Transformation of group A streptococci by electroporation

Abstract The introduction, via electroporation, of free plasmid DNA into three strains of Streptococcus pyogenes is described. The method is very simple and rapid and efficiencies vary from 1 × 10³ to 4 × 10⁴ per μg of DNA. The method was also used to introduce an integrative plasmid and transformants were obtained, albeit at a somewhat lower frequency (2 × 10²). Some of the plasmids used in this study are derivatives of the Lactococcus lactis subsp. cremoris Wg2 plasmid pWV01. These broad host range vectors replicate in Gram-positives as well as Gram-negatives (viz. Escherichia coli ). Here we show that they also replicate in S. pyogenes and S. sanguis .     

Energy conservation characterizes sleep in sharks

Sharks represent the earliest group of jawed vertebrates and as such, they may provide original insight for understanding the evolution of sleep in more derived animals. Unfortunately, beyond a single behavioural investigation, very little is known about sleep in these ancient predators. As such, recordings of physiological indicators of sleep in sharks have never been reported. Reduced energy expenditure arising from sustained restfulness and lowered metabolic rate during sleep have given rise to the hypothesis that sleep plays an important role for energy conservation. To determine whether this idea applies also to sharks, we compared metabolic rates of draughtsboard sharks (Cephaloscyllium isabellum) during periods ostensibly thought to be sleep, along with restful and actively swimming sharks across a 24 h period. We also investigated behaviours that often characterize sleep in other animals, including eye closure and postural recumbency, to establish relationships between physiology and behaviour. Overall, lower metabolic rate and a flat body posture reflect sleep in draughtsboard sharks, whereas eye closure is a poorer indication of sleep. Our results support the idea for the conservation of energy as a function of sleep in these basal vertebrates.     

Status and distribution of jaguarundi in Texas and Northeastern México: Making the case for extirpation and initiation of recovery in the United States

The jaguarundi (Puma yagouaroundi) is a small felid with a historical range from central Argentina through southern Texas. Information on the current distribution of this reclusive species is needed to inform recovery strategies in the United States where its last record was in 1986 in Texas. From 2003 to 2021, we conducted camera‐trap surveys across southern Texas and northern Tamaulipas, México to survey for medium‐sized wild cats (i.e., ocelots [Leopardus pardalis], bobcats [Lynx rufus], and jaguarundi). After 350,366 trap nights at 685 camera sites, we did not detect jaguarundis at 16 properties or along 2 highways (1050 km²) in Texas. However, we recorded 126 jaguarundi photographic detections in 15,784 trap nights on 2 properties (125.3 km²) in the northern Sierra of Tamaulipas, Tamaulipas, México. On these properties, latency to detection was 72 trap nights, with a 0.05 probability of detection per day and 0.73 photographic event rate every 100 trap nights. Due to a lack of confirmed class I sightings (e.g., specimen, photograph) in the 18 years of this study, and no other class I observations since 1986 in the United States, we conclude that the jaguarundi is likely extirpated from the United States. Based on survey effort and results from México, we would have expected to detect jaguarundis over the course of the study if still extant in Texas. We recommend that state and federal agencies consider jaguarundis as extirpated from the United States and initiate recovery actions as mandated in the federal jaguarundi recovery plan. These recovery actions include identification of suitable habitat in Texas, identification of robust populations in México, and re‐introduction of the jaguarundi to Texas.     

Phylodynamic Inference of Bacterial Outbreak Parameters Using Nanopore Sequencing

Nanopore sequencing and phylodynamic modeling have been used to reconstruct the transmission dynamics of viral epidemics, but their application to bacterial pathogens has remained challenging. Cost-effective bacterial genome sequencing and variant calling on nanopore platforms would greatly enhance surveillance and outbreak response in communities without access to sequencing infrastructure. Here, we adapt random forest models for single nucleotide polymorphism (SNP) polishing developed by Sanderson and colleagues (2020. High precision Neisseria gonorrhoeae variant and antimicrobial resistance calling from metagenomic nanopore sequencing. Genome Res. 30(9):1354–1363) to estimate divergence and effective reproduction numbers (R_e) of two methicillin-resistant Staphylococcus aureus (MRSA) outbreaks from remote communities in Far North Queensland and Papua New Guinea (PNG; n = 159). Successive barcoded panels of S. aureus isolates (2 × 12 per MinION) sequenced at low coverage (>5× to 10×) provided sufficient data to accurately infer genotypes with high recall when compared with Illumina references. Random forest models achieved high resolution on ST93 outbreak sequence types (>90% accuracy and precision) and enabled phylodynamic inference of epidemiological parameters using birth–death skyline models. Our method reproduced phylogenetic topology, origin of the outbreaks, and indications of epidemic growth (R_e > 1). Nextflow pipelines implement SNP polisher training, evaluation, and outbreak alignments, enabling reconstruction of within-lineage transmission dynamics for infection control of bacterial disease outbreaks on portable nanopore platforms. Our study shows that nanopore technology can be used for bacterial outbreak reconstruction at competitive costs, providing opportunities for infection control in hospitals and communities without access to sequencing infrastructure, such as in remote northern Australia and PNG.     

Optimization of α-ketoamide based p38 inhibitors through modifications to the region that binds to the allosteric site

We have optimized a novel series of potent p38 MAP kinase inhibitors based on an α-ketoamide scaffold through structure based design that due to their extended molecular architecture bind, in addition to the ATP site, to an allosteric pocket. In vitro ADME, in vivo PK and efficacy studies show these compounds to have drug-like characteristics and have resulted in the nomination of a development candidate which is currently in phase II clinical trials for the oral treatment of inflammatory conditions.     

Can a stench be beautiful? – Osmophores in stem-succulent stapeliads (Apocynaceae-Asclepiadoideae-Ceropegieae-Stapeliinae)

Carrion flower stapeliads are examples of olfactory mimicry, forming sapromyiophilous flowers, which mimic food sources or oviposition sites to attract fly pollinators. The aim of this work was to investigate the ultrastructure of osmophores involved in the release of the carrion odor of Orbea variegata and Boucerosia indica flowers. In spite of their similar architecture (epidermal epithelium+subepidermal secretory layers), the osmophores of stapeliads feature some differences in morphology and ultrastructure. The epidermal epithelial cells of O. variegata and B. indica differ in shape, but both are extremely rich in endoplasmic reticulum and flocculent material in the vacuole. Unlike the Orbea, Boucerosia has starchless leucoplasts in the epidermal epithelium. Orbea features a cuticle with microchannels, while Boucerosia has a different mechanism for the pathway of scent substances to the cell exterior. They are released by rupturing of the outer layer of cuticle at the apex of the papillae. The epidermal cells of the adaxial corolla differ even between parts of the corolla, the corolla lobes and the annulus in the flower. This diversity may be connected with an odor gradient. The morphological and anatomical features of stapeliad (subtribe Stapeliinae) osmophores are generally similar to osmophores of members of subtribe Ceropegiinae (Ceropegia), thus, we suggest that this model of osmophores evolved before early diversification of Ceropegieae. The ultrastructural features of stapeliad osmophores are generally similar to those of Araceae, Orchidaceae and Passifloraceae.     

Artificial sowing of endangered dry grassland species into disused basalt quarries

Steppe-like dry (semi) natural grasslands are valuable and endangered habitats in central Europe. In the study area (the ?eské st?edoho?í Hills, Czech Republic), they occur in fragments on southern slopes of volcanic hills, often in the vicinity of quarries, which are numerous there. We performed sowing experiments and observed seedling recruitment of six species, typical for the dry grasslands, in 9 basalt quarries located in 3 different climatic regions over 3 yr. The sowing experiments were established in young successional stages, 5–12 yr after quarrying was stopped. The objectives of the experiments were to evaluate: (i) if seedling recruitment and survival of the target species are possible regarding different climatic regions and (ii) what is the influence of weather fluctuations among years on seedling survival. Seedlings of all studied species were able to recruit and survive in the early successional stages at least in some quarries. The species, except one, showed significant differences in recruitment among the climatic regions, with the best recruitment and survival in the driest and warmest region. Seedlings of two species did not recruit in the wettest and coldest region. All species survived in the driest and warmest region, while only one did so in the coldest and wettest region. Different weather conditions in the studied years significantly influenced recruitment of two species. The number of localities (floristic records) of particular species in the regions was the best predictor of species germination and survival. Thus a traditional floristic survey may help to predict success of species in restoration projects. It emerged that artificial sowing can be considered in restoration programs as a way of contributing to restoration of dry grasslands in disused quarries.     

Linoleamide, a brain lipid that induces sleep, increases cytosolic Ca2+ levels in MDCK renal tubular cells

Linoleamide is an endogenous lipid that has been shown to induce sleep in cats, rats and humans. However, its physiological function remains unclear. In this study the effect of linoleamide on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) tubular cells was examined, by using fura-2 as a Ca2+ probe. In a concentration-dependent manner, linoleamide induced increases in [Ca2+]i between 10-500 microM with an EC50 of 20 microM. The signal comprised a slow rise and a persistent phase, and was a result of internal Ca2+ release and external Ca2+ influx because it was partly inhibited by external Ca2+ removal. In Ca2+-free medium, depletion of the endoplasmic reticulum Ca2+ store with 1 microM thapsigargin abolished 100 microM linoleamide-induced internal Ca2+ release, and conversely, pretreatment with linoleamide prevented thapsigargin from releasing internal Ca2+. This demonstrates that the internal source of linoleamide-induced [Ca2+]i increase is located in the endoplasmic reticulum. This discharge of internal Ca2+ caused capacitative Ca2+ entry because after incubation with 100 microM linoleamide in Ca2+-free medium for 8 min readmission of 3 mM CaCl2 induced increases in [Ca2+]i. After the formation of inositol-1,4,5-trisphosphate (IP3) was blocked by the phospholipase C inhibitor U73122 (1 microM), linoleamide still induced an increase in [Ca2+]i but the shape of the increase was altered. Similar results were found for another sleep-inducing lipid 9,10-octadecenoamide. Together, the present study shows that the endogenous sleep-inducing lipid linoleamide was able to cause significant increases in [Ca2+]i in renal tubular cells, by releasing the endoplasmic reticulum Ca2+ store and triggering capacitative Ca2+ entry in a manner independent of IP3.     

Zur Problematik der baktericiden Wirkung von Antibiotica bei tiefen Temperaturen

Zusammenfassung Es wurde der Einfluß von Penicillin und Streptomycin auf das Absterben von E. coli und M. pyogenes unter Kältewirkung untersucht und mittels spezifischer Inaktivation der Antibiotica während des Auftauens bewiesen, daß deren zusätzliche baktericide Wirkung nicht in der Frostperiode, sondern erst beim Auftauen einsetzt. Die Abhängigkeit der zusätzlichen Abtötung von der Gefriertemperatur und Antibioticumkonzentration wird mit dem Maß des ausgefrorenen Zellwassers, bzw. der aufgesaugten Antibioticamenge während der Quellung erklärt. Einige auf die Ergebnisse quantitativer Studien des Kältetodes von Mikroben sich störend auswirkende Erscheinungen, insbesondere das durch Eisbildung eingeleitete Auseinandersprengen der Zellverbände werden erläutert.