Proteomic study uncovers molecular principles of single-cell-level phenotypic heterogeneity in lipid storage of Nannochloropsis oceanica

Abstract Background Nannochloropsis oceanica belongs to a large group of photoautotrophic eukaryotic organisms that play important roles in fixation and cycling of atmospheric CO2. Its capability of storing solar energy and carbon dioxide in the form of triacylglycerol (TAG) of up to 60% of total weight under nitrogen deprivation stress sparked interest in its use for biofuel production. Phenotypes varying in lipid accumulation among an N. oceanica population can be disclosed by single-cell analysis/sorting using fluorescence-activated cell sorting (FACS); yet the phenomenon of single cell heterogeneity in an algae population remains to be fully understood at the molecular level. In this study, combination of FACS and proteomics was used for identification, quantification and differentiation of these heterogeneities on the molecular level. Results For N. oceanica cultivated under nitrogen deplete (−N) and replete (+N) conditions, two groups differing in lipid content were distinguished. These differentiations could be recognized on the population as well as the single-cell levels; proteomics uncovered alterations in carbon fixation and flux, photosynthetic machinery, lipid storage and turnover in the populations. Although heterogeneity patterns have been affected by nitrogen supply and cultivation conditions of the N. oceanica populations, differentiation itself seems to be very robust against these factors: cultivation under +N, −N, in shaker bottles, and in a photo-bioreactor all split into two subpopulations. Intriguingly, population heterogeneity resumed after subpopulations were separately recultivated for a second round, refuting the possible development of genetic heterogeneity in the course of sorting and cultivation. Conclusions This work illustrates for the first time the feasibility of combining FACS and (prote)-omics for mechanistic understanding of phenotypic heterogeneity in lipid-producing microalgae. Such combinatorial method can facilitate molecular breeding and design of bioprocesses

Twenty-Four Hour Fasting (Basal Rate) Tests to Achieve Custom-Tailored, Hour-by-Hour Basal Insulin Infusion Rates in Patients With Type 1 Diabetes Using Insulin Pumps (CSII).

BACKGROUND: Twenty-four hour fasting periods are being used to scrutinize basal insulin infusion rates for pump-treated patients with type 1 diabetes. METHODS: Data from 339 consecutive in-patients with adult type 1 diabetes on insulin pump therapy undergoing a 24-hour fast as a basal rate test were retrospectively analyzed. Hourly programmed basal insulin infusion rates and plasma glucose concentrations within, below, or above arbitrarily defined target ranges were assessed for periods of the day of special interest (eg, 01:00-07:00 am, "dawn" period, 04:00-07:00 pm, and "dusk" period). Statistics: χ2-tests, paired t-tests were used. RESULTS: Basal rates (mean: 0.90 ± 0.02 IU/h) showed circadian variations with peaks corresponding to "dawn" (1.07 ± 0.02 IU/h from 01:00 to 07:00 am) and, less prominently, "dusk" (0.95 ± 0.02 IU/h from 03:00 to 07:00 pm). Individual mean plasma glucose concentrations averaged 6.6 ± 0.1 mmol/L, with 53.1% in the predefined "strict" (4.4-7.2 mmol/L) target range. Interestingly, during the "dawn" period, plasma glucose was significantly higher (by 0.5 ± 0.1 mmol/L [95% confidence interval: 0.3-0.8 mmol/L; P < .0001]) and the odds ratio for hypoglycemia was significantly lower compared to the reference period. INTERPRETATION: Twenty-four hour fasting periods as basal rate tests frequently unravel periods with inappropriate basal insulin infusion rates potentially responsible for fasting hyper- or hypoglycemia. Notably, the higher basal insulin infusion rate found during the "dawn" period seems to be justified and may need to be accentuated.status: publishe