The flexible N-terminus of BchL autoinhibits activity through interaction with its [4Fe-4S] cluster and released upon ATP binding.

A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide to chlorophyllide, catalyzed by dark-operative protochlorophyllide oxidoreductase. Dark-operative protochlorophyllide oxidoreductase contains two [4Fe-4S]-containing component proteins (BchL and BchNB) that assemble upon ATP binding to BchL to coordinate electron transfer and protochlorophyllide reduction. But the precise nature of the ATP-induced conformational changes is poorly understood. We present a crystal structure of BchL in the nucleotide-free form where a conserved, flexible region in the N-terminus masks the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions in this region produce a hyperactive enzyme complex, suggesting a role for the N-terminus in autoinhibition. Hydrogen-deuterium exchange mass spectrometry shows that ATP binding to BchL produces specific conformational changes leading to release of the flexible N-terminus from the docking interface. The release also promotes changes within the local environment surrounding the [4Fe-4S] cluster and promotes BchL-complex formation with BchNB. A key patch of amino acids, Asp-Phe-Asp (the 'DFD patch'), situated at the mouth of the BchL ATP-binding pocket promotes intersubunit cross stabilization of the two subunits. A linked BchL dimer with one defective ATP-binding site does not support protochlorophyllide reduction, illustrating nucleotide binding to both subunits as a prerequisite for the intersubunit cross stabilization. The masking of the [4Fe-4S] cluster by the flexible N-terminal region and the associated inhibition of the activity is a novel mechanism of regulation in metalloproteins. Such mechanisms are possibly an adaptation to the anaerobic nature of eubacterial cells with poor tolerance for oxygen.

Kidney transplant outcomes in children and adolescents with systemic lupus erythematosus.

BACKGROUND: Literature supports equivalent kidney transplant outcomes in adults with systemic lupus erythematosus (SLE) compared with those without SLE. However, there are conflicting and scant data on kidney transplant outcomes, as well as controversy over optimal timing of transplantation, in children and adolescents with SLE. METHODS: Analysis included kidney-only transplant recipients aged 2-21 years from 2000 to 2017 enrolled in the Organ Procurement and Transplant Network (OPTN). The relationship between diagnosis (SLE n = 457, non-SLE glomerular disease n = 4492, and non-SLE non-glomerular disease n = 5605) and transplant outcomes was evaluated. The association between dialysis time and outcomes was analyzed in the SLE group only. RESULTS: In adjusted models, SLE had higher mortality compared with non-SLE glomerular recipients (HR 1.24 CI 1.07-1.44) and non-glomerular recipients (HR 1.42 CI 1.20-1.70). SLE was associated with higher graft failure compared with non-SLE glomerular (HR 1.42 CI 1.20-1.69) and non-glomerular disease (HR 1.67 CI 1.22-2.28). SLE had a higher risk of acute rejection at 1 year compared with non-glomerular disease (HR 1.39 CI 1.03-1.88). There was a decreased risk of delayed graft function compared with non-SLE glomerular disease (HR 0.54, CI 0.36-0.82). There were no significant associations between dialysis time and transplant outcomes in the SLE group. CONCLUSION: SLE in children and adolescents is associated with worse patient and graft survival compared with non-SLE diagnoses. Outcomes in children and adolescents with SLE are not associated with dialysis time. Further studies are needed to assess implications of potential earlier transplantation and shorter time on dialysis prior to transplantation.

Substrate recognition induces sequential electron transfer across subunits in the nitrogenase-like DPOR complex.

A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), catalyzed by dark-operative protochlorophyllide oxidoreductase (DPOR). DPOR is made of electron donor (BchL) and acceptor (BchNB) component proteins. BchNB is further composed of two subunits each of BchN and BchB arranged as an α2ß2 heterotetramer with two active sites for substrate reduction. Such oligomeric architectures are found in several other electron transfer (ET) complexes, but how this architecture influences activity is unclear. Here, we describe allosteric communication between the two identical active sites in Rhodobacter sphaeroides BchNB that drives sequential and asymmetric ET. Pchlide binding to one BchNB active site initiates ET from the pre-reduced [4Fe-4S] cluster of BchNB, a process similar to the deficit spending mechanism observed in the structurally related nitrogenase complex. Pchlide binding in one active site is recognized in trans by an Asp-274 from the opposing half, which is positioned to serve as the initial proton donor. A D274A variant DPOR binds to two Pchlide molecules in the BchNB complex, but only one is bound productively, stalling Pchlide reduction in both active sites. A half-active complex combining one WT and one D274A monomer also stalled after one electron was transferred in the WT half. We propose that such sequential electron transfer in oligomeric enzymes serves as a regulatory mechanism to ensure binding and recognition of the correct substrate. The findings shed light on the functional advantages imparted by the oligomeric architecture found in many electron transfer enzymes.

Infiltrating Kaposi sarcoma presenting as acute kidney injury: An unexpected consequence of deliberate hepatitis C-positive organ transplantation.

Kaposi sarcoma (KS) following kidney transplantation can result from recipient reactivation of latent human herpesvirus 8 (HHV-8) infection or activation of donor-acquired HHV-8 infection. Post-transplant KS typically manifests with cutaneous pathology, but rare cases of renal allograft involvement have been reported. We describe two cases of donor-derived HHV-8 infection in two hepatitis C (HCV) viremia-negative transplant recipients who each received a kidney from a donor with HCV viremia. One recipient did not develop KS while the other presented with acute kidney injury caused by extensive KS infiltration of the renal parenchyma and metastatic disease. This report reviews the literature for cases of KS involving the renal allograft and highlights an unexpected consequence of deliberate HCV-positive organ transplantation.

Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein.

Structural and spectroscopic analysis of iron-sulfur [Fe-S] cluster-containing proteins is often limited by the occupancy and yield of recombinantly produced proteins. Here we report that Escherichia coli BL21(DE3), a strain routinely used to overproduce [Fe-S] cluster-containing proteins, has a nonfunctional Suf pathway, one of two E. coli [Fe-S] cluster biogenesis pathways. We confirmed that BL21(DE3) and commercially available derivatives carry a deletion that results in an in-frame fusion of sufA and sufB genes within the sufABCDSE operon. We show that this fusion protein accumulates in cells but is inactive in [Fe-S] cluster biogenesis. Restoration of an intact Suf pathway combined with enhanced suf operon expression led to a remarkable (∼3-fold) increase in the production of the [4Fe-4S] cluster-containing BchL protein, a key component of the dark-operative protochlorophyllide oxidoreductase complex. These results show that this engineered "SufFeScient" derivative of BL21(DE3) is suitable for enhanced large-scale synthesis of an [Fe-S] cluster-containing protein.IMPORTANCE Large quantities of recombinantly overproduced [Fe-S] cluster-containing proteins are necessary for their in-depth biochemical characterization. Commercially available E. coli strain BL21(DE3) and its derivatives have a mutation that inactivates the function of one of the two native pathways (Suf pathway) responsible for cluster biogenesis. Correction of the mutation, combined with sequence changes that elevate Suf protein levels, can increase yield and cluster occupancy of [Fe-S] cluster-containing enzymes, facilitating the biochemical analysis of this fascinating group of proteins.

Optical Detection of Interleukin-6 Using Liquid Janus Emulsions Using Hyperthermophilic Affinity Proteins.

When equal volumes of two immiscible liquids are mixed (e.g., a hydrocarbon and a fluorocarbon), Janus droplets can form in an aqueous solution. In a gravity-aligned Janus droplet, the boundary between the two phases is flat and thus optically transparent when viewed from above. When tipped due to interactions with an analyte (i.e., agglutination), the resulting change in refraction and reflection yields an optical signal that can be detected and quantified. This study reports the detection and quantitation of interleukin-6 (IL-6) using emulsions functionalized at the hydrocarbon:aqueous interface with engineered proteins that bind IL-6 at high affinity and specificity. Hyperthermophilic affinity proteins (rcSso7d) are derived from thermophiles, giving them excellent thermal stability. Two rcSso7d affinity protein variants were synthesized with a noncanonical azide-functionalized amino acid to enable click chemistry to novel polymeric anchors embedded in the hydrocarbon phase. The two binding proteins recognize different epitopes, enabling the detection of both monomeric and dimeric IL-6 via agglutination. It is noteworthy that the rsSso7d protein variants, in addition to having superior thermal stability and facile recombinant synthesis in E. coli, show superior performance when compared to commercial antibodies for IL-6.

Kidney Allocation Issues in Liver Transplantation Candidates with Chronic Kidney Disease and Severe Kidney Liver Injury.

The number of liver transplant candidates with concomitant renal disease has been steadily rising since the implementation of MELD-based allocation in 2002. Consequently, the number of simultaneous liver-kidney (SLK) transplants being performed each year has also increased. However, the establishment of well-defined criteria for when to choose SLK over liver transplant alone has lagged behind. The lack of clear guidelines has worsened an already large shortage of transplantable kidneys. This article further explores the rationale for and outlines the implementation of the SLK allocation policy.

Influence of Donor Race and Donor-recipient Race-matching on Pediatric Kidney Transplant Outcomes.

Existing literature has demonstrated the significant relationship between race and kidney transplant outcomes; however, there are conflicting and limited data on the influence of donor race or donor-recipient race-matching on pediatric kidney transplant outcomes. Methods: Analysis included kidney-only transplant recipients between ages 2 and 17 from 2000 to 2017 enrolled in the Organ Procurement and Transplantation Network and their associated donors. Multivariable regression models were used to compare outcomes by donor race and donor-recipient race-matched status. Results: Of the total 7343 recipients, 4458 (60.7%) recipients received a kidney from a White donor, 1009 (13.7%) from a Black donor, 1594 (21.7%) from Hispanic donor, and 169 (4.1%) from an Asian donor; 4089 (55.7%) were race-matched. No donor races were significantly associated with transplant outcomes (all P > 0.05). Race-matched status was not associated with graft failure (hazard ratio, 1.03; 95% confidence interval [CI] = 0.89-1.2; P = 0.68), mortality (hazard ratio, 1.1; 95% CI, 0.79-1.53; P = 0.56), acute rejection at 1 y (odds ratio, 0.94; 95% CI, 0.77-1.15; P = 0.53), or delayed graft function (odds ratio, 1.02; 95% CI, 0.80-1.29; P = 0.91). Conclusions: Neither donor race nor race-matched status is associated with better transplant outcomes. Further studies are necessary to confirm the impact of donor race and race-matching more fully on pediatric kidney transplant outcomes.

Methods for Heterologous Overproduction of Fe-S Proteins.

Proteins carrying iron-sulfur ([Fe-S]) clusters are critical to the basic metabolism of all organisms. Structural and biochemical investigations of many such [Fe-S] cluster proteins depend on recombinant overproduction using heterologous bacterial hosts such as Escherichia coli . Here, we describe a detailed procedure for the overproduction and purification of two oxygen-sensitive component proteins of the dark-operative protochlorophyllide oxidoreductase (DPOR) complex. The method relies on an engineered Escherichia coli cell line carrying a correction in its genome to restore the loss of a key [Fe-S] cluster biogenesis pathway. The method can also be potentially adapted for the overproduction of other Fe-S proteins.

Exploration of the antibiotic potentiating activity of indolglyoxylpolyamines.

A series of substituted di-indolglyoxylamido-spermine analogues were prepared and evaluated for intrinsic antimicrobial properties and the ability to enhance antibiotic action. As a compound class, intrinsic activity was typically observed towards Gram-positive bacteria and the fungus Cryptococcus neoformans, with notable exceptions being the 5-bromo- and 6-chloro-indole analogues which also exhibited modest activity (MIC 34-50 µM) towards the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae. Several analogues enhanced the activity of doxycycline towards the Gram-negative bacteria Pseudomonas aeruginosa, E. coli, K. pneumoniae and Acinetobacter baumannii. Of particular note was the identification of five antibiotic enhancing analogues (5-Br, 7-F, 5-Me, 7-Me, 7-OMe) which also exhibited low to no cytotoxicity and red blood cell haemolytic properties. The mechanisms of action of the 5-Br and 7-F analogues were attributed to the ability to disrupt the integrity of, and depolarize, bacterial membranes.

Dynamics and selective remodeling of the DNA-binding domains of RPA.

Replication protein A (RPA) coordinates important DNA metabolic events by stabilizing single-stranded DNA (ssDNA) intermediates, activating the DNA-damage response and handing off ssDNA to the appropriate downstream players. Six DNA-binding domains (DBDs) in RPA promote high-affinity binding to ssDNA yet also allow RPA displacement by lower affinity proteins. We generated fluorescent versions of Saccharomyces cerevisiae RPA and visualized the conformational dynamics of individual DBDs in the context of the full-length protein. We show that both DBD-A and DBD-D rapidly bind to and dissociate from ssDNA while RPA remains bound to ssDNA. The recombination mediator protein Rad52 selectively modulates the dynamics of DBD-D. These findings reveal how RPA-interacting proteins with lower ssDNA binding affinities can access the occluded ssDNA and remodel individual DBDs to replace RPA.

Renal Transplantation as a Platform for Teaching Residents Open Vascular Surgical Techniques: Effects on Early Graft Function.

BACKGROUND: Over the past decade, increases in vascular fellowships and the use of endovascular technology have decreased the general surgery residents' exposure to open vascular surgery. We sought to elucidate whether renal transplant is a safe way to teach general surgery residents the essential tenants of vascular surgery without adversely affecting early patient outcomes. METHODS: All solitary, adult deceased donor kidney transplants performed at the University of Wisconsin from 2011 through 2016 were identified and divided into a resident-assist (RA) and fellow-assist cohorts (FA). DGF, defined by the requirement of dialysis within 1 week of transplant, was the primary outcome. Early graft survival and postoperative complications were considered the secondary endpoints. RESULTS: Of the 774 total cases, there were 228 (29.5%) in the RA cohort and 546 (70.5%) in the FA cohort. The RA and FA cohorts had comparable characteristics, except for a nonclinically significant difference in mean donor creatinine (0.96 vs 0.88mg/dL, p = 0.03). RA cases had a similar DGF rate compared to FA cases (25% vs 26%, p = 0.93). Additionally, there was no difference in 2-year graft survival (93.7% vs 95.5%, p = 0.38), nor the rates of graft thromboses (0.4% vs 0.7%, p = 0.65), incisional hernias (0.9% vs 1.8%, p = 0.35), and ureteral strictures (2.2% vs 1.6%, p = 0.55) between the 2 cohorts. CONCLUSIONS: Resident involvement in renal transplantation has no effect on DGF and early allograft function. Though the procedural involvement of each resident in a case is variable, it seems to be a safe way to teach retroperitoneal vascular exposure and anastomotic techniques.

Activities performed and treatments conducted before consultation with a spine surgeon: are patients and clinicians following evidence-based clinical practice guidelines?

BACKGROUND CONTEXT: Clinical practice guidelines (CPGs) are designed to ensure that evidence-based treatment is easily put into action. Whether patients and clinicians follow these guidelines is equivocal. PURPOSE: The objectives of this study were to examine how many patients complaining of low back pain (LBP) underwent evidence-based medical interventional treatment in line with CPG recommendations before consultation with a spine surgeon, and to evaluate any associations between adherence to CPG recommendations and baseline factors. STUDY DESIGN/SETTING: This is a cross-sectional cohort analysis at a tertiary care center. PATIENT SAMPLE: A total of 229 patients were referred for surgical consultation for an elective lumbar spinal condition. OUTCOME MEASURES: The outcome measures include the number of CPG-recommended treatments undertaken by patients at or before the time of referral, the validated pain score, the EuroQol-5D (EQ-5D) health status, and the Oswestry Disability Index (ODI) score. METHODS: Questionnaires assessing demographic and functional characteristics as well as overall health care use were sent to patients immediately after their referral was received by the surgeon's office. RESULTS: Medications were the most common modality before consultation (74.2% of patients), of which 46.3% received opioids. The number of medications taken was significantly related to a higher ODI score (R=0.23, p=.0004), a higher pain score (R=0.15, p=.026), and a lower EQ-5D health status (R=-0.15, p=.024). In contrast, a lower pain score (7.2 vs. 7.7, p=.037) and a lower ODI score (26.6 vs. 29.9, p=.0023) were associated with performing adequate amounts of exercise. There was a significant association between lower numbers of treatments received and higher numerical pain rating scores (R=-0.14, p=.035). The majority (61.1%) of patients received two or less forms of treatment. CONCLUSIONS: Evidence-based medical interventional treatments for patients with LBP are not being taken advantage of before spine surgery consultation. If more patients were to undertake CPG-endorsed conservative modalities, it may result in fewer unnecessary referrals from primary care physicians, and patients might not deteriorate as much while lingering on long wait lists. Further studies incorporating knowledge translation or health system pathway changes are necessary.

An Evaluation of the Safety and Efficacy of Simultaneous Bilateral Nephrectomy and Renal Transplantation for Polycystic Kidney Disease: A 20-Year Experience.

BACKGROUND: Many strategies regarding timing of native nephrectomies exist for patients with symptomatic polycystic kidney disease (PCKD). METHODS: This is a single-center, retrospective study of 594 adults with PCKD who had renal transplants from 1994 to 2014. Three groups were analyzed: renal transplant-only recipients (tx alone), recipients of simultaneous bilateral nephrectomies and transplant (simultaneous), and recipients with pretransplant bilateral nephrectomies (pre). The primary outcome was graft survival. Secondary outcomes included postoperative complications. RESULTS: Five hundred sixty-five adults with PCKD received kidney transplants (303 tx alone, 161 simultaneous, 27 pre). Ten-year posttransplant graft survival was 68.5%, 63.6%, and 65.7% for tx alone, simultaneous, and precohorts (P = 0.86). No statistically significant differences were observed in rates of postoperative ileus, deep vein thrombosis, small bowel obstruction, urinary stricture, urine leak, hernia formation, and delayed graft function. More wound complications were seen in prepatients (25.9% vs 11.1% tx alone, 5.1% simultaneous; P = 0.03), whereas simultaneous patients had a lower incidence of lymphocele (1.3% vs 11.1% pre, 10.2% tx-alone; P = 0.002). Importantly, simultaneous patients had more renal vascular thromboses (4.4% vs 1.3% tx alone, 0% pre; P = 0.04). 16.3% of renal transplant alone patients required nephrectomy at 10 years follow-up. Twenty-nine patients were referred for transplant having had nephrectomies and were ultimately not transplanted. In 4 of these patients who had data available for analysis, the mean panel-reactive antibody significantly increased after nephrectomy was performed. CONCLUSIONS: Simultaneous bilateral nephrectomy can be safely performed at the time of renal transplantation, however, carries a significantly increased risk of renal vascular thrombosis.

Immunologic studies in two patients with persistent lymphocytic thyroiditis, thyrotoxicosis, and low radioactive iodine uptake.

Two patients with persistent lymphocytic thyroiditis and thyrotoxicosis were studied. Both patients presented with severe hyperthyroidism of nine months' duration and had nontender, small thyroid glands. Uptake of radioactive iodine (131I) was consistently low. Serum thyroxine and triiodothyronine levels remained elevated without remission until thyroidectomy. The serum thyroglobulin level was normal, but testing for microsomal antibody gave weakly positive results in one case. Thyroglobulin and thyroid stimulatory antibodies were not found. The ratio of helper to suppressor T cells was elevated in one case. Neither patient showed response to propranolol, prednisone, or iodine. Light microscopic and immunohistologic studies showed severe lymphocytic thyroiditis with formation of secondary lymphoid follicles. Lymphocytes were predominately T cells (OKT11-positive), primarily helper/inducer T cells (OKT4-positive). Hyperplastic nodules contained high immunoreactive thyroglobulin and thyroxine levels. Aberrant thymus was seen within the thyroid. These studies suggest the possibility of intrathyroidal stimulation and hydrolysis of thyroglobulin within thyroid cells and also support the hypothesis that T and B cell immunoregulatory defects are important in the pathogenesis of this disease.

Activation of human gingival collagenase.

To study the regulation of collagen degradation in periodontium, human gingival homogenate was incubated at 36 degrees C and the release of hydroxyproline was assayed as a measure of collagenase activity. Phenylmethylsulfonyl fluoride, soybean trypsin inhibitor and serum albumin inhibited the in vitro collagenolysis while p-aminophenylmercuric acetate, a sulfhydryl reagent, increased the degradation. When latent collagenase obtained from gingival fibroblast culture was added to the incubation a marked increase in the collagen degradation was found. This increase was prevented by phenylmethylsulfonyl fluoride. The data suggests that collagenase may exist in gingiva partly in a latent form and its activation may be brought about by 2 mechanisms. A serine proteinase present in tissue may activate collagenase by producing a limited cleavage, or the activation may occur through a reaction that involves the sulfhydryl groups of the collagenase molecule.