Analysis of risk factors and changes in myocardial biomarker levels in 122 cases of early neonatal anemia.

Background/aim: Anemia in the first week after birth, which could affect growth, development, and organ function, should be an important warning sign to clinicians. The aim of this study was to assess the related risk factors of early neonatal anemia and to analyze the effect of anemia on the expression levels of myocardial markers in newborns. Materials and methods: Clinical data from 122 confirmed cases of anemic newborns and 108 nonanemic newborns were collected to analyze the independent risk factors for early anemia using logistic regression analyses. Blood samples were collected from both groups for the detection of myocardial markers, including the protein marker cardiac troponin T (cTnT), as well as enzyme markers creatine kinase isoenzyme MB (CK-MB) and lactate dehydrogenase (LDH). Results: Multivariate logistic regression analysis revealed that preterm birth (OR: 3.589 [1.119-11.506], p < 0.05), multiple pregnancy (OR: 4.117 [1.021-16.611], p < 0.05), and abnormal placenta (OR: 4.712 [1.077-20.625], p < 0.05) were independent risk factors for early neonatal anemia. The levels of myocardial markers, including cTnT (303.1 ± 244.7 vs. 44.2 ± 55.41 ng/L), CK-MB (6.803 ± 8.971 vs. 2.5326 ± 2.927 µkat/L), and LDH (32.42 ± 35.26 vs. 19.73 ± 17.13 µkat/L), were significantly higher in the anemic group than in the nonanemic group. Conclusion: Multiple pregnancy, preterm birth, and abnormal placenta were identified as risk factors for early neonatal anemia. The occurrence of early neonatal anemia was associated with increased levels of myocardial markers.

Improvement of Inflammation and Abnormal Vascularization by TSP1 Treatment Combined with ADSCs Transplantation in Mice with Induced Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a common gynecological endocrine disease with a certain degree of chronic inflammation and abnormal ovarian angiogenesis in reproductive women. Mesenchymal stem cells (MSCs) have potent immunomodulatory properties to regulate ovarian function, while thrombospondin 1 (TSP1) improves the abnormal formation of ovarian vessels. The present study investigated the efficacy of the combined use of adipose-derived mesenchymal stem cells (ADSCs) and TSP1 in PCOS mice. The PCOS model is established using dehydroepiandrosterone (DHEA) by subcutaneous injection. Ovarian apoptosis is assessed using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Real-time quantitative PCR (RT-PCR) and western blotting are used to detect the expression of inflammatory factors and the levels of angiogenesis-related factors in ovarian tissues. Inflammatory cells count and ovarian angiogenesis are evaluated by immunofluorescence staining. This research shows that TSP1 and ADSCs treatment can significantly reduce the inflammatory state of PCOS mice, relieve the degree of ovarian cell apoptosis, optimize the ovarian histological manifestations, and restore the levels of related hormones. The proportion of CD31-positive cells in PCOS mice returned to near-normal levels. The synergistic use of ADSCs and TSP1 therapy can exert a more impressive effect by inhibiting the ovarian inflammatory response and regulating the balance of angiogenesis than the single application in PCOS mice.

Publisher Correction: Human DEF6 deficiency underlies an immunodeficiency syndrome with systemic autoimmunity and aberrant CTLA-4 homeostasis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

RETRACTED: HBV-encoded miR-2 functions as an oncogene by downregulating TRIM35 but upregulating RAN in liver cancer cells.

This article has been retracted: please see Elsevier Policy on Article Withdrawal https://www.elsevier.com/about/our-business/policies/article-withdrawal This article has been retracted at the request of the editor because following publication concerns were raised by a reader with respect to images presented in multiple figures. Specifically, the reader suspected partial image duplications between the migration/pTRIM35 panel and the invasion/pcDNA3 panel in Fig. 4e, the migration/pshR-RAN panel and the migration/pcDNA3 panel in Fig. 5e, the invasion/pRAN panel in Fig. 5e and the invasion/pHBV-miR-2+pcDNA3 panel in Fig. 6e, the migration/pcDNA3+pcDNA3 panel in Fig. 6e and the migration/pcDNA3+pSilencer panel in Fig. 7e, and finally, the migration/pBHV-miR-2+pTRIM3 panel in Fig. 6e with the migration/pcDNA3+pSilencer panel in Fig. 7e. These image duplication allegations were referred to the Office of the Academic Ethics Committee at Basic Medical School, Tianjin Medical University, China, for independent investigation. The Committee received all the original data related to the paper from corresponding author and first author, and confirmed that the authors had done all experiments supporting the reported results. The independent investigation concluded the laboratory led by the corresponding author had serious problems with data management and supervision, which may have accounted for the errors in the paper. The committee did not detect deliberate fraud, but requested the corresponding author should withdraw the paper based on the nature of multiple misuses of figures. The authors subsequently requested retraction of the article. The editors no longer have confidence in the integrity of these data. All authors agree to this retraction.

Human DEF6 deficiency underlies an immunodeficiency syndrome with systemic autoimmunity and aberrant CTLA-4 homeostasis.

Immune responses need to be controlled tightly to prevent autoimmune diseases, yet underlying molecular mechanisms remain partially understood. Here, we identify biallelic mutations in three patients from two unrelated families in differentially expressed in FDCP6 homolog (DEF6) as the molecular cause of an inborn error of immunity with systemic autoimmunity. Patient T cells exhibit impaired regulation of CTLA-4 surface trafficking associated with reduced functional CTLA-4 availability, which is replicated in DEF6-knockout Jurkat cells. Mechanistically, we identify the small GTPase RAB11 as an interactor of the guanine nucleotide exchange factor DEF6, and find disrupted binding of mutant DEF6 to RAB11 as well as reduced RAB11+CTLA-4+ vesicles in DEF6-mutated cells. One of the patients has been treated with CTLA-4-Ig and achieved sustained remission. Collectively, we uncover DEF6 as player in immune homeostasis ensuring availability of the checkpoint protein CTLA-4 at T-cell surface, identifying a potential target for autoimmune and/or cancer therapy.

Phosphoproteomics Reveals Regulatory T Cell-Mediated DEF6 Dephosphorylation That Affects Cytokine Expression in Human Conventional T Cells.

Regulatory T cells (Tregs) control key events of immune tolerance, primarily by suppression of effector T cells. We previously revealed that Tregs rapidly suppress T cell receptor (TCR)-induced calcium store depletion in conventional CD4+CD25- T cells (Tcons) independently of IP3 levels, consequently inhibiting NFAT signaling and effector cytokine expression. Here, we study Treg suppression mechanisms through unbiased phosphoproteomics of primary human Tcons upon TCR stimulation and Treg-mediated suppression, respectively. Tregs induced a state of overall decreased phosphorylation as opposed to TCR stimulation. We discovered novel phosphosites (T595_S597) in the DEF6 (SLAT) protein that were phosphorylated upon TCR stimulation and conversely dephosphorylated upon coculture with Tregs. Mutation of these DEF6 phosphosites abrogated interaction of DEF6 with the IP3 receptor and affected NFAT activation and cytokine transcription in primary Tcons. This novel mechanism and phosphoproteomics data resource may aid in modifying sensitivity of Tcons to Treg-mediated suppression in autoimmune disease or cancer.

Stabilization of F-actin by tropomyosin isoforms regulates the morphology and mechanical behavior of red blood cells.

The short F-actins in the red blood cell (RBC) membrane skeleton are coated along their lengths by an equimolar combination of two tropomyosin isoforms, Tpm1.9 and Tpm3.1. We hypothesized that tropomyosin's ability to stabilize F-actin regulates RBC morphology and mechanical properties. To test this, we examined mice with a targeted deletion in alternatively spliced exon 9d of Tpm3 (Tpm3/9d-/- ), which leads to absence of Tpm3.1 in RBCs along with a compensatory increase in Tpm1.9 of sufficient magnitude to maintain normal total tropomyosin content. The isoform switch from Tpm1.9/Tpm3.1 to exclusively Tpm1.9 does not affect membrane skeleton composition but causes RBC F-actins to become hyperstable, based on decreased vulnerability to latrunculin-A-induced depolymerization. Unexpectedly, this isoform switch also leads to decreased association of Band 3 and glycophorin A with the membrane skeleton, suggesting that tropomyosin isoforms regulate the strength of F-actin-to-membrane linkages. Tpm3/9d-/- mice display a mild compensated anemia, in which RBCs have spherocytic morphology with increased osmotic fragility, reduced membrane deformability, and increased membrane stability. We conclude that RBC tropomyosin isoforms directly influence RBC physiology by regulating 1) the stability of the short F-actins in the membrane skeleton and 2) the strength of linkages between the membrane skeleton and transmembrane glycoproteins.

USP14 de-ubiquitinates vimentin and miR-320a modulates USP14 and vimentin to contribute to malignancy in gastric cancer cells.

Vimentin plays important roles in the epithelial-to-mesenchymal transition (EMT). In this study, we found that vimentin was highly expressed in human gastric cancer (GC) tissues and cell lines and significantly promoted cell growth, migration and invasion. Ubiquitin-specific protease 14 (USP14) interacted with the vimentin protein, which led to its de-ubiquitination. miR-320a was found to bind to the 3'UTR of both vimentin and USP14 transcripts and downregulate the expression of both proteins. The downregulation of miR-320a upregulates vimentin expression by directly binding to the 3'UTR of vimentin to derepress expression and indirectly by augmenting USP14 to increase vimentin stability in GC cells. Taken together, these results provide new insight into malignancy in gastric cancers.

Regulation of actin polymerization by tropomodulin-3 controls megakaryocyte actin organization and platelet biogenesis.

The actin cytoskeleton is important for platelet biogenesis. Tropomodulin-3 (Tmod3), the only Tmod isoform detected in platelets and megakaryocytes (MKs), caps actin filament (F-actin) pointed ends and binds tropomyosins (TMs), regulating actin polymerization and stability. To determine the function of Tmod3 in platelet biogenesis, we studied Tmod3(-/-) embryos, which are embryonic lethal by E18.5. Tmod3(-/-) embryos often show hemorrhaging at E14.5 with fewer and larger platelets, indicating impaired platelet biogenesis. MK numbers are moderately increased in Tmod3(-/-) fetal livers, with only a slight increase in the 8N population, suggesting that MK differentiation is not significantly affected. However, Tmod3(-/-) MKs fail to develop a normal demarcation membrane system (DMS), and cytoplasmic organelle distribution is abnormal. Moreover, cultured Tmod3(-/-) MKs exhibit impaired proplatelet formation with a wide range of proplatelet bud sizes, including abnormally large proplatelet buds containing incorrect numbers of von Willebrand factor-positive granules. Tmod3(-/-) MKs exhibit F-actin disturbances, and Tmod3(-/-) MKs spreading on collagen fail to polymerize F-actin into actomyosin contractile bundles. Tmod3 associates with TM4 and the F-actin cytoskeleton in wild-type MKs, and confocal microscopy reveals that Tmod3, TM4, and F-actin partially colocalize near the membrane of proplatelet buds. In contrast, the abnormally large proplatelets from Tmod3(-/-) MKs show increased F-actin and redistribution of F-actin and TM4 from the cortex to the cytoplasm, but normal microtubule coil organization. We conclude that F-actin capping by Tmod3 regulates F-actin organization in mouse fetal liver-derived MKs, thereby controlling MK cytoplasmic morphogenesis, including DMS formation and organelle distribution, as well as proplatelet formation and sizing.

Differential actin-regulatory activities of Tropomodulin1 and Tropomodulin3 with diverse tropomyosin and actin isoforms.

Tropomodulins (Tmods) are F-actin pointed end capping proteins that interact with tropomyosins (TMs) and cap TM-coated filaments with higher affinity than TM-free filaments. Here, we tested whether differences in recognition of TM or actin isoforms by Tmod1 and Tmod3 contribute to the distinct cellular functions of these Tmods. We found that Tmod3 bound ~5-fold more weakly than Tmod1 to α/ßTM, TM5b, and TM5NM1. However, surprisingly, Tmod3 was as effective as Tmod1 at capping pointed ends of skeletal muscle α-actin (αsk-actin) filaments coated with α/ßTM, TM5b, or TM5NM1. Tmod3 only capped TM-coated αsk-actin filaments more weakly than Tmod1 in the presence of recombinant αTM2, which is unacetylated at its NH2 terminus, binds F-actin weakly, and has a disabled Tmod-binding site. Moreover, both Tmod1 and Tmod3 were similarly effective at capping pointed ends of platelet ß/cytoplasmic γ (γcyto)-actin filaments coated with TM5NM1. In the absence of TMs, both Tmod1 and Tmod3 had similarly weak abilities to nucleate ß/γcyto-actin filament assembly, but only Tmod3 could sequester cytoplasmic ß- and γcyto-actin (but not αsk-actin) monomers and prevent polymerization under physiological conditions. Thus, differences in TM binding by Tmod1 and Tmod3 do not appear to regulate the abilities of these Tmods to cap TM-αsk-actin or TM-ß/γcyto-actin pointed ends and, thus, are unlikely to determine selective co-assembly of Tmod, TM, and actin isoforms in different cell types and cytoskeletal structures. The ability of Tmod3 to sequester ß- and γcyto-actin (but not αsk-actin) monomers in the absence of TMs suggests a novel function for Tmod3 in regulating actin remodeling or turnover in cells.

Calpain-mediated proteolysis of tropomodulin isoforms leads to thin filament elongation in dystrophic skeletal muscle.

Duchenne muscular dystrophy (DMD) induces sarcolemmal mechanical instability and rupture, hyperactivity of intracellular calpains, and proteolytic breakdown of muscle structural proteins. Here we identify the two sarcomeric tropomodulin (Tmod) isoforms, Tmod1 and Tmod4, as novel proteolytic targets of m-calpain, with Tmod1 exhibiting ∼10-fold greater sensitivity to calpain-mediated cleavage than Tmod4 in situ. In mdx mice, increased m-calpain levels in dystrophic soleus muscle are associated with loss of Tmod1 from the thin filament pointed ends, resulting in ∼11% increase in thin filament lengths. In mdx/mTR mice, a more severe model of DMD, Tmod1 disappears from the thin filament pointed ends in both tibialis anterior (TA) and soleus muscles, whereas Tmod4 additionally disappears from soleus muscle, resulting in thin filament length increases of ∼10 and ∼12% in TA and soleus muscles, respectively. In both mdx and mdx/mTR mice, both TA and soleus muscles exhibit normal localization of α-actinin, the nebulin M1M2M3 domain, Tmod3, and cytoplasmic γ-actin, indicating that m-calpain does not cause wholesale proteolysis of other sarcomeric and actin cytoskeletal proteins in dystrophic skeletal muscle. These results implicate Tmod proteolysis and resultant thin filament length misspecification as novel mechanisms that may contribute to DMD pathology, affecting muscles in a use- and disease severity-dependent manner.

Tropomodulin3-null mice are embryonic lethal with anemia due to impaired erythroid terminal differentiation in the fetal liver.

Tropomodulin (Tmod) is a protein that binds and caps the pointed ends of actin filaments in erythroid and nonerythoid cell types. Targeted deletion of mouse tropomodulin3 (Tmod3) leads to embryonic lethality at E14.5-E18.5, with anemia due to defects in definitive erythropoiesis in the fetal liver. Erythroid burst-forming unit and colony-forming unit numbers are greatly reduced, indicating defects in progenitor populations. Flow cytometry of fetal liver erythroblasts shows that late-stage populations are also decreased, including reduced percentages of enucleated cells. Annexin V staining indicates increased apoptosis of Tmod3(-/-) erythroblasts, and cell-cycle analysis reveals that there are more Ter119(hi) cells in S-phase in Tmod3(-/-) embryos. Notably, enucleating Tmod3(-/-) erythroblasts are still in the process of proliferation, suggesting impaired cell-cycle exit during terminal differentiation. Tmod3(-/-) late erythroblasts often exhibit multilobular nuclear morphologies and aberrant F-actin assembly during enucleation. Furthermore, native erythroblastic island formation was impaired in Tmod3(-/-) fetal livers, with Tmod3 required in both erythroblasts and macrophages. In conclusion, disruption of Tmod3 leads to impaired definitive erythropoiesis due to reduced progenitors, impaired erythroblastic island formation, and defective erythroblast cell-cycle progression and enucleation. Tmod3-mediated actin remodeling may be required for erythroblast-macrophage adhesion, coordination of cell cycle with differentiation, and F-actin assembly and remodeling during erythroblast enucleation.

Overexpression of the phosphatidylinositol synthase gene from Zea mays in tobacco plants alters the membrane lipids composition and improves drought stress tolerance.

Phosphatidylinositol (PtdIns) is an important lipid because it serves as a key membrane constituent and is the precursor of the inositol-containing lipids that are found in all plants and animals. It is synthesized from cytidine-diphosphodiacylglycerol (CDP-DG) and myo-inositol by PtdIns synthase (PIS). We have previously reported that two putative PIS genes from maize (Zea mays L.), ZmPIS and ZmPIS2, are transcriptionally up-regulated in response to drought (Sui et al., Gene, 426:47-56, 2008). In this work, we report on the characterization of ZmPIS in vitro and in vivo. The ZmPIS gene successfully complemented the yeast pis mutant BY4743, and the determination of PIS activity in the yeast strain further confirmed the enzymatic function of ZmPIS. An ESI-MS/MS-based lipid profiling approach was used to identify and quantify the lipid species in transgenic and wild-type tobacco plants before and after drought treatment. The results show that the overexpression of ZmPIS significantly increases lipid levels in tobacco leaves under drought stress compared to those of wild-type tobacco, which correlated well with the increased drought tolerance of the transgenic plants. Further analysis showed that, under drought stress conditions, ZmPIS overexpressors were found to exhibit increased membrane integrity, thereby enabling the retention of more solutes and water compared with the wild-type and the vector control transgenic lines. Our findings give us new insights into the role of the ZmPIS gene in the response of maize to drought/osmotic stress and the mechanisms by which plants adapt to drought stress.

'A life or death decision' for pollen tubes in S-RNase-based self-incompatibility.

Mate choice is an essential process during sexual plant reproduction, in which self-incompatibility (SI) is widely adopted as an intraspecific reproductive barrier to inhibit self-fertilization by many flowering plants. Genetic studies show that a single polymorphic S-locus, encoding at least two components from both the pollen and pistil sides, controls the discrimination of self and non-self pollen. In the Solanaceae, Plantaginaceae, and Rosaceae, an S-RNase-based SI mechanism is involved in such a discrimination process. Recent studies have provided some important clues to how a decision is made to accept cross pollen or specifically to reject self pollen. In this review, the molecular features of the pistil and pollen S-specificity factors are briefly summarized and then our current knowledge of the molecular control of cross-pollen compatibility (CPC) and self-pollen incompatibility (SPI) responses, respectively, is presented. The possible biochemical mechanisms of the specificity determinant between the pistil and pollen S factors are discussed and a hypothetical S-RNase endosome sorting model is proposed to illustrate the distinct destinies of pollen tubes following compatible and incompatible pollination.

Cloning and expression analysis of some genes involved in the phosphoinositide and phospholipid signaling pathways from maize (Zea mays L.).

Previous studies have indicated the phosphoinositide and phospholipid signaling pathways play a key role in plant growth, development and responses to environmental stresses. However, little is known about the phosphoinositide and phospholipid signaling pathways in maize (Zea mays L.). To better understand the function of genes involved in the phosphoinositide and phospholipid signaling pathways in maize, the cDNA sequences of ZmPIS2, ZmPLC2, ZmDGK1, ZmDGK2 and ZmDGK3 were obtained by RACE (rapid amplification of cDNA ends) or in silico cloning combined with PCR. RT-PCR analysis of cDNA from five tissues (roots, stems, leaves, tassels, and ears) indicated that the expression patterns of the five cDNAs we isolated as well as ZmPIS, ZmPLC, ZmPLD varied in different tissues. To determine the effects of different environmental conditions such as cold, drought and various phytohormones (abscisic acid, indole-3-acetic acid and gibberellic acid) on gene expression, we analyzed expression by Real-Time (RT-PCR), and found that the different isoforms of these gene families involved in the phosphoinositide and phospholipid signaling pathways have specific expression patterns. Our results suggested that these genes may be involved in the responses to environmental stresses, but have different functions. The isolation and analysis of expression patterns of genes involved in the phosphoinositide and phospholipid signaling pathways provides a good basis for further research of the phosphoinositide and phospholipid signaling pathways in maize and is a novel supplement to our comprehension of these pathways in plants.

Molecular cloning and characterization of a novel H+-translocating pyrophosphatase gene in Zea mays.

A cDNA encoding a putative H+-translocating pyrophosphatase (H+-PPase) has been cloned from Zea mays by suppression subtractive hybridization (SSH) coupled with in silico cloning approach. The isolated 2974 bp full-length cDNA named ZmGPP contains a single 2400 bp open reading frame encoding a putative protein of 799 amino acids. The predicted protein has 16 transmembrane domains and is significantly similar to Golgi apparatus resident type-II H+-PPase from Arabidopsis thaliana. DNA gel blotting analysis shows that ZmGPP is a low-copy gene. Organ expression pattern analysis reveals that ZmGPPexpressed highly in leaf and tassel, followed by in stem, root, and ear. The Real-time RT-PCR assays showed that the expression of ZmGPP was up-regulated both in shoots and roots of maize seedlings under dehydration, cold and high salt stresses. Those results suggest that the ZmGPP product may play an important role in abiotic stress tolerance of Z. mays.

Characterization of a novel phosphoinositide-specific phospholipase C from Zea mays and its expression in Escherichia coli.

A cDNA encoding a phosphoinositide-specific phospholipase C (PI-PLC) has been isolated from Zea mays by screening a cDNA library. The cDNA, designated ZmPLC, encodes a polypeptide of 586 amino acids, containing the catalytic X, Y and C2 domains found in all PI-PLCs from plants. Northern blot analysis showed that the expression of the ZmPLC gene in roots is up-regulated under conditions of high salt, dehydration, cold or low osmotic stress conditions. Recombinant ZmPLC protein was expressed in Esch- erichia coli, purified and used to produce polyclonal antibody, this polyclonal antibody is important for further studies to assess the ultimate function of the ZmPLC gene in plants.