Human Activin Research Articles

Identification of ActivinβA and Gonadotropin Regulation of the Activin System in the Ovary of Chinese Sturgeon Acipenser sinensis.

Activin is a dimeric growth factor with diverse biological activities in vertebrates. This study aimed to investigate the regulatory role of the activin signaling pathway in the ovary of the endangered, cultured sturgeon species Acipenser sinensis. One activinβA subunit was identified, with a full-length complementary DNA (cDNA) sequence of 1572 base pairs. Multiple sequence alignment suggested that ActivinβA shared high sequence identities with its counterparts in four other sturgeon species. Phylogenetic analysis indicated the conserved evolution of ActivinβA among vertebrates from mammals to fish species. Transcripts of activinβA were distributed ubiquitously in the liver, kidney, intestine, ovary, midbrain, hypothalamus, and pituitary, with the highest transcription found in the pituitary. In Chinese sturgeon ovarian cells, in vitro human recombinant Activin A incubation stimulated the activin system-related gene transcriptions of activinβA, follistatin, its receptors -activinRIIA and activinRIIB, and drosophila mothers against decapentaplegic proteins (smads) smad2, smad3, and smad4. Ovary development-related mRNA levels of cyp19a1a and aromatase receptors of erα and erβ were enhanced by Activin A or human chorionic gonadotropin (hCG) incubation. Furthermore, 15 IU/mL hCG treatment increased the transcription levels of activinβA, follistatin, activinRIIA, and smad2. This suggested that the activin system was functional for the regulation of ovary development in Chinese sturgeon, possibly under the regulation of gonadotropin, by recruiting activinβA, follistatin, activinRIIA, and smad2. These results were helpful for the molecular exploration of activin signaling in fish species, as well as the ovarian maturation regulation of A. sinensis.

Activin E upregulates uncoupling protein 1 and fibroblast growth factor 21 in brown adipocytes

Activin E activates brown and beige adipocytes and has been controversially implicated as a factor that induces obesity and fatty liver. Here, we sought to address this controversial issue by producing recombinant human activin E to evaluate its effects on HB2 brown adipocytes in vitro. Activin E increased uncoupling protein 1 (Ucp1) and fibroblast growth factor 21 (Fgf21) mRNA expression in the adipocytes. This upregulation was suppressed by SB431542, an inhibitor of activin receptor-like kinase (Alk) TGF-β type I receptors. SB431542 also inhibited the activin E-induced phosphorylation of Smad2/3. A promoter assay using a CAGA-Luc reporter and Alk expression vectors revealed that activin E activated the TGF-β/activin pathway via Alk7. The upregulation of Ucp1 and Fgf21 mRNA might be mediated through Alk7 and Smad2/3 phosphorylation. Activin E is a potential stimulator of energy expenditure by activating brown adipocytes and highlights its potential as a therapeutic target for treating obesity.

Production of Mature Recombinant Human Activin A in Transgenic Rice Cell Suspension Culture.

Activin A belongs to the transforming growth factor (TGF) family member, which exhibits a wide range of biological activities, including the regulation of cellular proliferation and differentiation and the promotion of neuronal survival. The isolation of AA from natural sources can only produce limited quantities of this bioactive protein. In this study, the whole gene of the precursor form of recombinant human activin A (rhAA) contains a signal peptide, and a pro-region and a mature region were cloned into an expression vector under the control of the rice α-amylase 3D (RAmy3D) promoter. To obtain the mature (active) form of rhAA, an enterokinase cleavage site was inserted between the pro-region and mature region of rhAA. The rice seed (Oryza sativa L. cv. Dongjin) was transformed with recombinant vectors by the Agrobacterium-mediated method, and the integration of the target gene into the plant genome was confirmed by genomic PCR. The transcript expression of rhAA in transgenic rice calli was confirmed by a Northern blot analysis of mRNA. The production of rhAA was verified by Western blot analysis and ELISA. The accumulation of secreted rhAA in the culture medium was purified by Ni2+-NTA. The mature form of AA was released from the precursor form of rhAA after proteolytically processing with enterokinase. Western blot shows that the mature AA was split into monomer and homodimer with molecular weights of 14 kDa and 28 kDa under reducing and non-reducing conditions, respectively. These results suggest that the mature form of rhAA could be produced and purified using transgenic rice cell suspension culture.

Preeclampsia and Its Impact on Human Milk Activin A Concentration.

It is known that preeclampsia affects lactogenesis. However, data on the effects of this pathology on human milk neurobiomarker composition are not available. The aim of this study is to investigate the effects of this gestational pathology on activin A levels, a neurobiomarker known to play an important role in the development and protection of the central nervous system. The women recruited were divided in two different study groups: preeclamptic or normotensive women. All the human milk samples were collected using the same procedure. Activin A was quantified using an Enzyme-linked immunosorbent assay (ELISA) test. To investigate the effect of preeclampsia on the activin A concentration in the three lactation phases, a mixed linear model with a unistructural covariance structure, with the mother as the random effect, and fixed effects were performed. Activin A was detected in all samples. There were no significant differences between preeclamptic and normotensive women. The only significant effect is related to the lactation phase: the difference between colostrum and mature milk (p < 0.01) was significant. In conclusion, these results allow us to affirm that breast milk's beneficial properties are maintained even if preeclampsia occurs.

Renal injury, biomarkers, and myositis, an understudied aspect of disease: prospective study in the MyoCite cohort.

The mechanisms leading to chronic kidney disease (CKD) in patients with idiopathic inflammatory myopathies (IIMs) are poorly understood. We assessed the prevalence of subclinical renal injury in patients with IIMs, through elevation in biomarker levels of tubular injury and fibrosis (NGAL, KIM1, Activin A, CD163, and Cys-c), and assessed differences between subtypes of IIMs, and the effect of disease activity and duration. Clinical data, core set measures, sera and urine were prospectively collected from all patients enrolled in the MyoCite cohort from 2017 to 2021. Twenty healthy subjects (HC) and 16 patients with acute kidney injury (AKI) were included as controls. Baseline and follow up data for IIMs were included. Enzyme-linked immunosorbent assay (ELISA) was used to measure urine NGAL (Human Lipocalin-2/NGAL Duoset ELISA, Cat no: DY1757), KIM1 (Human TIM-1/KIM 1/HAVCR Duoset ELISA, Cat.no: DY1750B), Activin A (Human Activin A Duoset ELISA, Cat no: DY338), CD163 (Human CD163 Duoset ELISA,Cat no: DY1607-05), and Cys-c (Human Cystatin C Duoset ELISA, Cat. no.: DY1196) levels, while eGFR (unit mL/min/1.73 m2) was calculated by the Cockcroft-Gault formula and CKD-EPI formula. Analysis of 201 visits of 110 adult patients with IIMs indicated higher normalized biomarker levels compared to HCs, and comparable to patients with AKI, with the exception of NGAL, which was higher in the AKI group. Notably 72 (49%) patients with IIMs had eGFR<90; the levels of the 5 biomarkers were comparable between active and inactive IIMs, and different subtypes of IIMs. Similarly, a poor correlation between urine biomarker levels and core set measures of activity and damage was found. Changes in biomarker levels on follow-up did not correlate with eGFR changes. This exploratory analysis of urinary biomarkers identified low eGFR and elevated biomarkers of CKD in nearly half of the patients with IIMs, comparable to patients with AKI and higher than HCs, indicative of potential renal damage in IIMs that may have a lead to complications in other systems.

High-level production and purification of bioactive recombinant human activin A in Chinese hamster ovary cells

Activin A, a member of the TGF-β superfamily, is a homodimer of the inhibin βΑ subunit that plays a diversity of roles in biological processes. Because of its multiple functions, significant efforts have been made to produce activin A, however, unsatisfactory results were obtained due to its low level of expression. In this study, a stable CHO cell line exhibiting high expression of rhActivin A was isolated and production of rhActivin A was achieved using the cell line from 11-day fed-batch cultures in a 7.5 L bioreactor. The production rate was 0.22 g/L, substantially higher than those reported in previous studies. The culture supernatant of the bioreactor was used to purify rhActivin A (purity: >99%, recovery rate: 47%). The purified rhActivin A exhibited biological activity, with an EC50 of 3.893 ng/mL and a specific activity of 1.38 × 103 IU/mg. The control of process-related impurities in the purified rhActivin A was successful and met the USP recommendations for use in cell therapy. Thus, our production and purification methods were appropriate for large-scale GMP-grade rhActivin A production, which can be used for various purposes including cell therapy.

IL-12 drives the differentiation of human T follicular regulatory cells

Abstract T follicular regulatory (Tfr) cells can counteract the B cell-helper activity of T follicular helper (Tfh) cells and hinder the production of antibodies against self-antigens or allergens. A mechanistic understanding of the cues initiating the differentiation of T regulatory (Treg) cells into Tfr cells, which is instrumental for the therapeutic manipulation of diseases associated with a Tfr cell imbalance, is still missing. Despite their opposed roles, Tfr and Tfh cell differentiation appears to be controlled by partially overlapping signals, including TCR stimulation and costimulatory molecules. However, it is still unknown if cytokines that have been shown to control the biology of human Tfh cells, including IL-12 and activin A, can influence the differentiation of Tfr cells. To address this question, we evaluated the impact of these two cytokines on the in vitro differentiation of Tfr cells. Herein, we report a role for IL-12 in driving, on activated Treg cells, the induction of molecules that belong to the Tfr cell program, including CXCR5, PD-1, BCL6 and ICOS meanwhile preserving Tfr regulatory function. Conversely, activin A presented a largely negligible effect on the in vitro differentiation of Tfr-like cells and only modestly strengthened the IL-12-driven differentiation of Tfr-like cells. Importantly, patients with inborn errors of immunity in IL12RB1 gene presented with a severe decrease in circulating Tfr cells and produced higher levels of anti-actin autoantibodies in vivo. Overall, this study unveils IL-12 as an inducer of Tfr cell differentiation in vivo and provides a novel approach for the in vitro generation of human Tfr-like cells. AI123738

Serum activin-A as a predictive marker for postacne scarring

Background Postacne scar is the most distressing sequelae of inflammatory acne vulgaris. It develops owing to abnormal wound healing process in response to inflammation. Activins are members of the family named transforming growth factors-b that are involved in inflammation, immunity, and wound healing.Objective To evaluate human activin-A as a predictive marker for postacne scarring and the effect of treatment with oral isotretinoin on its serum level.Patients and methods A total of 40 patients who presented with either moderate or severe acne vulgaris were selected for this case–control study. Patients were classified into two groups: group A had no scarring and group B had postacne scarring. Group B patients were treated with oral isotretinoin therapy. Measurement of serum activin-A levels was done using the enzyme-linked immunosorbent assay technique.Results The mean serum level of human activin-A was significantly higher in patients with postacne scarring (189.47±59.63 ng/ml) than patients without scarring (155.4±41.19 ng/ml). Moreover, the mean serum level of human activin-A in group B was significantly decreased after treatment with oral isotretinoin. Patients with serum human activin-A level of more than 144.8 ng/ml are more liable to develop postacne scarring with 80.0% sensitivity and 55% specificity.Conclusion Serum level of activin-A could be a good and reliable marker for the prediction of those patients liable to develop postacne scarring, but more in-depth studies are still required to detect the exact pathogenic action of activin-A in the development of acne scars and to test the targeting of activin-A in an attempt to prevent postacne scarring.

First Single-Center Experience with Luspatercept Therapy in Low-Risk Myelodysplastic Syndrome (LR-MDS) Patients with Transfusion Dependence Refractory to Erythropoietin Therapy

Introduction: In the treatment of low-risk myelodysplastic syndromes (MDS) patients, anemia is the predominant problem. A recent breakthrough in the therapy of anemia in low-risk MDS patients are drugs involved in TGFβ pathway blockade. Luspatercept is a recombinant fusion protein derived from human activin receptor type IIB. It binds TGFβ superfamily ligands and inhibits the SMAD2/3 pathway leading to potentiation of red cell line differentiation and proliferation. Luspatercept was approved last year by the FDA and EMEA in the treatment of anemic low-risk MDS patients with ring sideroblasts (MDS-RS-MLD, MDS-RS-SLD) and/or SF3B1 mutated patients who are not eligible for erythropoiesis-stimulating agents (ESA) or for patients who failed on this therapy. Luspatercept showed high clinical activity for the treatment of anemia in the phase 2 trial (PACE-MDS) and subsequently in the phase 3 trial (MEDALIST). It has led to transfusion independence (TI) in about 40 % of patients. Methods: From January 2021 to July 2022, 27 patients were treated with luspatercept at the hematology department of Charles University Hospital in Prague, Czech Republic. The median age of the patients was 77 years (range 57-89) with a male to female ratio 17:10. WHO 2016 diagnosis were: MDS-RS-MLD (N = 23) and MDS/MPN-RS-T (N = 4). Median of IPSS-R was 2.5 (range 1-4). Median of IPSS-R cytogenetic score scaled from 0-2 with the median of 1. Median follow-up was 7.8 months (range 1-38). Luspatercept was administered in subcutaneous injections once every 3 weeks with an initial dose of 1 mg/kg. Dose escalation was done according to the level of response to 1.33 and 1.75 mg/kg. The median number of cycles administered was 7 (2-17). Transfusion dependency before luspatercept initiation ranged from 2 transfusion units (TU)/8 weeks to 12 TU/8 weeks, 11 patients had low transfusion burden (LTB), 16 high transfusion burden (HTB). Baseline erythropoietin (EPO) level was 374 IU/l in average (range 150-770) and ferritin level was 1307 μg/l in average (range 234-3832). 9 patients received chelation therapy. 22 patients were tested for SF3B1 mutation with 21 positive results (95.5 %) using Sanger sequencing (N = 3) or NGS approach (N = 18). Isolated mutation of SF3B1 was present in 8 patients (44 %). The most frequent mutations besides SF3B1 were TET2 (N = 8; 44 %), ASXL1 (N = 3; 17 %), DNMT3A and RUNX1 (N = 2; 11 %). Average mutations count per one patient was 1.7 (range 0-5). We did not observe any TP53 mutation. Results: There were 24 evaluable patients in terms of response. Thus far 11 (45 %) patients have responded by achieving TI, 5 (20 %) patients have had a reduction in transfusion dependence (HI, according to IWG criteria 2006), 8 (33 %) patients have not responded. One patient progressed to the higher risk MDS, and one transformed to acute myeloid leukemia. Median duration of response was 7.4 months (range: 0.7-31.8). Median number of cycles to achieve response was 4. Eleven patients responded to the basic dose 1 mg/kg, 2 additional patients on the dose 1.33 mg/kg and 2 more on the dose 1.75 mg/kg. There were differences in response according to transfusion burden, in LTB group achieved TI 8 patients and HI 1 patient, in HTB group we observed TI in 3 patients and HI in 4 patients. In 9 patients, we added ESA to luspatercept therapy, as they act both at different levels of erythropoiesis, which led to the improvement of response in 5 cases. In general, patients with lower initial baseline EPO level were better responders. We did not observe decrease in ferritin level during therapy. There was no significant effect of mutation burden on patient's OS, although patients with single mutated SF3B1 achieved better ORR (75 %), than patients bearing mutations in multiple genes (ORR = 40 %). There were no adverse effects of Grade II or more. Conclusion: In conclusion, luspatercept is certainly the next major step in the treatment of anemia in MDS patients. The advantage of the drug is very good tolerance and easy administration. We observed better responses in patients bearing single mutation in SF3B1, patients with LTB and lower initial baseline EPO levels. The higher response rate in our follow-up may be influenced by the combination with ESA and rapid dose escalation. This work was supported by the following grants: grant AZV 2021-2024 NU21-08-00312, Grant AZV 2019-2023 NV19-08-00144, UNCE/MED/016

Selection of culture media for chicken PGC long‐term cultivation

Primordial germ cells (PGCs) are precursors of eggs and spermatozoa and serve as an effective means for the transgenic chickens production, including using CRISPR / Cas9 technology. The aim study is selection of PGCs culture media suitable for long‐term cultivation. All experiments on chickens were conducted with ethical approval of the Russian Research Institute of Farm Animal Genetics and Breeding ‐ Branch of the L.K. Ernst Federal Research Center for Animal Husbandry (Protocol Number: 2020‐3). For the work, eggs of the Pushkin breed chickens (n = 21) were selected from the Bioresource collection RRIFAGB (Pushkin, St. Petersburg). PGCs were selected from the embryos dorsal aorta for 3‐4 days of incubation (stage Hamburger‐Hamilton 14) using a microinjector (Narishige IM‐11‐2, Japan). Cells were cultured for 21 days at 37 ° C with 5% CO2. Three variants of the culture medium were used. The first basic medium was Opti‐MEM (Reduced Serum Medium, GlutaMAX Supplement) (Gibco, Thermo Fisher) with the addition of sodium pyruvate 1M, nucleosides 100X ‐ 2.5 ml, Chicken Serum (Gibco, Thermo Fisher) ‐ 2%, 2‐mercaptoethanol (NF, VWR) ‐ 3.9 μl. On day 21, the concentration of cells in the first culture reached 1,2х103, and some of the wells with cells were overgrown. The second base medium was similar to the first base medium, but with the addition of antimycotic antibiotic (Thermo Fisher) up to 1X. The medium with the use of the antibiotic‐antimycotic was less susceptible to overgrowth, on day 21, the concentration of cells in the culture reached 3.5x104. The third basic medium was KnockOut DMEM / F‐12, without L‐glutamine (Gibco, ThermoFisher) with the addition sodium pyruvate 1M, nucleosides 100X ‐ 2.5 ml, Human Activin A Recombinant Protein (Gibco, Thermo Fisher) ‐ 25 ng / μl, Human FGF‐basic (FGF‐2 / bFGF) Recombinant Protein (Gibco, Thermo Fisher) ‐ 10 ng / μl, Chicken Serum (Gibco, Thermo Fisher) ‐ 2%, 2‐mercaptoethanol (NF, VWR) ‐ 3.9 μl), antimycotic antibiotic (Thermo Fisher) up to 1X. The third medium with the addition of Activin A and FGF‐2 / bFGF supported the cell culture better, overgrowth was practically not observed, cells were actively proliferating, оn day 21, the concentration of cells in the culture reached 2.7x105. The PGCs presence was confirmed by expression high level (p≤0.01) specific marker genes PGCs such as CXCR4 (chemokine receptor CXC type 4) and PIWIL1 (Piwi‐like protein 1) in cultured cells. In a control tissue small intestine sample of an adult chicken (330 days old), the above genes expression was completely absent.

Luspatercept in MDS with Ring Sideroblasts: A Real-Life Multicenter Experience from a Named Patient Program

BackgroundLow risk Myelodysplastic syndromes (LR MDS) are a heterogeneous group of clonal disorders of the hematopoietic stem cell characterized by ineffective hematopoiesis, peripheral cytopenias and relative increase of bone marrow blasts [List 2004]. The most common cytopenia is anemia, which occurs in 85-90% of cases (isolated in 35% of cases) [Fenaux 2013], and ranges from mild/asymptomatic to transfusion dependent. The standard of care for anemia remains supportive care and about 70% of cases benefits from erythropoiesis-stimulating agents (ESAs).According to the FAB classification, a low-risk group of MDS characterized by the presence of ring sideroblasts (RS) was recognized. In the 2017 WHO classification, the nosologic entities of MDS uni-linear and multi-linear dysplasia with RS (MDS-UD-RS, MDS-MD-RS) were established, to emphasize the importance of identifying forms with a potential therapeutic target [Arber 2016].Luspatercept is a recombinant fusion protein consisting of the modified extracellular domain of human activin receptor type IIB (ActRIIB) linked to a domain of human immunoglobulin Fc G1. The drug showed efficacy in re-establishing erythropoiesis and was recently approved for the treatment of adult patients with very low- to intermediate-risk MDS-RS failing ESAs[Platzbecker 2017; Fenaux 2020].AimsTo retrospectively assess the efficacy and/or clinical benefit (defined as a reduction in red blood cells transfusions [RBCT] or increase in hemoglobin levels) of luspatercept in patients with LR MDS with RS in a real-life setting. Safety issues were also addressed and clinical and hematological predictors of outcome analyzed.MethodsWe considered all IPSS-R lower-risk MDS with RS patients with unsatisfactory response or unsuitable for ESAs, treated with luspatercept as monotherapy through an expanded access program since November 2020, in 2 tertiary hematologic centers in Milan, Italy. Luspatercept was administered according to the published schedule: starting dose 1,0 mg/kg every three weeks, with the possibility to increase up to 1,33 mg/kg and 1,75 mg/kg if patients did not reach transfusion independence or decreased transfusion burden after two consecutive infusions. Response to luspatercept was evaluated according to IWG 2018 criteria: Hb increase by > 1.5 g/dL and/or relevant reduction of units of RBC transfusions by an absolute number of at least 4 RBC transfusions/8 wk compared with the pretreatment transfusion number in the previous 8 wk.ResultsWe included a total of 17 pts (70% males, 30% females), with a median age of 68 years (range 45-89). The median follow-up from the start of the Luspatercept was 4 months (range 3-6).According to WHO 2017 we included 13 MLD (76%) and 4 ULD (24%); in all of them SF3B1 mutations was confirmed. Moreover, According to IPSS-R classification: 5 (27%)cases were intermediate and 12 (73%) low risk.All patients received at least three doses of luspatercept. Nine patients (53%) needed a dose escalation to 1,33 mg/kg, and 7 (41%) required the maximal dose of 1,75 mg/kg. Interestingly, not all patients received a dose escalation, and 8 (47%) obtained prolonged efficacy with 1 mg/kg dose.Hemoglobin levels increased in 9 pts (53%): 4 (24%), 3 (18%) and 2 (12%) cases at 1 mg/Kg, 1,33 mg/kg and 1,75 mg/dl, respectively. Medina increase was 1,5 g/dl (range 0,2 - 2,2) and these levels were maintained for a median of 4 months (range 3-6).In addition, considering transfusion burden, the independence was reached in 3 pts (18%), after a median time of 6 weeks from treatment start. Importantly, off the 14 patients remaining transfusion-dependent, 6 (35%) reduced the transfusion burden.The most prominent toxicity was increased blood pressure (grade 2) occurring in 3 pts (18%), and dizziness (grade 2) in 2 pts (12%); though the drug was generally well tolerated. No serious adverse events (SAEs) were reported. Therefore, patients were treated as outpatients in all cases.At the last follow up, all patients are alive and free from leukemic evolution.Conclusions:Our real-world experience confirms the efficacy of luspatercept in improving erythropoiesis and decreasing transfusion dependency in very low, low or intermediate risk MDS with RS, with an acceptable toxicity profile. DisclosuresFattizzo: Amgen: Honoraria, Speakers Bureau; Apellis: Speakers Bureau; Annexon: Consultancy; Momenta: Honoraria, Speakers Bureau; Novartis: Speakers Bureau; Alexion: Speakers Bureau; Kira: Speakers Bureau. Barcellini: Agios: Honoraria, Research Funding; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals: Honoraria; Novartis: Honoraria.

Isolation of the canine inhibin βB subunit gene and characterization of signalling mediated by canine inhibin βB.

Activin B, a homodimer of the inhibin βB subunit, acts as a regulator of gonadal function and as an adipokine. To clarify the role of activin B in dogs, we characterized the canine inhibin βB gene and signalling pathways regulated by the canine inhibin βB. Using 5'- and 3'-rapid amplification of cDNA end (RACE) and RT-PCR on RNA isolated from the ovary of dogs, we identified short and long forms of the inhibin βB gene. Immunoreactive inhibin βB molecules were detected at ~25 and ~14 kDa under nonreducing and reducing conditions, respectively, in culture supernatants from HEK293 cells transfected with a plasmid containing the long form of the inhibin βB gene, indicating activin B production and secretion. Similar to human and murine activin B, the canine activin B-stimulated transcriptions of reporter genes, CAGA-luc and Hepcidin-luc, regulated by the canonical activin/transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) pathway, respectively. Activin B-induced CAGA-luc transcription was not detected in ALK7-deficient MDCK canine-derived cells; however, the forced expression of ALK7 resulted in the activin B-dependent expression in MDCK cells. Unexpectedly, the activin B-induced activation of the BMP pathway was partially blocked by the inhibition of endogenous activin/TGF-β receptor activity. The present study identified an experimentally isolated long form of the canine inhibin βB gene producing activin B that transactivates BMP- and activin/TGF-β-regulated gene expression.

Potential of Activin B as a Clinical Biomarker in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).

Reliable serum biomarkers are of immense need for diagnostic purposes of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)—a disabling and complex disease for which diagnosis is mainly based on clinical symptoms. The aim of this study was to evaluate a possible diagnostic potential of activin B by directly comparing 134 cases of ME/CFS with 54 healthy controls. Analyses of human activin B level in plasma samples were performed using a validated human activin B ELISA assay. The results of the study show that activin B levels did not differ statistically significantly between ME/CFS patients and healthy controls (p = 0.6511). No gender or age-related differences in activin B levels were observed in the ME/CFS group and healthy controls. The level of activin B tended to decrease with increasing visual analogue scale score (r = −0.2004; p = 0.5085) nevertheless the results obtained so far does not support the clinical utility of activin B as a biomarker for ME/CFS.

The effect of Activin pathway modulation on the expression of both pluripotency and differentiation markers during early zebrafish development compared with other vertebrates.

Activin-like factors control many developmental processes, including pluripotency maintenance and differentiation. Although Activin-like factors' action in mesendoderm induction has been demonstrated in zebrafish, their involvement in preserving the stemness remains unknown. To investigate the role of maternal Activin-like factors, their effects were promoted or blocked using synthetic human Activin A or SB-431542 treatments respectively until the maternal to zygotic transition. To study the role of zygotic Activin-like factors, SB-431542 treatment was also applied after the maternal to zygotic transition. The effect of the pharmacological modulations of the Activin/Smad pathway was then studied on the mRNA expressions of the ndr1, ndr2, tbxta (no tail/ntl) as the differentiation index, mych, nanog, and oct4 (pou5f3) as the pluripotency markers of the zebrafish embryonic cells as well as sox17 as a definitive endoderm marker. Expression of the target genes was measured at the 16-cell, 256-cell, 1K-cell, oblong, dome, and shield stages using the real-time quantitative polymerase chain reaction (RT-qPCR). Activation of the maternal Activin signaling pathway led to an increase in zygotic expression of the tbxta, particularly marked at the oblong stage. In other words, promotion of the maternal Activin/Smad pathway induced differentiation by advancing the major peaks of ndr1 and nanog, thereby eliciting tbxta expression. Whereas suppression of the maternal or zygotic Activin/Smad pathway sustained the pluripotency by preventing the major peaks of ndr1 and nanog as well as tbxta encoding.

Bimagrumab to improve recovery after hip fracture in older adults: a multicentre, double-blind, randomised, parallel-group, placebo-controlled, phase 2a/b trial

Older adult patients (ie, those aged ≥60 years) undergoing surgery for hip fracture repair frequently experience loss of muscle mass and strength due to poor mobility and delayed functional recovery. No proven treatment is currently available to enhance recovery of physical function in this growing patient population. This study aimed to investigate whether bimagrumab, a human monoclonal antibody targeting activin type 2 receptors, can improve post-surgical recovery. This multicentre, double-blind, randomised, parallel-group, placebo-controlled, phase 2a/b trial was done at 50 clinical research centres in 18 countries. Participants aged 60 years or older with a body-mass index of 15-35 kg/m2 who had undergone internal fixation or hemiarthroplasty for a proximal femoral fracture (confirmed by radiography) in the previous 6 weeks were eligible. Patients with a history of a high-energy subtrochanteric fracture or any other lower limb fracture in the past 6 months, or any major surgery of the lower limbs in the past 3 months were excluded. Participants were randomly assigned (2:1:2:2) via interactive response technology to receive intravenous treatment with placebo, bimagrumab 70 mg, bimagrumab 210 mg, or bimagrumab 700 mg every 4 weeks for 24 weeks. Participants, investigators, site personnel, and study sponsor personnel in participating countries were masked to treatment assignment. The primary endpoint was the change from baseline in total lean body mass, measured by dual-energy x-ray absorptiometry, at week 24 in the full analysis set, which included all randomised participants who had received at least one dose of the assigned treatment. Key secondary endpoints included changes in habitual gait speed (measured in m/s) and short physical performance battery score between baseline and 24 weeks. Safety and tolerability were assessed by recording adverse events and vital signs on weeks 4, 8, 12, 24, and 48, and by laboratory assessments and electrocardiography at the screening visit and on days 1, 84, and 168. Safety was assessed in all randomised participants who had received at least one dose of study drug, analysed according to treatment received. This study was registered with ClinicalTrials.gov, NCT02152761. Between Sept 16, 2014, and Dec 15, 2017, 384 patients were screened, of whom 250 patients were enrolled and randomly assigned to the placebo group (n=72), the bimagrumab 70 mg group (n=34), the bimagrumab 210 mg group (n=69), or the bimagrumab 700 mg group (n=75). A total of 207 (83%) participants completed the 24-week treatment period. There was a significant absolute increase in lean body mass from baseline compared with placebo (0·2 kg [SD 2·0]) in the bimagrumab 210 mg group (1·9 kg [1·7]; p<0·0001) and in the bimagrumab 700 mg group 2·8 kg [2·2]; p<0·0001) but not in the bimagrumab 70 mg group (0·6 kg [SD 2·2]; significance not assessed). Changes in habitual gait speed and short physical performance battery scores between baseline and week 24 were not significantly different across the treatment groups, suggesting no enhancement of physical recovery with bimagrumab over placebo. Bimagrumab was safe and well tolerated. The most frequently reported treatment-emergent adverse events were falls (six [18%] of 34 participants in the bimagrumab 70 mg group; 12 [17%] of 69 participants in the bimagrumab 210 mg group; 14 [19%] of 75 participants in the bimagrumab 700 mg group; and 13 [18%] of 72 participants in the placebo group), muscle spasms (two [6%] in the bimagrumab 70 mg group; 17 [25%] in the bimagrumab 210 mg group; 12 [16%] in the bimagrumab 700 mg group; and six [8%] in the placebo group), and arthralgia (five [15%] in the bimagrumab 70 mg group; six [9%] in the bimagrumab 210 mg group; nine [12%] in the bimagrumab 700 mg group; and five [7%] in the placebo group). Six deaths were reported during the study, none of which were considered by investigators as related to the study drug. Bimagrumab treatment for 24 weeks led to dose-dependent, significant increases in lean body mass in older patients recovering from hip fracture surgery when compared with placebo. However, no functional benefit was observed in recovery of mobility or lower extremity function following bimagrumab treatment compared with placebo. Novartis Pharma.

Maternal and zygotic activin signaling promotes adequate pattern and differentiation of mesoderm through regulation of pluripotency genes during zebrafish development.

To investigate the role of maternal Activin-like factors in the preservation of stemness and mesendoderm induction, their effects were promoted and inhibited using synthetic human Activin A or SB-505124 treatments, respectively, before the maternal to zygotic transition (MZT). To study the role of zygotic Activin-like factors, SB-505124 treatment was also used after the MZT. Promoting the signaling intensity of maternal Activin-like factors led to premature differentiation, loss of stemness, and no mesendoderm malformation, while its alleviation delayed the differentiation and caused various malformations. Inhibition of the zygotic Activin-like factors was associated with suppressing the ndr1, ndr2, oct4 (pou5f3), mycb and notail transcription as well as differentiation retardation at the oblong stage, and a broad spectrum of anomalies in a dose-dependent manner. Together, promoting the signal intensity of maternal Activin-like factors drove development along with mesendodermal differentiation, while suppression of the maternal or zygotic ones maintained the pluripotent state and delayed differentiation.

Human and mouse activin genes: Divergent expression of activin A protein variants and identification of a novel heparan sulfate-binding domain in activin B

Activins are members of the transforming growth factor-β (TGF-β) superfamily of signaling proteins and were originally identified as components of follicular fluid. The proteins are now known to play critical roles in numerous normal and pathological processes and conditions, but less is clear about the relationships between their gene organization and protein variant expression and structure. The four human and mouse activin (Act) genes, termed INHβA, INHβB, INHβC and INHβE, differ in exon numbers. Human INHβA is the most complex with 7 exons and elicits production of three Act A variants (Act A X1, X2 and X3) differing in their pro-region, as we showed previously. Here we further analyzed the mouse INHβA gene and found that its 4 exons encode for a single open reading frame (mouse Act A), corresponding to the shortest human Act A X3 variant. Activins are synthesized and secreted as large complexes made of a long pro-region and a short mature C- terminal ligand and are known to interact with the heparan sulfate (HS) chains of cell surface and matrix proteoglycans. Human Act A X1 and X2 variants do have a HS-binding domain (HBD) with Cardin/Weintraub traits in their pro-region, while the X3 variant does not as shown previously. We found that the mouse Act A lacks a HBD as well. However, we identified a typical HBD in the pro-region of both mouse and human Act B, and synthetic peptides containing that domain interacted with immobilized HS and cell surface with nanomolar affinity. In sum, human and mouse Act A genes elicit expression of different variant sets, while there is concordance in Act B protein expression, reflecting possible evolutionary diversity in function of, and responses to, these signaling proteins in the two species.

Association of Body Mass Index with Activin-A and Inhibin-A in Gestational Diabetic Women

Objective: The objective of the study was to assess the association of body mass index (BMI) with Activin-A and Inhibin-A (biomarkers) irrespective to age in women with gestational diabetes mellitus (GDM).Methods: This study was a case-controlled study, in which sixty (60) diagnosed cases of gestational diabetic women with and without obesity and thirty (30) pregnant women without diabetes or any other complications were included in the study after taking informed written consent. The duration of the study was from January 2018 to June 2018. The enrolled participants were ninety (90) who were fulfilling the criteria in the duration of the study. Participants were placed into three groups A, B and C. In group A, thirty (30) pregnant females without complications, in group B thirty (30) non-obese pregnant women with gestationaldiabetes and in group C thirty (30) obese pregnant females with gestational diabetes mellitus (GDM). The biophysical parameters including weight, height and blood pressure were measured through standard protocols. Blood tests for biomarkers Activin-A and Inhibin-A levels were measured through Human Activin-A enzyme-linked immunosorbent assay(ELISA) kit and Human Inhibin-A ELISA kit respectively.Results: The results of the study revealed that the biophysical markers Actin-A and Inhibin-A had associations with gestational diabetes, the significant (p-value&lt;0.05) association was observed between Activin-A and body mass index but Inhibin-A had an insignificant association with it. However, the levels of bothbiomarkers (Activin-A and Inhibin-A) were found to be raised in both obese and non-obese gestational diabetic women when compared with normal pregnant women. The secondary outcome of the study is, there is significant (p-value&gt;0.05) relation between age and body mass index of obese GDM.Conclusion: The study concluded that the level of Activin-A was raised significantly (p-value&gt;0.05) in obese and non-obese gestational diabetic women as compared to normal pregnant women and non-significant increases in inhibin-A were observed in obese and non-obese gestational diabetic women as compared to normal pregnant women. A secondary outcome was, that there is significant (p-value&gt;0.05) association found between body mass index and age of the pregnant women.

Denovo designing: a novel signal peptide for tat translocation pathway to transport activin A to the periplasmic space of E. coli.

The twin-arginine translocation (Tat) pathway is one of the bacterial secretory strategies which exports folded proteins across the cytoplasmic membrane. In the present study, we designed a novel Tat-signal peptide for secretion of human activin A used as a recombinant protein model here. In doing so, Haloferax volcanii, Halobacterium salinarum, and Escherichia coli Tat specific signal peptides were aligned by ClustalW program to determine conserved and more frequently used residues. After making the initial signal peptide sequence and doing some mutations, efficiency of this designed signal peptide was evaluated using a set of well-known software programs such as TatP, PRED-TAT, and Phobius. Then the best complex between TatC as an initiator protein in Tat secretory machine and the new designed signal peptide connected to activin A with the lowest binding energy was constructed by HADDOCK server, and ΔΔG value of - 5.5kcal/mol was calculated by FoldX module. After that, efficiency of this novel signal peptide for secretion of human activin A to the periplasmic space of E. coli Rosetta-gami (DE3) strain was experimentally evaluated; to scrutinize the activity of the novel signal peptide, Iranian Bacillus Licheniformis α-Amylase enzyme signal peptide as a Sec pathway signal peptide was used as a positive control. The quantitative analysis of western blotting bands by ImageJ software confirmed the high secretion ability of the new designed signal peptide; translocation of 69% of the produced recombinant activin A to the periplasmic space of E. coli. Circular Dichroism (CD) spectroscopy technique also approved the proper secondary structure of activin A secreted to the periplasmic space. The biological activity of activin A was also confirmed by differentiation of K562 erythroleukemia cells to the red blood cell by measuring the amount of hemoglobin or Fe2+ ion using ICP method. In conclusion, this novel designed signal peptide can be used to secrete any other recombinant proteins to the periplasmic space of E. coli efficiently.

Enhanced periplasmic expression of human activin A in Escherichia coli using a modified signal peptide

Activin A is a member of the transforming growth factor-beta (TGF-β) protein superfamily, which acts as a hormone in regulating cell proliferation and differentiation. Structurally, activin is a dimer of two subunits linked by a disulfide bond. Since the correct folding of this protein is essential for its function, we aimed to use a modified signal peptide to target the expressed recombinant protein to the periplasm of Escherichia coli as an effective strategy to produce correctly-folded activin A. Therefore, the coding sequence of native Iranian Bacillus licheniformis α-amylase signal peptide was modified and its efficiency was checked by SignalP bioinformatics tool. Then its final sequence was cloned upstream of the activin A mature cDNA. Protein expression was done using 1 mM of isopropyl thio-β-D-galactoside (IPTG) and a post-induction time of 8 hr. Additionally, following purification of recombinant activin A, circular dichroism spectroscopy was used to determine the accuracy of secondary structure of the protein. Importantly, differentiation of K562 cells to the red blood cell was confirmed by measuring the amount of Fe+2 ions after treatment with recombinant activin A. The results indicated that the produced recombinant activin A had the same secondary structure as the commercial human activin A and was fully functional.