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Background: 2-Naphthol, a carbocation scavenger, is known to mitigate lignin condensation during the acidic processing of lignocellulosic biomass, which may benefit downstream processing of the resulting materials. Consequently, various raw materials have demonstrated improved enzymatic saccharification yields for substrates pretreated through autohydrolysis and dilute acid hydrolysis in the presence of 2-naphthol. However, 2-naphthol is toxic to ethanol-producing organisms, which may hinder its potential application. Little is known about the implications of 2-naphthol in combination with the pretreatment of softwood bark during continuous steam explosion in an industrially scalable system., Results: The 2-naphthol-pretreated softwood bark was examined through spectroscopic techniques and subjected to separate hydrolysis and fermentation along with a reference excluding the scavenger and a detoxified sample washed with ethanol. The extractions of the pretreated materials with water resulted in a lower aromatic content in the extracts and stronger FTIR signals, possibly related to guaiacyl lignin, in the nonextractable residue when 2-naphthol was used during pretreatment. In addition, cyclohexane/acetone (9:1) extraction revealed the presence of pristine 2-naphthol in the extracts and increased aromatic content of the nonextractable residue detectable by NMR for the scavenger-pretreated materials. Whole-slurry enzymatic saccharification at 12% solids loading revealed that elevated saccharification recoveries after 48 h could not be achieved with the help of the scavenger. Glucose concentrations of 16.9 (reference) and 15.8 g/l (2-naphthol) could be obtained after 48 h of hydrolysis. However, increased inhibition during fermentation of the scavenger-pretreated hydrolysate, indicated by yeast cell growth, was slight and could be entirely overcome by the detoxification stage. The ethanol yields from fermentable sugars after 24 h were 0.45 (reference), 0.45 (2-naphthol), and 0.49 g/g (2-naphthol, detoxified)., Conclusion: The carbocation scavenger 2-naphthol did not increase the saccharification yield of softwood bark pretreated in an industrially scalable system for continuous steam explosion. On the other hand, it was shown that the scavenger's inhibitory effects on fermenting microorganisms can be overcome by controlling the pretreatment conditions to avoid cross-inhibition or detoxifying the substrates through ethanol washing. This study underlines the need to jointly optimize all the main processing steps., (© 2024. The Author(s).)

The present work elucidates facile one-pot synthesis from biomass forestry waste (Norway spruce bark) and its chemical activation yielding high specific surface area ( S BET ) biochars as efficient lithium- and sodium-ion storage anodes. The chemically activated biochar using ZnCl 2 (Biochar-1) produced a highly mesoporous carbon containing 96.1% mesopores in its structure as compared to only 56.1% mesoporosity from KOH-activated biochars (Biochar-2). The latter exhibited a lower degree of graphitization with disordered and defective carbon structures, while the former presented more formation of ordered graphite sheets in its structure as analyzed from Raman spectra. In addition, both biochars presented a high degree of functionalities on their surfaces but Biochar-1 presented a pyridinic-nitrogen group, which helps improve its electrochemical response. When tested electrochemically, Biochar-1 showed an excellent rate capability and the longest capacity retentions of 370 mA h g -1 at 100 mA g -1 (100 cycles), 332.4 mA h g -1 at 500 mA g -1 (1000 cycles), and 319 mA h g -1 at 1000 mA g -1 after 5000 cycles, rendering as an alternative biomass anode for lithium-ion batteries (LIBs). Moreover, as a negative electrode in sodium-ion batteries, Biochar-1 delivered discharge capacities of 147.7 mA h g -1 at 50 mA g -1 (140 cycles) and 126 mA h g -1 at 100 mA g -1 after 440 cycles., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

This study utilized pulp and paper mill sludge as a carbon source to produce activated biochar adsorbents. The response surface methodology (RSM) application for predicting and optimizing the activated biochar preparation conditions was investigated. Biochars were prepared based on a Box-Behnken design (BBD) approach with three independent factors (i.e., pyrolysis temperature, holding time, and KOH:biomass ratio), and the responses evaluated were specific surface area (SSA), micropore area ( S micro ), and mesopore area ( S meso ). According to the RSM and BBD analysis, a pyrolysis temperature of 800 °C for 3 h of holding and an impregnation ratio of 1:1 (biomass:KOH) are the optimum conditions for obtaining the highest SSA (885 m 2 g -1 ). Maximized S micro was reached at 800 °C, 1 h and the ratio of 1:1, and for maximizing S meso (569.16 m 2 g -1 ), 800 °C, 2 h and ratio 1:1.5 (445-473 m 2 g -1 ) were employed. The biochars presented different micro- and mesoporosity characteristics depending on pyrolysis conditions. Elemental analysis showed that biochars exhibited high carbon and oxygen content. Raman analysis indicated that all biochars had disordered carbon structures with structural defects, which can boost their properties, e.g., by improving their adsorption performances. The hydrophobicity-hydrophilicity experiments showed very hydrophobic biochar surfaces. The biochars were used as adsorbents for diclofenac and amoxicillin. They presented very high adsorption performances, which could be explained by the pore filling, hydrophobic surface, and π-π electron-donor-acceptor interactions between aromatic rings of both adsorbent and adsorbate. The biochar with the highest surface area (and highest uptake performance) was subjected to regeneration tests, showing that it can be reused multiple times., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

A biomass amino silica-functionalized material was successfully prepared by a simple sol-gel method. 3-Aminopropyltriethoxysilane (APTES) was added to a tannin-rich grape residue to improve its physicochemical properties and enhance the adsorption performance. The APTES functionalization led to significant changes in the material's characteristics. The functionalized material was efficiently applied in the removal of methyl orange (MO) due to its unique characteristics, such as an abundance of functional groups on its surface. The adsorption process suggests that the electrostatic interactions were the main acting mechanism of the MO dye removal, although other interactions can also take place. The functionalized biomass achieved a very high MO dye maximum adsorption capacity ( Q max ) of 361.8 mg g -1 . The temperature positively affected the MO removal, and the thermodynamic studies indicated that the adsorption of MO onto APTES-functionalized biomass was spontaneous and endothermic, and enthalpy is driven in the physisorption mode. The regeneration performance revealed that the APTES-functionalized biomass material could be easily recycled and reused by maintaining very good performance even after five cycles. The adsorbent material was also employed to treat two simulated dye house effluents, which showed 48% removal. At last, the APTES biomass-based material may find significant applications as a multifunctional adsorbent and can be used further to separate pollutants from wastewater., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Preparing sustainable and highly efficient biochars as electrodes remains a challenge for building green energy storage devices. In this study, efficient carbon electrodes for supercapacitors were prepared via a facile and sustainable single-step pyrolysis method using spruce bark as a biomass precursor. Herein, biochars activated by KOH and ZnCl 2 are explored as templates to be applied to prepare electrodes for supercapacitors. The physical and chemical properties of biochars for application as supercapacitors electrodes were strongly affected by factors such as the nature of the activators and the meso/microporosity, which is a critical condition that affects the internal resistance and diffusive conditions for the charge accumulation process in a real supercapacitor. Results confirmed a lower internal resistance and higher phase angle for devices prepared with ZnCl 2 in association with a higher mesoporosity degree and distribution of Zn residues into the matrix. The ZnCl 2 -activated biochar electrodes' areal capacitance reached values of 342 mF cm -2 due to the interaction of electrical double-layer capacitance/pseudocapacitance mechanisms in a matrix that favors hydrophilic interactions and the permeation of electrolytes into the pores. The results obtained in this work strongly suggest that the spruce bark can be considered a high-efficiency precursor for biobased electrode preparation to be employed in SCs.

In this work, Norway spruce bark was used as a precursor to prepare activated biochars (BCs) via chemical activation with potassium hydroxide (KOH) as a chemical activator. A Box-Behnken design (BBD) was conducted to evaluate and identify the optimal conditions to reach high specific surface area and high mass yield of BC samples. The studied BC preparation parameters and their levels were as follows: pyrolysis temperature (700, 800, and 900 °C), holding time (1, 2, and 3 h), and ratio of the biomass: chemical activator of 1: 1, 1.5, and 2. The planned BBD yielded BC with extremely high SSA values, up to 2209 m 2 ·g -1 . In addition, the BCs were physiochemically characterized, and the results indicated that the BCs exhibited disordered carbon structures and presented a high quantity of O-bearing functional groups on their surfaces, which might improve their adsorption performance towards organic pollutant removal. The BC with the highest SSA value was then employed as an adsorbent to remove Evans blue dye (EB) and colorful effluents. The kinetic study followed a general-order (GO) model, as the most suitable model to describe the experimental data, while the Redlich-Peterson model fitted the equilibrium data better. The EB adsorption capacity was 396.1 mg·g -1 . The employment of the BC in the treatment of synthetic effluents, with several dyes and other organic and inorganic compounds, returned a high percentage of removal degree up to 87.7%. Desorption and cyclability tests showed that the biochar can be efficiently regenerated, maintaining an adsorption capacity of 75% after 4 adsorption-desorption cycles. The results of this work pointed out that Norway spruce bark indeed is a promising precursor for producing biochars with very promising properties.

In this study, bio-adhesives from natural rubber latex (NRL) were combined with starch and formic acid to fabricate jute stick-based particleboards (JSPs). Different blends of NRL, starch, and formic acid, i.e. , 6 : 1 : 1, 2 : 1 : 1, and 2 : 3 : 3, were used to produce particleboards using a pressing temperature of 180 °C and applied pressure of 5 MPa using a 5 min pressing time. The particleboards were tested for physical, mechanical, and thermal properties according to ANSI standards. Based on initial screening, the best formula (NRL/starch/formic acid of 2 : 3 : 3) was used to optimize the temperature and pressing time for the highest board performance. The highest density, tensile strength, modulus of elasticity, and modulus of rupture were 830 g cm -3 , 10.51, 2380, and 20.05 N mm -2 , respectively. Thermo-gravimetric analysis indicated that thermal decomposition of samples primarily occurred in a temperature range of 265 to 399 °C, indicating good thermal performance. The measured physical and mechanical properties of the produced JSPs fulfilled the production standards. However, fulfilling the water absorption and thickness swelling criteria was a challenge. The results indicate that NRL is a promising alternative binder when blended with starch and formic acid., Competing Interests: 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. It is here mentionable that several authors from the article author list have applied for a provisional patent (Application Number: P/BD/2018/000291) related to the research work and the final patent application is now in processing., (This journal is © The Royal Society of Chemistry.)

Inadequate flowability of powders in industries during handling can cause many problems. For example, lack of flow from hoppers, poor tablet weight consistency, and low production rate in tableting. Many factors are known to commonly affect flow properties of powders, such as temperature, humidity and conditioning duration. In this paper, flow properties of a mannitol powder, which was conditioned between 24 and 72 h at various high relative humidities and temperature, were measured using a shear tester. A statistical model was developed to investigate the relative importance of these variables on the mannitol flow properties. The developed model showed all independent variables are significant in estimating bulk cohesion. Two separate approaches were used to evaluate inter-particle forces in the bulk, and how these changed with environmental conditions. First, inter-particle forces were inferred from the measured bulk properties using the Rumpf model approach. Secondly, inter-particle forces were predicted based on a model of moisture present on the particle surface using a combination of Kelvin model with the Laplace-Young (KLY) equation. The second approach also involved a new method to measure surface energy of mannitol powder based on measurements using Finite Dilution Inverse Gas Chromatography (FD-IGC). The surface energies of the mannitol powder were measured at high temperature (35 °C) and at different range of relative humidities. In spite of the fundamentally different approaches to the two ways of inferring inter-particles forces, these forces came out within less than 1.5:1 in magnitude. The Rumpf approach from bulk behaviour data obviously reflected the measured change in behaviour with humidity in particular, but this was not predicted from the KLY approach, however the likely reasons for this are postulated and recommendations for improvement are made., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cheap and efficient carbon electrodes (CEs) for energy storage systems (ESS) such as supercapacitors (SCs) and batteries are an increasing priority issue, among other things, due to a globally increasing share of intermittent electricity production (solar and wind) and electrification of transport. The increasing consumption of portable and non-portable electronic devices justifies research that enables environmentally and economically sustainable production (materials, processing techniques, and product design) of products with a high electrochemical performance at an acceptable cost. Among all the currently explored CEs materials, biomass-based activated carbons (AC) present enormous potential due to their availability and low-cost, easy processing methods, physicochemical stability, and methods for self-doping. Nitrogen doping methods in CEs for SCs have been demonstrated to enhance its conductivities, surface wettability, and induced pseudocapacitance effect, thereby delivering improved energy/power densities with versatile properties. Herein, a short review is presented, focusing on the different types of natural carbon sources for preparing CEs towards the fabrication of SCs with high electrochemical performance. The influences of ACs' pore characteristics (micro and mesoporosity) and nitrogen doping on the overall electrochemical performance (EP) are addressed.

Some recent developments in the preparation of biomass carbon electrodes (CEs) using various biomass residues for application in energy storage devices, such as batteries and supercapacitors, are presented in this work. The application of biomass residues as the primary precursor for the production of CEs has been increasing over the last years due to it being a renewable source with comparably low processing cost, providing prerequisites for a process that is economically and technically sustainable. Electrochemical energy storage technology is key to the sustainable development of autonomous and wearable electronic devices. This article highlights the application of various types of biomass in the production of CEs by using different types of pyrolysis and experimental conditions and denotes some possible effects on their final characteristics. An overview is provided on the use of different biomass types for the synthesis of CEs with efficient electrochemical properties for batteries and supercapacitors. This review showed that, from different biomass residues, it is possible to obtain CEs with different electrochemical properties and that they can be successfully applied in high-performance batteries and supercapacitors. As the research and development of producing CEs still faces a gap by linking the type and composition of biomass residues with the carbon electrodes' electrochemical performances in supercapacitor and battery applications, this work tries to diminish this gap. Physical and chemical characteristics of the CEs, such as porosity, chemical composition, and surface functionalities, are reflected in the electrochemical performances. It is expected that this review not only provides the reader with a good overview of using various biomass residues in the energy storage applications, but also highlights some goals and challenges remaining in the future research and development of this topic.

Activated reduced graphene oxide (a-rGO) is a material with a rigid 3D porous structure and high specific surface area (SSA). Using variation of activation parameters and post-synthesis mechanical treatment we prepared two sets of materials with a broad range of BET (N2) SSA ∼1000-3000 m2 g-1, and significant differences in pore size distribution and oxygen content. The performance of activated graphene as an electrode in a supercapacitor with KOH electrolyte was correlated with the structural parameters of the materials and water sorption properties. a-rGO is a hydrophobic material as evidenced by the negligibly small BET (H2O) SSA determined using analysis of water vapor sorption isotherms. However, the total pore volume determined using water vapor sorption and sorption of liquid water is almost the same as the one found by analysis of nitrogen sorption isotherms. Ball milling is found to provide an improved bulk density of activated graphene and collapse of all pores except the smallest ones (<2 nm). A decrease in the activation temperature from 850 °C to 550 °C is found to result in materials with a narrow micropore size distribution and increased oxygen content. Elimination of mesopores using ball milling or a lower activation temperature provided materials with better specific capacitance despite a significant decrease (by ∼30%) of the BET (N2) SSA. The best gravimetric and volumetric capacitances in KOH electrolyte were achieved not for samples with the highest value of the BET (N2) SSA but for materials with 80-90% of the total pore volume in micropores and an increased BET (H2O) SSA. Comparing the performance of electrodes prepared using rGO and a-rGO shows that a more hydrophilic surface is favorable for charge storage in supercapacitors with KOH electrolyte.

Hot-air (75-100 °C) pasteurisation (HAP) of birch-wood-based substrate was compared to conventional autoclaving (steam at 121 °C) with regard to shiitake growth and yield, chemical composition of heat-pretreated material and spent mushroom substrate (SMS), enzymatic digestibility of glucan in SMS, and theoretical bioethanol yield. Compared to autoclaving, HAP resulted in faster mycelial growth, earlier fructification, and higher or comparable fruit-body yield. The heat pretreatment methods did not differ regarding the fractions of carbohydrate and lignin in pretreated material and SMS, but HAP typically resulted in lower fractions of extractives. Shiitake cultivation, which reduced the mass fraction of lignin to less than half of the initial without having any major impact on the mass fraction of glucan, enhanced enzymatic hydrolysis of glucan about four-fold. The choice of heating method did not affect enzymatic digestibility. Thus, HAP could substitute autoclaving and facilitate combined shiitake mushroom and bioethanol production., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Hydrothermal carbonization (HTC) can be used to break down sludge structure and generate carbonaceous hydrochar suitable for solid fuel or value-added material applications. The separation of char and the reaction medium however generates a filtrate, which needs to treated before potential discharge. Thus, this work determined filtrate properties based on HTC temperature and sludge moisture content and estimated the discharge emissions and the potential increase in analyte loads to an industrial wastewater treatment plant based on derived regression models. Direct discharge of HTC filtrate would significantly increase effluent emissions at the mill, indicating the filtrate treatment is crucial for the future implementation of HTC at pulp and paper mills. Recycling the HTC filtrate to the wastewater plant would lead to only a nominal increase in effluent flow, but would increase the suspended solids, BOD, COD and total nitrogen loads by 0.1-0.8%, 3.8-5.3%, 2.7-3.1% and 42-67%, respectively, depending on HTC temperature., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Despite numerous studies of geographic variation in healthcare cost and utilization at the local, regional, and state levels across the U.S., a comprehensive characterization of geographic variation in outcomes has not been published. Our objective was to quantify variation in US health outcomes in an all-payer population before and after risk-adjustment., Methods and Findings: We used information from 16 independent data sources, including 22 million all-payer inpatient admissions from the Healthcare Cost and Utilization Project (which covers regions where 50% of the U.S. population lives) to analyze 24 inpatient mortality, inpatient safety, and prevention outcomes. We compared outcome variation at state, hospital referral region, hospital service area, county, and hospital levels. Risk-adjusted outcomes were calculated after adjusting for population factors, co-morbidities, and health system factors. Even after risk-adjustment, there exists large geographical variation in outcomes. The variation in healthcare outcomes exceeds the well publicized variation in US healthcare costs. On average, we observed a 2.1-fold difference in risk-adjusted mortality outcomes between top- and bottom-decile hospitals. For example, we observed a 2.3-fold difference for risk-adjusted acute myocardial infarction inpatient mortality. On average a 10.2-fold difference in risk-adjusted patient safety outcomes exists between top and bottom-decile hospitals, including an 18.3-fold difference for risk-adjusted Central Venous Catheter Bloodstream Infection rates. A 3.0-fold difference in prevention outcomes exists between top- and bottom-decile counties on average; including a 2.2-fold difference for risk-adjusted congestive heart failure admission rates. The population, co-morbidity, and health system factors accounted for a range of R2 between 18-64% of variability in mortality outcomes, 3-39% of variability in patient safety outcomes, and 22-70% of variability in prevention outcomes., Conclusion: The amount of variability in health outcomes in the U.S. is large even after accounting for differences in population, co-morbidities, and health system factors. These findings suggest that: 1) additional examination of regional and local variation in risk-adjusted outcomes should be a priority; 2) assumptions of uniform hospital quality that underpin rationale for policy choices (such as narrow insurance networks or antitrust enforcement) should be challenged; and 3) there exists substantial opportunity for outcomes improvement in the US healthcare system., Competing Interests: We have the following interests: BLR, JAK, AL, DHMM, MR, PV, RL and SHL are employees of The Boston Consulting Group, a global management consulting firm serving healthcare companies including hospitals, payers, medtech and pharma. HM is principal of Alerion Institute and Alerion Advisors that advises corporations and non-profit entities. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Douglas fir (Pseudotsuga menziesii L.) particles were exposed to high pressure saturated steam (200 and 220 °C for 5 and 10 min) to improve the durability and hydrophobicity of pellets produced from them. Depending on treatment severity, the moisture content of the particles increased from 10% to 36% (wet basis). Douglas fir particles steam-treated at 220 °C for 10 min had the fastest drying rate of 0.014 min(-1). The equilibrium moisture content (EMC) of steam-treated samples decreased with increasing steam temperature and treatment time. The Giggnheim-Anderson-deBoer (GAB) equilibrium model gave a good fit with the equilibrium data with R(2) = 0.99. The adsorption rate of untreated pellets exposed to humid air (30 °C, 90% RH) for 72 h was 0.0152 min(-1) while that of steam-treated pellets ranged from 0.0125 to 0.0135 min(-1) without a clear trend with steam treatment severity. These findings are critical to develop durable and less hygroscopic pellets., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In this study, pre-compaction was evaluated as a method to enhance stable reed canary grass pellet production. An experimental design of the factors raw material moisture content, steam addition, raw material bulk density, and die temperature was used to find production conditions for high quality pellets by multiple linear regression modelling of responses. Response variables being modelled were variability of pelletizer current (as a measurement of uneven production), pellet bulk density, and pellet durability. By pre-compacting the raw material from a bulk density of 150 kg/m3 to 270kg/m3, continuous production could be obtained at minimum raw material moisture content of 13.8%. Bulk density and durability were both highly correlated to raw material moisture content, but showed different optima. Multiple response optimization was used to target process settings for production of high quality reed canary grass pellets with bulk density >650kg/m3 and durability >97.5%.

WT1 is essential for normal kidney development, and genetic alterations are associated with Wilms' tumor, Denys Drash (DDS), and Frasier syndromes. Although generally considered a transcription factor this study has revealed that WT1 interacts with an essential splicing factor, U2AF65, and associates with the splicing machinery. WT1 is alternatively spliced and isoforms that include three amino acids, KTS, show stronger interaction with U2AF65 in vitro and better colocalization with splicing factors in vivo. Interestingly a mutation associated with DDS enhanced both -KTS WT1 binding to U2AF65 and splicing-factor colocalization. These data illustrate the functional importance of WT1 isoforms and suggest that WT1 plays a role in pre-mRNA splicing.

Phosphorylation of the alpha-1 subunit of rat Na+,K(+)-ATPase by protein kinase C has been shown previously to decrease the activity of the enzyme in vitro. We have now undertaken an investigation of the mechanism by which this inhibition occurs. Analysis of the phosphorylation of recombinant glutathione S-transferase fusion proteins containing putative cytoplasmic domains of the protein, site-directed mutagenesis, and two-dimensional peptide mapping indicated that protein kinase C phosphorylated the alpha-1 subunit of the rat Na+,K(+)-ATPase within the extreme NH2-terminal domain, on serine-23. The phosphorylation of this residue resulted in a shift in the equilibrium toward the E1 form, as measured by eosin fluorescence studies, and this was associated with a decrease in the apparent K+ affinity of the enzyme, as measured by ATPase activity assays. The rate of transition from E2 to E1 was apparently unaffected by phosphorylation by protein kinase C. These results, together with previous studies that examined the effects of tryptic digestion of Na+,K(+)-ATPase, suggest that the NH2-terminal domain of the alpha-1 subunit, including serine-23, is involved in regulating the activity of the enzyme.

The Wilms tumor suppressor gene WT1 is implicated in the ontogeny of genito-urinary abnormalities, including Denys-Drash syndrome and Wilms tumor of the kidney. WT1 encodes Kruppel-type zinc finger proteins that can regulate the expression of several growth-related genes, apparently by binding to specific DNA sites located within 5' untranslated leader regions as well as 5' promoter sequences. Both WT1 and a closely related early growth response factor, EGR1, can bind the same DNA sequences from the mouse gene encoding insulin-like growth factor 2 (Igf-2). We report that WT1, but not EGR1, can bind specific Igf-2 exonic RNA sequences, and that the zinc fingers are required for this interaction. WT1 zinc finger 1, which is not represented in EGR1, plays a more significant role in RNA binding than zinc finger 4, which does have a counterpart in EGR1. Furthermore, the normal subnuclear localization of WT1 proteins is shown to be RNase, but not DNase, sensitive. Therefore, WT1 might, like the Kruppel-type zinc finger protein TFIIIA, regulate gene expression by both transcriptional and posttranscriptional mechanisms.

WT1 is a tumor suppressor gene with a key role in urogenital development and the pathogenesis of Wilms' tumor. Two alternative splice sites in the WT1 transcript allow the gene to encode four proteins. These carry four Krüppel-type zinc fingers and to date have primarily been implicated in transcriptional control of genes involved in growth regulation. However, here we demonstrate colocalization of WT1 with splicing factors in the fetal kidney and testis and in expressing cell lines. Using immunoprecipitation, we show that two WT1 isoforms directly associate with one or a limited number of components in the spliceosomes and coiled bodies. Moreover, COS cell expression studies suggest that alternative splicing within the WT1 zinc finger region determines whether the protein localizes mainly with splicing factors or with DNA in transcription factor domains in the nucleus. We propose that WT1 plays roles in posttranscriptional processing of RNA as well as in transcription.

We have previously demonstrated that leukotriene B4 (LTB4) induces in vitro a transient state of hyperadhesiveness in cultured human umbilical vein endothelial cells (HUVEC) for neutrophils (PMN). The magnitude of this response is intermediate of that conferred by thrombin and by platelet-activating factor (PAF). This report shows that the LTB4 response was neither related to HUVEC expression of PAF (because it could not be blocked by the PAF receptor antagonist WEB-2086), nor to access to LTB4 receptors on neutrophils (as shown by LTB4 receptor desensitization experiments). However, it could be partly blocked by treating HUVEC with an LTB4 receptor antagonist (SC-41930). LTB4 evoked a rise of intracellular calcium concentrations, [Ca2+]i, in the HUVEC, and the hyperadhesive HUVEC response to LTB4 was abrogated by buffering of [Ca2+]i by Quin-2. The response was not inhibited by treating HUVEC with pertussis toxin before LTB4. Neutrophils showed no signs of activation when adhering to LTB4-treated HUVEC because they did not i) release lactoferrin, or ii) react with an increase of [Ca2+]i, and iii) they bound equally well to the stimulated endothelial cells after having been treated with pertussis toxin so that up-regulation of PMN adhesion to LTB4 was abolished. LTB4-treated HUVEC did not shed factors that modulated neutrophil adherence or chemotaxis. Thus, LTB4 promotes HUVEC hyperadhesiveness for PMN, and the transduction mechanism involves calcium ions, may depend on a surface receptor for LTB4, but does not involve pertussis toxin-sensitive G proteins or PMN activation.

This is a review of recent work based on an in vitro model which has allowed us to investigate the postnatal maturation of renal epithelial cells. Renal proximal tubule cells from 8- to 40-day-old Sprague-Dawley rats were studied after 48 h of primary culture. The regulation of intracellular pH (pHi) was measured by quantitative fluorescence microscopy using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Recordings were made under basal conditions and after imposing a cytoplasmic alkalosis or acidosis using 15 mM ammonium salt. The ability of the cells to recover from both acidosis and alkalosis improved during post-natal maturation. The improvement in recovery from intracellular acidosis could be entirely accounted for by an increase in the rate of Na+/H+ exchange. The capacity for Na+/H+ exchange was independent of the cellular growth rate, but depended on cellular differentiation. A developmental increase in the activity of Cl-/HCO3- exchange between 12 and 14 days of age was also demonstrated. No developmental change was seen in either steady-state pHi (7.27-7.35) or in cytoplasmic buffer capacity (37.6-44.4 mM/pHi). The characteristics of transporter maturation revealed by these experiments are very similar to those observed in isolated perfused proximal tubules of developing rabbits.

To gain more information about the growth regulation of renal epithelial cells, we examined the growth stimulatory effect of serum and intracellular sodium in the renal epithelial cell line, LLC-PK1. In subconfluent LLC-PK1 cells serum-starved for 5 days and exposed to [3H]thymidine for 24 h, 22.9% of the cells synthesized DNA. Stimulation with 10% foetal calf serum (FCS) caused an almost three-fold increase in the fraction of labelled nuclei (62.2%). Serum-starved LLC-PK1 cells exposed to 10% FCS responded with an increased abundance of c-jun transcripts. The maximal expression of the c-jun transcripts occurred at 60 min and declined 120 min after serum stimulation. It has been suggested that an increase in Na+ influx plays a role in the growth regulation of renal epithelial cells. This prompted us to study the effect of intracellular Na+ loading on the growth response of LLC-PK1 cells. Serum-starved LLC-PK1 cells were incubated in a low K+ medium or exposed to Nystatin. Incubation in a low K+ medium or with Nystatin resulted in a marked increase in intracellular Na after only 5 min. A low K+ medium did not significantly influence the intracellular pH. No effect was observed on DNA synthesis or the abundance of c-jun transcripts in LLC-PK1 cells. Nor did Na+ loading enhance the growth stimulatory effect of serum. The results suggest that an increase in intracellular sodium does not directly regulate the growth of renal epithelial cells.

This study examines the ontogeny of cellular pH regulation in renal proximal tubule cells (RPTC). RPTC from 8- to 40-day-old Sprague-Dawley rats (RPTC-8 to RPTC-40) were studied after 48 h of primary culture. Intracellular pH (pHi) was measured by quantitative fluorescence microscopy using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Recordings were made under basal conditions and after imposing a cytoplasmic alkalosis and acidosis using 15 mM NH4+ salt. The net recovery rate (dpHi/dt) from intracellular acidosis increases significantly between 10 and 12 days of age from 0.39 +/- 0.04 to 0.54 +/- 0.06 pH units/min (P < 0.05, n = 10 vs. 6). This increase can be completely accounted for by an increase in the rate of amiloride (100 microM)-inhibitable Na(+)-H+ exchange (0.29 +/- 0.04 vs. 0.42 +/- 0.05 pH units/min, P < 0.05, n = 6 vs. 6). The rate of Na(+)-H+ exchange increases similarly in RPTC-10 and RPTC-40 when the transmembrane Na+ gradient is increased by Na+ depleting the cells (48 and 49%, respectively). The amiloride-insensitive recovery is Na+ independent and insensitive to 4-acetamido-4'-isothiocyanostilbene-2-2'-disulfonic acid (SITS, 500 microM) (range 0.08-0.14 pH units/min). The net recovery rate from intracellular alkalosis is significantly lower in RPTC-10 than in RPTC-40 (0.16 +/- 0.02 vs. 0.28 +/- 0.02 pH units/min, P < 0.01, n = 4 vs. 5). SITS (500 microM) inhibits the recovery by 27 +/- 8 and 26 +/- 9%, respectively, whereas amiloride has no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

During the peri- and early postnatal period, nephrogenesis is completed and kidney growth is accomplished both by cellular proliferation and enlargement. The number of nephrons in a given species is predetermined, whereas cellular growth can be influenced by environmental factors in an age-dependent manner. Unilateral nephrectomy or a high-protein diet stimulates renal growth more in the young than in the adult. Conversely, pyelonephritis inhibits renal growth in infancy but not in adulthood. The relative importance of hyperplasia and hypertrophy for renal growth also changes with renal maturation. The mechanisms behind these developmental changes in regulation of renal growth are largely unknown, but age-dependent changes in the expression of several proto-oncogene products have been demonstrated. These include growth factor receptors as well as components of the intracellular system that transfers the signal from an activated growth factor receptor to the cell nucleus. Studies on rat proximal tubule cells in primary culture might be of great value in expanding our knowledge of growth regulation in the developing kidney. Such studies have already shown that under identical environmental conditions the basal proliferative rate is age dependent, that the proliferative response to growth stimulation changes postnatally, and that this is associated with changes of both the response of the Na+/H(+)-exchanger and the expression of the c-fos proto-oncogene.

Recent methodological developments have made primary cultures of renal cells a promising model for studies of renal growth regulation. By minimal culture time and single-cell techniques, homogenous populations of highly differentiated cells can be studied. This report reviews the model and recent studies on growth regulation in rat proximal tubule cells (PTC). It is demonstrated that no correlation exists between proliferative rate and intracellular pH (pHi) in PTC, a topic of previous debate. The rate of proliferation in cultured PTC is similar to in vivo and dependent on the postnatal age of the animal. Recent evidence demonstrates that the model is an important tool for studies of postnatal changes in renal growth regulation.

Kidney epithelial cells in short-term primary culture have been studied with regard to proliferative rate and expression on the c-fos protooncogene. The experiments were performed on subconfluent renal proximal tubule cells isolated from infant and adolescent rats. Proliferation was determined by 3H-thymidine autoradiography and nuclear content of c-fos protein by semiquantitative immunofluorescence. The basal proliferative rates in infant and adolescent renal proximal tubule cells were the same after 48 h of primary culture in Dulbecco's modified Eagle's medium with 10% FCS. Serum deprivation for 24 h caused a significant growth inhibition in both infant and adolescent cells. C-fos was expressed to the same extent in infant and adolescent serum-deprived cells. The rapid response to the addition of serum was markedly different in infant and adolescent cells. In adolescent cells, addition of serum led to a transient significant increase in the nuclear expression of c-fos protein, reaching a peak at 60 min. No increase in c-fos was seen in infant cells. In adolescent cells, the rate of proliferation increased 11-fold and 3H-thymidine labeling index reached 26.7 +/- 4.3%. In infant cells, the proliferative response to serum addition was significantly lower; the labeling index reached only 4.2 +/- 1.2%. It could be excluded that the attenuated response in infant cells was due to cell death or impaired metabolic function. The results imply that the principles of growth regulation change postnatally.

Neurokinin A, a member of the tachykinin family of neuropeptides, has been identified as a mitogen for cultured smooth muscle cells. Tachykinin-induced DNA synthesis has previously been shown to be mediated by a receptor-specific mechanism and to correlate with accumulation of phosphatidylinositol 4,5-bisphosphate breakdown products. In the present experiments, we have studied intracellular pH and expression of the proto-oncogenes c-myc, c-jun and c-fos in smooth muscle cells exposed to mitogenic concentrations of neurokinin A. Growth-arrested smooth muscle cells stimulated with neurokinin A responded with an amiloride-sensitive intracellular alkalinization, indicating Na+/H+ antiport activation. c-myc and c-jun mRNA expression was only slightly elevated by neurokinin A, while c-fos expression underwent a more pronounced increase. Maximal levels of c-fos transcripts were found after 15 min and 30 min following neurokinin A stimulation. The results demonstrate that neuropeptides may influence proto-oncogene expression in smooth muscle cells and suggest a mechanism by which peripheral neurons may modulate differentiation and growth of these cells.

Ouabain-sensitive (OS) O2 consumption was determined in proximal tubular cells from weanling rats fed 21% (normal-protein, NP) or 50% (high-protein, HP) protein diet for 4 days. Butyric acid 10(-3)M was added as a substrate for mitochondrial respiration and the ionophore amphotericin B (10 micrograms ml-1) was used to sodium-load the cells. OS respiration was higher in HP than in NP cells in both DME and amino acid-free electrolyte solution (ES). Amphotericin B significantly increased OS respiration in both NP and HP cells, implying that the Na-K pump was activated by increased intracellular Na. In cells incubated in ES, addition of amino acids stimulated OS respiration significantly in HP cells (16.9 +/- 1.4 vs 21.2 +/- 1.1 nmol min-1 mg-1 protein) and in NP cells (13.9 +/- 0.3 vs 14.9 +/- 0.6 nmol min-1 mg-1 protein). Stimulation was significantly higher in HP cells (26 +/- 4%) than in NP cells (7 +/- 4%) (P less than 0.001). The amino acids did not stimulate ouabain-insensitive respiration. The results indicate that an HP diet to weanling rats will increase proximal tubule cell Na, K-ATPase-dependent respiration by enhancing Na entry via the Na-amino acid symports.

Renal proximal tubule (PT) cells from adult rats will maintain much of their functional characteristics in short-term primary culture [S. Larsson, A. Aperia, and C. Lechene. Am. J. Physiol. 251 (Cell Physiol. 20): C455-C464, 1986]. This study examines the growth regulation of these highly differentiated cells with particular reference to cell density, intracellular pH (pHi), and the expression of the Na(+)-H+ exchanger. PT cells were obtained from young adult rats and studied after 48 h in culture. The mitotic rate was determined as the labeling index (LI) after [3H]thymidine autoradiography, and pHi was determined by 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein quantitative fluorescence microscopy in single cells. Cells were grown either continuously in serum (S) or were serum deprived after 24 h (D). The cells were nonconfluent and grew in colonies. We defined the two peripheral layers of cells in a colony as peripheral (P) cells and the remaining cells as central (C). In C cells LI/h and pHi were in the range of what has been observed under in vivo conditions. In S condition LI/h was 2.2 +/- 0.3% and in D condition was 0.3 +/- 0.1%. LI was significantly higher in P than in C cells both under S (2.5 +/- 0.4-fold) and D conditions (5.6 +/- 0.8-fold). The rapidly growing P cells had a significantly lower pHi than the growth-retarded C cells both under S (7.25 +/- 0.02 vs. 7.30 +/- 0.01, P less than 0.05) and D conditions (7.21 +/- 0.02 vs. 7.28 +/- 0.01, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

We have studied the ontogeny of Na-K ATPase-mediated Na and K transport in rat renal proximal tubular cells using electron probe analysis. The cells were cultured from kidneys of 10-day-old, young (Y), and 40-day-old, adult (A) rats. Before an experiment cells were Na-loaded and K-depleted by incubation in K-free medium. The maximum rate of ouabain-sensitive Na and K transport was measured after reactivating the Na-K pump by transferring the cells from K-free medium to medium containing 5 mM K. In cells cultured for 2 days, ouabain-sensitive Na and K net initial transport rates were significantly higher in A than in Y cells. Between 2 and 4 days in culture there was a significant decrease in ouabain-sensitive Na and K transport rates in both Y and A cells. From 2 to 4 days of culture there was, in Y but not in A cells, a significant decrease in K/Na ratio. The decrease in K/Na ratio was due to a significant increase in Na content. After incubation in K-free medium, net intracellular solute accumulation was observed in A and Y cells cultured for 4 days but not in A and Y cells cultured for 2 days. In conclusion, maximal Na- and K-pump-mediated transport increases during terminal differentiation. This increase can be measured in cells cultured for 2 days. With longer time in culture, Na-K pump activity decreases and the difference between A and Y cells is not measurable.(ABSTRACT TRUNCATED AT 250 WORDS)

There are several lines of indirect evidence suggesting that the renal tubule cells have not yet reached terminal differentiation at birth. Methods used in cell biology can now be applied to study renal ontogeny. This review describes how primary cultures of proximal tubule cells from rats can be used to investigate developmental changes in Na permeability and Na-K-ATPase-mediated transport.