Fatigue Analysis of a Jacket-Supported Offshore Wind Turbine at Block Island Wind Farm.

Offshore wind-turbine (OWT) support structures are subjected to cyclic dynamic loads with variations in loadings from wind and waves as well as the rotation of blades throughout their lifetime. The magnitude and extent of the cyclic loading can create a fatigue limit state controlling the design of support structures. In this paper, the remaining fatigue life of the support structure for a GE Haliade 6 MW fixed-bottom jacket offshore wind turbine within the Block Island Wind Farm (BIWF) is assessed. The fatigue damage to the tower and the jacket support structure using stress time histories at instrumented and non-instrumented locations are processed. Two validated finite-element models are utilized for assessing the stress cycles. The modal expansion method and a simplified approach using static calculations of the responses are employed to estimate the stress at the non-instrumented locations-known as virtual sensors. It is found that the hotspots at the base of the tower have longer service lives than the jacket. The fatigue damage to the jacket leg joints is less than 20% and 40% of its fatigue capacity during the 25-year design lifetime of the BIWF OWT, using the modal expansion method and the simplified static approach, respectively.

Update on the Selection Criteria of Plastic Surgery Residents: A Survey of Program Directors and Associate Program Directors.

BACKGROUND: Plastic and reconstructive surgery (PRS) is recognized as a highly competitive specialty. Since the first assessment of resident selection criteria in 2007, PRS residency programs have adopted holistic review processes and adapted to changes such as a decline in medical schools participating in the Alpha Omega Alpha Honor Medical Society as well as the recent transition to pass/fail grading for the United States Medical Licensing Examination (USMLE) step 1 examination (Schultz et al. Plast Reconstr Surg Glob Open . 2020;8:e2892; Tadisina et al. Plast Reconstr Surg . 2017;139:330e-331e). This study was devised to evaluate current PRS residency criteria in light of these changes. METHODS: An anonymous, 12-item, electronic survey was generated and distributed using Alchemer. An email was sent to 171 program directors (PDs) and associate program directors (APDs) of PRS residency programs. Survey questions were developed to collect data regarding respondent demographics and their desired criteria when assessing residency applicants. Complete responses were collected and analyzed with summary statistics and multivariate logistic regression using RStudio (version 1.3.109). RESULTS: In total, 44 (25.7% response rate) of the 171 PDs and APDs completed the survey. Of the 16 programs (36.4%) with a USMLE cutoff score, 7 (43.8%) reported a range of 230 to 239 and 6 (37.5%) reported a range of 240 to 249. Without a score for step 1, the majority (48.8%) of respondents believe that step 2 scores will replace step 1 scores in terms of assessment criteria, and the content of recommendation letters was selected as the criterion with the greatest increase in weight (66.7%). In addition, 27.3% of programs require a step 2 score at the time of interview. The top 3 academic criteria in order of decreasing importance were the content of recommendation letters, clinical grades, and letter writers, whereas the top 3 nonacademic criteria were subinternship performance, maturity, and interview performance. CONCLUSIONS: Plastic and reconstructive surgery remains a highly competitive specialty for residency applicants. Our findings suggest that Alpha Omega Alpha membership remains diminished in importance, whereas USMLE cutoff scores have increased. With recent changes in the step 1 grading system, PDs and APDs will rely more heavily on step 2 scores and the content of recommendation letters.

Hemodynamics in Distally Based Sural Flaps for Lower Leg Reconstruction: A Literature Review.

Coverage of posttraumatic and chronic wounds at the distal leg is a difficult problem due to limited soft tissue available for local flaps. The sural flap is a versatile and effective method for reconstruction in this area since it does not need a significant amount of time or assistance to complete. Improving the survival of these flaps is critically dependent on understanding the basics of flap circulation and why recent modifications were introduced. This review will serve as a much-needed comprehensive analysis of these topics for surgeons looking to increase the reliability of their sural flaps.

Perspectives on Cybersecurity and Plastic Surgery: A Survey of Plastic Surgeons and Scoping Review of the Literature.

BACKGROUND: Data breach costs in the United States are among the highest in the world, making robust cybersecurity an important bulwark of national defense. Healthcare is a popular target for cyber threats, and there is increasing emphasis on cybersecurity safeguards to protect sensitive patient data. OBJECTIVES: The objective of this national survey and scoping review is to (1) identify cybersecurity awareness, preparedness, and practices among plastic surgeons, and (2) to provide guidelines to mitigate the threat of cyberattacks. METHODS: A 16-question, anonymous online survey was developed and distributed to The Aesthetic Society registrants to ascertain plastic surgeons' cybersecurity practices. Utilizing PubMed, CINAHL, and Embase databases, eligible articles were identified as part of this scoping review. RESULTS: Of 89 individuals who began the survey, 69 completed it (77.5%). Sixty respondents agreed or strongly agreed that cybersecurity is an important issue in plastic surgery. The greatest perceived limitations for protection against cyberattacks were insufficient expertise (41.7%), followed by lack of funding and insufficient time to dedicate to this goal. Most respondents (78.7%) had cybersecurity policies incorporated into their practice. Those who agreed or strongly agreed they had technology to prevent data theft/breach were significantly more likely to be older than 54 years of age (P < .001). No articles identified in the literature specifically addressed cybersecurity in plastic surgery; however, 12 articles detailing cybersecurity in healthcare were identified and included. CONCLUSIONS: Despite possessing adequate technology and procedures in place to prevent cyberattacks, plastic surgeons perceive significant barriers to cybersecurity protection, including insufficient expertise and lack of dedicated funding. It is imperative that our field establishes standards and protocols to protect our patients.

The Impact of Navigation Grid Overlay on Performance of Camera Assistants during Laparoscopic Abdominal Procedures: A Randomized Controlled Trial.

OBJECTIVE: Camera assistance is important for proper visualization of the operative field in laparoscopic surgery. Navigation grid (NG) has been designed to help the camera assistants focus the camera on the target operative field. This is a randomized, controlled trial to study the effect of the NG on performance of camera assistants. DESIGN: Minimally invasive operations were randomized (1:1) to either with or without use of NG for the camera assistant. The operations were recorded and the time spent inside and outside of the target area were reported. SETTING: A tertiary care teaching hospital. RESULTS: Fifty-eight operations (30 with and 28 without NG) were recorded. Sixteen camera assistants participated. Time spent outside the target area was significantly less with the use of NG (64.5 ± 63 seconds vs 396 ± 226.5 seconds; p < 0.0001). This impact of NG on performance of the camera assistants was significant regardless of their level of training. CONCLUSIONS: NG improved performance of the camera assistant during laparoscopic abdominal procedures. This is a feasible tool that can help camera holders better assist the operating surgeons.

Fluid type influences acute hydration and muscle performance recovery in human subjects.

BACKGROUND: Exercise and heat trigger dehydration and an increase in extracellular fluid osmolality, leading to deficits in exercise performance and thermoregulation. Evidence from previous studies supports the potential for deep-ocean mineral water to improve recovery of exercise performance post-exercise. We therefore wished to determine whether acute rehydration and muscle strength recovery was enhanced by deep-ocean mineral water following a dehydrating exercise, compared to a sports drink or mountain spring water. We hypothesized that muscle strength would decrease as a result of dehydrating exercise, and that recovery of muscle strength and hydration would depend on the type of rehydrating fluid. METHODS: Using a counterbalanced, crossover study design, female (n = 8) and male (n = 9) participants performed a dehydrating exercise protocol under heat stress until achieving 3% body mass loss. Participants rehydrated with either deep-ocean mineral water (Deep), mountain spring water (Spring), or a carbohydrate-based sports drink (Sports) at a volume equal to the volume of fluid loss. We measured relative hydration using salivary osmolality (Sosm) and muscle strength using peak torque from a leg extension maneuver. RESULTS: Sosm significantly increased (p < 0.0001) with loss of body mass during the dehydrating exercise protocol. Males took less time (90.0 ± 18.3 min; P < 0.0034) to reach 3% body mass loss when compared to females (127.1 ± 20.0 min). We used a mono-exponential model to fit the return of Sosm to baseline values during the rehydrating phase. Whether fitting stimulated or unstimulated Sosm, male and female participants receiving Deep as the hydrating fluid exhibited the most rapid return to baseline Sosm (p < 0.0001) regardless of the fit parameter. Males compared to females generated more peak torque (p = 0.0005) at baseline (308.3 ± 56.7 Nm vs 172.8 ± 40.8 Nm, respectively) and immediately following 3% body mass loss (276.3 ± 39.5 Nm vs 153.5 ± 35.9 Nm). Participants experienced a loss. We also identified a significant effect of rehydrating fluid and sex on post-rehydration peak torque (p < 0.0117). CONCLUSION: We conclude that deep-ocean mineral water positively affected hydration recovery after dehydrating exercise, and that it may also be beneficial for muscle strength recovery, although this, as well as the influence of sex, needs to be further examined by future research. TRIAL REGISTRATION: clincialtrials.gov PRS, NCT02486224 . Registered 08 June 2015.

Proteomic analysis of the peritrophic matrix from the gut of the caterpillar, Helicoverpa armigera.

The peritrophic matrix from the midgut of the caterpillar, Helicovera armigera, was solubilized by treatment with anhydrous trifluoromethanesulfonic acid, apparently by depolymerisation of its chitin component. This allowed the efficient extraction of proteins in a technique that may be broadly applicable to the analysis of other structures containing chitin. Gel electrophoresis and mass spectrometry of tryptic peptides were used to identify the extracted proteins with gut-expressed cDNA sequences. The major proteins of this cohesive, digestion-resistant structure are chitin deacetylase-like and mucin-like proteins, the latter with multiple chitin-binding domains that may cross-link chitin fibrils to provide a barrier against abrasive food particles and parasites, one of the major functions of the matrix. Other proteins found in the H. armigera gut peritrophic matrix suggest that the matrix is a dynamic, complex structure that may participate in the immobilization of digestive enzymes, actively protect the gut from parasite invasion and intercept toxins such as lectins and Bacillus thuringiensis crystal proteins.

Acquisition algorithm for direct-detection ladars with Geiger-mode avalanche photodiodes.

An optimal algorithm for detecting a target using a ladar system employing Geiger-mode avalanche photodiodes (GAPDs) is presented. The algorithm applies to any scenario where a ranging direct detection ladar is used to determine the presence of a target against a sky background within a specified range window. A complete statistical model of the detection process for GAPDs is presented, including GAPDs that are inactive for a fixed period of time each time they fire. The model is used to develop a constant false alarm rate detection algorithm that minimizes acquisition time. Numerical performance predictions, simulation results, and experimental results are presented.

The role of tumor necrosis factor alpha in down-regulation of osteoblast Phex gene expression in experimental murine colitis.

BACKGROUND & AIMS: Reduced bone mass is a common complication of inflammatory bowel disease (IBD), although the mechanisms that contribute to osteopenia are not completely understood. Tumor necrosis factor alpha (TNF-alpha) is up-regulated in patients with IBD and has detrimental effects on osteoblasts. Phex gene is expressed predominantly in osteoblasts, and its disruption results in defective bone mineralization. The aim of this study was to evaluate whether TNF-alpha regulates Phex gene expression thus contributing to the abnormal bone metabolism observed in IBD. METHODS: Phex gene expression was evaluated in calvaria of 6-7-week-old mice administered with trinitrobenzene sulfonic acid (TNBS) with or without neutralizing anti-TNF-alpha antibody, dietary curcumin, or systemically with recombinant TNF-alpha. TNF-alpha-treated UMR-106 osteoblasts were also examined. Phex promoter activity was assayed in transiently transfected TNF-alpha-treated UMR-106 cells. RESULTS: Compared with control animals, Phex messenger RNA (mRNA) expression decreased by 40%-50% in both TNBS colitis and TNF-alpha-injected mice. Dietary curcumin and anti-TNF-alpha antibody counteracted the detrimental effect of TNBS on Phex gene expression. TNF-alpha-treated UMR-106 cells showed a concentration-dependent and transcriptionally mediated decrease in Phex mRNA and gene promoter activity, with the -133 to -74 bp region of the Phex promoter likely involved in the mechanism of TNF-alpha action. Coinciding with decreased Phex protein level, TNF-alpha drastically reduced mineralization in UMR-106 osteoblasts. CONCLUSIONS: Acute colitis and TNF-alpha decrease Phex mRNA and protein expression via a transcriptional mechanism. TNF-alpha-mediated reduction in Phex protein is at least in part responsible for inhibition of osteoblast mineralization, and the described mechanism may contribute to the abnormal bone metabolism associated with IBD.

1alpha,25-Dihydroxyvitamin D3 upregulates FGF23 gene expression in bone: the final link in a renal-gastrointestinal-skeletal axis that controls phosphate transport.

Fibroblast growth factor (FGF)23 is a phosphaturic hormone that decreases circulating 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and elicits hypophosphatemia, both of which contribute to rickets/osteomalacia. It has been shown recently that serum FGF23 increases after treatment with renal 1,25(OH)(2)D(3) hormone, suggesting that 1,25(OH)(2)D(3) negatively feedback controls its levels by inducing FGF23. To establish the tissue of origin and the molecular mechanism by which 1,25(OH)(2)D(3) increases circulating FGF23, we administered 1,25(OH)(2)D(3) to C57BL/6 mice. Within 24 h, these mice displayed a dramatic elevation in serum immunoreactive FGF23, and the expression of FGF23 mRNA in bone was significantly upregulated by 1,25(OH)(2)D(3), but there was no effect in several other tissues. Furthermore, we treated rat UMR-106 osteoblast-like cells with 1,25(OH)(2)D(3), and real-time PCR analysis revealed a dose- and time-dependent stimulation of FGF23 mRNA concentrations. The maximum increase in FGF23 mRNA was 1,024-fold at 10(-7) M 1,25(OH)(2)D(3) after 24-h treatment, but statistically significant differences were observed as early as 4 h after 1,25(OH)(2)D(3) treatment. In addition, using cotreatment with actinomycin D or cycloheximide, we observed that 1,25(OH)(2)D(3) regulation of FGF23 gene expression occurs at the transcriptional level, likely via the nuclear vitamin D receptor, and is dependent on synthesis of an intermediary transfactor. These results indicate that bone is a major site of FGF23 expression and source of circulating FGF23 after 1,25(OH)(2)D(3) administration or physiological upregulation. Our data also establish FGF23 induction by 1,25(OH)(2)D(3) in osteoblasts as a feedback loop between these two hormones that completes a kidney-intestine-bone axis that mediates phosphate homeostasis.

Neuropeptide co-localisation in the lepidopteran frontal ganglion studied by electron-microscopic gold-labelling immunocytochemistry.

An immunogold-labelling electron-microscopic study of the frontal ganglion of two noctuids, Lacanobia oleracea and Helicoverpa armigera, has been carried out with antisera directed against three neuropeptides; allatostatins of the Y/FXFGL-NH2 type, Manduca sexta allatostatin (Mas-AS) and M. sexta allatotropin. The ganglion of both noctuids has two pairs of large peptidergic neurones with many clusters of electron-dense granules, one pair being situated anteriorly and the other posteriorly. By means of a double-labelling ("flip-flop") technique, with different sizes of gold particles, all possible paired combinations of the three different types of peptide have been visualised within granules of the anterior neurones, leading to the conclusion that the three peptides are co-packaged and co-stored in these cells. Within the posterior neurones of L. oleracea, gold labelling of granules is only linked to the Y/FXFGL-NH2 allatostatin antisera and, in contrast to the anterior cells of this species in which double gold labelling results in a sparse accumulation of gold particles for any one peptide type, single labelling gives a more intense, uniform pattern of gold particles. In contrast to L. oleracea, the gold-labelling pattern seen in the posterior neurones of H. armigera reflects the co-localisation of allatostatins of the Y/FXFGL-NH2 type with Mas-AS in this species. Allatotropin is absent in the posterior neurones of both species.

1,25-dihydroxyvitamin D3 down-regulation of PHEX gene expression is mediated by apparent repression of a 110 kDa transfactor that binds to a polyadenine element in the promoter.

The PHEX gene encodes an endopeptidase expressed in osteoblasts that inactivates an uncharacterized peptide hormone, phosphatonin, which suppresses bone mineralization as well as renal phosphate reabsorption and vitamin D bioactivation. We demonstrate that 1alpha-25-dihydroxyvitamin D (1,25(OH)2D3), the, active renal vitamin D metabolite, decreases PHEX mRNA in the rat osteoblastic cell line, UMR-106, as well as in mouse calvaria. Promoter/reporter construct analysis of the murine PHEX gene in transfected UMR-106 cells localized the repressive effect of 1,25(OH)2D3 to the -133 to -74 bp region, and gel mobility shift experiments revealed that 1,25(OH)2D3 treatment of the cells diminished the binding of a nuclear protein(s) to a stretch of 17 adenines from bp -116 to -100 in the proximal PHEX promoter. Either overexpression of a dominant-negative vitamin D receptor (VDR) or deletion of this sequence of 17 A-T base pairs abolished the repressive effect of 1,25(OH)2D3 by attenuating basal promoter activity, indicating that this region mediates the 1,25(OH)2D3 response and is involved in basal transcription. South-western blot analysis and DNA affinity purification show that an unidentified 110 kDa nuclear protein binds to the poly(A) element. Because 1,25(OH)2D3-liganded VDR neither binds to the polyadenine region of the PHEX promoter nor directly influences the association of the 110 kDa transfactor, we conclude that 1,25(OH)2D3 indirectly decreases PHEX expression via VDR-mediated repression (or modification) of this novel transactivator. Thus, we have identified a cis-element required for PHEX gene transcription that participates in negative feedback control of PHEX expression and thereby modulates the actions of phosphatonin.

A novel secreted protein toxin from the insect pathogenic bacterium Xenorhabdus nematophila.

The bacterium Xenorhabdus nematophila is an insect pathogen that produces several proteins that enable it to kill insects. Screening of a cosmid library constructed from X. nematophila strain A24 identified a gene that encoded a novel protein that was toxic to insects. The 42-kDa protein encoded by the toxin gene was expressed and purified from a recombinant system, and was shown to kill the larvae of insects such as Galleria mellonella and Helicoverpa armigera when injected at doses of around 30-40 ng/g larvae. Sequencing and bioinformatic analysis suggested that the toxin was a novel protein, and that it was likely to be part of a genomic island involved in pathogenicity. When the native bacteria were grown under laboratory conditions, a soluble form of the 42-kDa toxin was secreted only by bacteria in the phase II state. Preliminary histological analysis of larvae injected with recombinant protein suggested that the toxin primarily acted on the midgut of the insect. Finally, some of the common strategies used by the bacterial pathogens of insects, animals, and plants are discussed.

Transcriptional regulation of the human NaPi-IIb cotransporter by EGF in Caco-2 cells involves c-myb.

The type IIb sodium-phosphate (NaP(i)-IIb) cotransporter mediates intestinal phosphate absorption. Previous work in our laboratory has shown that EGF inhibited NaP(i)-IIb cotransporter expression through transcriptional regulation. To understand this regulation, progressively shorter human NaP(i)-IIb promoter constructs were used to define the EGF response region, and gel mobility shift assays (GMSAs) were used to characterize DNA-protein interactions. Promoter analysis determined that the EGF response region was located between -784 and -729 base pair (bp) of the promoter. GMSAs and overexpression studies revealed an interaction between this promoter region and c-myb transcription factor. Inhibition of EGF receptor activation restored promoter function. Further studies suggested that MAPK, PKC, and/or PKA pathways are involved in this regulation. In conclusion, these studies suggest that EGF decreases human NaP(i)-IIb gene expression by modifying the c-myb protein such that it inhibits transcriptional activation. We further conclude that this downregulation of promoter function is mediated by EGF-activated PKC/PKA and MAPK pathways. This is the first study that demonstrates involvement of c-myb in the regulation of intestinal nutrient absorption.

Infection of its lepidopteran host by the Helicoverpa armigera stunt virus (Tetraviridae).

Techniques of microscopy and histopathology were employed to study the positive-sense, single-stranded RNA virus, the Helicoverpa armigera stunt virus (HaSV; omegatetravirus, Tetraviridae) infecting its caterpillar host. Infection of the virus per os during the first three instars of larval development is virulent and leads to rapid stunting and mortality. In contrast, no detectable symptoms occur in later larval development, signifying a high degree of developmental resistance. A quantitative study of cell populations in the host midgut during this time showed that increased cell numbers during development alone could not account for the increase in resistance. HaSV infection was restricted to the midgut and three of its four cell types. In younger larvae, the virus initiated its infection in closely situated foci that appeared to expand to link with others to cover larger areas of the midgut. The midgut cells of the infected larvae responded with an increased rate of sloughing to an extent rendering the midgut incapable of maintenance or recovery of normal function. In contrast, infection of older larvae by HaSV did not lead to overt pathology although foci of HaSV infection were detected in their midguts. However, the foci were more sparsely situated, failed to expand, and eventually disappeared, presumably due to cell sloughing. These observations indicate that cell sloughing is an immune response existing throughout larval development but midguts of older larvae have an additional mechanism to account for the increased resistance. This second mechanism results in midgut cells becoming more refractory to infection and, combined with cell sloughing, allows the midguts of older larvae to recover more readily from HaSV infection. These two mechanisms are similar to those seen with host responses to baculoviruses, which display developmental resistance to a lesser degree against more general infections. HaSV remaining in the midgut appears to amplify the degree of developmental resistance.

Glucocorticoid regulation of the murine PHEX gene.

The phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) is a member of the neutral endopeptidase family, which is expressed predominantly on the plasma membranes of mature osteoblasts and osteocytes. Although it is known that the loss of PHEX function results in X-linked hypophosphatemic rickets, characterized by abnormal bone matrix mineralization and renal phosphate wasting, little is known about how PHEX is regulated. We therefore sought to determine whether the murine PHEX gene is regulated by glucocorticoids (GCs), which are known to influence phosphate homeostasis and bone metabolism. Northern blot analysis revealed increased PHEX mRNA expression in GC-treated suckling mice (1.5-fold) and in rat osteogenic sarcoma (UMR-106) cells (2.5-fold). An increase was also seen in PHEX promoter activity in transiently transfected UMR-106 cells with GC treatment. Analysis of nested promoter deletions revealed that an atypical GC response element was located between -337 and -315 bp. Mutational analysis and electrophoretic mobility shift assays further identified -326 to -321 bp as a site involved in GC regulation. Supershift analyses and electrophoretic mobility shift assay competition studies indicated that the core binding factor alpha1-subunit transcription factor is able to bind to this region and may therefore play a role in the GC response of the murine PHEX gene.

Glucocorticoid regulation and glycosylation of mouse intestinal type IIb Na-P(i) cotransporter during ontogeny.

We sought to characterize expression of an apically expressed intestinal Na-P(i) cotransporter (Na-P(i)-IIb) during mouse ontogeny and to assess the effects of methylprednisolone (MP) treatment. In control mice, Na-P(i) uptake by intestinal brush-border membrane vesicles was highest at 14 days of age, lower at 21 days, and further reduced at 8 wk and 8-9 mo of age. Na-P(i)-IIb mRNA and immunoreactive protein levels in 14-day-old animals were markedly higher than in older groups. MP treatment significantly decreased Na-P(i) uptake and Na-P(i)-IIb mRNA and protein expression in 14-day-old mice. Additionally, the size of the protein was smaller in 14-day-old mice. Deglycosylation of protein from 14-day-old and 8-wk-old animals with peptide N-glycosidase reduced the molecular weight to the predicted size. We conclude that intestinal Na-P(i) uptake and Na-P(i)-IIb expression are highest at 14 days and decrease with age. Furthermore, MP treatment reduced intestinal Na-P(i) uptake approximately threefold in 14-day-old mice and this reduction correlates with reduced Na-P(i)-IIb mRNA and protein expression. We also demonstrate that Na-P(i)-IIb is an N-linked glycoprotein and that glycosylation is age dependent.

Role of GlnK in NifL-mediated regulation of NifA activity in Azotobacter vinelandii.

In several diazotrophic species of Proteobacteria, P(II) signal transduction proteins have been implicated in the regulation of nitrogen fixation in response to NH(4)(+) by several mechanisms. In Azotobacter vinelandii, expression of nifA, encoding the nif-specific activator, is constitutive, and thus, regulation of NifA activity by the flavoprotein NifL appears to be the primary level of nitrogen control. In vitro and genetic evidence suggests that the nitrogen response involves the P(II)-like GlnK protein and GlnD (uridylyltransferase/uridylyl-removing enzyme), which reversibly uridylylates GlnK in response to nitrogen limitation. Here, the roles of GlnK and GlnK-UMP in A. vinelandii were studied to determine whether the Nif (-) phenotype of glnD strains was due to an inability to modify GlnK, an effort previously hampered because glnK is an essential gene in this organism. A glnKY51F mutation, encoding an unuridylylatable form of the protein, was stable only in a strain in which glutamine synthetase activity is not inhibited by NH(4)(+), suggesting that GlnK-UMP is required to signal adenylyltransferase/adenylyl-removing enzyme-mediated deadenylylation. glnKY51F strains were significantly impaired for diazotrophic growth and expression of a nifH-lacZ fusion. NifL interacted with GlnK and GlnKY51F in a yeast two-hybrid system. Together, these data are consistent with those obtained from in vitro experiments (Little et al., EMBO J., 19:6041-6050, 2000) and support a model for regulation of NifA activity in which unmodified GlnK stimulates NifL inhibition and uridylylation of GlnK in response to nitrogen limitation prevents this function. This model is distinct from one proposed for the related bacterium Klebsiella pneumoniae, in which unmodified GlnK relieves NifL inhibition instead of stimulating it.