Dynamic regulation of oxygenic photosynthesis in cyanobacteria by flavodiiron proteins

The ability of oxygenic photosynthetic organisms to develop protective mechanisms for the regulation of the photosynthetic apparatus is crucial for their survival in continuously changing environmental conditions. The flavodiiron proteins (FDPs) represent a remarkable regulatory electron transport pathway evolved in all photosynthetic organisms, apart from angiosperms, red and brown algae. The FDPs act as photoprotective electron sinks in directing excess electrons from the photosynthetic electron chain to O2, via the so-called Mehler-like reaction. In this thesis work, I focused on the regulatory mechanism, physiological relevance and functional regulation of FDPs in cyanobacteria. In the unicellular cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis), the Flv1/Flv3 heterodimer has long been regarded as solely responsible for the Mehler-like reaction downstream of PSI under both high (HC) and air level (low, LC) levels of CO2. In this work, I revealed, for the first time, the contribution of the Flv2/Flv4 heterodimer to the Mehler-like reaction in vivo. Moreover, I directly compare the Mehler-like reaction kinetics under HC and LC conditions. My results demonstrate that, contrary to the apparent futile contribution of FDPs under HC, WT cells display a strong and steady-state Mehler-like reaction (biphasic kinetics) mediated by low amounts of the Flv1/Flv3 heterodimer. Whereas, under LC (at pH 6–8.2), the expression of FDPs is induced and WT cells show triphasic kinetics (induction, quenching and steady-state) of O2 photoreduction driven by Flv1/Flv3 and Flv2/Flv4 functioning downstream of PSI. Furthermore, I was able to unravel the contribution of Flv1/Flv3 and Flv2/Flv4 to the O2 photoreduction kinetics: Flv1/Flv3 was shown to be the main responsible for the strong but transient phase upon illumination, while Flv2/Flv4 mostly contributes to the slow steady-state phase under LC. Importantly, the mutants with defective NDH-1 complexes lacked the quenching phase of O2 photoreduction after the initial induction, suggesting that the transient activity of Flv1/Flv3, under LC, is due to competition for electrons with the NDH-1 complex via reduced Fd. A more thorough examination demonstrated a partial functional redundancy between Flv1/Flv3 and NDH-11/2. Under constant illumination in LC, cells devoid of NDH-11/2 prioritize the oxidation of PSI by enhancing the accumulation and activity of all FDPs, over the efficient induction of CO2 fixation. Under the same conditions, the absence of the transient activity of Flv1/Flv3 can be compensated for by the joint work of NDH 11/2 and Flv2/Flv4 to maintain PSI oxidation. The absence of both Flv1/Flv3 and NDH-11/2 resulted in a diminished ability to oxidize PSI and, albeit at a high cost, this allowed the allocation of reductants towards CO2 fixation. These results demonstrated a dynamic coordination between both pathways for the efficient oxidation of PSI and CO2 fixation. In this work, I also demonstrated that the essential role of Flv1/Flv3 under fluctuating light (FL) conditions cannot be compensated for by neither Flv2/Flv4 nor NDH-1. Additionally, I demonstrated that Flv1/Flv1 and Flv3/Flv3 homodimers contribute to the acclimation of Synechocystis to FL, mediating an oxygen-independent reaction. The filamentous N2-fixing cyanobacterium Anabaena sp. PCC 7120 (hereafter Anabaena) contains, along with Flv2 and Flv4, two copies of genes encoding for Flv1 and Flv3 proteins: Flv1A and Flv3A are expressed in vegetative cells, whereas Flv1B and Flv3B are localized in mature heterocysts. My results indicate that Flv3A has an important role during light acclimation regardless of the of the nitrogen and CO2 availability. Moreover, I demonstrated that unlike Flv3 in Synechocystis, Flv3A is capable of mediating, to some extent, O2 photoreduction independently of Flv1A. My results suggest that Flv3A functions in coordination with Flv2 and Flv4, likely forming various oligomeric arrangement. Furthermore, I examined the physiological relevance of the vegetative cell-specific Flv1A and Flv3A on the diazotrophic metabolism of Anabaena and demonstrated that the deletion of the vegetative cellspecific Flv3A resulted in downregulation of the heterocyst specific-protein uptake hydrogenase (Hup) which led to enhanced H2 photoproduction under both oxic and micro-oxic conditions. These results revealed a complex regulatory network between the Mehler-like reaction in the vegetative cells and the H2 metabolism in the heterocysts of Anabaena that will need to be further elucidated in future studies.Happea tuottavaa fotosynteesiä hyödyntävien organismien kyky kehittää suojamekanismeja fotosynteettisen koneiston säätelemiseksi on ratkaisevan tärkeää, jotta eliöt selviytyisivät jatkuvasti muuttuvissa ympäristöolosuhteissa. Flavodiironiproteiinit (FDP:t) toimivat elektroninsiirtoreitissä, joka on kehittynyt kaikissa fotosynteettisissä organismeissa, lukuun ottamatta koppisiemenisiä kasveja sekä puna- ja ruskoleviä. FDP:t suojaavat eliötä liialta valoenergialta toimimalla elektroninieluina, jotka ohjaavat ylimääräisiä elektroneja fotosynteettisestä elektroninsiirtoketjusta hapelle ns. Mehlerin kaltaisen reaktion välityksellä. Väitöskirjassani selvitin syanobakteerien FDP:en säätelymekanismeja, fysiologista merkitystä ja toiminnallista säätelyä. Yksisoluisessa syanobakteerissa Synechocystis sp. PCC 6803:ssa (tästä eteenpäin Synechocystis) Flv1/Flv3-heterodimeerin on pitkään ajateltu olevan yksin vastuussa PSI:n vastaanottajapuolella tapahtuvasta Mehlerin kaltaisesta reaktiosta sekä korkeassa (HC) että normaalissa (LC) ilman hiilidioksidipitoisuudessa. Väitöskirjassani osoitin ensimmäistä kertaa, että Flv2/Flv4-heterodimeeri vaikuttaa Mehlerin kaltaiseen reaktioon in vivo. Lisäksi vertasin Mehlerin kaltaisen reaktion kinetiikkaa HC- ja LC-olosuhteissa. Tulokseni osoittavat, että HC-olosuhteissa, joissa FDP:n merkitys on ennen ajateltu olevan pieni, villityyppisissä (WT) soluissa oleva pieni määrä Flv1/Flv3-heterodimeeriä saa aikaan merkittävän ja vakaan Mehlerin kaltaisen reaktion (kaksivaiheinen kinetiikka). Sitä vastoin LColosuhteissa (pH 6–8,2) FDP:en ilmentyminen indusoituu, ja WT-solujen hapen valopelkistys noudattaa kolmivaiheista reaktiokinetiikkaa (induktio, vaimeneminen ja vakaa tila), jonka saavat aikaan PSI:n vastaanottajapuolella sijaitsevat Flv1/Flv3 ja Flv2/Flv4. Lisäksi pystyin selvittämään Flv1/Flv3:n ja Flv2/Flv4:n vaikutuksen hapen valopelkistyksen kinetiikkaan: Flv1/Flv3 on pääasiallisesti vastuussa sen voimakkaasta, mutta ohimenevästä vaiheesta valojakson aikana LC-olosuhteissa, kun taas Flv2/Flv4 vaikuttaa enimmäkseen hitaan ja vakaan vaiheen aikana. On tärkeää huomata, että mutanttikannat, joilta puuttuu toiminnallinen NDH-1-kompleksi, eivät osoittaneet hapen valopelkistyksen vaimenemista induktion jälkeen, mikä viittaa siihen, että Flv1/Flv3:n ohimenevä aktiivisuus LC-olosuhteissa johtuu kilpailusta NDH-1-kompleksin kanssa pelkistyneeltä ferredoksiinilta tulevista elektroneista. Perusteellisempi tutkimus osoitti osittaisen toiminnallisen päällekkäisyyden Flv1/Flv3:n ja NDH-11/2:n välillä. Jatkuvassa valaistuksessa LC olosuhteissa solut, joissa ei ole NDH-11/2:ta, asettavat etusijalle PSI:n hapettumisen tehostamalla kaikkien FDP:en kertymistä ja aktiivisuutta verrattuna tehokkaaseen hiilensidonnan indusointiin. Samoissa olosuhteissa Flv1/Flv3:n ohimenevä aktiivisuuden puuttuminen voidaan korvata NDH-11/2:n ja Flv2/Flv4:n yhteistyöllä PSI:n hapettumisen ylläpitämiseksi. Sekä Flv1/Flv3:n että NDH-11/2:n samanaikainen puuttuminen johti heikentyneeseen kykyyn hapettaa PSI ja, vaikkakin resursseja uhraten, tämä mahdollisti pelkistysvoimaa tarjoavien yhdisteiden kohdistamisen hiilensidontaan. Nämä tulokset osoittivat dynaamisen koordinoinnin molempien elektroninsiirtoreittien välillä PSI:n tehokkaan hapettumisen ja hiilensidonnan varmistamiseksi. Tulokseni myös osoittavat, että Flv2/Flv4 tai NDH- 1 ei kykene korvaamaan Flv1/Flv3:a vaihtelevissa valo-olosuhteissa (FL) korostaen Flv1/Flv3:n olennaista merkitystä FL:n aikana. Lisäksi osoitin, että Flv1/Flv1- ja Flv3/Flv3- homodimeerit välittävät hapesta riippumattomia elektroninsiirtoreaktioita, jotka myötävaikuttavat Synechocystiksen sopeutumiseen FL-olosuhteisiin. Filamenttisella, ilmakehän typpeä sitovalla syanobakteerilla Anabaena sp. PCC 7120 (tästä lähtien Anabaena) on Flv2:n ja Flv4:n lisäksi kaksi kopiota Flv1- ja Flv3 -proteiineja koodaavista geeneistä: kasvulliset solut ilmentävät Flv1A- ja Flv3Aproteiineja, kun taas Flv1B- ja Flv3B- proteiinit löytyvät kypsistä heterokysteistä. Tulokseni osoittavat, että Flv3A:lla on tärkeä rooli solujen sopeutuessa valoon riippumatta typen ja hiilidioksidin saatavuudesta. Olen myöskin osoittanut, että toisin kuin Synechocystiksen Flv3, Anabaenan Flv3A kykenee jossain määrin itsenäisesti välittämään hapen valopelkistystä Flv1A:lle. Tulokseni viittaavat siihen, että Flv3A toimii yhdessä Flv2:n ja Flv4:n kanssa muodostaen todennäköisesti erilaisia oligomeerisiä konformaatioita. Lisäksi tarkastelin vegetatiivisille soluille spesifisten Flv1A:n ja Flv3A:n fysiologista merkitystä Anabaenan typpiaineenvaihdunnassa ja osoitin, että vegetatiivisille soluille spesifisen Flv3A:n deleetio sai aikaan heterokystispesifisen uptake hydrogenase (Hup) -proteiinin ilmenemisen vähenemisen, mikä johti lisääntyneeseen vedyn tuotantoon valossa sekä ilmakehän normaalissa happipitoisuudessa että vähähappisissa olosuhteissa. Väitöskirjatyöni osoitti, että Anabaena-syanobakteerin kasvullisissa soluissa tapahtuvan Mehlerin kaltaisen reaktion ja heterokysteissä tapahtuvan vetyaineenvaihdunnan välillä toimii monimutkainen säätelyverkosto. Jatkotutkimuksissa selvitetään tämän säätelyverkoston rakennetta ja merkitystä syanobakteerin aineenvaihdunnalle

Nordic cyanobacterial and algal lipids: Triacylglycerol accumulation, chemotaxonomy and bioindustrial potential

The ability to capture and convert sunlight, water and nutrients into useful compounds make photosynthetic microbes ideal candidates for the bio-industrial factories of the future. However, the suitability of isolates from temperate regions to grow under Nordic conditions is questionable. In this work, we explore the chemotaxonomy of Nordic strains of cyanobacteria and one green alga and evaluate their potential as raw materials for the production of lipid-based bio-industrial compounds. Thin-layer chromatography was used to identify the presence of triacylglycerol, which were detected in the majority of strains. Fatty acid methyl ester profiles were analysed to determine the suitability of strains for the production of biodiesel or the production of polyunsaturated fatty acids for the nutraceutical industry. The Nordic Synechococcus strains were unique in demonstrating fatty acid profiles comprised mostly C14:0, C16:0 and C16:1 and lacking polyunsaturated fatty acids. These properties translated to superior predicted biodiesel qualities, including cetane number, cold filter plugging point and oxidative stability compared to the other evaluated strains. Polyunsaturated fatty acids were detected at high levels (38-53%), with Calothrix sp. 336/3 being abundant in two essential fatty acids, linoleic and alpha-linolenic acid (21 and 17%, respectively). Gamma-linoleic acid was the predominant polyunsaturated fatty acid for the remaining strains (13-21%). In addition to assessing the potential of Nordic strains for bio-industrial production, this work also discusses issues such as taxonomy and predictive modelling, which can affect the identification of prospective high-performing strains

Flv3A facilitates O2 photoreduction and affects H2 photoproduction independently of Flv1A in diazotrophic Anabaena filaments

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Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to &lt;90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], &gt;300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of &lt;15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P&lt;0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P&lt;0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Flv3A facilitates O2 photoreduction and affects H2 photoproduction independently of Flv1A in diazotrophic Anabaena filaments.

The model heterocyst-forming filamentous cyanobacterium Anabaena sp. PCC 7120 (Anabaena) is a typical example of a multicellular organism capable of simultaneously performing oxygenic photosynthesis in vegetative cells and O2 -sensitive N2 -fixation inside heterocysts. The flavodiiron proteins have been shown to participate in photoprotection of photosynthesis by driving excess electrons to O2 (a Mehler-like reaction). Here, we performed a phenotypic and biophysical characterization of Anabaena mutants impaired in vegetative-specific Flv1A and Flv3A in order to address their physiological relevance in the bioenergetic processes occurring in diazotrophic Anabaena under variable CO2 conditions. We demonstrate that both Flv1A and Flv3A are required for proper induction of the Mehler-like reaction upon a sudden increase in light intensity, which is likely important for the activation of carbon-concentrating mechanisms and CO2 fixation. Under ambient CO2 diazotrophic conditions, Flv3A is responsible for moderate O2 photoreduction, independently of Flv1A, but only in the presence of Flv2 and Flv4. Strikingly, the lack of Flv3A resulted in strong downregulation of the heterocyst-specific uptake hydrogenase, which led to enhanced H2 photoproduction under both oxic and micro-oxic conditions. These results reveal a novel regulatory network between the Mehler-like reaction and the diazotrophic metabolism, which is of great interest for future biotechnological applications

The risk of COVID-19 death is much greater and age dependent with type I IFN autoantibodies

International audienceSignificance There is growing evidence that preexisting autoantibodies neutralizing type I interferons (IFNs) are strong determinants of life-threatening COVID-19 pneumonia. It is important to estimate their quantitative impact on COVID-19 mortality upon SARS-CoV-2 infection, by age and sex, as both the prevalence of these autoantibodies and the risk of COVID-19 death increase with age and are higher in men. Using an unvaccinated sample of 1,261 deceased patients and 34,159 individuals from the general population, we found that autoantibodies against type I IFNs strongly increased the SARS-CoV-2 infection fatality rate at all ages, in both men and women. Autoantibodies against type I IFNs are strong and common predictors of life-threatening COVID-19. Testing for these autoantibodies should be considered in the general population

The risk of COVID-19 death is much greater and age dependent with type I IFN autoantibodies

International audienceSignificance There is growing evidence that preexisting autoantibodies neutralizing type I interferons (IFNs) are strong determinants of life-threatening COVID-19 pneumonia. It is important to estimate their quantitative impact on COVID-19 mortality upon SARS-CoV-2 infection, by age and sex, as both the prevalence of these autoantibodies and the risk of COVID-19 death increase with age and are higher in men. Using an unvaccinated sample of 1,261 deceased patients and 34,159 individuals from the general population, we found that autoantibodies against type I IFNs strongly increased the SARS-CoV-2 infection fatality rate at all ages, in both men and women. Autoantibodies against type I IFNs are strong and common predictors of life-threatening COVID-19. Testing for these autoantibodies should be considered in the general population