scholarly journals A Novel Crosstalk Between Iron Homeostasis and mTOR Mediated By the Immunophilin FKBP12

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 261-261 ◽  
Author(s):  
Laura Silvestri ◽  
Silvia Colucci ◽  
Alessia Pagani ◽  
Irene Artuso ◽  
Mariatesera Pettinato ◽  
...  
Keyword(s):  
Iron Homeostasis ◽  
Activin A ◽  
Hepatoma Cells ◽  
Transplant Proc ◽  
Type I ◽  
Type Ii ◽  
Transfected Cells ◽  
Hepcidin Expression ◽  
Bmp Pathway

Abstract Introduction Hepcidin, the liver hormone that regulates iron homeostasis, is mainly activated via the BMP/SMAD pathway. Among other stimuli, as erythropoiesis expansion, inflammation and hypoxia, hepcidin expression is influenced by drugs, including the mTOR inhibitor rapamycin (RAPA) (Mleczko-Sanecka et al., Blood 2014). In the liver, BMP type I receptors, ALK2 and ALK3, are both essential for hepcidin regulation (Steinbiecker et al., Blood 2012), whereas BMP type II receptors, BMPR2 and ACVR2A, have a redundant role (Mayeur et al., Blood 2014). The signaling is activated when BMP type II receptors phosphorylate residues of the glycine/serine rich (GS) domain of BMP type I receptors, which then activate SMAD1/5/8. Treatment with RAPA increases hepcidin expression in murine hepatocytes and may cause microcytic anemia in patients (Przybylowski P. et al., Transplant Proc. 2013). However, the mechanisms involved in hepcidin and mTOR crosstalk are unknown. Methods Hepcidin, BMP-SMAD and mTOR target genes were analyzed in human hepatoma cell lines and murine primary hepatocytes (HCs), treated with RAPA (100 nM), Torin1 (100 nM), tacrolimus (TAC, 1 μg/ml) in the presence or absence of the BMP pathway inhibitor DMH1 (0.5 μg/ml) or of the ligands BMP6, BMP2, Activin B (ActB) and Activin A (ActA) (1-100 ng/ml). ALK2wt was mutagenized in the GS domain (R206H, Q207E) or close to the GS domain (R258S) to generate ALK2mut with reduce binding to FKBP12 (Taylor et al., Cancer Res. 2014). SMAD1/5/8 phosphorylation was analyzed by Western Blotting in cells transfected with SMAD1 and ALK2wt or ALK2mut and treated or not with TAC, BMP6 and ActA. Binding of FKBP12 to ALK2wt and ALK2mut was assessed by coimmunoprecipitation of tagged proteins in transfected cells treated as above. Eight weeks old C57BL/6 wild type male mice were treated with a single dose of TAC (10 mg/kg) or vehicle and sacrificed at different time points (3-18 hrs). Hepcidin expression, LIC, SIC, serum iron and hematological parameters were analyzed by standard methods. Results We analyzed hepcidin expression in hepatoma cells and primary HCs treated with RAPA, an inhibitor of mTORC1, and Torin1, an ATP-competitive inhibitor of mTORC1 and 2. Hepcidin is increased by RAPA, but not Torin1, in a SMAD1/5/8 dependent way since DMH1 abrogates the effect. RAPA inhibits mTORC1 when complexed with FKBP12, an immunophilin that binds BMP receptors to avoid leakage activation of the pathway. To investigate whether hepcidin upregulation by RAPA is mediated by FKBP12 sequestration, we used genetic and pharmacologic approaches. First, we confirmed by coimmunoprecipitation that ALK2mut have a reduced ability to bind FKBP12. Then we transfected hepatoma cells with ALK2wt and ALK2mut and analyzed hepcidin and BMP pathway activation. Overexpression of ALK2mut increases hepcidin through SMAD1/5/8 as shown by high levels of SMAD1 phosphorylation, an effect abrogated by DMH1. Second, we treated hepatoma cells and primary HCs with TAC, a calcineurin inhibitor that binds FKBP12. This treatment increases hepcidin through SMAD1/5/8, suggesting a mechanism shared with RAPA. The same effect is observed in vivo since hepcidin is increased at 6 hrs post-injection in TAC-treated wt mice. Our results identify FKBP12 as a novel regulator of hepcidin. In addition, FKBP12 displacement alters the BMP receptor selectivity to ligands. Despite ALK2wt preferentially binds BMP6, ALK2mut become responsive to ActA, a TGF-β ligand that signals through SMAD2/3. Hepcidin activation by BMP6, BMP2 and ActB is comparable in ALK2wt and ALK2mut expressing cells. However, ActA upregulates hepcidin (through SMAD1/5/8) only in ALK2mut transfected cells. This effect is due to the impaired ability of ALK2mut to bind FKBP12, since it is observed even in ALK2wt transfected cells pretreated with TAC. Conclusions FKBP12 contributes to hepcidin regulation both in vitro and in vivo, thus adding a new player to the BMP-dependent hepcidin activation and a potential pharmacologic target for disorders characterized by low hepcidin and iron overload. Furthermore the ability of ALK2 to respond to Activin A, which is released in inflammation, links the BMP pathway-hepcidin activation to the inflammatory response. Disclosures No relevant conflicts of interest to declare.

scholarly journals Functional redundancy of type I and type II receptors in the regulation of skeletal muscle growth by myostatin and activin A

2020 ◽  
Vol 117 (49) ◽  
pp. 30907-30917 ◽  
Author(s):  
Se-Jin Lee ◽  
Adam Lehar ◽  
Yewei Liu ◽  
Chi Hai Ly ◽  
Quynh-Mai Pham ◽  
...  
Keyword(s):  
Family Member ◽  
Muscle Mass ◽  
Transforming Growth Factor ◽  
Muscle Growth ◽  
Functional Redundancy ◽  
Activin A ◽  
Metabolic Effects ◽  
Type I ◽  
Type Ii

Myostatin (MSTN) is a transforming growth factor-β (TGF-β) family member that normally acts to limit muscle growth. The function of MSTN is partially redundant with that of another TGF-β family member, activin A. MSTN and activin A are capable of signaling through a complex of type II and type I receptors. Here, we investigated the roles of two type II receptors (ACVR2 and ACVR2B) and two type I receptors (ALK4 and ALK5) in the regulation of muscle mass by these ligands by genetically targeting these receptors either alone or in combination specifically in myofibers in mice. We show that targeting signaling in myofibers is sufficient to cause significant increases in muscle mass, showing that myofibers are the direct target for signaling by these ligands in the regulation of muscle growth. Moreover, we show that there is functional redundancy between the two type II receptors as well as between the two type I receptors and that all four type II/type I receptor combinations are utilized in vivo. Targeting signaling specifically in myofibers also led to reductions in overall body fat content and improved glucose metabolism in mice fed either regular chow or a high-fat diet, demonstrating that these metabolic effects are the result of enhanced muscling. We observed no effect, however, on either bone density or muscle regeneration in mice in which signaling was targeted in myofibers. The latter finding implies that MSTN likely signals to other cells, such as satellite cells, in addition to myofibers to regulate muscle homeostasis.

scholarly journals The Immunophilin FKBP12 Inhibits Hepcidin By Modulating BMP Type I-Type II Receptors Interaction and Ligand Responsiveness

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 430-430
Author(s):  
Alessandro Dulja ◽  
Alessia Pagani ◽  
Mariateresa Pettinato ◽  
Antonella Nai ◽  
Clara Camaschella ◽  
...  
Keyword(s):  
Hepatoma Cell Line ◽  
Type I ◽  
Type Ii ◽  
Smad Pathway ◽  
Hepcidin Expression ◽  
Receptor Complexes ◽  
Bmp Receptors ◽  
Pathway Activation ◽  
Huh7 Cells

Introduction The liver hormone hepcidin is the master regulator of iron metabolism that modulates iron release into the circulation by binding and blocking the iron exporter ferroportin (Nemeth et al., 2004). Hepcidin expression is under the control of the BMP-SMAD pathway (Babitt et al., 2006), whose activation requires the formation of a hexameric complex composed of a dimer of BMP receptors type I (BMPR-Is), a dimer of BMPR type II (BMPR-IIs) and dimeric ligands. ALK2 and ALK3, as BMPR-Is (Steinbiecker et al., 2011), BMPR2 and ACVR2A, as BMPR-IIs (Mayeur et al., 2014), and BMP2 (Koch et al, 2017) and BMP6 (Meynard et al., 2009), as ligands, control hepcidin expression in vivo. We previously demonstrated that the immunophilin FKBP12 limits hepcidin expression in hepatocytes by binding ALK2 (Colucci et al., 2017). However, the molecular mechanism whereby FKBP12 regulates ALK2 and its relationship with BMPR-IIs and ligands in the regulation of the BMP-SMAD pathway and hepcidin expression are still unclear. Methods: BMPR-Is dimerization was evaluated by co-immunoprecipitation (CoIP) experiments performed in the HuH7 human hepatoma cell line. BMP-SMAD pathway and hepcidin promoter activation were analyzed by using a reporter vector with the luciferase under the control of BMP responsive elements or of the human hepcidin promoter, respectively. Endogenous hepcidin expression was measured by qRT-PCR. Results: Since BMPRIs act as dimers, we first tested whether FKBP12 modulates the dimerization process. MYC- and FLAG-tagged ALK2 or ALK3 were transfected in HuH7 cells in the presence of FKBP12. Cells were treated or not with tacrolimus (TAC), an immunosuppressive drug that sequesters FKBP12 from ALK2. FKBP12 promotes ALK2 homodimers, functionally inactive in the absence of ligands, with no effect on ALK3 homodimerization. TAC promotes increased ALK2 homodimerization and SMAD1/5/8 phosphorylation, demonstrating that in the absence of FKBP12, ALK2 homodimers are stabilized and functionally active. We next focused on BMP6, the physiologic ligand that binds preferentially ALK2 and plays a fundamental role in hepcidin regulation in vivo. In HuH7 cells transfected with FKBP12 and ALK2, BMP6 treatment reduced FKBP12-ALK2 binding and increased ALK2 homodimers. In agreement, SMAD1/5/8 phosphorylation was increased, indicating that FKBP12 displacement allows the formation of functional receptor complexes responsive to BMP6. BMPR-Is activate SMAD1/5/8 following BMPR-IIs phosphorylation. Since TAC induces SMAD1/5/8 phosphorylation in the absence of ligands, we hypothesized that FKBP12 displacement also affects the formation of BMPR-I/BMPR-II oligomers. HuH7 cells were transfected with ALK2, BMPR2 or ACVR2A and FKBP12, and treated or not with TAC. FKBP12 sequestration by TAC enhances the ALK2-BMPR2 and ALK2-ACVR2A interaction and accordingly activates SMAD1/5/8 signaling. Given that FKBP12 modulates BMP receptor interaction, we wondered how this functionally impacts on the response to BMP ligands, as BMP2, that guarantees basal hepcidin levels by binding ALK3, and BMP6, upregulated in iron overload that signals preferentially through ALK2. ALK3 upregulates the BMP pathway and hepcidin expression in a similar way in response to BMP2 and BMP6, in agreement with the evidence that both ligands bind ALK3. ALK2, which failed to activate the pathway in the absence ligands, leads to a greater response to BMP6, consistent with the fact that it is the BMP6 receptor. Thus FKBP12 quantitatively, rather than qualitatively, modulates the BMP-SMAD pathway activation in response to BMP ligands. Conclusions: Altogether our results clarify the molecular mechanisms of hepcidin regulation demonstrating that: 1) FKBP12 limits hepcidin expression by inducing the formation of inactive ALK2 homodimers in the absence of ligands. 2) Decreased FKBP12 binding to ALK2, by TAC or BMP6, favors the formation of active ALK2 homodimers. 3) FKBP12 sequestration increases the binding of ALK2 with the BMPR-IIs, thus favoring SMAD1/5/8 phosphorylation and pathway activation. 4) FKBP12 quantitatively modulates the response of BMPRIs to the ligands BMP2 and BMP6. Disclosures Camaschella: Vifor Iron Core: Consultancy; Celgene: Consultancy; Novartis: Consultancy.

A delayed rectifier conductance in type I hair cells of the mouse utricle

1996 ◽  
Vol 76 (2) ◽  
pp. 995-1004 ◽  
Author(s):  
A. Rusch ◽  
R. A. Eatock
Keyword(s):  
Hair Cells ◽  
Time Course ◽  
Dissociation Constants ◽  
Resting Potential ◽  
Delayed Rectifier ◽  
Type I ◽  
Type Ii ◽  
Voltage Range ◽  
Average Maximum

1. Membrane currents of hair cells in acutely excised or cultured mouse utricles were recorded with the whole cell voltage-clamp method at temperatures between 23 and 36 degrees C. 2. Type I and II hair cells both had delayed rectifier conductances that activated positive to -55 mV. 3. Type I, but not type II, hair cells had an additional delayed rectifier conductance (gK,L) with an activation range that was unusually negative and variable. At 23-25 degrees C, V(1/2) values ranged from -88 to -62 mV in 57 cells. 4. gK,L was very large. At 23-25 degrees C, the average maximum chord conductance was 75 +/- 65 nS (mean +/- SD, n = 57; measured at -54 mV), or approximately 21 nS/pF of cell capacitance. 5. gK,L was highly selective for K+ over Na+ (permeability ratio PNa+/PK+:0.006), but unlike other delayed rectifiers, gK,L was significantly permeable to Cs+ (PCs+/PK+:0.31). gK,L was independent of extracellular Ca2+. 6. At -64 mV, Ba2+ and 4-aminopyridine blocked gK,L with apparent dissociation constants of 2.0 mM and 43 microM, respectively. Extracellular Cs+ (5 mM) blocked gK,L by 50% at -124 mV. Apamin (100 nM) and dendrotoxin (10 nM) has no effect. 7. The kinetic data of gK,L are consistent with a sequential gating model with at least two closed states and one open state. The slow activation kinetics (principal time constants at 23-25 degrees C:600-200 ms) had a thermal Q10 of 2.1. Inactivation (Q10:2.7) was partial at all temperatures. Deactivation followed a double-exponential time course and had a Q10 of 2.0. 8. At 23-25 degrees C, gK,L was appreciably activated at the mean resting potential of type I hair cells (-77 +/- 3.1 mV, n = 62), so that input conductances were often more than an order of magnitude larger than those of type II cells. If these conditions hold in vivo, type I cells would produce unusually small receptor potentials. Warming the cells to 36 degrees C produced parallel shifts in gK,L's activation range (0.8 +/- 0.3 mV/degrees C, n = 8), and in the resting potential (0.6 +/- 0.3 mV/degrees C, n = 4). Thus the high input conductances were not an artifact of unphysiological temperatures but remained high near body temperature. It remains possible that in vivo gK,L's activation range is less negative and input conductances are lower; the large variance in the voltage range of activation suggests that it may be subject to modulation.

scholarly journals Biological Relevance of Colony Morphology and Phenotypic Switching by Burkholderia pseudomallei

2006 ◽  
Vol 189 (3) ◽  
pp. 807-817 ◽  
Author(s):  
Narisara Chantratita ◽  
Vanaporn Wuthiekanun ◽  
Khaemaporn Boonbumrung ◽  
Rachaneeporn Tiyawisutsri ◽  
Mongkol Vesaratchavest ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Vaccine Development ◽  
Colony Morphology ◽  
Phenotypic Switching ◽  
Type I ◽  
Type Ii ◽  
Altered Expression ◽  
Replication Fitness

ABSTRACT Melioidosis is a notoriously protracted illness and is difficult to cure. We hypothesize that the causative organism, Burkholderia pseudomallei, undergoes a process of adaptation involving altered expression of surface determinants which facilitates persistence in vivo and that this is reflected by changes in colony morphology. A colony morphotyping scheme and typing algorithm were developed using clinical B. pseudomallei isolates. Morphotypes were divided into seven types (denoted I to VII). Type I gave rise to other morphotypes (most commonly type II or III) by a process of switching in response to environmental stress, including starvation, iron limitation, and growth at 42°C. Switching was associated with complex shifts in phenotype, one of which (type I to type II) was associated with a marked increase in production of factors putatively associated with in vivo concealment. Isogenic types II and III, derived from type I, were examined using several experimental models. Switching between isogenic morphotypes occurred in a mouse model, where type II appeared to become adapted for persistence in a low-virulence state. Isogenic type II demonstrated a significant increase in intracellular replication fitness compared with parental type I after uptake by epithelial cells in vitro. Isogenic type III demonstrated a higher replication fitness following uptake by macrophages in vitro, which was associated with a switch to type II. Mixed B. pseudomallei morphologies were common in individual clinical specimens and were significantly more frequent in samples of blood, pus, and respiratory secretions than in urine and surface swabs. These findings have major implications for therapeutics and vaccine development.

scholarly journals Innate triggering and antiviral effector functions of activin A

2021 ◽  
Author(s):  
Kinda Al-Hourani ◽  
Narayan Ramamurthy ◽  
Emanuele Marchi ◽  
Ruth M Eichinger ◽  
Lian N Lee ◽  
...  
Keyword(s):  
Viral Infection ◽  
Influenza A ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Activin A ◽  
Type I ◽  
Type I Ifn ◽  
Effector Functions

First-line defence against viral infection is contingent upon rapid detection of conserved viral structural and genomic motifs by germline-encoded pattern recognition receptors, followed by activation of the type I IFN system and establishment of an intracellular antiviral state. Novel antiviral functions of bone morphogenetic protein and related activin cytokines, acting in conjunction with, and independently of, type I IFN, have recently been described. Activin A mediates multiple innate and adaptive immune functions, including antiviral effects. However, how such effects are mediated and how activin might be triggered by viral infection have not been defined. Here we addressed this in vivo and in vitro, in humans and mice. Transcriptomic analyses delineated strikingly congruent patterns of gene regulation in hepatocytes stimulated with recombinant activin A and IFNα in vitro. Activin A mRNA, encoded by INHBA, is induced upon activation of RIG-I, MDA5 and TLR7/8 viral nucleic acid sensors in vitro, across multiple cell lines and in human peripheral blood mononuclear cells. In vivo, infection of mice with influenza A also upregulated Inhba mRNA in the lung; this local upregulation of Inhba is retained in MAVS knockout mice, indicating a role for non-RIG-I-like receptors in its induction. Activin induction and signalling were also detectable in patients with chronic viral hepatitis. Together, these data suggest Activin A is triggered in parallel with type I IFN responses and can trigger related antiviral effector functions. This model has implications for the development of targeted antiviral therapies, in addition to revealing novel facets of activin biology.

Dye coupling between rat striatal neurons recorded in vivo: compartmental organization and modulation by dopamine

1994 ◽  
Vol 71 (5) ◽  
pp. 1917-1934 ◽  
Author(s):  
S. P. Onn ◽  
A. A. Grace
Keyword(s):  
Intravenous Administration ◽  
D2 Receptor ◽  
Type I ◽  
Type Ii ◽  
Dye Coupling ◽  
Striatal Neurons ◽  
Receptor Stimulation ◽  
D2 Agonist ◽  
Response Profile

1. The presence of dye coupling between striatal neurons was investigated using in vivo intracellular recording and dye injection in adult rats. In 17% of the cases in which a single striatal neuron was injected with Lucifer yellow, more than one labeled neuron was recovered. In control rats, this dye coupling was observed only between single pairs of medium spiny neurons and only when the neuron injected exhibited the Type II response profile as defined by paired-pulse stimulation of corticostriatal afferents. 2. After intravenous administration of the D1/D2 agonist apomorphine at a behaviorally effective dose (i.e., 0.1–0.3 mg/kg), an increase in the incidence (from 17% to 82% of injected cells) and extent (from 2 cells to 3–7 cells labeled per injection) of dye coupling was observed. This effect was mediated by D2 receptor stimulation because administration of the D2 agonist quinpirole caused similar alterations in the incidence and extent of dye coupling (66% coupled). In contrast, administration of the D1 agonist SKF 38393 or the D1 antagonist SCH 23390 did not result in any significant alteration in dye coupling. 3. In control rats, the entire somatodendritic regions of dye-coupled neurons were found to be localized within single matrix compartments of the striatum. However, after intravenous administration of apomorphine or quinpirole, clusters of dye-coupled neurons were found to extend across the patch/matrix boundary. Moreover, dye coupling was observed after injecting cells exhibiting either the Type I or the Type II response profile. 4. In response to D2 receptor stimulation, both the extent and the pattern of coupling between striatal neurons is altered, resulting in direct coupling between neurons that are otherwise functionally and anatomically segregated in the control animal.

scholarly journals LeuRS can leucylate type I and type II tRNALeus in Streptomyces coelicolor

2019 ◽  
Vol 47 (12) ◽  
pp. 6369-6385
Author(s):  
Jia-Yi Fan ◽  
Qian Huang ◽  
Quan-Quan Ji ◽  
En-Duo Wang
Keyword(s):  
Tertiary Structure ◽  
Streptomyces Coelicolor ◽  
Catalytic Efficiency ◽  
Type I ◽  
Type Ii ◽  
Specific Domain ◽  
Recognition Mechanism ◽  
Variable Loop

Abstract Transfer RNAs (tRNAs) are divided into two types, type I with a short variable loop and type II with a long variable loop. Aminoacylation of type I or type II tRNALeu is catalyzed by their cognate leucyl-tRNA synthetases (LeuRSs). However, in Streptomyces coelicolor, there are two types of tRNALeu and only one LeuRS (ScoLeuRS). We found that the enzyme could leucylate both types of ScotRNALeu, and had a higher catalytic efficiency for type II ScotRNALeu(UAA) than for type I ScotRNALeu(CAA). The results from tRNA and enzyme mutagenesis showed that ScoLeuRS did not interact with the canonical discriminator A73. The number of nucleotides, rather than the type of base of the variable loop in the two types of ScotRNALeus, was determined as important for aminoacylation. In vitro and in vivo assays showed that the tertiary structure formed by the D-loop and TψC-loop is more important for ScotRNALeu(UAA). We showed that the leucine-specific domain (LSD) of ScoLeuRS could help LeuRS, which originally only leucylates type II tRNALeu, to aminoacylate type I ScotRNALeu(CAA) and identified the crucial amino acid residues at the C-terminus of the LSD to recognize type I ScotRNALeu(CAA). Overall, our findings identified a rare recognition mechanism of LeuRS to tRNALeu.

scholarly journals Fucoidan from Ascophyllum nodosum Suppresses Postprandial Hyperglycemia by Inhibiting Na+/Glucose Cotransporter 1 Activity

Marine Drugs ◽  
2020 ◽  
Vol 18 (9) ◽  
pp. 485
Author(s):  
Xindi Shan ◽  
Xueliang Wang ◽  
Hao Jiang ◽  
Chao Cai ◽  
Jiejie Hao ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Leptin Receptor ◽  
Specific Binding ◽  
Fasting Blood Glucose ◽  
Postprandial Hyperglycemia ◽  
Oral Glucose ◽  
Type I ◽  
Type Ii ◽  
Everted Gut Sac

We previously demonstrated that fucoidan with a type II structure inhibited postprandial hyperglycemia by suppressing glucose uptake, but the mechanism remains elusive. Here, we aimed to assess whether the effect of glucose absorption inhibition was related to the basic structure of fucoidans and preliminarily clarified the underlying mechanism. Fucoidans with type II structure and type I structure were prepared from Ascophyllumnodosum (AnF) or Laminariajaponica (LjF) and Kjellmaniellacrassifolia (KcF), respectively. The effects of various fucoidans on suppressing postprandial hyperglycemia were investigated using in vitro (Caco-2 monolayer model), semi-in vivo (everted gut sac model), and in vivo (oral glucose tolerance test, OGTT) assays. The results showed that only AnF with a type II structure, but not LjF or KcF with type I structure, could inhibit the glucose transport in the Caco-2 monolayer and everted gut sac models. A similar result was seen in the OGTT of Kunming mice and leptin receptor-deficient (db/db) mice, where only AnF could effectively inhibit glucose transport into the bloodstream. Furthermore, AnF (400 mg/kg/d) treatment decreased the fasting blood glucose, HbA1c, and fasting insulin levels, while increasing the serum glucagon-like peptide-1 (GLP-1) level in obese leptin receptor-deficient (db/db) mice. Furthermore, surface plasmon resonance (SPR) analysis revealed the specific binding of AnF to Na+/glucose cotransporter 1 (SGLT1), which indicated the effect of AnF on postprandial hyperglycemia could be due to its suppression on SGLT1 activity. Taken together, this study suggests that AnF with a type II structure can be a promising candidate for hyperglycemia treatment.

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