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Abstract Background Benzoic acid is one of the most useful aromatic compounds. Despite its versatility and simple structure, benzoic acid production using microbes has not been reported previously. Streptomyces are aerobic, Gram-positive, mycelia-forming soil bacteria, and are known to produce various kinds of antibiotics composed of many aromatic residues. S. maritimus possess a complex amino acid modification pathway and can serve as a new platform microbe to produce aromatic building-block compounds. In this study, we carried out benzoate fermentation using S. maritimus. In order to enhance benzoate productivity using cellulose as the carbon source, we constructed endo-glucanase secreting S. maritimus. Results After 4 days of cultivation using glucose, cellobiose, or starch as a carbon source, the maximal level of benzoate reached 257, 337, and 460 mg/l, respectively. S. maritimus expressed β-glucosidase and high amylase-retaining activity compared to those of S. lividans and S. coelicolor. In addition, for effective benzoate production from cellulosic materials, we constructed endo-glucanase-secreting S. maritimus. This transformant efficiently degraded the phosphoric acid swollen cellulose (PASC) and then produced 125 mg/l benzoate. Conclusions Wild-type S. maritimus produce benzoate via a plant-like β-oxidation pathway and can assimilate various carbon sources for benzoate production. In order to encourage cellulose degradation and improve benzoate productivity from cellulose, we constructed endo-glucanase-secreting S. maritimus. Using this transformant, we also demonstrated the direct fermentation of benzoate from cellulose. To achieve further benzoate productivity, the L-phenylalanine availability needs to be improved in future.

Abstract Background Despite efforts to improve surgical techniques, serious complications still sometimes occur. Use of a physiological posterior mediastinal pathway has increased given advances such as automated anastomotic devices and a reduction in the incidence of anastomotic sufficiency. Until now the gastric conduit created has been protected by an echo probe cover and, sown to the ventral side of polyester tape placed through the abdomen to the neck, and then blindly elevated to the neck. We report on a new method of gastric conduit elevation. Methods Two 60-cm lengths polyester tape are ligated at both ends to form a loop. An echo probe cover of 10 cm in diameter and 50 cm in length is prepared and the tip cut off, forming a cylinder. The knots in the previously looped polyester tape are inserted into the echo probe cover. The looped polyester tape and echo probe cover is ligated with silk approximately 5 cm in front of the knots on both sides. After dissection is carried out according to practice, the previously crafted polyester tape is inserted into the chest cavity. One end of polyester tape is fixed to the distal esophageal stump with the clips, with the opposite end fixed to the proximal esophageal stump. The echo probe cover that connects the proximal esophagus and distal esophagus is monitored for the presence of creases along the long axis to ensure there are no twists in the echo probe cover. We carry out a laparoscopic-assisted perigastric lymph node dissection, make a small skin incision, and guide part of the thoracic esophagus and stomach outside the body. Either one of the two lengths of polyester tape is connected to the gastric conduit. By pulling up this length of polyester tape from the neck, the gastric conduit can pass through the echo probe cover and be elevated to the neck. Results No perioperative complications such as bleeding or difficulty of the gastric conduit elevation were recognized with this method. Conclusions This method is considered to serve as a useful technique for gastric conduit elevation.

Abstract Here we report the method of anastomosis based on double stapling technique (hereinafter, DST) using a trans-oral anvil delivery system (EEATM OrVilTM) for reconstructing the esophagus and lifted jejunum following laparoscopic total gastrectomy or proximal gastric resection. As a basic technique, laparoscopic total gastrectomy employed Roux-en-Y reconstruction, laparoscopic proximal gastrectomy employed double tract reconstruction, and end-to-side anastomosis was used for the cut-off stump of the esophagus and lifted jejunum. We used EEATM OrVilTM as a device that permitted mechanical purse-string suture similarly to conventional EEA, and endo-Surgitie. After the gastric lymph node dissection, the esophagus was cut off using an automated stapler. EEATM OrVilTM was orally and slowly inserted from the valve tip, and a small hole was created at the tip of the obliquely cut-off stump with scissors to let the valve tip pass through. Yarn was cut to disconnect the anvil from a tube and the anvil head was retained in the esophagus. The end-Surgitie was inserted at the right subcostal margin, and after the looped-shaped thread was wrapped around the esophageal stump opening, assisting Maryland forceps inserted at the left subcostal and left abdomen were used to grasp the left and right esophageal stump. The surgeon inserted anvil grasping forceps into the right abdomen, and after grasping the esophagus with the forceps, tightened the end Surgitie, thereby completing the purse-string suture on the esophageal stump. The main unit of the automated stapler was inserted from the cut-off stump of the lifted jejunum, and a trocar was made to pass through. To prevent dropout of the small intestines from the automated stapler, the automated stapler and the lifted jejunum were fastened with silk thread, the abdomen was again inflated, and the lifted jejunum was led into the abdominal cavity. When it was confirmed that the automated stapler and center rod were made completely linear, the anvil and the main unit were connected with each other and firing was carried out. Then, DST-based anastomosis was completed with no dog-ear. The method may facilitate safe laparoscopic anastomosis between the esophagus and reconstructed intestine. This is also considered to serve as a useful anastomosis technique for upper levels of the esophagus in laparotomy.

Abstract Background Hydrolysis of cellulose requires the action of the cellulolytic enzymes endoglucanase, cellobiohydrolase and β-glucosidase. The expression ratios and synergetic effects of these enzymes significantly influence the extent and specific rate of cellulose degradation. In this study, using our previously developed method to optimize cellulase-expression levels in yeast, we constructed a diploid Saccharomyces cerevisiae strain optimized for expression of cellulolytic enzymes, and attempted to improve the cellulose-degradation activity and enable direct ethanol production from rice straw, one of the most abundant sources of lignocellulosic biomass. Results The engineered diploid strain, which contained multiple copies of three cellulase genes integrated into its genome, was precultured in molasses medium (381.4 mU/g wet cell), and displayed approximately six-fold higher phosphoric acid swollen cellulose (PASC) degradation activity than the parent haploid strain (63.5 mU/g wet cell). When used to ferment PASC, the diploid strain produced 7.6 g/l ethanol in 72 hours, with an ethanol yield that achieved 75% of the theoretical value, and also produced 7.5 g/l ethanol from pretreated rice straw in 72 hours. Conclusions We have developed diploid yeast strain optimized for expression of cellulolytic enzymes, which is capable of directly fermenting from cellulosic materials. Although this is a proof-of-concept study, it is to our knowledge, the first report of ethanol production from agricultural waste biomass using cellulolytic enzyme-expressing yeast without the addition of exogenous enzymes. Our results suggest that combining multigene expression optimization and diploidization in yeast is a promising approach for enhancing ethanol production from various types of lignocellulosic biomass.

Abstract Background We assessed the expression of cytokeratin (CK) and apomucin (MUC) in ampullary carcinoma (AC) to develop a system for the classification of ACs on the basis of their clinical significance. Method We studied the expressions of CK7, CK20, MUC1, MUC2, MUC5AC, and MUC6 in 43 patients with ACs. Clinical data were obtained retrospectively by examining surgically resected ACs of the patients. Results We classified the cases into 3 groups: tumors expressing CK20 and lacking MUC1 (intestinal type [I-type], 26%), tumors expressing MUC1 and lacking CK20 (pancreatobiliary type [PB-type], 35%), and those expressing or lacking both CK20 and MUC1 (other type [O-type], 39%). Eight (73%) of 11 I-type carcinomas, 3 (20%) of 15 PB-type carcinomas, and 4 (24%) of 17 O-type carcinomas were classified as pT1. The number of I-type carcinomas in the early tumor stages was significantly higher than the number of PB- and O-type carcinomas (p = 0.014 and p = 0.018, respectively). The 5-year survival rates for pT1, pT2, and pT3 tumors were 76%, 33%, and 22%, respectively (p < 0.001). Rates of MUC5AC and MUC6 coexpression for I-type, PB-type, and O-type tumors were 18%, 13%, and 53%, respectively. There was a significant correlation between MUC5AC and MUC6 coexpression and O-type characteristics (p = 0.031). The five-year survival rates for O-type ACs with and without MUC5AC and MUC6 coexpression were 71% and 17%, respectively (p = 0.048). Conclusions The immunohistochemical subtypes based on CK and MUC expression correlated with tumor progression. Gastric MUC5AC and MUC6 coexpression correlated with better prognosis for O-type ACs.

Abstract Background The filamentous fungus T. reesei effectively degrades cellulose and is known to produce various cellulolytic enzymes such as β-glucosidase, endoglucanase, and cellobiohydrolase. The expression levels of each cellulase are controlled simultaneously, and their ratios and synergetic effects are important for effective cellulose degradation. However, in recombinant Saccharomyces cerevisiae, it is difficult to simultaneously control many different enzymes. To construct engineered yeast with efficient cellulose degradation, we developed a simple method to optimize cellulase expression levels, named cocktail δ-integration. Results In cocktail δ-integration, several kinds of cellulase expression cassettes are integrated into yeast chromosomes simultaneously in one step, and strains with high cellulolytic activity (i.e., expressing an optimum ratio of cellulases) are easily obtained. Although the total integrated gene copy numbers of cocktail δ-integrant strain was about half that of a conventional δ-integrant strain, the phosphoric acid swollen cellulose (PASC) degradation activity (64.9 mU/g-wet cell) was higher than that of a conventional strain (57.6 mU/g-wet cell). This suggests that optimization of the cellulase expression ratio improves PASC degradation activity more so than overexpression. Conclusions To our knowledge, this is the first report on the expression of cellulase genes by δ-integration and optimization of various foreign genes by δ-integration in yeast. This method should be very effective and easily applied for other multi-enzymatic systems using recombinant yeast.

Abstract Background Combination chemotherapy with oxaliplatin plus 5-fluorouracil/leucovorin (FOLFOX) has become a standard regimen for colorectal cancer. An increase of adverse events with combination chemotherapy is predicted in elderly patients, and it remains controversial whether they should receive the same chemotherapy as younger patients. Accordingly, this study of modified FOLFOX6 (mFOLFOX6) therapy was performed to compare its safety between elderly and non-elderly patients. Methods We prospectively studies 14 non-elderly patients aged Results The main adverse events were neutropenia and peripheral neuropathy, which occurred in 62.5% (≥ grade 3) and 87.5% (≥ grade 1) of elderly patients. The grade and frequency of adverse events were similar in the elderly and non-elderly groups. In some patients with neutropenia, treatment could be continued without reducing the dose of oxaliplatin by deleting bolus 5-fluorouracil. A correlation was found between the cumulative dose of oxaliplatin and the severity of neuropathy, and there were 2 elderly and 3 younger patients in whom discontinuation of treatment was necessary due to peripheral neuropathy. The median number of treatment cycles was 10.0 and 9.5 in the non-elderly and elderly groups, respectively. The response rate was 60.0% in the non-elderly and 50.0% in the elderly group, while the disease control rate was 100% and 83.3% respectively, showing no age-related difference. Conclusion mFOLFOX6 therapy was well-tolerated and effective in both non-elderly and elderly patients. However, discontinuation of treatment was sometimes necessary due to peripheral neuropathy, which is dose-limiting toxicity of this therapy.

Men who have sex with men (MSM) and transgender women (TW) are disproportionally affected by HIV infection. This cross-sectional study evaluated the HIV-1/2 prevalence, risk factors and HIV molecular features of MSM and TW from Midwest Brazil. Four hundred and thirty participants (278 MSM and 152 TW) from Mato Grosso do Sul, Brazil, were interviewed and tested for HIV-1/2 infection between November 2011 and September 2013. Participants who were assigned male at birth, older than 18 years old and self-declared as MSM or TW were recruited from LGBT+ associations, as well as public (parks, square, streets, etc) and private [nightclubs, saunas, brothels, etc] places. The prevalence of HIV-1 was 14.4% (9.0% among MSM and 24% among TW; p<0.001). The factor independently associated with HIV-1 infection among MSM was being 30 years-old or older. Among TW, having suffered sexual coercion, lifetime syphilis infection and hepatitis C virus exposure were associated with HIV-1 infection. Phylogenetic analyses classified 65% sequences as subtype B and 35% as possible recombinants. All but one recombinant sample were from TW individuals. High HIV-1 prevalences were observed in both groups, highlighting the urgent need to devise specific HIV interventions targeting these key populations. Notably, TWs are more vulnerable to HIV infection, which was associated with sexual violence and co-infection with other STIs. With regard to MSM, being 30 years old or older was significanty associated to HIV, reinforcing the idea that MSM are less exposed [or exposed later] to STIs than TWs, although MSM are clearly more vulnerable than the general population., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Cesar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Hepatitis A virus (HAV) infection is transmitted by the fecal-oral route, through interpersonal contact and ingestion of contaminated food or water. Prisoners are at higher risk of acquiring HAV infection mainly due to the environment of closed penal institutions and socioeconomic conditions. This study aims to determine the seroprevalence of anti-HAV and its associated risk factors among inmates from twelve prisons in Central Brazil. A cross-sectional study was conducted between March 2013 and March 2014. A total of 580 prisoners participated in the study. The participant's samples were tested for Total and IgM anti-HAV antibodies by electrochemiluminescence immunoassay (ECLIA). Risk factors associated with anti-HAV seropositivity were also analyzed. The prevalence rate of HAV exposure was 88.1% (95% CI: 85.5-90.7). No sample had a positive reaction to IgM anti-HAV. Increasing age, low level of education, and being imprisoned in Corumbá city were independently associated with HAV exposure among prisoners. To prevent the burden of the disease, vaccination strategies should be considered for susceptible prisoners in Central Brazil., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Castro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Asymptomatic individuals apparently able for blood donation, could be infected with Leishmania imposing risks for immunologically vulnerable recipients. Reverse transcribed conventional PCR targeting the 28S ribosomal subunit was conducted, in order to confirm the viability of the parasite in blood donors positive for Leishmania infection., (Copyright © 2019. Published by Elsevier B.V.)

Background/aim: To review the long-term outcomes of penetrating keratoplasty (PKP) for corneal complications of herpes zoster ophthalmicus (HZO)., Methods: We reviewed the medical records of 53 eyes of 53 patients who underwent PKP due to corneal complications of HZO at the Kellogg Eye Center., Results: The mean age of patients at the time of PKP was 68.0±16.4 years, with a follow-up of 4.0±3.8 years and quiescent period of 6.5±5.3 years from active HZO to PKP. Preoperatively, 25 (47.2%) eyes were completely anaesthetic, while 16 (30.2%) had deep corneal neovascularisation in four quadrants. Comorbid ocular disease, including cataract, glaucoma and macular disease, was present in 25 (47.2%) eyes. Twenty patients (37.8%) received acyclovir for the entire postoperative period. There were no recurrences of zoster keratitis in any eye. The most common complications were difficulty healing the ocular surface (12/53, 22.6%) and glaucoma (14/53, 26.4%). Thirty per cent of the eyes required one or more additional postoperative procedures, most commonly tarsorrhaphy (10/53, 18.9%) and amniotic membrane graft (6/53, 11.3%). At 1, 2-4 and ≥5 years, 94%, 82% and 70% grafts remained clear, respectively. Visual acuity improved at 1 year postoperatively (p<0.0001), but this improvement was not sustained. There was no significant benefit of long-term acyclovir on visual acuity (p=0.2132) or graft survival (p=0.241)., Conclusions: Even in eyes with significant preoperative risk factors, PKP for the corneal complications of HZO can achieve favourable tectonic and visual results. Although most grafts remained clear, long-term visual potential may be limited by comorbid ocular diseases. Prophylactic postoperative oral acyclovir did not improve outcomes., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Histone citrullination regulates diverse cellular processes. Here, we report that SMARCAD1 preferentially associates with H3 arginine 26 citrullination (H3R26Cit) peptides present on arrays composed of 384 histone peptides harboring distinct post-transcriptional modifications. Among ten histone modifications assayed by ChIP-seq, H3R26Cit exhibited the most extensive genomewide co-localization with SMARCAD1 binding. Increased Smarcad1 expression correlated with naive pluripotency in pre-implantation embryos. In the presence of LIF, Smarcad1 knockdown (KD) embryonic stem cells lost naive state phenotypes but remained pluripotent, as suggested by morphology, gene expression, histone modifications, alkaline phosphatase activity, energy metabolism, embryoid bodies, teratoma, and chimeras. The majority of H3R26Cit ChIP-seq peaks occupied by SMARCAD1 were associated with increased levels of H3K9me3 in Smarcad1 KD cells. Inhibition of H3Cit induced H3K9me3 at the overlapping regions of H3R26Cit peaks and SMARCAD1 peaks. These data suggest a model in which SMARCAD1 regulates naive pluripotency by interacting with H3R26Cit and suppressing heterochromatin formation., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The aim of this multicenter, cross sectional study was to assess the prevalence, incidence and associated risk factors among incarcerated populations from twelve Brazilian prisons. The total of 3,368 individuals from twelve prisons was randomly recruited between March 2013 and March 2014. Participants were interviewed, and provided blood samples which were tested for antibodies to Hepatitis C (HCV ab). One year after the first investigation, a cohort study was conducted with 1,656 inmates who participated the cross sectional study. Positive samples were tested for the presence of HCV RNA. Out of 3,368 inmates, 520 (15.4%) were females, and 2,848 (84.6%) were males. The overall prevalence of HCV was 2.4% (95% CI: 1.9 to 2.9), with 0.6% (95% CI: 0.4 to 0.8) in females, and 2.7% (95% CI: 2.1 to 3.3) in males (p<0.01). HCV RNA was detected in 51/80 (63.7%) samples. Among men prisoners, multivariate analysis of associated factors showed independent associations between HCV exposure and increasing age, inject drug use, length of incarceration, smoking hashish, sharing needle and syringe and HIV positivity. During the cohort study, 7/1,656 new cases of HCV infection were detected, and the incidence rate was 0.4/100 person-year. Once high frequency rates of specific HCV risk behaviors and new HCV infections have been identified inside prisons, effective interventions strategies such as screening, clinical evaluation and treatment to reduce the spread of HCV infection are essential., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Cryopreserved ultra-thick human amniotic membrane (AM) is used for glaucoma surgery. We evaluated the use of cryopreserved ultra-thick human AM for conjunctival surface reconstruction after excision of a conjunctival tumor., Methods: We retrospectively reviewed the medical records of 28 patients who underwent conjunctival surface reconstruction with cryopreserved ultra-thick human AM after excision of the tumor. The AM was secured to the surrounding conjunctiva and underlying sclera with interrupted 8-0 Vicryl sutures. Clinical data regarding demographics, diagnosis, size and location of conjunctival tumors, patient outcome, and complications were gathered., Results: Of 28 patients, 6 (21.4%) had malignant melanoma, 4 (14.3%) had squamous cell carcinoma, 6 (21.4%) had conjunctival intraepithelial neoplasia, 1 (3.6%) had sebaceous carcinoma, 1 (3.6%) had mucoepidermoid carcinoma, 1 (3.6%) had conjunctival intraepithelial dysplasia, 5 (17.9%) had pterygium, 2 (7.1%) had compound nevus, 1 (3.6%) had a large epithelial inclusion cyst, and 1 (3.6%) patient had a granuloma. The mean area of graft size was 156 ± 120 mm2. Postoperatively, the graft was well tolerated with no failure, discomfort, or dehiscence. During the 17-month mean follow-up, symblepharon, which was clinically nonsignificant, developed in 3 (11%) patients and partial stem cell deficiency was noted in 5 (18%) patients., Conclusions: Cryopreserved ultra-thick human AM is a well-tolerated, effective graft material that is easy to handle. It is a viable alternative for conjunctival surface reconstruction after excision of a conjunctival tumor.

In recent decades, constructed wetlands dominated by common reeds [Phragmites australis (CAV.) Trin. ex Steudel] have been utilized for treating nitrogen-rich wastewaters. Although plant harvest is a vegetation management in constructed wetlands for the purpose of improving nutrient removal, harvested biomass has become a problem in many places. The reed has attracted increasing interest for its potential as high-quality roughage for ruminants. Therefore, it is crucial to understand the effect of reed harvest timing on subsequent regrowth, reconstruction of canopy structure, and nutritive value of regrown biomass for roughage when defining an appropriate vegetation management in constructed wetlands. The shoots of common reeds were harvested in January (winter), March (spring), and May (early summer) in a free-water surface constructed wetland in southwest China. Harvesting in winter enhanced the shoot regrowth and concentrations of total digestible nutrients (TDN), probably due to vigorous translocations of nonstructural carbohydrates from rhizomes. Harvesting in spring and early summer decreased aboveground biomass, nitrogen (N) standing stock, and concentrations of TDN. From fifty to 110 days after harvest, the TDN had sharply declined to values similar to non-harvested stands. Thus, to obtain high-quality roughage, it is recommended that regrown shoots be harvested again within a year in the early growing stage after the first harvest in winter., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

This chapter describes standard techniques to (1) maintain mouse embryonic stem cell culture, (2) deliver transgenes into mouse embryonic stem cells mediated by electroporation, nucleofection, lipofection, and retro/lentiviruses, and (3) assess the pluripotency of mouse embryonic stem cells. The last part of this chapter presents induction of random cell differentiation followed by the alkaline phosphatase and embryoid body formation assays, immunofluorescence microscopy, and the teratoma formation assay.

Background: Human T-lymphotropic virus type 1 (HTLV-1) has worldwide distribution and is considered endemic in many world regions, including southwestern Japan and Brazil. Japanese immigrants and their descendants have a high risk of acquiring this infection due to intense population exchange between Brazil and Japan., Objective: This cross-sectional study aimed to estimate the prevalence of HTLV, analyze the main risk factors associated with this infection, identify the main circulating types and subtypes of HTLV in Japanese immigrants and descendants living in Campo Grande-MS (Middle-West Brazil), as well as analyze the phylogenetic relationship among isolates of HTLV., Study Design: A total of 219 individuals were interviewed and submitted to blood collection. All collected blood samples were submitted for detection of anti-HTLV-1/2 using the immunoassay ELISA and confirmed by immunoblot method. The proviral DNA of the 14 samples HTLV- 1 positive were genotyped by nucleotide sequencing., Results: The overall prevalence of HTLV-1 was 6.8% (IC 95%: 3,5-10,2). Descriptive analysis of behavioral risk factors showed statistical association between HTLV-1 and age greater than or equal to 45 years. The proviral DNA of HTLV-1 was detected in all HTLV-1 positive samples. Of these, 14 were sequenced and classified as Cosmopolitan subtype, and 50% (7/14) belonged to subgroup A (transcontinental) and 50% (7/14) to the subgroup B (Japanese)., Conclusion: The high prevalence of HTLV-1 found evidence of the importance of early diagnosis and counseling of individuals infected with HTLV-1 for the control and prevention of the spread of this infection among Japanese immigrants and their descendants in Central Brazil.

Purpose: To report a solitary epibulbar neurofibroma in 2 elderly patients without systemic neurofibromatosis., Methods: Case reports and literature review., Results: A 67-year-old man presented with a 2-month history of left epibulbar mass. On slit-lamp examination, an 8- × 8-mm amelanotic, translucent, gelatinous, circumscribed lesion was present deep to the nasal bulbar conjunctiva. A 60-year-old otherwise healthy man presented with a 6-week history of a subconjunctival growth causing pain and photophobia in his right eye. On slit-lamp examination, an 8- × 8-mm amelanotic, translucent, gelatinous, circumscribed subconjunctival lesion, causing a delle was present near the nasal limbus. In each case, excisional biopsy was performed and histopathologic study revealed the diagnosis of neurofibroma. In both cases, after 12 months of follow-up, there was no evidence of recurrence or systemic disease., Conclusions: Solitary epibulbar neurofibroma in older adult patients without systemic neurofibromatosis is rare but should be considered in the differential diagnosis of epibulbar tumors.

Objectives: Men who have sex with men (MSM) and transgender women (TW) are highly vulnerable groups to sexually transmitted infections (STIs). This study aims to assess the prevalence of syphilis infection, sexual behaviour and identify factors associated with syphilis in MSM and TW in Campo Grande, Central Brazil., Methods: Between 2009 and 2011, 430 MSM/TW participants were interviewed and tested for syphilis. Univariable and multivariable regression analyses were done to assess associations with syphilis infection., Results: A total of 430 MSM/TW (278 MSM and 152 TW) were included in the study. The overall prevalence of lifetime syphilis and active syphilis was 34.7% (26.3% among MSM; 50.0% among TW) and 17.5% (12.3% among MSM; 27.0% among TW), respectively (p<0.001). In multivariable regression analysis, being 20-24 years and ≥30 years, having engaged in a variety of sexual practices and with a history of genital/anal ulcer in the last 12 months were associated with lifetime syphilis infection in the MSM group. Among TW participants, being ≥30 years of age, having more than 10 male sexual partners in last week and being infected with HIV were associated with lifetime syphilis. Factors associated with active syphilis among MSM were massage parlour/sauna recruitment and alcohol consumption at least once a week. Having sex with female partners in the past 12 months was predictive for active syphilis among TW., Conclusions: The prevalence of syphilis infection and risk sexual behaviour were high in the two samples, especially among TW. High levels of bisexual behaviours and low rates of consistent condom use indicate potential HIV/STIs transmission into the heterosexual population. This finding indicates the need and urgency for implementing more effective integrated programmes targeting MSM/TW for the prevention of syphilis and other STIs., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Plant harvest is one of the most important management practices in constructed wetlands. In this study, we evaluated the impact of harvesting Phragmites australis (Cav.) Trin. ex Steudel on the activity and community structure of nitrifiers and denitrifiers in a free-water surface constructed wetland. The nitrifiers were targeted using bacterial and archaeal-amoA that encode ammonia monooxygenase, and the denitrifiers were targeted using nirK and nirS that encode the nitrite reductase. The community structures were evaluated using denaturing gradient gel electrophoresis. The potential nitrification and nitrate reduction rates were shown to be significantly higher in the harvested plant rhizosphere than in a non-harvested control plot. The potential nitrification rate positively correlated with the potential nitrate reduction rate and influenced the community structure of nirK. In addition, plant canopy developed differently after harvest and simultaneously changed the microclimate beneath the plant community. These results suggest that plant harvest management could change subsequent plant development and associated microenvironments, thereby affecting the function and community structure of nitrifiers and denitrifiers. Our study highlights the importance of plant harvest management within constructed wetlands to regulate the functions of nitrification and denitrification., (© FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Purpose: To report a case of corneal hydrops with perforation in a patient with ectasia after undergoing laser in situ keratomileusis (LASIK)., Methods: An observational study with clinical, optical coherence tomographic, and histopathologic findings., Results: A 41-year-old woman had an acute onset of blurry vision, pain, photophobia, tearing, and foreign body sensation in the right eye 10 years after undergoing unilateral LASIK in Jordan. According to her, the surgeon elected not to operate on the left eye because of a "corneal abnormality." On slit-lamp examination, a tear in Descemet membrane with a stromal cleft extending to the overlying LASIK flap interface was noted. The flap was partially dehisced by a diffuse channel of aqueous humor draining from the cleft and streaming out the temporal flap edge. When leakage failed to stop after 2 weeks of treatment with a bandage contact lens, the patient underwent penetrating keratoplasty. Histopathological examination of the host button showed a fluid-filled cleft connecting the flap interface. Slit-lamp examination and corneal topography of the contralateral left eye were consistent with keratoconus., Conclusions: Corneal hydrops with perforation in the setting of post-LASIK ectasia is extremely rare and may be associated with flap dehiscence requiring penetrating keratoplasty.

A cross-sectional study on prevalence, associated factors and genotype distribution of HCV infection was conducted among 848 HIV-infected patients recruited at reference centers in the Midwest Region of Brazil. The prevalence rate of HIV-HCV coinfection was 6.9% (95% CI: 5.2 to 8.6). In multivariable analysis, increasing age, use of illicit drugs (injection and non-injection), a history of blood transfusion before 1994, and the absence of a steady partnership were significant independent associated factors for HIV-HCV coinfection. The phylogenetic analysis based on the NS5B region revealed the presence of two major circulating genotypes of HCV: genotypes 1 (58.3%) and 3 (41.7%). The prevalence of HIV-HCV coinfection was lower than those reported in studies conducted with HIV-infected patients in different regions of Brazil, due to the fact that illicit drug use is not a frequent mode of HIV transmission in this region of Brazil. Serologic screening of HIV-patients for HCV before initiating antiretroviral treatment, a comprehensive identification of associated factors, and the implementation of effective harm reduction programs are highly recommended to provide useful information for treatment and to prevent HCV coinfection in these patients.

Objectives: A cross-sectional study on prevalence of HBV and HDV infection, risk factors and genotype distribution of HBV infection was conducted among 848 HIV-infected patients in Mato Grosso do Sul, Central Brazil., Methods: Serum samples of 848 participants were tested for hepatitis B surface antigen (HBsAg), hepatitis B core antibody (anti-HBc) and hepatitis surface antibody (anti-HBs). HBsAg positive samples were tested for anti-HBc IgM, HBeAg, anti-HBe, anti-HCV, and total anti-HDV. HBsAg and anti-HBc positive were subjected to DNA extraction. Viral DNA was amplified by semi-nested PCR for the regions pre-S/S and then purified and genotyped/subgenotyped by direct sequencing. Student's t-test, chi-square test and Fisher's exact test were used to compare variables and to evaluate association between HBV positivity (defined as anti-HBc and/or HBsAg positivity) and risk factors., Results: Among the 848 HIV infected patients investigated 222 had serological markers of HBV infection. The prevalence rate of HIV-HBV coinfection was 2.5% (21/848; 95% CI: 1.4-3.5%); 484 (57.1%) patients were susceptible for HBV infection. There were no cases of anti-HDV positive and only one (0.1%) anti-HCV-positive case among the HIV-HBV coinfected patients. Male gender, increasing age, family history of hepatitis, use of illicit drug, and homosexual activity were independent factors associated with HBV exposure. The phylogenetic analysis based on the S gene region revealed the presence of genotypes D (76.9%), F (15.4%) and A (7.7%) in the study sample., Conclusion: This study demonstrates the low prevalence of HIV-HBV infection and also highlights the need for early vaccination against HBV as well as testing for HBV, HCV and HDV in all HIV-infected individuals., (Copyright © 2014 Elsevier Editora Ltda. All rights reserved.)

Mammalian inner cell mass cells undergo lineage-specific differentiation into germ layers of endoderm, mesoderm and ectoderm during gastrulation. It has been a long-standing challenge in developmental biology to replicate these organized germ layer patterns in culture. Here we present a method of generating organized germ layers from a single mouse embryonic stem cell cultured in a soft fibrin matrix. Spatial organization of germ layers is regulated by cortical tension of the colony, matrix dimensionality and softness, and cell-cell adhesion. Remarkably, anchorage of the embryoid colony from the 3D matrix to collagen-1-coated 2D substrates of ~1 kPa results in self-organization of all three germ layers: ectoderm on the outside layer, mesoderm in the middle and endoderm at the centre of the colony, reminiscent of generalized gastrulating chordate embryos. These results suggest that mechanical forces via cell-matrix and cell-cell interactions are crucial in spatial organization of germ layers during mammalian gastrulation. This new in vitro method could be used to gain insights on the mechanisms responsible for the regulation of germ layer formation.

Female sex workers (FSWs) are considered a high-risk group for human immunodeficiency virus (HIV) infection due to their social vulnerability and factors associated with their work. We estimated the prevalence of HIV, and identified viral subtypes and risk factors among FSWs. A cross-sectional study using respondent-driven sampling (RDS) method was conducted among 402 FSWs in Campo Grande city, Brazil, from 2009 to 2011. Participants were interviewed using a standardized questionnaire about sociodemograpic characteristics and risk behavior. Blood samples were collected for serological testing of HIV. Of the 402 FSWs, median age and age of initiating sex work were 25 years (Interquartile range [IQR]: 9) and 20 years (IQR: 6), respectively. The majority reported use of alcohol (88.5%), had 5-9 years (median: 9; IQR: 3) of schooling (54.5%), 68.6% had tattoos/body piercings, and 45.1% had more than seven clients per week (median: 7; IQR: 10). Only 32.9% of FSW reported using a condom with nonpaying partners in the last sexual contact. Prevalence of HIV infection was 1.0% (95% CI: 0.1-2.6%). Genotyping for HIV-1 performed on three samples detected subtypes B, C, and F1. Sex work in the Midwestern region of Brazil is characterized by reduced education, large numbers of clients per week, and inconsistent condom use, mainly with nonpaying partners. Although prevalence of HIV infection is currently low, elevated levels of high-risk sexual behavior confirm a need to implement prevention measures. Specific interventions targeting FSWs must emphasize the risk associated with both clients and nonpaying partners while providing knowledge about HIV prevention.

We examined the transcriptional activity of Oct3/4 (Pou5f1) in mouse embryonic stem cells (mESCs) maintained under standard culture conditions to gain a better understanding of self-renewal in mESCs. First, we built an expression vector in which the Oct3/4 promoter drives the monocistronic transcription of Venus and a puromycin-resistant gene via the foot-and-mouth disease virus self-cleaving peptide T2A. Then, a genetically-engineered mESC line with the stable integration of this vector was isolated and cultured in the presence or absence of puromycin. The cultures were subsequently subjected to Illumina expression microarray analysis. We identified approximately 4600 probes with statistically significant differential expression. The genes involved in nucleic acid synthesis were overrepresented in the probe set associated with mESCs maintained in the presence of puromycin. In contrast, the genes involved in cell differentiation were overrepresented in the probe set associated with mESCs maintained in the absence of puromycin. Therefore, it is suggested with these data that the transcriptional activity of Oct3/4 fluctuates in mESCs and that Oct3/4 plays an essential role in sustaining the basal transcriptional activities required for cell duplication in populations with equal differentiation potential. Heterogeneity in the transcriptional activity of Oct3/4 was dynamic. Interestingly, we found that genes involved in the hedgehog signaling pathway showed unique expression profiles in mESCs and validated this observation by RT-PCR analysis. The expression of Gli2, Ptch1 and Smo was consistently detected in other types of pluripotent stem cells examined in this study. Furthermore, the Gli2 protein was heterogeneously detected in mESC nuclei by immunofluorescence microscopy and this result correlated with the detection of the Oct3/4 protein. Finally, forced activation of Gli2 in mESCs increased their proliferation rate. Collectively, it is suggested with these results that Gli2 may play a novel role in the self-renewal of pluripotent stem cells., (© 2013.)

Background: The aim of the present study was to estimate hepatitis B virus seroprevalence among first-time blood donors in the city of Campo Grande, Mato Grosso do Sul State, in the central-western region of Brazil., Findings: A retrospective analysis of first-time voluntary blood donor records, from January 2010 to December 2010, was conducted at the Hematology Center of Mato Grosso do Sul. The prevalence of the HBsAg and anti-HBc serological markers and their respective 95% confidence intervals were calculated. Chi-square analysis was performed between the seroprevalence previously found in 2001 and the one determined by the current study. Results were considered statistically significant if p < 0.05. Among 8,840 subjects, 269 (3.04%, 95% CI: 2.7-3.4) were positive for HBV markers. The prevalence rate of HBsAg was 0.19% (95% CI: 0.1-0.3) and anti-HBc alone was 2.85% (95% CI: 2.5-3.2)., Conclusions: There was no statistically significant difference regarding gender. However, an important association was observed between HBV infection and older age (p < 0.01). The seroprevalence of HBV infection in first-time blood donors diminished from 2001 to 2010 (p < 0.01). Such decrease suggests an improvement in the recruitment of safe donors, the positive impact of vaccination programs and the decreasing of HBV infection prevalence in the general population.

Increasing evidence suggests that mechanical factors play a critical role in fate decisions of stem cells. Recently we have demonstrated that a local force applied via Arg-Gly-Asp (RGD) peptides coated magnetic beads to mouse embryonic stem (ES) cells increases cell spreading and cell stiffness and decreases Oct3/4 (Pou5f1) gene expression. However, it is not clear whether the effects of the applied stress on these functions of ES cells can be extended to natural extracellular matrix proteins or cell-cell adhesion molecules. Here we show that a local cyclic shear force applied via fibronectin or laminin to integrin receptors increased cell spreading and stiffness, downregulated Oct3/4 gene expression, and decreased cell proliferation rate. In contrast, the same cyclic force applied via cell-cell adhesion molecule E-cadherin (Cdh1) had no effects on cell spreading, Oct3/4 gene expression, and the self-renewal of mouse ES cells, but induced significant cell stiffening. Our findings demonstrate that biological responses of ES cells to force applied via integrins are different from those to force via E-cadherin, suggesting that mechanical forces might play different roles in different force transduction pathways to shape early embryogenesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

CTRP3/cartducin, a novel C1q family protein, is expressed in proliferating chondrocytes in the growth plate and has an important role in regulating the growth of both chondrogenic precursors and chondrocytes in vitro. We examined the expression of CTRP3/cartducin mRNA in Meckel's cartilage and in condylar cartilage of the fetal mouse mandible. Based on in situ hybridization studies, CTRP3/cartducin mRNA was not expressed in the anlagen of Meckel's cartilage at embryonic day (E)11.5, but it was strongly expressed in Meckel's cartilage at E14.0, and then reduced in the hypertrophic chondrocytes at E16.0. CTRP3/cartducin mRNA was not expressed in the condylar anlagen at E14.0, but was expressed in the upper part of newly formed condylar cartilage at E15.0. At E16.0, CTRP3/cartducin mRNA was expressed from the polymorphic cell zone to the upper part of the hypertrophic cell zone, but was reduced in the lower part of the hypertrophic cell zone. CTRP3/cartducin-antisense oligodeoxynucleotide (AS-ODN) treatment of Meckel's cartilage and condylar anlagen from E14.0 using an organ culture system indicated that, after 4-day culture, CTRP3/cartducin abrogation induced curvature deformation of Meckel's cartilage with loss of the perichondrium and new cartilage formation. Aggrecan, type I collagen, and tenascin-C were simultaneously immunostained in this newly formed cartilage, indicating possible transformation from the perichondrium into cartilage. Further, addition of recombinant mouse CTRP3/cartducin protein to the organ culture medium with AS-ODN tended to reverse the deformation. These results suggest a novel function for CTRP3/cartducin in maintaining the perichondrium. Moreover, AS-ODN induced a deformation of the shape, loss of the perichondrium/fibrous cell zone, and disorder of the distinct architecture of zones in the mandibular condylar cartilage. Additionally, AS-ODN-treated condylar cartilage showed reduced levels of mRNA expression of aggrecan, collagen types I and X, and reduced BrdU-incorporation. These results suggest that CTRP3/cartducin is not only involved in the proliferation and differentiation of chondrocytes, but also contributes to the regulation of mandibular condylar cartilage., (© 2011 The Authors. Journal of Anatomy © 2011 Anatomical Society of Great Britain and Ireland.)

Here, we present evidence that the tumor-like growth of mouse embryonic stem cells (mESCs) is suppressed by short-term serum-free culture, which is reversed by pharmacological inhibition of Gsk3β. Mouse ESCs maintained under standard conditions using fetal bovine serum (FBS) were cultured in a uniquely formulated chemically-defined serum-free (CDSF) medium, namely ESF7, for three passages before being subcutaneously transplanted into immunocompromised mice. Surprisingly, the mESCs failed to produce teratomas for up to six months, whereas mESCs maintained under standard conditions generated well-developed teratomas in five weeks. Mouse ESCs cultured under CDSF conditions maintained the expression of Oct3/4, Nanog, Sox2 and SSEA1, and differentiated into germ cells in vivo. In addition, when mESCs were cultured under CDSF conditions supplemented with FBS, or when the cells were cultured under CDSF conditions followed by standard culture conditions, they consistently developed into teratomas. Thus, these results validate that the pluripotency of mESCs was not compromised by CDSF conditions. Mouse ESCs cultured under CDSF conditions proliferated significantly more slowly than mESCs cultured under standard conditions, and were reminiscent of Eras-null mESCs. In fact, their slower proliferation was accompanied by the downregulation of Eras and c-Myc, which regulate the tumor-like growth of mESCs. Remarkably, when mESCs were cultured under CDSF conditions supplemented with a pharmacological inhibitor of Gsk3β, they efficiently proliferated and developed into teratomas without upregulation of Eras and c-Myc, whereas mESCs cultured under standard conditions expressed Eras and c-Myc. Although the role of Gsk3β in the self-renewal of ESCs has been established, it is suggested with these data that Gsk3β governs the tumor-like growth of mESCs by means of a mechanism different from the one to support the pluripotency of ESCs.

Rhox6 is one of the Reproductive Homeobox genes on the X chromosome (Rhox) that is expressed in the placenta and the post-migratory primordial germ cells (PGCs) in the nascent gonad. Despite its novel expression pattern, the significance of Rhox6 expression in the differentiation of these cell types remains unknown. To investigate the role that Rhox6 plays in PGCs, cDNA encoding Rhox6 and short-hairpin (sh) RNA directed against Rhox6 transcripts were introduced by unique expression vectors into a genetically engineered mouse embryonic stem cell (ESC) line. This ESC line expresses enhanced green fluorescent protein (EGFP) under the Oct3/4 promoter, thereby allowing us to monitor the presence of undifferentiated ESCs and PGCs in culture in real time. This ESC line was used to isolate clones that stably expressed Rhox6 cDNA, shRNA against Rhox6 transcripts, or controls. Quantitative RT-PCR results validated that overexpression had been achieved, as well as knockdown of Rhox6 transcripts in these ESC clones. However, these clones exhibited a normal appearance of undifferentiated ESCs and expressed EGFP. Next, these ESC clones were induced to differentiate into PGCs by generating embryoid bodies (EBs) in culture medium without leukemia inhibitory factor. Detection of EGFP expression by fluorescence microscopy and germ cell markers by RT-PCR validated the differentiation of PGCs in EBs. The Rhox6 transgene had little, if any, effect on EGFP expression in EBs, whereas Rhox6 knockdown significantly decreased EGFP expression in EBs. Thus, it is suggested with these results that Rhox6 is necessary for determination of the germ cell lineage.

Maintaining undifferentiated mouse embryonic stem cell (mESC) culture has been a major challenge as mESCs cultured in Leukemia Inhibitory Factor (LIF) conditions exhibit spontaneous differentiation, fluctuating expression of pluripotency genes, and genes of specialized cells. Here we show that, in sharp contrast to the mESCs seeded on the conventional rigid substrates, the mESCs cultured on the soft substrates that match the intrinsic stiffness of the mESCs and in the absence of exogenous LIF for 5 days, surprisingly still generated homogeneous undifferentiated colonies, maintained high levels of Oct3/4, Nanog, and Alkaline Phosphatase (AP) activities, and formed embryoid bodies and teratomas efficiently. A different line of mESCs, cultured on the soft substrates without exogenous LIF, maintained the capacity of generating homogeneous undifferentiated colonies with relatively high levels of Oct3/4 and AP activities, up to at least 15 passages, suggesting that this soft substrate approach applies to long term culture of different mESC lines. mESC colonies on these soft substrates without LIF generated low cell-matrix tractions and low stiffness. Both tractions and stiffness of the colonies increased with substrate stiffness, accompanied by downregulation of Oct3/4 expression. Our findings demonstrate that mESC self-renewal and pluripotency can be maintained homogeneously on soft substrates via the biophysical mechanism of facilitating generation of low cell-matrix tractions.

Human embryonic stem cells (ESCs [hESCs]) proliferate as colonies wherein individual cells are strongly adhered to one another. This architecture is linked to hESC self-renewal, pluripotency, and survival and depends on epithelial cadherin (E-cadherin), NMMIIA (nonmuscle myosin IIA), and p120-catenin. E-cadherin and p120-catenin work within a positive feedback loop that promotes localized accumulation of E-cadherin at intercellular junctions. NMMIIA stabilizes p120-catenin protein and controls E-cadherin-mediated intercellular adhesion. Perturbations of this signaling network disrupt colony formation, destabilize the transcriptional regulatory circuitry for pluripotency, and impair long-term survival of hESCs. Furthermore, depletion of E-cadherin markedly reduces the efficiency of reprogramming of human somatic cells to an ESC-like state. The feedback regulation and mechanical-biochemical integration provide mechanistic insights for the regulation of intercellular adhesion and cellular architecture in hESCs during long-term self-renewal. Our findings also contribute to the understanding of microenvironmental regulation of hESC identity and somatic reprogramming.

We have reported elsewhere that offspring from the No. 65 female of Xenopus laevis cleaved normally, but their development was arrested at the onset of gastrulation, like the Ambystoma ova-deficient (o) mutant, irrespective of mating with different wild-type males, and that an acidic, 38 kDa protein present in wild-type eggs was lacking in eggs of the female. In the current study, we first determined the partial amino acid sequence (VANLE) of one of the well-separated tryptic peptides from the protein, which was found in elongation factor 1 delta (Ef1delta) in Xenopus, and finally identified the protein as one of the Ef1delta isoforms, Ef1delta2, by peptide mass spectrometry. RT-PCR analyses for Ef1delta2 and its close homolog Ef1delta1 in wild-type oocytes and embryos demonstrated that both transcripts are maternal and Ef1delta1 is present more abundantly than Ef1delta2 throughout the stages examined. Importantly, the amount of the Ef1delta2 transcript per embryo decreased gradually after gastrulation, in accordance with the gradual decrease of the 38 kDa protein per embryo reported in our earlier study. Because pharmacological inhibition of translation induces gastrulation arrest in wild-type embryos, it is reasonable to conclude that the mutant embryos arrest in development due to the lack of Ef1delta2 that is indispensable for translation. Thus, the present study provides the first molecular information on the cause of the gastrulation-defective mutation in Amphibia.

Growing evidence suggests that physical microenvironments and mechanical stresses, in addition to soluble factors, help direct mesenchymal-stem-cell fate. However, biological responses to a local force in embryonic stem cells remain elusive. Here we show that a local cyclic stress through focal adhesions induced spreading in mouse embryonic stem cells but not in mouse embryonic stem-cell-differentiated cells, which were ten times stiffer. This response was dictated by the cell material property (cell softness), suggesting that a threshold cell deformation is the key setpoint for triggering spreading responses. Traction quantification and pharmacological or shRNA intervention revealed that myosin II contractility, F-actin, Src or cdc42 were essential in the spreading response. The applied stress led to oct3/4 gene downregulation in mES cells. Our findings demonstrate that cell softness dictates cellular sensitivity to force, suggesting that local small forces might have far more important roles in early development of soft embryos than previously appreciated.

The embryonic stem (ES) cell is a stem cell derived from early embryos that can indefinitely repeat self-renewing cell division cycles as an undifferentiated cell in vitro and give rise to all specialised cell types in the body. However, manipulating ES cell differentiation in vitro is a challenge due to, at least in part, heterogeneous gene induction. Recent experimental evidence has demonstrated that undifferentiated mouse ES cells maintained in culture exhibit heterogeneous expression of Dppa3, Nanog, Rex1, Pecam1 and Zscan4 as well as genes (Brachyury/T, Rhox6/9 and Twist2) normally expressed in specialised cell types. The Nanog-negative, Rex1-negative or T-positive ES cell subpopulation has a unique differentiation potential. Thus, studying the mechanism that generates ES cell subpopulations will improve manipulation of ES cell fate and help our understanding of the nature of embryonic development.

Summary: Combining high-content screening (HCS) with random gene-trap mutagenesis could be a powerful tool to investigate transcriptional networks, cell signaling, chemical genetics, and developmental processes. However, a critical limitation has been poor quantification of reporter expression per cell. To overcome this hurdle, we generated a variety of Gtx-based expression cassettes and re-evaluated translational enhancement of arrayed Gtx segments in tandem by HCS. We then modified the cassette into a new polyA trap vector, which consists of a variant of yellow fluorescent protein, Venus, in combination with the Gtx segments. Expression of Venus was detected in about 60% of trapped genes assayed in embryonic stem cell (ESC) cultures, comparable to expression screening of LacZ-based vectors. Furthermore, tetraploid aggregations using a clone encoding a gene-trap insertion into Twist2 demonstrated identical spatiotemporal expression between Venus and Twist2. This highly sensitive reporter system is amenable to high-throughput expression-based real-time HCS including single cell analyses.

Stem cell fate is governed by the integration of intrinsic and extrinsic positive and negative signals upon inherent transcriptional networks. To identify novel embryonic stem cell (ESC) regulators and assemble transcriptional networks controlling ESC fate, we performed temporal expression microarray analyses of ESCs after the initiation of commitment and integrated these data with known genome-wide transcription factor binding. Effects of forced under- or overexpression of predicted novel regulators, defined as differentially expressed genes with potential binding sites for known regulators of pluripotency, demonstrated greater than 90% correspondence with predicted function, as assessed by functional and high-content assays of self-renewal. We next assembled 43 theoretical transcriptional networks in ESCs, 82% (23 out of 28 tested) of which were supported by analysis of genome-wide expression in Oct4 knockdown cells. By using this integrative approach, we have formulated novel networks describing gene repression of key developmental regulators in undifferentiated ESCs and successfully predicted the outcomes of genetic manipulation of these networks.

In our earlier attempt to identify genes involved in the maintenance of cellular pluripotency, we found that KH-domain protein Embryonal stem cell-specific gene 1 (Esg1) showed similar expression patterns to those of Oct3/4 (Pou5f1), whereas the forced repression of Oct3/4 in mouse embryonic stem cells immediately downregulated the expression of Esg1. Here we further confirm this overlap by in situ hybridization and immunohistochemical analyses. Both Esg1 transcript and protein exist in the egg and preimplantation embryos. At embryonic day 3.5, blastocyst stage, however, ESG1 protein was more abundant in the inner cell mass (ICM) than in trophectoderm (TE), whereas Esg1 transcript was detected in both the ICM and the TE, particularly in the polar trophectoderm. The presence of an RNA-binding KH-domain in ESG1 led us to search for and identify 902 target transcripts by microarray analysis of immunoprecipitated ESG1 complex. Interaction of 20 target mRNA with ESG1, including Cdc25a, Cdc42, Ezh2, Nfyc and Nr5a2, was further validated by reverse transcriptase-polymerase chain reaction of the immunoprecipitation material, supporting the notion that ESG1 is an RNA-binding protein which associates with specific target transcripts.

Interferon regulatory factor-8 (IRF-8)/interferon consensus sequence-binding protein (ICSBP) is a transcription factor that controls myeloid-cell development. Microarray gene expression analysis of Irf-8-/- myeloid progenitor cells expressing an IRF-8/estrogen receptor chimera (which differentiate into macrophages after addition of estradiol) was used to identify 69 genes altered by IRF-8 during early differentiation (62 up-regulated and 7 down-regulated). Among them, 4 lysosomal/endosomal enzyme-related genes (cystatin C, cathepsin C, lysozyme, and prosaposin) did not require de novo protein synthesis for induction, suggesting that they were direct targets of IRF-8. We developed a reporter assay system employing a self-inactivating retrovirus and analyzed the cystatin C and cathepsin C promoters. We found that a unique cis element mediates IRF-8-induced activation of both promoters. Similar elements were also found in other IRF-8 target genes with a consensus sequence (GAAANN[N]GGAA) comprising a core IRF-binding motif and an Ets-binding motif; this sequence is similar but distinct from the previously reported Ets/IRF composite element. Chromatin immunoprecipitation assays demonstrated that IRF-8 and the PU.1 Ets transcription factor bind to this element in vivo. Collectively, these data indicate that IRF-8 stimulates transcription of target genes through a novel cis element to specify macrophage differentiation.

Given that thrombin is ubiquitously expressed at sites of vascular injury, and that osteogenic cells express receptors for thrombin, we questioned whether thrombin could attract osteogenic cells to a bony wound. Using a scratch wound assay, thrombin stimulated a significant increase in migration of osteogenic cultures of primary marrow cells. This effect was dependent on thrombin proteolytic activity; however, thrombin was unable to stimulate the migration of a more differentiated marrow-derived osteogenic cell line. To better understand the role of thrombin in osteoprogenitor migration, we developed an osteoprogenitor migration assay that combines a modified Boyden chamber with a bone nodule assay. Primary cells that migrated through the transwell filter in the presence of thrombin formed significantly more bone nodules compared to the condition without thrombin. This was not due to proliferation or differentiation effects of thrombin. In contrast, thrombin was unable to stimulate an increase in the number of nodules for the more differentiated osteogenic cell line. Thus, our results suggest that thrombin exhibits differential motogenic effects on osteogenic cells depending on their differentiation state. The cell migration/bone nodule assay described here is the first assay that can be specifically used to examine the effects of factors on the migration of osteoprogenitor cells, particularly those derived from primary populations.

As a first step to understand preimplantation development, we performed global gene-expression profiling of morula and blastocyst using the NIA 15k mouse cDNA microarray. Gene expression levels were measured four times for blastocyst and five times for morula. Student's t-test at the 5% significance level identified 428 genes upregulated and 748 downregulated in blastocyst compared to morula. This trend was consistent with semi-quantative RT-PCR analysis of sample genes. The upregulated genes known to be involved in critical regulatory processes, included Mist1, Id2, Hd1, and Requiem; the downregulated genes included CREB-binding protein, Per3, zinc finger protein 217, Krox-25, and miwi1. Such well-characterized genes and many novel genes provide markers for early stages in development and starting materials for further functional studies.

A catalog of mouse genes expressed in early embryos, embryonic and adult stem cells was assembled, including 250000 ESTs, representing approximately 39000 unique transcripts. The cDNA libraries, enriched in full-length clones, were condensed into the NIA 15 and 7.4K clone sets, freely distributed to the research community, providing a standard platform for expression studies using microarrays. They are essential tools for studying mammalian development and stem cell biology, and to provide hints about the differential nature of embryonic and adult stem cells.

WNT signaling plays a major role in patterning the dermomyotome of the somitic mesoderm. However, knowledge of downstream target genes and their regulation is limited. To identify new genes involved in the development and early patterning of the somite, we performed a comparison of gene expression by microarray between the presomitic mesoderm and the 5 most recently formed somites of the mouse at embryonic day 9.5. We identified 207 genes upregulated and 120 genes downregulated in somite formation. Expression analysis and functional categorization of these genes demonstrate this to be a diverse pool that provides a valuable resource for studying somite development. Thus far, we have found three genes expressed in the dermomyotome of the early somite. Consistent with their expression patterns, these genes are transcriptional targets of WNT signals, but display differential activation by different WNTs. We further demonstrate that 1 of these genes, Troy, is a direct target of canonical WNT signaling, while the other 2 genes, Selp and Arl4, are not. Thus, our microarray study using microdissected tissues not only provides global information on gene expression during somite development, it also provides novel targets to study the inductive signaling pathways that direct somite patterning.

Microarray expression profiling of a collection of 15,000 mouse genes with placental and embryonic RNAs revealed candidates for placental-enriched genes, three of which we have confirmed and further characterized. One, Plac1, strongly expressed in all trophoblast-derived cells in the placenta, has been described earlier (Genomics 68 (2000) 305). Here we report that of the other two, Plac8 expression is restricted to the spongiotrophoblast layer during development, whereas Plac9 is weakly expressed though highly enriched in placenta. For both, cDNAs with complete open reading frames were recovered and exon-intron structures inferred from comparisons of mouse cDNA and genomic sequence. The predicted proteins (112 and 108 amino acids) both contain putative signal peptides, with a coiled-coil segment of mPLAC9 as the only other detected motif. Genomic sequence comparisons reveal that in addition to an apparent pseudogene on chromosome 1, Plac8 is expressed at mouse cytoband 5e3. It is tightly conserved in human in a syntenically equivalent ortholog at 4q21.23. Plac9 is present in a single copy on chromosome 14, with a syntenically equivalent human ortholog at 10q22.3. Putative promoter regions up to 10 kb 5' of the transcription units for Plac1, Plac8, and Plac9 contain sites for widely-expressed transcription factors which, by analogy to other instances, may be sufficient to explain placental enrichment.

Large-scale gene expression profiling was performed on embryo-derived stem cell lines to identify molecular signatures of pluripotency and lineage specificity. Analysis of pluripotent embryonic stem (ES) cells, extraembryonic-restricted trophoblast stem (TS) cells, and terminally-differentiated mouse embryo fibroblast (MEF) cells identified expression profiles unique to each cell type, as well as genes common only to ES and TS cells. Whereas most of the MEF-specific genes had been characterized previously, the majority (67%) of the ES-specific genes were novel and did not include known differentiated cell markers. Comparison with microarray data from embryonic material demonstrated that ES-specific genes were underrepresented in all stages sampled, whereas TS-specific genes included known placental markers. Investigation of four novel TS-specific genes showed trophoblast-restricted expression in cell lines and in vivo, whereas one uncharacterized ES-specific gene, Esg-1, was found to be exclusively associated with pluripotency. We suggest that pluripotency requires a set of genes not expressed in other cell types, whereas lineage-restricted stem cells, like TS cells, express genes predictive of their differentiated lineage.