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Competing Interests: Disclosures The authors have no conflicts of interest to disclose.

Background: Esophagectomy is an important, but potentially morbid, operation used to treat benign and malignant conditions that may significantly impact patient quality of life (QOL). Patient-reported outcomes (PROs) are measures of QOL that come directly from patient self-report. This study characterizes patterns of change and recovery in PROs in the first year after esophagectomy., Methods: Longitudinal QOL scores measuring physical function, pain, and dyspnea were obtained from esophagectomy patients during all clinic visits. PRO scores were obtained using the National Institutes of Health-sponsored Patient-Reported Outcomes Measurement Information System from April 2018 to February 2021. Mean PRO scores over 100 days after surgery were compared with baseline PRO scores using mixed-effects modeling with compound symmetry correlational structure., Results: One hundred three patients with PRO results were identified. Reasons for esophagectomy were malignancy (87.4%), achalasia (5.8%), stricture (5.8%), and dysplasia (1.0%). When comparing mean PRO scores at visits ≤ 50 days after surgery with preoperative PRO scores, physical function scores declined by 27.3% (P < .001), whereas dyspnea severity and pain interference scores had increased by 24.5% (P < .001) and 17.1% (P < .001), respectively. Although recovery occurred over the course of the 100 days after surgery, mean physical function scores and dyspnea scores were still 12.7% (P = .02) and 26.4% (P = .001) worse, respectively, than mean preoperative levels., Conclusions: Despite declines in QOL scores immediately after esophagectomy, recovery back toward baseline was observed during the first 100 days. These findings are of considerable importance when counseling patients regarding esophagectomy, tracking recovery, and implementing quality improvement initiatives. Further long-term follow-up is needed to determine recovery beyond 100 days., (Copyright © 2023 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Betanin, a natural food color and the only betalain, is approved for use in pharmaceutical and food industries as natural antioxidative and preservative agent, respectively. However, the antioxidant power and health-promoting properties of betanin have been disregarded due to its low stability in physiological conditions. Therefore, this study is designed to synthesize and evaluate in vitro pharmacological characteristics of betanin-encapsulated chitosan nanoparticles (ChBetNPs). ChBetNPs were synthesized by ionic gelation method and characterized by DLS, UV, FTIR, SEM and zeta potential analysis. The encapsulation efficiency (EE) and in vitro release kinetics were analyzed using spectrophotometric technique for quantifying the encapsulated amount of betanin in ChBetNPs as a function of time. The antioxidant activity of ChBetNPs was analyzed by DPPH and H 2 O 2 radical scavenging assays, anti-inflammatory activity by protein denaturation and human RBCs stabilization assays, and anti-acetylcholinesterase activity using standard protocol with minor modifications. Unloaded chitosan nanoparticles (CSNPs) were found to be sized at 161.4 ± 5.75 nm while an increase in the size to 270.3 ± 8.50 nm was noticed upon encapsulating betanin. EE of ChBetNPs was measured to be ∼87.5%. The IC 50 of ChBetNPs depicted significant free radical scavenging activities as compared to CSNPs. Similarly, a strong anti-inflammatory activity of ChBetNPs was noted. Significant decrease in acetylcholinesterase activity by ChBetNPs was measured (IC 50 0.5255 μg/mL vs. control 26.09 μg/mL). The vegetables coated with 3% ChBetNPs showed decreased weight loss as compared to uncoated control. ChBetNPs was shown to exhibit strong antioxidant, anti-inflammatory and anti-acetylcholinesterase activities thus making it a significant therapeutic agent for the management of Alzheimer's disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Gallbladder cancer (GBC) is the most common biliary malignancy frequently metastatic at diagnosis with poor prognosis. While surgery remains the standard for early-stage GBC, the role of surgery in patients with metastatic gastrointestinal cancers is expanding due to improvements in systemic therapies. We sought to evaluate the survival of patients with stage IV GBC undergoing surgery in an era of improved multi-agent systemic therapy., Methods: A retrospective review of the National Cancer Database was performed. Patients with stage IV GBC who underwent systemic therapy were included. Patients who received radiation therapy, palliative therapy or had missing survival data were excluded. Univariable and multivariable analysis was performed., Results: 4,145 patients were identified between 2004 and 2016. Mean age was 69. Surgery combined with systemic therapy predicted improved median survival compared with chemotherapy alone (11.1mo versus 6.8mo, HR 0.65, p < 0.001). Additionally, receipt of treatment after 2011 predicted improved survival (HR 0.86, p < 0.001). Patients treated with multi-agent chemotherapy in combination with surgery were associated with the greatest hazard ratio benefit (0.40, p < 0.001) versus single agent therapy alone., Conclusion: Patients with stage IV gallbladder cancer treated with a combination of surgery and chemotherapy are associated with an improved overall survival compared to chemotherapy alone. Patients receiving care during the more recent era demonstrated improved survival. These results support a role for surgery in selected patients with stage IV gallbladder cancer receiving chemotherapy., Competing Interests: Declaration of competing interest Research reported in this publication was supported in part by the Biostatistics Shared Resource of Winship Cancer Institute of Emory University and NIH/NCI under award number P30CA138292. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health., (Copyright © 2022 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Ecological restoration of coal mine degraded soils across arid and semi-arid environments worldwide remains particularly challenging. We used a combination of greenhouse and field experiments to assess the potential role of a woody species, Ulmus pumila, in the restoration of degraded soils associated with coal-mining activities in the northwest China. We investigated how various combinations of water-nitrogen-phosphorus (W-N-P) resources affect multiple growth parameters in U. pumila. We found that several plant growth traits significantly improved with W-N applications, regardless of P inputs. Moderate-to-highest W-N-P doses increased net photosynthesis and transpiration rates, water use efficiency, stomatal conductance, chlorophyll and carotenoid contents under greenhouse conditions. A combination of high W together with low N-P applications led to high relative water content and net photosynthetic rates under field conditions. Increasing of N-P doses under W-shortage condition, aided U. pumila to enhance osmotic adjustments by increasing contents of proline and soluble sugar and also boost the activity of superoxide dismutase, peroxidase and catalase in leaf tissues to reduce accumulation of reactive oxygen species and malondialdehyde content in all conditions of greenhouse and field. Our study is the first to assess the optimum W-N-P resources in U. pumila and demonstrate that optimum growth performance could be obtained under W supplements corresponding to 90 mm year -1 , N and P at 110 and 45 kg ha -1 , respectively, under field condition. These findings can have far reaching implications for vegetation restoration of degraded areas associated with coal-mining activities across arid and semi-arid regions worldwide., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Postoperative emergency department (ED) visits represent fragmented care, are costly, and often evolve into readmission. Readmission rates after pancreatoduodenectomy (PD) are defined, while ED visits following PD are not. We examined the pattern of 30-day post-discharge ED visits for PD patients., Methods: A quaternary institutional database analysis of adult patients who underwent PD between 2010-2017 was reviewed for ED utilization within 30 days from discharge., Results: Of the 1,004 patients who underwent PD, 12% (N = 117) patients sought care in the ED within 30 days from postoperative discharge. The median time to ED presentation was 5 days post-discharge (IQR 3-9). Half of ED visits occurred during nights and weekends (N = 59, 50%). Of ED-utilizing patients, 64% (N = 76) were admitted to the hospital and 29% (N = 34) were discharged from the ED. ED visits were associated with a Clavien-Dindo Classification of 0 in 10.2% (N = 13) of patients, I-II in 62.4% (N = 73), and III-V in 26.5% (N = 31)., Discussion: Post-discharge ED utilization is a novel quality metric and represents a potential target population for reducing hospital readmissions. Over two-thirds (72%) of ED visits were associated with low acuity complications, and promoting institutional strategies addressing postoperative ED visits may improve patient care and efficient utilization of healthcare resources., (Copyright © 2019 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

In the past few years, biologically synthesized silver nanoparticles (AgNPs) have been standout amongst the most utilized nanoparticles both in the field of therapeutics and clinical practices. Therefore, the current study aimed to synthesize AgNPs for the first time using aqueous root extracts of important plants of Pakistan i.e. Bergenia ciliata, Bergenia stracheyi, Rumex dantatus and Rumex hastatus and characterize them. In addition, antibacterial activity of synthesized AgNPs at 30-150 μg/well was assessed using well diffusion method against Staphylococcus aureus, Staphylococcus haemolyticus, Bacillus cereus, Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa bacterial strains that are considered most harmful bacteria for human beings. The characterization of synthesized AgNPs showed the absorption maxima ranged from 434 to 451 nm and XRD confirmed the crystalline nature of AgNPs as well as FTIR elucidated the involvement of biomolecules for reduction and capping of AgNPs. SEM determined the average size of AgNPs ranging from 25 to 73 nm and strong signals of silver were captured in EDX images. The result of antibacterial activity showed that only aqueous root extracts of all selected plants were inactive against all the tested bacterial strains. However, importantly, direct relationship between zone of inhibition of S. aureus, S. typhi and P. aeruginosa was found with increasing concentration of AgNPs of each selected plant. Moreover, S. haemolyticus was only inhibited by R. hastatus based AgNPs at only high concentrations and E. coli was inhibited by R. dantatus and R. hastatus based AgNPs. However, B. cereus was not inhibited by any AgNPs except R. hastatus and R. hastatus based AgNPs have greater antibacterial potential among all the synthesized AgNPs. These results suggest that synthesized AgNPs have improved antibacterial potential of root extracts of each selected plant and these synthesized AgNPs could be used in pharmaceutical and homeopathic industry for the cure of human diseases., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Chronic myeloid leukemia (CML) stem/progenitor cells (SPCs) express a transcriptional program characteristic of proliferation, yet can achieve and maintain quiescence. Understanding the mechanisms by which leukemic SPCs maintain quiescence will help to clarify how they persist during long-term targeted treatment. We have identified a novel BCR-ABL1 protein kinase-dependent pathway mediated by the upregulation of hsa-mir183 , the downregulation of its direct target early growth response 1 (EGR1), and, as a consequence, upregulation of E2F1. We show here that inhibition of hsa-mir183 reduced proliferation and impaired colony formation of CML SPCs. Downstream of this, inhibition of E2F1 also reduced proliferation of CML SPCs, leading to p53-mediated apoptosis. In addition, we demonstrate that E2F1 plays a pivotal role in regulating CML SPC proliferation status. Thus, for the first time, we highlight the mechanism of hsa-mir183 /EGR1-mediated E2F1 regulation and demonstrate this axis as a novel, critical factor for CML SPC survival, offering new insights into leukemic stem cell eradication., (© 2018 by The American Society of Hematology.)

Comprehensive description with illustrations of the 4 biliary reconstruction options for bile duct injury in patients with history of Roux-en-Y gastric bypass., (Copyright © 2017 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.)

Purpose: To determine the frequency of emergency department (ED) visits for nonurgent and urgent ocular conditions and risk factors associated with ED use for nonurgent and urgent ocular problems., Design: Retrospective, longitudinal cohort analysis., Participants: All enrollees aged 21 years or older in a United States managed care network during 2001-2014., Methods: We identified all enrollees visiting an ED for ocular conditions identified by International Classification of Diseases, billing codes. Diagnosis is well-described as urgent, nonurgent, or other. We assessed the frequency of ED visits for urgent and nonurgent ocular conditions and how they changed over time. Next, we performed multivariable Cox regression modeling to determine factors associated with visiting an ED for urgent or nonurgent ocular conditions., Main Outcome Measures: Hazard ratios (HRs) with 95% confidence intervals (CIs) of visiting an ED for urgent or nonurgent ocular conditions., Results: Of the 11 160 833 enrollees eligible for this study, 376 680 (3.4%) had 1 or more ED visit for an eye-related problem over a mean ± standard deviation of 5.4±3.3 years' follow-up. Among these enrolled, 86 473 (23.0%) had 1 or more ED visits with a nonurgent ocular condition and 25 289 (6.7%) had at least 1 ED visit with an urgent ocular condition. Use of the ED for nonurgent ocular problems was associated with younger age (P < 0.0001 for all comparisons), black race or Latino ethnicity (P < 0.0001 for both), male sex (P < 0.0001), lower income (P < 0.0001 for all comparisons), and those who frequently sought treatment at an ED for nonophthalmologic medical problems in a given year (P < 0.0001). Enrollees with established eye care professionals had a 10% reduced hazard of visiting the ED for nonurgent ocular conditions (adjusted HR, 0.90; 95% CI, 0.88-0.92; P < 0.0001)., Conclusions: Nearly one-quarter of enrollees who visited the ED for an ocular problem received a diagnosis of a nonurgent condition. Better educating and incentivizing patients to seek care for nonurgent ocular diseases in an office-based setting could yield considerable cost savings without adversely affecting health outcomes and could allow EDs to better serve patients with more severe conditions., (Copyright © 2017 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Heparan sulfate proteoglycans (HSPGs) are found in the basement membrane and at the cell-surface where they modulate the binding and activity of a variety of growth factors and other molecules. Most of the functions of HSPGs are mediated by the variable sulfated glycosaminoglycan (GAG) chains attached to a core protein. Sulfation of the GAG chain is key as evidenced by the renal agenesis phenotype in mice deficient in the HS biosynthetic enzyme, heparan sulfate 2-O sulfotransferase (Hs2st; an enzyme which catalyzes the 2-O-sulfation of uronic acids in heparan sulfate). We have recently demonstrated that this phenotype is likely due to a defect in induction of the metanephric mesenchyme (MM), which along with the ureteric bud (UB), is responsible for the mutually inductive interactions in the developing kidney (Shah et al., 2010). Here, we sought to elucidate the role of variable HS sulfation in UB branching morphogenesis, particularly the role of 6-O sulfation. Endogenous HS was localized along the length of the UB suggesting a role in limiting growth factors and other molecules to specific regions of the UB. Treatment of cultures of whole embryonic kidney with variably desulfated heparin compounds indicated a requirement of 6O-sulfation in the growth and branching of the UB. In support of this notion, branching morphogenesis of the isolated UB was found to be more sensitive to the HS 6-O sulfation modification when compared to the 2-O sulfation modification. In addition, a variety of known UB branching morphogens (i.e., pleiotrophin, heregulin, FGF1 and GDNF) were found to have a higher affinity for 6-O sulfated heparin providing additional support for the notion that this HS modification is important for robust UB branching morphogenesis. Taken together with earlier studies, these findings suggest a general mechanism for spatio-temporal HS regulation of growth factor activity along the branching UB and in the developing MM and support the view that specific growth factor-HSPG interactions establish morphogen gradients and function as developmental switches during the stages of epithelial organogenesis (Shah et al., 2004)., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We describe the efforts of the Maternal Health Team, which was formed to address the needs of pregnant and breastfeeding women during the Centers for Disease Control and Prevention's (CDC's) 2009 pandemic influenza A (2009 H1N1) emergency response. We examined the team's activities, constructed a timeline of key pandemic events, and analyzed the Maternal Health 2009 H1N1 inquiry database. During the pandemic response, 9 guidance documents that addressed the needs of pregnant and breastfeeding women and their providers were developed by the Maternal Health Team. The Team received 4661 maternal health-related inquiries that came primarily from the public (75.5%) and were vaccine related (69.3%). Peak inquiry volume coincided with peak hospitalizations (October-November 2009). The Maternal Health 2009 H1N1 inquiry database proved useful to identify information needs of the public and health care providers during the pandemic., (Copyright © 2011 Mosby, Inc. All rights reserved.)

Embryonic kidney development begins with the outgrowth of the ureteric bud (UB) from the Wolffian duct (WD) into the adjacent metanephric mesenchyme (MM). Both a GDNF-dependent and GDNF-independent (Maeshima et al., 2007) pathway have been identified. In vivo and in vitro, the GDNF-dependent pathway is inhibited by BMPs, one of the factors invoked to explain the limitation of UB formation in the unbudded regions of the WD surrounding the UB. However, the exact mechanism remains unknown. Here a previously described in vitro system that models UB budding from the WD was utilized to study this process. Because Protein kinase A (PKA) activation has been shown to prevent migration, morphogenesis and tubulogenesis of epithelial cells (Santos et al., 1993), its activity in budded and non-budded portions of the GDNF-induced WD was analyzed. The level of PKA activity was 15-fold higher in the unbudded portions of the WD compared to budded portions, suggesting that PKA activity plays a key role in controlling the site of UB emergence. Using well-characterized PKA agonists and antagonists, we demonstrated that at various levels of the PKA-signaling hierarchy, PKA regulates UB outgrowth from the WD by suppressing budding events. This process appeared to be PKA-2 isoform specific, and mediated by changes in the duct rather than the surrounding mesenchyme. In addition, it was not due to changes in either the sorting of junctional proteins, cell death, or cell proliferation. Furthermore, the suppressive effect of cAMP on budding did not appear to be mediated by spread to adjacent cells via gap junctions. Conversely, antagonism of PKA activity stimulated UB outgrowth from the WD and resulted in both an increase in the number of buds per unit length of WD as well as a larger surface area per bud. Using microarrays, analysis of gene expression in GDNF-treated WDs in which the PKA pathway had been activated revealed a nearly 14-fold decrease in Ret, a receptor for GDNF. A smaller decrease in GFRα1. a co-receptor for GDNF, was also observed. Using Ret-null WDs, we were able to demonstrate that PKA regulated GDNF-dependent budding but not GDNF-independent pathway for WD budding. We also found that BMP2 was higher in unbudded regions of the GDNF-stimulated WD. Treatment of isolated WDs with BMP2 suppressed budding and resulted in a 3-fold increase in PKA activity. The data suggests that the suppression of budding by BMPs and possibly other factors in non-budded zones of the WD may be regulated in part by increased PKA activity, probably partially through downregulation of Ret/GFRα1 coreceptor expression., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Heparan sulfate proteoglycans (HSPGs) are central modulators of developmental processes likely through their interaction with growth factors, such as GDNF, members of the FGF and TGFbeta superfamilies, EGF receptor ligands and HGF. Absence of the biosynthetic enzyme, heparan sulfate 2-O-sulfotransferase (Hs2st) leads to kidney agenesis. Using a novel combination of in vivo and in vitro approaches, we have reanalyzed the defect in morphogenesis of the Hs2st(-)(/)(-) kidney. Utilizing assays that separately model distinct stages of kidney branching morphogenesis, we found that the Hs2st(-/-) UB is able to undergo branching and induce mesenchymal-to-epithelial transformation when recombined with control MM, and the isolated Hs2st null UB is able to undergo branching morphogenesis in the presence of exogenous soluble pro-branching growth factors when embedded in an extracellular matrix, indicating that the UB is intrinsically competent. This is in contrast to the prevailing view that the defect underlying the renal agenesis phenotype is due to a primary role for 2-O sulfated HS in UB branching. Unexpectedly, the mutant MM was also fully capable of being induced in recombination experiments with wild-type tissue. Thus, both the mutant UB and mutant MM tissue appear competent in and of themselves, but the combination of mutant tissues fails in vivo and, as we show, in organ culture. We hypothesized a 2OS-dependent defect in the mutual inductive process, which could be on either the UB or MM side, since both progenitor tissues express Hs2st. In light of these observations, we specifically examined the role of the HS 2-O sulfation modification on the morphogenetic capacity of the UB and MM individually. We demonstrate that early UB branching morphogenesis is not primarily modulated by factors that depend on the HS 2-O sulfate modification; however, factors that contribute to MM induction are markedly sensitive to the 2-O sulfation modification. These data suggest that key defect in Hs2st null kidneys is the inability of MM to undergo induction either through a failure of mutual induction or a primary failure of MM morphogenesis. This results in normal UB formation but affects either T-shaped UB formation or iterative branching of the T-shaped UB (possibly two separate stages in collecting system development dependent upon HS). We discuss the possibility that a disruption in the interaction between HS and Wnts (e.g. Wnt 9b) may be an important aspect of the observed phenotype. This appears to be the first example of a defect in the MM preventing advancement of early UB branching past the first bifurcation stage, one of the limiting steps in early kidney development., (Copyright 2010 Elsevier Inc. All rights reserved.)

In search of guiding principles involved in the branching of epithelial tubes in the developing kidney, we analyzed branching of the ureteric bud (UB) in whole kidney culture as well as in isolated UB culture independent of mesenchyme but in the presence of mesenchymally derived soluble factors. Microinjection of the UB lumen (both in the isolated UB and in the whole kidney) with fluorescently labeled dextran sulfate demonstrated that branching occurred via smooth tubular epithelial outpouches with a lumen continuous with that of the original structure. Epithelial cells within these outpouches cells were wedge-shaped with actin, myosin-2 and ezrin localized to the luminal side, raising the possibility of a "purse-string" mechanism. Electron microscopy and decoration of heparan sulfates with biotinylated FGF2 revealed that the basolateral surface of the cells remained intact, without the type of cytoplasmic extensions (invadopodia) that are seen in three-dimensional MDCK, mIMCD, and UB cell culture models of branching tubulogenesis. Several growth factor receptors (i.e., FGFR1, FGFR2, c-Ret) and metalloproteases (i.e., MT1-MMP) were localized toward branching UB tips. A large survey of markers revealed the ER chaperone BiP to be highly expressed at UB tips, which, by electron microscopy, are enriched in rough endoplasmic reticulum and Golgi, supporting high activity in the synthesis of transmembrane and secretory proteins at UB tips. After early diffuse proliferation, proliferating and mitotic cells were mostly found within the branching ampullae, whereas apoptotic cells were mostly found in stalks. Gene array experiments, together with protein expression analysis by immunoblotting, revealed a differential spatiotemporal distribution of several proteins associated with epithelial maturation and polarization, including intercellular junctional proteins (e.g., ZO-1, claudin-3, E-cadherin) and the subapical cytoskeletal/microvillar protein ezrin. In addition, Ksp-cadherin was found at UB ampullary cells next to developing outpouches, suggesting a role in epithelial-mesenchymal interactions. These data from the isolated UB culture system support a model where UB branching occurs through outpouching possibly mediated by wedge-shaped cells created through an apical cytoskeletal purse-string mechanism. Additional potential mechanisms include (1) differential localization of growth factor receptors and metalloproteases at tips relative to stalks; (2) creation of a secretory epithelium, in part manifested by increased expression of the ER chaperone BiP, at tips relative to stalks; (3) after initial diffuse proliferation, coexistence of a balance of proliferation vs. apoptosis favoring tip growth with a very different balance in elongating stalks; and (4) differential maturation of the tight and adherens junctions as the structures develop. Because, without mesenchyme, both lateral and bifid branching occurs (including the ureter), the mesenchyme probably restricts lateral branching and provides guidance cues in vivo for directional branching and elongation as well as functioning to modulate tubular caliber and induce differentiation. Selective cadherin, claudin, and microvillar protein expression as the UB matures likely enables the formation of a tight, polarized differentiated epithelium. Although, in vivo, metanephric mesenchyme development occurs simultaneously with UB branching, these studies shed light on how (mesenchymally derived) soluble factors alone regulate spatial and temporal expression of morphogenetic molecules and processes (proliferation, apoptosis, etc.) postulated to be essential to the UB branching program as it forms an arborized structure with a continuous lumen.

Glycosaminoglycans in the form of heparan sulfate proteoglycans (HSPG) and chondroitin sulfate proteoglycans (CSPG) are required for normal kidney organogenesis. The specific roles of HSPGs and CSPGs on ureteric bud (UB) branching morphogenesis are unclear, and past reports have obtained differing results. Here we employ in vitro systems, including isolated UB culture, to clarify the roles of HSPGs and CSPGs on this process. Microarray analysis revealed that many proteoglycan core proteins change during kidney development (syndecan-1,2,4, glypican-1,2,3, versican, decorin, biglycan). Moreover, syndecan-1, syndecan-4, glypican-3, and versican are differentially expressed during isolated UB culture, while decorin is dynamically regulated in cultured isolated metanephric mesenchyme (MM). Biochemical analysis indicated that while both heparan sulfate (HS) and chondroitin sulfate (CS) are present, CS accounts for approximately 75% of the glycosaminoglycans (GAG) in the embryonic kidney. Selective perturbation of HS in whole kidney rudiments and in the isolated UB resulted in a significant reduction in the number of UB branch tips, while CS perturbation has much less impressive effects on branching morphogenesis. Disruption of endogenous HS sulfation with chlorate resulted in diminished FGF2 binding and proliferation, which markedly altered kidney area but did not have a statistically significant effect on patterning of the ureteric tree. Furthermore, perturbation of GAGs did not have a detectable effect on FGFR2 expression or epithelial marker localization, suggesting the expression of these molecules is largely independent of HS function. Taken together, the data suggests that nonselective perturbation of HSPG function results in a general proliferation defect; selective perturbation of specific core proteins and/or GAG microstructure may result in branching pattern defects. Despite CS being the major GAG synthesized in the whole developing kidney, it appears to play a lesser role in UB branching; however, CS is likely to be integral to other developmental processes during nephrogenesis, possibly involving the MM. A model is presented of how, together with growth factors, heterogeneity of proteoglycan core proteins and glycosaminoglycan sulfation act as a switching mechanism to regulate different stages of the branching process. In this model, specific growth factor-HSPG combinations play key roles in the transitioning between stages and their maintenance.

Gap junction channels formed by the connexin43 protein are considered to play crucial roles in development and function because they allow the direct cell-to-cell exchange of molecules that mediate multiple signaling events. Previous results have shown that connexin43 channels are intricately gated by phosphorylation and that disruption of this regulation gives rise to severe heart malformations and defects of laterality in human, chick and frog. Here we report the identification of connexin43 gene mutations that represent a minor population of connexin43 alleles, which could be reliably detected by using denaturing gradient gel electrophoresis (DGGE) to visualize normal and mutant DNAs that were separately sequenced. In contrast, sequencing of total PCR products without DGGE-pre-selection failed to consistently identify these mutations. Forty-six controls and 20 heart transplant recipients were examined in this study. In the latter group, 14 children had hypoplastic left heart syndrome (HLHS) in which connexin43 gene defects were detected in eight. The remaining six transplant patients with HLHS and all controls showed no defects. All eight HLHS children with gene defects had the same four substitutions: two that were silent polymorphisms, and two that were missense, replacing arginine codons at positions 362 and 376 with codons for glutamines. All four of these substitutions are identical to the nucleotide sequence of the connexin43 pseudogene, suggesting the possibility of an illicit recombination. A breakpoint region was identified 5' to the mutation site in a 63bp domain that is 100% identical in the gene and pseudogene. Results from in vitro phosphorylation indicate that the absence of arginines 362 and 376 completely abolishes phosphorylation in the connexin43 channel regulation domain suggesting a possible mechanism for the pathologies associated with HLHS.

Nitrobenzene was reduced in a solution containing ferredoxin NADP oxidoreductase (FNR) from spinach leaves and NADPH generating system. The product of nitrobenzene was identified as phenylhydroxylamine (PHA) on 1:1 basis.

Nitroreductase enzymes generally catalyze the reduction of nitroaromatic compounds to the corresponding amines. In contrast, ferredoxin NADP oxidoreductase (FNR), glutathione reductase, xanthine oxidase, and cytochrome c reductase catalyze the NADPH dependent elimination of the nitramine nitro group from 2,4,6-trinitrophenylmethylnitramine to form N-methylpicramide (NMP). Nitrite elimination was inhibited under aerobic conditions. Our results suggest that under aerobic conditions, tetryl is enzymatically reduced to the nitroanion radical which is then involved in the reduction of molecular oxygen. Under anaerobic conditions, the radical is reduced to NMP and nitrite is eliminated.

A manganese-dependent peroxidase (MnP) from Phanerochaete chrysosporium catalyzed the reduction of cytochrome c in a reaction mixture containing H2O2, Mn(II)-tartrate, and p-hydroquinone. Electron spin resonance studies have shown that the hydroquinone-dependent reductive activity of MnP is due to the benzosemiquinone formed upon the one-electron oxidation of p-hydroquinone by Mn(III)-tartrate, which is formed upon the oxidation of Mn(II) by MnP. The reductive activity increased linearly with an increase in the concentration of p-hydroquinone. The reductive activity was also observed using other hydroquinones such as methylhydroquinone, 2,5-dimethylhydroquinone, and trimethylhydroquinone. The apparent Km values for Mn(II) and H2O2 for the hydroquinone-dependent reductive activity were similar to those for oxidative reactions of MnP. A stoichiometry study showed that about 1.5 mol of cytochrome c was reduced per mole of H2O2 consumed. The stoichiometry decreased with an increase in the concentration of H2O2. The optimal pH for the reductive activity was 5.0, approximately the physiological pH of the fungus. The reduction of cytochrome c was also observed using a quinone and cellobiose:quinone oxidoreductase isolated from the extracellular medium of the fungus.

We were able to spin trap the trichloromethyl radical with PBN in a reaction mixture containing lignin peroxidase H2, H2O2, CCl4, veratryl alcohol and EDTA. Upon removal of CCl4, EDTA radicals were detected. Based on a previously reported study (Shah et al., 1992, J. Biol. Chem. 267, 21564-21569), we propose that the oxidation of EDTA to EDTA derived radicals is mediated by the veratryl alcohol cation radical, and the reduction of CCl4 to the trichloromethyl radical is caused by the EDTA derived radicals. The novel finding here is that CCl4, which is neither a substrate for the enzyme nor a good reductant, is degraded via free radicals generated by lignin peroxidases under reducing conditions.

The iodide oxidase activity and iodide-dependent pseudocatalatic activity of lignin peroxidase H2, an extracellular enzyme of the white rot fungus Phanerochaete chrysosporium, was inhibited by EDTA. The inhibition of iodide oxidase activity by EDTA was reversed at higher concentrations of iodide. Similar results were observed with a number of peroxidases. On further investigation, it was found that EDTA was decarboxylated in a reaction mixture containing a peroxidase, iodide, H2O2, and EDTA. EDTA was also decarboxylated by hypoiodite, a possible intermediate during oxidation of iodide by peroxidases. Iodide-dependent pseudocatalatic activity was increased with an increase in the concentration of H2O2 and inhibited at higher concentrations of iodide. EDTA was also oxidized by horseradish peroxidase, lactoperoxidase, and myeloperoxidase using iodide or bromide as a mediator. However, only myeloperoxidase was able to decarboxylate EDTA using chloride as a mediator. It is proposed that halide is oxidized to hypohalite by peroxidases. The hypohalite is then reduced by EDTA, H2O2, or halide. Reduction is associated with the decarboxylation of EDTA, oxidation of H2O2 to molecular oxygen, or oxidation of halide.

White rot fungi have been shown to mineralize a wide variety of environmental pollutants. These fungi secrete a number of enzymes that are involved in its unique ability to degrade lignin, the structural component of woody plants. Lignin is a very complex heteropolymer that can only be degraded by white rot fungi. Degradation is complete without energy value to the fungus. The evolution of this ability has apparently given the organism the ability to degrade structurally diverse and normally very recalcitrant environmental pollutants such as DDT, PCB, benzo(a)pyrene, TNT, etc. Some of the major enzymes that are secreted by the fungi are peroxidases with unique properties. In addition to their ability to catalyze a wide variety of oxidations, they can also catalyze indirect oxidations and reductions. The fungi synthesize and secrete hydrogen peroxide to activate the peroxidases, veratryl alcohol to serve as a free radical intermediate for indirect oxidations, and electron donors, such as oxalate, which with veratryl alcohol catalyze reductions. Reductions are often required for subsequent oxidation of chemicals by the peroxidases. The enzymes can also reduce molecular oxygen.

The mechanism for the production of hydroxyl radical by lignin peroxidase from the white rot fungus Phanerochaete chrysosporium was investigated. Ferric iron reduction was demonstrated in reaction mixtures containing lignin peroxidase isozyme H2 (LiPH2), H2O2, veratryl alcohol, oxalate, ferric chloride, and 1,10-phenanthroline. The rate of iron reduction was dependent on the concentration of oxalate and was inhibited by the addition of superoxide dismutase. The addition of ferric iron inhibited oxygen consumption in reaction mixtures containing LiPH2, H2O2, veratryl alcohol, and oxalate. Thus, the reduction of ferric iron was thought to be dependent on the LiPH2-catalyzed production of superoxide in which veratryl alcohol and oxalate serve as electron mediators. Oxalate production and degradation in nutrient nitrogen-limited cultures of P. chrysosporium was also studied. The concentration of oxalate in these cultures decreased during the period in which maximum lignin peroxidase activity (veratryl alcohol oxidation) was detected. Electron spin resonance studies using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide were used to obtain evidence for the production of the hydroxyl radical in reaction mixtures containing LiPH2, H2O2, veratryl alcohol, EDTA, and ferric chloride. It was concluded that the white rot fungus might produce hydroxyl radical via a mechanism that includes the secondary metabolites veratryl alcohol and oxalate. Such a mechanism may contribute to the ability of this fungus to degrade environmental pollutants.

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 (LiP H2) from the white rot fungus Phanerochaete chrysosporium was strongly inhibited by sodium cyanide. The I50 was estimated to be about 2-3 microM. In contrast, sodium cyanide binds to the native enzyme with an apparent sodium cyanide dissociation constant Kd of about 10 microM. Inhibition of the veratryl alcohol oxidase activity of LiP H2 by cyanide was reversible. Ligninolytic cultures of P. chrysosporium mineralized cyanide at a rate that was proportional to the concentration of cyanide to 2 mM. The N-tert-butyl-alpha-phenylnitrone-cyanyl radical adduct was observed by ESR spin trapping upon incubation of LiP H2 with H2O2 and sodium cyanide. The identity of the spin adduct was confirmed using 13C-labeled cyanide. Six-day-old cultures of the fungus were more tolerant to sodium cyanide toxicity than spores. Toxicity measurements were based on the effect of sodium cyanide on respiration of the fungus as determined by the metabolism of [14C]glucose to [14C]CO2. We propose that this tolerance of the mature fungus was due to its ability to mineralize cyanide and that this fungus might be effective in treating environmental pollution sites contaminated with cyanide.

A rapid enzyme linked immunoassay for pregnancy specific beta 1-glycoprotein is described. The method is sensitive and precise (within batch C.V. = 6.5-7.4%) and should be suitable for the biochemical monitoring of pregnancy and the investigation of patients suffering from various malignancies.