A Phase 2 Trial of Sibeprenlimab in Patients with IgA Nephropathy.

BACKGROUND: A proliferation-inducing ligand (APRIL) is implicated in the pathogenesis of IgA nephropathy. Sibeprenlimab is a humanized IgG2 monoclonal antibody that binds to and neutralizes APRIL. METHODS: In this phase 2, multicenter, double-blind, randomized, placebo-controlled, parallel-group trial, we randomly assigned adults with biopsy-confirmed IgA nephropathy who were at high risk for disease progression, despite having received standard-care treatment, in a 1:1:1:1 ratio to receive intravenous sibeprenlimab at a dose of 2, 4, or 8 mg per kilogram of body weight or placebo once monthly for 12 months. The primary end point was the change from baseline in the log-transformed 24-hour urinary protein-to-creatinine ratio at month 12. Secondary end points included the change from baseline in the estimated glomerular filtration rate (eGFR) at month 12. Safety was also assessed. RESULTS: Among 155 patients who underwent randomization, 38 received sibeprenlimab at a dose of 2 mg per kilogram, 41 received sibeprenlimab at a dose of 4 mg per kilogram, 38 received sibeprenlimab at a dose of 8 mg per kilogram, and 38 received placebo. At 12 months, the geometric mean ratio reduction (±SE) from baseline in the 24-hour urinary protein-to-creatinine ratio was 47.2±8.2%, 58.8±6.1%, 62.0±5.7%, and 20.0±12.6% in the sibeprenlimab 2-mg, 4-mg, and 8-mg groups and the placebo group, respectively. At 12 months, the least-squares mean (±SE) change from baseline in eGFR was -2.7±1.8, 0.2±1.7, -1.5±1.8, and -7.4±1.8 ml per minute per 1.73 m2 in the sibeprenlimab 2-mg, 4-mg, and 8-mg groups and the placebo group, respectively. The incidence of adverse events that occurred after the start of administration of sibeprenlimab or placebo was 78.6% in the pooled sibeprenlimab groups and 71.1% in the placebo group. CONCLUSIONS: In patients with IgA nephropathy, 12 months of treatment with sibeprenlimab resulted in a significantly greater decrease in proteinuria than placebo. (Funded by Visterra; ENVISION ClinicalTrials.gov number, NCT04287985; EudraCT number, 2019-002531-29.).

l-Theanine Prevents Long-Term Affective and Cognitive Side Effects of Adolescent Δ-9-Tetrahydrocannabinol Exposure and Blocks Associated Molecular and Neuronal Abnormalities in the Mesocorticolimbic Circuitry.

Chronic adolescent exposure to Δ-9-tetrahydrocannabinol (THC) is linked to elevated neuropsychiatric risk and induces neuronal, molecular and behavioral abnormalities resembling neuropsychiatric endophenotypes. Previous evidence has revealed that the mesocorticolimbic circuitry, including the prefrontal cortex (PFC) and mesolimbic dopamine (DA) pathway are particularly susceptible to THC-induced pathologic alterations, including dysregulation of DAergic activity states, loss of PFC GABAergic inhibitory control and affective and cognitive abnormalities. There are currently limited pharmacological intervention strategies capable of preventing THC-induced neuropathological adaptations. l-Theanine is an amino acid analog of l-glutamate and l-glutamine derived from various plant sources, including green tea leaves. l-Theanine has previously been shown to modulate levels of GABA, DA, and glutamate in various neural regions and to possess neuroprotective properties. Using a preclinical model of adolescent THC exposure in male rats, we report that l-theanine pretreatment before adolescent THC exposure is capable of preventing long-term, THC-induced dysregulation of both PFC and VTA DAergic activity states, a neuroprotective effect that persists into adulthood. In addition, pretreatment with l-theanine blocked THC-induced downregulation of local GSK-3 (glycogen synthase kinase 3) and Akt signaling pathways directly in the PFC, two biomarkers previously associated with cannabis-related psychiatric risk and subcortical DAergic dysregulation. Finally, l-theanine powerfully blocked the development of both affective and cognitive abnormalities commonly associated with adolescent THC exposure, further demonstrating functional and long-term neuroprotective effects of l-theanine in the mesocorticolimbic system.SIGNIFICANCE STATEMENT With the increasing trend of cannabis legalization and consumption during adolescence, it is essential to expand knowledge on the potential effects of adolescent cannabis exposure on brain development and identify potential pharmacological strategies to minimize Δ-9-tetrahydrocannabinol (THC)-induced neuropathology. Previous evidence demonstrates that adolescent THC exposure induces long-lasting affective and cognitive abnormalities, mesocorticolimbic dysregulation, and schizophrenia-like molecular biomarkers that persist into adulthood. We demonstrate for the first time that l-theanine, an amino acid analog of l-glutamate and l-glutamine, is capable of preventing long-term THC side effects. l-Theanine prevented the development of THC-induced behavioral aberrations, blocked cortical downregulation of local GSK-3 (glycogen synthase kinase 3) and Akt signaling pathways, and normalized dysregulation of both PFC and VTA DAergic activity, demonstrating powerful and functional neuroprotective effects against THC-induced developmental neuropathology.

Adolescent Nicotine Exposure Induces Dysregulation of Mesocorticolimbic Activity States and Depressive and Anxiety-like Prefrontal Cortical Molecular Phenotypes Persisting into Adulthood.

Considerable evidence demonstrates strong comorbidity between nicotine dependence and mood and anxiety disorders. Nevertheless, the neurobiological mechanisms linking adolescent nicotine exposure to mood and anxiety disorders are not known. Disturbances in the mesocorticolimbic dopamine (DA) system, comprising the prefrontal cortex (PFC), ventral tegmental area (VTA), and nucleus accumbens (NAc), are correlates of mood and anxiety-related symptoms and this circuitry is strongly influenced by acute or chronic nicotine exposure. Using a combination of behavioral pharmacology, in vivo neuronal electrophysiology and molecular analyses, we examined and compared the effects of chronic nicotine exposure in rats during adolescence versus adulthood to characterize the mechanisms by which adolescent nicotine may selectively confer increased risk of developing mood and anxiety-related symptoms in later life. We report that exposure to nicotine, selectively during adolescence, induces profound and long-lasting neuronal, molecular and behavioral disturbances involving PFC DA D1R and downstream extracellular-signal-related kinase 1-2 (ERK 1-2) signaling. Remarkably, adolescent nicotine induced a persistent state of hyperactive DA activity in the ventral tegmental area (VTA) concomitant with hyperactive neuronal activity states in the PFC. Our findings identify several unique neuronal and molecular biomarkers that may serve as functional risk mechanisms for the long-lasting neuropsychiatric effects of adolescent smoking behaviors.

A Proliferation Inducing Ligand (APRIL) targeted antibody is a safe and effective treatment of murine IgA nephropathy.

IgA nephropathy (IgAN) is the most prevalent primary chronic glomerular disease for which no safe disease-specific therapies currently exist. IgAN is an autoimmune disease involving the production of autoantigenic, aberrantly O-glycosylated IgA1 and ensuing deposition of nephritogenic immune complexes in the kidney. A Proliferation Inducing Ligand (APRIL) has emerged as a key B-cell-modulating factor in this pathogenesis. Using a mouse anti-APRIL monoclonal antibody (4540), we confirm both the pathogenic role of APRIL in IgAN and the therapeutic efficacy of antibody-directed neutralization of APRIL in the grouped mouse ddY disease model. Treatment with 4540 directly translated to a reduction in relevant pathogenic mechanisms including suppressed serum IgA levels, reduced circulating immune complexes, significantly lower kidney deposits of IgA, IgG and C3, and suppression of proteinuria compared to mice receiving vehicle or isotype control antibodies. Furthermore, we translated these findings to the pharmacological characterization of VIS649, a highly potent, humanized IgG2κ antibody targeting and neutralizing human APRIL through unique epitope engagement, leading to inhibition of APRIL-mediated B-cell activities. VIS649 treatment of non-human primates showed dose-dependent reduction of serum IgA levels of up to 70%. A reduction of IgA+, IgM+, and IgG+ B cells was noted in the gut-associated mucosa of VIS649-treated animals. Population-based modeling predicted a favorable therapeutic dosing profile for subcutaneous administration of VIS649 in the clinical setting. Thus, our data highlight the potential therapeutic benefit of VIS649 for the treatment of IgAN.

Engineering Escherichia coli coculture systems for the production of biochemical products.

Engineering microbial consortia to express complex biosynthetic pathways efficiently for the production of valuable compounds is a promising approach for metabolic engineering and synthetic biology. Here, we report the design, optimization, and scale-up of an Escherichia coli-E. coli coculture that successfully overcomes fundamental microbial production limitations, such as high-level intermediate secretion and low-efficiency sugar mixture utilization. For the production of the important chemical cis,cis-muconic acid, we show that the coculture approach achieves a production yield of 0.35 g/g from a glucose/xylose mixture, which is significantly higher than reported in previous reports. By efficiently producing another compound, 4-hydroxybenzoic acid, we also demonstrate that the approach is generally applicable for biosynthesis of other important industrial products.

Efficient utilization of pentoses for bioproduction of the renewable two-carbon compounds ethylene glycol and glycolate.

The development of lignocellulose as a sustainable resource for the production of fuels and chemicals will rely on technology capable of converting the raw materials into useful compounds; some such transformations can be achieved by biological processes employing engineered microorganisms. Towards the goal of valorizing the hemicellulose fraction of lignocellulose, we designed and validated a set of pathways that enable efficient utilization of pentoses for the biosynthesis of notable two-carbon products. These pathways were incorporated into Escherichia coli, and engineered strains produced ethylene glycol from various pentoses, including simultaneously from D-xylose and L-arabinose; one strain achieved the greatest reported titer of ethylene glycol, 40 g/L, from D-xylose at a yield of 0.35 g/g. The strategy was then extended to another compound, glycolate. Using D-xylose as the substrate, an engineered strain produced 40 g/L glycolate at a yield of 0.63 g/g, which is the greatest reported yield to date.

Biosynthesis of poly(glycolate-co-lactate-co-3-hydroxybutyrate) from glucose by metabolically engineered Escherichia coli.

Metabolically engineered Escherichia coli strains were constructed to effectively produce novel glycolate-containing biopolymers from glucose. First, the glyoxylate bypass pathway and glyoxylate reductase were engineered such as to generate glycolate. Second, glycolate and lactate were activated by the Megasphaera elsdenii propionyl-CoA transferase to synthesize glycolyl-CoA and lactyl-CoA, respectively. Third, ß-ketothiolase and acetoacetyl-CoA reductase from Ralstonia eutropha were introduced to synthesize 3-hydroxybutyryl-CoA from acetyl-CoA. At last, the Ser325Thr/Gln481Lys mutant of polyhydroxyalkanoate (PHA) synthase from Pseudomonas sp. 61-3 was over-expressed to polymerize glycolyl-CoA, lactyl-CoA and 3-hydroxybutyryl-CoA to produce poly(glycolate-co-lactate-co-3-hydroxybutyrate). The recombinant E. coli was able to accumulate the novel terpolymer with a titer of 3.90g/l in shake flask cultures. The structure of the resulting polymer was chemically characterized by proton NMR analysis. Assessment of thermal and mechanical properties demonstrated that the produced terpolymer possessed decreased crystallinity and improved toughness, in comparison to poly(3-hydroxybutyrate) homopolymer. This is the first study reporting efficient microbial production of poly(glycolate-co-lactate-co-3-hydroxybutyrate) from glucose.

Engineering a novel biosynthetic pathway in Escherichia coli for production of renewable ethylene glycol.

Ethylene glycol (EG) is an important commodity chemical with broad industrial applications. It is presently produced from petroleum or natural gas feedstocks in processes requiring consumption of significant quantities of non-renewable resources. Here, we report a novel pathway for biosynthesis of EG from the renewable sugar glucose in metabolically engineered Escherichia coli. Serine-to-EG conversion was first achieved through a pathway comprising serine decarboxylase, ethanolamine oxidase, and glycolaldehyde reductase. Serine provision in E. coli was then enhanced by overexpression of the serine-biosynthesis pathway. The integration of these two parts into the complete EG-biosynthesis pathway in E. coli allowed for production of 4.1 g/L EG at a cumulative yield of 0.14 g-EG/g-glucose, establishing a foundation for a promising biotechnology.

Engineering E. coli-E. coli cocultures for production of muconic acid from glycerol.

BACKGROUND: cis, cis-Muconic acid is an important chemical that can be biosynthesized from simple substrates in engineered microorganisms. Recently, it has been shown that engineering microbial cocultures is an emerging and promising approach for biochemical production. In this study, we aim to explore the potential of the E. coli-E. coli coculture system to use a single renewable carbon source, glycerol, for the production of value-added product cis, cis-muconic acid. RESULTS: Two coculture engineering strategies were investigated. In the first strategy, an E. coli strain containing the complete biosynthesis pathway was co-cultivated with another E. coli strain containing only a heterologous intermediate-to-product biosynthetic pathway. In the second strategy, the upstream and downstream pathways were accommodated in two separate E. coli strains, each of which was dedicated to one portion of the biosynthesis process. Compared with the monoculture approach, both coculture engineering strategies improved the production significantly. Using a batch bioreactor, the engineered coculture achieved a 2 g/L muconic acid production with a yield of 0.1 g/g. CONCLUSIONS: Our results demonstrate that coculture engineering is a viable option for producing muconic acid from glycerol. Moreover, microbial coculture systems are shown to have the potential for converting single carbon source to value-added products.

Safety, Pharmacokinetics, and Pharmacodynamics of Subcutaneous Sibeprenlimab in Healthy Participants.

Sibeprenlimab blocks the cytokine "A Proliferation-Inducing Ligand" (APRIL), which may play a key role in immunoglobulin A nephropathy pathogenesis. A phase 1 study of subcutaneous (SC) sibeprenlimab evaluated preliminary safety, tolerability, pharmacokinetics, and pharmacodynamics in healthy participants. This was an open-label, single-ascending-dose study. Twelve participants in each of 4 sequential dosing cohorts received 1 SC dose of sibeprenlimab (200 mg [1×1 mL injection], 400 mg [2×1 mL injections], 400 mg [1×2 mL injection], or 600 mg [1 mL+2 mL injections]) and underwent 16-week follow-up for adverse events, pharmacokinetics, and pharmacodynamics (serum APRIL, immunoglobulin [Ig] levels). Sibeprenlimab in single SC doses of 200-600 mg was slowly absorbed into the systemic circulation, with a median time to maximum serum concentration of approximately 6-10.5 days, and a mean elimination half-life of approximately 8-10 days. Serum APRIL, IgA, IgM, and, to a lesser extent, IgG decreased in a dose-dependent and reversible manner. Maximal reduction in serum IgA was approximately 60% at the 400- and 600-mg doses and 40% at 200 mg. Serum APRIL rapidly decreased to near the lower limit of quantification, and duration of suppression was dose-dependent, with near complete suppression until weeks 4-6 at the 400-mg dose and week 8 at the 600-mg dose. Adverse events occurred in 30/48 (62.5%) participants; none were serious or led to study discontinuation. Sibeprenlimab rapidly and sustainably reduced target APRIL and Ig biomarkers in a dose-dependent and reversible manner, with acceptable preliminary safety and pharmacokinetics.

Factors affecting response and tolerability to ferumoxytol in nondialysis chronic kidney disease patients.

BACKGROUND: Ferumoxytol is a unique intravenous (i.v.) iron therapy. This report examines factors affecting hemoglobin response to i.v. ferumoxytol, and the relationship between hematologic parameters, concomitant erythropoiesis-stimulating agents (ESA), and adverse events (AEs) in nondialysis CKD patients. METHODS: A series of post-hoc efficacy and safety analyses were performed using pooled data from two identically designed Phase III studies in 608 nondialysis CKD patients randomized to receive two 510 mg i.v. injections of ferumoxytol within 5 ± 3 days versus oral iron. RESULTS: Ferumoxytol resulted in a significant increase in hemoglobin in the presence and absence of ESA, and across a range of baseline hemoglobin, transferrin saturation, ferritin, and reticulocyte hemoglobin content levels. Adverse event rates with ferumoxytol were similar across quartiles of change in hemoglobin; there were no trends suggesting an increased rate of cardiovascular AEs with higher maximum achieved hemoglobin or faster rate of hemoglobin rise. There was no meaningful difference in the rate of AEs, serious AEs, and cardiovascular AEs between patients receiving or not receiving ESA. CONCLUSIONS: These analyses add to the knowledge of predictors of response and safety outcomes associated with i.v. iron therapy in nondialysis CKD patients.

Electronic structure of neighboring extein residue modulates intein C-terminal cleavage activity.

Protein splicing is an autocatalytic reaction where an intervening element (intein) is excised and the remaining two flanking sequences (exteins) are joined. The reaction requires specific conserved residues, and activity may be affected by both the intein and the extein sequence. Predicting how sequence will affect activity is a challenging task. Based on first-principles density functional theory and multiscale quantum mechanics/molecular mechanics, we report C-terminal cleavage reaction rates for five mutations at the first residue of the C-extein (+1), and describe molecular properties that may be used as predictors for future mutations. Independently, we report on experimental characterization of the same set of mutations at the +1 residue resulting in a wide range of C-terminal cleavage activities. With some exceptions, there is general agreement between computational rates and experimental cleavage, giving molecular insight into previous claims that the +1 extein residue affects intein catalysis. These data suggest utilization of attenuating +1 mutants for intein-mediated protein manipulations because they facilitate precursor accumulation in vivo for standard purification schemes. A more detailed analysis of the "+1 effect" will also help to predict sequence-defined effects on insertion points of the intein into proteins of interest.

Mitochondrial DNA injury and mortality in hemodialysis patients.

The role of mitochondrial injury in the pathogenesis of complications of uremia is incompletely defined, although diminished bioenergetic capacity and the accumulation of mitochondrial DNA (mtDNA) mutations have been reported. This study was undertaken to evaluate the prevalence of mtDNA injury in 180 patients who had ESRD and were enrolled into the baseline phase of the HEMO study and to relate these markers to all-cause mortality. The mitochondrial injury markers studied in peripheral blood mononuclear cells were the mtDNA copy number per cell, measured by quantitative PCR, and the presence of the mtDNA(4977) mutation. After frequency-matching healthy control subjects for age, mtDNA copy number was lower among older dialysis patients compared with older healthy subjects (P = 0.01). A one-log increase in mtDNA copy number was independently associated with a decreased hazard for mortality (adjusted hazard ratio 0.64; 95% confidence interval 0.46 to 0.89). The mtDNA(4977) deletion was present in 48 (31%) patients and was independently associated with a decreased hazard for mortality (adjusted hazard ratio 0.33; 95% confidence interval 0.19 to 0.56). In summary, the mtDNA(4977) seems to predict survival in ESRD, but a reduced mitochondrial copy number seems to predict a poor outcome. Although further exploration of these associations is needed, evaluation of mitochondrial DNA copy number and somatic mtDNA mutations may provide simple genomic biomarkers to predict clinical outcomes among patients with ESRD.

Highly conserved histidine plays a dual catalytic role in protein splicing: a pKa shift mechanism.

Protein splicing is a precise autocatalytic process in which an intein excises itself from a precursor with the concomitant ligation of the flanking sequences. Protein splicing occurs through acid-base catalysis in which the ionization states of active site residues are crucial to the reaction mechanism. In inteins, several conserved histidines have been shown to play important roles in protein splicing, including the most conserved "B-block" histidine. In this study, we have combined NMR pK(a) determination with quantum mechanics/molecular mechanics (QM/MM) modeling to study engineered inteins from Mycobacterium tuberculosis (Mtu) RecA intein. We demonstrate a dramatic pK(a) shift for the invariant B-block histidine, the most conserved residue among inteins. The B-block histidine has a pK(a) of 7.3 +/- 0.6 in a precursor and a pK(a) of <3.5 in a spliced intein. The pK(a) values and QM/MM data suggest that the B-block histidine has a dual role in the acid-base catalysis of protein splicing. This histidine likely acts as a general base to initiate splicing with an acyl shift and then as a general acid to cause the breakdown of the scissile bond at the N-terminal splicing junction. The proposed pK(a) shift mechanism accounts for the biochemical data supporting the essential role for the B-block histidine and for the near absolute sequence conservation of this residue.

Ferumoxytol for treating iron deficiency anemia in CKD.

Iron deficiency is an important cause of anemia in patients with chronic kidney disease (CKD), but intravenous iron is infrequently used among patients who are not on dialysis. Ferumoxytol is a novel intravenous iron product that can be administered as a rapid injection. This Phase III trial randomly assigned 304 patients with CKD in a 3:1 ratio to two 510-mg doses of intravenous ferumoxytol within 5 +/- 3 d or 200 mg of elemental oral iron daily for 21 d. The increase in hemoglobin at day 35, the primary efficacy end point, was 0.82 +/- 1.24 g/dl with ferumoxytol and 0.16 +/- 1.02 g/dl with oral iron (P < 0.0001). Among patients who were not receiving erythropoiesis-stimulating agents, hemoglobin increased 0.62 +/- 1.02 g/dl with ferumoxytol and 0.13 +/- 0.93 g/dl with oral iron. Among patients who were receiving erythropoiesis-stimulating agents, hemoglobin increased 1.16 +/- 1.49 g/dl with ferumoxytol and 0.19 +/- 1.14 g/dl with oral iron. Treatment-related adverse events occurred in 10.6% of patients who were treated with ferumoxytol and 24.0% of those who were treated with oral iron; none was serious. In summary, a regimen of two doses of 510 mg of intravenous ferumoxytol administered rapidly within 5 +/- 3 d was well tolerated and had the intended therapeutic effect. This regimen may offer a new, efficient option to treat iron deficiency anemia in patients with CKD.

The Bivalent Rewarding and Aversive properties of Δ9-tetrahydrocannabinol are Mediated Through Dissociable Opioid Receptor Substrates and Neuronal Modulation Mechanisms in Distinct Striatal Sub-Regions.

The primary psychoactive compound in cannabis, Δ9-tetrahydrocannabinol (THC), is capable of producing bivalent rewarding and aversive affective states through interactions with the mesolimbic system. However, the precise mechanisms underlying the dissociable effects of THC are not currently understood. In the present study, we identify anatomically dissociable effects of THC within the rat nucleus accumbens (NAc), using an integrative combination of behavioral pharmacology and in vivo neuronal electrophysiology. We report that the rewarding vs. aversive stimulus properties of THC are both anatomically and pharmacologically dissociable within distinct anterior vs. posterior sub-regions of the NAc. While the rewarding effects of THC were dependent upon local µ-opioid receptor signaling, the aversive effects of THC were processed via a κ-opioid receptor substrate. Behaviorally, THC in the posterior NASh induced deficits in social reward and cognition whereas THC in the anterior NAc, potentiated opioid-related reward salience. In vivo neuronal recordings demonstrated that THC decreased medium spiny neuron (MSN) activity in the anterior NAc and increased the power of gamma (γ) oscillations. In contrast, THC increased MSN activity states in the posterior NASh and decreased γ-oscillation power. These findings reveal critical new insights into the bi-directional neuronal and pharmacological mechanisms controlling the dissociable effects of THC in mesolimbic-mediated affective processing.

Δ-9-Tetrahydrocannabinol and Cannabidiol produce dissociable effects on prefrontal cortical executive function and regulation of affective behaviors.

The use of cannabis for therapeutic and recreational purposes is growing exponentially. Nevertheless, substantial questions remain concerning the potential cognitive and affective side-effects associated with cannabis exposure. In particular, the effects of specific marijuana-derived phytocannabinoids on neural regions such as the prefrontal cortex (PFC) are of concern, given the role of the PFC in both executive cognitive function and affective processing. The main biologically active phytocannabinoids, ∆-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), interact with multiple neurotransmitter systems important for these processes directly within the PFC. Considerable evidence has demonstrated that acute or chronic THC exposure may induce psychotomimetic effects, whereas CBD has been shown to produce potentially therapeutic effects for both psychosis and/or anxiety-related symptoms. Using an integrative combination of cognitive and affective behavioral pharmacological assays in rats, we report that acute intra-PFC infusions of THC produce anxiogenic effects while producing no impairments in executive function. In contrast, acute infusions of intra-PFC CBD impaired attentional set-shifting and spatial working memory, without interfering with anxiety or sociability behaviors. In contrast, intra-PFC CBD reversed the cognitive impairments induced by acute glutamatergic antagonism within the PFC, and blocked the anxiogenic properties of THC, suggesting that the therapeutic properties of CBD within the PFC may be present only during pathologically aberrant states within the PFC. Interestingly, the effects of PFC THC vs. CBD were found to be mediated through dissociable CB1 vs. 5-HT1A-dependent receptor signaling mechanisms, directly in the PFC.

Crystallographic and mutational studies of Mycobacterium tuberculosis recA mini-inteins suggest a pivotal role for a highly conserved aspartate residue.

The 440 amino acid Mtu recA intein consists of independent protein-splicing and endonuclease domains. Previously, removal of the central endonuclease domain of the intein, and selection for function, generated a 168 residue mini-intein, DeltaI-SM, that had splicing activity similar to that of the full-length, wild-type protein. A D422G mutation (DeltaI-CM) increased C-terminal cleavage activity. Using the DeltaI-SM mini-intein structure (presented here) as a guide, we previously generated a highly active 139 residue mini-intein, DeltaDeltaI(hh)-SM, by replacing 36 amino acid residues in the residual endonuclease loop with a seven-residue beta-turn from the autoprocessing domain of Hedgehog protein. The three-dimensional structures of DeltaI-SM, DeltaDeltaI(hh)-SM, and two variants, DeltaDeltaI(hh)-CM and DeltaDeltaI(hh), have been determined to evaluate the effects of the minimization on intein integrity and to investigate the structural and functional consequences of the D422G mutation. These structural studies show that Asp422 is capable of interacting with both the N and C termini. These interactions are lacking in the CM variant, but are replaced by contacts with water molecules. Accordingly, additional mutagenesis of residue 422, combined with mutations that isolate N-terminal and C-terminal cleavage, showed that the side-chain of Asp422 plays a role in both N and C-terminal cleavage, thereby suggesting that this highly conserved residue regulates the balance between the two reactions.

KDOQI US commentary on the KDIGO clinical practice guideline for the prevention, diagnosis, evaluation, and treatment of hepatitis C in CKD.

KDIGO (Kidney Disease: Improving Global Outcomes) is an international initiative with a key mission of developing clinical practice guidelines in the area of chronic kidney disease (CKD). KDIGO recently published evidence-based clinical practice guidelines for the prevention, diagnosis, evaluation, and treatment of hepatitis C virus infection in individuals with CKD. The process of adaptation of international guidelines is an important task that, although guided by general principles, needs to be individualized for each region and country. Therefore, the National Kidney Foundation-Kidney Disease Outcomes Quality Initiative (KDOQI) convened a multidisciplinary group to comment on the application and implementation of the KDIGO guidelines for patients with CKD in the United States. This commentary summarizes the process undertaken by this group in considering the guidelines in the context of health care delivery in the United States. Guideline statements are presented, followed by a succinct discussion and annotation of the rationale for the statements. Research recommendations that are of particular interest to the United States are then summarized to highlight future areas of inquiry that would enable updating of the guidelines.

Role of prohepcidin, inflammatory markers and iron status in resistance to rhEPO therapy in hemodialysis patients.

The aim of our study was to assess possible relations between prohepcidin, iron status and inflammatory markers in hemodialysis (HD) patients, as well as its association with resistance to recombinant human erythropoietin (rhEPO) therapy. Fifty HD patients and 25 healthy controls were enrolled in the study. Among HD patients, 25 were non-responders and 25 were responders to rhEPO therapy. Complete blood cell count, reticulocyte count, and circulating levels of ferritin, iron, transferrin saturation, C-reactive protein (CRP), soluble interleukin (IL)-2 receptor (s-IL2R), soluble transferrin receptor (s-TfR), IL-6 and prohepcidin were measured in all patients and controls. HD patients showed higher circulating levels of ferritin, s-TfR, CRP, IL-6, s-IL2R and prohepcidin, and lower levels of transferrin compared to healthy controls. Higher levels of s-TfR, CRP and lower levels prohepcidin were observed among non-responders compared to responders. Prohepcidin levels correlated negatively with s-TfR and reticulocyte count. The weekly rhEPO/kg dose was found to be positively correlated with CRP, hemoglobin and s-TfR. In conclusion, our data show that a close interaction exists between inflammation, iron status and prohepcidin serum levels that ultimately regulate intracellular iron availability. Prohepcidin and s-TfR, together with CRP, may prove to be good markers of resistance to rhEPO therapy in HD patients.