Nonidentifiability in the presence of factorization for truncated data.

A time to event, [Formula: see text], is left-truncated by [Formula: see text] if [Formula: see text] can be observed only if [Formula: see text]. This often results in oversampling of large values of [Formula: see text], and necessitates adjustment of estimation procedures to avoid bias. Simple risk-set adjustments can be made to standard risk-set-based estimators to accommodate left truncation when [Formula: see text] and [Formula: see text] are quasi-independent. We derive a weaker factorization condition for the conditional distribution of [Formula: see text] given [Formula: see text] in the observable region that permits risk-set adjustment for estimation of the distribution of [Formula: see text], but not of the distribution of [Formula: see text]. Quasi-independence results when the analogous factorization condition for [Formula: see text] given [Formula: see text] holds also, in which case the distributions of [Formula: see text] and [Formula: see text] are easily estimated. While we can test for factorization, if the test does not reject, we cannot identify which factorization condition holds, or whether quasi-independence holds. Hence we require an unverifiable assumption in order to estimate the distribution of [Formula: see text] or [Formula: see text] based on truncated data. This contrasts with the common understanding that truncation is different from censoring in requiring no unverifiable assumptions for estimation. We illustrate these concepts through a simulation of left-truncated and right-censored data.

Overexpression of NBS1 induces epithelial-mesenchymal transition and co-expression of NBS1 and Snail predicts metastasis of head and neck cancer.

Major causes of head and neck squamous cell carcinoma (HNSCC)-related deaths are cervical node and distant metastasis. We previously demonstrated that overexpression of the DNA double-strand break repair protein Nijmegen breakage syndrome 1 (NBS1) is a prognostic marker of advanced HNSCCs. Epithelial-mesenchymal transition (EMT) was demonstrated to be the major mechanism responsible for mediating invasiveness and metastasis of late-stage cancers. We therefore investigated the role of NBS1 overexpression in mediating EMT and metastasis. NBS1 overexpression was associated with metastasis of HNSCC patients using tissue microarray-immunohistochemistry approach. Induction of EMT was observed in an NBS1-overexpressing HNSCC cell line (FADUNBS), whereas short-interference RNA (siRNA)-mediated repression of endogenous NBS1 reversed the shift of EMT markers. Increased migration/invasiveness of FADUNBS was shown by in vitro and in vivo assays. NBS1 overexpression upregulated the expression of an EMT regulator Snail and its downstream target matrix metalloproteinase-2. EMT phenotypes and increased migration/invasiveness of FADUNBS cells were reversed by siRNA-mediated repression of Snail expression or a phosphatidylinositol 3-kinase-specific inhibitor. In HNSCC samples, co-expression of NBS1/Snail in primary tumors correlated with metastasis and the worst prognosis. These results indicate that NBS1 overexpression induces EMT through the upregulation of Snail expression, and co-expression of NBS1/Snail predicts metastasis in HNSCCs.

Isolation and characterization of mesenchymal stromal cells from human anterior cruciate ligament.

BACKGROUND: The anterior cruciate ligament (ACL) is one of the most commonly injured ligaments of the knee. Because the torn ACL is always discarded during ACL reconstruction, it may be a potential source for isolating mesenchymal stromal cells (MSC). METHODS: To characterize MSC from human ACL, cells were enzymatically released from the ACL of adult human donors and seeded in plastic dishes with serial passages at confluence. At different passages, ACL-derived cells were subjected to in vitro assays to investigate their multilineage potential. Upon treatment, the phenotypes of the cell cultures were analyzed by histo- and immunohistochemistry and semi-quantitative reverse transcription-polymerase chain reaction for the expression of lineage-specific genes. RESULTS: Six independent cell lines from individual donors showed diversity in multilineage potential. Interestingly, five of the six lines displayed adipogenic potential, four had osteogenic and adipogenic potential, and only one cell line was tripotent. Both bone marrow (BM)- and ACL-derived MSC expressed marker genes for ligament fibroblasts, whereas the mRNA levels of collagen I and III were more abundant in ACL-derived MSC. DISCUSSION: Our study demonstrates that human MSC can be isolated from ACL with diversity in the potential to form bone, fat and cartilage and an increase as compared to BM MSC, in the potential to form ligament fibroblasts.

Characterization of a novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39.

AIMS: The aims of this study were to identify and characterize the novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39. METHODS AND RESULTS: Strain B39 was closely related to Paenibacillus cookii in 16S rRNA gene sequence. Nonetheless, this isolate can be identified as a novel Paenibacillus sp. with respect to its physiological characteristics, biochemical reactions, and profiles of fatty acid compositions. A cellulase with both CMCase and avicelase activities was secreted from strain B39 and purified by ion-exchange chromatography. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the molecular weight of B39 cellulase was determined as 148 kDa, which was much higher than other cellulases currently reported from Paenibacillus species. The enzyme showed a maximum CMCase activity at 60 degrees C and pH 6.5. Addition of 1 mmol l(-1) of Ca(2+) markedly enhanced both CMCase and avicelase activities of the enzyme. CONCLUSIONS: We have identified and characterized a novel thermophilic Paenibacillus sp. strain B39 which produced a high-molecular weight cellulase with both CMCase and avicelase activities. SIGNIFICANCE AND IMPACT OF THE STUDY: Based on the ability to hydrolyse CMC and avicel, the cellulase produced by Paenibacillus sp. strain B39 would have potential applications in cellulose biodegradation.

Transcripts of enriched germ cells responding to heat shock as potential markers for porcine semen quality.

A cDNA microarray-assisted experiment was conducted to survey genes that respond early to heat shock in enriched immature porcine germ cells; the 5'-UTR flanking the highest upregulated gene, heat shock 105/110 kDa protein 1 (Hsph1 or Hsp105), in response to heat shock was also investigated. We established a porcine testis cDNA microarray with 9944 transcripts from two libraries constructed from the testes of mature boars, with or without heat shock. After a mild heat shock treatment (39 degrees C for 1h and recovered at 34 degrees C for 2h), 380 transcripts demonstrated significant gene expression in enriched immature germ cells; 326 were upregulated and 54 were downregulated. Ten transcripts of interest exhibiting significance analysis of microarrays (SAM) scores higher than the median were subjected to quantitative real-time PCR; three (Hsp105, Hspa4l and Thap4) were upregulated >1.5-fold. The sequence of the 5'-UTR of Hsp105, the highest upregulated transcript, was cloned and analyzed. A single nucleotide polymorphism (SNP) was found at position -762 (C or T) upstream of the translational start site (ATG codon). Only two genotypes (CC or TC) were found in the mature boars that were studied (n=31). A heterozygous genotype (TC) at this SNP site revealed an elevated percentage of morphologically normal sperm during hot and cold seasons; this SNP may be a useful marker for semen quality in boars. Furthermore, the cell-model established from enriched primitive germ cells has potential for the study of reproduction in mature animals.

Fibronectin suppresses lipopolysaccharide-induced liver damage and promotes the cytoprotection abilities of hepatocyte-like cells derived from human bone marrow mesenchymal stem cells.

We isolated mesenchymal stem cells (MSCs) from adult human bone marrow. By using reverse-transcription polymerase chain reactions, we confirmed that MSCs possessed the potential to differentiate into hepatocyte-like cells (MSC-HLCs) with the expression of hepatocyte-specific marker genes. We further observed that fibronectin (FN) treatment significantly inhibited lipopolysaccharide (LPS)-induced apoptotic activities in FN-treated MSC-HLCs, as detected by caspase 3 enzyme-linked immunosorbent (ELISA) and terminal dUTP nick-end labeling (TUNEL) assays (P<.05). The FN-treated MSC-HLCs were transplanted into SCID mice with or without LPS injection. This study demonstrated that FN treatment improved liver function repair and survival rates among LPS-treated SCID mice.

Using gelatin scaffold with coated basic fibroblast growth factor as a transfer system for transplantation of human neural stem cells.

Gelatin scaffolds for ex vivo cell cultures are a promising development. These scaffolds can be used as three-dimensional skeletons for cell attachment and culture before transplantation. In this study, we isolated and cultivated neural stem cells from human brain tissues in serum-free medium (DMEM+F12 nutrient). Better neuron growth was observed using the tetrazolium assay (MTT) in the group when basic fibroblast growth factor (bFGF) was coated on the gelatin polymer scaffold. Further development of this nontoxic system may help the future development of transplantation of human neural stem cells.

Evaluation of wound healing effect on skin-defect nude mice by using human dermis-derived mesenchymal stem cells.

We report that human dermis-derived mesenchymal stem cells (hDMSCs) possess differentiation potential of epidermis facilitating wound healing in skin-defect nude mice in combination with the treatment using gelatin/thermosensitive poly N-isopropylacrylamide (pNIPAAm)/polypropylene (PP). The results showed that the rate of cell growth and wound recovery in the hDMSC and gelatin/pNIPAAm/PP-treated group was significantly greater than those in the gelatin/pNIPAAm/PP-treated only group (P < .01). The reepithelialization marker of human pan-cytokeratin was also significantly increased on days 14 and day 21 in the wound site of hDMSCs and gelatin/pNIPAAm/PP-treated group. Furthermore, the stem cell marker of human CD13 gradually decreased during the period of wound healing. In sum, this novel method provided a transferring system for stem cell therapy, maintaining its temperature-sensitive property of easy peeling by lower temperature treatment.

Use of a recombinant protein containing major epitopes of hnRNP G to detect anti-hnRNP G antibodies in dogs with systemic lupus erythematosus.

The objective of this study was to express major epitopes of heterogeneous nuclear ribonucleoprotein G (hnRNP G) for detecting anti-hnRNP G antibodies in dogs with systemic lupus erythematosus (SLE). HnRNP G cDNA clone was isolated from HEp-2 cells, and a DNA fragment encoding immunodominant region (residues 189-272) of hnRNP G (hnRNP Gi) was subcloned into pET32 vector to construct a prokaryotic expression plasmid named pEThnRNPGi. After induction, Escherichia coli carrying pEThnRNPGi expressed a recombinant protein of 28 kDa, comprising recombinant hnRNP Gi and fusion tag. Purified recombinant hnRNP Gi protein was further analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and its identity was confirmed. Western blot analysis showed that recombinant hnRNP Gi was specifically recognized by anti-hnRNP G positive sera of SLE dogs, and not by negative control sera. In conclusion, recombinant hnRNP Gi protein expressed in this study may serve as a useful reagent to assist in the immunological diagnosis of canine SLE.

T-cell factor-4 and MHC upregulation in pigs receiving a live attenuated classical swine fever virus (CSFV) vaccine strain with interferon-gamma adjuvant.

The effect of co-administration of interferon (IFN)-γ in pigs undergoing vaccination with an attenuated strain (LPC) of classical swine fever virus (CSFV) was investigated. Unvaccinated pigs demonstrated pyrexia and died 7-9 days after challenge with virulent CSFV. Pigs receiving the attenuated vaccine remained healthy after virus challenge, except for mild, transient pyrexia, whereas pigs receiving IFN-γ simultaneously with the vaccine demonstrated normal body temperatures after virus challenge. Examination by nested RT-PCR revealed greater viral load in the spleens of the pigs vaccinated with the attenuated CSFV, compared with those that had additionally received IFN-γ. Expression of major histocompatibility complex (MHC) class I and MHC class II molecules was upregulated in the spleens of the IFN-γ treated vaccinated pigs, demonstrated by immunohistochemistry. Based on Western blot analysis, anti-CSFV IgG2 antibodies were elevated in vaccinated pigs by co-administration of IFN-γ (IFN-γ(Hi): P < 0.01; IFN-γ(Lo): P <0.05). By employing the suppression subtractive hybridization technique, RT-PCR, in situ hybridization, and immunohistochemistry, T-cell factor-4 (Tcf-4) mRNA and protein expression were found to be upregulated in the spleens of vaccinated pigs that had received IFN-γ. This study suggests involvement of Tcf-4 in IFN-γ-mediated immune regulation following CSFV vaccination.

Biochemical comparison of lens crystallins from three reptilian species.

Lens crystallins were isolated from the homogenates of reptilian eye lenses derived from three different species by gel-permeation chromatography and characterized by gel electrophoresis, amino-acid analysis, N-terminal sequence analysis and circular dichroism. Four fractions corresponding to alpha-, delta/epsilon/beta-, beta- and gamma-crystallins were obtained for the crystallins from caiman lenses, whereas delta- and gamma-crystallin fraction were present in lesser amounts or missing in the turtle and snake lenses, respectively. The native molecular masses for these purified fractions and their polypeptide compositions were determined by gel filtration and SDS-gel electrophoresis, respectively, revealing the typical subunit compositions for each classified crystallin. The spectra of circular dichroism indicate a predominant beta-sheet structure in alpha-, beta- and gamma-crystallins, and a major contribution of alpha-helical structure in delta/epsilon-crystallin fraction, which bears a resemblance to the secondary structure of delta-crystallin from the chicken lenses. Comparison of the amino-acid contents of each orthologous class of reptilian crystallins with those of evolutionary distant species still exhibited similarity in their amino-acid compositions. N-terminal sequence analysis of the crystallin fractions revealed that all fractions except that of gamma-crystallin are N-terminally blocked. Extensive sequence similarity between the reptilian gamma-crystallin polypeptides and those from other vertebrate species were found, which establish the close relatedness of gamma-crystallins amongst the major classes of vertebrates.

Biochemical characterization of crystallins from pigeon lenses: structural and sequence analysis of pigeon delta-crystallin.

Crystallins from pigeon eye lenses were isolated and purified by gel-permeation chromatography and characterized by gel electrophoresis, amino-acid composition and sequence analysis. Alpha- and beta-crystallins could be obtained in relatively pure forms by single-step size-exclusion chromatography whereas an extra step of ion-exchange chromatography was needed for the separation of delta-crystallin from the beta-crystallin fraction. In contrast to most characterized vertebrate species, a large amount of glycogen is eluted as a high molecular form in the first peak of the gel filtration column. Pigeon delta-crystallin, similar to duck and reptilian delta-crystallins, exists as a tetrameric structure of about 200 kDa in the native form and is composed of one major subunit of 50 kDa with heterogeneous isoelectric points spreading in a range of 4.7 to 6.8. In contrast to those obtained from duck, goose and caiman, delta-crystallin isolated from the pigeon lens possessed very little argininosuccinate lyase activity. However, pigeon delta-crystallin can still cross-react with the antibody against enzymically active duck delta-crystallin as revealed by the sensitive immunoblotting technique. It was also shown that the delta-crystallin content of the total pigeon soluble proteins decreased with the age of the animal. Structural analysis of purified delta-crystallin fraction was made with respect to its amino-acid composition and protein primary sequence. N-terminal sequence analysis indicated the presence of blocked amino-termini in all crystallin fractions of pigeon lenses. Therefore, a sequence analysis of PCR (polymerase chain reaction) amplified delta-crystallin cDNA was employed to deduce the protein sequence of this crystallin. Structural comparison of delta-crystallin sequences from pigeon, chicken and duck lenses casts some doubts on the recent claim that His-89-->Gln mutation in the chicken delta-crystallin may account for the loss of argininosuccinate lyase activity in this avian species, as compared to high enzymic activity in the duck crystallin (Barbosa et al. (1991) J. Biol. Chem. 266, 5286-5290).

Carp gamma-crystallins with high methionine content: cloning and sequencing of the complementary DNA.

The nucleotide sequences of gamma-crystallin cDNAs cloned from the common carp (Cyprinus carpio) have been determined. The amino-acid sequences derived consist of two polypeptides with 177 and 172 amino-acid residues for gamma-m1 and gamma-m2, respectively. They exhibit unusually high methionine contents: 12.4% for gamma-m1 and 14% for gamma-m2. Comparison of both fish gamma-crystallins with bovine gamma-II crystallin reveals that they are similar in structure. The striking features of both fish gamma-crystallins are as follows. (1) Both of them retain the 'conserved' residues, i.e., Tyr-6, Glu-7, Gly-13, Ser-34 and their equivalents in other motifs. (2) they possess the second aromatic residue at position 11. Both of these structural features are considered to be the major factors in stabilizing the folded hairpin structure of the protein. (3) The variable residues in the core region of C-terminal domain are almost all sulfur-containing amino acids, i.e., methionine or cysteine. (4) 30% of the surface hydrophobic groups are composed of methionine. The last two unusual features have been found so far only in these two fish gamma-crystallins. The high methionine content may make an important contribution to the protein stability of fish gamma-crystallins.

A re-evaluation of the molecular size of duck epsilon-crystallin and its comparison with avian lactate dehydrogenases.

A biochemical comparison of epsilon-crystallin isolated from the duck lens and lactate dehydrogenases of chicken heart has been made in order to establish the structural and functional identities of these two proteins. The native molecular weight of epsilon-crystallin was re-examined by combining sedimentation and gel-filtration data. It was found that epsilon-crystallin is 150 kDa in contrast to the 120 kDa reported previously for this crystallin. Subunit cross-linking experiments corroborated that lactate dehydrogenase and epsilon-crystallin both exist as tetramers of four identical subunits in their native quaternary structures. Amino acid compositions plus N-terminal analyses revealed no differences between the two proteins. Duck epsilon-crystallin exhibited high enzymatic activity of lactate dehydrogenases even after a long period of storage, and showed characteristic thermostability at 50 degrees C for several hours. Comparison of the enzyme activity of duck lens homogenate with those of heart, liver and muscle tissues revealed that duck lens is a much richer source than other tissues for the isolation and characterization of this important enzyme which appears also as a structural protein in the lens.

Characterization of lens crystallins and their mRNA from the carp lenses.

Crystallins from carp eye lenses have been isolated and characterized by gel permeation chromatography, SDS-gel electrophoresis, immunodiffusion and amino acid analysis. gamma-Crystallin is the most abundant class of crystallins and constitutes over 55% of the total lens cytoplasmic proteins. It is immunologically distinct from the alpha- and beta-crystallins isolated from the same lens and its antiserum shows a very weak cross-reaction to total pig lens antigens. Comparison of the amino acid compositions of carp gamma-crystallin with those of bovine gamma-II, haddock gamma- and squid crystallins indicates that gamma-crystallin from the carp is very closely related to that of the haddock, and probably also related to the invertebrate squid crystallin. In vitro translation of total mRNAs isolated from carp lenses confirms the predominant existence of gamma-crystallin. The genomic characterization of carp crystallin genes should provide some insight into the mechanism of crystallin evolution in general.

Using human plasma supplemented medium to cultivate human bone marrow-derived mesenchymal stem cell and evaluation of its multiple-lineage potential.

The objective of this study was to evaluate the proliferation and the multiple-lineage differentiation capacity when bone marrow mesenchymal stem cells (BMSCs) were cultured short-term in autologous serum/plasma instead of fetal calf serum (FCS). The BMSCs from 12 donors were cultivated individually in 10% autogenic plasma or serum, with or without bFGF and EGF growth factors. Cell proliferation was examined by a Tetrazolium assay (MTT) after passages 1, 3, and 5. A medium supplemented with 10% human plasma or serum was sufficient to propagate BMSCs. However, no significant proliferation was shown when bFGF and EGF (20 ng/mL each) were added into the medium with autologous serum/plasma. We examined, inductions of adipogenesis, osteogenesis, and chondrocytogenesis, as capacities of multiple-lineage differentiation of cultivated BMSCs (passages 8). Differentiation was investigated by both RT-PCR and immunohistochemistry staining (IHC). Qualitative evidence demonstrated the differentiation capacity was preserved in cultivated BMSCs with autologous serum/plasma.

Progranulin promotes Temozolomide resistance of glioblastoma by orchestrating DNA repair and tumor stemness.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults with a dismal prognosis. Current therapy of surgical removal combined with Temozolomide (TMZ) and radiation therapy only slightly prolongs the survival of GBM patients. Thus, it is essential to elucidate mechanism underlying its highly malignant properties in order to develop efficacious therapeutic regimens. In this study, we showed that progranulin (PGRN) was overexpressed in most GBM cell lines and the majority of human tumor samples. PGRN overexpression conferred GBM cells with tumorigenic properties and TMZ resistance by upregulating DNA repair (PARP, ATM, BRCA1, Rad51, XRCC1 and so on) and cancer stemness (CD133, CD44, ABCG2) genes, in part via an AP-1 transcription factor, specifically cFos/JunB. Curcumin, an AP-1 inhibitor, was also found to regulate PGRN promoter activity and expression including its downstream effectors aforementioned. These data suggested a feedforward loop between PGRN signaling and AP-1. PGRN depletion significantly decreased unlimited self-renewal and multilineage differentiation and the malignant properties of GBMs cells S1R1, and enhanced their vulnerability to TMZ. In addition, S1R1 depleted of PGRN also lost the ability to form tumor in an orthotopic xenograft mouse model. In conclusion, PGRN had a critical role in the pathogenesis and chemoresistance of GBM and functioned at the top of the hierarchy of cellular machinery that modulates both DNA repair pathways and cancer stemness. Our data suggest that a new strategy combining current regimens with compounds targeting PGRN/AP-1 loop like curcumin may significantly improve the therapeutic outcome of GBM.

Phylogenetic comparison of lens crystallins from the vertebrate and invertebrate--convergent or divergent evolution?

A systematic biochemical comparison has been made of the crystallins isolated from the lenses of five different species belonging to the five major classes of vertebrates. Gel-permeation chromatography of the lens homogenates on Fractogel TSK HW-55(S) revealed well-defined elution patterns with a characteristic distribution of different classes of crystallins from each species. SDS gel electrophoresis and statistical comparison of the amino acid contents indicated that all crystallin groups from different classes share some common subunits and similarity in their amino acid compositions. The results coupled with the relatedness shown in the amino acid compositions of fish gamma-crystallin with those of mammalian gamma-crystallin and the squid crystallin from the invertebrate pointed to the possibility of the existence of a common ancestral protein for all crystallins. This is in favor of the divergent rather than convergent evolution of lens crystallins as commonly assumed in the literature.

Characterization of gamma-crystallins from eye lenses of shark: closer structural similarity to mammalian than other piscine gamma-crystallins?

Lens crystallins were isolated and characterized from sharks of the cartilaginous fishes. Four crystallin fractions corresponding to alpha-, beta H-, beta L- and gamma-crystallins, similar to those of mammalian crystallins, were obtained. The native molecular masses and subunit structures of these purified fractions were analyzed by gel permeation chromatography, SDS gel electrophoresis and isoelectric focusing, revealing the typical subunit compositions with various extents of heterogeneity in each orthologous crystallin class. Amino acid and N-terminal sequence analyses corroborate the identification and classification of crystallin classes based on electrophoresis. Unexpectedly, it was found that the amino acid composition and N-terminal sequence of shark gamma-crystallin are more closely related to those of bovine than carp gamma-crystallin. This finding may have some bearing on the divergence and specification of gamma-crystallins between the phylogenetic lines of mammals and fishes.

A novel crystallin from octopus lens.

Lens crystallins were isolated from cephalopods, octopus and squid. Two protein fractions were obtained from the octopus in contrast to only one crystallin from the squid. The native molecular mass for these purified fractions and their polypeptide compositions were determined by gel filtration, sedimentation analysis, and SDS-gel electrophoresis. Octopod and decapod lenses share one common major squid-type crystallin of 29 kDa, with one additional novel crystallin present only in the octopus lens. This newly-characterized crystallin (termed omega-crystallin) exists as a tetrameric protein of 230 kDa, consisting of 4 identical subunits of approx. 59 kDa. It is distinct from the previously known crystallins both in amino acid composition and subunit structure. N-terminal sequence analysis indicated that the omega-crystallin is N-terminally blocked, whereas the major octopus crystallin is identical to the reported squid crystallin with regard to the first 25 residues of protein sequence. Sequence similarity between this major cephalopod crystallin and glutathione S-transferase were found, which suggested some enzymatic role of crystallins inside the cephalopod lens.