Self-organization of a human organizer by combined Wnt and Nodal signalling.

In amniotes, the development of the primitive streak and its accompanying 'organizer' define the first stages of gastrulation. Although these structures have been characterized in detail in model organisms, the human primitive streak and organizer remain a mystery. When stimulated with BMP4, micropatterned colonies of human embryonic stem cells self-organize to generate early embryonic germ layers 1 . Here we show that, in the same type of colonies, Wnt signalling is sufficient to induce a primitive streak, and stimulation with Wnt and Activin is sufficient to induce an organizer, as characterized by embryo-like sharp boundary formation, markers of epithelial-to-mesenchymal transition and expression of the organizer-specific transcription factor GSC. Moreover, when grafted into chick embryos, human stem cell colonies treated with Wnt and Activin induce and contribute autonomously to a secondary axis while inducing a neural fate in the host. This fulfils the most stringent functional criteria for an organizer, and its discovery represents a milestone in human embryology.

Author Correction: Self-organization of a human organizer by combined Wnt and Nodal signalling.

Ref. 7 from Benvenisty and colleagues was inadvertently omitted; this has now been cited in the text and added to the reference list, and subsequent references have been renumbered. The Letter has been corrected online.

What are the risk factors for postoperative infections of third molar extraction surgery: A retrospective clinical study?

BACKGROUND: This study aimed to identify (1) the predilection site of postoperative infection after third molar extraction surgery, (2) risk factors associated with postoperative infection, and (3) the cause of the difference between delayed- and early-onset infections. MATERIALS AND METHODS: This retrospective study included 1010 patients (396 male, 614 female) who had ≥1 third molars extracted (2407; 812 maxilla, 1595 mandible). The risk factors were classified as attributes, general health, anatomic, and operative. Outcome variables were delayed- and early-onset infections. RESULTS: Postoperative infection was completely absent in the maxilla, and all infections occurred in the mandible, with a probability of 1.94% (31/1595). Bivariate analysis for postoperative infection showed depth of inclusion and intraoperative hemostatic treatment to be significantly associated with the development of infections. Bivariate analysis for delayed- and early-onset infections showed simultaneous extraction of the left and right mandibular third molars to be prominent risk factors. CONCLUSIONS: Postoperative infection occurs mainly in the mandible, and that in the maxilla is very rare. The risk of postoperative infection in the mandible was found to be related to the depth of inclusion and intraoperative hemostatic treatment. Simultaneous extraction of the left and right mandibular third molars appear to increase the risk of delayed-onset postoperative infection.

Impact of prestorage leucoreduction of autologous whole blood on length of hospital stay with a subgroup analysis in bilateral hip arthroplasty.

BACKGROUND: Although prestorage leucoreduction (LR) of blood components for transfusion has gained favour around the world, evidence of its beneficial clinical effects is ambiguous. STUDY DESIGN AND METHODS: To reveal whether leucocytes and/or platelets in transfused blood are related to transfusion-related adverse effects, a prospective randomized crossover study was performed on patients who donated autologous blood prior to elective surgery. Among 1487 primary enrolees, a total of 192 patients undergoing two-stage, bilateral total hip arthroplasty were randomized to receive autologous blood that was either prestorage leucoreduced, or not, for the first procedure. For the second procedure, each patient was crossed over to receive alternatively processed autologous blood. Length of hospital stay served as a primary end-point, with perioperative infectious/thrombotic complications, pre- and postoperative laboratory values, and body temperature serving as secondary endpoints. RESULTS: No significant differences emerged between prestorage LR and non-LR cohorts in length of hospital stay, as well as perioperative infectious/thrombotic complications, postoperative body temperature and duration of fever. Postoperative laboratory values including white blood cell counts and C-reactive protein levels had no significant differences. CONCLUSION: This study could not prove any superiority of prestorage LR over non-LR for autologous whole blood among patients who underwent total hip arthroplasty.

Analysis of the essentiality of ROM2 genes in the pathogenic yeasts Candida glabrata and Candida albicans using temperature-sensitive mutants.

AIMS: To analyse the essentiality of the ROM2 genes originating from the pathogenic yeasts Candida glabrata and Candida albicans by using temperature-sensitive (ts) mutants. METHODS AND RESULTS: Based on the general concepts that ts mutations are generated by virtue of point mutation within essential genes, we have previously established a novel method (termed 'ETS system' for screening and identification of essential genes using ts mutants of C. glabrata). According to this ETS system, the present study successfully identified a putative C. glabrata ROM2 homologue as an essential gene that complements its point mutation (Cys-1275/Tyr substitution). The C. albicans ROM2 mutant (Cys-1281/Tyr), constructed patterned after this point mutation, also displayed ts phenotype. Both ts mutants recovered colony-forming ability, with concomitant suppression of lysis phenotype, at the elevated temperature in the presence of 1 mol l(-1) sorbitol as an osmotic stabilizer. Sequence alignment revealed that human genome possesses relatively low homology against Rom2 homologues, which are highly conserved among yeast species. CONCLUSIONS: ROM2 genes of C. glabrata and C. albicans are essential for viability, probably involved in cell wall integrity. SIGNIFICANCE AND IMPACT OF THE STUDY: ROM2 genes essential for both Candida species may be a potentially useful antifungal targets from chemotherapeutic viewpoint.

Barriers and motivators to blood donation among university students in Japan: development of a measurement tool.

BACKGROUND AND OBJECTIVES: Despite growing demand for transfusion, the number of voluntary young blood donors has steadily decreased over recent years in Japan. This study aimed to develop an easy-to-use survey tool to assess barriers and motivators to blood donation among Japanese university students. MATERIALS AND METHODS: We conducted cross-sectional studies at two universities in Fukushima Prefecture, Japan, in December 2011 (Stage 1) and February 2012 (Stage 2) using self-administered questionnaires. A short list of motivators and barriers to blood donation was developed from the open-ended questions asked of 50 students in Stage 1. In the Stage 2, we asked 105 students how important these items were when they decided whether or not to donate blood. Items showing a significant difference between donors and non-donors were kept in the final list. RESULTS: Overall, 56% of the 100 participants analysed in Stage 2 were men, and ages ranged from 19 to 24 with a median of 20 years. Comparison of motivators and barriers between donors and non-donors revealed that only barrier item 8 ('Frightened by blood donation') showed a significant difference (P = 0·0006) in an expected direction and with a consistency between two universities. CONCLUSIONS: This study identified fear as being the most significant barrier to blood donation among Japanese university students, which could be used as a single convenient indicator to assess their readiness to donate. More academic and clinical efforts are needed to understand and address students' fear towards blood donation in order to increase the donor pool in Japan.

Impact of amino acid 233 in Tax on bovine leukemia virus infection in Japanese Black cattle.

Bovine leukemia virus (BLV) is an economically important pathogen that both causes fatal enzootic bovine leukosis (EBL) and reduces lifetime milk production, reproductive efficiency, carcass weight, and longevity in dairy cows. The virus can be divided into two categories based on the amino acid at position 233 in Tax protein, which activates viral transcription and probably plays crucial roles in leukemogenesis. We recently reported that early-onset EBL in Japanese Black (JB) cattle was frequently caused by L233-Tax-carrying virus. This study examined the impact of BLV infection, the proviral load (PVL), and amino acid 233 in Tax on the outcomes of JB cattle. We measured PVL in cattle enrolled between February 2016 and December 2018, determined the Tax type of the isolates, and performed follow-up until March 2022. The results demonstrated that BLV infection increased the risk of involuntary culling and mortality in JB cattle in a PVL-dependent manner. Infection with L233-Tax-carrying virus increased the likelihood of mortality by 1.6-fold compared with the effects of P233-Tax-carrying virus infection. Intrauterine and perinatal infections were frequently caused by L233-Tax-carrying virus, and these infections were likely to influence the early onset of EBL in JB cattle. Conversely, breeding cows infected with P233-Tax-carrying virus were often eliminated by involuntary culling. These findings indicate that amino acid 233 in Tax has importance in terms of preventing economic loss attributable to EBL in JB cattle.

Characteristic endoscopic findings of gastrointestinal malignant lymphomas other than mucosa-associated lymphoid tissue lymphoma.

Background and study aims: The gastrointestinal (GI) tract is the most common site of extra-nodal involvement for non-Hodgkin's lymphoma (NHL). The features of GI NHLs remain unclear. The aim of this study was to clarify endoscopic characteristics of GI NHLs. Patients and methods: We retrospectively analyzed the morphological characteristics of 63 GI malignant lymphomas other than mucosa-associated lymphoid tissue lymphoma. Lesions were diagnosed between 2005 and 2020. Macroscopic findings were classified into five subtypes: superficial (S); protruding without ulcer (P); protruding with ulcer (PU); fungating (F); and multiple nodules (MN). Results: Thirty-one lesions in the stomach were classified as S type in 3 cases (9.6%), P type in 6 (19%), PU type in 13 (42%), and F type in 9 (29%). In the stomach, the ulcerated phenotype was more frequent for diffuse large B-cell lymphoma (DLBCL) (89.5%) than for other histological types (41.7%; P = 0.01). In the intestine, 23 tumors were classified as S type in 4 cases (17%), P type in 1 (4%), PU type in 6 (26%), F type in 1 (4%), and MN in 11 (48%). Eleven of the 14 cases (78.6%) of intestinal follicular lymphoma lesions showed MN type. In the colon, eight tumors were classified as S type in 2 cases (25%), P type in 2 (25%), PU type in 1 (13%), and F type in 3 (38%). Conclusion: We have clarified the endoscopic features of GI NHL using macroscopic classifications. The ulcerated phenotype was the most frequent endoscopic finding for DLBCL.

Splenectomy and antiviral treatment for thrombocytopenic patients with chronic hepatitis C virus infection.

Thrombocytopenic patients with chronic hepatitis C virus (HCV) infection are poor candidates for antiviral treatment with interferon (IFN), but no standard treatment for thrombocytopenia has yet been established. We evaluated the safety of splenectomy and its efficacy for the initiation and continuation of antiviral therapy. From March 2003 to April 2006, 10 patients (mean age 62.5 years) with HCV-related cirrhosis, low platelet count (<==106 000/mm(3)) and splenomegaly (spleen size >==10 cm) underwent splenectomy. Platelet counts significantly increased at 4-8 weeks after splenectomy [pre: 64 200 +/- 6900/mm(3)vs post 209 000 +/- 40 600/mm(3) (P = 0.004)]. No severe operative complications were observed. All patients subsequently received antiviral therapy. Of the eight patients who were infected with HCV genotype 1 and had a high viral load (>==100 KIU/mL), four received combination therapy with pegylated IFNalpha-2b plus ribavirin, and the other four received standard IFNalpha-2b plus ribavirin. One patient infected with HCV genotype 2 and another with HCV genotype 1 and a low viral load (<100 KIU/mL) were treated with pegylated IFNalpha-2a. Six patients achieved sustained virologic response (SVR). Among four patients who failed to achieve SVR, one was given retreatment with pegylated IFN plus ribavirin, and the other three received low-dose long-term IFN therapy. Although this study was small, the treatment results were similar to those for patients without thrombocytopenia and suggested that splenectomy would not reduce the antiviral efficacy of IFNalpha-based treatment.

Significance of pre-storage leucoreduction for autologous blood.

BACKGROUND AND OBJECTIVES: To reveal the associations between cytokines in blood and transfusion-related adverse events, we studied whether pre-storage leucoreduction of autologous blood could reduce the degree of inflammatory responses, infection rates, or the duration of hospitalizations. MATERIALS AND METHODS: Patients scheduled to donate autologous blood for elective orthopaedic surgery were assigned to receive either leucoreduced (LR) or non-leucoreduced (N-LR) autologous blood based on their date of birth. Levels of cytokines in the autologous blood, values for C-reactive protein, complete blood count and body temperature of the patients, as well as adverse clinical events, were evaluated periodically. RESULTS: Four hundred patients entered this study (LR group: 196, N-LR group: 204). The production of cytokines, excluding interleukin 1beta (IL-1beta), was suppressed for the LR group. However, for unknown reasons, IL-1beta actually increased during storage for the LR group. There were no differences between the two groups in the length of hospital stay, postoperative C-reactive protein changes, leucocyte count, or body temperature, and no clinical problems associated with blood transfusion were observed in either group. CONCLUSION: Pre-storage leucoreduction for autologous blood may be effective to suppress cytokine accumulation. However, clinical benefits such as prevention of febrile non-haemolytic reactions could not be demonstrated.

Survival and recovery of apheresis platelets stored in a polyolefin container with high oxygen permeability.

BACKGROUND AND OBJECTIVES: Oxygen permeability is important in platelet storage media. We compared a new polyolefin container with enhanced oxygen permeability (PO-80; Kawasumi, Tokyo, Japan) to a widely used alternative (PL2410; Baxter Healthcare, Deerfield, IL, USA). MATERIALS AND METHODS: In vitro characteristics of paired platelet concentrates (PCs; mean 4.2 x 10(11)/250 ml plasma/bag) stored in PO-80 or PL2410 were assessed through 9 days of storage. In vivo recovery and survival of 7-day-old autologous PCs were assessed according to the Murphy method. RESULTS: Laboratory assessment of platelet quality favoured PO-80 during 9 days of storage with statistically significant differences in glucose consumption (2.75 vs. 4.93 mmol/10(12)/24 h in the interval 120-168 h), lactate generation (4.37 vs. 8.11 mmol/10(12)/24 h in the interval 120-168 h), pressure of oxygen (pO(2)) (59.3 vs. 38.1 mmHg at day 1), and HCO(3)(-) (14.7 vs. 13.4 mmol/l at day 1). Statistically significant differences were not seen in aggregation, hypotonic shock response or pH. In vivo assessment of autologous platelets stored 7 days in the PO-80 container revealed that recovery was 82.1% and survival was 81.0% of fresh control. Seven-day stored PCs in PO-80 were shown in vivo to be non-inferior to fresh platelets, with upper confidence limits (UCL(95)) in recovery and survival of stored PCs below the maximum acceptable difference (MAD); 15.3% UCL(95) < 20.4% MAD and 2.1 days UCL(95) < 2.1 days MAD. CONCLUSIONS: The in vitro characteristics of PCs stored in a highly oxygen-permeable container were stable at least 7 days. The in vivo study supports the suitability of PO-80 for 7-day platelet storage.

Endotracheal tube stabilization using an orthodontic skeletal anchor in a patient with facial burns.

Stabilizing the endotracheal tube is of vital importance in patients suffering facial burns or trauma in the intensive care unit, as well as during a general anaesthetic procedure. Here is presented a secure method using a simple orthodontic skeletal anchorage system on the maxilla and 0.4-mm stainless steel wire that does not require any work or place any burden on the teeth or gingival tissue, and does not require extensive surgery.

Is the removal of osteosynthesis plates after orthognathic surgery necessary? Retrospective long-term follow-up study.

The removal of titanium miniplates is a controversial topic in oral and maxillofacial surgery. This retrospective study examined the timing of and reasons for titanium plate removal after orthognathic surgery. The study included 240 orthognathic surgery patients (71 male, 169 female; age range 16-55 years, mean 25.0±8.8 years) who had maxillofacial osteosynthesis plates inserted or inserted and then removed at the Division of Oral and Maxillofacial Surgery, Kagawa Prefectural Central Hospital, between April 2003 and March 2017. During the study period, a total of 717 miniplates were inserted in the 240 patients, and 71 of the patients (29.6%) had 236 plates (32.9%) removed. Ten patients (14.1%) had their plates removed within a year due to early complications. Although no patient had their plate removed due to complications at 1-5 years postoperative, a further 14 patients (19.7%) had their plates removed after more than 5 years of long-term follow-up due to plate-related complications. Complications requiring plate removal were evidently biphasic, occurring within 1 year after the operation and at ≥5 years after the operation. Therefore, after confirming postoperative bone healing, it is necessary to explain to patients the risks of plate removal and the importance of long-term follow-up.

Potentiation of cholecystokinin-induced exocrine secretion by both exogenous and endogenous insulin in isolated and perfused rat pancreata.

Using an isolated perfused rat pancreas preparation, the interrelationship between the endocrine and exocrine portions of the pancreas were studied. Addition of exogenous rat insulin (1-20 mU/ml) to the perfusing solution potentiated the action of cholecystokinin (CCK) (1 mU/ml) to increase both pancreatic juice flow and the release of the enzyme, amylase. Raising the glucose concentration in the perfusing solution from 2.5 to 17.5 mM both increased endogenous insulin release and potentiated the CCK-induced exocrine secretory response. Two lines of evidence indicated that this effect of glucose on the exocrine pancreas was mediated by endogenous insulin release. First, the addition of comparable amounts of xylose or galactose to the perfusion medium neither released insulin nor potentiated the CCK-induced response. Second, epinephrine blocked the effect of high glucose on both insulin release and potentiation of CCK action. Epinephrine alone did not affect the action of CCK. The magnitude of the exocrine response induced by high glucose was comparable to that of 2.5 mU/ml exogenous insulin. It seems possible that pancreatic acinar cells can be exposed to insulin levels of this magnitude in situ.

Kinetic analysis of human T-cell leukemia virus type I Tax-mediated activation of NF-kappa B.

The human T-cell leukemia virus type I (HTLV-I) Tax protein induces the expression of cellular genes, at least in part, by activating the endogenous NF-kappa B transcription factors. Induced expression of cellular genes is thought to be important for transformation of T cells to continued growth, a prelude to the establishment of adult T-cell leukemia. However, neither underlying mechanisms nor kinetics of the Tax-mediated activation of NF-kappa B are understood. We have analyzed a permanently transfected Jurkat T-cell line in which the expression of Tax is entirely dependent on addition of heavy metals. The initial NF-kappa B binding activity seen after induction of Tax is due almost exclusively to p50/p65 heterodimers. At later times, NF-kappa B complexes containing c-Rel and/or p52 accumulate. The early activation of p50/p65 complexes is a posttranslational event, since neither mRNA nor protein levels of NF-kappa B subunits had increased at that time. We demonstrate for the first time a Tax-induced proteolytic degradation of the NF-kappa B inhibitor, I kappa B-alpha, which may trigger the initial nuclear translocation of NF-kappa B. As nuclear NF-kappa B rapidly and potently stimulates resynthesis of I kappa B-alpha, the steady-state level of I kappa B-alpha does not significantly change. Thus, the dramatic Tax-induced increase in the I kappa B-alpha turnover may continually weaken inhibition and activate NF-kappa B. Additional, distinct actions of Tax may contribute further to the high levels of NF-kappa B activity seen.

Cytoplasmic sequestration of the polyomavirus enhancer binding protein 2 (PEBP2)/core binding factor alpha (CBFalpha) subunit by the leukemia-related PEBP2/CBFbeta-SMMHC fusion protein inhibits PEBP2/CBF-mediated transactivation.

The polyomavirus enhancer binding protein 2 (PEBP2)/core binding factor (CBF) is a transcription factor composed of two subunits, alpha and beta. The gene encoding the beta subunit is disrupted by inv(16), resulting in the formation of a chimeric protein, beta-SMMHC, which is associated with acute myelogenous leukemia. To understand the effect of beta-SMMHC on PEBP2-mediated transactivation, we used a luciferase assay system in which contribution of both the alpha and beta subunits was absolutely required to activate transcription. Using this system, we found that the minimal region of the beta subunit required for transactivation resides between amino acid 1 and 135, which is known to dimerize with the alpha subunit. In contrast, beta-SMMHC, despite having this minimal region for dimerization and transactivation, failed to support transcription with the alpha subunit. Furthermore beta-SMMHC blocked the synergistic transcription achieved by PEBP2 and CCAAT/enhancer binding protein alpha. By using a construct in which the PEBP2 alpha subunit was fused to the glucocorticoid receptor ligand binding domain, we demonstrated that coexpressed beta-SMMHC tightly sequestered the alpha subunit in the cytoplasm and blocked dexamethasone-dependent nuclear translocation of the alpha subunit. Thus, the result suggess that beta-SMMHC inhibits PEBP2-mediated transcription via cytoplasmic sequestration of the alpha subunit. Lastly proliferation of ME-1 cells that harbor inv(16) was blocked by an antisense oligonucleotide complementary to the junction of the chimeric mRNA, suggesting that beta-SMMHC contributes to leukemogenesis by blocking the differentiation of myeloid cells.

Intrinsic transcriptional activation-inhibition domains of the polyomavirus enhancer binding protein 2/core binding factor alpha subunit revealed in the presence of the beta subunit.

A member of the polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF) is composed of PEBP2 alphaB1/AML1 (as the alpha subunit) and a beta subunit. It plays an essential role in definitive hematopoiesis and is frequently involved in the chromosomal abnormalities associated with leukemia. In the present study, we report functionally separable modular structures in PEBP2 alphaB1 for DNA binding and for transcriptional activation. DNA binding through the Runt domain of PEBP2 alphaB1 was hindered by the adjacent carboxy-terminal region, and this inhibition was relieved by interaction with the beta subunit. Utilizing a reporter assay system in which both the alpha and beta subunits are required to achieve strong transactivation, we uncovered the presence of transcriptional activation and inhibitory domains in PEBP2 alphaB1 that were only apparent in the presence of the beta subunit. The inhibitory domain keeps the full transactivation potential of full-length PEBP2 alphaB1 below its maximum potential. Fusion of the transactivation domain of PEBP2 alphaB1 to the yeast GAL4 DNA-binding domain conferred transactivation potential, but further addition of the inhibitory domain diminished the activity. These results suggest that the activity of the alpha subunit as a transcriptional activator is regulated intramolecularly as well as by the beta subunit. PEBP2 alphaB1 and the beta subunit were targeted to the nuclear matrix via signals distinct from the nuclear localization signal. Moreover, the transactivation domain by itself was capable of associating with the nuclear matrix, which implies the existence of a relationship between transactivation and nuclear matrix attachment.

A novel transcript encoding an N-terminally truncated AML1/PEBP2 alphaB protein interferes with transactivation and blocks granulocytic differentiation of 32Dcl3 myeloid cells.

The gene AML1/PEBP2 alphaB encodes the alpha subunit of transcription factor PEBP2/CBF and is essential for the establishment of fetal liver hematopoiesis. Rearrangements of AML1 are frequently associated with several types of human leukemia. Three types of AML1 cDNA isoforms have been described to date; they have been designated AML1a, AML1b, and AML1c. All of these isoforms encode the conserved-Runt domain, which harbors the DNA binding and heterodimerization activities. We have identified a new isoform of the AML1 transcript, termed AML1 deltaN, in which exon 1 is directly connected to exon 4 by alternative splicing. The AML1 deltaN transcript was detected in various hematopoietic cell lines of lymphoid to myeloid cell origin, as revealed by RNase protection and reverse transcriptase PCR analyses. The protein product of AML1 deltaN lacks the N-terminal region of AML1, including half of the Runt domain, and neither binds to DNA nor heterodimerizes with the beta subunit. However, AML1 deltaN was found to interfere with the transactivation activity of PEBP2, and the molecular region responsible for this activity was identified. Stable expression of AML1 deltaN in 32Dcl3 myeloid cells blocked granulocytic differentiation in response to granulocyte colony-stimulating factor. These results suggest that AML1 deltaN acts as a modulator of AML1 function and serves as a useful tool to dissect the functional domains in the C-terminal region of AML1.

Reconstruction of upper limb motor function using functional electrical stimulation (FES).

Functional electrical stimulation (FES) techniques progress by adopting the developments in computers and engineering, but complete functional reconstruction is not yet possible to be achieved. The attachment of the devices to the body can be complex, and training to handle FES is not easy. FES systems are expensive and their coverage by medical insurance is limited with the exception of a few systems. Hence, recognition of FES by the medical community is limited and as a result, it is not a common therapy. However, FES is the main method available for reconstruction of motor function, at present. The improvement in activities of daily living (ADL) of patients using FES may not only improve the patient's quality of life (QOL) but also reduce the burden to persons who look after them, and hence, secure a valuable work force. The medical insurance should support the use of FES and reduce the patients' financial burden. Studies and developments based on a close collaboration of users (patients and care-givers), persons involved in therapy (doctors and nurses), and manufactures (engineers and technicians) are necessary. In addition to FES, other methods such as therapeutic electrical stimulation (TES) for prevention of atrophy and spasms of paralytic limbs show the therapeutic potential of neuromodulation.

Dorsal column stimulation for persistent vegetative state.

Dorsal column stimulation (DCS) is described as a therapy for persistent deterioration of consciousness. The mechanism of its effect has not yet been elucidated. Various other methods, such as deep brain stimulation of the CM-p f complex, vagus nerve stimulation, and musical functional therapy, are being investigated as potential treatments of this problem. We present our series of DCS for persistent vegetative state and review the potential mechanisms of action and the relevant literature.