Lyoprotectant Constituents Suited for Lyophilization and Reconstitution of Stem-Cell-Derived Extracellular Vesicles.

Stem-cell-derived extracellular vesicles (EVs) are emerging as an alternative approach to stem cell therapy. Successful lyophilization of EVs could enable convenient storage and distribution of EV medicinal products at room temperature for long periods, thus considerably increasing the accessibility of EV therapeutics to patients. In this study, we aimed to identify an appropriate lyoprotectant composition for the lyophilization and reconstitution of stem-cell-derived EVs. MSC-derived EVs were lyophilized using different lyoprotectants, such as dimethyl sulfoxide, mannitol, trehalose, and sucrose, at varying concentrations. Our results revealed that a mixture of trehalose and sucrose at high concentrations could support the formation of amorphous ice by enriching the amorphous phase of the solution, which successfully inhibited the acceleration of buffer component crystallization during lyophilization. Lyophilized and reconstituted EVs were thoroughly evaluated for concentration and size, morphology, and protein and RNA content. The therapeutic effects of the reconstituted EVs were examined using a tube formation assay with human umbilical vein endothelial cells. After rehydration of the lyophilized EVs, most of their generic characteristics were well-maintained, and their therapeutic capacity recovered to levels similar to those of freshly collected EVs. The concentrations and morphologies of the lyophilized EVs were similar to the initial features of the fresh EV group until day 30 at room temperature, although their therapeutic capacity appeared to decrease after 7 days. Our study suggests an appropriate composition of lyoprotectants, particularly for EV lyophilization, which could encourage the applications of stem-cell-derived EV therapeutics in the health industry.

Mesenchymal Stem Cell-Extracellular Vesicle Therapy for Stroke: Scalable Production and Imaging Biomarker Studies.

A major clinical hurdle to translate MSC-derived extracellular vesicles (EVs) is the lack of a method to scale-up the production of EVs with customized therapeutic properties. In this study, we tested whether EV production by a scalable 3D-bioprocessing method is feasible and improves neuroplasticity in animal models of stroke using MRI study. MSCs were cultured in a 3D-spheroid using a micro-patterned well. The EVs were isolated with filter and tangential flow filtration and characterized using electron microscopy, nanoparticle tracking analysis, and small RNA sequencing. Compared to conventional 2D culture, the production-reproduction of EVs (the number/size of particles and EV purity) obtained from 3D platform were more consistent among different lots from the same donor and among different donors. Several microRNAs with molecular functions associated with neurogenesis were upregulated in EVs obtained from 3D platform. EVs induced both neurogenesis and neuritogenesis via microRNAs (especially, miR-27a-3p and miR-132-3p)-mediated actions. EV therapy improved functional recovery on behavioral tests and reduced infarct volume on MRI in stroke models. The dose of MSC-EVs of 1/30 cell dose had similar therapeutic effects. In addition, the EV group had better anatomical and functional connectivity on diffusion tensor imaging and resting-state functional MRI in a mouse stroke model. This study shows that clinical-scale MSC-EV therapeutics are feasible, cost-effective, and improve functional recovery following experimental stroke, with a likely contribution from enhanced neurogenesis and neuroplasticity.

Discrimination of Brassica juncea Varieties Using Visible Near-Infrared (Vis-NIR) Spectroscopy and Chemometrics Methods.

Brown mustard (Brassica juncea (L.) is an important oilseed crop that is mostly used to produce edible oils, industrial oils, modified lipids and biofuels in subtropical nations. Due to its higher level of commercial use, the species has a huge array of varieties/cultivars. The purpose of this study is to evaluate the use of visible near-infrared (Vis-NIR) spectroscopy in combination with multiple chemometric approaches for distinguishing four B. juncea varieties in Korea. The spectra from the leaves of four different growth stages of four B. juncea varieties were measured in the Vis-NIR range of 325-1075 nm with a stepping of 1.5 nm in reflectance mode. For effective discrimination, the spectral data were preprocessed using three distinct approaches, and eight different chemometric analyses were utilized. After the detection of outliers, the samples were split into two groups, one serving as a calibration set and the other as a validation set. When numerous preprocessing and chemometric approaches were applied for discriminating, the combination of standard normal variate and deep learning had the highest classification accuracy in all the growth stages achieved up to 100%. Similarly, few other chemometrics also yielded 100% classification accuracy, namely, support vector machine, generalized linear model, and the random forest. Of all the chemometric preprocessing methods, Savitzky-Golay filter smoothing provided the best and most convincing discrimination. The findings imply that chemometric methods combined with handheld Vis-NIR spectroscopy can be utilized as an efficient tool for differentiating B. juncea varieties in the field in all the growth stages.

Characteristics and Fitness Analysis through Interspecific Hybrid Progenies of Transgenic Brassica napus and B. rapa L. ssp.

Interspecific hybridization between transgenic crops and their wild relatives is a major concern for transgene dispersal in the environment. Under controlled conditions, artificial hand pollination experiments were performed in order to assess the hybridization potential and the fitness of interspecific hybrids between Brassica rapa and genetically modified (GM) Brassica napus. Initially, six subspecies of B. rapa were hybridized with GM B. napus through hand pollination. In the resulting F1 hybrids, the combination of B. rapa ssp. narinosa (♀) × GM B. napus (♂) had the highest crossability index (16.9 ± 2.6). However, the F1 selfing progenies of B. rapa ssp. rapa (♀) × GM B. napus were found to be more effective in producing viable future generations with the highest crossability index (1.6 ± 0.69) compared to other subspecies. Consequently, they were used for the generation of F2 and F3 progenies. The 18 different morphological characteristics among the parental cross-combinations and F1 hybrid progenies were measured and visualized through hierarchical clustering. Different generations were found to be grouped based on their different morphological characteristics. The chromosome numbers among the interspecific hybrids ranged from 2n = 29 to 2n = 40. Furthermore, the SSR markers revealed the presence of genomic portions in the hybrids in comparison with their parental lines. There is a high possibility of transgene flow between GM B. napus and B. rapa. The study concluded that the interspecific hybrids between B. napus and B. rapa can be viable and can actively hybridize up to F3 generations and more. This suggests that the GM B. napus can disperse the transgene into B. rapa, and that it can pass through for several generations by hand pollination in a greenhouse environment.

Interspecific Hybridization of Transgenic Brassica napus and Brassica rapa-An Overview.

In nature, interspecific hybridization occurs frequently and can contribute to the production of new species or the introgression of beneficial adaptive features between species. It has great potential in agricultural systems to boost the process of targeted crop improvement. In the advent of genetically modified (GM) crops, it has a disadvantage that it involves the transgene escaping to unintended plants, which could result in non-specific weedy crops. Several crop species in the Brassica genus have close kinship: canola (Brassica napus) is an ancestral hybrid of B. rapa and B. oleracea and mustard species such as B. juncea, B. carinata, and B. nigra share common genomes. Hence, intraspecific hybridization among the Brassica species is most common, especially between B. napus and B. rapa. In general, interspecific hybrids cause numerous genetic and phenotypic changes in the parental lines. Consequently, their fitness and reproductive ability are also highly varied. In this review, we discuss the interspecific hybridization and reciprocal hybridization studies of B. napus and B. rapa and their potential in the controlled environment. Further, we address the fate of transgenes (herbicide resistance) and their ability to transfer to their progenies or generations. This could help us to understand the environmental influence of interspecific hybrids and how to effectively manage their transgene escape in the future.

A Review of the Unintentional Release of Feral Genetically Modified Rapeseed into the Environment.

Globally, the cultivation area of genetically modified (GM) crops is increasing dramatically. Despite their well-known benefits, they may also pose many risks to agriculture and the environment. Among the various GM crops, GM rapeseed (Brassica napus L.) is widely cultivated, mainly for oil production. At the same time, B. napus possesses a number of characteristics, including the ability to form feral populations and act as small-seeded weeds, and has a high potential for hybridization with other species. In this review, we provide an overview of the commercialization, approval status, and cultivation of GM rapeseed, as well as the status of the feral rapeseed populations. In addition, we highlight the case studies on the unintentional environmental release of GM rapeseed during transportation in several countries. Previous studies suggest that the main reason for the unintentional release is seed spillage during transport/importing of rapeseed in both GM rapeseed-cultivating and -non-cultivating countries. Despite the fact that incidents of unintentional release have been recorded often, there have been no reports of serious detrimental consequences. However, since rapeseed has a high potential for hybridization, the possibilities of gene flow within the genus, especially with B. rapa, are relatively significant, and considering their weedy properties, effective management methods are needed. Hence, we recommend that specific programs be used for the effective monitoring of environmental releases of GM rapeseed as well as management to avoid environmental and agricultural perturbations.

An Overview of Near Infrared Spectroscopy and Its Applications in the Detection of Genetically Modified Organisms.

Near-infrared spectroscopy (NIRS) has become a more popular approach for quantitative and qualitative analysis of feeds, foods and medicine in conjunction with an arsenal of chemometric tools. This was the foundation for the increased importance of NIRS in other fields, like genetics and transgenic monitoring. A considerable number of studies have utilized NIRS for the effective identification and discrimination of plants and foods, especially for the identification of genetically modified crops. Few previous reviews have elaborated on the applications of NIRS in agriculture and food, but there is no comprehensive review that compares the use of NIRS in the detection of genetically modified organisms (GMOs). This is particularly important because, in comparison to previous technologies such as PCR and ELISA, NIRS offers several advantages, such as speed (eliminating time-consuming procedures), non-destructive/non-invasive analysis, and is inexpensive in terms of cost and maintenance. More importantly, this technique has the potential to measure multiple quality components in GMOs with reliable accuracy. In this review, we brief about the fundamentals and versatile applications of NIRS for the effective identification of GMOs in the agricultural and food systems.

Dynamics of Bacterial Community Structure in the Rhizosphere and Root Nodule of Soybean: Impacts of Growth Stages and Varieties.

Bacterial communities in rhizosphere and root nodules have significant contributions to the growth and productivity of the soybean (Glycine max (L.) Merr.). In this report, we analyzed the physiological properties and dynamics of bacterial community structure in rhizosphere and root nodules at different growth stages using BioLog EcoPlate and high-throughput sequencing technology, respectively. The BioLog assay found that the metabolic capability of rhizosphere is in increasing trend in the growth of soybeans as compared to the bulk soil. As a result of the Illumina sequencing analysis, the microbial community structure of rhizosphere and root nodules was found to be influenced by the variety and growth stage of the soybean. At the phylum level, Actinobacteria were the most abundant in rhizosphere at all growth stages, followed by Alphaproteobacteria and Acidobacteria, and the phylum Bacteroidetes showed the greatest change. But, in the root nodules Alphaproteobacteria were dominant. The results of the OTU analysis exhibited the dominance of Bradyrhizobium during the entire stage of growth, but the ratio of non-rhizobial bacteria showed an increasing trend as the soybean growth progressed. These findings revealed that bacterial community in the rhizosphere and root nodules changed according to both the variety and growth stages of soybean in the field.

Discrimination of Transgenic Canola (Brassica napus L.) and their Hybrids with B. rapa using Vis-NIR Spectroscopy and Machine Learning Methods.

In recent years, the rapid development of genetically modified (GM) technology has raised concerns about the safety of GM crops and foods for human health and the ecological environment. Gene flow from GM crops to other crops, especially in the Brassicaceae family, might pose a threat to the environment due to their weediness. Hence, finding reliable, quick, and low-cost methods to detect and monitor the presence of GM crops and crop products is important. In this study, we used visible near-infrared (Vis-NIR) spectroscopy for the effective discrimination of GM and non-GM Brassica napus, B. rapa, and F1 hybrids (B. rapa X GM B. napus). Initially, Vis-NIR spectra were collected from the plants, and the spectra were preprocessed. A combination of different preprocessing methods (four methods) and various modeling approaches (eight methods) was used for effective discrimination. Among the different combinations, the Savitzky-Golay and Support Vector Machine combination was found to be an optimal model in the discrimination of GM, non-GM, and hybrid plants with the highest accuracy rate (100%). The use of a Convolutional Neural Network with Normalization resulted in 98.9%. The same higher accuracy was found in the use of Gradient Boosted Trees and Fast Large Margin approaches. Later, phenolic acid concentration among the different plants was assessed using GC-MS analysis. Partial least squares regression analysis of Vis-NIR spectra and biochemical characteristics showed significant correlations in their respective changes. The results showed that handheld Vis-NIR spectroscopy combined with chemometric analyses could be used for the effective discrimination of GM and non-GM B. napus, B. rapa, and F1 hybrids. Biochemical composition analysis can also be combined with the Vis-NIR spectra for efficient discrimination.

Horse-riding accidents and injuries in historical records of Joseon Dynasty, Korea.

Only a few osteological reports describe bone injuries thought to have been caused by falls from horses. Nevertheless, anthropological study alone is insufficient for establishing the correlates of such equestrian accidents. We therefore reviewed the records in Seungjeongwon ilgi (Diaries of the Royal Secretariat) and Joseon wangjo silrok (Annals of the Joseon Dynasty) of the Korea's Joseon period (1392-1910 CE). Although the mechanisms of trauma were diverse, the Joseon documents recorded many injuries caused by horse-riding accidents. During 1625-1872 CE, equestrian-related accidents occurred almost every year, overwhelming other causes of trauma. In all horse-riding accidents (n=142), 37.77% of the records offer detailed data about the traumatic mechanism. Injuries occurred most frequently to the extremities (79.58%), which were followed by the trunk (34.5%) and head (4.92%). Although we do not think that this attempt can explain every paleopathological case, our historical review shows that equestrian-related injuries could be considered as one of the major causes for the bone trauma observed among ancient equestrian people.

Efficient scalable production of therapeutic microvesicles derived from human mesenchymal stem cells.

Microvesicles (MVs) released by cells are involved in a multitude of physiological events as important mediators of intercellular communication. MVs derived from mesenchymal stem cells (MSCs) contain various paracrine factors from the cells that primarily contribute to their therapeutic efficacy observed in numerous clinical trials. As nano-sized and bi-lipid layered vesicles retaining therapeutic potency equivalent to that of MSCs, MSC-derived MVs have been in focus as ideal medicinal candidates for regenerative medicine, and are preferred over MSC infusion therapy with their improved safety profiles. However, technical challenges in obtaining sufficient amounts of MVs have limited further progress in studies and clinical application. Of the multiple efforts to reinforce the therapeutic capacity of MSCs, few studies have reportedly examined the scale-up of MSC-derived MV production. In this study, we successfully amplified MV secretion from MSCs compared to the conventional culture method using a simple and efficient 3D-bioprocessing method. The MSC-derived MVs produced in our dynamic 3D-culture contained numerous therapeutic factors such as cytokines and micro-RNAs, and showed their therapeutic potency in in vitro efficacy evaluation. Our results may facilitate diverse applications of MSC-derived MVs from the bench to the bedside, which requires the large-scale production of MVs.

Is WHODAS 2.0 Useful for Colorectal Cancer Survivors?

OBJECTIVE: To compare the disability level of colorectal cancer survivors with and without stoma by using the Korean version of the 12-item, interview-administered World Health Organization Disability Assessment Schedule 2.0 (Korean version of WHODAS 2.0). METHODS: This is a multicenter (five tertiary university hospitals and the Korea Ostomy Association) and cross-sectional survey. Colorectal cancer survivors with and without stoma were interviewed. Survey measured disability level using the Korean version of WHODAS 2.0 and health-related quality of life using the SF-36. RESULTS: A significant difference was observed between patients with and without a stoma in two subdomains: getting around (31.1 vs. 20.3; p=0.013) and participation in society (32.3 vs. 22.2; p=0.028). After adjusting for age, gender, and time since surgery, having a stoma was associated with severe to extreme disabilities in participation (OR=2.72, p=0.045). The Korean version of WHODAS 2.0 showed satisfactory internal consistency (r=0.96) and convergent validity. CONCLUSION: Patients with stoma participated less in society than those without stoma. The Korean version of WHODAS 2.0 is a reliable and valid instrument for measuring disability in Korean colorectal cancer patients.

A novel cylindrical microwell featuring inverted-pyramidal opening for efficient cell spheroid formation without cell loss.

Spheroid cultures have been often used to simulate and understand in situ biological occurrences with potential to be further applied to therapeutic approaches, such as cell transplantation. However, traditional lab-scale techniques hardly reached the needed large scale production of cell spheroids, thus limiting their versatility in many biomedical fields. Microscale technologies have rapidly improved in the last decade, and contributed to the large scale production of cell spheroids with high controllability and reproducibility. Nonetheless, the existing microwell culture platforms are problematic due to unwanted cellular adhesion to the substrate as well as due to substantial amounts of cell loss. In this study, we have developed a novel configuration of cylindrical type polyethylene glycol (PEG) hydrogel microwells featuring inverted-pyramidal openings (iPO). Highly refined microstructures of our novel microwell could be fabricated by our optimized micro-electro mechanical system protocols consisting of a silicon (Si) wet/dry etching, Si-to-polydimethylsiloxane substrate bonding, and the established soft-lithography techniques. The iPO, the PEG hydrogel, and the cylindrical geometry of our microwell successfully (1) avoided inefficient washing steps after cell seeding, (2) achieved the complete resistance to cellular adhesion on the microwell substrate, and (3) made all seeded cells readily gathered and jam-packed to form cell spheroids with uniform size, respectively. The maximal sizes of cell spheroids were confined to below 200 µm according to the size of microwells used in this study. The efficiency testing for cell spheroid formation was conducted in comparison with other types of microwells that have been often used in the fields. The results showed that our novel microwell platform effectively reached almost zero percent of cell loss while mass-producing human mesenchymal stem cell spheroids with highly precise control over spheroid's size and cell number. We believe that this study could deliver an effective method to extend the practical usability of cell spheroids in a variety of biomedical applications.

Psychometric properties of scale constructed from the International Classification of Functioning, Disability and Health (ICF) core set for breast cancer based on Rasch analysis.

OBJECTIVES: The purpose of this study was to construct a clinical instrument to measure functioning in breast cancer survivors using the International Classification of Functioning, Disability and Health (ICF) categories for body functions, activity and participation, and environmental factors, based on a Rasch analysis. METHODS: Items were generated from the brief ICF core set for breast cancer and in-depth interviews from eight oncologists. Psychometric properties were evaluated in 158 female Korean patients with breast cancer using Rasch analysis, such as fit of the ICF categories, targeting between the ICF categories and a person's abilities, unidimensionality, and reliability. RESULTS: The Rasch refinement led to a change from the original 43-item, 5-level scale to a 30-item, 3- or 4-level scale. Rasch reliabilities were 0.89 (body function scale), 0.96 (activity and participation scale), and 0.93 (environmental scale). The item-difficulty hierarchy was stable across age (<50 or ≥50 years) and had no non-fitting items or gaps (all information weighted fit (infit)/outlier sensitive fit (outfit) mean square error of 0.7-1.3, n = 140). CONCLUSION: The Brief Core Set Breast Cancer Questionnaire for Screening is a reliable and valid 30-item questionnaire based on the brief ICF core set. It allows measurement of functioning as a unidimensional construct in patients with breast cancer.

Oestrogen up-regulates interleukin-21 production by CD4(+) T lymphocytes in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease in which abnormal immune responses are mediated by tissue-binding autoantibodies and immune complex deposition. Because most SLE patients are women of child-bearing age, oestrogen has been suggested to play an important role in SLE pathogenesis. One proposed role is to induce B-cell activation, culminating in increased autoantibody production. Interleukin-21 (IL-21) has been shown to be crucial in the differentiation of activated B cells into plasma cells. We therefore hypothesized that oestrogen up-regulates IL-21 production and induces subsequent B-cell activation in SLE patients. Peripheral blood was obtained from 22 SLE patients and 16 healthy controls. Expression levels of IL-21 and its receptor in serum, peripheral blood mononuclear cells, and CD4(+) T cells were higher in SLE patients than in healthy controls. Exposure of CD4(+) T cells from SLE patients to 17ß-oestradiol led to a dose- and time-dependent increase in IL-21 expression, which was abolished in the presence of mitogen-activated protein kinase (MAPK) (MAPK kinase, p38, Jun N-terminal kinase) inhibitors. B cells from healthy controls showed increased antibody production when they were co-cultured with oestrogen-treated CD4(+) T cells from SLE patients. Treatment with IL-21 antibody abrogated the increased antibody production of the co-culture systems. This study revealed the association between oestrogen and IL-21 in SLE patients. Oestrogen up-regulates IL-21 expression of CD4(+) T cells via MAPK-dependent pathways in SLE patients, which in turn induces increased antibody production by B cells.

Ethyl pyruvate ameliorates albuminuria and glomerular injury in the animal model of diabetic nephropathy.

Pyruvate is an endogenous antioxidant and anti-inflammatory substance. The present study was implemented to investigate the protective effect of ethyl pyruvate (EP) against the development and progression of diabetic nephropathy in an in vivo and in vitro model. Diabetic rats were prepared by injecting streptozotocin (65 mg/kg). Those that developed diabetes after 72 h were treated with EP (40 mg/kg) intraperitoneally. Diabetic rats without pyruvate treatment and nondiabetic rats were used for control. As an in vitro experiment, rat mesangial cells cultured primarily from Sprague-Dawley rats were treated in high-glucose (HG; 50 mM) or normal-glucose (NG; 5 mM) conditions and with or without pyruvate. Pyruvate-treated diabetic rats exhibited decreased albuminuria and attenuated NADPH-dependent reactive oxygen species generation. Immunohistochemistry showed reduced laminin, type IV collagen, and fibronectin deposition in the glomeruli compared with nontreated diabetic rats. Parallel changes were shown in tissue mRNA and protein expression levels of monocyte chemoattractant protein-1, transforming growth factor-ß1, laminin, fibronectin, and type IV collagen in the kidney. Concordantly, protective effects were also exhibited in the mesangial cell culture system. These findings suggest that pyruvate protects against kidney injury via NADPH oxidase inhibition. The present study established that activation of NADPH oxidase plays a crucial role in diabetes-induced oxidative stress, glomerular hypertrophy, and ECM molecule expression. Pyruvate exhibited a renoprotective effect in the progression of experimental diabetic nephropathy. Future research is warranted to investigate the protective mechanism of pyruvate more specifically in relation to NADPH oxidase in diabetic nephropathy.

Role of lung apolipoprotein A-I in idiopathic pulmonary fibrosis: antiinflammatory and antifibrotic effect on experimental lung injury and fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is caused by alterations in expression of proteins involved in multiple pathways, including matrix deposition, inflammation, injury, and repair. OBJECTIVES: To understand the pathogenic changes in lung protein expression in IPF and to evaluate apolipoprotein (Apo) A-I as a candidate therapeutic molecule. METHODS: Two-dimensional electrophoresis was adopted for differential display proteomics. Reverse-transcriptase polymerase chain reaction, Western blotting, immunohistochemical staining, and ELISA were performed for identification and quantitative measurement of Apo A-I in bronchoalveolar lavage fluids from subjects with IPF and experimental bleomycin-induced mice. MEASUREMENTS AND MAIN RESULTS: Sixteen protein spots showed differences in relative intensity between IPF (n = 14) and healthy control subjects (n = 8). Nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed increase of haptoglobulin and decrease of alpha(1)-antitrypsin, alpha(1)-antichymotrypsin, macrophage capping protein, angiotensinogen, hemoglobin chain B, Apo A-I, clusterin, protein disulfide isomerase A3, immunoglobulin, and complement C4A in IPF compared with normal control subjects (P = 0.006-0.044). Apo A-I concentrations were lower in bronchoalveolar lavage fluids from subjects with IPF (n = 28) than in normal control subjects (n = 18; P < 0.01). In bleomycin-treated mice, Apo A-I protein in BALF was lower than that in sham-treated control animals. Immunohistochemical analysis showed positive staining on intraalveolar macrophages and epithelial cells of the lungs. Intranasal treatment with Apo A-I protein reduced the bleomycin-induced increases in number of inflammatory cells and collagen deposition in sham-treated mice in a dose-dependent manner. CONCLUSIONS: Alterations of several inflammatory and antiinflammatory proteins in the lungs may be related to the pathogenesis of IPF, and local treatment with Apo A-I is very effective against the development of experimental lung injury and fibrosis.

Immunocytochemical staining of p16(ink4a) protein as an adjunct test in equivocal liquid-based cytology.

For cervical cancer screening, HPV-DNA test is expensive and is not easily available in all clinical situations. Thus, we investigated the role of p16(ink4a) immunostaining as another adjunct test to diagnose cervical neoplasia in equivocal liquid based cytology. Eighty-seven patients were randomly selected for this study (3 patients with normal, 84 patients with abnormal including 24 ASCUS, 30 LSIL, and 30 HSIL). We performed p16(ink4a) immunostaining on ThinPrep slide and on each case from the corresponding cervical biopsy tissues. High-risk HPV-DNA testing was also performed on all the subjects. We found that the immunoreactivity of p16(ink4a) is strongly correlated with the grade of cytologic and histologic diagnoses as well as with Hybrid Capture 2. In comparing the p16(ink4a) immunostaining with the Hybrid Capture 2 for accuracy of the diagnosis of CIN II/III or a higher-grade disease in the case of ASCUS/LSIL on ThinPrep, no significant differences were observed. Our data implies that p16(ink4a) immunocytochemical staining in liquid-based cytology specimens might be used as a good adjunct test to predict cervical histology in equivocal ThinPrep tests.

Heterogeneous rRNAs are differentially expressed during the morphological development of Streptomyces coelicolor.

It is generally assumed that all mature rRNA molecules assembled into ribosomes within a single cell are identical. However, sequence analysis of Streptomyces coelicolor genome revealed that it harbors six copies of divergent rRNA operons that may express and constitute three and five different kinds of small subunit (SSU) and large subunit (LSU) rRNA molecules, respectively, in a single cell. Phylogenetic analyses of the LSU rRNA genes and the internal transcribed spacer between SSU and LSU genes indicated that the LSU gene of rrnA and rrnE operons might be the result of interspecies recombination between rRNA genes in closely related streptomycetes. Profiling of rRNA species using primer extension analysis showed that heterogeneous rRNA transcripts are expressed and assembled into ribosomes in the cell. As the cells developed from germination to sporulation, the relative amount of LSU rRNA molecules derived from three rRNA operons (rrnA, D, and E) gradually decreased from approximately 85% to approximately 60%, whereas the distribution of LSU rRNA molecules from two other operons (rrnB and F) and rrnC operon gradually increased from approximately 10% to approximately 20% of the total LSU rRNA. These findings indicate that heterogeneous rRNA molecules are differentially expressed during the life cycle of this developmentally complex microorganism.

Characterization and pathogenic significance of Vibrio vulnificus antigens preferentially expressed in septicemic patients.

Many important virulence genes of pathogenic bacteria are preferentially expressed in vivo. We used the recently developed in vivo-induced antigen technology (IVIAT) to identify Vibrio vulnificus genes induced in vivo. An expression library of V. vulnificus was screened by colony blot analysis by using pooled convalescent-phase serum that had been thoroughly adsorbed with in vitro-expressed V. vulnificus whole cells and lysates. Twelve clones were selected, and the sequences of the insert DNAs were analyzed. The DNA sequences showed homologies with genes encoding proteins of diverse functions: these functions included chemotaxis (a methyl-accepting chemotaxis protein), signaling (a GGDEF-containing protein and a putative serine/threonine kinase), biosynthesis and metabolism (PyrH, PurH, and IlvC), secretion (TatB and plasmid Achromobacter secretion [PAS] factor), transcriptional activation (IlvY and HlyU), and the activity of a putative lipoprotein (YaeC). In addition, one identified open reading frame encoded a hypothetical protein. Isogenic mutants of the 12 in vivo-expressed (ive) genes were constructed and tested for cytotoxicity. Cytotoxic activity of the mutant strains, as measured by lactate dehydrogenase release from HeLa cells, was nearly abolished in pyrH, purH, and hlyU mutants. The intraperitoneal 50% lethal dose in mice increased by ca. 10- to 50-fold in these three mutants. PyrH and PurH seem to be essential for in vivo growth. HlyU appears to be one of the master regulators of in vivo virulence expression. The successful identification of ive genes responsible for the in vivo bacterial virulence, as done in the present study, demonstrates the usefulness of IVIAT for the detection of new virulence genes.