A Simple Method for Isolating DNA of High Yield and Quality from Cotton (shape Gossypium hirsutum L.) Leaves

An easy, reproducible and fast procedure to isolate DNA from cotton leaves is described. The addition of 0.5 M glucose in the extraction buffer avoids browning by polyphenolic compounds and improves the quality of DNA for molecular analysis. The DNA yield ranged between 150–400 mg per gram of fresh tissue. The DNA was suitable for digestion by restriction enzymes and amplificatiion by Taq DNA polymerase.     

Synthesis and evaluation of novel daunomycin-phosphate-sulfate -β-glucuronide and -β-glucoside prodrugs for application in adept

The synthesis, antiproliferative effect and enzymatic hydrolysis of daunomycin-3′-N- and -4′-O-phosphate and -sulfate derivatives and of daunomycin-3′-N-CO-β-glucuronide and -β-glucoside, designed to be prodrugs in ADEPT are described. The phosphate derivatives were almost as toxic as the parent drug whereas the sulfates were not hydrolyzed by aryl sulfatases. Glucuronyl and glucosyl prodrugs were found to be useful for application in ADEPT.     

Aerobic and anaerobic metabolism during activity in snakes

Summary Oxygen consumption and blood lactate concentration in the snakesColuber constrictor, Crotalus viridis, Lichanura roseofusca andMasticophis flagellum and whole body lactate concentration inCrotalus viridis andMasticophis flagellum were determined under standard conditions and after a bout of maximal activity induced by a 5 min period of mechanical stimulation. Observations were made atT _b=35°C inColuber, Crotalus, andMasticophis and 32°C inLichanura. Maximal oxygen consumption inColuber andMasticophis was twice that ofCrotalus and 4 x that ofLichanura (Fig. 1). Post-active whole body lactate concentration inMasticophis was twice that ofCrotalus (Fig. 2). Immediately post-active and 30 min post-active blood lactate concentration inColuber andMasticophis was 1.5 x and 3.5 x that ofCrotalus andLichanura, respectively (Fig. 3). These data support conclusions that: (a) maximal energy production by these snakes correlates well with their respective modes of predation and defense, the highly active predatorsColuber andMasticophis being capable of the greatest net energy production during activity; (b)Coluber andMasticophis exhibit aerobic scopes as high or higher than any other comparably sized reptile heretofore investigated; (c) weight specific anaerobic metabolism probably does not decrease with increasing body size in reptiles; (d) anaerobic metabolism provides >50% of net energy production during five minutes of activity in all species examined (Table 1).     

Dracula’s ménagerie: A multispecies occupancy analysis of lynx,wildcat, and wolf in the Romanian Carpathians

1. The recovery of terrestrial carnivores in Europe is a conservation success story. Initiatives focused on restoring top predators require information on how resident species may interact with the re‐introduced species as their interactions have the potential to alter food webs, yet such data are scarce for Europe.
2. In this study, we assessed patterns of occupancy and interactions between three carnivore species in the Romanian Carpathians. Romania houses one of the few intact carnivore guilds in Europe, making it an ideal system to assess intraguild interactions and serve as a guide for reintroductions elsewhere.
3. We used camera trap data from two seasons in Transylvanian forests to assess occupancy and co‐occurrence of carnivores using multispecies occupancy models.
4. Mean occupancy in the study area was highest for lynx (Ψ_winter = 0.76 95% CI: 0.42–0.92; Ψ_autumn = 0.71 CI: 0.38–0.84) and wolf (Ψ_winter = 0.60 CI: 0.34–0.78; Ψ_autumn = 0.81 CI: 0.25–0.95) and lowest for wildcat (Ψ_winter = 0.40 CI: 0.19–0.63; Ψ_autumn = 0.52 CI: 0.17–0.78)
5. We found that marginal occupancy predictors for carnivores varied between seasons. We also found differences in predictors of co‐occurrence between seasons for both lynx‐wolf and wildcat‐wolf co‐occurrence. For both seasons, we found that conditional occupancy probabilities of all three species were higher when another species was present.
6. Our results indicate that while there are seasonal differences in predictors of occupancy and co‐occurrence of the three species, co‐occurrence in our study area is high.
7. Terrestrial carnivore recovery efforts are ongoing worldwide. Insights into interspecific relations between carnivore species are critical when considering the depauperate communities they are introduced in. Our work showcases that apex carnivore coexistence is possible, but dependent on protection afforded to forest habitats and their prey base.

     

Diversity and distribution of reptiles in Romania

The reptile fauna of Romania comprises 23 species, out of which 12 species reach here the limit of their geographic range. We compiled and updated a national database of the reptile species occurrences from a variety of sources including our own field surveys, personal communication from specialists, museum collections and the scientific literature. The occurrence records were georeferenced and stored in a geodatabase for additional analysis of their spatial patterns. The spatial analysis revealed a biased sampling effort concentrated in various protected areas, and deficient in the vast agricultural areas of the southern part of Romania. The patterns of species richness showed a higher number of species in the warmer and drier regions, and a relatively low number of species in the rest of the country. Our database provides a starting point for further analyses, and represents a reliable tool for drafting conservation plans.     

Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage

CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels. By contrast, concurrent nicking of the genomic locus and targeting vector, known as in-trans paired nicking (ITPN), stimulates efficient HDR-mediated genome editing to generate large knock-ins without introducing DSBs. Here, we show that ITPN allows for fast, highly efficient, and indel-free fluorescent gene tagging in human normal and cancer organoids. Highlighting the ease and efficiency of ITPN, we generate triple fluorescent knock-in organoids where 3 genomic loci were simultaneously modified in a single round of targeting. In addition, we generated model systems with allele-specific readouts by differentially modifying maternal and paternal alleles in one step. ITPN using our palette of targeting vectors, publicly available from Addgene, is ideally suited for generating error-free heterozygous knock-ins in human organoids.

A major downside of double-strand break-mediated genome editing is the need to verify the genomic integrity of the targeted locus and confirm the absence of off-target indels. This study shows that in-trans paired nicking is a mutation-free CRISPR strategy to introduce precise knock-ins into human organoids; its genomic fidelity allows all knock-in cells to be pooled, accelerating the establishment of new organoid models.