Generation and characterization of a novel shoulder contracture mouse model

Frozen shoulder is a relatively common disorder that leads to severe pain and stiffness in the shoulder joint. Although this disorder is self‐limiting in nature, the symptoms often persist for years, resulting in severe disability. Recent studies using human specimens and animal models have shown distinct changes in the gene expression patterns in frozen shoulder tissue, indicating that novel therapeutic intervention could be achieved by controlling the genes that are potentially involved in the development of frozen shoulder. To achieve this goal, it is imperative to develop a reliable animal joint contracture model in which gene expression can be manipulated by gene targeting and transgenic technologies. Here, we describe a novel shoulder contracture mouse model. We found that this model mimics the clinical presentation of human frozen shoulder and recapitulates the changes in the gene expression pattern and the histology of frozen shoulder and joint contracture in humans and other larger animal models. The model is highly reproducible, without any major complications. Therefore, the present model may serve as a useful tool for investigating frozen shoulder etiology and for identifying its potential target genes. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1732–1738, 2015.     

Multicolour fluorescence in situ hybridization analysis of t(14;18)-positive follicular lymphoma and correlation with gene expression data and clinical outcome

In order fully to identify secondary chromosomal alterations, such as duplications, additions and marker chromosomes that remained unresolved by G banding, 60 cases of t(14;18)-positive follicular lymphoma (FL) were analysed by multicolour karyotyping techniques [multicolour fluorescence in situ hybridization (MFISH)/multicolour banding for chromosome 1 (MBAND1)]. A total of 165 additional structural chromosomal aberrations were delineated. An increased frequency of chromosomal gains involving X, 1q, 2, 3q27-q29, 5, 6p11-p21, 7, 8, 11, 12, 14q32, 17q, 18 and 21 and deletions of 1p36, 3q28-q29, 6q, 10q22-q24 and 17p11-p13 was revealed by the MFISH/MBAND1 analysis. Balanced translocations other than t(14;18) were uncommon, whereas unbalanced translocations were numerous. Deletion of 1p36 and duplication of 1p33-p35, 1p12-p21 and 1q21-q41 were regularly involved in chromosome 1 alterations, seen in 53% of the cases. A strong correlation was demonstrated between gains of individual chromosomal bands and increased gene expression, including 1q22/MNDA, 6p21/CDKN1A, 12q13-q14/SAS, 17q23/ZNF161, 18q21/BCL2 and Xq13/IL2RG. Unfavourable overall survival was associated with del(1)(p36) and dup(18q). These data support the notion that translocation events are primarily responsible for FL disease initiation, whereas the unbalanced chromosomal gains and losses that mirror the gene expression patterns characterize clonal evolution and disease progression, and thus provide further insights into the biology of FL.     

乙肝病毒PreS2基因在大肠埃希菌中的表达

目的在大肠埃希菌(Escherichiacoli)中表达乙肝病毒前S2(HBVPreS2)蛋白,并对其进行鉴定和纯化。方法利用DNA重组技术,将全长的HBVPreS2基因克隆至pMAL-C2x[融合表达载体含麦芽糖结合蛋白(maltosebindingprotein,MBP)标签蛋白]质粒中,构建pMAL-C2/S2表达载体,转化至E.coli,经IPTG诱导表达,SDS-PAGE分析目的蛋白的表达形式,依据其不同的表达形式采取适当的纯化方案,纯化产物经Westernblot和ELISA法检测反应原性,经Xa因子切割去除MBP标签蛋白。结果成功构建了表达载体pMAL-C2/S2,目的蛋白为PreS2-MBP,以包涵体和可溶性2种形式表达。纯化产物能与anti-HepatitisBvirusPreS2Antigen发生特异性反应。融合蛋白经Xa因子切割去除了MBP标签蛋白。结论在E.coli中成功表达了HBVPreS2蛋白,为进一步研制新型HBV预防及免疫治疗制剂打下了基础。     

T淋巴细胞活化衔接子及其调控因子在支气管哮喘患者T淋巴细胞中的表达

目的 探讨T淋巴细胞活化衔接子(LAT)及其上游调控因子(Syk、Lck和ZAP-70)在支气管哮喘(简称哮喘)患者外周血T淋巴细胞中的转录表达水平是否存在异常.方法 对20例哮喘患者(哮喘组)及20例非特应症对照者(对照组)采用逆转录-聚合酶链反应(RT-PCR)法检测外周静脉血T淋巴细胞的LAT及Lck、Syk和ZAP-70 mRNA的表达,有关LAT基因转录的结果通过实时定量RT-PCR法进行验证.统计学处理采用SPSS 11.5软件.数据以-x±s表示.组间比较采用t检验.结果 哮喘组患者外周血T淋巴细胞LAT基因的mRNA表达水平为0.54±0.14,对照组为0.72±0.17,两组比较差异有统计学意义(t=3.11,P＜0.01);实时定量RT-PCR法(0.0065±0.0066)证实哮喘患者外周血T淋巴细胞LAT转录水平较对照组(0.0124±0.0045)下调(t=0.0022,P＜0.01).20例哮喘患者Lek和ZAP-70基因mRNA表达水平分别为0.71±0.16、1.05±0.41,对照组分别为0.53±0.17、0.82±0.27.两组比较差异有统计学意义(t值分别为3.18、2.10,P分别＜0.01、＜0.05);哮喘患者Syk基因mRNA表达水平为1.16±0.42,对照组为1.24±0.34,两组间Syk基因转录水平比较差异无统计学意义(t=0.22,P＞0.05).结论 哮喘患者外周血T淋巴细胞LAT基因转录水平下调可能与上游调控因子Lck和ZAP-70基因转录水平上调有关,LAT及上游调控因子Lck和ZAP-70转录水平异常可能是哮喘发病机制之一.     

The use of a novel bone allograft wash process to generate a biocompatible,mechanically stable and osteoinductive biological scaffold for use in bone tissue engineering

Fresh‐frozen biological allograft remains the most effective substitute for the ‘gold standard’ autograft, sharing many of its osteogenic properties but, conversely, lacking viable osteogenic cells. Tissue engineering offers the opportunity to improve the osseointegration of this material through the addition of mesenchymal stem cells (MSCs). However, the presence of dead, immunogenic and potentially harmful bone marrow could hinder cell adhesion and differentiation, graft augmentation and incorporation, and wash procedures are therefore being utilized to remove the marrow, thereby improving the material's safety. To this end, we assessed the efficiency of a novel wash technique to produce a biocompatible, biological scaffold void of cellular material that was mechanically stable and had osteoinductive potential. The outcomes of our investigations demonstrated the efficient removal of marrow components (~99.6%), resulting in a biocompatible material with conserved biomechanical stability. Additionally, the scaffold was able to induce osteogenic differentiation of MSCs, with increases in osteogenic gene expression observed following extended culture. This study demonstrates the efficiency of the novel wash process and the potential of the resultant biological material to serve as a scaffold in bone allograft tissue engineering. © 2014 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons Ltd.     

Placental genomic risk scores and early neurodevelopmental outcomes

Tracing the early paths leading to developmental disorders is critical for prevention. In previous work, we detected an interaction between genomic risk scores for schizophrenia (GRSs) and early-life complications (ELCs), so that the liability of the disorder explained by genomic risk was higher in the presence of a history of ELCs, compared with its absence. This interaction was specifically driven by loci harboring genes highly expressed in placentae from normal and complicated pregnancies [G. Ursini et al., Nat. Med. 24, 792–801 (2018)]. Here, we analyze whether fractionated genomic risk scores for schizophrenia and other developmental disorders and traits, based on placental gene-expression loci (PlacGRSs), are linked with early neurodevelopmental outcomes in individuals with a history of ELCs. We found that schizophrenia’s PlacGRSs are negatively associated with neonatal brain volume in singletons and offspring of multiple pregnancies and, in singletons, with cognitive development at 1 y and, less strongly, at 2 y, when cognitive scores become more sensitive to other factors. These negative associations are stronger in males, found only with GRSs fractionated by placental gene expression, and not found in PlacGRSs for other developmental disorders and traits. The relationship of PlacGRSs with brain volume persists as an anlage of placenta biology in adults with schizophrenia, again selectively in males. Higher placental genomic risk for schizophrenia, in the presence of ELCs and particularly in males, alters early brain growth and function, defining a potentially reversible neurodevelopmental path of risk that may be unique to schizophrenia.

Understanding the deviations from normal trajectories of brain development may be crucial for predicting illness and for prevention. Epidemiological studies have consistently identified early antecedents, including complications during pregnancy (1–4) and delays in developmental milestones (5–8). The incidence of many developmental disorders tends to be higher in males (9), and risk is typically highly heritable (10). While rare and moderately penetrant genetic variations account for a minority of cases, genome-wide association studies (GWASs) show that most risk is attributable to common variants across the genome (11, 12). Genomic risk scores (GRSs) from GWASs allow a much greater prediction of liability of the disorder than single common variant genotypes, but GRSs per se are not useful in predicting individual risk (13).We previously identified an environmental context in early life in which genomic risk for schizophrenia may enhance disease susceptibility (14). We found that the liability of schizophrenia explained by genomic risk (that is, schizophrenia GRS, also referred to as polygenic risk score; PRS) was more than five times higher in individuals with a history of obstetrical complications (here, early-life complications; ELCs; i.e., during pregnancy, at labor/delivery, and early in neonatal life) compared with its absence (14). Such interaction was exclusive of the GRSs constructed from the loci with the most significant associations with schizophrenia (GRS1: GWAS P < 5 × 10⁻⁸; GRS2: GWAS P < 1 × 10⁻⁶). Genes in the GRS1 and GRS2 loci were more highly expressed in placental tissue compared with genes in GWAS loci not interacting with ELCs (GRS3 to 10); they were up-regulated in placentae from complicated pregnancies and strongly correlated within placenta with expression of immune response genes (14), consistent with previous evidence linking placenta, inflammation, and brain development (15, 16).To investigate the role of placenta biology in the interaction between schizophrenia GRSs and ELCs, we derived sets of GRSs based on single-nucleotide polymorphisms (SNPs) marking schizophrenia-GWAS loci containing genes highly expressed in placenta and differentially expressed in placentae from complicated compared with normal pregnancies (PlacGRSs; placental genomic risk scores) and also from the remaining GWAS loci (NonPlacGRSs; nonplacental genomic risk scores). We found that only PlacGRSs interacted with ELCs on schizophrenia-case control status, while NonPlacGRSs did not, implicating genes involved in placenta stress as driving the interaction between genomic risk and ELCs. These interactions were specifically related to placental gene expression, in that they were not detected when calculating GRSs based on SNPs marking loci highly expressed or epigenetically regulated in other tissues, including various adult and fetal tissues/embryonic cells, and fetal brain. Finally, we detected a much stronger enrichment of expression of the schizophrenia-risk genes in placentae from male compared with female offspring, suggesting a role of placenta in the higher incidence of schizophrenia in males (14).We here investigate whether placental genomic risk for schizophrenia as well as several other developmental disorders and traits is linked with early neurodevelopmental outcomes in individuals with a history of ELCs associated with placenta pathophysiology. Abundant evidence shows that ELCs have implications for early developmental trajectories, including brain size, intellectual development, and neuromotor function as well as for schizophrenia later in life (3, 5, 17–19). Based on these prior observations and our earlier findings (14), we hypothesized that schizophrenia PlacGRSs, in contrast to NonPlacGRSs, have a negative effect on early developmental outcomes, especially in males. Further consistent with our earlier findings, we hypothesized that this negative relationship is characteristic of the PlacGRSs constructed from the placental schizophrenia-GWAS loci with the strongest association with the disorder (PlacGRS1: GWAS P < 5 × 10⁻⁸; PlacGRS2: GWAS P < 1 × 10⁻⁶). We studied the relationship of PlacGRSs and NonPlacGRSs with brain volume in a unique sample of neonates who underwent MRI scanning shortly after birth, and analyzed the relationship with neurocognitive development at 1 and 2 y of age in the same subjects. Finally, we analyzed the relationship of PlacGRSs and NonPlacGRSs with brain volume in a sample of adult controls and patients with schizophrenia.     

De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins

Enzymes that bear a nonnative or artificially introduced metal center can engender novel reactivity and enable new spectroscopic and structural studies. In the case of metal-organic cofactors, such as metalloporphyrins, no general methods exist to build and incorporate new-to-nature cofactor analogs in vivo. We report here that a common laboratory strain, Escherichia coli BL21(DE3), biosynthesizes cobalt protoporphyrin IX (CoPPIX) under iron-limited, cobalt-rich growth conditions. In supplemented minimal media containing CoCl₂, the metabolically produced CoPPIX is directly incorporated into multiple hemoproteins in place of native heme b (FePPIX). Five cobalt-substituted proteins were successfully expressed with this new-to-nature cobalt porphyrin cofactor: myoglobin H64V V68A, dye decolorizing peroxidase, aldoxime dehydratase, cytochrome P450 119, and catalase. We show conclusively that these proteins incorporate CoPPIX, with the CoPPIX making up at least 95% of the total porphyrin content. In cases in which the native metal ligand is a sulfur or nitrogen, spectroscopic parameters are consistent with retention of native metal ligands. This method is an improvement on previous approaches with respect to both yield and ease-of-implementation. Significantly, this method overcomes a long-standing challenge to incorporate nonnatural cofactors through de novo biosynthesis. By utilizing a ubiquitous laboratory strain, this process will facilitate spectroscopic studies and the development of enzymes for CoPPIX-mediated biocatalysis.

The identity of a metal center often defines enzymatic activity, and swapping the native metal for an alternative one or introducing a new metal center has profound effects. More generally, the chemical utility of natural cofactors has inspired decades of study into synthetic analogs with distinct properties, and researchers have subsequently sought straightforward ways to put these novel cofactors back into proteins (1). Substituted metalloenzymes constitute one of the simplest cases. Changing the identity of the metal ion in metalloproteins has enabled powerful spectroscopic and functional studies of these proteins (2–10) in addition to new biocatalytic activities (11–20). However, most methods for producing such proteins with new-to-nature cofactors are limited by the inability to produce the novel protein–cofactor complex in vivo.Hemoproteins, in particular, have been studied through metal substitution because of their important biological functions and utility as biocatalysts. Heme is a ubiquitous and versatile cofactor in biology, and heme-dependent proteins serve essential gas sensing functions (21), metabolize an array of xenobiotic molecules (22), and perform synthetically useful oxygen activation and radical-based chemistry (23). Metal-substituted hemoproteins have enabled key spectroscopic studies of hemoprotein function and the development of biocatalysts with novel reactivity. For example, electron paramagnetic resonance (EPR) studies on cobalt-substituted sperm whale myoglobin (CoMb) enabled detailed characterization of the paramagnetic CoMbO₂ complex (3, 4, 24, 25). In analogous oxygen-binding studies in CoMb and cobalt-substituted hemoglobin (5, 6, 26), resonance Raman was used to identify the O–O stretching mode because cobalt-substituted proteins exhibit enhancement of this vibrational mode compared to the native iron proteins.Metal substitution has a profound effect on catalytic activity of hemoproteins, enabling numerous synthetic applications. Substitution of the native iron for cobalt in several hemoproteins, including a thermostable cytochrome c variant, enabled the reduction of water to H₂ under aerobic, aqueous conditions (27–29). Reconstitution of apoprotein with selected metalloporphyrins has been used to generate metal-substituted myoglobin and cytochrome P450s variants. These enzymes were effective as biocatalysts for C–H activation and carbene insertion reactions (11–14). In a tour de force of directed evolution, which required purification and cofactor reconstitution of each individual variant, Hartwig and coworkers generated a cytochrome P450 variant that utilizes a nonnative Ir(Me)mesoporphyrin cofactor to perform desirable C–H activation chemistry (14). These activities may not be unique to the Ir-substituted protein, as synthetic cobalt porphyrin complexes have been shown to mediate a variety of Co(III)-aminyl and -alkyl radical transformations, including C–H activation (30–32). Indeed, a number of cobalt porphyrin carbene complexes display significant carbon-centered radical character (33–35), whereas the corresponding Fe-porphyrin complexes are closed shell species (36, 37), indicating that cobalt porphyrins may possess distinct, complementary modes of reactivity (38–40).Inspired by these applications, researchers have sought strategies for generating metal-substituted hemoproteins. For many metalloproteins, metal substitution is carried out by removal of the native metal with a chelator and replacement with an alternate metal of similar coordination preference. This method is inapplicable to hemoproteins, as porphyrins do not readily exchange metal ions. Consequently, diverse methods have been employed to make metal-substituted hemoproteins (41–46). Early on, copper, cobalt, nickel, and manganese-substituted horseradish peroxidase (HRP) were prepared by a multistep process that subjected protein to strong acid and organic solvents (41, 42). Variations of this method have been used repeatedly (24, 43, 47–49). However, this method is applicable only to a narrow range of hemoproteins that tolerate the harsh treatment. With the advent of overexpression methods, significant improvement of metalloporphyrin-substituted protein yield was achieved by direct expression of the apoprotein and reconstitution with the desired metalloporphyrin in lysate prior to purification (50). Although this approach has many virtues, direct expression of apoprotein is ineffective for many hemoproteins, again limiting the utility of this method.As an alternative to the above in vitro approaches, researchers have pursued systems for direct in vivo expression of metal substituted hemoproteins. Two specialty strains of Escherichia coli (E. coli) were engineered to incorporate metalloporphyrin analogs from the growth medium into hemoproteins during protein expression. The engineered RP523 strain cannot biosynthesize heme and bears an uncharacterized heme permeability phenotype. Together, these two features enable this strain to assimilate and incorporate various metalloporphyrins into overexpressed hemoproteins with no background heme incorporation (44, 51–53). However, heme auxotrophy makes RP523 cells exceedingly sensitive to O₂, and, in many situations, RP523 cultures must be grown anaerobically. An alternative BL21(DE3)-based engineered strain harbors a plasmid bearing the heme transporter ChuA, which facilitates import of exogenous heme analogs (45). Production of metalloporphyrin-substituted protein with this ChuA-containing strain relies on growth in iron-limited minimal media, thereby diminishing heme biosynthesis. This method was used successfully to express metal-substituted versions of the heme domain of cytochrome P450 BM3 (45) and several myoglobin variants (11, 12). Because these cells biosynthesize a small quantity of their own heme, they are far more robust than the RP523 cells. Unfortunately, this advantage comes at the cost of increased heme contamination in the product protein (2 to 5%) (45).A set of intriguing papers reported the production of cobalt-substituted human cystathionine β-synthase (CoCBS) that relies on the de novo biosynthesis of CoPPIX from CoCl₂ and δ-aminolevulinic acid (δALA), a biosynthetic precursor to heme (46, 54). This method yielded significant amounts of CoCBS—albeit with modest heme contamination (7.4%)—sufficient for spectroscopic and functional characterization of the CoPPIX-substituted protein (8, 46). As cobalt is known to be toxic to E. coli, the researchers passaged the CBS expression strain through cobalt-containing minimal media for 12 d, enabling the cells to adapt to high concentrations of cobalt prior to protein expression. It is plausible that this serial passaging alters the E. coli cells, enabling the biosynthesis of CoPPIX and in vivo production of metal-substituted protein. The adaptation process is slow (>10 d), and it is unknown how genomic instability under these mutagenic conditions affects the reproducibility of this passaging approach.The possibility of facile CoPPIX production is particularly attractive for future biocatalysis efforts. As described above, synthetic cobalt porphyrins have been shown to perform a range of radical-mediated reactions. The ability to produce a CoPPIX center in vivo may enable engineering these unusual reactivities via directed evolution in addition to spectroscopic applications. We therefore set out to explore the unusual phenotype of CoPPIX production by E. coli and to ascertain whether it was possible to efficiently biosynthesize cobalt-containing hemoproteins in vivo from a single “generalist” cell line. Our goal was to achieve an efficient and facile method of cobalt-substituted hemoprotein production with minimal contamination of the native cofactor. Herein, we report the surprising discovery that native E. coli BL21(DE3) can biosynthesize a new-to-nature CoPPIX cofactor (Fig. 1). We use this insight to produce cobalt-substituted hemoproteins in vivo without requirement for complex expression methods or specialized strains.Open in a separate windowFig. 1.Chemical structures of iron protoporphyrin IX (FePPIX or heme b), cobalt protoporphyrin IX (CoPPIX), and free base protoporphyrin IX (H₂PPIX).