Pan-Arctic soil moisture control on tundra carbon sequestration and plant productivity.

Long-term atmospheric CO2 concentration records have suggested a reduction in the positive effect of warming on high-latitude carbon uptake since the 1990s. A variety of mechanisms have been proposed to explain the reduced net carbon sink of northern ecosystems with increased air temperature, including water stress on vegetation and increased respiration over recent decades. However, the lack of consistent long-term carbon flux and in situ soil moisture data has severely limited our ability to identify the mechanisms responsible for the recent reduced carbon sink strength. In this study, we used a record of nearly 100 site-years of eddy covariance data from 11 continuous permafrost tundra sites distributed across the circumpolar Arctic to test the temperature (expressed as growing degree days, GDD) responses of gross primary production (GPP), net ecosystem exchange (NEE), and ecosystem respiration (ER) at different periods of the summer (early, peak, and late summer) including dominant tundra vegetation classes (graminoids and mosses, and shrubs). We further tested GPP, NEE, and ER relationships with soil moisture and vapor pressure deficit to identify potential moisture limitations on plant productivity and net carbon exchange. Our results show a decrease in GPP with rising GDD during the peak summer (July) for both vegetation classes, and a significant relationship between the peak summer GPP and soil moisture after statistically controlling for GDD in a partial correlation analysis. These results suggest that tundra ecosystems might not benefit from increased temperature as much as suggested by several terrestrial biosphere models, if decreased soil moisture limits the peak summer plant productivity, reducing the ability of these ecosystems to sequester carbon during the summer.

Aneuploidy rates and morphokinetic parameters of embryos cultured in distinct culture media: a sibling oocyte study.

STUDY QUESTION: Do embryos from sibling oocytes assigned to distinct single-step media culture systems demonstrate differences in early embryo development, morphokinectics or aneuploidy rates? SUMMARY ANSWER: Embryo quality, morphokinetic parameters and aneuploidy rates from trophectoderm biopsy were similar between sibling embryos cultured in distinct media systems from the time of gamete isolation. WHAT IS KNOWN ALREADY: Studies on the effect of commercially available embryo culture media systems have demonstrated inconsistent impact on human embryonic development, morphokinetics, aneuploidy rates and clinical outcomes. In addition, these studies have been primarily randomized at the level of the embryo or the patient to culture media. STUDY DESIGN, SIZE, DURATION: Prospective sibling oocyte cohort derived from 200 subjects undergoing IVF at a tertiary academic medical center between February 2018 and November 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sibling oocytes were allocated to Global® or SAGE® media system based upon laterality of ovary from which they were retrieved. All embryos were cultured in a time-lapse incubator. Blastocysts underwent trophectoderm biopsy for preimplantation genetic testing for aneuploidy using next-generation sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: One hundred twenty-seven subjects (n = 127) had paired blastocysts for biopsy in each culture media system. There was no difference in top quality blastocyst formation (47.1 ± 31.0 vs 48.1 ± 27.2%; P = 0.87) nor aneuploidy rate (62.3 ± 34.0 vs 56.1 ± 34.4%; P = 0.07) for sibling embryos cultured in Global versus SAGE media system. Embryo morphokinetic parameters including time to each cell division from two cells (t2) to eight cells (t8), time to morula stage (tM), time to blastocele formation (tSB), time to fully formed blastocyst (tB) and time to expansion of the blastocyst (tEB) were similar between paired blastocysts from each culture media system. LIMITATIONS, REASONS FOR CAUTION: Pregnancy outcomes and offspring health data were not available for analysis. WIDER IMPLICATIONS OF THE FINDINGS: Commercially available culture media may not have a differential impact on embryo development and blastocyst aneuploidy rate when patient and stimulation-related factors are held constant. STUDY FUNDING/COMPETING INTEREST(S): There was no external funding for this study. C.H. is owner of a consultancy company, IVF Professionals, Chief Scientific Officer at Apricity, Executive Director at TMRW and co-owner and shareholder of Aria Fertility. She has received speaker fees, consulting fees and travel support from Cooper Surgical and Vitrolife. J.B. is an employee and shareholder of vitrolife. TRIAL REGISTRATION NUMBER: N/A.

Regional air transportation of ovarian tissue for cryopreservation in a prepubertal female with cancer.

Ovarian tissue cryopreservation is the only fertility preservation (FP) option available to prepubescent females receiving gonadotoxic therapy, but it has limited availability. A 6-year-old female was diagnosed with high-risk rhabdomyosarcoma, and the planned treatment carried an 80% risk of ovarian failure. Her parents desired FP, but the nearest center was 500 miles away. The patient underwent oophorectomy at the cancer center with air transport of the tissue to the oncofertility center, where it was successfully cryopreserved. Formation of networks between full-service and limited oncofertility centers in a hub-and-spoke model would increase access to FP services, particularly in children.

Increased high-latitude photosynthetic carbon gain offset by respiration carbon loss during an anomalous warm winter to spring transition.

Arctic and boreal ecosystems play an important role in the global carbon (C) budget, and whether they act as a future net C sink or source depends on climate and environmental change. Here, we used complementary in situ measurements, model simulations, and satellite observations to investigate the net carbon dioxide (CO2 ) seasonal cycle and its climatic and environmental controls across Alaska and northwestern Canada during the anomalously warm winter to spring conditions of 2015 and 2016 (relative to 2010-2014). In the warm spring, we found that photosynthesis was enhanced more than respiration, leading to greater CO2 uptake. However, photosynthetic enhancement from spring warming was partially offset by greater ecosystem respiration during the preceding anomalously warm winter, resulting in nearly neutral effects on the annual net CO2 balance. Eddy covariance CO2 flux measurements showed that air temperature has a primary influence on net CO2 exchange in winter and spring, while soil moisture has a primary control on net CO2 exchange in the fall. The net CO2 exchange was generally more moisture limited in the boreal region than in the Arctic tundra. Our analysis indicates complex seasonal interactions of underlying C cycle processes in response to changing climate and hydrology that may not manifest in changes in net annual CO2 exchange. Therefore, a better understanding of the seasonal response of C cycle processes may provide important insights for predicting future carbon-climate feedbacks and their consequences on atmospheric CO2 dynamics in the northern high latitudes.

Impact of Clustering Oral Symptoms in the Pathogenesis of Radiation Caries: A Systematic Review.

Radiation-related caries (RRC) is a disease with a high potential for destruction of the dentition, which impairs quality of life in head-and-neck (HN) cancer (HNC) patients who undergo radiotherapy. In light of the recently described "clustering of oral symptoms theory," the present systematic review (PROSPERO CRD42019132709) aims to assess HN and gastrointestinal (GI) symptom clusters among HNC patients and discusses how these indirect effects of cancer therapy play a pivotal role in the pathophysiology of RRC. The search was performed at PubMed, Scopus, and Embase and resulted in 11 studies that met the inclusion criteria. Data extraction was performed with respect to the presence of HN/GI symptom clusters among HNC patients. The methodological data of the studies included were assessed using the MAStARI and GRADE instruments. The most prevalent reported HN symptoms were dysphagia, xerostomia, and pain. Taste alterations and fatigue were also commonly reported by the patients. Loss of appetite and weight loss were regularly reported in the studies, as well as nausea and vomiting. The results of the present study suggest that HNC treatment generates clusters of oral symptoms, leading to dietary changes, impaired oral hygiene, enamel fragility, and a highly cariogenic oral environment, which may impact the risk for RRC. A better understanding of oral symptom clustering could be of considerable clinical significance for the oral health and quality of life of HNC patients. Therefore, contemporary protocols of RRC prevention must take this broader treatment scenario of symptom clusters such as oral side effects into account.

The molecular basis of protein toxin HicA-dependent binding of the protein antitoxin HicB to DNA.

Toxin-antitoxin (TA) systems are present in many bacteria and play important roles in bacterial growth, physiology, and pathogenicity. Those that are best studied are the type II TA systems, in which both toxins and antitoxins are proteins. The HicAB system is one of the prototypic TA systems, found in many bacterial species. Complex interactions between the protein toxin (HicA), the protein antitoxin (HicB), and the DNA upstream of the encoding genes regulate the activity of this system, but few structural details are available about how HicA destabilizes the HicB-DNA complex. Here, we determined the X-ray structures of HicB and the HicAB complex to 1.8 and 2.5 Å resolution, respectively, and characterized their DNA interactions. This revealed that HicB forms a tetramer and HicA and HicB form a heterooctameric complex that involves structural reorganization of the C-terminal (DNA-binding) region of HicB. Our observations indicated that HicA has a profound impact on binding of HicB to DNA sequences upstream of hicAB in a stoichiometric-dependent way. At low ratios of HicA:HicB, there was no effect on DNA binding, but at higher ratios, the affinity for DNA declined cooperatively, driving dissociation of the HicA:HicB:DNA complex. These results reveal the structural mechanisms by which HicA de-represses the HicB-DNA complex.

Changing the Paradigm of Daily Prevention to Achieve Whole Mouth Health in the 21st Century.

A comprehensive mechanical plaque control program - designed, monitored, and reinforced by dental professionals - can help patients achieve excellent oral hygiene and oral health. However, this approach to prevention is impractical for many individuals, so dental caries and periodontal disease are highly prevalent globally. Experts recently agreed that a toothpaste with fluoride and a plaque control agent can augment mechanical procedures to simultaneously prevent caries and periodontal disease. Notwithstanding this, it is timely to rethink prevention and oral health promotion. A new definition of oral health raises awareness of its different dimensions and empowers dentistry to move from treating disease to supporting prevention. In addition, a deeper understanding of the relationship between oral biofilms and the host facilitates new opportunities for disease prevention. The knowledge that health-associated biofilms help prevent establishment of pathogenic species, regulate the potentially damaging host response, and provide essential benefits to health and well-being is paramount to changing the paradigm of prevention of dental disease. Ecological approaches to biofilm control can reduce plaque sufficiently to reduce disease risk, while creating and supporting the beneficial functions of biofilms consistent with health. The knowledge that the oral soft tissues are the primary reservoir of bacteria for tooth recolonization and that reducing bacteria on soft tissues results in improved plaque control and consequently better oral health is also salient. Indeed, a toothpaste that delivers 12-hour protection to the hard and soft tissues (whole mouth protection) and multiple oral health benefits (whole mouth health) could become the future cornerstone of prevention. An innovative fluoride toothpaste with a Dual Zinc plus Arginine multi-functional therapeutic agent offers whole mouth protection against daily oral challenges and whole mouth health for the patient. Within a patient-centered preventive program, next to messaging that motivates towards improved self-care, this toothpaste empowers patients to achieve effective prevention of common oral diseases and better oral health compared to just brushing with an ordinary toothpaste.

Role of microbial biofilms in the maintenance of oral health and in the development of dental caries and periodontal diseases. Consensus report of group 1 of the Joint EFP/ORCA workshop on the boundaries between caries and periodontal disease.

BACKGROUND AND AIMS: The scope of this working group was to review (1) ecological interactions at the dental biofilm in health and disease, (2) the role of microbial communities in the pathogenesis of periodontitis and caries, and (3) the innate host response in caries and periodontal diseases. RESULTS AND CONCLUSIONS: A health-associated biofilm includes genera such as Neisseria, Streptococcus, Actinomyces, Veillonella and Granulicatella. Microorganisms associated with both caries and periodontal diseases are metabolically highly specialized and organized as multispecies microbial biofilms. Progression of these diseases involves multiple microbial interactions driven by different stressors. In caries, the exposure of dental biofilms to dietary sugars and their fermentation to organic acids results in increasing proportions of acidogenic and aciduric species. In gingivitis, plaque accumulation at the gingival margin leads to inflammation and increasing proportions of proteolytic and often obligately anaerobic species. The natural mucosal barriers and saliva are the main innate defence mechanisms against soft tissue bacterial invasion. Similarly, enamel and dentin are important hard tissue barriers to the caries process. Given that the present state of knowledge suggests that the aetiologies of caries and periodontal diseases are mutually independent, the elements of innate immunity that appear to contribute to resistance to both are somewhat coincidental.

A Flow Cytometry Method for Rapidly Assessing Mycobacterium tuberculosis Responses to Antibiotics with Different Modes of Action.

Current methods for assessing the drug susceptibility of Mycobacterium tuberculosis are lengthy and do not capture information about viable organisms that are not immediately culturable under standard laboratory conditions as a result of antibiotic exposure. We have developed a rapid dual-fluorescence flow cytometry method using markers for cell viability and death. We show that the fluorescent marker calcein violet with an acetoxy-methyl ester group (CV-AM) can differentiate between populations of M. tuberculosis growing at different rates, while Sytox green (SG) can differentiate between live and dead mycobacteria. M. tuberculosis was exposed to isoniazid or rifampin at different concentrations over time and either dual stained with CV-AM and SG and analyzed by flow cytometry or plated to determine the viability of the cells. Although similar trends in the loss of viability were observed when the results of flow cytometry and the plate counting methods were compared, there was a lack of correlation between these two approaches, as the flow cytometry analysis potentially captured information about cell populations that were unable to grow under standard conditions. The flow cytometry approach had an additional advantage in that it could provide insights into the mode of action of the drug: antibiotics targeting the cell wall gave a flow cytometry profile distinct from those inhibiting intracellular processes. This rapid drug susceptibility testing method could identify more effective antimycobacterials, provide information about their potential mode of action, and accelerate their progress to the clinic.

Sorafenib in the treatment of hepatocellular carcinoma: a multi-centre real-world study.

OBJECTIVE: Sorafenib is an oral multikinase inhibitor that improves survival in advanced hepatocellular carcinoma (HCC). In the absence of alternative therapies, sorafenib is often continued despite advancing liver disease or tumour progression. Real world studies are important to better characterise outcomes in these populations. Our aim was to review patterns of sorafenib use across eight Australian tertiary hospitals, defining variables associated with clinical outcomes. MATERIAL AND METHODS: Retrospective cohort study of medical records of 320 patients treated with sorafenib for HCC. Baseline clinical parameters, dosage, adverse effects, and survival from initiation of treatment were collected. Time to radiological progression and 3-month alpha-fetoprotein (AFP) levels were available for a subset of patients. RESULTS: Adverse effects occurred in 79% of patients, requiring dose reduction in 31% of patients. Multivariate analysis identified an increased rate of mortality with Child-Pugh C (HR 5.52, p = 0.012), ECOG performance status 2-3 (HR 2.84, p = 0.001), and extrahepatic metastases (HR 1.54, p = 0.04), and decreased rate of mortality with an AFP reduction of at least 20% at 3 months (HR 0.38, p = 0.001). An increased rate of radiological progression was associated with ECOG performance status 2-3 (HR 2.34, p = 0.041), whilst a decreased rate of radiological progression was associated with development of on-treatment diarrhoea (HR 0.55, p = 0.015). CONCLUSIONS: Survival in patients with Child-Pugh C liver function or advanced functional impairment treated with sorafenib is poor and thus routine use of this agent in these patients does not appear justified, particularly given the high rate of adverse effects. AFP concentration on therapy may help identify favourable response to treatment.

Influence of saliva on the oral microbiota.

Saliva plays a major role in determining the composition and activity of the oral microbiota, via a variety of mechanisms. Molecules, mainly from saliva, form a conditioning film on oral surfaces, thus providing receptors for bacterial attachment. The attached cells use saliva components, such as glycoproteins, as their main source of nutrients for growth. Oral bacteria work sequentially and in a concerted manner to catabolize these structurally complex molecules. Saliva also buffers the pH in the biofilm to around neutrality, creating an environment which is conducive to the growth of many oral bacteria that provide important benefits to the host. Components of the adaptive and innate host defences are delivered by saliva, and these often function synergistically, and at sublethal concentrations, so a complex relationship develops between the host and the resident microbiota. Dysbiosis can occur rapidly if the flow of saliva is perturbed.

The effect of growth rate on pyrazinamide activity in Mycobacterium tuberculosis - insights for early bactericidal activity?

BACKGROUND: Pyrazinamide (PZA) plays an essential part in the shortened six-month tuberculosis (TB) treatment course due to its activity against slow-growing and non-replicating organisms. We tested whether PZA preferentially targets slow growing cells of Mycobacterium tuberculosis that could be representative of bacteria that remain after the initial kill with isoniazid (INH), by observing the response of either slow growing or fast growing bacilli to differing concentrations of PZA. METHODS: M. tuberculosis H37Rv was grown in continuous culture at either a constant fast growth rate (Mean Generation Time (MGT) of 23.1 h) or slow growth rate (69.3 h MGT) at a controlled dissolved oxygen tension of 10 % and a controlled acidity at pH 6.3 ± 0.1. Cultures were exposed to step-wise increases in the concentration of PZA (25 to 500 µgml(-1)) every two MGTs, and bacterial survival was measured. PZA-induced global gene expression was explored for each increase in PZA-concentration, using DNA microarray. RESULTS: At a constant pH 6.3, actively dividing mycobacteria were susceptible to PZA, with similar responses to increasing concentrations of PZA at both growth rates. Three distinct phases of drug response could be distingished for both slow growing (69.3 h MGT) and fast growing (23.1 h MGT) bacilli. A bacteriostatic phase at a low concentration of PZA was followed by a recovery period in which the culture adapted to the presence of PZA and bacteria were actively dividing in steady-state. In contrast, there was a rapid loss of viability at bactericidal concentrations. There was a notable delay in the onset of the recovery period in quickly dividing cells compared with those dividing more slowly. Fast growers and slow growers adapted to PZA-exposure via very similar mechanisms; through reduced gene expression of tRNA, 50S, and 30S ribosomal proteins. CONCLUSIONS: PZA had an equivalent level of activity against fast growing and slow growing M. tuberculosis. At both growth rates drug-tolerance to sub-lethal concentrations may have been due to reduced expression of tRNA, 50S, and 30S ribosomal proteins. The findings from this study show that PZA has utility against more than one phenotypic sub-population of bacilli and could be re-assessed for its early bactericidal activity, in combination with other drugs, during TB treatment.

Thiopurine metabolite testing in inflammatory bowel disease.

BACKGROUND: Thiopurine use in inflammatory bowel disease (IBD) is limited by drug toxicity and lack of therapeutic efficacy. We assessed the utility of thiopurine metabolite testing and the relationship between disease activity, dose, and metabolite levels in a real world setting. METHODS: Patients identified from pathology databases (2007-2012) at two tertiary IBD centers were included if they had thiopurines for at least four weeks. Demographics, dose, test indication, clinical status, action taken, and outcome were obtained by retrospective medical record review. RESULTS: A total of 169 patients were included. 6-Thioguanine (TGN) levels were sub-therapeutic in 52%, therapeutic in 34%, and supratherapeutic in 14%. Test indication was active disease (79%), adverse effect (11%), or adherence assessment (7%). TGN trended lower in the active disease group compared to those with adverse effects (273 (+/- 23.2) versus 447 (+/- 117.7) pmol/8 × 10(8) RBC, P = 0.05). Weight-based dosing did not improve rates of therapeutic TGN levels (under-dosed 31.5% vs standard dose 35.4%), but was significantly associated with shunting toward 6-MMP (23.1% vs 6.8%, P = 0.008, OR = 4.1). Testing resulted in a change in patient treatment in 86% of patients with active disease and subtherapeutic levels and in 68% of tested patients overall. CONCLUSIONS: Metabolite testing resulted in a change in management in most patients not responding to thiopurines or experiencing adverse events. Weight-based dosing did not increase rates of therapeutic levels but was associated with increased 6MMP shunting.

Mortality and use of the auxiliary score in extensive toxic epidermal necrolysis patients admitted to an adult burns referral centre.

BACKGROUND: Toxic epidermal necrolysis (TEN) is a rare but fatal condition characterised by cutaneous exfoliation of the dermoepidermal layer and mucosal surfaces. Extensive TEN with epidermal detachment >30% of the total body surface area has been associated with a high mortality. OBJECTIVE: This study aims to evaluate factors associated with mortality in extensive TEN. In the absence of data to qualify scoring systems such as SCORTEN, this study also aims to evaluate the use of the auxiliary score as a tool for calculating expected mortality. METHODS: A retrospective chart review of all patients presenting to our burns service with extensive TEN was undertaken. Application and evaluation of the auxiliary score was also undertaken for this patient population. RESULTS: In extensive TEN, age and delay in admission to a burns centre were factors associated with mortality. Applying the auxiliary score to our patient population, there were no significant differences between expected mortality and observed mortality. CONCLUSION: Mortality was associated with age and delay in definitive treatment in extensive TEN. Whilst SCORTEN is the gold standard prognostic tool for patients with TEN, in the absence of SCORTEN values, the auxiliary score provides an alternative scoring system to evaluate expected mortality.

Hemin availability induces coordinated DNA methylation and gene expression changes in Porphyromonas gingivalis.

Periodontal disease is a chronic inflammatory disease in which the oral pathogen Porphyromonas gingivalis plays an important role. Porphyromonas gingivalis expresses virulence determinants in response to higher hemin concentrations, but the underlying regulatory processes remain unclear. Bacterial DNA methylation has the potential to fulfil this mechanistic role. We characterized the methylome of P. gingivalis, and compared its variation to transcriptome changes in response to hemin availability. Porphyromonas gingivalis W50 was grown in chemostat continuous culture with excess or limited hemin, prior to whole-methylome and transcriptome profiling using Nanopore and Illumina RNA-Seq. DNA methylation was quantified for Dam/Dcm motifs and all-context N6-methyladenine (6mA) and 5-methylcytosine (5mC). Of all 1,992 genes analyzed, 161 and 268 were respectively over- and under-expressed with excess hemin. Notably, we detected differential DNA methylation signatures for the Dam "GATC" motif and both all-context 6mA and 5mC in response to hemin availability. Joint analyses identified a subset of coordinated changes in gene expression, 6mA, and 5mC methylation that target genes involved in lactate utilization and ABC transporters. The results identify altered methylation and expression responses to hemin availability in P. gingivalis, with insights into mechanisms regulating its virulence in periodontal disease. IMPORTANCE DNA methylation has important roles in bacteria, including in the regulation of transcription. Porphyromonas gingivalis, an oral pathogen in periodontitis, exhibits well-established gene expression changes in response to hemin availability. However, the regulatory processes underlying these effects remain unknown. We profiled the novel P. gingivalis epigenome, and assessed epigenetic and transcriptome variation under limited and excess hemin conditions. As expected, multiple gene expression changes were detected in response to limited and excess hemin that reflect health and disease, respectively. Notably, we also detected differential DNA methylation signatures for the Dam "GATC" motif and both all-context 6mA and 5mC in response to hemin. Joint analyses identified coordinated changes in gene expression, 6mA, and 5mC methylation that target genes involved in lactate utilization and ABC transporters. The results identify novel regulatory processes underlying the mechanism of hemin regulated gene expression in P. gingivalis, with phenotypic impacts on its virulence in periodontal disease.

Arctic soil methane sink increases with drier conditions and higher ecosystem respiration.

Arctic wetlands are known methane (CH4) emitters but recent studies suggest that the Arctic CH4 sink strength may be underestimated. Here we explore the capacity of well-drained Arctic soils to consume atmospheric CH4 using >40,000 hourly flux observations and spatially distributed flux measurements from 4 sites and 14 surface types. While consumption of atmospheric CH4 occurred at all sites at rates of 0.092 ± 0.011 mgCH4 m-2 h-1 (mean ± s.e.), CH4 uptake displayed distinct diel and seasonal patterns reflecting ecosystem respiration. Combining in situ flux data with laboratory investigations and a machine learning approach, we find biotic drivers to be highly important. Soil moisture outweighed temperature as an abiotic control and higher CH4 uptake was linked to increased availability of labile carbon. Our findings imply that soil drying and enhanced nutrient supply will promote CH4 uptake by Arctic soils, providing a negative feedback to global climate change.

Thermodynamic basis for the demarcation of Arctic and alpine treelines.

At the edge of alpine and Arctic ecosystems all over the world, a transition zone exists beyond which it is either infeasible or unfavorable for trees to exist, colloquially identified as the treeline. We explore the possibility of a thermodynamic basis behind this demarcation in vegetation by considering ecosystems as open systems driven by thermodynamic advantage-defined by vegetation's ability to dissipate heat from the earth's surface to the air above the canopy. To deduce whether forests would be more thermodynamically advantageous than existing ecosystems beyond treelines, we construct and examine counterfactual scenarios in which trees exist beyond a treeline instead of the existing alpine meadow or Arctic tundra. Meteorological data from the Italian Alps, United States Rocky Mountains, and Western Canadian Taiga-Tundra are used as forcing for model computation of ecosystem work and temperature gradients at sites on both sides of each treeline with and without trees. Model results indicate that the alpine sites do not support trees beyond the treeline, as their presence would result in excessive CO[Formula: see text] loss and extended periods of snowpack due to temperature inversions (i.e., positive temperature gradient from the earth surface to the atmosphere). Further, both Arctic and alpine sites exhibit negative work resulting in positive feedback between vegetation heat dissipation and temperature gradient, thereby extending the duration of temperature inversions. These conditions demonstrate thermodynamic infeasibility associated with the counterfactual scenario of trees existing beyond a treeline. Thus, we conclude that, in addition to resource constraints, a treeline is an outcome of an ecosystem's ability to self-organize towards the most advantageous vegetation structure facilitated by thermodynamic feasibility.

Molecular basis for avirulence of spontaneous variants of Porphyromonas gingivalis: Genomic analysis of strains W50, BE1 and BR1.

The periodontal pathogen Porphyromonas gingivalis is genetically heterogeneous. However, the spontaneous generation of phenotypically different sub-strains has also been reported. McKee et al. (1988) cultured P. gingivalis W50 in a chemostat during investigations into the growth and properties of this bacterium. Cell viability on blood agar plates revealed two types of non-pigmenting variants, W50 beige (BE1), and W50 brown (BR1), in samples grown in a high-hemin medium after day 7, and the population of these variants increased to approximately 25% of the total counts by day 21. W50, BE1 and BR1 had phenotypic alterations in pigmentation, reduced protease activity and haemagglutination and susceptibility to complement killing. Furthermore, the variants exhibited significant attenuation in a mouse model of virulence. Other investigators showed that in BE1, the predominant extracellular Arg-gingipain was RgpB, and no reaction with an A-lipopolysaccharide-specific MAb 1B5 (Collinson et al., 1998; Slaney et al., 2006). In order to determine the genetic basis for these phenotypic properties, we performed hybrid DNA sequence long reads using Oxford Nanopore and the short paired-end DNA sequence reads of Illumina HiSeq platforms to generate closed circular genomes of the parent and variants. Comparative analysis indicated loss of intact kgp in the 20 kb region of the hagA-kgp locus in the two variants BE1 and BR1. Deletions in hagA led to smaller open reading frames in the variants, and BR1 had incurred a major chromosomal DNA inversion. Additional minor changes to the genomes of both variants were also observed. Given the importance of Kgp and HagA to protease activity and haemagglutination, respectively, in this bacterium, genomic changes at this locus may account for most of the phenotypic alterations of the variants. The homologous and repetitive nature of hagA and kgp and the features at the inverted junctions are indicative of specific and stable homologous recombination events, which may underlie the genetic heterogeneity of this species.

A deep dive into the morphokinetics and ploidy of low-quality blastocysts.

Objective: To describe morphokinetic parameters and ploidy among low-quality blastocysts not meeting the criteria for clinical use. Design: Prospective cohort study. Setting: Academic medical center. Patients: Two hundred patients undergoing in vitro fertilization between February 2018 and November 2019. Interventions: All embryos were cultured in a time-lapse incubator. All expanded blastocysts underwent preimplantation genetic testing for aneuploidy using next-generation sequencing. Main Outcome Measures: Static blastocyst morphology grading; morphokinetic parameters, including time to each cell division (2-cell formation to 8-cell formation); time to morula formation; time to the start of blastulation; time to blastocyst formation; and preimplantation genetic testing for aneuploidy results. Results: A total of 1,306 embryos progressed to the expanded blastocyst stage; of these, 935 embryos met the criteria for clinical use and were designated as high quality, whereas 371 embryos were graded as low quality and did not meet the criteria for use. In morphokinetic evaluation, low-quality embryos developed more quickly to 5-cell formation (t5) 48.4 [42.4-48.7) vs 50.2 [46.3-50.1] hours, but progressed more slowly thereafter with tM 91.5 [85.9-92.3] vs 88.3 [82.1-88.3] and tB 114.0 [106.4-113.9] vs 106.9 [101.3-107.4] hours. Among the low-quality embryos, 75.5% were aneuploid, 22.4% were euploid, and 2.2% had undetermined chromosome copy number results. Morphokinetic parameters did not differ between the euploid and aneuploid low-quality embryos. Conclusions: Morphokinetic analysis did not distinguish between euploid and aneuploid low-quality embryos.

Optimization of conditions for in vitro modeling of subgingival normobiosis and dysbiosis.

Modeling subgingival microbiome in health and disease is key to identifying the drivers of dysbiosis and to studying microbiome modulation. Here, we optimize growth conditions of our previously described in vitro subgingival microbiome model. Subgingival plaque samples from healthy and periodontitis subjects were used as inocula to grow normobiotic and dysbiotic microbiomes in MBEC assay plates. Saliva supplemented with 1%, 2%, 3.5%, or 5% (v/v) heat-inactivated human serum was used as a growth medium under shaking or non-shaking conditions. The microbiomes were harvested at 4, 7, 10 or 13 days of growth (384 microbiomes in total) and analyzed by 16S rRNA gene sequencing. Biomass significantly increased as a function of serum concentration and incubation period. Independent of growth conditions, the health- and periodontitis-derived microbiomes clustered separately with their respective inocula. Species richness/diversity slightly increased with time but was adversely affected by higher serum concentrations especially in the periodontitis-derived microbiomes. Microbial dysbiosis increased with time and serum concentration. Porphyromonas and Alloprevotella were substantially enriched in higher serum concentrations at the expense of Streptococcus, Fusobacterium and Prevotella. An increase in Porphyromonas, Bacteroides and Mogibacterium accompanied by a decrease in Prevotella, Catonella, and Gemella were the most prominent changes over time. Shaking had only minor effects. Overall, the health-derived microbiomes grown for 4 days in 1% serum, and periodontitis-derived microbiomes grown for 7 days in 3.5%-5% serum were the most similar to the respective inocula. In conclusion, normobiotic and dysbiostic subgingival microbiomes can be grown reproducibly in saliva supplemented with serum, but time and serum concentration need to be adjusted differently for the health and periodontitis-derived microbiomes to maximize similarity to in vivo inocula. The optimized model could be used to identify drivers of dysbiosis, and to evaluate interventions such as microbiome modulators.