The economic burden of individuals living with generalized myasthenia gravis and facing social determinants of health challenges.

Objective: Better understanding the impact of social determinants of health (SDOH) barriers from the patient perspective is crucial to improve holistic patient support in generalized myasthenia gravis (gMG), a rare autoimmune disorder with high disease and treatment burden. The objective of this study was to identify economic challenges experienced by individuals living with gMG and SDOH barriers to better address current unmet needs. Methods: Adults (18-75 years) living with gMG and experiencing SDOH barriers in the United States were recruited to a mixed-methods study including qualitative interviews and a web-based quantitative survey. Quotas were implemented to include a balanced spread of baseline demographic categories including insurance type, living environment, and employment status among the study sample. Direct and indirect economic challenges were identified by degree of concern. Results: The survey was completed by 38 individuals living with gMG, the majority of whom were enrolled in public insurance and not employed. The most commonly reported major economic concerns were managing funds for emergency care (66%), loss of income (61%), and non-medical expenses (58%), highlighting the diversity of economic challenges. Individuals who were using public insurance plans, living in non-urban environments, and unemployed experienced pronounced challenges around managing non-medical costs and accessing government assistance. Conclusion: Both direct and indirect costs were emphasized as major concerns among individuals living with gMG and SDOH barriers. Increasing access to relevant, personalized, and holistic resources, including care management, should be prioritized to improve disease management and outcomes for individuals living with gMG.

Effects of haemodynamically atrio-ventricular optimized His bundle pacing on heart failure symptoms and exercise capacity: the His Optimized Pacing Evaluated for Heart Failure (HOPE-HF) randomized, double-blind, cross-over trial.

AIMS: Excessive prolongation of PR interval impairs coupling of atrio-ventricular (AV) contraction, which reduces left ventricular pre-load and stroke volume, and worsens symptoms. His bundle pacing allows AV delay shortening while maintaining normal ventricular activation. HOPE-HF evaluated whether AV optimized His pacing is preferable to no-pacing, in a double-blind cross-over fashion, in patients with heart failure, left ventricular ejection fraction (LVEF) ≤40%, PR interval ≥200 ms and either QRS ≤140 ms or right bundle branch block. METHODS AND RESULTS: Patients had atrial and His bundle leads implanted (and an implantable cardioverter-defibrillator lead if clinically indicated) and were randomized to 6 months of pacing and 6 months of no-pacing utilizing a cross-over design. The primary outcome was peak oxygen uptake during symptom-limited exercise. Quality of life, LVEF and patients' holistic symptomatic preference between arms were secondary outcomes. Overall, 167 patients were randomized: 90% men, 69 ± 10 years, QRS duration 124 ± 26 ms, PR interval 249 ± 59 ms, LVEF 33 ± 9%. Neither peak oxygen uptake (+0.25 ml/kg/min, 95% confidence interval [CI] -0.23 to +0.73, p = 0.3) nor LVEF (+0.5%, 95% CI -0.7 to 1.6, p = 0.4) changed with pacing but Minnesota Living with Heart Failure quality of life improved significantly (-3.7, 95% CI -7.1 to -0.3, p = 0.03). Seventy-six percent of patients preferred His bundle pacing-on and 24% pacing-off (p < 0.0001). CONCLUSION: His bundle pacing did not increase peak oxygen uptake but, under double-blind conditions, significantly improved quality of life and was symptomatically preferred by the clear majority of patients. Ventricular pacing delivered via the His bundle did not adversely impact ventricular function during the 6 months.

Randomized controlled trial demonstrates nutritional management is superior to metronidazole for treatment of acute colitis in dogs.

OBJECTIVE: To describe the outcome of dietary management of canine noninfectious acute colitis with or without concurrent oral administration of metronidazole using a randomized controlled clinical trial. ANIMALS: 59 client-owned dogs with noninfectious acute colitis. PROCEDURES: Dogs with acute noninfectious colitis were enrolled in a 30-day diet trial after exclusion of parasitic infectious etiologies (fecal centrifugation floatation, Giardia/Cryptosporidium antigen testing) and systemic disease (CBC, biochemistry, urinalysis). Dogs were randomized into 3 placebo-controlled groups: group 1, easily digestible diet + placebo tablet; group 2, easily digestible diet + metronidazole tablet; and group 3, psyllium-enhanced easily digestible diet + placebo tablet. Dogs were evaluated serially using fecal scoring for time to remission, average fecal score, relapse after remission, and dysbiosis index. RESULTS: Median remission time was significantly different among the 3 groups (P < .01) with median times of 5 days (range, 4 to 10) for group 1, 8.5 days (range, 7 to 12) for group 2, and 5 days (range, 3 to 6) for group 3. Metronidazole addition affected the fecal dysbiosis index negatively at days 7 to 10. No adverse effects or complications were noted throughout the study. CLINICAL RELEVANCE: For canine noninfectious acute colitis, dietary management with an easily digestible diet with or without psyllium enhancement proved a superior management strategy compared to metronidazole. The omission of metronidazole reduced the adverse impact significantly on intestinal microbiota. Longitudinal clinical trials are necessary to compare the long-term response, stability, and complications associated with dietary management alone versus combined dietary and antimicrobial therapy for canine acute colitis.

Incidence, survival time, and surgical treatment of parathyroid carcinomas in dogs: 100 cases (2010-2019).

OBJECTIVE: To evaluate outcomes of dogs with parathyroid carcinoma (PTC) treated by surgical excision and to describe the incidence of postoperative hypocalcemia, degree of hypocalcemia, duration of hospitalization, duration of calcium supplementation, and survival time. ANIMALS: 100 client-owned dogs with PTC admitted to academic, referral veterinary institutions. PROCEDURES: In a retrospective multi-institutional study, medical records of dogs undergoing surgical excision of PTC between 2010 to 2019 were reviewed. Signalment, relevant medical history, clinical signs, clinicopathologic testing, imaging, surgical findings, intraoperative complications, histologic examination, and survival time were recorded. RESULTS: 100 dogs with PTC were included, and 96 dogs had clinical or incidental hypercalcemia. Common clinical signs included polyuria (44%), polydipsia (43%), hind limb paresis (22%), lethargy (21%), and hyporexia (20%). Cervical ultrasonography detected a parathyroid nodule in 91 of 91 dogs, with a single nodule in 70.3% (64/91), 2 nodules in 25.3% (23/91), and ≥ 3 nodules in 4 (4/91)% of dogs. Hypercalcemia resolved in 89 of 96 dogs within 7 days after surgery. Thirty-four percent of dogs developed hypocalcemia, on the basis of individual analyzer ranges, within 1 week after surgery. One dog had metastatic PTC to the prescapular lymph node, and 3 dogs were euthanized for refractory postoperative hypocalcemia. Estimated 1-, 2-, and 3-year survival rates were 84%, 65%, and 51% respectively, with a median survival time of 2 years. CONCLUSIONS AND CLINICAL RELEVANCE: Excision of PTC results in resolution of hypercalcemia and excellent long-term tumor control. Surgical excision of PTC is recommended because of resolution of hypercalcemia and a good long-term prognosis. Future prospective studies and long-term follow-up are needed to further assess primary tumor recurrence, metastasis, and incidence of postoperative hypocalcemia.

Quantification of Electromechanical Coupling to Prevent Inappropriate Implantable Cardioverter-Defibrillator Shocks.

OBJECTIVES: This study sought to test specialized processing of laser Doppler signals for discriminating ventricular fibrillation (VF) from common causes of inappropriate therapies. BACKGROUND: Inappropriate implantable cardioverter-defibrillator (ICD) therapies remain a clinically important problem associated with morbidity and mortality. Tissue perfusion biomarkers, implemented to assist automated diagnosis of VF, sometimes mistake artifacts and random noise for perfusion, which could lead to shocks being inappropriately withheld. METHODS: The study tested a novel processing algorithm that combines electrogram data and laser Doppler perfusion monitoring as a method for assessing circulatory status. Fifty patients undergoing VF induction during ICD implantation were recruited. Noninvasive laser Doppler and continuous electrograms were recorded during both sinus rhythm and VF. Two additional scenarios that might have led to inappropriate shocks were simulated for each patient: ventricular lead fracture and T-wave oversensing. The laser Doppler was analyzed using 3 methods for reducing noise: 1) running mean; 2) oscillatory height; and 3) a novel quantification of electromechanical coupling which gates laser Doppler relative to electrograms. In addition, the algorithm was tested during exercise-induced sinus tachycardia. RESULTS: Only the electromechanical coupling algorithm found a clear perfusion cut off between sinus rhythm and VF (sensitivity and specificity of 100%). Sensitivity and specificity remained at 100% during simulated lead fracture and electrogram oversensing. (Area under the curve running mean: 0.91; oscillatory height: 0.86; electromechanical coupling: 1.00). Sinus tachycardia did not cause false positive results. CONCLUSIONS: Quantifying the coupling between electrical and perfusion signals increases reliability of discrimination between VF and artifacts that ICDs may interpret as VF. Incorporating such methods into future ICDs may safely permit reductions of inappropriate shocks.

Optimum lesion set and predictors of outcome in persistent atrial fibrillation ablation: a meta-regression analysis.

AIMS: Ablation of persistent atrial fibrillation (PsAF) has been performed by many techniques with varying success rates. This may be due to ablation techniques, patient demographics, comorbidities, and trial design. We conducted a meta-regression of studies of PsAF ablation to elucidate the factors affecting atrial fibrillation (AF) recurrence. METHODS AND RESULTS: Databases were searched for prospective studies of PsAF ablation. A meta-regression was performed. Fifty-eight studies (6767 patients) were included. Complex fractionated atrial electrogram (CFAE) ablation reduced freedom from AF by 8.9% [95% confidence interval (CI) -15 to -2.3, P = 0.009). Left atrial appendage [LAA isolation (three study arms)] increased freedom from AF by 39.5% (95% CI 9.1-78.4, P = 0.008). Posterior wall isolation (PWI) (eight study arms) increased freedom from AF by 19.4% (95% CI 3.3-38.1, P = 0.017). Linear ablation or ganglionated plexi ablation resulted in no significant effect on freedom from AF. More extensive ablation increased intraprocedural AF termination; however, intraprocedural AF termination was not associated with improved outcomes. Increased left atrial diameter was associated with a reduction in freedom from AF by 4% (95% CI -6.8% to -1.1%, P = 0.007) for every 1 mm increase in diameter. CONCLUSION: Linear ablation, PWI, and CFAE ablation improves intraprocedural AF termination, but such termination does not predict better long-term outcomes. Study arms including PWI or LAA isolation in the lesion set were associated with improved outcomes in terms of freedom from AF; however, further randomized trials are required before these can be routinely recommended. Left atrial size is the most important marker of AF chronicity influencing outcomes.

Is There a Role for Preoperative Iron Supplementation in Patients Preparing for a Total Hip or Total Knee Arthroplasty?

BACKGROUND: Several treatment modalities exist for the treatment of perioperative anemia. We determined the effect of oral iron supplementation on preoperative anemia, and the use of blood-conserving interventions before total hip arthroplasty (THA) and total knee arthroplasty (TKA). METHODS: A total of 3435 total joint arthroplasties (1461 THAs and 1974 TKAs) were analyzed during 2 phases of a blood conservation program. The first phase used erythropoietin alfa (EPO) or intravenous (IV) iron for patients at risk for perioperative anemia. The second phase included these interventions, as well as preoperative iron supplementation. The effect on preoperative hemoglobin (Hb) and serum ferritin, as well as EPO and IV iron utilization, was determined. RESULTS: Oral iron therapy increased preoperative Hb level by 6 g/L (P < .001) and 7 g/L (P < .001) in the hip and knee cohorts, respectively. Serum ferritin level rose by 80 µg/L (P < .001) and 52 µg/L (P < .001) in the hip and knee cohorts, respectively. The number of patients with an Hb level <130 g/L was significantly reduced (P < .001 for both cohorts), as were patients with serum ferritin levels <35 µg/L (P = .002 for hip and P < .001 for knee cohorts). Utilization of EPO reduced from 16% to 6% (P < .001) and 18% to 6% (P < .001) in the hip and knee cohorts, respectively. Utilization of IV iron reduced from 4% to 2% (P = .05) and 5% to 2% (P < .001) in the hip and knee cohorts, respectively. CONCLUSION: Oral iron therapy reduced the burden of perioperative anemia and reduced utilization of other blood-conserving therapies before THA and TKA. Future research should delineate the cost-effectiveness of oral iron therapy.

Minimally invasive surgery versus radiotherapy/chemoradiotherapy for small-volume primary oropharyngeal carcinoma.

BACKGROUND: More than 400,000 cases of oropharyngeal squamous cell carcinoma (OPSCC) are diagnosed each year worldwide and the incidence is rising, partly as a result of human papillomavirus. Human papillomavirus-associated OPSCC affects younger patients and often presents at a higher stage; however, it is associated with a better prognosis.Until recently, first-line management of OPSCC involved chemoradiotherapy, as research had demonstrated comparable survival outcomes when compared with open surgery, with significantly decreased morbidity. However, interventions have now evolved with computerised planning and intensity-modulated radiotherapy, and the advent of endoscopic head and neck surgery, which provide the potential for decreased treatment-associated morbidity.The oropharynx plays an essential role in swallowing, speech and protecting the airway as it is situated at the bifurcation of the respiratory and digestive tracts. Treatment modality recommendations are based on survival outcomes. Given the younger patient demographic, establishing the safety of modalities that potentially have better functional outcome is becoming increasingly important. OBJECTIVES: To assess the efficacy of endoscopic head and neck surgery (transoral robotic surgery or transoral laser microsurgery) for small-volume, primary (T1-2, N0-2) oropharyngeal squamous cell carcinoma (OPSCC) in comparison to radiotherapy/chemoradiotherapy. SEARCH METHODS: The Cochrane ENT Information Specialist searched the ENT Trials Register; Central Register of Controlled Trials (CENTRAL 2016, Issue 10); PubMed; EMBASE; CINAHL; Web of Science; ClinicalTrials.gov; ICTRP and additional sources for published and unpublished trials. The date of the search was 8 November 2016. SELECTION CRITERIA: Randomised controlled trials in patients with carcinoma in the oropharynx subsite (as defined by the World Health Organization classification C09, C10). Cancers included were primary squamous cell carcinomas arising from the oropharyngeal mucosa. The tumours were classified as T1-T2 with or without nodal disease and with no evidence of distant metastatic spread. The intervention was transoral, minimally invasive surgery with or without adjuvant radiotherapy or adjuvant chemoradiotherapy. The comparator was primary radiotherapy with or without induction or concurrent chemotherapy for the tumour. The treatments received and compared were of curative intent and patients had not undergone prior intervention, other than diagnostic biopsy. DATA COLLECTION AND ANALYSIS: We used the standard methodological procedures expected by Cochrane. Our primary outcomes were overall survival (disease-related mortality was to be studied where possible), locoregional control, disease-free survival and progression-free survival or time to recurrence. All outcomes were to be measured at two, three and five years after diagnosis. Our secondary outcomes included quality of life, harms associated with treatment, patient satisfaction and xerostomia score. MAIN RESULTS: No completed studies met the inclusion criteria for the review. Two ongoing trials fulfilled the selection criteria, however neither are complete.'Early-stage squamous cell carcinoma of the oropharynx: radiotherapy versus trans-oral robotic surgery (ORATOR)' is a phase II randomised controlled trial comparing primary radiation therapy with primary transoral robotic surgery for small-volume primary (T1-2, N0-2) OPSCC. It is currently in progress with an estimated completion date of June 2021.'European Organisation for Research and Treatment of Cancer 1420 (EORTC 1420-HNCG-ROG)' is a phase III, randomised study assessing the "best of" radiotherapy compared to transoral robotic surgery/transoral laser microsurgery in patients with T1-T2, N0 squamous cell carcinoma of the oropharynx and base of tongue. It was due to start accrual mid-2016. AUTHORS' CONCLUSIONS: The role of endoscopic head and neck surgery in the management of OPSCC is clearly expanding as evidenced by its more overt incorporation into the current National Comprehensive Cancer Network guidelines. Data are mounting regarding its outcomes both in terms of survival and lower morbidity. As confidence increases, it is being used in the management of more advanced OPSCC.Based on this review, there is currently no high-quality evidence from randomised controlled trials regarding clinical outcomes for patients with oropharyngeal cancer receiving endoscopic head and neck surgery compared with primary chemoradiotherapy.

Task-related functional connectivity of the caudate mediates the association between trait mindfulness and implicit learning in older adults.

Accumulating evidence shows a positive relationship between mindfulness and explicit cognitive functioning, i.e., that which occurs with conscious intent and awareness. However, recent evidence suggests that there may be a negative relationship between mindfulness and implicit types of learning, or those that occur without conscious awareness or intent. Here we examined the neural mechanisms underlying the recently reported negative relationship between dispositional mindfulness and implicit probabilistic sequence learning in both younger and older adults. We tested the hypothesis that the relationship is mediated by communication, or functional connectivity, of brain regions once traditionally considered to be central to dissociable learning systems: the caudate, medial temporal lobe (MTL), and prefrontal cortex (PFC). We first replicated the negative relationship between mindfulness and implicit learning in a sample of healthy older adults (60-90 years old) who completed three event-related runs of an implicit sequence learning task. Then, using a seed-based connectivity approach, we identified task-related connectivity associated with individual differences in both learning and mindfulness. The main finding was that caudate-MTL connectivity (bilaterally) was positively correlated with learning and negatively correlated with mindfulness. Further, the strength of task-related connectivity between these regions mediated the negative relationship between mindfulness and learning. This pattern of results was limited to the older adults. Thus, at least in healthy older adults, the functional communication between two interactive learning-relevant systems can account for the relationship between mindfulness and implicit probabilistic sequence learning.

Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor.

Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32-1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis.

The effect of sacral neuromodulation on pudendal nerve function and female sexual function.

AIMS: To quantify changes in pudendal nerve function with sacral neuromodulation (SNM). To understand the relationship of pudendal nerve function to SNM treatment response for overactive bladder. To assess the relationship between female sexual function and pudendal nerve function after SNM. METHODS: Women undergoing SNM between January 2010 and May 2011 were enrolled. Baseline pudendal nerve terminal motor latencies (PNTML) were measured bilaterally. Subjects underwent peripheral nerve evaluation (PNE) prior to SNM therapy. PNTML was measured at 1 and 6 weeks after sacral neuromodulator implant. Women who did not undergo permanent implantation were reassessed at the end of the 1-week PNE testing phase. Pelvic floor and sexual function questionnaires were administered at baseline and follow-up to assess pelvic floor and sexual function. RESULTS: Of 34 women enrolled, 31 were evaluated. Mean age was 67.4 ± 14.2 years with 29/34 (85.3%) treated for refractory overactive bladder. Thirty of 31 (96.7%) went on to a permanent implant. PNE success rate was 73.5% (25/33). Mean PNTML changed from 2.74 ± 0.52 msec at baseline to 2.57 ± 0.50 msec at 6 weeks postop (P = 0.198). Baseline amplitude remained stable at 1 and 6 weeks. At 6 weeks PISQ-12 scores showed improved sexual function (P = 0.034) and PFDI-20 and PFIQ-7 scores showed improved pelvic floor, colorectal and urinary symptoms (P < 0.05). CONCLUSIONS: Women with refractory overactive bladder and non-obstructive urinary retention have abnormal pudendal nerve function, which showed a non-significant trend toward improvement after SNM. Sexually active women undergoing sacral neuromodulation experienced improvement in sexual function. Quality of life improved due to improvement in urinary and colorectal function.

Dispositional mindfulness is associated with reduced implicit learning.

Behavioral and neuroimaging evidence suggest that mindfulness exerts its salutary effects by disengaging habitual processes supported by subcortical regions and increasing effortful control processes supported by the frontal lobes. Here we investigated whether individual differences in dispositional mindfulness relate to performance on implicit sequence learning tasks in which optimal learning may in fact be impeded by the engagement of effortful control processes. We report results from two studies where participants completed a widely used questionnaire assessing mindfulness and one of two implicit sequence learning tasks. Learning was quantified using two commonly used measures of sequence learning. In both studies we detected a negative relationship between mindfulness and sequence learning, and the relationship was consistent across both learning measures. Our results, the first to show a negative relationship between mindfulness and implicit sequence learning, suggest that the beneficial effects of mindfulness do not extend to all cognitive functions.

Attention to odor modulates thalamocortical connectivity in the human brain.

It is widely assumed that the thalamus is functionally irrelevant for the sense of smell. Although animal studies suggest that the mediodorsal (MD) thalamus links primary olfactory (piriform) cortex to olfactory neocortical projection sites in orbitofrontal cortex (OFC), this transthalamic route is regarded to be inconsequential, particularly compared with a direct monosynaptic pathway linking piriform cortex and OFC. In this study, we combined functional magnetic resonance imaging with novel effective connectivity techniques to measure attention-dependent network coherence within direct (nonthalamic) and indirect (transthalamic) olfactory pathways. Human subjects were presented with (or without) an odor and with (or without) a tone, while selectively attending to either modality. Attention to odor significantly modulated neural coupling within the indirect pathway, strengthening MD thalamus-OFC connectivity. Critically, these effects were modality specific (odor > tone attention), directionally sensitive (forward > backward connections), and selective to route (indirect > direct pathway). Our findings support the idea that the human transthalamic pathway is an active modulatory target of olfactory attention. The results imply that olfaction, like all other sensory modalities, requires a thalamic relay, if only to consciously analyze a smell.

Implicit sequence learning without motor sequencing in young and old adults.

The ability to detect patterns and organize individual events into complex sequences is a fundamental cognitive skill that is often learned implicitly. The serial response time (SRT) task has been widely used to investigate implicit sequence learning, but it remains unclear whether people learn a perceptual or motor sequence in this task. This study reports three experiments that build on previous research by Goschke and colleagues using an auditory SRT task in which the stimulus-to-response mapping changes on every trial to eliminate spatio-motor sequencing. The current study extends earlier work in three ways. First, healthy young and older adults were tested rather than the neuropsychological patients used in previous research. Second, sequences of different structural complexity were investigated including first- and second-order repeating sequences as well as higher-order probabilistic sequences. Third, the potential role of explicit knowledge was examined using three separate tests of declarative knowledge. Results indicate that young and old adults are able to learn purely perceptual auditory sequences, but that explicit knowledge contributes to learning of repeating sequences by young adults.

Tumor response and toxicity with multiple infusions of high dose 131I-MIBG for refractory neuroblastoma.

BACKGROUND: (131)I Metaiodobenzylguanidine ((131)I-MIBG) is an effective targeted radiotherapeutic for neuroblastoma with response rates greater than 30% in refractory disease. Toxicity is mainly limited to myelosuppression. The aim of this study was to determine the response rate and hematologic toxicity of multiple infusions of (131)I-MIBG. PROCEDURE: Patients received two to four infusions of (131)I-MIBG at activity levels of 3-19 mCi/kg per infusion. Criteria for subsequent infusions were neutrophil recovery without stem cell support and lack of disease progression after the first infusion. RESULTS: Sixty-two infusions were administered to 28 patients, with 24 patients receiving two infusions, two patients receiving three infusions, and two patients receiving four infusions. All patients were heavily pre-treated, including 16 with prior myeloablative therapy. Eleven patients (39%) had overall disease response to multiple therapies, including eight patients with measurable responses to each of two or three infusions, and three with a partial response (PR) after the first infusion and stable disease after the second. The main toxicity was myelosuppression, with 78% and 82% of patients requiring platelet transfusion support after the first and second infusion, respectively, while only 50% had grade 4 neutropenia, usually transient. Thirteen patients did not recover platelet transfusion independence after their final MIBG infusion; stem cell support was given in ten patients. CONCLUSIONS: Multiple therapies with (131)I-MIBG achieved increasing responses, but hematologic toxicity, especially to platelets, was dose limiting. More effective therapy might be given using consecutive doses in rapid succession with early stem cell support.

Biochemical and structural characterization of the cross-linked complex of nitrogenase: comparison to the ADP-AlF4(-)-stabilized structure.

The transient formation of a complex between the component Fe- and MoFe-proteins of nitrogenase represents a central event in the substrate reduction mechanism of this enzyme. Previously, we have isolated an N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide (EDC) cross-linked complex of these proteins stabilized by a covalent isopeptide linkage between Glu 112 and Lys beta400 of the Fe-protein and MoFe-protein, respectively [Willing, A., et al. (1989) J. Biol. Chem. 264, 8499-8503; Willing, A., and Howard, J. B. (1990) J. Biol. Chem. 265, 6596-6599]. We report here the biochemical and structural characterization of the cross-linked complex to assess the mechanistic relevance of this species. Glycinamide inhibits the cross-linking reaction, and is found to be specifically incorporated into Glu 112 of the Fe-protein, without detectable modification of either of the neighboring residues (Glu 110 and Glu 111). This modified protein is still competent for substrate reduction, demonstrating that formation of the cross-linked complex is responsible for the enzymatic inactivation, and not the EDC reaction or the modification of the Fe-protein. Crystallographic analysis of the EDC-cross-linked complex at 3.2 A resolution confirms the site of the isopeptide linkage. The nature of the protein surfaces around the cross-linking site suggests there is a strong electrostatic component to the formation of the complex, although the interface area between the component proteins is small. The binding footprints between proteins in the cross-linked complex are adjacent, but with little overlap, to those observed in the complex of the nitrogenase proteins stabilized by ADP-AlF(4)(-). The results of these studies suggest that EDC cross-linking traps a nucleotide-independent precomplex of the nitrogenase proteins driven by complementary electrostatic interactions that subsequently rearranges in a nucleotide-dependent fashion to the electron transfer competent state observed in the ADP-AlF(4)(-) structure.

Nutrient recovery from swine lagoon water by Spirodela punctata.

Spirodela punctata 7776, the best duckweed strain in total protein production selected from in vitro screening experiments with synthetic swine lagoon water medium was examined for N and P recovery. It has shown a capability to grow in and to remove N and P from synthetic swine lagoon water with high N (240 mg NH4 N/l) and P (31.0 mg PO4 P/l) levels. A lag period of approximately 96 h was observed before the duckweed started to grow. During the lag period, utilization of N and P by the duckweed was very slow. The rates of N and P uptake, and duckweed growth increased with the increase of the initial N and P concentrations in the medium. The highest rates of N and P uptakes, and duckweed growth observed in this study were 0.955. 0.129 mg/l-h, and 1.33 g/m2-h (or 31.92 g/m2-day), respectively. The N:P ratio in swine lagoon water is adequate for growing the duckweed.