The gut microbiome-Does stool represent right?

Many stool-based gut microbiome studies have highlighted the importance of the microbiome. However, we hypothesized that stool is a poor proxy for the inner-colonic microbiome and that studying stool samples may be inadequate to capture the true inner-colonic microbiome. To test this hypothesis, we conducted prospective clinical studies with up to 20 patients undergoing an FDA-cleared gravity-fed colonic lavage without oral purgative pre-consumption. The objective of this study was to present the analysis of inner-colonic microbiota obtained non-invasively during the lavage and how these results differ from stool samples. The inner-colonic samples represented the descending, transverse, and ascending colon. All samples were analyzed for 16S rRNA and shotgun metagenomic sequences. The taxonomic, phylogenetic, and biosynthetic gene cluster analyses showed a distinctive biogeographic gradient and revealed differences between the sample types, especially in the proximal colon. The high percentage of unique information found only in the inner-colonic effluent highlights the importance of these samples and likewise the importance of collecting them using a method that can preserve these distinctive signatures. We proposed that these samples are imperative for developing future biomarkers, targeted therapeutics, and personalized medicine.

Light-dependent signal transduction in the marine diatom Phaeodactylum tricornutum.

Unlike most higher plants, unicellular algae can acclimate to changes in irradiance on time scales of hours to a few days. The process involves an enigmatic signaling pathway originating in the plastid that leads to coordinated changes in plastid and nuclear gene expression. To deepen our understanding of this process, we conducted functional studies to examine how the model diatom, Phaeodactylum tricornutum, acclimates to low light and sought to identify the molecules responsible for the phenomenon. We show that two transformants with altered expression of two putative signal transduction molecules, a light-specific soluble kinase and a plastid transmembrane protein, that appears to be regulated by a long noncoding natural antisense transcript, arising from the opposite strand, are physiologically incapable of photoacclimation. Based on these results, we propose a working model of the retrograde feedback in the signaling and regulation of photoacclimation in a marine diatom.

High-Volume Colonic Lavage Is a Safe and Preferred Colonoscopy Preparation for Patients With Inflammatory Bowel Disease.

Background: Colonoscopies provide a crucial diagnostic and surveillance tool for inflammatory bowel disease (IBD). Accordingly, IBD patients undergo repeated and frequent colonoscopies. The oral purgative bowel prep (BP) is often burdensome on patients, resulting in delayed or missed colonoscopies due to patient noncompliance. Additionally, oral BP has been noted to possibly induce colon mucosal inflammatory changes in some patients, which may be misleading when assessing actual disease activity. Methods: In this retrospective clinical study, we evaluated the use of an FDA cleared, defecation-inducing high-volume colon irrigation (>40 L) BP to prepare IBD patients for colonoscopy. Data were collected at 4 US Hygieacare centers from September 2016 to March 2021. The IBD patient population consisted of 314 patients that underwent 343 BPs. The BPs were prescribed by 65 physicians and performed by 16 nurses and technicians. Results: Patient ages were 20-85 years old, 76% females, 24% males, and 97% of the patients were adequately prepared for their colonoscopy (n = 309). Patient satisfaction with the BP was very high, as reflected in postprocedure surveys and open-ended responses text analyses, and there were no serious adverse events. Conclusions: We present data supporting that the defecation-inducing high-volume colon irrigation BP for colonoscopy is safe, effective, and preferred for IBD patients. Using this BP for IBD patients can allow earlier interventions, significantly impacting disease management and future outcomes.

Structural and functional analyses of photosystem II in the marine diatom Phaeodactylum tricornutum.

A descendant of the red algal lineage, diatoms are unicellular eukaryotic algae characterized by thylakoid membranes that lack the spatial differentiation of stroma and grana stacks found in green algae and higher plants. While the photophysiology of diatoms has been studied extensively, very little is known about the spatial organization of the multimeric photosynthetic protein complexes within their thylakoid membranes. Here, using cryo-electron tomography, proteomics, and biophysical analyses, we elucidate the macromolecular composition, architecture, and spatial distribution of photosystem II complexes in diatom thylakoid membranes. Structural analyses reveal 2 distinct photosystem II populations: loose clusters of complexes associated with antenna proteins and compact 2D crystalline arrays of dimeric cores. Biophysical measurements reveal only 1 photosystem II functional absorption cross section, suggesting that only the former population is photosynthetically active. The tomographic data indicate that the arrays of photosystem II cores are physically separated from those associated with antenna proteins. We hypothesize that the islands of photosystem cores are repair stations, where photodamaged proteins can be replaced. Our results strongly imply convergent evolution between the red and the green photosynthetic lineages toward spatial segregation of dynamic, functional microdomains of photosystem II supercomplexes.

Overexpression of a diacylglycerol acyltransferase gene in Phaeodactylum tricornutum directs carbon towards lipid biosynthesis.

Under nutrient deplete conditions, diatoms accumulate between 15% to 25% of their dry weight as lipids, primarily as triacylglycerols (TAGs). As in most eukaryotes, these organisms produce TAGs via the acyl-CoA dependent Kennedy pathway. The last step in this pathway is catalyzed by diacylglycerol acyltransferase (DGAT) that acylates diacylglycerol (DAG) to produce TAG. To test our hypothesis that DGAT plays a major role in controlling the flux of carbon towards lipids, we overexpressed a specific type II DGAT gene, DGAT2D, in the model diatom Phaeodactylum tricornutum. The transformants had 50- to 100-fold higher DGAT2D mRNA levels and the abundance of the enzyme increased 30- to 50-fold. More important, these cells had a 2-fold higher total lipid content and incorporated carbon into lipids more efficiently than the wild type (WT) while growing only 15% slower at light saturation. Based on a flux analysis using 13 C as a tracer, we found that the increase in lipids was achieved via increased fluxes through pyruvate and acetyl-CoA. Our results reveal overexpression of DAGT2D increases the flux of photosynthetically fixed carbon towards lipids, and leads to a higher lipid content than exponentially grown WT cells.

An RNA interference knock-down of nitrate reductase enhances lipid biosynthesis in the diatom Phaeodactylum tricornutum.

When diatoms are stressed for inorganic nitrogen they remodel their intermediate metabolism and redirect carbon towards lipid biosynthesis. However, this response comes at a significant cost reflected in decreased photosynthetic energy conversion efficiency and growth. Here we explore a molecular genetics approach to restrict the assimilation of inorganic nitrogen by knocking down nitrate reductase (NR). The transformant strain, NR21, exhibited about 50% lower expression and activity of the enzyme but simultaneously accumulated over 40% more fatty acids. However, in contrast to nitrogen-stressed wild-type (WT) cells, which grow at about 20% of the rate of nitrogen-replete cells, growth of NR21 was only reduced by about 30%. Biophysical analyses revealed that the photosynthetic energy conversion efficiency of photosystem II was unaffected in NR21; nevertheless, the plastoquinone pool was reduced by 50% at the optimal growth irradiance while in the WT it was over 90% oxidized. Further analyses reveal a 12-fold increase in the glutamate/glutamine ratio and an increase NADPH and malonyl-CoA pool size. Transcriptomic analyses indicate that the knock down resulted in changes in the expression of genes for lipid biosynthesis, as well as the expression of specific transcription factors. Based on these observations, we hypothesize that the allocation of carbon and reductants in diatoms is controlled by a feedback mechanism between intermediate metabolites, the redox state of the plastid and the expression and binding of transcription factors related to stress responses.

Remodeling of intermediate metabolism in the diatom Phaeodactylum tricornutum under nitrogen stress.

Diatoms are unicellular algae that accumulate significant amounts of triacylglycerols as storage lipids when their growth is limited by nutrients. Using biochemical, physiological, bioinformatics, and reverse genetic approaches, we analyzed how the flux of carbon into lipids is influenced by nitrogen stress in a model diatom, Phaeodactylum tricornutum. Our results reveal that the accumulation of lipids is a consequence of remodeling of intermediate metabolism, especially reactions in the tricarboxylic acid and the urea cycles. Specifically, approximately one-half of the cellular proteins are cannibalized; whereas the nitrogen is scavenged by the urea and glutamine synthetase/glutamine 2-oxoglutarate aminotransferase pathways and redirected to the de novo synthesis of nitrogen assimilation machinery, simultaneously, the photobiological flux of carbon and reductants is used to synthesize lipids. To further examine how nitrogen stress triggers the remodeling process, we knocked down the gene encoding for nitrate reductase, a key enzyme required for the assimilation of nitrate. The strain exhibits 40-50% of the mRNA copy numbers, protein content, and enzymatic activity of the wild type, concomitant with a 43% increase in cellular lipid content. We suggest a negative feedback sensor that couples photosynthetic carbon fixation to lipid biosynthesis and is regulated by the nitrogen assimilation pathway. This metabolic feedback enables diatoms to rapidly respond to fluctuations in environmental nitrogen availability.

Trichodesmium's strategies to alleviate phosphorus limitation in the future acidified oceans.

Global warming may exacerbate inorganic nutrient limitation, including phosphorus (P), in the surface waters of tropical oceans that are home to extensive blooms of the marine diazotrophic cyanobacterium, Trichodesmium. We examined the combined effects of P limitation and pCO(2), forecast under ocean acidification scenarios, on Trichodesmium erythraeum IMS101 cultures. We measured nitrogen acquisition,glutamine synthetase activity, C uptake rates, intracellular Adenosine Triphosphate (ATP) concentration and the pool sizes of related key proteins. Here, we present data supporting the idea that cellular energy re-allocation enables the higher growth and N(2) fixation rates detected in Trichodesmium cultured under high pCO(2). This is reflected in altered protein abundance and metabolic pools. Also modified are particulate organic carbon and nitrogen production rates,enzymatic activities, and cellular ATP concentrations. We suggest that adjusting these cellular pathways to changing environmental conditions enables Trichodesmium to compensate for low P availability and to thrive in acidified oceans. Moreover, elevated pCO(2) could provide Trichodesmium with a competitive dominance that would extend its niche, particularly in P-limited regions of the tropical and subtropical oceans.

Using natural selection to explore the adaptive potential of Chlamydomonas reinhardtii.

Improving feedstock is critical to facilitate the commercial utilization of algae, in particular in open pond systems where, due to the presence of competitors and pests, high algal growth rates and stress tolerance are beneficial. Here we raised laboratory cultures of the model alga Chlamydomonas reinhardtii under serial dilution to explore the potential of crop improvement using natural selection. The alga was evolved for 1,880 generations in liquid medium under continuous light (EL population). At the end of the experiment, EL cells had a growth rate that was 35% greater than the progenitor population (PL). The removal of acetate from the medium demonstrated that EL growth enhancement largely relied on efficient usage of this organic carbon source. Genome re-sequencing uncovered 1,937 polymorphic DNA regions in the EL population with 149 single nucleotide polymorphisms resulting in amino acid substitutions. Transcriptome analysis showed, in the EL population, significant up regulation of genes involved in protein synthesis, the cell cycle and cellular respiration, whereas the DNA repair pathway and photosynthesis were down regulated. Like other algae, EL cells accumulated neutral lipids under nitrogen depletion. Our work demonstrates transcriptome and genome-wide impacts of natural selection on algal cells and points to a useful strategy for strain improvement.

Diatoms: a fossil fuel of the future.

Long-term global climate change, caused by burning petroleum and other fossil fuels, has motivated an urgent need to develop renewable, carbon-neutral, economically viable alternatives to displace petroleum using existing infrastructure. Algal feedstocks are promising candidate replacements as a 'drop-in' fuel. Here, we focus on a specific algal taxon, diatoms, to become the fossil fuel of the future. We summarize past attempts to obtain suitable diatom strains, propose future directions for their genetic manipulation, and offer biotechnological pathways to improve yield. We calculate that the yields obtained by using diatoms as a production platform are theoretically sufficient to satisfy the total oil consumption of the US, using between 3 and 5% of its land area.

The influence of pCO2 and temperature on gene expression of carbon and nitrogen pathways in Trichodesmium IMS101.

Growth, protein amount, and activity levels of metabolic pathways in Trichodesmium are influenced by environmental changes such as elevated pCO(2) and temperature. This study examines changes in the expression of essential metabolic genes in Trichodesmium grown under a matrix of pCO(2) (400 and 900 µatm) and temperature (25 and 31°C). Using RT-qPCR, we studied 21 genes related to four metabolic functional groups: CO(2) concentrating mechanism (bicA1, bicA2, ccmM, ccmK2, ccmK3, ndhF4, ndhD4, ndhL, chpX), energy metabolism (atpB, sod, prx, glcD), nitrogen metabolism (glnA, hetR, nifH), and inorganic carbon fixation and photosynthesis (rbcL, rca, psaB, psaC, psbA). nifH and most photosynthetic genes exhibited relatively high abundance and their expression was influenced by both environmental parameters. A two to three orders of magnitude increase was observed for glnA and hetR only when both pCO(2) and temperature were elevated. CO(2) concentrating mechanism genes were not affected by pCO(2) and temperature and their expression levels were markedly lower than that of the nitrogen metabolism and photosynthetic genes. Many of the CO(2) concentrating mechanism genes were co-expressed throughout the day. Our results demonstrate that in Trichodesmium, CO(2) concentrating mechanism genes are constitutively expressed. Co-expression of genes from different functional groups were frequently observed during the first half of the photoperiod when oxygenic photosynthesis and N(2) fixation take place, pointing at the tight and complex regulation of gene expression in Trichodesmium. Here we provide new data linking environmental changes of pCO(2) and temperature to gene expression in Trichodesmium. Although gene expression indicates an active metabolic pathway, there is often an uncoupling between transcription and enzyme activity, such that transcript level cannot usually be directly extrapolated to metabolic activity.

Combined effects of CO2 and light on the N2-fixing cyanobacterium Trichodesmium IMS101: a mechanistic view.

The marine diazotrophic cyanobacterium Trichodesmium responds to elevated atmospheric CO(2) partial pressure (pCO(2)) with higher N(2) fixation and growth rates. To unveil the underlying mechanisms, we examined the combined influence of pCO(2) (150 and 900 microatm) and light (50 and 200 micromol photons m(-2) s(-1)) on Trichodesmium IMS101. We expand on a complementary study that demonstrated that while elevated pCO(2) enhanced N(2) fixation and growth, oxygen evolution and carbon fixation increased mainly as a response to high light. Here, we investigated changes in the photosynthetic fluorescence parameters of photosystem II, in ratios of the photosynthetic units (photosystem I:photosystem II), and in the pool sizes of key proteins involved in the fixation of carbon and nitrogen as well as their subsequent assimilation. We show that the combined elevation in pCO(2) and light controlled the operation of the CO(2)-concentrating mechanism and enhanced protein activity without increasing their pool size. Moreover, elevated pCO(2) and high light decreased the amounts of several key proteins (NifH, PsbA, and PsaC), while amounts of AtpB and RbcL did not significantly change. Reduced investment in protein biosynthesis, without notably changing photosynthetic fluxes, could free up energy that can be reallocated to increase N(2) fixation and growth at elevated pCO(2) and light. We suggest that changes in the redox state of the photosynthetic electron transport chain and posttranslational regulation of key proteins mediate the high flexibility in resources and energy allocation in Trichodesmium. This strategy should enable Trichodesmium to flourish in future surface oceans characterized by elevated pCO(2), higher temperatures, and high light.

Combined effects of CO2 and light on the N2-fixing cyanobacterium Trichodesmium IMS101: physiological responses.

Recent studies on the diazotrophic cyanobacterium Trichodesmium erythraeum (IMS101) showed that increasing CO(2) partial pressure (pCO(2)) enhances N(2) fixation and growth. Significant uncertainties remain as to the degree of the sensitivity to pCO(2), its modification by other environmental factors, and underlying processes causing these responses. To address these questions, we examined the responses of Trichodesmium IMS101 grown under a matrix of low and high levels of pCO(2) (150 and 900 microatm) and irradiance (50 and 200 micromol photons m(-2) s(-1)). Growth rates as well as cellular carbon and nitrogen contents increased with increasing pCO(2) and light levels in the cultures. The pCO(2)-dependent stimulation in organic carbon and nitrogen production was highest under low light. High pCO(2) stimulated rates of N(2) fixation and prolonged the duration, while high light affected maximum rates only. Gross photosynthesis increased with light but did not change with pCO(2). HCO(3)(-) was identified as the predominant carbon source taken up in all treatments. Inorganic carbon uptake increased with light, but only gross CO(2) uptake was enhanced under high pCO(2). A comparison between carbon fluxes in vivo and those derived from (13)C fractionation indicates high internal carbon cycling, especially in the low-pCO(2) treatment under high light. Light-dependent oxygen uptake was only detected under low pCO(2) combined with high light or when low-light-acclimated cells were exposed to high light, indicating that the Mehler reaction functions also as a photoprotective mechanism in Trichodesmium. Our data confirm the pronounced pCO(2) effect on N(2) fixation and growth in Trichodesmium and further show a strong modulation of these effects by light intensity. We attribute these responses to changes in the allocation of photosynthetic energy between carbon acquisition and the assimilation of carbon and nitrogen under elevated pCO(2). These findings are supported by a complementary study looking at photosynthetic fluorescence parameters of photosystem II, photosynthetic unit stoichiometry (photosystem I:photosystem II), and pool sizes of key proteins in carbon and nitrogen acquisition.

Regulation of nitrogen metabolism in the marine diazotroph Trichodesmium IMS101 under varying temperatures and atmospheric CO2 concentrations.

We examined the influence of forecasted changes in global temperatures and pCO(2) on N(2) fixation and assimilation in the ecologically important cyanobacterium Trichodesmium spp. Changes of mRNA transcripts (nifH, glnA, hetR, psbA, psaB), protein (nitrogenase, glutamine synthetase) pools and enzymatic activity (nitrogenase) were measured under varying pCO(2) and temperatures. High pCO(2) shifted transcript patterns of all genes, resulting in a more synchronized diel expression. Under the same conditions, we did not observe any significant changes in the protein pools or in total cellular allocations of carbon and nitrogen (i.e. C : N ratio remained stable). Independently of temperature, high pCO(2) (900 microatm) elevated N(2) fixation rates. Levels of the key enzymes, nitrogenase and glutamine synthetase that mediate nitrogen assimilation did not increase, implying that the high pCO(2) allowed higher reaction turnover rates through these key enzymes. Moreover, increased temperatures and high pCO(2) resulted in higher C : P ratios. The plasticity in phosphorous stoichiometry combined with higher enzymatic efficiencies lead to higher growth rates. In cyanobacteria photosynthesis, carbon uptake, respiration, N(2) fixation and nitrogen assimilation share cellular components. We propose that shifted cellular resource and energy allocation among those components will enable Trichodesmium grown at elevated temperatures and pCO(2) to extend its niche in the future ocean, through both tolerance of a broader temperature range and higher P plasticity.

Iron limitation in the marine cyanobacterium Trichodesmium reveals new insights into regulation of photosynthesis and nitrogen fixation.

* As iron (Fe) deficiency is a main limiting factor of ocean productivity, its effects were investigated on interactions between photosynthesis and nitrogen fixation in the marine nonheterocystous diazotrophic cyanobacterium Trichodesmium IMS101. * Biophysical methods such as fluorescence kinetic microscopy, fast repetition rate (FRR) fluorimetry, and in vivo and in vitro spectroscopy of pigment composition were used, and nitrogenase activity and the abundance of key proteins were measured. * Fe limitation caused a fast down-regulation of nitrogenase activity and protein levels. By contrast, the abundance of Fe-requiring photosystem I (PSI) components remained constant. Total levels of phycobiliproteins remained unchanged according to single-cell in vivo spectra. However, the regular 16-kDa phycoerythrin band decreased and finally disappeared 16-20 d after initiation of Fe limitation, concomitant with the accumulation of a 20-kDa protein cross-reacting with the phycoerythrin antibody. Concurrently, nitrogenase expression and activity increased. Fe limitation dampened the daily cycle of photosystem II (PSII) activity characteristic of diazotrophic Trichodesmium cells. Further, it increased the number and prolonged the time period of occurrence of cells with elevated basic fluorescence (F(0)). Additionally, it increased the effective cross-section of PSII, probably as a result of enhanced coupling of phycobilisomes to PSII, and led to up-regulation of the Fe stress protein IsiA. * Trichodesmium survives short-term Fe limitation by selectively down-regulating nitrogen fixation while maintaining but re-arranging the photosynthetic apparatus.

Coupling between autocatalytic cell death and transparent exopolymeric particle production in the marine cyanobacterium Trichodesmium.

Extracellular polysaccharide aggregates, operationally defined as transparent exopolymeric particles (TEP), are recognized as an important conduit for carbon recycling and export in aquatic systems. Yet, the factors controlling the build-up of the TEP pool are not well characterized. Here we show that increased TEP production by Trichodesmium, an oceanic bloom-forming nitrogen-fixing (diazotrophic) cyanobacterium, is coupled with autocatalytic programmed cell death (PCD) process. We demonstrate that PCD induction, in both laboratory cultures and natural populations, is characterized by high caspase-like activity, correlates with enhanced TEP production, and occurs under iron and phosphorus starvation, as well as under high irradiance and oxidative stress. Enhanced TEP production was not observed in actively growing populations. We provide further evidence that iron is a key trigger for the induction of PCD. We demonstrate, for the first time, the concomitant enhanced build-up of the TEP pool when Trichodesmium is Fe-stressed. These results suggest a functional linkage between activation of caspases and PCD in Trichodesmium and regulation of vertical carbon and nitrogen fluxes. We hypothesize that modulation of TEP formation and its qualities by different mortality pathways could regulate the fate of phytoplankton blooms and particulate organic matter in aquatic ecosystems.