Structure of Chlorella ohadii Photosystem II Reveals Protective Mechanisms against Environmental Stress.

Green alga Chlorella ohadii is known for its ability to carry out photosynthesis under harsh conditions. Using cryogenic electron microscopy (cryoEM), we obtained a high-resolution structure of PSII at 2.72 Å. This structure revealed 64 subunits, which encompassed 386 chlorophylls, 86 carotenoids, four plastoquinones, and several structural lipids. At the luminal side of PSII, a unique subunit arrangement was observed to protect the oxygen-evolving complex. This arrangement involved PsbO (OEE1), PsbP (OEE2), PsbB, and PsbU (a homolog of plant OEE3). PsbU interacted with PsbO, PsbC, and PsbP, thereby stabilizing the shield of the oxygen-evolving complex. Significant changes were also observed at the stromal electron acceptor side. PsbY, identified as a transmembrane helix, was situated alongside PsbF and PsbE, which enclosed cytochrome b559. Supported by the adjacent C-terminal helix of Psb10, these four transmembrane helices formed a bundle that shielded cytochrome b559 from the surrounding solvent. Moreover, the bulk of Psb10 formed a protective cap, which safeguarded the quinone site and likely contributed to the stacking of PSII complexes. Based on our findings, we propose a protective mechanism that prevents QB (plastoquinone B) from becoming fully reduced. This mechanism offers insights into the regulation of electron transfer within PSII.

A PSII photosynthetic control is activated in anoxic cultures of green algae following illumination.

Photosynthetic hydrogen production from microalgae is considered to have potential as a renewable energy source. Yet, the process has two main limitations holding it back from scaling up; (i) electron loss to competing processes, mainly carbon fixation and (ii) sensitivity to O2 which diminishes the expression and the activity of the hydrogenase enzyme catalyzing H2 production. Here we report a third, hitherto unknown challenge: We found that under anoxia, a slow-down switch is activated in photosystem II (PSII), diminishing the maximal photosynthetic productivity by three-fold. Using purified PSII and applying in vivo spectroscopic and mass spectrometric techniques on Chlamydomonas reinhardtii cultures, we show that this switch is activated under anoxia, within 10 s of illumination. Furthermore, we show that the recovery to the initial rate takes place following 15 min of dark anoxia, and propose a mechanism in which, modulation in electron transfer at the acceptor site of PSII diminishes its output. Such insights into the mechanism broaden our understanding of anoxic photosynthesis and its regulation in green algae and inspire new strategies to improve bio-energy yields.

CryoEM PSII structure reveals adaptation mechanisms to environmental stress in Chlorella ohadii.

Performing photosynthesis in the desert is a challenging task since it requires a fast adaptation to extreme illumination and temperature changes. To understand adaptive mechanisms, we purified Photosystem II (PSII) from Chlorella ohadii , a green alga from the desert soil surface, and identified structural elements that might enable the photosystem functioning under harsh conditions. The 2.72 Å cryogenic electron-microscopy (cryoEM) structure of PSII exhibited 64 subunits, encompassing 386 chlorophylls, 86 carotenoids, four plastoquinones, and several structural lipids. At the luminal side of PSII, the oxygen evolving complex was protected by a unique subunit arrangement - PsbO (OEE1), PsbP (OEE2), CP47, and PsbU (plant OEE3 homolog). PsbU interacted with PsbO, CP43, and PsbP, thus stabilising the oxygen evolving shield. Substantial changes were observed on the stromal electron acceptor side - PsbY was identified as a transmembrane helix situated alongside PsbF and PsbE enclosing cytochrome b559, supported by the adjacent C-terminal helix of Psb10. These four transmembrane helices bundled jointly, shielding cytochrome b559 from the solvent. The bulk of Psb10 formed a cap protecting the quinone site and probably contributed to the PSII stacking. So far, the C. ohadii PSII structure is the most complete description of the complex, suggesting numerous future experiments. A protective mechanism that prevented Q B from rendering itself fully reduced is proposed.

A simple procedure to identify children with B-lineage acute lymphoblastic leukemia who can be successfully treated with low or moderate intensity: Sequential versus single-point minimal residual disease measurement.

Sequential monitoring of minimal residual disease (MRD) by molecular techniques or multicolor flow cytometry (MFC) has emerged over the past two decades as the primary tool to optimize treatment in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The aim of our study was to compare the prognostic power of repeated MFC-MRD measurement with single-point MRD assessment in children with BCP-ALL treated with the reduced-intensity protocol ALL-MB 2008. Data from consecutive MFC-MRD at day 15 and day 36 (end of induction, EOI) were available for 507 children with Philadelphia-negative BCP-ALL. They were stratified into standard risk (SR, n = 265), intermediate risk (ImR, n = 211), and high risk (HR, n = 31) according to the initial clinical characteristics defined in the ALL-MB 2008 protocol. Quantitative (relative to quantitative thresholds) and kinetic (logarithmic reduction) assessments of MFC-MRD at both time points effectively separated patients into three groups with different risk of recurrence. On the other hand, starting with low (for the SR group) and moderate (for the ImR group) induction therapy, a single MFC-MRD measurement at EOI proved sufficient to unequivocally identify patients in whom this therapy is highly effective and distinguish them from those who cannot be successfully treated with such therapy. Therefore, initiating treatment with low or moderate treatment from the start, together with careful consideration of initial clinical risk factors and just one EOI-MFC-MRD measurement is simple, inexpensive, and entirely sufficient for treatment optimization. Furthermore, for a large proportion of patients, this approach allows better adjustment, in particular also reduction of therapy intensity than sequential MRD measurements.

Structure of Dunaliella photosystem II reveals conformational flexibility of stacked and unstacked supercomplexes.

Photosystem II (PSII) generates an oxidant whose redox potential is high enough to enable water oxidation , a substrate so abundant that it assures a practically unlimited electron source for life on earth . Our knowledge on the mechanism of water photooxidation was greatly advanced by high-resolution structures of prokaryotic PSII . Here, we show high-resolution cryogenic electron microscopy (cryo-EM) structures of eukaryotic PSII from the green alga Dunaliella salina at two distinct conformations. The conformers are also present in stacked PSII, exhibiting flexibility that may be relevant to the grana formation in chloroplasts of the green lineage. CP29, one of PSII associated light-harvesting antennae, plays a major role in distinguishing the two conformations of the supercomplex. We also show that the stacked PSII dimer, a form suggested to support the organisation of thylakoid membranes , can appear in many different orientations providing a flexible stacking mechanism for the arrangement of grana stacks in thylakoids. Our findings provide a structural basis for the heterogenous nature of the eukaryotic PSII on multiple levels.

Targeted Therapy With Venetoclax and Daratumumab as Part of HSCT Preparative Regimen in Children With Chemorefractory Acute Myeloid Leukemia.

The long-term outcome of allogeneic hematopoietic stem cell transplantation (HSCT) in chemorefractory acute myeloid leukemia (AML) remains suboptimal because of a high relapse rate. Enhancement of conditioning regimens by the incorporation of targeted anti-leukemia agents is a potential approach to improve the efficacy of HSCT. In a pilot trial and extended access cohort, we evaluated the safety and potential value of adding combinations of venetoclax and daratumumab to a preparative regimen among children with chemorefractory acute myeloid leukemia grafted with αß T-cell-depleted peripheral blood stem cells. All 20 patients had active disease status of AML at the time of transplantation. The preparative regimen included myeloablative conditioning based on either total body irradiation or treosulfan. A haploidentical related donor was used as a graft source for all patients. Engraftment was not compromised, and no excess toxicity was noted. Minimal residual disease-negative complete remission was achieved in 17 patients (85%). The cumulative incidence of grade II to IV acute graft-versus-host disease (GVHD) was 17%, and the cumulative incidence of chronic GVHD was 7%. At 2 years, nonrelapse mortality was 10%, relapse incidence was 46%, event-free survival was 44%, and overall survival was 65%. Our data show the possibility of safely adding targeted agents to conditioning regimens; however, no evidence of a significant improvement in long-term transplantation outcomes in this cohort of patients was observed.

One-point flow cytometric MRD measurement to identify children with excellent outcome after intermediate-risk BCP-ALL: results of the ALL-MB 2008 study.

BACKGROUND:  Measurement of minimal residual disease (MRD) with multicolor flow cytometry (MFC) has become an important tool in childhood acute lymphoblastic leukemia (ALL), mainly to identify rapid responders and reduce their therapy intensity. Protocols of the Moscow-Berlin (MB) group use a comparatively low (for standard risk; SR) or moderate (for intermediate risk; ImR) treatment intensity from the onset, based on initial patient characteristics. Recently, we reported that 90% of SR patients-50% B cell precursor (BCP-ALL)-MFC-MRD negative at end of induction (EOI)-had 95% event-free survival (EFS).  METHODS: In the present study, we applied this method to children with initial ImR features. RESULTS:  In study MB 2008, 1105 children-32% of BCP-ALL patients-were assigned to the ImR group. Of these, 227 were treated in clinics affiliated with MFC laboratories of the MB group network, and included in this MFC-MRD pilot study. A single-point MFC-MRD measurement at the EOI with the threshold of 0.01% identified 65% of patients-20% of all BCP-ALL patients-with EFS of 93.5%. CONCLUSION:  Taking both studies together, the combination of clinical parameters and a one-point MRD measurement identifies 70% of BCP-ALL patients with an excellent outcome after low- or moderate-intensity therapy and avoids overtreatment of a significant proportion of patients.

Cryo-EM photosystem I structure reveals adaptation mechanisms to extreme high light in Chlorella ohadii.

Photosynthesis in deserts is challenging since it requires fast adaptation to rapid night-to-day changes, that is, from dawn's low light (LL) to extreme high light (HL) intensities during the daytime. To understand these adaptation mechanisms, we purified photosystem I (PSI) from Chlorella ohadii, a green alga that was isolated from a desert soil crust, and identified the essential functional and structural changes that enable the photosystem to perform photosynthesis under extreme high light conditions. The cryo-electron microscopy structures of PSI from cells grown under low light (PSILL) and high light (PSIHL), obtained at 2.70 and 2.71 Å, respectively, show that part of light-harvesting antenna complex I (LHCI) and the core complex subunit (PsaO) are eliminated from PSIHL to minimize the photodamage. An additional change is in the pigment composition and their number in LHCIHL; about 50% of chlorophyll b is replaced by chlorophyll a. This leads to higher electron transfer rates in PSIHL and might enable C. ohadii PSI to act as a natural photosynthesiser in photobiocatalytic systems. PSIHL or PSILL were attached to an electrode and their induced photocurrent was determined. To obtain photocurrents comparable with PSIHL, 25 times the amount of PSILL was required, demonstrating the high efficiency of PSIHL. Hence, we suggest that C. ohadii PSIHL is an ideal candidate for the design of desert artificial photobiocatalytic systems.

T-cell tracking, safety, and effect of low-dose donor memory T-cell infusions after αß T cell-depleted hematopoietic stem cell transplantation.

The delayed recovery of adaptive immunity underlies transplant-related mortality (TRM) after αß T cell-depleted hematopoietic stem cell transplantation (HSCT). We tested the use of low-dose memory donor lymphocyte infusions (mDLIs) after engraftment of αß T cell-depleted grafts.A cohort of 131 pediatric patients (median age 9 years) were grafted with αß T cell-depleted products from either haplo (n = 79) or unrelated donors (n = 52). After engraftment, patients received mDLIs prepared by CD45RA depletion. Cell dose was escalated monthly from 25 × 103 to 100 × 103/kg (haplo) and from 100 × 103 to 300 × 103 /kg (MUD). In a subcohort of 16 patients, T-cell receptor (TCR) repertoire profiling with deep sequencing was used to track T-cell clones and to evaluate the contribution of mDLI to the immune repertoire.In total, 343 mDLIs were administered. The cumulative incidence (CI) of grades II and III de novo acute graft-versus-host disease (aGVHD) was 5% and 2%, respectively, and the CI of chronic graft-versus-host disease was 7%. Half of the patients with undetectable CMV-specific T cells before mDLI recovered CMV-specific T cells. TCR repertoire profiling confirmed that mDLI-derived T cells significantly contribute to the TCR repertoire up to 1 year after HSCT and include persistent, CMV-specific T-cell clones.

Structure and energy transfer pathways of the Dunaliella Salina photosystem I supercomplex.

Oxygenic photosynthesis evolved more than 3 billion years ago in cyanobacteria. The increased complexity of photosystem I (PSI) became apparent from the high-resolution structures that were obtained for the complexes that were isolated from various organisms, ranging from cyanobacteria to plants. These complexes are all evolutionarily linked. In this paper, the researchers have uncovered the increased complexity of PSI in a single organism demonstrated by the coexistance of two distinct PSI compositions. The Large Dunaliella PSI contains eight additional subunits, six in PSI core and two light harvesting complexes. Two additional chlorophyll a molecules pertinent for efficient excitation energy transfer in state II transition were identified in PsaL and PsaO. Short distances between these newly identified chlorophylls correspond with fast excitation transfer rates previously reported during state II transition. The apparent PSI conformations could be a coping mechanism for the high salinity.

Alternative pyrimidine biosynthesis protein ApbE is a flavin transferase catalyzing covalent attachment of FMN to a threonine residue in bacterial flavoproteins.

Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) contains two flavin residues as redox-active prosthetic groups attached by a phosphoester bond to threonine residues in subunits NqrB and NqrC. We demonstrate here that flavinylation of truncated Vibrio harveyi NqrC at Thr-229 in Escherichia coli cells requires the presence of a co-expressed Vibrio apbE gene. The apbE genes cluster with genes for Na(+)-NQR and other FMN-binding flavoproteins in bacterial genomes and encode proteins with previously unknown function. Experiments with isolated NqrC and ApbE proteins confirmed that ApbE is the only protein factor required for NqrC flavinylation and also indicated that the reaction is Mg(2+)-dependent and proceeds with FAD but not FMN. Inactivation of the apbE gene in Klebsiella pneumoniae, wherein the nqr operon and apbE are well separated in the chromosome, resulted in a complete loss of the quinone reductase activity of Na(+)-NQR, consistent with its dependence on covalently bound flavin. Our data thus identify ApbE as a novel modifying enzyme, flavin transferase.

Redox properties of the prosthetic groups of Na(+)-translocating nadh:quinone oxidoreductase. 1. Electron paramagnetic resonance study of the enzyme.

Redox properties of all EPR-detectable prosthetic groups of Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from Vibrio harveyi were studied at pH 7.5 using cryo-EPR spectroelectrochemistry. Titration shows five redox transitions. One with E(m) = -275 mV belongs to the reduction of the [2Fe-2S] cluster, and the four others reflect redox transitions of flavin cofactors. Two transitions (E(m)(1) = -190 mV and E(m)(2) = -275 mV) originate from the formation of FMN anion radical, covalently bound to the NqrC subunit, and its subsequent reduction. The remaining two transitions arise from the two other flavin cofactors. A high potential (E(m) = -10 mV) transition corresponds to the reduction of riboflavin neutral radical, which is stable at rather high redox potentials. An E(m) = -130 mV transition reflects the formation of FMN anion radical from a flavin covalently bound to the NqrB subunit, which stays as a radical down to very low potentials. Taking into account the EPR-silent, two-electron transition of noncovalently bound FAD located in the NqrF subunit, there are four flavins in Na(+)-NQR all together. Defined by dipole-dipole magnetic interaction measurements, the interspin distance between the [2Fe-2S](+) cluster and the NqrB subunit-bound FMN anion radical is found to be 22.5 +/- 1.5 A, which means that for the functional electron transfer between these two centers another cofactor, most likely FMN bound to the NqrC subunit, should be located.

Catalytic properties of Na+-translocating NADH:quinone oxidoreductases from Vibrio harveyi, Klebsiella pneumoniae, and Azotobacter vinelandii.

The catalytic properties of sodium-translocating NADH:quinone oxidoreductases (Na+-NQRs) from the marine bacterium Vibrio harveyi, the enterobacterium Klebsiella pneumoniae, and the soil microorganism Azotobacter vinelandii have been comparatively analyzed. It is shown that these enzymes drastically differ in their affinity to sodium ions. The enzymes also possess different sensitivity to inhibitors. Na+-NQR from A. vinelandii is not sensitive to low 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) concentrations, while Na+-NQR from K. pneumoniae is fully resistant to either Ag+ or N-ethylmaleimide. All the Na+-NQR-type enzymes are sensitive to diphenyliodonium, which is shown to modify the noncovalently bound FAD of the enzyme.

Regulation of expression of Na+ -translocating NADH:quinone oxidoreductase genes in Vibrio harveyi and Klebsiella pneumoniae.

The expression of genes encoding sodium-translocating NADH:quinone oxidoreductase (Na(+)-NQR) was studied in the marine bacterium Vibrio harveyi and in the enterobacterium Klebsiella pneumoniae. It has been shown that such parameters as NaCl concentration, pH value, and presence of an uncoupler in the growth media do not influence significantly the level of nqr expression. However, nqr expression depends on the growth substrates used by these bacteria. Na(+)-NQR is highly repressed in V. harveyi during anaerobic growth, and nqr expression is modulated by electron acceptors and values of their redox potentials. The latter effect was shown to be independent of the ArcAB regulatory system.