The Management of Persistent Distal Occlusions after Mechanical Thrombectomy and Thrombolysis: An Inter- and Intrarater Agreement Study.

BACKGROUND AND PURPOSE: The best management of patients with persistent distal occlusion after mechanical thrombectomy with or without IV thrombolysis remains unknown. We sought to evaluate the variability and agreement in decision-making for persistent distal occlusions. MATERIALS AND METHODS: A portfolio of 60 cases was sent to clinicians with varying backgrounds and experience. Responders were asked whether they considered conservative management or rescue therapy (stent retriever, aspiration, or intra-arterial thrombolytics) a treatment option as well as their willingness to enroll patients in a randomized trial. Agreement was assessed using κ statistics. RESULTS: The electronic survey was answered by 31 physicians (8 vascular neurologists and 23 interventional neuroradiologists). Decisions for rescue therapies were more frequent (n = 1116/1860, 60%) than for conservative management (n = 744/1860, 40%; P < .001). Interrater agreement regarding the final management decision was "slight" (κ = 0.12; 95% CI, 0.09-0.14) and did not improve when subgroups of clinicians were studied according to background, experience, and specialty or when cases were grouped according to the level of occlusion. On delayed re-questioning, 23 of 29 respondents (79.3%) disagreed with themselves on at least 20% of cases. Respondents were willing to offer trial participation in 1295 of 1860 (69.6%) cases. CONCLUSIONS: Individuals did not agree regarding the best management of patients with persistent distal occlusion after mechanical thrombectomy and IV thrombolysis. There is sufficient uncertainty to justify a dedicated randomized trial.

A Pragmatic Randomized Trial Comparing Surgical Clipping and Endovascular Treatment of Unruptured Intracranial Aneurysms.

BACKGROUND AND PURPOSE: Surgical clipping and endovascular treatment are commonly used in patients with unruptured intracranial aneurysms. We compared the safety and efficacy of the 2 treatments in a randomized trial. MATERIALS AND METHODS: Clipping or endovascular treatments were randomly allocated to patients with one or more 3- to 25-mm unruptured intracranial aneurysms judged treatable both ways by participating physicians. The study hypothesized that clipping would decrease the incidence of treatment failure from 13% to 4%, a composite primary outcome defined as failure of aneurysm occlusion, intracranial hemorrhage during follow-up, or residual aneurysms at 1 year, as adjudicated by a core lab. Safety outcomes included new neurologic deficits following treatment, hospitalization of >5 days, and overall morbidity and mortality (mRS > 2) at 1 year. There was no blinding. RESULTS: Two hundred ninety-one patients were enrolled from 2010 to 2020 in 7 centers. The 1-year primary outcome, ascertainable in 290/291 (99%) patients, was reached in 13/142 (9%; 95% CI, 5%-15%) patients allocated to surgery and in 28/148 (19%; 95% CI, 13%-26%) patients allocated to endovascular treatments (relative risk: 2.07; 95% CI, 1.12-3.83; P = .021). Morbidity and mortality (mRS >2) at 1 year occurred in 3/143 and 3/148 (2%; 95% CI, 1%-6%) patients allocated to surgery and endovascular treatments, respectively. Neurologic deficits (32/143, 22%; 95% CI, 16%-30% versus 19/148, 12%; 95% CI, 8%-19%; relative risk: 1.74; 95% CI, 1.04-2.92; P = .04) and hospitalizations beyond 5 days (69/143, 48%; 95% CI, 40%-56% versus 12/148, 8%; 95% CI, 5%-14%; relative risk: 0.18; 95% CI, 0.11-0.31; P < .001) were more frequent after surgery. CONCLUSIONS: Surgical clipping is more effective than endovascular treatment of unruptured intracranial aneurysms in terms of the frequency of the primary outcome of treatment failure. Results were mainly driven by angiographic results at 1 year.

Stent-Assisted Coiling in the Treatment of Unruptured Intracranial Aneurysms: A Randomized Clinical Trial.

BACKGROUND AND PURPOSE: Stent-assisted coiling may improve angiographic results of endovascular treatment of unruptured intracranial aneurysms compared with coiling alone, but this has never been shown in a randomized trial. MATERIALS AND METHODS: The Stenting in the Treatment of Aneurysm Trial was an investigator-led, parallel, randomized (1:1) trial conducted in 4 university hospitals. Patients with intracranial aneurysms at risk of recurrence, defined as large aneurysms (≥10 mm), postcoiling recurrent aneurysms, or small aneurysms with a wide neck (≥4 mm), were randomly allocated to stent-assisted coiling or coiling alone. The composite primary efficacy outcome was "treatment failure," defined as initial failure to treat the aneurysm; aneurysm rupture or retreatment during follow-up; death or dependency (mRS > 2); or an angiographic residual aneurysm adjudicated by an independent core laboratory at 12 months. The primary hypothesis (revised for slow accrual) was that stent-assisted coiling would decrease treatment failures from 33% to 15%, requiring 200 patients. Primary analyses were intent to treat. RESULTS: Of 205 patients recruited between 2011 and 2021, ninety-four were allocated to stent-assisted coiling and 111 to coiling alone. The primary outcome, ascertainable in 203 patients, was reached in 28/93 patients allocated to stent-assisted coiling (30.1%; 95% CI, 21.2%-40.6%) compared with 30/110 (27.3%; 95% CI, 19.4%-36.7%) allocated to coiling alone (relative risk = 1.10; 95% CI, 0.7-1.7; P = .66). Poor clinical outcomes (mRS >2) occurred in 8/94 patients allocated to stent-assisted coiling (8.5%; 95% CI, 4.0%-16.6%) compared with 6/111 (5.4%; 95% CI, 2.2%-11.9%) allocated to coiling alone (relative risk = 1.6; 95% CI, 0.6%-4.4%; P = .38). CONCLUSIONS: The STAT trial did not show stent-assisted coiling to be superior to coiling alone for wide-neck, large, or recurrent unruptured aneurysms.

Surgical or Endovascular Treatment of MCA Aneurysms: An Agreement Study.

BACKGROUND AND PURPOSE: MCA aneurysms are still commonly clipped surgically despite the recent development of a number of endovascular tools and techniques. We measured clinical uncertainty by studying the reliability of decisions made for patients with middle cerebral artery (MCA) aneurysms. MATERIALS AND METHODS: A portfolio of 60 MCA aneurysms was presented to surgical and endovascular specialists who were asked whether they considered surgery or endovascular treatment to be an option, whether they would consider recruitment of the patient in a randomized trial, and whether they would provide their final management recommendation. Agreement was studied using κ statistics. Intrarater reliability was assessed with the same, permuted portfolio of cases of MCA aneurysm sent to the same specialists 1 month later. RESULTS: Surgical management was the preferred option for neurosurgeons (n = 844/1320; [64%] responses/22 raters), while endovascular treatment was more commonly chosen by interventional neuroradiologists (1149/1500 [76.6%] responses/25 raters). Interrater agreement was only "slight" for all cases and all judges (κ = 0.094; 95% CI, 0.068-0.130). Agreement was no better within specialties or with more experience. On delayed requestioning, 11 of 35 raters (31%) disagreed with themselves on at least 20% of cases. Surgical management and endovascular treatment were always judged to be a treatment option, for all patients. Trial participation was offered to patients 65% of the time. CONCLUSIONS: Individual clinicians did not agree regarding the best management of patients with MCA aneurysms. A randomized trial comparing endovascular with surgical management of patients with MCA aneurysms is in order.

Flow Diversion in the Treatment of Intracranial Aneurysms: A Pragmatic Randomized Care Trial.

BACKGROUND AND PURPOSE: Flow diversion is a recent endovascular treatment for intracranial aneurysms. We compared the safety and efficacy of flow diversion with the alternative standard management options. MATERIALS AND METHODS: A parallel group, prerandomized, controlled, open-label pragmatic trial was conducted in 3 Canadian centers. The trial included all patients considered for flow diversion. A Web-based platform 1:1 randomly allocated patients to flow diversion or 1 of 4 alternative standard management options (coiling with/without stent placement, parent vessel occlusion, surgical clipping, or observation) as prespecified by clinical judgment. Patients ineligible for alternative standard management options were treated with flow diversion in a registry. The primary safety outcome was death or dependency (mRS > 2) at 3 months. The composite primary efficacy outcome included the core lab-determined angiographic presence of a residual aneurysm, aneurysm rupture, progressive mass effect during follow-up, or death or dependency (mRS > 2) at 3-12 months. RESULTS: Between May 2011 and November 2020, three hundred twenty-three patients were recruited: Two hundred seventy-eight patients (86%) had treatment randomly allocated (139 to flow diversion and 139 to alternative standard management options), and 45 (14%) received flow diversion in the registry. Patients in the randomized trial frequently had unruptured (83%), large (52% ≥10 mm) carotid (64%) aneurysms. Death or dependency at 3 months occurred in 16/138 patients who underwent flow diversion and 12/137 patients receiving alternative standard management options (relative risk, 1.33; 95% CI, 0.65-2.69; P = .439). A poor primary efficacy outcome was found in 30.9% (43/139) with flow diversion and 45.6% (62/136) of patients receiving alternative standard management options, with an absolute risk difference of 14.7% (95% CI, 3.3%-26.0%; relative risk, 0.68; 95% CI, 0.50-0.92; P = .014). CONCLUSIONS: For patients with mostly unruptured, large, anterior circulation (carotid) aneurysms, flow diversion was more effective than the alternative standard management option in terms of angiographic outcome.

Transcranial Doppler Velocities and Angiographic Vasospasm after SAH: A Diagnostic Accuracy Study.

BACKGROUND AND PURPOSE: After aneurysmal SAH, transcranial Doppler is commonly used to monitor cerebral vasospasm. The diagnostic accuracy of transcranial Doppler flow velocity values in detecting angiographic vasospasm in patients requiring urgent endovascular intervention has not been established. MATERIALS AND METHODS: We performed a retrospective analysis of a consecutive series of patients with aneurysmal SAH who underwent transcranial Doppler (index test) within 24 hours of conventional angiography (reference test). The judgment of 33%, 50%, and 66% degree of vessel narrowing on angiography was independently established by multiple neuroendovascular clinicians. Vessel-specific per-segment and per-patient transcranial Doppler velocities were studied using receiver operating characteristic curves, the Youden index, and minimal acceptable sensitivity models. Optimal mean flow-velocity thresholds were explored to calculate sensitivity and specificity using a per-patient judgment of vasospasm of at least 50% angiographic narrowing in any large arterial segment except A1. RESULTS: In 221 patients, vasospasm was found in 15%, 8%, and 4% of arteries when the degree of reference angiographic luminal narrowing was 33%, 50%, and 66%, respectively. Mean flow velocities were significantly higher in vasospastic segments (P = . 001), but per-segment exploratory analyses yielded unsound mean flow velocity thresholds. The Youden and minimal acceptable sensitivity models proposed mean flow velocity thresholds of approximately 160 cm/s for the anterior circulation and 80 cm/s for the posterior circulation in the per-patient diagnosis of angiographic vasospasm (≥50%), yielding a sensitivity of 80%-90% (95% CI, 0.77-0.96), but with a corresponding specificity of 50% (95% CI, 0.40-0.56). CONCLUSIONS: In this study, a threshold transcranial Doppler mean flow-velocity value that would accurately diagnose ≥50% angiographic vasospasm remained elusive.

Reliability of the Diagnosis of Cerebral Vasospasm Using Catheter Cerebral Angiography: A Systematic Review and Inter- and Intraobserver Study.

BACKGROUND AND PURPOSE: Conventional angiography is the benchmark examination to diagnose cerebral vasospasm, but there is limited evidence regarding its reliability. Our goals were the following: 1) to systematically review the literature on the reliability of the diagnosis of cerebral vasospasm using conventional angiography, and 2) to perform an agreement study among clinicians who perform endovascular treatment. MATERIALS AND METHODS: Articles reporting a classification system on the degree of cerebral vasospasm on conventional angiography were systematically searched, and agreement studies were identified. We assembled a portfolio of 221 cases of patients with subarachnoid hemorrhage and asked 17 raters with different backgrounds (radiology, neurosurgery, or neurology) and experience (junior ≤10 and senior >10 years) to independently evaluate cerebral vasospasm in 7 vessel segments using a 3-point scale and to evaluate, for each case, whether findings would justify endovascular treatment. Nine raters took part in the intraobserver reliability study. RESULTS: The systematic review showed a very heterogeneous literature, with 140 studies using 60 different nomenclatures and 21 different thresholds to define cerebral vasospasm, and 5 interobserver studies reporting a wide range of reliability (κ = 0.14-0.87). In our study, only senior raters reached substantial agreement (κ ≥ 0.6) on vasospasm of the supraclinoid ICA, M1, and basilar segments and only when assessments were dichotomized (presence or absence of ≥50% narrowing). Agreement on whether to proceed with endovascular management of vasospasm was only fair (κ ≤ 0.4). CONCLUSIONS: Research on cerebral vasospasm would benefit from standardization of definitions and thresholds. Dichotomized decisions by experienced readers are required for the reliable angiographic diagnosis of cerebral vasospasm.

Effects of nutrient enrichment on surface microbial community gene expression in the oligotrophic North Pacific Subtropical Gyre.

Marine microbial communities are critical for biogeochemical cycles and the productivity of ocean ecosystems. Primary productivity in the surface ocean is constrained by nutrients which are supplied, in part, by mixing with deeper water. Little is known about the time scales, frequency, or impact of mixing on microbial communities. We combined in situ sampling using the Environmental Sample Processor and a small-scale mixing experiment with lower euphotic zone water to determine how individual populations respond to mixing. Transcriptional responses were measured using the MicroTOOLs (Microbiological Targets for Ocean Observing Laboratories) microarray, which targets all three domains of life and viruses. The experiment showed that mixing substantially affects photosynthetic taxa as expected, but surprisingly also showed that populations respond differently to unfiltered deep water which contains particles (organisms and detritus) compared to filtered deep water that only contains nutrients and viruses, pointing to the impact of biological interactions associated with these events. Comparison between experimental and in situ population transcription patterns indicated that manipulated populations can serve as analogs for natural populations, and that natural populations may be frequently or continuously responding to nutrients from deeper waters. Finally, this study also shows that the microarray approach, which is complementary to metatranscriptomic sequencing, is useful for determining the physiological status of in situ microbial communities.

Female Mice Lacking Estrogen Receptor-α in Hypothalamic Proopiomelanocortin (POMC) Neurons Display Enhanced Estrogenic Response on Cortical Bone Mass.

Estrogens are important regulators of bone mass and their effects are mainly mediated via estrogen receptor (ER)α. Central ERα exerts an inhibitory role on bone mass. ERα is highly expressed in the arcuate (ARC) and the ventromedial (VMN) nuclei in the hypothalamus. To test whether ERα in proopiomelanocortin (POMC) neurons, located in ARC, is involved in the regulation of bone mass, we used mice lacking ERα expression specifically in POMC neurons (POMC-ERα(-/-)). Female POMC-ERα(-/-) and control mice were ovariectomized (OVX) and treated with vehicle or estradiol (0.5 µg/d) for 6 weeks. As expected, estradiol treatment increased the cortical bone thickness in femur, the cortical bone mechanical strength in tibia and the trabecular bone volume fraction in both femur and vertebrae in OVX control mice. Importantly, the estrogenic responses were substantially increased in OVX POMC-ERα(-/-) mice compared with the estrogenic responses in OVX control mice for cortical bone thickness (+126 ± 34%, P < .01) and mechanical strength (+193 ± 38%, P < .01). To test whether ERα in VMN is involved in the regulation of bone mass, ERα was silenced using an adeno-associated viral vector. Silencing of ERα in hypothalamic VMN resulted in unchanged bone mass. In conclusion, mice lacking ERα in POMC neurons display enhanced estrogenic response on cortical bone mass and mechanical strength. We propose that the balance between inhibitory effects of central ERα activity in hypothalamic POMC neurons in ARC and stimulatory peripheral ERα-mediated effects in bone determines cortical bone mass in female mice.

Prenatal androgen blockade accelerates pubertal development in male rhesus monkeys.

Aspects of the prenatal environment, including steroid hormones, modulate the timing of puberty onset in many mammalian species. This study tested whether prenatal androgen manipulations altered pubertal development in male rhesus macaques (Macaca mulatta). Pregnant females received testosterone enanthate (TE), the androgen receptor blocker flutamide, or vehicle during one of two periods of gestation, and their male offspring were observed for morphological, endocrine, and behavioral development from 3 to 4.5 years of age. Males exposed to flutamide early in gestation had a greater response to exogenous GnRH prepubertally, and greater testes volume, elevated testosterone, and elevated LH at age 3.5 than did control subjects. Males exposed to flutamide late in gestation also had greater testes volumes at age 3.5 than did control males. However, these differences between flutamide treated males and control males did not persist postpubertally. By 4.5 years of age, development in control males had reached comparable levels to that of flutamide-treated males. Late gestation treatment with TE had no effect on morphological pubertal development but early TE treatment altered some aspects of endocrine function during puberty. None of the prenatal androgen manipulations affected sexual behavior. These findings suggest that prenatal androgens, in conjunction with social factors, masculinize pubertal timing in rhesus monkey males.

Vertical distribution of nitrogen-fixing phylotypes in a meromictic, hypersaline lake.

We investigated the diversity of nitrogenase genes in the alkaline, moderately hypersaline Mono Lake, California to determine (1) whether nitrogen-fixing (diazotrophic) populations were similar to those in other aquatic environments and (2) if there was a pattern of distribution of phylotypes that reflected redox conditions, as well as (3) to identify populations that could be important in N dynamics in this nitrogen-limited lake. Mono Lake has been meromictic for almost a decade and has steep gradients in oxygen and reduced compounds that provide a wide range of aerobic and anaerobic habitats. We amplified a fragment of the nitrogenase gene (nifH) from planktonic DNA samples collected at three depths representing oxygenated surface waters, the oxycline, and anoxic, ammonium-rich deep waters. Forty-three percent of the 90 sequences grouped in nifH Cluster I. The majority of clones (57%) grouped in Cluster III, which contains many known anaerobic bacteria. Cluster I and Cluster III sequences were retrieved at every depth indicating little vertical zonation in sequence types related to the prominent gradients in oxygen and ammonia. One group in Cluster I was found most often at every depth and accounted for 29% of all the clones. These sequences formed a subcluster that contained other environmental clones, but no cultivated representatives. No significant nitrogen fixation was detected by the 15N2 method after 48 h of incubation of surface, oxycline, or deep waters, suggesting that pelagic diazotrophs were contributing little to nitrogen fluxes in the lake. The failure to measure any significant nitrogen fixation, despite the detection of diverse and novel nitrogenase genes throughout the water column, raises interesting questions about the ecological controls on diazotrophy in Mono Lake and the distribution of functional genes in the environment.

Nitrogen-fixing phylotypes of Chesapeake Bay and Neuse River estuary sediments.

Sediments often exhibit low rates of nitrogen fixation, despite the presence of elevated concentrations of inorganic nitrogen. The organisms that potentially fix nitrogen in sediments have not previously been identified. Amplification of nifH genes with degenerate primers was used to assess the diversity of diazotrophs in two distinct sediment systems, anoxic muds of Chesapeake Bay and shallow surficial sediments of the Neuse River. Phylogenetic analysis revealed that sequences obtained from mid-Chesapeake Bay, which receive high organic loading and are highly reducing, clustered closely with each other and with known anaerobic microorganisms, suggesting a low abundance of aerobic or facultative diazotrophs in these sediments. Sulfate reduction dominates in the surface, but methanogenesis becomes more important with depth. A thin (<1 cm) oxidized layer is present only in the spring. No archaeal nifH sequences were obtained from Chesapeake Bay. Sequences of nifH amplified from surficial sediments of the Neuse River were distant from Chesapeake Bay sequences and included nif phylotypes related to sequences previously reported from marine mats and the Spartina rhizosphere. Differences in environmental site characteristics appear to select for different types of sediment diazotrophs, which is reflected in the phylogenetic composition of amplified nifH sequences.

Diversity and detection of nitrate assimilation genes in marine bacteria.

A PCR approach was used to construct a database of nasA genes (called narB genes in cyanobacteria) and to detect the genetic potential for heterotrophic bacterial nitrate utilization in marine environments. A nasA-specific PCR primer set that could be used to selectively amplify the nasA gene from heterotrophic bacteria was designed. Using seawater DNA extracts obtained from microbial communities in the South Atlantic Bight, the Barents Sea, and the North Pacific Gyre, we PCR amplified and sequenced nasA genes. Our results indicate that several groups of heterotrophic bacterial nasA genes are common and widely distributed in oceanic environments.

Unicellular cyanobacteria fix N2 in the subtropical North Pacific Ocean.

Fixed nitrogen (N) often limits the growth of organisms in terrestrial and aquatic biomes, and N availability has been important in controlling the CO2 balance of modern and ancient oceans. The fixation of atmospheric dinitrogen gas (N2) to ammonia is catalysed by nitrogenase and provides a fixed N for N-limited environments. The filamentous cyanobacterium Trichodesmium has been assumed to be the predominant oceanic N2-fixing microorganism since the discovery of N2 fixation in Trichodesmium in 1961 (ref. 6). Attention has recently focused on oceanic N2 fixation because nitrogen availability is generally limiting in many oceans, and attempts to constrain the global atmosphere-ocean fluxes of CO2 are based on basin-scale N balances. Biogeochemical studies and models have suggested that total N2-fixation rates may be substantially greater than previously believed but cannot be reconciled with observed Trichodesmium abundances. It is curious that there are so few known N2-fixing microorganisms in oligotrophic oceans when it is clearly ecologically advantageous. Here we show that there are unicellular cyanobacteria in the open ocean that are expressing nitrogenase, and are abundant enough to potentially have a significant role in N dynamics.

Microbiological, molecular biological and stable isotopic evidence for nitrogen fixation in the open waters of Lake Michigan.

We have used a combination of microbiological, molecular biological and stable isotope methods to relate specific microbial populations to elemental cycling at an offshore site in Lake Michigan. Several lines of evidence suggest that atmospheric N2 may be a significant source of nitrogen to the lake. Particulate organic nitrogen (PON) at approximately equals 10-15m depth in July and October had a delta15N of 0.5-1.5%o. These values closely reflect the 15N composition of atmospheric N2, suggesting biological nitrogen fixation. Historical data show a developing late-summer N:P minimum at approximately equals 15 m; low abundance of inorganic nitrogen relative to phosphorus favours species able to acquire atmospheric nitrogen. Microscopic examination of October water samples revealed abundant heterocystous cyanobacteria, including Nodularia sp. Potentially nitrogen-fixing Anabaena spp. have been found in Lake Michigan before but, to our knowledge, this is the first report of Nodularia. Finally, we have amplified both cyanobacterial and non-cyanobacterial nifH sequences (encoding the nitrogenase iron protein) from lakewater samples, evidence for the presence of bacteria capable of nitrogen fixation. The surface waters of Lake Michigan are considered to be phosphate limited in the stratified season and, under these conditions, energetically expensive nitrogen fixation is expected to be uncompetitive with assimilation of combined nitrogen. Our results suggest that, from both microbiological and biogeochemical perspectives, this may be an oversimplification.

Variation in reproductive traits is associated with short anogenital distance in female rats.

Anogenital distance was used as a biomarker for natural variation in prenatal androgenization of female rats and was associated with individual differences in a suite of reproductive characteristics. Females with short anogenital distances were likely to have first vaginal estrus earlier than females with longer anogenital distances and to have first vaginal estrus on the same day as vaginal opening. In young adulthood, females with short anogenital distances had shorter ovarian cycles and less intense lordosis reflexes in response to manual palpation than those with longer anogenital distances, but only when living individually, not in groups. Taken together, these findings indicate that prenatal androgenization affects reproductive traits throughout the life span.

Distribution of Nitrogen-Fixing Microorganisms along the Neuse River Estuary, North Carolina.

Nitrogen fixation genes (nifH) were amplified and sequenced from DNA extracted from surface water samples collected from six stations along the length of the Neuse River Estuary, North Carolina, in order to determine the distribution of nitrogen-fixing organisms in the transition from fresh- to saltwater. Nitrogenase genes were detected in all samples by a nested polymerase chain reaction method, and the amplification products from the upriver, midriver, and downriver stations were cloned, sequenced, and used for phylogenetic analysis. The composition of nifH clone libraries from upriver, midriver, and downriver stations (each composed of 14 randomly selected clones) were very diverse (samples from upriver and midriver stations were composed of 14 unique sequences, downriver station composed of 7 unique sequences) and differed among the stations. Some phylotypes were found at more than one station, but were usually found in the upriver and midriver stations or in the midriver and downriver stations, indicating that the phylotypes were probably transported along the river. Cyanobacterial nifH were not found at the most upriver site, but were a large fraction of sequences (50%) recovered from the downriver station, where nitrate concentration was an order of magnitude lower and salinity was higher. In contrast, g proteobacteria nifH sequences were much more common at the midriver and upriver sites (58% and 64%, respectively), compared to the downriver site (14%). Results indicate that substantially different nitrogen-fixing assemblages are present along the river, reflecting differential watershed hydrological inputs, sedimentation, and environmental selection pressures, along the salinity gradient.

Expression of nifH genes in natural microbial assemblages in Lake George, New York, detected by reverse transcriptase PCR.

A modified nested reverse transcriptase PCR (RT-PCR) method was used to detect the expression of nitrogenase genes in meso-oligotrophic Lake George, New York. Net (>20-microm pore size) plankton samples collected from two sites (Dome Island and Hague Marina) were extracted for total RNA and genomic DNA to determine the identity of diazotrophic organisms that were present and those that were actively expressing nitrogenase genes. Phylogenetic analysis of individual sequences cloned from PCR amplifications showed that there were phylogenetically diverse groups of bacteria that possessed a nifH gene, including representatives of unicellular and filamentous cyanobacteria, the alpha- and gamma-subdivisions of the division Proteobacteria (alpha- and gamma-proteobacteria), and a previously undefined group of bacteria. The phylotypes cloned from RT-PCR amplifications, which were actively expressing nifH transcripts, clustered with the unicellular and filamentous cyanobacteria, alpha-proteobacteria, and the novel bacterial cluster. No bacterial sequences were found which clustered with sequences from cluster II (alternative nitrogenases), III (nitrogenases in strict anaerobes), or IV (nifH-like sequences). These results indicate that there were several distinct groups of nitrogen-fixing microorganisms in the net plankton from both sampling sites and that most of the groups had representative phylotypes that were actively expressing nitrogenase genes.

New perspectives on nitrogen-fixing microorganisms in tropical and subtropical oceans.

New molecular and microscopic evidence indicates that the open ocean harbors a diverse range of novel free-living and symbiotic nitrogen-fixing microorganisms. Although the extent to which these microorganisms fix nitrogen is currently unclear, ongoing research indicates that they might make a substantial contribution to the open ocean nitrogen budget.