Bacteriohopanetetrol-<i>x</i>: constraining its application as a lipid biomarker for marine anammox using the water column oxygen gradient of the Benguela upwelling system

Interpreting lipid biomarkers in the sediment archive requires a good understanding of their application and limitations in modern systems. Recently it was discovered that marine bacteria performing anaerobic ammonium oxidation (anammox), belonging to the genus Ca. Scalindua, uniquely synthesize a stereoisomer of bacteriohopanetetrol (“BHT-x”). The ratio of BHT-x over total bacteriohopanetetrol (BHT, ubiquitously synthesized by diverse bacteria) has been suggested as a proxy for water column anoxia. As BHT has been found in sediments over 50 Myr old, BHT-x has the potential to complement and extend the sedimentary biomarker record of marine anammox, conventionally constructed using ladderane lipids. Yet, little is known about the distribution of BHT-x in relation to the distribution of ladderanes and to the genetic evidence of Ca. Scalindua in modern marine systems. Here, we investigate the distribution of BHT-x and the application of the BHT-x ratio in relation to distributions of ladderane intact polar lipids (IPLs), ladderane fatty acids (FAs) and Ca. Scalindua 16S rRNA genes in suspended particulate matter (SPM) from the water column of the Benguela upwelling system (BUS), sampled across a large oxygen gradient. In BUS SPM, high BHT-x abundances were restricted to the oxygen-deficient zone on the continental shelf (at [O2] < 45 µmol L−1, in all but one case). High BHT-x abundances co-occurred with high abundances of the Ca. Scalindua 16S rRNA gene (relative to the total number of bacterial 16S rRNA genes) and ladderane IPLs. At shelf stations with [O2] > 50 µmol L−1, the BHT-x ratio was < 0.04 (in all but one case). In apparent contradiction, ladderane FAs and low abundances of BHT and BHT-x (resulting in BHT-x ratios > 0.04) were also detected in oxygenated offshore waters ([O2] up to 180 µmol L−1), whereas ladderane IPLs were undetected. The index of ladderane lipids with five cyclobutane rings (NL5) correlates with in situ temperature. NL5-derived temperatures suggested that ladderane FAs in the offshore waters were not synthesized in situ but were transported down-slope from warmer shelf waters. Thus, in sedimentary archives of systems with known lateral organic matter transport, such as the BUS, relative BHT and BHT-x abundances should be carefully considered. In such systems, a higher BHT-x ratio may act as a safer threshold for deoxygenation and/or Ca. Scalindua presence: our results and previous studies indicate that a BHT-x ratio of ≥ 0.2 is a robust threshold for oxygen-depleted waters ([O2] < 50 µmol kg−1). In our data, ratios of ≥ 0.2 coincided with Ca. Scalindua 16S rRNA genes in all samples (n=62), except one. Lastly, when investigating in situ anammox, we highlight the importance of using ladderane IPLs over BHT-x and/or ladderane FAs; these latter compounds are more recalcitrant and may derive from transported fossil anammox bacteria remnants.

Millimeter-scale vertical partitioning of nitrogen cycling in hypersaline mats reveals prominence of genes encoding multi-heme and prismane proteins

Microbial mats are modern analogues of the first ecosystems on the Earth. As extant representatives of microbial communities where free oxygen may have first been available on a changing planet, they offer an ecosystem within which to study the evolution of biogeochemical cycles requiring and inhibited by oxygen. Here, we report the distribution of genes involved in nitrogen metabolism across a vertical oxygen gradient at 1 mm resolution in a microbial mat using quantitative PCR (qPCR), retro-transcribed qPCR (RT-qPCR) and metagenome sequencing. Vertical patterns in the presence and expression of nitrogen cycling genes, corresponding to oxygen requiring and non-oxygen requiring nitrogen metabolism, could be seen across gradients of dissolved oxygen and ammonium. Metagenome analysis revealed that genes annotated as hydroxylamine dehydrogenase (proper enzyme designation EC 1.7.2.6, hao) and hydroxylamine reductase (hcp) were the most abundant nitrogen metabolism genes in the mat. The recovered hao genes encode hydroxylamine dehydrogenase EC 1.7.2.6 (HAO) proteins lacking the tyrosine residue present in aerobic ammonia oxidizing bacteria (AOB). Phylogenetic analysis confirmed that those proteins were more closely related to ɛHao protein present in Campylobacterota lineages (previously known as Epsilonproteobacteria) rather than oxidative HAO of AOB. BLAST analysis of some transcribed proteins indicated that they likely functioned as a nitrate reductase. The presence of hao sequences related with ɛHao protein, as well as numerous hcp genes encoding a prismane protein, suggest the presence of a nitrogen cycling pathway previously described in Nautilia profundicola as ancestral to the most commonly studied present day nitrogen cycling pathways.

Use of Red Blood Cell Phenotypes for Second Line Therapy Selection in Sickle Cell Disease

INTRODUCTION In sickle cell disease (SCD), abnormal red blood cells (RBCs) sickle upon deoxygenation due to polymerization of hemoglobin S (HbS). Sickle RBCs exhibit poor deformability and increased viscosity, density, and microvascular adhesion. These rheological properties can be measured using existing devices. An oxygen-gradient ektacytometer measures the deformability of sickle RBCs under normoxic (maximum elongation index, or EImax) and hypoxic conditions (minimum elongation index, or EImin), and the pO 2 level at which sickling beings (point of sickling, or PoS). Dense RBCs are measured using a commercially available hematology analyzer. The hematocrit-to-viscosity ratio (HVR), an oxygen delivery index, is calculated based on the viscosity as measured by a cone and plate viscometer. RBC adhesion in the microvasculature can be modeled using a laminin-lined microfluidics device. These rheological biomarkers correlate with clinical complications such as pain events and acute chest syndrome, and are modified by known, clinically effective therapies such as hydroxyurea (HU) and transfusion (TF). HU is the standard of care for most individuals with SCD and positively modifies EImin, EImax, PoS, HVR, adhesion, and %DRBC. Recently, new agents to treat SCD have emerged including voxelotor, crizanlizumab, and pyruvate kinase activators like etavopivat, which have more targeted effects. It is essential to pair the appropriate novel agent to the patient, addressing their most prominent RBC abnormality remaining after HU therapy. We hypothesize that there is significant variability of rheology biomarkers between individuals with SCD on standard of care therapy, and that the most severe aspects of their RBC pathophysiology can be identified and targeted by novel second line therapies for clinical optimization. METHODS We collected peripheral blood in EDTA under an IRB approved protocol from 312 pediatric patients with SCD ranging in age from 2 to 21 years, 70% on HU. Subjects on chronic TF therapy were excluded. We measured whole blood viscosity at 45s -1 shear and calculated the HVR. %DRBC and complete blood counts were obtained using an ADVIA hematology analyzer (Siemens) and EImin, EImax, and PoS obtained using oxygen gradient ektacytometry (Lorrca, RR Mechatronics). RBCs from 17 HbSS subjects were analyzed for adhesion index to a laminin-lined microfluidics device. Values were assembled for each biomarker into histograms to demonstrate distribution, and quartile ranked. Venn diagrams were constructed comparing overlap between top 25% most severe rheology biomarkers to demonstrate effectiveness of a targeted, precision medicine approach to adding second line therapies to individuals with SCD. RESULTS Distribution of biomarkers in a typical pediatric SCD population in a US academic center are shown in Figure 1. High PoS, adhesion index, %DRBC, and low EImin and EImax, low HVR, are associated with disease severity and clinical complications in SCD; biomarker values were stratified from high to low severity association. The most severe quartile subjects from each biomarker were compared, and percentage of overlap noted (Figure 2). CONCLUSION Our rheologic assessment of a large pediatric cohort heavily treated with HU indicates a broad distribution of RBC phenotypes. Even on HU, patients exhibited loss of deformability, sickling, adhesion, or RBC density abnormalities, with little overlap of unrelated biomarkers associated with disease severity in an individual, i.e. the individuals with very high PoS did not have low HVR (16% of highest severity quartile subjects in common), compared to related biomarkers PoS and EImin or EImax (48% of highest severity quartile subjects in common). Only four subjects, all on HU, were in the quartile associated with highest severity for all biomarkers. Given the lack of overlapping pathology between different red cell abnormalities, selection of the appropriate agent should be straightforward. With three new FDA approved therapies for SCD and novel therapies in clinical trials, it is possible to choose the appropriate second agent to be added to HU based on individual patient RBC phenotype according to the principles of precision medicine. Future goals include CLIA certification for novel devices like the oxygen gradient ektacytometer and adhesive microfluidics at major academic SCD centers and use of these biomarkers in routine patient care. Figure 1 Figure 1. Disclosures Rab: Agios Pharmaceuticals: Research Funding; Axcella Health: Research Funding. Lam: Sanguina, Inc.: Current holder of individual stocks in a privately-held company. Wijk: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Axcella health: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Sheehan: Beam Therapeutics: Research Funding; Forma Therapeutics: Research Funding; Novartis: Research Funding.

Bacteriohopanetetrol-x: constraining its application as a lipid biomarker for marine anammox using the water column oxygen gradient of the Benguela upwelling system

Interpreting lipid biomarkers in the sediment archive requires a good understanding of their application and limitations in modern systems. Recently it was discovered that marine bacteria performing anaerobic ammonium oxidation (anammox), belonging to the genus Ca. Scalindua, uniquely synthesize a stereoisomer of bacteriohopanetetrol (‘BHT-x’). The ratio of BHT-x over total bacteriohopanetetrol (BHT; ubiquitously synthesized by diverse bacteria) has been suggested as a proxy for water column anoxia. As BHT has been found in sediments over 50 Myr old, BHT-x has the potential to complement and extend the sedimentary biomarker record of marine anammox, conventionally constructed using ladderane lipids. Yet, little is known about the distribution of BHT-x in relation to the distribution of ladderanes and to the genetic evidence of Ca. Scalindua in modern marine systems. Here, we investigate the distribution of BHT-x and the application of the BHT-x ratio in relation to distributions of intact polar (IPL) ladderane lipids, ladderane fatty acids (FAs) and Ca. Scalindua 16S rRNA genes in suspended particulate matter (SPM) from the water column, sampled across a large oxygen gradient in the Benguela upwelling system (BUS). In BUS SPM, high BHT-x abundances were constrained to the oxygen deficient zone on the continental shelf (at [O2] < 45 µmol L−1, in all but one case). High BHT-x abundances co-occurred with high abundances of the Ca. Scalindua 16S rRNA gene (relative to the total number of bacterial 16S rRNA genes) and ladderane IPLs. At shelf stations with [O2] > 50 µmol L−1, the BHT-x ratio was < 0.04 (in all but one case). In apparent contradiction, ladderane FAs and low abundances of BHT and BHT-x (resulting in BHT-x ratio’s > 0.04) were also detected in oxygenated offshore waters ([O2] up to 180 µmol L−1), whereas ladderane IPLs were undetected. NL5-derived temperatures suggested that ladderane FAs in the offshore waters were not synthesized in situ but derived from warmer shelf waters. Thus, in sedimentary archives of systems with known lateral organic matter transport, such as the BUS, relative BHT and BHT-x abundances should be carefully considered. In such systems, a higher BHT-x ratio may act as a safer threshold for deoxygenation and/or Ca. Scalindua presence: in the BUS, at [O2] > 50 µmol L−1, the BHT-x ratio was < 0.18 at both off -and onshore sites (in all but one case) and a ratio > 0.18 corresponded in all cases (except one) with the presence of Ca. Scalindua 16S rRNA genes. Lastly, when investigating in situ anammox, we highlight the importance of using ladderane IPLs over BHT-x and/or ladderane FAs; these latter compounds are more recalcitrant and may derive from transported fossil anammox bacteria remnants.

Hepatopulmonary Syndrome. Review

Liver cirrhosis is often accompanied by complications from the pulmonary system. These include hydrothorax, portopulmonary hypertension and hepatopulmonary syndrome. Hepatic hydrothorax affects about 6-10% of patients with end-stage disease, which results in the passage of ascetic fluid into the pleural space through diaphragm defects. The common cause of the hepatopulmonary syndrome and portopulmonary hypertension is portal hypertension and portosystemic shunting, indicating that vasoactive and angiogenetic factors originating from the liver normally control the pulmonary circulation. Portopulmonary hypertension is like pulmonary arterial hypertension, which develops against the background of portal hypertension as a result of chronic liver disease or without other causes of increased pressure in the pulmonary vessels. The prevalence of portopulmonary hypertension ranges from 2% to 8.5% among patients with portal hypertension and is associated with a poor prognosis. Hepatopulmonary syndrome is characterized by intrapulmonary dilatation of microvessels, which causes intrapulmonary shunting and leads to impaired gas exchange in liver diseases, and is associated with a decrease in the quality and duration of life in patients with cirrhosis. Nitric oxide overproduction and angiogenesis seem to be the hallmarks of a complicated pathogenetic mechanism, leading to intrapulmonary shunting and ventilation-perfusion mismatch. A classification of hepatopulmonary syndrome according to the severity of hypoxemia has been suggested. Hepatopulmonary syndrome includes a triad: hepatic dysfunction and / or portal hypertension, dilatation of intrapulmonary vessels, and increased alveolar-arterial oxygen gradient. The prevalence of hepatopulmonary syndrome varies depending on the study groups from 5% to 30%. The most common symptom of the complication is shortness of breath, but in most cases, hepatopulmonary syndrome is asymptomatic. A decrease in oxygen saturation less than 96% corresponds to a decrease in PaO2<70 mm Hg and testifies to the possible development of hepatopulmonary syndrome. In the case of a positive screening, the patient should undergo arterial blood gas analysis, which helps to determine PaO2 and alveolar to arterial oxygen gradient. Conclusion. Contrast-enhanced echocardiography with agitated saline is the gold standard in the diagnosis of intrapulmonary dilatation. The only effective treatment for hepatopulmonary syndrome is liver transplantation. Complete recovery of hepatopulmonary syndrome after liver transplantation is observed within a year in most patients with cirrhosis and hepatopulmonary syndrome