Analysis of living S. cerevisiae cell states--a three color approach.

BACKGROUND: Biosyntheses often fluctuate with the state of the cell in the cell cycle and on the capacity of the cell to access and metabolize a carbon source. Visualization of substrate uptake by individual cells, together with the simultaneous analysis of proliferation activity and the proportion of dead cells, facilitate reliable and quasi-online process optimization. METHODS: Flow cytometry and Hoechst 33342 staining were used to follow proliferation activity of living Saccharomyces cerevisiae cells, whereas 2-NBD-glucose was employed to analyze the cells' substrate affinity. Propidium iodide was used to determine the proportion of dead cells. Calibration and verification experiments were performed with cells grown batch-wise as well as in transient state regimes. RESULTS: A new and rapid three-color assay was developed and tested under varying microenvironmental conditions. CONCLUSIONS: Live/dead cell states and the affinity to 2-NBD-glucose vs. proliferation states were determined during respiratory and/or fermentative modes of metabolism.     

Completion of isoniazid–rifapentine (3HP) for tuberculosis prevention among people living with HIV: Interim analysis of a hybrid type 3 effectiveness–implementation randomized trial

BackgroundScaling up shorter regimens for tuberculosis (TB) prevention such as once weekly isoniazid–rifapentine (3HP) taken for 3 months is a key priority for achieving targets set forth in the World Health Organization’s (WHO) END TB Strategy. However, there are few data on 3HP patient acceptance and completion in the context of routine HIV care in sub-Saharan Africa.Methods and findingsThe 3HP Options Trial is a pragmatic, parallel type 3 effectiveness–implementation randomized trial comparing 3 optimized strategies for delivering 3HP—facilitated directly observed therapy (DOT), facilitated self-administered therapy (SAT), or informed choice between DOT and SAT using a shared decision-making aid—to people receiving care at a large urban HIV clinic in Kampala, Uganda. Participants and healthcare providers were not blinded to arm assignment due to the nature of the 3HP delivery strategies. We conducted an interim analysis of participants who were enrolled and exited the 3HP treatment period between July 13, 2020 and April 30, 2021. The primary outcome, which was aggregated across trial arms for this interim analysis, was the proportion who accepted and completed 3HP (≥11 of 12 doses within 16 weeks of randomization). We used Bayesian inference analysis to estimate the posterior probability that this proportion would exceed 80% under at least 1 of the 3HP delivery strategies, a coprimary hypothesis of the trial. Through April 2021, 684 participants have been enrolled, and 479 (70%) have exited the treatment period. Of these 479 participants, 309 (65%) were women, mean age was 41.9 years (standard deviation (SD): 9.2), and mean time on antiretroviral therapy (ART) was 7.8 years (SD: 4.3). In total, 445 of them (92.9%, 95% confidence interval (CI): [90.2 to 94.9]) accepted and completed 3HP treatment. There were no differences in treatment acceptance and completion by sex, age, or time on ART. Treatment was discontinued due to a documented adverse event (AE) in 8 (1.7%) patients. The probability that treatment acceptance and completion exceeds 80% under at least 1 of the three 3HP delivery strategies was greater than 99%. The main limitations are that the trial was conducted at a single site, and the interim analysis focused on aggregate outcome data to maintain blinding of investigators to arm-specific outcomes.Conclusions3HP was widely accepted by people living with HIV (PLHIV) in Uganda, and very high levels of treatment completion were achieved in a programmatic setting. These findings show that 3HP can enable effective scale-up of tuberculosis preventive therapy (TPT) in high-burden countries, particularly when delivery strategies are tailored to target known barriers to treatment completion.Trial registrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT03934931","term_id":"NCT03934931"}}NCT03934931.

In this interim analysis, Adithya Cattamanchi and colleagues report their findings on 3HP patient acceptance and completion in a routine HIV care context.     

Key sites for the conservation of waterbirds in Uganda

Arinaitwe, J., Byaruhanga, A. & Mafabi, P. 2000. Key sites for the conservation of waterbirds in Uganda. Ostrich 71 (1 &2): 102–105.

Uganda has a rich bird diversity with 1 008 species including 28 listed species (Collar et al. 1994) and 207 species of regional conservation concern (Bennun & Njoroge 1996). Over 240 species are waterfowl, of which nine are globally threatened, 45 are regionally threatened and five are considered to be of national conservation interest. This high waterbird diversity corresponds to a large wetland area covering about 10% of the land area of Uganda. However, only 6% of the wetlands fall under gazetted protected areas and only one site is recognised as of global importance under the terms of the Ramsar Convention. Three recent activities of JA, the National Wetlands Programme and East Africa Natural History Society (Uganda), have produced new information on distribution and abundance of waterbirds in Uganda. Concurrent with these activities, criteria for the identification of globally threatened species (Collar et al. 1994), species of regional conservation concern (Bennun & Njoroge 1996) and nationally important species (National Wetlands Programme, in press) have been developed. Also, criteria for selection of sites at the global (Ramsar and IBA), regional (IBA) and national (National Wetlands Programme) levels have become available. This paper reviews the advances in the knowledge of waterbirds in Uganda and applies the above criteria to highlight sites that are crucial for conservation at global, regional and national levels.     

Five decades of rapid forest spread in the Pieria Mountains (N. Greece) reconstructed by means of high-resolution pollen analysis and aerial photographs

The rapid spread of pine forests on the western side of the Pieria Mountains (N. Greece) began in 1945 and was facilitated by the recession of human activity and the siliceous geological substrate of the area. The pine expansion is visible not only in the vegetation maps for the years 1945, 1960 and 1992 but is also reflected in pollen data acquired from the high resolution analysis of a peat sequence covering the period 1945–1997, according to ²¹⁰Pb and ¹³⁷Cs dating. A possible inflation of the calculated PAR values due to discrepancies in the dating model is suspected and indirectly traced from the comparison of the Pinus mean PAR values for the most recent eight years of the monolith with those of a Tauber trap placed in the same mire. The presence of a high pollen producer such as pine enables the calculation of reliable PAR values, despite any discrepancies in the age-depth model. The Pinus PAR values “quantify” better the expansion of this timberline species, while pollen percentages reflect the same event in a “qualitative” way, i.e. as part of the general vegetation changes that take place in the area.     

Brace technology thematic series: the dynamic derotation brace

Background

The curveback lineage of guppy is characterized by heritable idiopathic-type spinal curvature that develops during growth. Prior work has revealed several important developmental similarities to the human idiopathic scoliosis (IS) syndrome. In this study we investigate structural and histological aspects of the vertebrae that are associated with spinal curvature in the curveback guppy and test for sexual dimorphism that might explain a female bias for severe curve magnitudes in the population.

Methods

Vertebrae were studied from whole-mount skeletal specimens of curved and non-curved adult males and females. A series of ratios were used to characterize structural aspects of each vertebra. A three-way analysis of variance tested for effects of sex, curvature, vertebral position along the spine, and all 2-way interactions (i.e., sex and curvature, sex and vertebra position, and vertebra position and curvature). Histological analyses were used to characterize micro-architectural changes in affected vertebrae and the intervertebral region.

Results

In curveback, vertebrae that are associated with curvature demonstrate asymmetric shape distortion, migration of the intervertebral ligament, and vertebral thickening on the concave side of curvature. There is sexual dimorphism among curved individuals such that for several vertebrae, females have more slender vertebrae than do males. Also, in the region of the spine where lordosis typically occurs, curved and non-curved females have a reduced width at the middle of their vertebrae, relative to males.

Conclusions

Based on similarities to human spinal curvatures and to animals with induced curves, the concave-convex biases described in the guppy suggest that there is a mechanical component to curve pathogenesis in curveback. Because idiopathic-type curvature in curveback is primarily a sagittal deformity, it is structurally more similar to Scheuermann kyphosis than IS. Anatomical differences between teleosts and humans make direct biomechanical comparisons difficult. However, study of basic biological systems involved in idiopathic-type spinal curvature in curveback may provide insight into the relationship between a predisposing aetiology, growth, and biomechanics. Further work is needed to clarify whether observed sex differences in vertebral characteristics are related to the female bias for severe curves that is observed in the population.     

Eleven‐year Prevalence Trends of Obesity in Greek Children: First Evidence that Prevalence of Obesity Is Leveling Off

We examined 11‐year (1997–2007) trends in underweight, overweight, and obesity in Greek children. Population data derived from a yearly, school‐based health survey carried out between 1997 and 2007 in >80% of all Greek schools. Height and weight measurements from 651,582 children, aged 8–9 years (boys: 51.2%) were analyzed. The gender‐ and age‐specific BMI cutoff points by the International Obesity Task Force (IOTF) were used in order to define underweight, normal weight, overweight, and obesity. Trend analysis showed an increase in the prevalence of obesity from 7.2 ± 0.2% in 1997 to 11.3 ± 0.2% in 2004 for girls (P < 0.001) and from 8.1 ± 0.2% in 1997 to 12.3 ± 0.2% in 2004 for boys (P < 0.001). An apparent leveling off in obesity rates was observed during 2004–2007 for both boys and girls. The prevalence of overweight rose between 1997 and 2007 from 20.2 ± 0.2% to 26.7 ± 0.2% for girls (P < 0.001) and from 19.6 ± 0.2% to 26.5 ± 0.2% for boys (P < 0.001). The overall prevalence of thinness in the same period remained constant in both sexes. The presented population‐based data revealed that the prevalence of overweight and obesity among 8‐ to 9‐year‐old Greek children is alarmingly elevated, with the overweight rates rising continuously. However, an apparent leveling off in obesity rates for the past 4 consecutive years was documented for the first time in both genders.     

Role of P2X purinergic receptors in the rescue of ischemic heart failure

Evidence is accumulating to support the presence of P2X purinergic receptors in the heart. However, the biological role of this receptor remains to be defined. The objectives here were to determine the role of cardiac P2X receptors in modulating the progression of post-myocardial infarction ischemic heart failure and to investigate the underlying mechanism. The P2X4 receptor (P2X4R) is an important subunit of native cardiac P2X receptors, and the cardiac-specific transgenic overexpression of P2X4R (Tg) was developed as a model. Left anterior descending artery ligation resulted in similar infarct size between Tg and wild-type (WT) mice (P > 0.1). However, Tg mice showed an enhanced cardiac contractile performance at 7 days, 1 mo, and 2 mo after infarction and an increased survival at 1 and 2 mo after infarction (P < 0.01). The enhanced intact heart function was manifested by a greater global left ventricular developed pressure and rate of contraction of left ventricular pressure in vitro and by a significantly increased fractional shortening and systolic thickening in the noninfarcted region in vivo (P < 0.05). The salutary effects on the ischemic heart failure phenotype were seen in both sexes and were not the result of any difference in infarct size in Tg versus WT hearts. An enhanced contractile function of the noninfarcted area in the Tg heart was likely an important rescuing mechanism. The cardiac P2X receptor is a novel target to treat post-myocardial infarction ischemic heart failure.     

Affinity of single S. cerevisiae cells to 2-NBDglucose under changing substrate concentrations.

BACKGROUND: Saccharomyces cerevisiae is a widely employed microorganism in biotechnological processes. Since proliferation and product formation depend on the capacity of the cell to access and metabolize a carbon source, a technique was developed to enable for analyzing the S. cerevisiae H155 cells' affinity to extracellular glucose concentrations. METHODS: The fluorescent glucose analogue 2-NBDglucose was employed as a functional parameter to analyze the cells' affinity to glucose. Structural parameters (proliferation, neutral lipid content, granularity, and cell size) were also investigated. Cells were grown both in batches and in chemostat regimes. RESULTS: The 2-NBDglucose uptake in individual cells proceeds in a time- and concentration-dependent manner and is affected by respiratory and respirofermentative modes of growth. The process is inhibited by D-glucose, D-fructose, D-mannose, and sucrose, but not L-glucose, D-galactose or lactose; maltose is a weak inhibitor. The affinity of the individual cells to 2-NBDglucose was found to be high at low extracellular glucose concentrations, and weak at high concentrations. An additional, underlying pattern in the cells' affinity to glucose was detected, illustrated by the recurrent appearance of two subpopulations showing distinctly differing quantities of this substrate. CONCLUSIONS: A multiparameter flow cytometry approach is presented that enables, for the first time, for analysis of the affinity of individual S. cerevisiae cells to glucose. Besides the adjustment of the yeast cell metabolism to extracellular glucose concentrations by altering their affinity to glucose, at least one further mechanism is clearly involved. Two subpopulations of cells were resolved, with different affinities not correlated with other cellular parameters measured.