Identification of pattern recognition receptor genes in peripheral blood mononuclear cells and monocytes as biomarkers for the diagnosis of lupus nephritis.

BACKGROUND: The study aimed to investigate the diagnostic value of lupus-related pattern recognition receptors (PRRs) genes in peripheral blood mononuclear cells (PBMCs) and monocytes (MONs) for lupus nephritis (LN). METHODS: PBMCs were isolated from a cohort with 37 LN patients and 39 healthy controls (HCs), and MONs were derived from another cohort with 70 LN patients and 66 HCs. Q-PCR was used to measure the mRNA levels of CGAS, IFNB1, AIM2, IL1Β, NLRC4, NLRP3, NLRP12 and ZBP1 in the PBMCs and MONs. The Mann-Whitney U test was used to compare the data in LN patients and HCs. Eleven GEO datasets of SLE/LN were used to perform differentially expressed genes (DEGs) analysis to these PRR genes. Receiver operating characteristic (ROC) curve analysis was employed to assess the performance of individual genes or the disease prediction model established by combining multiple genes in LN diagnosis. Spearman correlation method was done to analyze the correlation between these PRRs and other clinical characteristics. RESULTS: The mRNA levels of five genes (AIM2, NLRC4, IL1B, NLRP12 and ZBP1) in PBMCs, and seven genes (CGAS, IFNB1, AIM2, IL1B, NLRP3, NLRP12 and ZBP1) in MONs of LN patients were significantly higher than those of HCs (P < 0.05). DEGs analysis based on the GEO datasets showed that ZBP1, AIM2 and IL1B were significantly increased in several datasets. The ROC curve analysis indicated that the area under curve (AUC) of the LN prediction models derived from PBMCs or MONs were 0.82 or 0.91 respectively. In addition, the expression levels of these PRRs were correlated with other clinical features in LN patients, including Anti-Sm, ESR, serum Cr, and C3. CONCLUSION: Our study suggests that these lupus-related PRRs might be served as potential biomarkers of LN.

A nomogram for predicting the risk of preeclampsia in women with intrahepatic cholestasis of pregnancy based on prenatal monitoring time: a multicenter retrospective cohort study.

BACKGROUND AND AIMS: Intrahepatic cholestasis of pregnancy (ICP) is a special liver disease during pregnancy, characterized by abnormal bile acid metabolism. However, there is no consensus on how to group women with ICP based on the time of diagnosis worldwide. This study aimed to adopt a new grouping model of women with ICP, and the time from diagnosis to delivery was defined as the monitoring period. METHODS: This retrospective real-world data study was conducted across multiple centers and included 3172 women with ICP. The study first evaluated the significant difference in medication and nonmedication during different monitoring times. The least absolute shrinkage and selection operator (LASSO) model was then used to screen nine risk factors based on the predictors. The model's discrimination, clinical usefulness, and calibration were assessed using the area under the receiver operating characteristic (ROC) curve, decision curve, and calibration analysis. RESULTS: The incidence of preeclampsia risk in ICP patients without drug intervention increased with the extension of the monitoring period. However, the risk of preeclampsia decreased in ICP patients treated with ursodeoxycholic acid. A predictive nomogram and risk score model was developed based on nine risk factors. The area under the ROC curve of the nomogram was 0.765 [95% confidence interval (CI): 0.724-0.807] and 0.812 (95% CI: 0.736-0.889) for the validation cohort. CONCLUSIONS: This study found that a longer ICP monitoring period could lead to adverse pregnancy outcomes in the absence of drug intervention, especially preeclampsia. A predictive nomogram and risk score model was developed to better manage ICP patients, maintain pregnancy to term delivery, and minimize the risk of severe adverse maternal and fetal outcomes.

[Gene Mutation Types of Thalassemia in Chongzuo Childbearing-age Population of Guangxi Zhuang Autonomous Region of China].

OBJECTIVE: To investigate the gene mutation and genotype distribution of thalassemia in the population of childbearing age in Chongzuo area of Guangxi. METHODS: Six α-thalassemia and 17 ß-thalassemia gene mutations common in Chinese were detected by gap-polymerase chain reaction (gap-PCR) combined with agarose gel eletrophoresis and reserve dot bolt hybridization in 29 266 cases of child-bearing age suspected of thalassemia. RESULTS: A total of 19 128 (65.36%) cases were identified with thalassemia. The detection rate of α-thalassemia, ß-thalassemia and α-combining ß-thalassemia was 45.25% (13 242/29 266), 15.47% (4 526/29 266) and 4.65% (1 360/29 266), respectively. A total carrying rate of 8 kinds of α-thalassemia gene mutations was 26.74% (15 649/58 532), including 12.51% for --SEA, followed by 5.70% for -α3.7, and 0.24% for --Thai. Among 32 α-thalassemia genotypes, the most common five were --SEA/αα, -α3.7/αα, αCSα/αα, -α4.2/αα and αWSα/αα, accounting for 47.27%, 18.31%, 8.56%, 8.52% and 7.91%, respectively, as well as 0.97% for --Thai/αα. A total carrying rate of 13 kinds of ß-thalassemia gene mutations was 10.07% (5 897/58 532), including 3.63% for CD41-42, followed by 2.55% for CD17, and 0.003% for -50 (G>A). Among 17 ß-thalassemia genotypes, the most common six were CD41-42/N, CD17/N, CD71-72/N, CD26/N, 28/N and IVSI-1/N, accounting for 36.15%, 25.81%, 9.43%, 8.18%, 8.09% and 7.75%. The homozygous genotype CD26/CD26 [hemoglobin (Hb): 121 g/L] and -28/-28 (Hb: 56 g/L) were respectively detected in one case, and double heterozygous genotype were detected in 5 cases, including 3 cases of CD41-42/CD26 (Hb: 41 g/L, 51 g/L, 63 g/L, respectively), 1 case of -28/IVSI-1 (Hb: 53 g/L), and 1 case of CD71-72/CD26 (Hb: 89 g/L), in which patients with moderate or severe anemia had a history of blood transfusion. Among 104 α-combining ß-thalassemia genotypes, the most common were --SEA/αα, -α3.7/αα combining CD41-42/N and --SEA/αα combining CD17/N, accounting for 12.13%, 9.63% and 9.26%, respectively. In addition, 1 case of --SEA/-α3.7 combining -28/IVSI-1 (Hb: 83 g/L) and 1 case of -α3.7/αα combining CD41-42/ CD41-42 (Hb: 110 g/L) were detected without history of blood transfusion, while 1 case of αWSα/αα combining CD41-42/CD17 (Hb: 79 g/L) and 1 case of --SEA/αα combining CD17/-28 (Hb: 46 g/L) were detected with history. CONCLUSIONS: The detection rate of thalassemia genes is high and the mutations are diverse in the population of childbearing age in Chongzuo area of Guangxi. The common deletion genotype is --SEA/αα in α-thalassemia and CD41-42/N in ß-thalassemia, and deletion genotype --Thai is not rare. There is a certain incidence of intermediate and severe ß-thalassemia, and most patients require transfusion therapy. The results are beneficial for genetic consultation and intervention of thalassemia.

[Analysis of HKαα Genotype and Hematological Characteristics in South Guangxi].

OBJECTIVE: To investigate the detection rate and hematologic phenotype of HKαα thalassemia in south Guangxi, in order to provide reference for the prevention and control of thalassemia and prenatal and postnatal care consultation in this region. METHODS: Gene testing was performed on pre-marital medical examinations, pre-pregnancy eugenic health examinations, prenatal examinations and hospitalized thalassemia-positive persons in south of Guangxi, and the results were analyzed. RESULTS: A total of 183 190 thalassemia patients were included in this study, the age was mainly concentrated in 26-35 years old (101 709 cases, accounting for 55.521%), and 40 HKαα mutations were detected, detection rate was 0.022%, including 5 cases in Nanning, 22 cases in Qinzhou, 2 cases in Fangchenggang, 11 cases in Beihai. A total of 29 ethnic groups were included in the survey, but HKαα gene was observed only in Han nationality (0.0380%) and Zhuang nationality (0.0068%). A total of 8 genotypes carrying HKαα mutations were detected in this study ( HKαα/--SEA, ßN/ ßN, HKαα/αα, ß-28/ ßN, HKαα/αα, ß-50/ ßN, HKαα/αα, ßCD17/ ßN, HKαα/αα, ßCD27/28/ß N, HKαα/αα, ßCD41-42/ ßN, HKαα/αα, ßCD71-72/ ßN, and HKαα/αα, ßN/ ßN). Except for most cases with HKαα/αα, ßN/ ßN genotypes with no significant changes in the hematological indexes, mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) of other genotypes were decreased, showing microcytic hypochromic performance, mild anemia or no anemia. CONCLUSION: HKαα carrier is often misdiagnosed as -α3.7 carrier, which easily leads to missed diagnosis or misdiagnosis. Therefore, it is necessary to continuously improve the diagnostic level of laboratory testing personnels and genetic counselors to avoid unnecessary interventional puncture operations and birth of children with moderate and severe thalassemia.

IMM-BCP-01, a patient-derived anti-SARS-CoV-2 antibody cocktail, is active across variants of concern including Omicron BA.1 and BA.2.

Monoclonal antibodies are an efficacious therapy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, rapid viral mutagenesis led to escape from most of these therapies, outlining the need for an antibody cocktail with a broad neutralizing potency. Using an unbiased interrogation of the memory B cell repertoire of patients with convalescent COVID-19, we identified human antibodies with broad antiviral activity in vitro and efficacy in vivo against all tested SARS-CoV-2 variants of concern, including Delta and Omicron BA.1 and BA.2. Here, we describe an antibody cocktail, IMM-BCP-01, that consists of three patient-derived broadly neutralizing antibodies directed at nonoverlapping surfaces on the SARS-CoV-2 Spike protein. Two antibodies, IMM20184 and IMM20190, directly blocked Spike binding to the ACE2 receptor. Binding of the third antibody, IMM20253, to its cryptic epitope on the outer surface of RBD altered the conformation of the Spike Trimer, promoting the release of Spike monomers. These antibodies decreased Omicron SARS-CoV-2 infection in the lungs of Syrian golden hamsters in vivo and potently induced antiviral effector response in vitro, including phagocytosis, ADCC, and complement pathway activation. Our preclinical data demonstrated that the three-antibody cocktail IMM-BCP-01 could be a promising means for preventing or treating infection of SARS-CoV-2 variants of concern, including Omicron BA.1 and BA.2, in susceptible individuals.

An All-in-One Nanomedicine Consisting of CRISPR-Cas9 and an Autoantigen Peptide for Restoring Specific Immune Tolerance.

Nanotechnology has shown great promise in treating diverse diseases. However, developing nanomedicines that can cure autoimmune diseases without causing systemic immunosuppression is still quite challenging. Herein, we propose an all-in-one nanomedicine comprising an autoantigen peptide and CRISPR-Cas9 to restore specific immune tolerance by engineering dendritic cells (DCs) into a tolerogenic phenotype, which can expand autoantigen-specific regulatory T (Treg) cells. In brief, we utilized cationic lipid-assisted poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-PLGA) nanoparticles to simultaneously encapsulate an autoimmune diabetes-relevant peptide (2.5mi), a CRISPR-Cas9 plasmid (pCas9), and three guide RNAs (gRNAs) targeting costimulatory molecules (CD80, CD86, and CD40). We demonstrated that the all-in-one nanomedicine was able to effectively codeliver these components into DCs, followed by simultaneous disruption of the three costimulatory molecules and presentation of the 2.5mi peptide on the genome-edited DCs. The resulting tolerogenic DCs triggered the generation and expansion of autoantigen-specific Treg cells by presenting the 2.5mi peptide to CD4+ T cells in the absence of costimulatory signals. Using autoimmune type 1 diabetes (T1D) as a typical disease model, we demonstrated that our nanomedicine prevented autoimmunity to islet components and inhibited T1D development. Our all-in-one nanomedicine achieved codelivery of CRISPR-Cas9 and the peptide to DCs and could be easily applied to other autoimmune diseases by substitution of different autoantigen peptides.

Intracellular conversion and in vivo dose response of favipiravir (T-705) in rodents infected with Ebola virus.

During the 2013-2016 Ebola virus (EBOV) outbreak in West Africa, our team at USAMRIID evaluated the antiviral activity of a number of compounds, including favipiravir (T-705), in vitro and in mouse and nonhuman primate (NHP) models of Ebola virus disease. In this short communication, we present our findings for favipiravir in cell culture and in mice, while an accompanying paper presents the results of NHP studies. We confirmed previous reports that favipiravir has anti-EBOV activity in mice. Additionally, we found that the active form of favipiravir is generated in mice in tissues relevant for the pathogenesis of EBOV infection. Finally, we observed that protection can be achieved in mice down to 8 mg/kg/day, which is lower than the dosing regimens previously reported. An accompanying paper reports the results of treating nonhuman primates infected with EBOV or with Marburg virus with oral or intravenous favipiravir.

Efficacy of favipiravir (T-705) in nonhuman primates infected with Ebola virus or Marburg virus.

Favipiravir is a broad-spectrum antiviral agent that has demonstrated efficacy against Ebola virus (EBOV) in rodents. However, there are no published reports of favipiravir efficacy for filovirus infection of nonhuman primates (NHPs). Here we evaluated the pharmacokinetic profile of favipiravir in NHPs, as well as in vivo efficacy against two filoviruses, EBOV and Marburg virus (MARV). While no survival benefit was observed in two studies employing once- or twice-daily oral dosing of favipiravir during EBOV infection of NHPs, an antiviral effect was observed in terms of extended time-to-death and reduced levels of viral RNA. However, oral dosing in biosafety level-4 (BSL-4) presents logistical and technical challenges, and repeated anesthesia events may potentially worsen survival outcome in animals. For the third study of treatment of MARV infection, we therefore made use of catheters, jackets, and tethers for intravenous (IV) dosing and blood collection, which minimized the requirement for repeated anesthesia events. When MARV infection was treated with IV favipiravir, five of six animals (83%) survived infection, while all untreated NHPs succumbed. An accompanying report presents the results of favipiravir treatment of EBOV infection in mice.

The distortions of care needs and medical professionalism: The ruling practices of migrant labor policy in Taiwan.

According to Taiwanese government policies and regulations, families planning to hire migrant care workers must apply for a medical assessment of the needs of elderly people destined to be cared for. The physician conducting this assessment acts as a gatekeeper who carries out her/his work with state and medical profession authority to identify, define, and regulate older people's needs. Using institutional ethnography as the method of inquiry, this article locates the problematic nature of the medical assessment as an entry point to an inquiry into how the care needs met by migrant workers are textually-mediated. This article begins by telling the daily story of an old woman and her live-in migrant worker to point out the standpoint of care recipients and their families where the inquiry anchors. I examine the physicians' daily working activities of medical assessment to discover how policy subordinates people's interests to the governmental purpose.

Spontaneous regression of retinopathy of prematurity: incidence and predictive factors.

AIM: To evaluate the incidence of spontaneous regression of changes in the retina and vitreous in active stage of retinopathy of prematurity(ROP) and identify the possible relative factors during the regression. METHODS: This was a retrospective, hospital-based study. The study consisted of 39 premature infants with mild ROP showed spontaneous regression (Group A) and 17 with severe ROP who had been treated before naturally involuting (Group B) from August 2008 through May 2011. Data on gender, single or multiple pregnancy, gestational age, birth weight, weight gain from birth to the sixth week of life, use of oxygen in mechanical ventilation, total duration of oxygen inhalation, surfactant given or not, need for and times of blood transfusion, 1,5,10-min Apgar score, presence of bacterial or fungal or combined infection, hyaline membrane disease (HMD), patent ductus arteriosus (PDA), duration of stay in the neonatal intensive care unit (NICU) and duration of ROP were recorded. RESULTS: The incidence of spontaneous regression of ROP with stage 1 was 86.7%, and with stage 2, stage 3 was 57.1%, 5.9%, respectively. With changes in zone III regression was detected 100%, in zone II 46.2% and in zone I 0%. The mean duration of ROP in spontaneous regression group was 5.65±3.14 weeks, lower than that of the treated ROP group (7.34±4.33 weeks), but this difference was not statistically significant (P=0.201). GA, 1min Apgar score, 5min Apgar score, duration of NICU stay, postnatal age of initial screening and oxygen therapy longer than 10 days were significant predictive factors for the spontaneous regression of ROP (P<0.05). Retinal hemorrhage was the only independent predictive factor the spontaneous regression of ROP (OR 0.030, 95%CI 0.001-0.775, P=0.035). CONCLUSION: This study showed most stage 1 and 2 ROP and changes in zone III can spontaneously regression in the end. Retinal hemorrhage is weakly inversely associated with the spontaneous regression.

A myostatin inhibitor (propeptide-Fc) increases muscle mass and muscle fiber size in aged mice but does not increase bone density or bone strength.

Loss of muscle and bone mass with age are significant contributors to falls and fractures among the elderly. Myostatin deficiency is associated with increased muscle mass in mice, dogs, cows, sheep and humans, and mice lacking myostatin have been observed to show increased bone density in the limb, spine, and jaw. Transgenic overexpression of myostatin propeptide, which binds to and inhibits the active myostatin ligand, also increases muscle mass and bone density in mice. We therefore sought to test the hypothesis that in vivo inhibition of myostatin using an injectable myostatin propeptide (GDF8 propeptide-Fc) would increase both muscle mass and bone density in aged (24 mo) mice. Male mice were injected weekly (20 mg/kg body weight) with recombinant myostatin propeptide-Fc (PRO) or vehicle (VEH; saline) for four weeks. There was no difference in body weight between the two groups at the end of the treatment period, but PRO treatment significantly increased mass of the tibialis anterior muscle (+ 7%) and increased muscle fiber diameter of the extensor digitorum longus (+ 16%) and soleus (+ 6%) muscles compared to VEH treatment. Bone volume relative to total volume (BV/TV) of the femur calculated by microCT did not differ significantly between PRO- and VEH-treated mice, and ultimate force (Fu), stiffness (S), toughness (U) measured from three-point bending tests also did not differ significantly between groups. Histomorphometric assays also revealed no differences in bone formation or resorption in response to PRO treatment. These data suggest that while developmental perturbation of myostatin signaling through either gene knockout or transgenic inhibition may alter both muscle and bone mass in mice, pharmacological inhibition of myostatin in aged mice has a more pronounced effect on skeletal muscle than on bone.

Immunolocalization of myostatin (GDF-8) following musculoskeletal injury and the effects of exogenous myostatin on muscle and bone healing.

The time course and cellular localization of myostatin expression following musculoskeletal injury are not well understood; therefore, the authors evaluated the temporal and spatial localization of myostatin during muscle and bone repair following deep penetrant injury in a mouse model. They then used hydrogel delivery of exogenous myostatin in the same injury model to determine the effects of myostatin exposure on muscle and bone healing. Results showed that a "pool" of intense myostatin staining was observed among injured skeletal muscle fibers 12-24 hr postsurgery and that myostatin was also expressed in the soft callus chondrocytes 4 days following osteotomy. Hydrogel delivery of 10 or 100 µg/ml recombinant myostatin decreased fracture callus cartilage area relative to total callus area in a dose-dependent manner by 41% and 80% (p<0.05), respectively, compared to vehicle treatment. Myostatin treatment also decreased fracture callus total bone volume by 30.6% and 38.8% (p<0.05), with the higher dose of recombinant myostatin yielding the greatest decrease in callus bone volume. Finally, exogenous myostatin treatment caused a significant dose-dependent increase in fibrous tissue formation in skeletal muscle. Together, these findings suggest that early pharmacological inhibition of myostatin is likely to improve the regenerative potential of both muscle and bone following deep penetrant musculoskeletal injury.

The 3'-terminal sequence of Bamboo mosaic virus minus-strand RNA interacts with RNA-dependent RNA polymerase and initiates plus-strand RNA synthesis.

A 3'-terminal, 77-nucleotide sequence of Bamboo mosaic virus (BaMV) minus-strand RNA (Ba-77), comprising a 5' stem-loop, a spacer and a 3'-CUUUU sequence, can be used to initiate plus-strand RNA synthesis in vitro. To understand the mechanism of plus-strand RNA synthesis, mutations were introduced in the 5' untranslated region of BaMV RNA, resulting in changes at the 3' end of minus-strand RNA. The results showed that at least three uridylate residues in 3'-CUUUU are required and the changes at the penultimate U are deleterious to viral accumulation in Nicotiana benthamiana protoplasts. Results from UV-crosslinking and in vitro RNA-dependent RNA polymerase competition assays suggested that the replicase preferentially interacts with the stem structure of Ba-77. Finally, CMV/83 + UUUUC, a heterologus RNA, which possesses about 80 nucleotides containing the 3'-CUUUU pentamer terminus, and which folds into a secondary structure similar to that of Ba-77, could be used as template for RNA production by the BaMV replicase complex in vitro.

Recombinant myostatin (GDF-8) propeptide enhances the repair and regeneration of both muscle and bone in a model of deep penetrant musculoskeletal injury.

BACKGROUND: Myostatin (GDF-8) is known as a potent inhibitor of muscle growth and development, and myostatin is also expressed early in the fracture healing process. The purpose of this study was to test the hypothesis that a new myostatin inhibitor, a recombinant myostatin propeptide, can enhance the repair and regeneration of both muscle and bone in cases of deep penetrant injury. METHODS: We used a fibula osteotomy model with associated damage to lateral compartment muscles (fibularis longus and brevis) in mice to test the hypothesis that blocking active myostatin with systemic injections of a recombinant myostatin propeptide would improve muscle and bone repair. Mice were assigned to two treatment groups after undergoing a fibula osteotomy: those receiving either vehicle (saline) or recombinant myostatin propeptide (20 mg/kg). Mice received one injection on the day of surgery, another injection 5 days after surgery, and a third injection 10 days after surgery. Mice were killed 15 days after the osteotomy procedure. Bone repair was assessed using microcomputed tomography (micro-CT) and histologic evaluation of the fracture callus. Muscle healing was assessed using Masson trichrome staining of the injury site, and image analysis was used to quantify the degree of fibrosis and muscle regeneration. RESULTS: Three propeptide injections over a period of 15 days increased body mass by 7% and increased muscle mass by almost 20% (p < 0.001). Micro-CT analysis of the osteotomy site shows that by 15 days postosteotomy, bony callus tissue was observed bridging the osteotomy gap in 80% of the propeptide-treated mice but only 40% of the control (vehicle)-treated mice (p < 0.01). Micro-CT quantification shows that bone volume of the fracture callus was increased by ∼ 30% (p < 0.05) with propeptide treatment, and the increase in bone volume was accompanied by a significant increase in cartilage area (p = 0.01). Propeptide treatment significantly decreased the fraction of fibrous tissue in the wound site and increased the fraction of muscle relative to fibrous tissue by 20% (p < 0.01). CONCLUSIONS: Blocking myostatin signaling in the injured limb improves fracture healing and enhances muscle regeneration. These data suggest that myostatin inhibitors may be effective for improving wound repair in cases of orthopaedic trauma and extremity injury.

[Nucleoplasmic viscosity of living cells investigated by fluorescence correlation spectroscopy].

In order to non-invasively investigate nucleoplasmic viscosity in real time with good temporal resolution, the present study firstly introduced a new method based on fluorescence correlation spectroscopy (FCS). FCS is a kind of single-molecule technique with high temporal and spatial resolution to analyze the dynamics of fluorescent molecules in nanomolar concentration. Through a time correlation analysis of spontaneous intensity fluctuations, this technique in conjunction with EGFP as a probe is capable of determining nucleoplasmic viscosity in terms of Stokes-Einstein equation as well as its corresponding analysis of the diffusion coefficient for EGFP in the nucleus. The results showed that nucleoplasmic viscosity of ASTC-a-1 cells and HeLa cells were respectively (2.55 +/- 0.61) cP and (2.04 +/- 0.49) cP at pH 7.4 and 37 degrees C, consistent with the results by traditional methods, and nucleoplasmic viscosity was found to be larger than cytoplasmic viscosity. Meanwhile, the real-time analysis of nucleoplasmic viscosity in living cells exposed to hypotonic media proved that FCS could be used to track the changing rheological characteristics of the nucleoplasm in living cells. Taken together, this study suggests that FCS provides an accurate and non-invasive method to investigate the microenvironment in living cells on the femtoliter scale and it can be used as a powerful tool in researches on the dynamical processes of intracellular molecules.

Regulation of muscle growth by multiple ligands signaling through activin type II receptors.

Myostatin is a secreted protein that normally functions as a negative regulator of muscle growth. Agents capable of blocking the myostatin signaling pathway could have important applications for treating human muscle degenerative diseases as well as for enhancing livestock production. Here we describe a potent myostatin inhibitor, a soluble form of the activin type IIB receptor (ACVR2B), which can cause dramatic increases in muscle mass (up to 60% in 2 weeks) when injected into wild-type mice. Furthermore, we show that the effect of the soluble receptor is attenuated but not eliminated in Mstn(-/-) mice, suggesting that at least one other ligand in addition to myostatin normally functions to limit muscle growth. Finally, we provide genetic evidence that these ligands signal through both activin type II receptors, ACVR2 and ACVR2B, to regulate muscle growth in vivo.

Myostatin signaling through Smad2, Smad3 and Smad4 is regulated by the inhibitory Smad7 by a negative feedback mechanism.

As a member of the TGF-beta superfamily, myostatin is a specific negative regulator of skeletal muscle mass. To identify the downstream components in the myostatin signal transduction pathway, we used a luciferase reporter assay to elucidate myostatin-induced activity. The myostatin-induced transcription requires the participation of regulatory Smads (Smad2/3) and Co-Smads (Smad4). Conversely, inhibitory Smad7, but not Smad6, dramatically reduces the myostatin-induced transcription. This Smad7 inhibition is enhanced by co-expression of Smurf1. We have also shown that Smad7 expression is stimulated by myostatin via the interaction between Smad2, Smad3, Smad4 and the SBE (Smad binding element) in the Smad7 promoter. These results suggest that the myostatin signal transduction pathway is regulated by Smad7 through a negative feedback mechanism.

Characterization and identification of the inhibitory domain of GDF-8 propeptide.

GDF-8 is a negative regulator of skeletal muscle mass. The mechanisms which regulate the biological activity of GDF-8 have not yet been elucidated. Analogous to the TGF-beta system, GDF-8 propeptide binds to and inhibits the activity of GDF-8. In these studies, we define the critical domain of the GDF-8 propeptide necessary for inhibitory activity. Two molecules of GDF-8 propeptide monomer inhibit the biological activity of one molecule of GDF-8 homodimer. Although the propeptide contains N-linked glycosylation when synthesized in mammalian cells, this glycosylation is not necessary for the inhibition of GDF-8. Taking advantage of the bacterial expression system, we express and purify GDF-8 propeptide which retains full inhibitory activity. To define the functional regions of the propeptide, we express a series of truncated GST-propeptide fusion proteins and examined their inhibitory activity. We observe that fusion proteins containing the C-terminal region (amino acid residues 99-266) are very stable, but do not exhibit inhibitory activity; while fusion proteins containing the N-terminal region (amino acid residues 42-115) are labile but contain essential inhibitory activity. The data suggest that the C-terminal region may play a role in the stability of the GDF-8 propeptide and that the inhibitory domain is located in the region between amino acids 42 and 115.

Mutation of a conserved hydrophobic patch prevents incorporation of ZP3 into the zona pellucida surrounding mouse eggs.

Three glycoproteins (ZP1, ZP2, and ZP3) are synthesized in growing mouse oocytes and secreted to form an extracellular zona pellucida that mediates sperm binding and fertilization. Each has a signal peptide to direct it into a secretory pathway, a "zona" domain implicated in matrix polymerization and a transmembrane domain from which the ectodomain must be released. Using confocal microscopy and enhanced green fluorescent protein (EGFP), the intracellular trafficking of ZP3 was observed in growing mouse oocytes. Replacement of the zona domain with EGFP did not prevent secretion of ZP3, suggesting the presence of trafficking signals and a cleavage site in the carboxyl terminus. Analysis of linker-scanning mutations of a ZP3-EGFP fusion protein in transient assays and in transgenic mice identified an eight-amino-acid hydrophobic region required for secretion and incorporation into the zona pellucida. The hydrophobic patch is conserved among mouse zona proteins and lies between a potential proprotein convertase (furin) cleavage site and the transmembrane domain. The cleavage site that releases the ectodomain from the transmembrane domain was defined by mass spectrometry of native zonae pellucidae and lies N-terminal to a proprotein convertase site that is distinct from the hydrophobic patch.

Conserved furin cleavage site not essential for secretion and integration of ZP3 into the extracellular egg coat of transgenic mice.

The extracellular zona pellucida surrounding mammalian eggs is formed by interactions of the ZP1, ZP2, and ZP3 glycoproteins. Female mice lacking ZP2 or ZP3 do not form a stable zona matrix and are sterile. The three zona proteins are synthesized in growing oocytes and secreted prior to incorporation into the zona pellucida. A well-conserved furin site upstream of a transmembrane domain near the carboxyl terminus of each has been implicated in the release of the zona ectodomains from oocytes. However, mutation of the furin site (RNRR --> ANAA) does not affect the intracellular trafficking or secretion of an enhanced green fluorescent protein (EGFP)-ZP3 fusion protein in heterologous somatic cells. After transient expression in growing oocytes, normal EGFP-ZP3 and mutant EGFP-ZP3 associate with the inner aspect of the zona pellucida, which is distinct from the plasma membrane. These in vitro results are confirmed in transgenic mice expressing EGFP-ZP3 with or without the mutant furin site. In each case, EGFP-ZP3 is incorporated throughout the width of the zona pellucida and the transgenic mice are fertile. These results indicate that the zona matrix accrues from the inside out and, unexpectedly, suggest that cleavage at the furin site is not required for formation of the extracellular zona pellucida surrounding mouse eggs.