Benchmarking DNA extraction methods for phylogenomic analysis of sub-Antarctic Rhodococcus and Williamsia species

Bacteria containing mycolic acids in their cell envelope are often recalcitrant to cell lysis, so extracting DNA of sufficient quality for third-generation sequencing and high-fidelity genome assembly requires optimization, even when using commercial kits with protocols for hard-to-lyse bacteria. We benchmarked three spin-column-based kits against a classical DNA extraction method employing lysozyme, proteinase K and SDS for six lysozyme-resistant, sub-Antarctic strains of Corynebaceriales. Prior cultivation in broths containing glycine at highly growth-inhibitory concentrations (4.0–4.5%) improved cell lysis using both classical and kit methods. The classical method produced DNA with average fragment sizes of 27–59 Kbp and tight fragment size ranges, meeting quality standards for genome sequencing, assembly and phylogenomic analyses. By 16S rRNA gene sequencing, we classified two strains as Williamsia and four strains as Rhodococcus species. Pairwise comparison of average nucleotide identity (ANI) and alignment fraction (AF), plus genome clustering analysis, confirmed Rhodococcus sp. 1163 and 1168 and Williamsia sp. 1135 and 1138 as novel species. Phylogenetic, lipidomic and biochemical analyses classified psychrotrophic strains 1139 and 1159 as R. qingshengii and R. erythropolis, respectively, using ANI similarity of >98% and AF >60% for species delineation. On this basis, some members of the R. erythropolis genome cluster groups, including strains currently named as R. enclensis, R. baikonurensis, R. opacus and R. rhodochrous, would be reclassified either as R. erythropolis or R. qingshengii

Factors influencing resilience to postoperative delirium in adults undergoing elective orthopaedic surgery

Introduction Delirium occurs after elective arthroplasty in 17 per cent of adults1, and is associated with poor outcomes, including cognitive decline2, dementia3,4, and death5. Predisposing and precipitating risk factors accumulate and interact to precipitate delirium6. Much of the current literature analyses delirium as a dichotomous outcome, inevitably placing many people with symptoms of delirium, but falling short of a diagnosis, into the no-delirium group. Freedom from delirium symptoms should be investigated as an outcome. As evidence accumulates that delirium symptoms can also be associated with negative outcomes, it is important to identify the resilient groups in these studies and establish modifiable resilience predictors. Studies have explored risk factors for postoperative delirium; however, none to date has defined or considered delirium resilience as an outcome or phenotype. Resilience may be broadly defined as ‘the ability to withstand or recover quickly from difficult conditions’7,8. The aim of this study was to identify predictors of delirium resilience in the perioperative setting

Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA

In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-γS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC-Cdc6 and Cdt1-MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC-Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. The conformation of ORC-Cdc6 undergoes a concerted change into a right-handed spiral with helical symmetry that is identical to that of the DNA double helix. The resulting ORC-Cdc6 helicase loader shows a notable structural similarity to the replication factor C clamp loader, suggesting a conserved mechanism of action

Damage to Broca's area does not contribute to long-term speech production outcome after stroke

Broca’s area in the posterior half of the left inferior frontal gyrus has long been thought to be critical for speech production. The current view is that long-term speech production outcome in patients with Broca’s area damage is best explained by the combination of damage to Broca’s area and neighbouring regions including the underlying white matter, which was also damaged in Paul Broca’s two historic cases. Here, we dissociate the effect of damage to Broca’s area from the effect of damage to surrounding areas by studying long-term speech production outcome in 134 stroke survivors with relatively circumscribed left frontal lobe lesions that spared posterior speech production areas in lateral inferior parietal and superior temporal association cortices. Collectively, these patients had varying degrees of damage to one or more of nine atlas-based grey or white matter regions: Brodmann areas 44 and 45 (together known as Broca’s area), ventral premotor cortex, primary motor cortex, insula, putamen, the anterior segment of the arcuate fasciculus, uncinate fasciculus and frontal aslant tract. Spoken picture description scores from the Comprehensive Aphasia Test were used as the outcome measure. Multiple regression analyses allowed us to tease apart the contribution of other variables influencing speech production abilities such as total lesion volume and time post-stroke. We found that, in our sample of patients with left frontal damage, long-term speech production impairments (lasting beyond 3 months post-stroke) were solely predicted by the degree of damage to white matter, directly above the insula, in the vicinity of the anterior part of the arcuate fasciculus, with no contribution from the degree of damage to Broca’s area (as confirmed with Bayesian statistics). The effect of white matter damage cannot be explained by a disconnection of Broca’s area, because speech production scores were worse after damage to the anterior arcuate fasciculus with relative sparing of Broca’s area than after damage to Broca’s area with relative sparing of the anterior arcuate fasciculus. Our findings provide evidence for three novel conclusions: (i) Broca’s area damage does not contribute to long-term speech production outcome after left frontal lobe strokes; (ii) persistent speech production impairments after damage to the anterior arcuate fasciculus cannot be explained by a disconnection of Broca’s area; and (iii) the prior association between persistent speech production impairments and Broca’s area damage can be explained by co-occurring white matter damage, above the insula, in the vicinity of the anterior part of the arcuate fasciculus

Properties of an acid-tolerant, persistent Cheddar cheese isolate, Lacticaseibacillus paracasei GCRL163

The distinctive flavours in hard cheeses are attributed largely to the activity of nonstarter lactic acid bacteria (NSLAB) which dominate the cheese matrix during maturation after lactose is consumed. Understanding how different strains of NSLAB survive, compete, and scavenge available nutrients is fundamental to selecting strains as potential adjunct starters which may influence product traits. Three Lacticaseibacillus paracasei isolates which dominated at different stages over 63-week maturation periods of Australian Cheddar cheeses had the same molecular biotype. They shared many phenotypic traits, including salt tolerance, optimum growth temperature, growth on N-acetylglucosamine and N-acetylgalactosamine plus delayed growth on D-ribose, carbon sources likely present in cheese due to bacterial autolysis. However, strains 124 and 163 (later named GCRL163) survived longer at low pH and grew on D-tagatose and D-mannitol, differentiating this phenotype from strain 122. When cultured on growth-limiting lactose (0.2%, wt/vol) in the presence of high concentrations of L-leucine and other amino acids, GCRL163 produced, and subsequently consumed lactate, forming acetic and formic acids, and demonstrated temporal accumulation of intermediates in pyruvate metabolism in long-term cultures. Strain GCRL163 grew in Tween 80-tryptone broths, a trait not shared by all L. casei-group dairy isolates screened in this study. Including citrate in this medium stimulated growth of GCRL163 above citrate alone, suggesting cometabolism of citrate and Tween 80. Proteomic analysis of cytosolic proteins indicated that growth in Tween 80 produced a higher stress state and increased relative abundance of three cell envelope proteinases (CEPs) (including PrtP and Dumpy), amongst over 230 differentially expressed proteins

The Ctf18 RFC-like complex positions yeast telomeres but does not specify their replication time

Peer reviewedPreprin

Documentation of best interest by intensivists: a retrospective study in an Ontario critical care unit

<p>Abstract</p> <p>Background</p> <p>Intensive care physicians often must rely on substitute decision makers to address all dimensions of the construct of "best interest" for incapable, critically ill patients. This task involves identifying prior wishes and to facilitate the substitute decision maker's understanding of the incapable patient's condition and their likely response to treatment. We sought to determine how well such discussions are documented in a typical intensive care unit.</p> <p>Methods</p> <p>Using a quality of communication instrument developed from a literature search and expert opinion, 2 investigators transcribed and analyzed 260 handwritten communications for 105 critically ill patients who died in the intensive care unit between January and June 2006. Cohen's kappa was calculated before analysis and then disagreements were resolved by consensus. We report results on a per-patient basis to represent documented communication as a process leading up to the time of death in the ICU. We report frequencies and percentages for discrete data, median (m) and interquartile range (IQR) for continuous data.</p> <p>Results</p> <p>Our cohort was elderly (m 72, IQR 58-81 years) and had high APACHE II scores predictive of a high probability of death (m 28, IQR 23-36). Length of stay in the intensive care unit prior to death was short (m 2, IQR 1-5 days), and withdrawal of life support preceded death for more than half (n 57, 54%). Brain death criteria were present for 18 patients (17%). Although intensivists' communications were timely (median 17 h from admission to critical care), the person consenting on behalf of the incapable patient was explicitly documented for only 10% of patients. Life support strategies at the time of communication were noted in 45% of charts, and options for their future use were presented in 88%. Considerations relevant to determining the patient's best interest in relation to the treatment plan were not well documented. While explicit survival estimates were noted in 50% of charts, physicians infrequently documented their own predictions of the patient's functional status (20%), anticipated need for chronic care (0%), or post ICU quality of life (3%). Similarly, documentation of the patient's own perspectives on these ranged from 2-18%.</p> <p>Conclusions</p> <p>Intensivists' documentation of their communication with substitute decision makers frequently outlined the proposed plan of treatment, but often lacked evidence of discussion relevant to whether the treatment plan was expected to improve the patient's condition. Legislative standards for determination of best interest, such as the Health Care Consent Act in Ontario, Canada, may provide guidance for intensivists to optimally document the rationales for proposed treatment plans.</p