Inherited bone marrow failure with macrothrombocytopenia due to germline tubulin beta class I (TUBB) variant.

Germline mutations in tubulin beta class I (TUBB), which encodes one of the ß-tubulin isoforms, were previously associated with neurological and cutaneous abnormalities. Here, we describe the first case of inherited bone marrow (BM) failure, including marked thrombocytopenia, morphological abnormalities, and cortical dysplasia, associated with a de novo p.D249V variant in TUBB. Mutant TUBB had abnormal cellular localisation in transfected cells. Following interferon/ribavirin therapy administered for transfusion-acquired hepatitis C, severe pancytopenia and BM aplasia ensued, which was unresponsive to immunosuppression. Acquired chromosome arm 6p loss of heterozygosity was identified, leading to somatic loss of the mutant TUBB allele.

Confocal microscopy analysis of breech face marks on fired cartridge cases from 10 consecutively manufactured pistol slides.

Recent publications from the National Academy of Sciences have called for additional foundational research in the field of firearm and toolmark analysis. We examined test fires from 10 pistol slides with consecutively manufactured breech faces. A total of nine test fires from each pistol slide, for a total of 90 test fired cartridge cases, were compared using confocal microscopy combined with three-dimensional cross-correlation analysis algorithms. A total of 8010 comparisons were performed (720 matches and 7290 nonmatches). The average score for matches was 0.82 with a standard deviation of 0.06. The average score for nonmatches was 0.20 with a standard deviation of 0.03. Additionally, subclass toolmarks were observed on the breech faces, but the presence of subclass was not detected in the correlation analysis. There was no overlap of scores between matching and nonmatching test fires. This provides objective data that support the AFTE (Association of Firearms and Tool Mark Examiners) theory of identification.

WITHDRAWN: Corrigendum to "Characterization of the nuclear import and export mechanisms of bovine herpesvirus-1 infected cell protein 27" [Virus Research 149 (2010) 95-103].

This article has been withdrawn at the request of the authors. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

WITHDRAWN: Corrigendum to "Characterization of the nuclear and nucleolar localization signals of bovine herpesvirus-1 infected cell protein 27" [Virus Res. 145 (2009) 312-320].

This article has been withdrawn at the request of the authors. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

The herpes simplex virus type 1 infected cell protein 22.

As one of the immediate-early (IE) proteins of herpes simplex virus type 1 (HSV-1), ICP22 is a multifunctional viral regulator that localizes in the nucleus of infected cells. It is required in experimental animal systems and some nonhuman cell lines, but not in Vero or HEp-2 cells. ICP22 is extensively phosphorylated by viral and cellular kinases and nucleotidylylated by casein kinase II. It has been shown to be required for efficient expression of early (E) genes and a subset of late (L) genes. ICP22, in conjunction with the UL13 kinase, mediates the phosphorylation of RNA polymerase II. Both ICP22 and UL13 are required for the activation of cdc2, the degradation of cyclins A and B and the acquisition of a new cdc2 partner, the UL42 DNA polymerase processivity factor. The cdc2-UL42 complex mediates postranscriptional modification of topoisomerase IIα in an ICP22-dependent manner to promote L gene expression. In addition, ICP22 interacts with cdk9 in a Us3 kinase dependent fashion to phosphorylate RNA polymerase II.

The nucleolus and viral infection.

The nucleolus is a subnuclear structure of eukaryocytes. It was thought that nucleolus only participates in the biogenesis and processing of rRNA. However, more and more evidence shows that it has many other functions, such as tRNA precursor processing, stress sensing and it is also involved in gene silencing, senescence and cell cycle regulation. Here, we summarize the recent understandings about the nucleolar functions, the regulation of nucleolar localization of proteins and the role that the nucleolus plays in virus infection, in which some related studies of Herpes simplex virus type 1 (HSV-1) US11, UL24 and bovine herpesvirus-1 infected cell protein 27 (BICP27) carried out in our lab will also be included.

Herpes simplex virus type 1 ICP27 protein: its expression, purification and specific antiserum production.

Herpes simplex virus type 1 (HSV-1) is the causative agent of cold sores and other more serious diseases. HSV-1 infected-cell protein 27 (ICP27) is an immediate-early regulatory phosphoprotein homologous to gene products identified in all classes of herpesviruses so far. To raise the antiserum to ICP27 for further characterization of its biological function, the ICP27 gene was cloned into the pET-28a (+) vector, then ICP27 protein was expressed in E. coli and purified by nickel-nitrilotriacetic acid (Ni(2+)-NTA) affinity resin column, finally the purified protein was used to raise antiserum. Western blot analysis demonstrated that the antiserum recognized the recombinant protein, and the antiserum was able to probe the ICP27 in HSV-1 infected cells with high specificity by immunofluorescence assay (IFA). Therefore, the specific antiserum will provide a valuable tool for further studies investigating ICP27's biological function during HSV-1 infection.

The nucleocytoplasmic transport of viral proteins.

Molecules can enter the nucleus by passive diffusion or active transport mechanisms, depending on their size. Small molecules up to size of 50-60 kDa or less than 10 nm in diameter can diffuse passively through the nuclear pore complex (NPC), while most proteins are transported by energy driven transport mechanisms. Active transport of viral proteins is mediated by nuclear localization signals (NLS), which were first identified in Simian Virus 40 large T antigen and had subsequently been identified in a large number of viral proteins. Usually they contain short stretches of lysine or arginine residues. These signals are recognized by the importin super-family (importin α and ß) proteins that mediate the transport across the nuclear envelope through Ran-GTP. In contrast, only one class of the leucine-rich nuclear export signal (NES) on viral proteins is known at present. Chromosome region maintenance 1 (CRM1) protein mediates nuclear export of hundreds of viral proteins through the recognition of the leucine-rich NES.

Expression, purification of the UL3 protein of herpes simplex virus type 1, and production of UL3 polyclonal antibody.

Herpes simplex virus type 1 (HSV-1) is a common pathogen which causes infections of the mucocutaneous membranes. The UL3 protein belongs to a group of HSV-1 late proteins. To date, the function of the UL3 protein in cell culture, animal models, and natural infection is unknown. To investigate further the function of the UL3 protein, this study was undertaken to express the UL3 protein and raise a polyclonal antibody. The UL3 gene was cloned in the prokaryotic expression vector pET-28a (+) to yield pET-28a (+)-UL3. The His6-tagged UL3 protein was expressed in Escherichia coli (E. coli) BL21 (DE3) cells and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After purification by nickel affinity chromatography and refolding, the recombinant protein was used to raise the anti-UL3 polyclonal antibody. Western blot analysis demonstrated that the UL3 protein was recognized by the polyclonal antibody, and immunofluorescent assay also showed that the antibody was able to recognize the UL3 protein in the cells infected with HSV-1.

Characterization of the nuclear import and export mechanisms of bovine herpesvirus-1 infected cell protein 27.

In previous study, we have identified a nuclear localization signal (NLS) and a nucleolar localization signal (NoLS) in bovine herpesvirus-1 (BHV-1) infected cell protein 27 (BICP27), which targets predominantly to the nucleolus. Furthermore, the C-terminal 300 amino acid residues targets exclusively to the cytoplasm, suggesting that BICP27 might contain a nuclear export signal (NES). Amino acid sequence analysis revealed that there is a cluster of leucine-rich residues resembling a NES. Heterokaryon assays demonstrated that BICP27 is capable of shuttling between the nucleus and the cytoplasm of the BHV-1 infected, BICP27 and BICP27-EYFP transfected cells. Deletion mutant analysis revealed that this property is attributed to the leucine-rich NES 299LEELCAARRLSL310. Moreover, the functional NES could mediate transport of a monomer EYFP and a dimer EYFP to the cytoplasm. The nucleocytoplasmic shuttling of BICP27 and the nuclear export of NES-EYFP and NES-dEYFP could be blocked by leptomycin LMB, an inhibitor of the chromosomal region maintenance 1 (CRM1), which is the receptor for exportin-1-dependent nuclear export. In addition, the nuclear import of BICP27 was inhibited by a dominant negative Ran-GTP, namely Ran-GTP Q69L, indicating that BICP27 localized to the nucleus by means of a classic Ran dependent nuclear import mechanism. In conclusion, these results demonstrate that BICP27 shuttles between the nucleus and the cytoplasm by the functional NES and NLS through a CRM1-dependent nuclear export pathway and a Ran dependent nuclear import pathway.

Expression, purification of herpes simplex virus type 1 UL4 protein, and production and characterization of UL4 polyclonal antibody.

The herpes simplex virus type 1 (HSV-1) UL4 protein is a late protein encoded by the UL4 gene. To date, the function of this protein is poorly understood. To aid further investigation of the function of this protein, the UL4 gene was cloned into the vector pET28a (+) to express His-tagged UL4 protein in Escherichia coli. The recombinant fusion protein was purified from inclusion body by histidine selected nickel affinity chromatography under denaturing conditions. After refolding, the purified recombinant protein was used to produce anti-UL4 polyclonal antibody. Western blot analysis demonstrated that the polyclonal sera could recognize the purified UL4 protein specifically, and in the immunofluorescence assay, the antibody was able to probe the UL4 protein with a punctate staining in HSV-1 infected cells.

Characterization of the nuclear and nucleolar localization signals of bovine herpesvirus-1 infected cell protein 27.

Bovine herpesvirus-1 infected cell protein 27 (BICP27) was detected predominantly in the nucleolus. The open reading frame of BICP27 was fused with the enhanced yellow fluorescent protein (EYFP) gene to investigate its subcellular localization in live cells and BICP27 was able to direct monomeric, dimeric or trimeric EYFP exclusively to the nucleolus. By constructing a series of deletion mutants, the putative nuclear localization signal (NLS) and nucleolar localization signal (NoLS) were mapped to (81)RRAR(84) and (86)RPRRPRRRPRRR(97) respectively. Specific deletion of the putative NLS, NoLS or both abrogated nuclear localization, nucleolar localization or both respectively. Furthermore, NLS was able to direct trimeric EYFP predominantly to the nucleus but excluded from the nucleolus, whereas NoLS targeted trimeric EYFP primarily to the nucleus, and enriched in the nucleolus with faint staining in the cytoplasm. NLS+NoLS directed trimeric EYFP predominantly to the nucleolus with faint staining in the nucleus. Moreover, deletion of NLS+NoLS abolished the transactivating activity of BICP27 on gC promoter, whereas deletion of either NLS or NoLS did not. The study demonstrated that BICP27 is a nucleolar protein, adding BICP27 to the growing list of transactivators which localize to the nucleolus.

NIST bullet signature measurement system for RM (Reference Material) 8240 standard bullets.

A bullet signature measurement system based on a stylus instrument was developed at the National Institute of Standards and Technology (NIST) for the signature measurements of NIST RM (Reference Material) 8240 standard bullets. The standard bullets are developed as a reference standard for bullet signature measurements and are aimed to support the recently established National Integrated Ballistics Information Network (NIBIN) by the Bureau of Alcohol, Tobacco and Firearms (ATF) and the Federal Bureau of Investigation (FBI). The RM bullets are designed as both a virtual and a physical bullet signature standard. The virtual standard is a set of six digitized bullet signatures originally profiled from six master bullets fired at ATF and FBI using six different guns. By using the virtual signature standard to control the tool path on a numerically controlled diamond turning machine at NIST, 40 RM bullets were produced. In this paper, a comparison parameter and an algorithm using auto-and cross-correlation functions are described for qualifying the bullet signature differences between the RM bullets and the virtual bullet signature standard. When two compared signatures are exactly the same (point by point), their cross-correlation function (CCF) value will be equal to 100%. The measurement system setup, measurement program, and initial measurement results are discussed. Initial measurement results for the 40 standard bullets, each measured at six land impressions, show that the CCF values for the 240 signature measurements are higher than 95%, with most of them even higher than 99%. These results demonstrate the high reproducibility for both the manufacturing process and the measurement system for the NIST RM 8240 standard bullets.