Empirical Characterization of False Discovery Rates of Differentially Expressed Genes and Transcriptomic Benchmark Concentrations in Zebrafish Embryos.

High-throughput transcriptomics (HTTr) is increasingly applied to zebrafish embryos to survey the toxicological effects of environmental chemicals. Before the adoption of this approach in regulatory testing, it is essential to characterize background noise in order to guide experimental designs. We thus empirically quantified the HTTr false discovery rate (FDR) across different embryo pool sizes, sample sizes, and concentration groups for toxicology studies. We exposed zebrafish embryos to 0.1% dimethyl sulfoxide (DMSO) for 5 days. Pools of 1, 5, 10, and 20 embryos were created (n = 24 samples for each pool size). Samples were sequenced on the TempO-Seq platform and then randomly assigned to mock treatment groups before differentially expressed gene (DEG), pathway, and benchmark concentration (BMC) analyses. Given that all samples were treated with DMSO, any significant DEGs, pathways, or BMCs are false positives. As expected, we found decreasing FDRs for DEG and pathway analyses with increasing pool and sample sizes. Similarly, FDRs for BMC analyses decreased with increasing pool size and concentration groups, with more stringent BMC premodel filtering reducing BMC FDRs. Our study provides foundational data for determining appropriate experiment designs for regulatory toxicity testing with HTTr in zebrafish embryos.

Dynamics of the enterohepatic circulation of bile acids in healthy humans.

Regulation of bile acid metabolism is normally discussed as the regulation of bile acid synthesis, which serves to compensate for intestinal loss in order to maintain a constant pool size. After a meal, bile acids start cycling in the enterohepatic circulation. Farnesoid X receptor-dependent ileal and hepatic processes lead to negative feedback inhibition of bile acid synthesis. When the intestinal bile acid flux decreases, the inhibition of synthesis is released. The degree of inhibition of synthesis and the mechanism and degree of activation are still unknown. Moreover, in humans, a biphasic diurnal expression pattern of bile acid synthesis has been documented, indicating maximal synthesis around 3 PM and 9 PM. Quantitative data on the hourly synthesis schedule as compensation for intestinal loss are lacking. In this review, we describe the classical view on bile acid metabolism and present alternative concepts that are based on the overlooked feature that bile acids transit through the enterohepatic circulation very rapidly. A daily profile of the cycling and total bile acid pool sizes and potential controlled and uncontrolled mechanisms for synthesis are predicted. It remains to be elucidated by which mechanism clock genes interact with the Farnesoid X receptor-controlled regulation of bile acid synthesis. This mechanism could become an attractive target to enhance bile acid synthesis at night, when cholesterol synthesis is high, thus lowering serum LDL-cholesterol.

Responses of isoprene emission and photochemical efficiency to severe drought combined with prolonged hot weather in hybrid Populus.

Isoprene emissions have been considered as a protective response of plants to heat stress, but there is limited information of how prolonged heat spells affect isoprene emission capacity, particularly under the drought conditions that often accompany hot weather. Under combined long-term stresses, presence of isoprene emission could contribute to the maintenance of the precursor pool for rapid synthesis of essential isoprenoids to repair damaged components of leaf photosynthetic apparatus. We studied changes in leaf isoprene emission rate, photosynthetic characteristics, and antioxidant enzyme activities in two hybrid Populus clones, Nanlin 1388 (relatively high drought tolerance) and Nanlin 895 (relatively high thermotolerance) that were subjected to long-term (30 d) soil water stress (25% versus 90% soil field capacity) combined with a natural heat spell (day-time temperatures of 35-40 °C) that affected both control and water-stressed plants. Unexpectedly, isoprene emissions from both the clones were similar and the overall effects of drought on the emission characteristics were initially minor; however, treatment effects and clonal differences increased with time. In particular, the isoprene emission rate only increased slightly in the Nanlin 895 control plants after 15 d of treatment, whereas it decreased by more than 5-fold in all treatment × clone combinations after 30 d. The reduction in isoprene emission rate was associated with a decrease in the pool size of the isoprene precursor dimethylallyl diphosphate in all cases at 30 d after the start of treatment. Net assimilation rate, stomatal conductance, the openness of PSII centers, and the effective quantum yield all decreased, and non-photochemical quenching and catalase activity increased in both control and water-stressed plants. Contrary to the hypothesis of protection of leaf photosynthetic apparatus by isoprene, the data collectively indicated that prolonged stress affected isoprene emissions more strongly than leaf photosynthetic characteristics. This primarily reflected the depletion of isoprene precursor pools under long-term severe stress.

Nitisinone causes acquired tyrosinosis in alkaptonuria.

For over two decades, nitisinone (NTBC) has been successfully used to manipulate the tyrosine degradation pathway and save the lives of many children with hereditary tyrosinaemia type 1. More recently, NTBC has been used to halt homogentisic acid accumulation in alkaptonuria (AKU) with evidence suggesting its efficacy as a disease modifying agent. NTBC-induced hypertyrosinaemia has been associated with cognitive impairment and potentially sight-threatening keratopathy. In the context of a non-lethal condition (ie, AKU), these serious risks call for an evaluation of the wider impact of NTBC on the tyrosine pathway. We hypothesised that NTBC increases the tyrosine pool size and concentrations in tissues. In AKU mice tyrosine concentrations of tissue homogenates were measured before and after treatment with NTBC. In humans, pulse injection with l-[13 C9 ]tyrosine and l-[d8 ]phenylalanine was used along with compartmental modelling to estimate the size of tyrosine pools before and after treatment with NTBC. We found that NTBC increased tyrosine concentrations in murine tissues by five to nine folds. It also significantly increased the tyrosine pool size in humans (P < .001), suggesting that NTBC increases tyrosine not just in serum but also in tissues (ie, acquired tyrosinosis). This study provides, for the first time, the experimental proof for the magnitude of NTBC-related acquired tyrosinosis which should be overcome to ensure the safe use of NTBC in AKU.

Noninvasive quantitative magnetization transfer MRI reveals tubulointerstitial fibrosis in murine kidney.

Excessive tissue scarring, or fibrosis, is a critical contributor to end stage renal disease, but current clinical tests are not sufficient for assessing renal fibrosis. Quantitative magnetization transfer (qMT) MRI provides indirect information about the macromolecular composition of tissues. We evaluated measurements of the pool size ratio (PSR, the ratio of immobilized macromolecular to free water protons) obtained by qMT as a biomarker of tubulointerstitial fibrosis in a well-established murine model with progressive renal disease. MR images were acquired from 16-week-old fibrotic hHB-EGFTg/Tg mice and normal wild-type (WT) mice (N = 12) at 7 T. QMT parameters were derived using a two-pool five-parameter fitting model. A normal range of PSR values in the cortex and outer stripe of outer medulla (CR + OSOM) was determined by averaging across voxels within WT kidneys (mean ± 2SD). Regions in diseased mice whose PSR values exceeded the normal range above a threshold value (tPSR) were identified and measured. The spatial distribution of fibrosis was confirmed using picrosirius red stains. Compared with normal WT mice, scattered clusters of high PSR regions were observed in the OSOM of hHB-EGFTg/Tg mouse kidneys. Moderate increases in mean PSR (mPSR) of CR + OSOM regions were observed across fibrotic kidneys. The abnormally high PSR regions (% area) detected by the tPSR were significantly increased in hHB-EGFTg/Tg mice, and were highly correlated with regions of fibrosis detected by histological fibrosis indices measured from picrosirius red staining. Renal tubulointerstitial fibrosis in OSOM can thus be assessed by qMT MRI using an appropriate analysis of PSR. This technique may be used as an imaging biomarker for chronic kidney diseases.

Assessment of renal fibrosis in murine diabetic nephropathy using quantitative magnetization transfer MRI.

PURPOSE: Renal fibrosis is a hallmark of progressive renal disease; however, current clinical tests are insufficient for assessing renal fibrosis. Here we evaluated the utility of quantitative magnetization transfer MRI in detecting renal fibrosis in a murine model of progressive diabetic nephropathy (DN). METHODS: The db/db eNOS-/- mice, a well-recognized model of progressive DN, and normal wild-type mice were imaged at 7T. The quantitative magnetization transfer data were collected in coronal plane using a 2D magnetization transfer prepared spoiled gradient echo sequence with a Gaussian-shaped presaturation pulse. Parameters were derived using a two-pool fitting model. A normal range of cortical pool size ratio (PSR) was defined as Mean±2SD of wild-type kidneys (N = 20). The cortical regions whose PSR values exceeded this threshold (threshold PSR) were assessed. The correlations between the PSR-based and histological (collagen IV or picrosirius red stain) fibrosis measurements were evaluated. RESULTS: Compared with wild-type mice, moderate increases in mean PSR values and scattered clusters of high PSR region were observed in cortex of DN mouse kidneys. Abnormally high PSR regions (% area) that were detected by the threshold PSR were significantly increased in renal cortexes of DN mice. These regions progressively increased on aging and highly correlated with histological fibrosis measures, while the mean PSR values correlated much less. CONCLUSION: Renal fibrosis in DN can be assessed by the quantitative magnetization transfer MRI and threshold analysis. This technique may be used as a novel imaging biomarker for DN and other renal diseases.

Assessment of unilateral ureter obstruction with multi-parametric MRI.

PURPOSE: Quantitative multi-parametric MRI (mpMRI) methods may allow the assessment of renal injury and function in a sensitive and objective manner. This study aimed to evaluate an array of MRI methods that exploit endogenous contrasts including relaxation rates, pool size ratio (PSR) derived from quantitative magnetization transfer (qMT), chemical exchange saturation transfer (CEST), nuclear Overhauser enhancement (NOE), and apparent diffusion coefficient (ADC) for their sensitivity and specificity in detecting abnormal features associated with kidney disease in a murine model of unilateral ureter obstruction (UUO). METHODS: MRI scans were performed in anesthetized C57BL/6N mice 1, 3, or 6 days after UUO at 7T. Paraffin tissue sections were stained with Masson trichrome following MRI. RESULTS: Compared to contralateral kidneys, the cortices of UUO kidneys showed decreases of relaxation rates R1 and R2 , PSR, NOE, and ADC. No significant changes in CEST effects were observed for the cortical region of UUO kidneys. The MRI parametric changes in renal cortex are related to tubular cell death, tubular atrophy, tubular dilation, urine retention, and interstitial fibrosis in the cortex of UUO kidneys. CONCLUSION: Measurements of multiple MRI parameters provide comprehensive information about the molecular and cellular changes produced by UUO. Magn Reson Med 79:2216-2227, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Effects of photosynthetic photon flux density, frequency, duty ratio, and their interactions on net photosynthetic rate of cos lettuce leaves under pulsed light: explanation based on photosynthetic-intermediate pool dynamics.

Square-wave pulsed light is characterized by three parameters, namely average photosynthetic photon flux density (PPFD), pulsed-light frequency, and duty ratio (the ratio of light-period duration to that of the light-dark cycle). In addition, the light-period PPFD is determined by the averaged PPFD and duty ratio. We investigated the effects of these parameters and their interactions on net photosynthetic rate (P n) of cos lettuce leaves for every combination of parameters. Averaged PPFD values were 0-500 µmol m-2 s-1. Frequency values were 0.1-1000 Hz. White LED arrays were used as the light source. Every parameter affected P n and interactions between parameters were observed for all combinations. The P n under pulsed light was lower than that measured under continuous light of the same averaged PPFD, and this difference was enhanced with decreasing frequency and increasing light-period PPFD. A mechanistic model was constructed to estimate the amount of stored photosynthetic intermediates over time under pulsed light. The results indicated that all effects of parameters and their interactions on P n were explainable by consideration of the dynamics of accumulation and consumption of photosynthetic intermediates.

Rapid and simultaneous measurement of phosphorus metabolite pool size ratio and reaction kinetics of enzymes in vivo.

PURPOSE: The metabolites phosphocreatine (PCr), adenosine triphosphate (ATP), and in-organic phosphate (Pi) are biochemically coupled. Their pool sizes, assessed by their magnetization ratios, have been extensively studied and reflect bioenergetics status in vivo. However, most such studies have ignored chemical exchange and T1 relaxation effects. In this work, we aimed to extend the T1nom method to simultaneously quantify the reaction rate constants as well as phosphorus metabolite pool size ratios under partially relaxed conditions. MATERIALS AND METHODS: Modified Bloch-McConnell equations were used to simulate the effects of chemical exchanges on T1 relaxation times and magnetization ratios among PCr, γ-ATP, and Pi. The T1nom method with iteration approach was used to measure both reaction constants and metabolite pool size ratios. To validate our method, in vivo data from rat brains (N = 8) at 9.4 Tesla were acquired under two conditions, i.e., approximately full relaxation (TR = 9 s) and partial relaxation (TR = 3 s). We compared metabolite pool size ratios and reaction constants before and after correcting the chemical exchange and T1 relaxation effects. RESULTS: There were significant errors in underestimation of PCr/γATP by 12 % (P = 0.03) and overestimation of ATP/Pi ratios by 14 % (P = 0.04) when not considering chemical exchange effects. These errors were minimized using our iteration approach, resulting in no significant differences (PCr/γATP, P = 0.47; ATP/Pi, P = 0.81) in metabolite pool size ratios and reaction constants between the two measurements (i.e., short versus long TR conditions). CONCLUSION: Our method can facilitate broad biomedical applications of 31 P magnetization saturation transfer spectroscopy, requiring high temporal and/or spatial resolution for assessment of altered bioenergetics. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2018;47:210-221.

Expanding the donor lung pool: how many donations after circulatory death organs are we missing?

BACKGROUND: The number of patients with end-stage pulmonary disease awaiting lung transplantation is at an all-time high, while the supply of available organs remains stagnant. Utilizing donation after circulatory death (DCD) donors may help to address the supply-demand mismatch. The objective of this study is to determine the potential donor pool expansion with increased procurement of DCD organs from patients who die at hospitals. MATERIAL AND METHODS: The charts of all patients who died at a single, rural, quaternary-care institution between August 2014 and June 2015 were reviewed for lung transplant candidacy. Inclusion criteria were age <65 y, absence of cancer and lung pathology, and cause of death other than respiratory or sepsis. RESULTS: A total of 857 patients died within a 1-year period and were stratified by age: pediatric <15 y (n = 32, 4%), young 15-64 y (n = 328, 38%), and old >65 y (n = 497, 58%). Those without cancer totaled 778 (90.8%) and 512 (59%) did not have lung pathology. This leaves 85 patients qualifying for DCD lung donation (pediatric n = 10, young n = 75, and old n = 0). Potential donors were significantly more likely to have clear chest X-rays (24.3% versus 10.0%, P < 0.0001) and higher mean PaO2/FiO2 (342.1 versus 197.9, P < 0.0001) compared with ineligible patients. CONCLUSIONS: A significant number of DCD lungs are available every year from patients who die within hospitals. We estimate the use of suitable DCD lungs could potentially result in a significant increase in the number of lungs available for transplantation.

Cyanobacterial carbon metabolism: Fluxome plasticity and oxygen dependence.

Synechocystis sp. strain PCC 6803 has been widely used as a photo-biorefinery chassis. Based on its genome annotation, this species contains a complete TCA cycle, an Embden-Meyerhof-Parnas pathway (EMPP), an oxidative pentose phosphate pathway (OPPP), and an Entner-Doudoroff pathway (EDP). To evaluate how Synechocystis 6803 catabolizes glucose under heterotrophic conditions, we performed 13 C metabolic flux analysis, metabolite pool size analysis, gene knockouts, and heterologous expressions. The results revealed a cyclic mode of flux through the OPPP. Small, but non-zero, fluxes were observed through the TCA cycle and the malic shunt. Independent knockouts of 6-phosphogluconate dehydrogenase (gnd) and malic enzyme (me) corroborated these results, as neither mutant could grow under dark heterotrophic conditions. Our data also indicate that Synechocystis 6803 metabolism relies upon oxidative phosphorylation to generate ATP from NADPH under dark or insufficient light conditions. The pool sizes of intermediates in the TCA cycle, particularly acetyl-CoA, were found to be several fold lower in Synechocystis 6803 (compared to E. coli metabolite pool sizes), while its sugar phosphate intermediates were several-fold higher. Moreover, negligible flux was detected through the native, or heterologous, EDP in the wild type or Δgnd strains under heterotrophic conditions. Comparing photoautotrophic, photomixotrophic, and heterotrophic conditions, the Calvin cycle, OPPP, and EMPP in Synechocystis 6803 possess the ability to regulate their fluxes under various growth conditions (plastic), whereas its TCA cycle always maintains at low levels (rigid). This work also demonstrates how genetic profiles do not always reflect actual metabolic flux through native or heterologous pathways. Biotechnol. Bioeng. 2017;114: 1593-1602. © 2017 Wiley Periodicals, Inc.

In vivo quantitative magnetization transfer imaging correlates with histology during de- and remyelination in cuprizone-treated mice.

The pool size ratio measured by quantitative magnetization transfer MRI is hypothesized to closely reflect myelin density, but their relationship has so far been confirmed mostly in ex vivo conditions. We investigate the correspondence between this parameter measured in vivo at 7.0 T, with Black Gold II staining for myelin fibres, and with myelin basic protein and beta-tubulin immunofluorescence in a hybrid longitudinal study of C57BL/6 and SJL/J mice treated with cuprizone, a neurotoxicant causing relatively selective myelin loss followed by spontaneous remyelination upon treatment suspension. Our results confirm that pool size ratio measurements correlate with myelin content, with the correlation coefficient depending on strain and staining method, and demonstrate the in vivo applicability of this MRI technique to experimental mouse models of multiple sclerosis.

Quantitative magnetization transfer imaging of rodent glioma using selective inversion recovery.

Magnetization transfer (MT) provides an indirect means to detect noninvasively variations in macromolecular contents in biological tissues, but, so far, there have been only a few quantitative MT (qMT) studies reported in cancer, all of which used off-resonance pulsed saturation methods. This article describes the first implementation of a different qMT approach, selective inversion recovery (SIR), for the characterization of tumor in vivo using a rodent glioma model. The SIR method is an on-resonance method capable of fitting qMT parameters and T1 relaxation time simultaneously without mapping B0 and B1 , which is very suitable for high-field qMT measurements because of the lower saturation absorption rate. The results show that the average pool size ratio (PSR, the macromolecular pool versus the free water pool) in rat 9 L glioma (5.7%) is significantly lower than that in normal rat gray matter (9.2%) and white matter (17.4%), which suggests that PSR is potentially a sensitive imaging biomarker for the assessment of brain tumor. Despite being less robust, the estimated MT exchange rates also show clear differences from normal tissues (19.7 Hz for tumors versus 14.8 and 10.2 Hz for gray and white mater, respectively). In addition, the influence of confounding effects, e.g. B1 inhomogeneity, on qMT parameter estimates is investigated with numerical simulations. These findings not only help to better understand the changes in the macromolecular contents of tumors, but are also important for the interpretation of other imaging contrasts, such as chemical exchange saturation transfer of tumors.

Cross-Sectional Melt Pool Geometry of Laser Scanned Tracks and Pads on Nickel Alloy 718 for the 2022 Additive Manufacturing Benchmark Challenges.

The Additive Manufacturing Benchmark Series (AM Bench) is a NIST-led organization that provides a continuing series of additive manufacturing benchmark measurements, challenge problems, and conferences with the primary goal of enabling modelers to test their simulations against rigorous, highly controlled additive manufacturing benchmark measurement data. To this end, single-track (1D) and pad (2D) scans on bare plate nickel alloy 718 were completed with thermography, cross-sectional grain orientation and local chemical composition maps, and cross-sectional melt pool size measurements. The laser power, scan speed, and laser spot size were varied for single tracks, and the scan direction was varied for pads. This article focuses on the cross-sectional melt pool size measurements and presents the predictions from challenge problems. Single-track depth correlated with volumetric energy density while width did not (within the studied parameters). The melt pool size for pad scans was greater than single tracks due to heat buildup. Pad scan melt pool depth was reduced when the laser scan direction and gas flow direction were parallel. The melt pool size in pad scans showed little to no trend against position within the pads. Uncertainty budgets for cross-sectional melt pool size from optical micrographs are provided for the purpose of model validation.

ARMC5 controls the degradation of most Pol II subunits, and ARMC5 mutation increases neural tube defect risks in mice and humans.

BACKGROUND: Neural tube defects (NTDs) are caused by genetic and environmental factors. ARMC5 is part of a novel ubiquitin ligase specific for POLR2A, the largest subunit of RNA polymerase II (Pol II). RESULTS: We find that ARMC5 knockout mice have increased incidence of NTDs, such as spina bifida and exencephaly. Surprisingly, the absence of ARMC5 causes the accumulation of not only POLR2A but also most of the other 11 Pol II subunits, indicating that the degradation of the whole Pol II complex is compromised. The enlarged Pol II pool does not lead to generalized Pol II stalling or a generalized decrease in mRNA transcription. In neural progenitor cells, ARMC5 knockout only dysregulates 106 genes, some of which are known to be involved in neural tube development. FOLH1, critical in folate uptake and hence neural tube development, is downregulated in the knockout intestine. We also identify nine deleterious mutations in the ARMC5 gene in 511 patients with myelomeningocele, a severe form of spina bifida. These mutations impair the interaction between ARMC5 and Pol II and reduce Pol II ubiquitination. CONCLUSIONS: Mutations in ARMC5 increase the risk of NTDs in mice and humans. ARMC5 is part of an E3 controlling the degradation of all 12 subunits of Pol II under physiological conditions. The Pol II pool size might have effects on NTD pathogenesis, and some of the effects might be via the downregulation of FOLH1. Additional mechanistic work is needed to establish the causal effect of the findings on NTD pathogenesis.

Bedrock outcrops weakly promote rather than inhibit soil carbon sequestration after vegetation restoration.

Vegetation restoration can increase soil carbon (C) content in karst regions characterized by highly exposed carbonate rocks; however, it remains unclear whether and how bedrock outcrops contribute to soil C-accumulation after vegetation restoration. We aimed to investigate the magnitude and mechanisms of bedrock outcrops on soil C-accumulation after vegetation restoration. Here, we selected 362 fixed locations to investigate changes in soil organic carbon (SOC) content and density before and after cropland restoration in a karst catchment with varying bedrock exposure ratios and initial soil C pools prior to restoration. Active vegetation restoration (i.e., cropland converted to forage grass, plantation forest, and a combination of grass and forest) and natural regeneration (cropland abandoned) were compared, with croplands maintained with no change as the control. Compared to croplands maintained with no change, SOC density significantly increased in the four vegetation restoration types. The SOC accumulation rate was higher for natural regeneration (39 g C m-2 yr-1) than for the three active restoration strategies (18-27 g C m-2 yr-1). SOC accumulation decreased with a higher initial pool size of soil C but increased with nitrogen accumulation and soil exchangeable calcium (Ca2+) concentration. Higher bedrock outcrops reduced soil volume but increased SOC content through their indirect effects on the initial pool size of soil C, external nitrogen inputs, and soil Ca2+ concentration. This weakly promoted rather than inhibited SOC sequestration. Our findings highlight the effectiveness of various restoration strategies in promoting SOC accumulation in karst areas, as well as the need to take bedrock outcrops and initial soil C pools into consideration when modeling SOC dynamics and maximizing C sinks for vegetation restoration.

Mitochondrial Coenzyme Q Redox Homeostasis and Reactive Oxygen Species Production.

Mitochondrial coenzyme Q (mtQ) of the inner mitochondrial membrane is a redox active mobile carrier in the respiratory chain that transfers electrons between reducing dehydrogenases and oxidizing pathway(s). mtQ is also involved in mitochondrial reactive oxygen species (mtROS) formation through the mitochondrial respiratory chain. Some mtQ-binding sites related to the respiratory chain can directly form the superoxide anion from semiubiquinone radicals. On the other hand, reduced mtQ (ubiquinol, mtQH2) recycles other antioxidants and directly acts on free radicals, preventing oxidative modifications. The redox state of the mtQ pool is a central bioenergetic patameter that alters in response to changes in mitochondrial function. It reflects mitochondrial bioenergetic activity and mtROS formation level, and thus the oxidative stress associated with the mitochondria. Surprisingly, there are few studies describing a direct relationship between the mtQ redox state and mtROS production under physiological and pathological conditions. Here, we provide a first overview of what is known about the factors affecting mtQ redox homeostasis and its relationship to mtROS production. We have proposed that the level of reduction (the endogenous redox state) of mtQ may be a useful indirect marker to assess total mtROS formation. A higher mtQ reduction level (mtQH2/mtQtotal) indicates greater mtROS formation. The mtQ reduction level, and thus the mtROS formation, depends on the size of the mtQ pool and the activity of the mtQ-reducing and mtQH2-oxidizing pathway(s) of respiratory chain. We focus on a number of physiological and pathophysiological factors affecting the amount of mtQ and thus its redox homeostasis and mtROS production level.

Pool Testing for COVID-19: Suitable Splitting Procedure and Pool Size for India.

OBJECTIVE: Coronavirus disease (COVID-19) has emerged as a global pandemic for public health due to the large scale outbreak, therefore there is an urgent need to detect the infected cases quickly and isolate them in order to suppress the further spread of the disease. This study tries to identify a suitable pool testing method and algorithm for COVID-19. METHODS: This study tries to derive a general equation for the number of tests required for a pooled sample to detect every infected individual in the specific pool. The gain in pool testing over the normal procedure is quantified by the percentage of tests required compared to individual testing. RESULTS: The percentage of tests required by the pool testing strategy varies according to the different splitting procedures, the size of the pooled sample, and the probability of an individual being infected in the population. If the probability of infection is 0.05, then for a pool size of 32, only 14 tests are sufficient to detect every infected individual. CONCLUSION: The number of tests required to detect infected individuals by using the pooling method is much lower than individual testing. This may help us with increasing our testing capacity for COVID-19 by testing a large number of individuals in less time with limited resources.

Re-Programing Glucose Catabolism in the Microalga Chlorella sorokiniana under Light Condition.

The microalga Chlorella sorokiniana has attracted much attention for lipid production and wastewater treatment. It can perform photosynthesis and organic carbon utilization concurrently. To understand its phototrophic metabolism, a biomass compositional analysis, a 13C metabolic flux analysis, and metabolite pool size analyses were performed. Under dark condition, the oxidative pentose phosphate pathway (OPP) was the major route for glucose catabolism (88% carbon flux) and a cyclic OPP-glycolytic route for glucose catabolism was formed. Under light condition, fluxes in the glucose catabolism, tricarboxylic acid (TCA) cycle, and anaplerotic reaction (CO2 fixation via phosphoenolpyruvate carboxylase) were all suppressed. Meanwhile, the RuBisCO reaction became active and the ratio of its carbon fixation to glucose carbon utilization was determined as 7:100. Moreover, light condition significantly reduced the pool sizes of sugar phosphate metabolites (such as E4P, F6P, and S7P) and promoted biomass synthesis (which reached 0.155 h-1). In addition, light condition increased glucose consumption rates, leading to higher ATP and NADPH production and a higher protein content (43% vs. 30%) in the biomass during the exponential growth phase.

Synaptic vesicle release during ribbon synapse formation of cone photoreceptors.

Mammalian cone photoreceptors enable through their sophisticated synapse the high-fidelity transfer of visual information to second-order neurons in the retina. The synapse contains a proteinaceous organelle, called the synaptic ribbon, which tethers synaptic vesicles (SVs) at the active zone (AZ) close to voltage-gated Ca2+ channels. However, the exact contribution of the synaptic ribbon to neurotransmission is not fully understood, yet. In mice, precursors to synaptic ribbons appear within photoreceptor terminals shortly after birth as free-floating spherical structures, which progressively elongate and then attach to the AZ during the following days. Here, we took advantage of the process of synaptic ribbon maturation to study their contribution to SV release. We performed whole-cell patch-clamp recordings from cone photoreceptors at three postnatal (P) development stages (P8-9, P12-13, >P30) and measured evoked SV release, SV replenishment rate, recovery from synaptic depression, domain organization of voltage-sensitive Ca2+ channels, and Ca2+-sensitivity of exocytosis. Additionally, we performed electron microscopy to determine the density of SVs at ribbon-free and ribbon-occupied AZs. Our results suggest that ribbon attachment does not organize the voltage-sensitive Ca2+ channels into nanodomains or control SV release probability. However, ribbon attachment increases SV density at the AZ, increases the pool size of readily releasable SVs available for evoked SV release, facilitates SV replenishment without changing the SV pool refilling time, and increases the Ca2+- sensitivity of glutamate release.