A general strategy for generating intact,full-length IgG antibodies that penetrate into the cytosol of living cells

Full-length IgG antibodies cannot cross cell membranes of living cells; this limits their use for direct targeting of cytosolic proteins. Here, we describe a general strategy for the generation of intact, full-length IgG antibodies, herein called cytotransmabs, which internalize into living cells and localize in the cytosol. We first generated a humanized light chain variable domain (VL) that could penetrate into the cytosol of living cells and was engineered for association with various subtypes of human heavy chain variable domains (VHs). When light chains with humanized VL were co-expressed with 3 heavy chains (HCs), including 2 HCs of the clinically approved adalimumab (Humira®) and bevacizumab (Avastin®), all 3 purified IgG antibodies were internalized into the cytoplasm of living cells. Cytotransmabs primarily internalized into living cells by the clathrin-mediated endocytic pathway through interactions with heparin sulfate proteoglycan that was expressed on the cell surface. The cytotransmabs escaped into the cytosol from early endosomes without being further transported into other cellular compartments, like the lysosomes, endoplasmic reticulum, Golgi apparatus, and nucleus. Furthermore, we generated a cytotransmab that co-localized with the targeted cytosolic protein when it was incubated with living cells, demonstrating that the cytotransmab can directly target cytosolic proteins. Internalized cytotransmabs did not show any noticeable cytotoxicity and remained in the cytosol for more than 6 h before being degraded by proteosomes. These results suggest that cytotransmabs, which efficiently enter living cells and reach the cytosolic space, will find widespread uses as research, diagnostic, and therapeutic agents.     

Association of Severe Thrombocytopenia and Poor Prognosis in Pregnancies with Aplastic Anemia

Purpose

We sought to estimate the risks of adverse obstetric outcomes and disease outcomes associated with severe thrombocytopenia in pregnant women with aplastic anemia (AA).

Methods

In a retrospective study, we compared demographics, clinical characteristics, laboratory results, and outcomes between severe thrombocytopenia (ST) and non-severe thrombocytopenia (non-ST) groups comprising pregnant women with AA.

Results

Of 61 AA patients, 43 (70%) were diagnosed as AA before pregnancy and 18 (30%) were AA during pregnancy. The ST group exhibited lower gestational age at nadir of platelet count (26.0 versus 37.0 weeks, p<0.001) and at delivery (37.3 versus 39.1 weeks, p = 0.008), and a higher rate of bleeding gums (33.8 versus 7.7%, p = 0.015) than the non-ST group. In addition, the ST group exhibited more transfusions during pregnancy (72.7 versus 15.4%, p<0.001) and postpartum period (45.0 versus 2.7%, p<0.001), and more bone marrow transplant after delivery (25.0 versus 0.0%, p<0.001) than the non-ST group. The ST group had a higher odds ratio of composite disease complications (OR, 9.63; 95% CI, 2.82–32.9; p<0.001) and composite obstetric complications (OR, 6.78; 95% CI, 2.11–21.8; p = 0.001) than the non-ST group.

Conclusions

Severe thrombocytopenia is more associated with obstetric and disease complications than is non-severe thrombocytopenia in pregnant women with AA.     

Integrated mRNA-MicroRNA Profiling of Human NK Cell Differentiation Identifies MiR-583 as a Negative Regulator of IL2Rγ Expression

Natural killer (NK) cells are innate immune effector cells that protect against cancer and some viral infections. Until recently, most studies have investigated the molecular signatures of human or mouse NK cells to identify genes that are specifically expressed during NK cell development. However, the mechanism regulating NK cell development remains unclear. Here, we report a regulatory network of potential interactions during in vitro differentiation of human NK cells, identified using genome-wide mRNA and miRNA databases through hierarchical clustering analysis, gene ontology analysis and a miRNA target prediction program. The microRNA (miR)-583, which demonstrated the largest ratio change in mature NK cells, was highly correlated with IL2 receptor gamma (IL2Rγ) expression. The overexpression of miR-583 had an inhibitory effect on NK cell differentiation. In a reporter assay, the suppressive effect of miR-583 was ablated by mutating the putative miR-583 binding site of the IL2Rγ 3′ UTR. Therefore, we show that miR-583 acts as a negative regulator of NK cell differentiation by silencing IL2Rγ. Additionally, we provide a comprehensive database of genome-wide mRNA and miRNA expression during human NK cell differentiation, offering a better understanding of basic human NK cell biology for the application of human NK cells in immunotherapy.     

Age-Related Association of Refractive Error with Intraocular Pressure in the Korea National Health and Nutrition Examination Survey

Background

To investigate the distribution of intraocular pressure (IOP) and refractive errors according to age group in a representative sample of non-glaucomatous Korean adults.

Methods

A total of 7,277 adults (≥19 years) who participated in the Korea National Health and Nutrition Examination Survey (KNHANES) from 2008 to 2011 underwent ophthalmic examination were divided into three groups according to age: the young- (19–39 years), middle- (40–59 years), and old- (≥60 years) age groups. Simple and multiple regression analyses between IOP and various parameters (including the refractive error) were conducted.

Results

The mean IOP of the total population was 14.0±0.1 mmHg [young: 13.9±0.1 mmHg; middle: 14.1±0.1 mmHg; old: 13.8±0.2 mmHg (P for trend = 0.085)]. Myopia and high myopia were more prevalent in the young- (70.8% and 16.1%, respectively), compared to the middle- (44.6% and 10.9%) and old- (8.9% and 2.2%) age groups. Univariate analysis in the total population showed that higher IOP was associated with myopic refractive error, the female gender, higher body mass index (BMI), diabetes, hypertension, and hypercholesterolemia (all P<0.05). In the young- and middle-age groups, higher IOP was associated with myopic refractive error, the female gender, higher BMI, hypercholesterolemia and diabetes (all P<0.05). In the old-age group, the association between IOP and refractive error was not significant (P = 0.828). In multiple linear regression analysis, similar significant relationships between the refractive error and IOP were found in the young- and middle-age groups (beta = −0.08 and −0.12; P = 0.002 and <0.001 for young- and middle-age group, respectively), but not in the old-age group (beta = 0.03; P = 0.728), after adjusting for age, gender, BMI, region of habitation, diabetes, hypertension, and hypercholesterolemia.

Conclusions

Myopic refractive error was an independent predictor of higher IOP in non- glaucomatous eyes, and the association between refractive error and IOP differed according to age.     

Development and function of CD94-deficient natural killer cells

The CD94 transmembrane-anchored glycoprotein forms disulfide-bonded heterodimers with the NKG2A subunit to form an inhibitory receptor or with the NKG2C or NKG2E subunits to assemble a receptor complex with activating DAP12 signaling proteins. CD94 receptors expressed on human and mouse NK cells and T cells have been proposed to be important in NK cell tolerance to self, play an important role in NK cell development, and contribute to NK cell-mediated immunity to certain infections including human cytomegalovirus. We generated a gene-targeted CD94-deficient mouse to understand the role of CD94 receptors in NK cell biology. CD94-deficient NK cells develop normally and efficiently kill NK cell-susceptible targets. Lack of these CD94 receptors does not alter control of mouse cytomegalovirus, lymphocytic choriomeningitis virus, vaccinia virus, or Listeria monocytogenes. Thus, the expression of CD94 and its associated NKG2A, NKG2C, and NKG2E subunits is dispensable for NK cell development, education, and many NK cell functions.     

Purification and biochemical characterization of a 17 kDa fibrinolytic enzyme from <Emphasis Type="Italic">Schizophyllum commune</Emphasis>

A fibrinolytic enzyme of the mushroom, Schizophyllum commune was purified with chromatographic methods, including a DEAE-Sephadex A-50 ion-exchange column and gel filtrations with Sephadex G-75 and Sephadex G-50 columns. The analysis of fibrin-zymography and SDS-PAGE showed that the enzyme was a monomeric subunit that was estimated to be approximately 17 kDa in size. The fibrinolytic activity of the enzyme in plasminogen-rich and plasminogen-free fibrin plates was 1.25 and 0.44 U/ml, respectively. The N-terminal amino acid sequence of the purified enzyme was identified as HYNIXNSWSSFID, which was highly distinguished from known fibrinolytic enzymes. The relative activity of the purified enzyme with an addition of 5 mM EDTA, Phosphoramidon, and Bestatin was about 76, 64, and 52%, respectively, indicating that it is a metalloprotease. The optimum temperature for the purified enzyme was approximately 45°C, and over 87% of the enzymatic activity was maintained as a stable state in a pH range from 4.0 to 6.0. Therefore, our results suggest that the potential thrombolytic agent from S. commune is a unique type of fibrinolytic enzyme.     

Construction of DNA-shuffled and incrementally truncated libraries by a mutagenic and unidirectional reassembly method: changing from a substrate specificity of phospholipase to that of lipase

A method of mutagenic and unidirectional reassembly (MURA) that can generate libraries of DNA-shuffled and randomly truncated proteins was developed. The method involved fragmenting the template gene(s) randomly by DNase I and reassembling the small fragments with a unidirectional primer by PCR. The MURA products were treated with T4 DNA polymerase and subsequently with a restriction enzyme whose site was located on the region of the MURA primer. The N-terminal-truncated and DNA-shuffled library of a Serratia sp. phospholipase A(1) prepared by this method had an essentially random variation of truncated size and also showed point mutations associated with DNA shuffling. After high-throughput screening on triglyceride-emulsified plates, several mutants exhibiting absolute lipase activity (NPL variants) were obtained. The sequence analysis and the lipase activity assay on the NPL variants revealed that N-terminal truncations at a region beginning with amino acids 61 to 71, together with amino acid substitutions, resulted in the change of substrate specificity from a phospholipase to a lipase. We therefore suggest that the MURA method, which combines incremental truncation with DNA shuffling, can contribute to expanding the searchable sequence space in directed evolution experiments.     

An optimal weighted aggregated association test for identification of rare variants involved in common diseases

The advent of next generation sequencing technologies allows one to discover nearly all rare variants in a genomic region of interest. This technological development increases the need for an effective statistical method for testing the aggregated effect of rare variants in a gene on disease susceptibility. The idea behind this approach is that if a certain gene is involved in a disease, many rare variants within the gene will disrupt the function of the gene and are associated with the disease. In this article, we present the rare variant weighted aggregate statistic (RWAS), a method that groups rare variants and computes a weighted sum of differences between case and control mutation counts. We show that our method outperforms the groupwise association test of Madsen and Browning in the disease-risk model that assumes that each variant makes an equally small contribution to disease risk. In addition, we can incorporate prior information into our method of which variants are likely causal. By using simulated data and real mutation screening data of the susceptibility gene for ataxia telangiectasia, we demonstrate that prior information has a substantial influence on the statistical power of association studies. Our method is publicly available at http://genetics.cs.ucla.edu/rarevariants.     

Protection by carnosine-related dipeptides against hydrogen peroxide-mediated ceruloplasmin modification

Carnosine, homocarnosine, and anserine are present in high concentrations in the muscle and brain of many animals and humans. Previous studies showed that these compounds have an antioxidant function. We investigated the protective effects of carnosine and related compounds on the modification of human ceruloplasmin that is induced by H2O2. Carnosine, homocarnosine, and anserine significantly inhibited the fragmentation and inactivation of ceruloplasmin that is induced by H2O2. All three compounds also inhibited the release of copper ion from protein, and the formation of hydroxyl radicals in the ceruloplasmin/H2O2 system. These compounds inhibited the fragmentation of human serum albumin that is induced by the copper-catalyzed oxidation system, as well as by the iron-catalyzed oxidation system. These results suggest that carnosine, homocarnosine, and anserine might protect ceruloplasmin against H2O2-mediated oxidative damage through a combination of copper chelation and free radical scavenging.