth and resistance of Manchurian ash to EAB, although it had little effect on resistance of the already highly susceptible white ash. High nutrient availability increased tree growth but had no effect on larval performance. These results show that the higher resistance of Manchurian ash to EAB is conferred by phloem traits that decrease larval performance, in addition to lower oviposition preference.
Role of Arachidonic Acid and COX Inhibitors in the Regulation of Reproduction in Freshwater Crab Oziothelphusa senex senex

Induced reproduction of cultured species helps to produce quantity and quality seed an important component for upright yield in crustacean aquaculture. The present study was aimed to investigate the role of arachidonic acid in the regulation of ovarian development in the freshwater crab, Oziothelphusa senex senex. Injection of AA significantly (p<0.001) increased the ovarian index, oocyte diameter and ovarian vitellogenin levels. Injection of COX inhibitors such as indomethacin and aspirin alone, and in combination with AA resulted in significant (p<0.001) reduction in ovarian index, oocyte diameter and ovarian vitellogenin levels in crabs. The results of the present study provide evidence that arachidonic acid and COX inhibitors involved in the regulation of female reproduction in the freshwater crab, Oziothelphusa senex senex.
First Identification of Nosema Ceranae (Microsporidia) Infecting Apis Mellifera in Venezuela

Abstract Nosema ceranae is a pathogen of Apis mellifera, which seems to have jumped from its original host Asiatic honey bee Apis ceranae. Nosemosis which affects the honey bee Apis mellifera is caused by two parasitic fungi described as etiologic agents of the disease. Nosema apis was the only microsporidian infection identified in A. mellifera until N. ceranae in Taiwan and Europe. Nosema spp. positive samples of adult worker bees from the Venezuelean state of Lara were determined through light microscopy of spores. Samples were then tested to determine Nosema species (N.apis/N.ceranae) using previously reported PCR primers for the 16S rRNA gene. A multiplex PCR assay was used to differentiate both N. apis and N. ceranae species. Only N. ceranae was found in the analyzed samples and the percentage of infected foragers fluctuated between 18% and 60%.
Altered Social Behavior and Neuroinflammation in a Mouse Model of Pten Mislocalization

by AMANDA KATHERINE KOMURO Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder affecting 1 in 68 children within the United States, whose high heritability is complicated by genetic heterogeneity. Studying the functions of genes mutated in syndromic forms of ASD is one method for identifying common pathogenic processes. In these studies, we used a murine model based on germline alterations to PTEN that can occur in patients with PTEN Hamartoma Tumor Syndrome, a cancer predisposition syndrome with high rates of ASD. In the Pten mouse, the localization of the Pten protein is shifted toward cytoplasmic predominance. In Chapter 2, we describe how this mislocalization leads to a range of cellular and behavioral phenotypes. The Pten brain overproduces glial cells, and displays hallmark signs of neuroinflammation. Behaviorally, the mice have increased social motivation and balance problems reminiscent of high functioning ASD. After discovering this connection to human ASD, we hypothesized that the neural transcriptome of the Pten mouse would show alterations that were relevant to those seen in idiopathic ASD. Examining gene expression in this model at 2 and 6 weeks of age revealed a progressive increase in expression differences that fell into categories related to increased neuroinflammation and decreased synaptic transmission – areas disrupted in