Ablation Index-guided point-by-point ablation versus Grid annotation-guided dragging for pulmonary vein isolation: A randomized controlled trial.

INTRODUCTION: Radiofrequency (RF) atrial fibrillation (AF) ablation using a catheter dragging technique may shorten procedural duration and improve durability of pulmonary vein isolation (PVI) by creating uninterrupted linear ablation lesions. We compared a novel AF ablation approach guided by Grid annotation allowing for "drag lesions" with a standard point-by-point ablation approach in a single-center randomized study. METHODS: Eighty-eight paroxysmal or persistent AF patients were randomized 1:1 to undergo RF-PVI with either a catheter dragging ablation technique guided by Grid annotation or point-by-point ablation guided by Ablation Index (AI) annotation. In the Grid annotation arm, ablation was visualized using 1 mm³ grid points coloring red after meeting predefined stability and contact force criteria. In the AI annotation arm, ablation lesions were created in a point-by-point fashion with AI target values set at 380 and 500 for posterior/inferior and anterior/roof segments, respectively. Patients were followed up for 12 months after PVI using ECGs, 24-h Holter monitoring and a mobile-based one-lead ECG device. RESULTS: Procedure time was not different between the two randomization arms (Grid annotation 71 ± 19 min, AI annotation 72 ± 26 min, p = .765). RF time was significantly longer in the Grid annotation arm compared with the AI annotation arm (49 ± 8 min vs. 37 ± 8 min, respectively, p < .001). Atrial tachyarrhythmia recurrence was documented in 10 patients (23%) in the Grid annotation arm compared with 19 patients (42%) in the AI annotation arm with time to recurrence not reaching statistical significance (p = .074). CONCLUSIONS: This study shows that a Grid annotation-guided dragging approach provides an alternative to point-by-point RF-PVI using AI annotation.

The Lulun Project's social marketing strategy in a trial to introduce eggs during complementary feeding in Ecuador.

The Lulun Project incorporated a social marketing strategy that accompanied a randomized controlled trial (RCT) of a food-based intervention that introduced eggs into the complementary feeding diet of Ecuadorian infants. This strategy was designed to promote behaviour change, in this case, egg consumption, through voluntary prosocial behaviour, empowerment, and brand loyalty. A three-phase social marketing strategy (design, campaigns, and evaluation) contributed to our successful RTC by applying techniques drawn from marketing, publicity, design, and communications. To develop the strategy, we conducted (a) market research focused on culturally based norms, values, and local expectations; (b) a situational assessment based on the four Ps of social marketing (people, product, place, and price); and (c) fostered a creative process to develop the project's brand and communication plan. The strategy combined a communication plan, brand, and activities that were implemented in four campaigns: outreach, recruitment, promotion, and closing. Our evaluation showed that the social marketing strategy was instrumental in promoting the RCT's objectives and responding to unforeseen events and community concerns regarding the RCT. The strategy resulted in high compliance, low attrition, and infant feeding policy change, including Ecuador's Ministry of Public Health new complementary feeding guidelines for introducing eggs early in complementary feeding. Use of social marketing techniques, like those in our study, could be key for scaling up this food-based intervention-or others like it-in Ecuador and beyond.

Defining Electron Bifurcation in the Electron-Transferring Flavoprotein Family.

Electron bifurcation is the coupling of exergonic and endergonic redox reactions to simultaneously generate (or utilize) low- and high-potential electrons. It is the third recognized form of energy conservation in biology and was recently described for select electron-transferring flavoproteins (Etfs). Etfs are flavin-containing heterodimers best known for donating electrons derived from fatty acid and amino acid oxidation to an electron transfer respiratory chain via Etf-quinone oxidoreductase. Canonical examples contain a flavin adenine dinucleotide (FAD) that is involved in electron transfer, as well as a non-redox-active AMP. However, Etfs demonstrated to bifurcate electrons contain a second FAD in place of the AMP. To expand our understanding of the functional variety and metabolic significance of Etfs and to identify amino acid sequence motifs that potentially enable electron bifurcation, we compiled 1,314 Etf protein sequences from genome sequence databases and subjected them to informatic and structural analyses. Etfs were identified in diverse archaea and bacteria, and they clustered into five distinct well-supported groups, based on their amino acid sequences. Gene neighborhood analyses indicated that these Etf group designations largely correspond to putative differences in functionality. Etfs with the demonstrated ability to bifurcate were found to form one group, suggesting that distinct conserved amino acid sequence motifs enable this capability. Indeed, structural modeling and sequence alignments revealed that identifying residues occur in the NADH- and FAD-binding regions of bifurcating Etfs. Collectively, a new classification scheme for Etf proteins that delineates putative bifurcating versus nonbifurcating members is presented and suggests that Etf-mediated bifurcation is associated with surprisingly diverse enzymes.IMPORTANCE Electron bifurcation has recently been recognized as an electron transfer mechanism used by microorganisms to maximize energy conservation. Bifurcating enzymes couple thermodynamically unfavorable reactions with thermodynamically favorable reactions in an overall spontaneous process. Here we show that the electron-transferring flavoprotein (Etf) enzyme family exhibits far greater diversity than previously recognized, and we provide a phylogenetic analysis that clearly delineates bifurcating versus nonbifurcating members of this family. Structural modeling of proteins within these groups reveals key differences between the bifurcating and nonbifurcating Etfs.

The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis.

The biological reduction of dinitrogen (N2) to ammonia (NH3) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (Em = -320 mV) coupled to reduction of flavodoxin semiquinone (Em = -460 mV) and reduction of coenzyme Q (Em = 10 mV). Knocking out fix genes rendered Δrnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Overall, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.

Spleen Behind the Heart Complicates Lung-to-Head Ratio Measurement in Left-Sided Congenital Diaphragmatic Hernia.

In fetuses with left-sided congenital diaphragmatic hernia, intrathoracic herniation of the spleen is a common occurrence. The herniated spleen can reside posterior to the left atrium of the heart in the right hemithorax and is increasingly differentiated from the lung with the use of newer sonographic equipment. Estimation of the neonatal prognosis relies on accurate measurement of fetal lung size, particularly with commonly used measurements such as the lung-to-head ratio. Here we describe how herniation of the spleen behind the heart can complicate measurement of the lung-to-head ratio on sonography and lead to overestimation, with implications for perinatal prognostication and management.

Fetal Stomach Position Predicts Neonatal Outcomes in Isolated Left-Sided Congenital Diaphragmatic Hernia.

INTRODUCTION: We sought to determine the relationship between the degree of stomach herniation by antenatal sonography and neonatal outcomes in fetuses with isolated left-sided congenital diaphragmatic hernia (CDH). MATERIALS AND METHODS: We retrospectively reviewed neonatal medical records and antenatal sonography of fetuses with isolated left CDH cared for at a single institution (2000-2012). Fetal stomach position was classified on sonography as follows: intra-abdominal, anterior left chest, mid-to-posterior left chest, or retrocardiac (right chest). RESULTS: Ninety fetuses were included with 70% surviving to neonatal discharge. Stomach position was intra-abdominal in 14% (n = 13), anterior left chest in 19% (n = 17), mid-to-posterior left chest in 41% (n = 37), and retrocardiac in 26% (n = 23). Increasingly abnormal stomach position was linearly associated with an increased odds of death (OR 4.8, 95% CI 2.1-10.9), extracorporeal membrane oxygenation (ECMO; OR 5.6, 95% CI 1.9-16.7), nonprimary diaphragmatic repair (OR 2.7, 95% CI 1.4-5.5), prolonged mechanical ventilation (OR 5.9, 95% CI 2.3-15.6), and prolonged respiratory support (OR 4.0, 95% CI 1.6-9.9). All fetuses with intra-abdominal stomach position survived without substantial respiratory morbidity or need for ECMO. DISCUSSION: Fetal stomach position is strongly associated with neonatal outcomes in isolated left CDH. This objective tool may allow for accurate prognostication in a variety of clinical settings.

Evolution of molybdenum nitrogenase during the transition from anaerobic to aerobic metabolism.

UNLABELLED: Molybdenum nitrogenase (Nif), which catalyzes the reduction of dinitrogen to ammonium, has modulated the availability of fixed nitrogen in the biosphere since early in Earth's history. Phylogenetic evidence indicates that oxygen (O2)-sensitive Nif emerged in an anaerobic archaeon and later diversified into an aerobic bacterium. Aerobic bacteria that fix N2 have adapted a number of strategies to protect Nif from inactivation by O2, including spatial and temporal segregation of Nif from O2 and respiratory consumption of O2. Here we report the complement of Nif-encoding genes in 189 diazotrophic genomes. We show that the evolution of Nif during the transition from anaerobic to aerobic metabolism was accompanied by both gene recruitment and loss, resulting in a substantial increase in the number of nif genes. While the observed increase in the number of nif genes and their phylogenetic distribution are strongly correlated with adaptation to utilize O2 in metabolism, the increase is not correlated with any of the known O2 protection mechanisms. Rather, gene recruitment appears to have been in response to selective pressure to optimize Nif synthesis to meet fixed N demands associated with aerobic productivity and to more efficiently regulate Nif under oxic conditions that favor protein turnover. Consistent with this hypothesis, the transition of Nif from anoxic to oxic environments is associated with a shift from posttranslational regulation in anaerobes to transcriptional regulation in obligate aerobes and facultative anaerobes. Given that fixed nitrogen typically limits ecosystem productivity, our observations further underscore the dynamic interplay between the evolution of Earth's oxygen, nitrogen, and carbon biogeochemical cycles. IMPORTANCE: Molybdenum nitrogenase (Nif), which catalyzes the reduction of dinitrogen to ammonium, has modulated the availability of fixed nitrogen in the biosphere since early in Earth's history. Nif emerged in an anaerobe and later diversified into aerobes. Here we show that the transition of Nif from anaerobic to aerobic metabolism was accompanied by both gene recruitment and gene loss, resulting in a substantial increase in the number of nif genes. While the observed increase in the number of nif genes is strongly correlated with adaptation to utilize O2 in metabolism, the increase is not correlated with any of the known O2 protective mechanisms. Rather, gene recruitment was likely a response to more efficiently regulate Nif under oxic conditions that favor protein turnover.

Fetal ultrasound markers of severity predict resolution of pulmonary hypertension in congenital diaphragmatic hernia.

OBJECTIVE: Congenital diaphragmatic hernia (CDH) results in morbidity and death from lung hypoplasia and persistent pulmonary hypertension (PH). We sought to define the relationship between fetal ultrasound markers of severity in CDH and the time to resolution of neonatal PH. STUDY DESIGN: We conducted a retrospective study of fetuses with an antenatal ultrasound scan and left-sided CDH cared for at the University of California San Francisco (2002-2012). Fetal liver position was classified on ultrasound scan as abdominal (entire liver within the abdomen) or thoracic (any portion of the liver within the thorax). Fetal stomach position was classified from least to most aberrant: abdominal, anterior left chest, mid-posterior left chest, or retrocardiac (right chest). Lung-to-head ratio (LHR) was determined from available scans at 20-29 weeks of gestational age (GA). Routine neonatal echocardiograms were performed weekly for up to 6 weeks or until PH resolved or until discharge. PH was assessed by echocardiogram with the use of a hierarchy of ductus arteriosus level shunt, interventricular septal position, and tricuspid regurgitant jet velocity. Days to PH-free survival was defined as the age at which pulmonary artery pressure was estimated to be <2/3 systemic blood pressure. Cox proportional hazards models adjusted for GA at birth, era of birth, fetal surgery, and GA at ultrasound scan (LHR model only), with censoring at 100 days. RESULTS: Of 118 patients, the following fetal markers were available: LHR (n = 53), liver position (n = 112), and stomach position (n = 80). Fewer infants experienced resolved PH if they had LHR <1 (P = .006), thoracic liver position (P = .001), or more aberrant stomach position (P < .001). There was also a decreased rate of resolution of PH in infants with LHR <1 (hazard ratio, 0.30; P = .007), thoracic liver position (hazard ratio, 0.38; P < .001), and more aberrant stomach position (hazard ratios, 0.28 [P = .002]; 0.1 [P < .001]; and 0.07 [P < .001]). CONCLUSION: Fetal ultrasound markers of CDH severity are predictive not only of death but also of significant morbidity. LHR <1, thoracic liver, and aberrant stomach position are associated with delayed time to resolution of PH in infants with CDH and may be used to identify fetuses at high risk of persistent PH.

Effect of etanercept therapy on psoriasis symptoms in patients from Latin America, Central Europe, and Asia: a subset analysis of the PRISTINE trial.

BACKGROUND: Psoriasis prevalence and characteristics in Asia, Central Europe, and Latin America have not been thoroughly investigated and there are no large trials for biologic treatments for patients from these regions. The goal of this analysis was to report clinical response to anti-tumor necrosis factor-alpha treatment in these patients. METHODS: Patients from Argentina, Czech Republic, Hungary, Mexico, Taiwan, and Thailand (N=171) were included in this subset analysis of the PRISTINE trial. Patients with stable moderate-to-severe plaque psoriasis were blinded and randomized to receive etanercept 50 mg once weekly (QW) or biweekly (BIW) for 12 weeks, followed by 12 weeks of open-label QW treatment with etanercept 50 mg through week 24 (QW/QW vs. BIW/QW). Concomitant methotrexate (≤20 mg/week) and mild topical corticosteroids or other agents were permitted at the physician's discretion, in accordance with therapeutic practice. RESULTS: As early as week 8, 26.7 % in the etanercept QW group and 44.0 % in the BIW group achieved Psoriasis Area and Severity Index (PASI) 75. At weeks 12 and 24, respectively, PASI 75 increased to 39.5 % and 62.8 % in the QW/QW group and 66.7 % and 83.3 % in the BIW/QW group. PASI 75 was significantly different between treatment groups from week 8 through the end of study (p<0.05). The Kaplan-Meier estimate of the proportions achieving PASI 75 in QW/QW and BIW/QW groups, respectively, was 27.4 % and 45.8 % through week 8; 41.9 % and 68.7 % through week 12; and 72.5 % and 95.2 % through week 24. CONCLUSIONS: Treatment with etanercept 50 mg provided rapid relief of psoriasis symptoms in patients from Asia, Central Europe, and Latin America. A more rapid response was observed in patients who received BIW treatment for the first 12 weeks which was sustained after reducing to QW dosing for the subsequent 12 weeks. Response rates were similar to those observed in the overall PRISTINE population. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT00663052 .

Scrotal swelling in the neonate.

Discovery of scrotal swelling in a neonate can be a source of anxiety for parents, clinicians, and sonologists alike. This pictorial essay provides a focused review of commonly encountered scrotal masses and mimics specific to the neonatal setting. Although malignancy is a concern, it is very uncommon, as most neonatal scrotal masses are benign. Key discriminating features and management options are highlighted to improve the radiologist's ability to diagnose neonatal scrotal conditions and guide treatment decisions. Neonatal scrotal processes ranging from common to uncommon will be discussed.

Acute exercise enhances the response to paired associative stimulation-induced plasticity in the primary motor cortex.

There is evidence that a single session of aerobic exercise can modulate intracortical inhibition. While decreases in inhibition appear to be a necessary precursor to the induction of long-term potentiation (LTP)-like plasticity, it is not known whether aerobic exercise can enhance the response to LTP induction. We investigated whether the addition of a preceding bout of exercise would modulate the response to paired associative stimulation (PAS) of the upper limb. It was hypothesized that exercise would enhance motor cortical (M1) excitability following PAS compared to a session of PAS alone. Ten healthy individuals underwent a control session involving PAS alone and an exercise session where PAS was preceded by 20 min of moderate-intensity stationary biking. PAS involved 180 pairs of stimuli (right median nerve, left M1) delivered at 0.1 Hz to the right abductor pollicis brevis representation. Excitability changes were measured by the area under a stimulus-response curve, and intracortical circuits were probed by testing short-interval intracortical inhibition (SICI), long-interval intracortical inhibition and intracortical facilitation. Two-way ANOVAs were conducted to compare excitability changes between sessions. PAS-induced increases in M1 excitability were enhanced in the exercise session (p < 0.026). In addition, SICI was differentially modulated between the two sessions, with greater decreases in SICI observed immediately after PAS when it was preceded by the exercise session (p < 0.03). Aerobic exercise enhances the effectiveness of PAS and may be a useful adjunct to traditional therapies and interventions that aim to promote neuroplasticity in cortical networks.

Polymorphisms of pro-inflammatory cytokine genes and the risk for acute suppurative or chronic nonsuppurative apical periodontitis in a Colombian population.

AIM: To determine the association of functional single nucleotide polymorphisms in genes of the pro-inflammatory cytokines tumour necrosis factor-α, interleukin-1ß, interleukin-8 and interleukin-12B with the development of two clinical forms of apical periodontitis (AP): acute suppurative and chronic nonsuppurative. METHODOLOGY: The study included 120 patients from Bucaramanga City, Colombia, 63 diagnosed with acute suppurative AP (ASAP) and 57 diagnosed with chronic nonsuppurative AP (CNAP). Genotyping for IL1B +3954 (rs1143634), IL8 / CXCL8 -251 (rs4073), IL12B +1188 (rs3212227) and TNFA -308 (rs1800629) was performed by the PCR-restriction fragment length polymorphisms method. The statistical analysis was performed using STATA 10.0 and PLINK V1.07 software. RESULTS: Significant differences in the distribution of IL8 / CXCL8 -251 A allele (P adjusted = 0.041; OR adjusted = 0.41, CI adjusted = 0.31-0.97) and IL8 / CXCL -251 TT genotype (P adjusted = 0.04; OR adjusted = 2.24, CI adjusted = 1.04-4.84) were observed comparing patients diagnosed with ASAP and CNAP. No association was observed in genotype and allele distribution for other genetic polymorphisms analysed. CONCLUSION: This study provides molecular epidemiological evidence that suggests in the present cohort that IL8 / CXCL8 -251 T allele, which is associated with higher production of IL8/CXCL8, is also associated with a higher risk of developing acute suppurative form of AP, whereas IL8 / CXCL8 -251 A allele, which is associated with lower production of IL8/CXCL8, is associated with chronic nonsuppurative form of AP. This suggests a pivotal role for IL-8/CXCL8 in periapical disease because of its ability to induce chemotaxis and modulating the directed migration of neutrophils to the site of inflammation in response to microbial infection of pulp.

[Prenylation: from bacteria to eukaryotes].

For their protection from host cell immune defense, intracellular eukaryotic parasites developed a variety of mechanisms, including secretion systems III and IV which inject bacterial effectors directly into eukaryotic cells. These effectors may be posttranslational modified by host cell machinery and may function inside the host cell. Recently, to the list of possible posttranslational modifications of bacterial proteins the prenylation was added. In this work we describe current state of the knowledge about the prenylation of eukaryotic and prokaryotic proteins and its inhibitors. The bioinformatics analyses suggest possibility of prenylation for a number of Francisella genus proteins.

Genome sequence of bacteriophage Aoka, a cluster FO phage isolated using Arthrobacter globiformis.

We report the discovery and genome sequence of bacteriophage Aoka, an actinobacteriophage isolated from a soil sample in Pueblo, Colorado using Arthrobacter globiformis, B2880-SEA. Its genome length is 36,744 base pairs with 54 protein-coding genes. Based on gene content similarity to other actinobacteriophages, Aoka is assigned to cluster FO.

COVID-19 Vaccination Personas in Syria: Evidence from a Cross-Sectional Survey.

Achieving a high level of COVID-19 vaccination coverage in a conflict-affected setting is challenging. The objective of this paper is to shed further light on the main determinants of vaccination coverage using a large, cross-sectional sample (October-November 2022) of over 17,000 adults in Syria. We find evidence that certain demographic and socioeconomic characteristics describe a core set of vaccination personas. Men, older respondents, and those who are more educated and trust information received from healthcare authorities are more likely to be vaccinated. Healthcare workers in this sample are highly vaccinated. Furthermore, respondents with more positive views towards COVID-19 vaccines are also more likely to be willing to be vaccinated. By contrast, respondents who believe that vaccines are associated with significant side effects are also more likely to refuse vaccination. In addition, younger respondents and women, as well as those with a lower level of education, are more likely to refuse to be vaccinated. Respondents with a neutral attitude towards vaccines are also more likely to be undecided, whereas respondents who are refusing to get vaccinated are more likely to trust the information received from private doctors, private clinics, as well as social media and, more broadly, the internet.

Species-specific FMRP regulation of RACK1 is critical for prenatal cortical development.

Fragile X messenger ribonucleoprotein 1 protein (FMRP) deficiency leads to fragile X syndrome (FXS), an autism spectrum disorder. The role of FMRP in prenatal human brain development remains unclear. Here, we show that FMRP is important for human and macaque prenatal brain development. Both FMRP-deficient neurons in human fetal cortical slices and FXS patient stem cell-derived neurons exhibit mitochondrial dysfunctions and hyperexcitability. Using multiomics analyses, we have identified both FMRP-bound mRNAs and FMRP-interacting proteins in human neurons and unveiled a previously unknown role of FMRP in regulating essential genes during human prenatal development. We demonstrate that FMRP interaction with CNOT1 maintains the levels of receptor for activated C kinase 1 (RACK1), a species-specific FMRP target. Genetic reduction of RACK1 leads to both mitochondrial dysfunctions and hyperexcitability, resembling FXS neurons. Finally, enhancing mitochondrial functions rescues deficits of FMRP-deficient cortical neurons during prenatal development, demonstrating targeting mitochondrial dysfunction as a potential treatment.

Identification of the bacteriochlorophylls, carotenoids, quinones, lipids, and hopanoids of "Candidatus Chloracidobacterium thermophilum".

"Candidatus Chloracidobacterium thermophilum" is a recently discovered chlorophototroph from the bacterial phylum Acidobacteria, which synthesizes bacteriochlorophyll (BChl) c and chlorosomes like members of the green sulfur bacteria (GSB) and the green filamentous anoxygenic phototrophs (FAPs). The pigments (BChl c homologs and carotenoids), quinones, lipids, and hopanoids of cells and chlorosomes of this new chlorophototroph were characterized in this study. "Ca. Chloracidobacterium thermophilum" methylates its antenna BChls at the C-8(2) and C-12(1) positions like GSB, but these BChls were esterified with a variety of isoprenoid and straight-chain alkyl alcohols as in FAPs. Unlike the chlorosomes of other green bacteria, "Ca. Chloracidobacterium thermophilum" chlorosomes contained two major xanthophyll carotenoids, echinenone and canthaxanthin. These carotenoids may confer enhanced protection against reactive oxygen species and could represent a specific adaptation to the highly oxic natural environment in which "Ca. Chloracidobacterium thermophilum" occurs. Dihydrogenated menaquinone-8 [menaquinone-8(H(2))], which probably acts as a quencher of energy transfer under oxic conditions, was an abundant component of both cells and chlorosomes of "Ca. Chloracidobacterium thermophilum." The betaine lipid diacylglycerylhydroxymethyl-N,N,N-trimethyl-ß-alanine, esterified with 13-methyl-tetradecanoic (isopentadecanoic) acid, was a prominent polar lipid in the membranes of both "Ca. Chloracidobacterium thermophilum" cells and chlorosomes. This lipid may represent a specific adaptive response to chronic phosphorus limitation in the mats. Finally, three hopanoids, diploptene, bacteriohopanetetrol, and bacteriohopanetetrol cyclitol ether, which may help to stabilize membranes during diel shifts in pH and other physicochemical conditions in the mats, were detected in the membranes of "Ca. Chloracidobacterium thermophilum."

Complete genome of Candidatus Chloracidobacterium thermophilum, a chlorophyll-based photoheterotroph belonging to the phylum Acidobacteria.

Candidatus Chloracidobacterium thermophilum, which naturally inhabits microbial mats of alkaline siliceous hot springs in Yellowstone National Park, is the only known chlorophototroph in the phylum Acidobacteria. The Ca. C. thermophilum genome was composed of two chromosomes (2,683,362 bp and 1,012,010 bp), and both encoded essential genes. The genome included genes to produce chlorosomes, the Fenna-Matthews-Olson protein, bacteriochlorophylls a and c as principal pigments, and type-1, homodimeric reaction centres. Ca. C. thermophilum is an aerobic photoheterotroph that lacks the ability to synthesize several essential nutrients. Key genes of all known carbon fixation pathways were absent, as were genes for assimilatory nitrate and sulfate reduction and vitamin B(12) synthesis. Genes for the synthesis of branched-chain amino acids (valine, isoleucine and leucine) were also absent, but genes for catabolism of these compounds were present. This observation suggested that Ca. C. thermophilum may synthesize branched-chain amino acids from an intermediate(s) of the catabolic pathway by reversing these reactions. The genome encoded an aerobic respiratory electron transport chain that included NADH dehydrogenase, alternative complex III and cytochrome oxidase. The chromosomes of the laboratory isolate were compared with assembled, metagenomic scaffolds from the major Ca. C. thermophilum population in hot-spring mats. The larger chromosomes of the two populations were highly syntenous but significantly divergent (~13%) in sequence. In contrast, the smaller chromosomes have undergone numerous rearrangements, contained many transposases, and might be less constrained by purifying selection than the large chromosomes. Some transposases were homologous to those of mat community members from other phyla.

Ultrastructural analysis and identification of envelope proteins of "Candidatus Chloracidobacterium thermophilum" chlorosomes.

Chlorosomes are sac-like, light-harvesting organelles that characteristically contain very large numbers of bacteriochlorophyll (BChl) c, d, or e molecules. These antenna structures occur in chlorophototrophs belonging to some members of the Chlorobi and Chloroflexi phyla and are also found in a recently discovered member of the phylum Acidobacteria, "Candidatus Chloracidobacterium thermophilum." "Ca. Chloracidobacterium thermophilum" is the first aerobic organism discovered to possess chlorosomes as light-harvesting antennae for phototrophic growth. Chlorosomes were isolated from "Ca. Chloracidobacterium thermophilum" and subjected to electron microscopic, spectroscopic, and biochemical analyses. The chlorosomes of "Ca. Chloracidobacterium thermophilum" had an average size of ∼100 by 30 nm. Cryo-electron microscopy showed that the BChl c molecules formed folded or twisted, sheet-like structures with a lamellar spacing of ∼2.3 nm. Unlike the BChls in the chlorosomes of the green sulfur bacterium Chlorobaculum tepidum, concentric cylindrical nanotubes were not observed. Chlorosomes of "Ca. Chloracidobacterium thermophilum" contained a homolog of CsmA, the BChl a-binding, baseplate protein; CsmV, a protein distantly related to CsmI, CsmJ, and CsmX of C. tepidum, which probably binds a single [2Fe-2S] cluster; and five unique polypeptides (CsmR, CsmS, CsmT, CsmU, and a type II NADH dehydrogenase homolog). Although "Ca. Chloracidobacterium thermophilum" is an aerobe, energy transfer among the BChls in these chlorosomes was very strongly quenched in the presence of oxygen (as measured by quenching of fluorescence emission). The combined analyses showed that the chlorosomes of "Ca. Chloracidobacterium thermophilum" possess a number of unique features but also share some properties with the chlorosomes found in anaerobic members of other phyla.

Genomic and Phenotypic Characterization of Chloracidobacterium Isolates Provides Evidence for Multiple Species.

Chloracidobacterium is the first and until now the sole genus in the phylum Acidobacteriota (formerly Acidobacteria) whose members perform chlorophyll-dependent phototrophy (i.e., chlorophototrophy). An axenic isolate of Chloracidobacterium thermophilum (strain B T ) was previously obtained by using the inferred genome sequence from an enrichment culture and diel metatranscriptomic profiling analyses in situ to direct adjustments to the growth medium and incubation conditions, and thereby a defined growth medium for Chloracidobacterium thermophilum was developed. These advances allowed eight additional strains of Chloracidobacterium spp. to be isolated from microbial mat samples collected from Mushroom Spring, Yellowstone National Park, United States, at temperatures of 41, 52, and 60°C; an axenic strain was also isolated from Rupite hot spring in Bulgaria. All isolates are obligately photoheterotrophic, microaerophilic, non-motile, thermophilic, rod-shaped bacteria. Chloracidobacterium spp. synthesize multiple types of (bacterio-)chlorophylls and have type-1 reaction centers like those of green sulfur bacteria. Light harvesting is accomplished by the bacteriochlorophyll a-binding, Fenna-Matthews-Olson protein and chlorosomes containing bacteriochlorophyll c. Their genomes are approximately 3.7 Mbp in size and comprise two circular chromosomes with sizes of approximately 2.7 Mbp and 1.0 Mbp. Comparative genomic studies and phenotypic properties indicate that the nine isolates represent three species within the genus Chloracidobacterium. In addition to C. thermophilum, the microbial mats at Mushroom Spring contain a second species, tentatively named Chloracidobacterium aggregatum, which grows as aggregates in liquid cultures. The Bulgarian isolate, tentatively named Chloracidobacterium validum, will be proposed as the type species of the genus, Chloracidobacterium. Additionally, Chloracidobacterium will be proposed as the type genus of a new family, Chloracidobacteriaceae, within the order Blastocatellales, the class Blastocatellia, and the phylum Acidobacteriota.