Tangentially migrating cells differentiate into stratum griseum central neurons to form tectofugal visual pathway in the developing chick optic tectum.

BACKGROUND: The optic tectum is the main visual processor of nonmammalian vertebrates and relays visual information from the eye to the telencephalon via the tectofugal pathway. In the development of the avian optic tectum, while the multipolar neurons are arranged by tangential migration, the behavior of individual cells in tangential migration, neural differentiation, and cell fate remain unclear. Here, we pursued the transition of tangentially migrating cells and their involvement in visual circuit formation during chick development. RESULTS: After tangential movement along the axons, the migrating cells relocated to the upper layers and turned back upon differentiation toward the multipolar neurons. The multipolar neurons are destined to differentiate into the stratum griseum central (SGC) neurons with the large dendritic field, which form the tectorotundal projection. Trans-synaptic virus labeling demonstrated that the tangentially migrating cells eventually participate in the tectofugal visual pathway. CONCLUSIONS: These results indicate that tangential migration is a crucial process in the formation of the tectofugal visual pathway during the development of the optic tectum.

Cadaveric and CT angiography study of vessels around the transverse colon mesentery.

BACKGROUND: Laparoscopic and robotic surgery for transverse colon cancer are difficult due to complex fusion of the foregut and midgut and variation of the vessels of the transverse colon. Although the vessels of the right colon have been investigated, middle colic artery (MCA) variation and the relationship with vessels around the transvers colon are unknown. We investigated variation of the MCA using computed tomography angiography (CTA) and cadaver specimen and the relationship between the superior mesenteric vein (SMV) and MCA using CTA. The classification of vessels around the transverse colon may lead to safer and reliable surgery. METHODS: This study included 505 consecutive patients who underwent CTA in our institution from 2014 to 2020 and 44 cadaver specimens. Vascular anatomical classifications and relationships were analyzed using CT images. RESULTS: The MCA was defined as the arteries arising from the superior mesenteric artery (SMA) that flowed into the transverse colon at the distal ends. The classifications were as follows: type I, branching right and left from common trunk; type II, the right and left branches bifurcated separately from the SMA; and type III, the MCA branched from a vessel other than the SMA. Type II was subclassified into two subtypes, type IIa with one left branch and type IIb with two or more left branches from SMA. In the CTA and cadaver studies, respectively, the classifications were as follows: type I, n = 290 and n = 31; type IIa, n = 211 and n = 13; type IIb, n = 3 and n = 0; and type III, n = 1 and n = 0. We classified the relationship between the MCA and left side of the SMV into three types: type A, a common trunk runs along the left edge of the SMV (n = 173; 59.7%); type B, a right branch of the MCA runs along the left edge of the SMV (n = 116; 40.0%); and type C, the MCA runs dorsal of the SMV (n = 1; 0.3%). CONCLUSIONS: This study revealed that The MCA branching classifications and relationship between the SMV and MCA. Preoperative CT angiography may be able to reliably identify vessel variation, which may be useful in clinical practice.

Impact of the hospital volume and setting on postoperative complications of surgery for gastroenterological cancers in a regional area of Western Japan.

PURPOSE: A research subgroup was established by the Japanese Society of Gastroenterological Surgery to improve the health care quality in the Chushikoku area of Western Japan. METHODS: The records of four surgical procedures were extracted from the Japanese National Clinical Database and analyzed retrospectively to establish the association between hospital characteristics, defined using a combination of hospital case-volume and patients' hospital travel distance, and the incidences of perioperative complications of ≥ Grade 3 of the Clavien-Dindo classification after gastroenterological surgery. RESULTS: This study analyzed 11,515 cases of distal gastrectomy for gastric cancer, 4,705 cases of total gastrectomy for gastric cancer, 4,996 cases of right hemicolectomy for colon cancer, and 5,243 cases of lower anterior resection for rectal cancer, with composite outcome incidences of 5.6%, 10.2%, 5.5%, and 10.7%, respectively. After adjusting for patient characteristics and surgical procedures, no association was identified between the hospital category and surgical outcomes. CONCLUSION: The findings of our study of the Chushikoku region did not provide positive support for the consolidation and centralization of hospitals, based solely on hospital case volume. Our grouping was unique in that we included patient travel distance in the analysis, but further investigations from other perspectives are needed.

Long-term prognosis of laparoscopic gastrectomy for patients on antithrombotic therapy: a retrospective cohort study.

PURPOSE: Few studies have reported on the interactions between gastrectomy and antithrombotic therapy, especially the long-term prognosis. We aimed to clarify the short- and long-term prognosis of gastrectomy for patients on antithrombotic therapy. METHODS: We reviewed the perioperative data and survival rate of patients who underwent laparoscopic distal gastrectomy (LDG) at our institute between 2010 and 2013. RESULTS: There were 119 patients enrolled in this retrospective study: 31 who were taking antithrombotic drugs (antithrombotic therapy (ATT) group), and 88 who were not (non-ATT group). The mean age was significantly higher in the ATT group than in the non-ATT group. No significant differences were observed in the amount of intraoperative bleeding or blood hemoglobin level after surgery between the groups. Bleeding complications occurred in only one patient from the ATT group, and the postoperative complication rate was comparable between the groups. During follow-up, cerebrovascular or cardiovascular events developed in 19.4% of the ATT group patients and 4.5% of the non-ATT group patients; however, there were no significant differences in the 5-year overall survival rates between the groups (ATT group, 76.9%; non-ATT group, 82.9%). CONCLUSIONS: Antithrombotic therapy did not affect the short-term or long-term prognosis of patients after LDG.

Perioperative coagulofibrinolytic responses in colorectal surgery patients without chemical thromboprophylaxis: a retrospective observational study.

PURPOSE: During the perioperative period, coagulofibrinolytic activation occurs, which occasionally results in thromboembolic complications. However, natural perioperative coagulofibrinolytic responses have not been well investigated. The present study examined perioperative coagulofibrinolytic changes and their association with the development of venous thromboembolism (VTE). METHODS: We retrospectively analyzed the changes in coagulofibrinolytic markers for 7 days in 70 patients undergoing elective colorectal surgery. To explore the natural coagulofibrinolytic response, we investigated patients not undergoing perioperative chemical thromboprophylaxis. RESULTS: Coagulation activation occurred from just after surgery to postoperative day (POD) 1, followed by a gradual decrease, but persisted to even POD 7. Fibrinolytic activity showed a tri-phasic response: activation, shutdown and reactivation. Consequently, fibrin/fibrinogen degradation product (FDP) and D-dimer levels continued to increase until POD 7. The development of deep vein thrombosis (DVT) was observed in 11 patients (15.7%). Postoperative sustained hyper-coagulation [soluble fibrin (SF) or thrombin-antithrombin complex (TAT) values on POD 7 > their normal limits] was significantly associated with the development of DVT (SF, p < 0.001; TAT, p = 0.001). CONCLUSION: We found initial coagulation activation and a tri-phasic response of fibrinolytic activity after colorectal surgery. Thus, physicians need to pay attention to these responses when attempting to prevent or treat VTE.

Endothelin receptor type A is involved in the development of oxaliplatin-induced mechanical allodynia and cold allodynia acting through spinal and peripheral mechanisms in rats.

Oxaliplatin, a platinum-based chemotherapeutic agent, frequently causes severe neuropathic pain typically encompassing cold allodynia and long-lasting mechanical allodynia. Endothelin has been shown to modulate nociceptive transmission in a variety of pain disorders. However, the action of endothelin varies greatly depending on many variables, including pain causes, receptor types (endothelin type A (ETA) and B (ETB) receptors) and organs (periphery and spinal cord). Therefore, in this study, we investigated the role of endothelin in a Sprague-Dawley rat model of oxaliplatin-induced neuropathic pain. Intraperitoneal administration of bosentan, a dual ETA/ETB receptor antagonist, effectively blocked the development or prevented the onset of both cold allodynia and mechanical allodynia. The preventive effects were exclusively mediated by ETA receptor antagonism. Intrathecal administration of an ETA receptor antagonist prevented development of long-lasting mechanical allodynia but not cold allodynia. In marked contrast, an intraplantar ETA receptor antagonist had a suppressive effect on cold allodynia but only had a partial and transient effect on mechanical allodynia. In conclusion, ETA receptor antagonism effectively prevented long-lasting mechanical allodynia through spinal and peripheral actions, while cold allodynia was prevented through peripheral actions.

Overexpression of Dok-7 in skeletal muscle enhances neuromuscular transmission with structural alterations of neuromuscular junctions: Implications in robustness of neuromuscular transmission.

Neuromuscular junctions (NMJs) are cholinergic synapses characterized by ultrastructural specializations, including the presynaptic active zones, the acetylcholine (ACh) release sites of the motor nerve terminal, and the postsynaptic junctional folds of muscle membrane, where ACh receptors (AChRs) cluster for efficient neuromuscular transmission. The formation and maintenance of NMJs are governed by the muscle-specific receptor tyrosine kinase MuSK. We had previously demonstrated that the muscle cytoplasmic protein Dok-7 is an essential activator of MuSK, and its activation and NMJ formation are enhanced in the Dok-7 transgenic (Tg) mice, in which Dok-7 is specifically overexpressed in skeletal muscle. Although Dok-7 Tg mice develop abnormally large NMJs but show normal motor function, the forced expression of Dok-7 in the muscle improves impaired motor activity in mouse models of neuromuscular disorders with NMJ defects. However, the effect of Dok-7 overexpression in skeletal muscle on ultrastructure and neuromuscular transmission of NMJs is yet to be studied. Here, we investigated the structural and electrophysiological properties of NMJs in the diaphragm muscle of 8-week-old Dok-7 Tg mice. The areas of the presynaptic motor nerve terminals and postsynaptic muscle membrane of NMJs were 2.7 and 4.3 times greater in Dok-7 Tg mice than in WT mice, respectively. Electrophysiological analyses revealed that neuromuscular transmission via NMJs in Dok-7 Tg mice was significantly enhanced but not proportionally with the increased size of the synaptic contact. Consistent with this, the densities of active zones and synaptic vesicles (ACh carriers) in the presynaptic motor nerve terminals were reduced. In addition, the density and size of postsynaptic junctional folds in the muscle membrane were also reduced. Moreover, terminal Schwann cells exhibited significantly greater penetration of their processes into the synaptic clefts, which connect the pre- and post-synaptic specializations. Together, our findings demonstrate that transgenic overexpression of Dok-7 in the skeletal muscle enhances neuromuscular transmission with significant enlargement and ultrastructural alterations of NMJs, the latter of which might prevent toxic overactivation of AChRs at the abnormally enlarged NMJs.

Dispersing movement of tangential neuronal migration in superficial layers of the developing chick optic tectum.

During embryonic brain development, groups of particular neuronal cells migrate tangentially to participate in the formation of a laminated structure. Two distinct types of tangential migration in the middle and superficial layers have been reported in the development of the avian optic tectum. Here we show the dynamics of tangential cell movement in superficial layers of developing chick optic tectum. Confocal time-lapse microscopy in organotypic slice cultures and flat-mount cultures revealed that vigorous cell migration continued during E6.5-E13.5, where horizontally elongated superficial cells spread out tangentially. Motile cells exhibited exploratory behavior in reforming the branched leading processes to determine their pathway, and intersected with each other for dispersion. At the tectal peripheral border, the cells retraced or turned around to avoid protruding over the border. The tangentially migrating cells were eventually distributed in the outer stratum griseum et fibrosum superficiale and differentiated into neurons of various morphologies. These results revealed the cellular dynamics for widespread neuronal distribution in the superficial layers of the developing optic tectum, which underline a mode of novel tangential neuronal migration in the developing brain.

SNX9 determines the surface levels of integrin ß1 in vascular endothelial cells: Implication in poor prognosis of human colorectal cancers overexpressing SNX9.

Angiogenesis, the formation of new blood vessels, is involved in a variety of diseases including the tumor growth. In response to various angiogenic stimulations, a number of proteins on the surface of vascular endothelial cells are activated to coordinate cell proliferation, migration, and spreading processes to form new blood vessels. Plasma membrane localization of these angiogenic proteins, which include vascular endothelial growth factor receptors and integrins, are warranted by intracellular membrane trafficking. Here, by using a siRNA library, we screened for the sorting nexin family that regulates intracellular trafficking and identified sorting nexin 9 (SNX9) as a novel angiogenic factor in human umbilical vein endothelial cells (HUVECs). SNX9 was essential for cell spreading on the Matrigel, and tube formation that mimics in vivo angiogenesis in HUVECs. SNX9 depletion significantly delayed the recycling of integrin ß1, an essential adhesion molecule for angiogenesis, and reduced the surface levels of integrin ß1 in HUVECs. Clinically, we showed that SNX9 protein was highly expressed in tumor endothelial cells of human colorectal cancer tissues. High-level expression of SNX9 messenger RNA significantly correlated with poor prognosis of the patients with colorectal cancer. These results suggest that SNX9 is an angiogenic factor and provide a novel target for the development of new antiangiogenic drugs.

Sudden onset of sleep caused by hypothalamic infarction: a case report.

BACKGROUND: Hypothalamic lesions, such as tumors and demyelinating diseases, reportedly cause abnormal sleepiness. However, stroke involving the hypothalamus has rarely been described. Here, we report a patient with infarction restricted to the hypothalamus who presented with sudden onset of sleep. CASE PRESENTATION: A 42-year-old woman with a history of migraine without aura presented with irresistible sleepiness and developed several episodes of sudden onset of sleep. Neurological examinations were unremarkable except for partial left Horner syndrome. Brain magnetic resonance imaging (MRI) revealed a high-intensity lesion restricted to the left hypothalamus on diffusion-weighted and fluid-attenuated inversion recovery MRI images. Cerebrospinal fluid (CSF) orexin-A levels obtained on hospital day 3 after her sleepiness had resolved were normal (337 pg/mL; normal > 200 pg/mL). Serum anti-nuclear and anti-aquaporin 4 (AQP4) antibodies and CSF myelin basic protein and oligoclonal band were negative. A small hypothalamic infarction was suspected, and the patient was treated with intravenous edaravone and argatroban, as well as oral clopidogrel. Three months later, there had been no clinical relapse, and the hypothalamic lesion had almost disappeared on follow-up MRI. No new lesion suggestive of demyelinating disease or tumor was observed. CONCLUSION: Hypothalamic stroke should be considered a cause of sudden onset of sleep.

Truncated CD200 stimulates tumor immunity leading to fewer lung metastases in a novel Wistar rat metastasis model.

CD200 mediates immunosuppression in immune cells that express its receptor, CD200R. There are two CD200 variants; truncated CD200 that lacks the part of N-terminal sequence necessary for CD200R binding (CD200S) and full-length CD200 (CD200L). We established a novel lung metastasis model by subcutaneously transplanting C6 glioma cells into the backs of neonatal Wistar rats. All transplanted rats developed large back tumors, nearly 90% of which bore lung metastases. To compare the effects of CD200S and CD200L on tumor immunity, CD200L (C6-L)- or CD200S (C6-S)-expressing C6 cells were similarly transplanted. The results showed that 100% of rats with C6-L tumors developed lung metastases, while metastases were found in only 44% of rats with C6-S tumors (n = 25). Tumors disappeared in approximately 20% of the C6-S-bearing rats, and these animals evaded death 180 d after transplantation, while all C6-L tumor-bearing rats died after 45 d. Next generation sequencing revealed that C6-S tumors expressed chemokines and granzyme B at much higher levels than C6-L tumors. Flow cytometry revealed that C6-S tumors contained more dead cells and more CD45+ cells, including natural killer cells and CD8+ lymphocytes. In particular, multiple subsets of dendritic cells expressing CD11c, MHC class II, CD8, and/or CD103 were more abundant in C6-S than in C6-L tumors. These results suggested that CD200S induced the accumulation of multiple dendritic cell subsets that activated cytotoxic T lymphocytes, leading to the elimination of metastasizing tumor cells.

Autonomic Symptoms Correlate with Non-Autonomic Non-Motor Symptoms and Sleep Problems in Patients with Parkinson's Disease.

OBJECTIVE: The objective of this study was to clarify the relationship between autonomic and non-autonomic non-motor symptoms in patients with Parkinson's disease (PD). METHODS: Sixty-five PD patients were included in this study (27 men and 38 women; aged 68.5 ± 10.0; Hoehn and Yahr (HY) stage 2.6 ± 1.1). The autonomic symptoms were evaluated by the Japanese version of the Scales for outcomes in PD autonomic (SCOPA-AUT) questionnaire. The patients were assessed with the mini-mental state examination (MMSE), PD sleep evaluation scale-2 (PDSS-2), Epworth sleepiness scale (ESS) and Beck's depression inventory II (BDI-II). The Non-Motor Symptom Scale (NMSS) total scores and subscores of non-autonomic non-motor symptom domains (sleep/fatigue, mood/cognition, perceptual problems/hallucination, and attention/memory) were evaluated. A dopamine transporter (DAT) scan, metaiodobenzylguanidine (MIBG) myocardial scintigraphy, and card type olfactory identification test (open essence [OE, Wako]) were performed. RESULTS: The SCOPA-AUT total score was positively correlated with the disease duration, HY stage, levodopa equivalent dose, PDSS-2, ESS, BDI-II and non-autonomic NMSS and inversely correlated with the MMSE. The high-SCOPA-AUT group (≥9) had lower MMSE scores and higher PDSS-2, ESS, BDI-II and non-motor NMSS scores than the low-SCOPA-AUT group (< 9). The DAT scan, MIBG uptake and OE score did not differ between the groups. In a stepwise linear regression analysis, which excluded possibly overlapping items among the scales, the subtotals of PDSS-2 items, except for item 8 (nocturia), (p < 0.0001) and non-autonomic NMSS domains (p = 0.00040) were significant predictors of the total SCOPA-AUT score. CONCLUSION: Our study shows significant correlations among autonomic symptoms, PD-related sleep problems and non-autonomic non-motor symptoms in PD patients.

NRP1-mediated Sema3A signals coordinate laminar formation in the developing chick optic tectum.

The optic tectum comprises multiple layers, which are formed by radial and tangential migration during development. Here, we report that Neuropilin 1 (NRP1)-mediated Sema3A signals are involved in the process of tectal laminar formation, which is elaborated by tangential migration. In the developing chick tectum, NRP1, a receptor for Sema3A, is expressed in microtubule-associated protein 2 (MAP2)-positive intermediate layers IV and V. Sema3A itself is a diffusible guidance factor and is expressed in the overlying layer VI. Using stable fluorescent labeling of tectal cells, we show that MAP2-positive intermediate layers are formed by the neurons that have been dispersed by tangential migration along the tectal efferent axons. When Sema3A was mis-expressed during laminar formation, local Sema3A repelled the tangential migrants, thus eliminating MAP2-positive neurons that expressed NRP1. Furthermore, in the absence of the MAP2-positive neurons, tectal layers were disorganized into an undulated form, indicating that MAP2-positive intermediate layers are required for proper laminar formation. These results suggest that NRP1-mediated Sema3A signals provide repulsive signals for MAP2-positive neurons to segregate tectal layers, which is important in order to coordinate laminar organization of the optic tectum.

Role of En2 in the tectal laminar formation of chick embryos.

The chick optic tectum consists of 16 laminae. Here, we report contribution of En2 to laminar formation in chick optic tecta. En2 is specifically expressed in laminae g-j of stratum griseum et fibrosum superficiale (SGFS). Misexpression of En2 resulted in disappearance of En2-expressing cells from the superficial layers (laminae a-f of SGFS), where endogenous En2 is not expressed. Misexpression of En2 before postmitotic cells had left the ventricular layer indicated that En2-misexpressing cells stopped at the laminae of endogenous En2 expression and that they did not migrate into the superficial layers. Induction of En2 misexpression using a tetracycline-inducible system after the postmitotic cells had reached superficial layers also resulted in disappearance of En2-expressing cells from the superficial layers. Time-lapse analysis showed that En2-misexpressing cells migrated back from the superficial layers towards the middle layers, where En2 is strongly expressed endogenously. Our results suggest a potential role of En2 in regulating cell migration and positioning in the tectal laminar formation.

Multiparametric voxel-based analyses of standardized uptake values and apparent diffusion coefficients of soft-tissue tumours with a positron emission tomography/magnetic resonance system: Preliminary results.

OBJECTIVES: To investigate the usefulness of voxel-based analysis of standardized uptake values (SUVs) and apparent diffusion coefficients (ADCs) for evaluating soft-tissue tumour malignancy with a PET/MR system. METHODS: Thirty-five subjects with either ten low/intermediate-grade tumours or 25 high-grade tumours were prospectively enrolled. Zoomed diffusion-weighted and fluorodeoxyglucose (18FDG)-PET images were acquired along with fat-suppressed T2-weighted images (FST2WIs). Regions of interest (ROIs) were drawn on FST2WIs including the tumour in all slices. ROIs were pasted onto PET and ADC-maps to measure SUVs and ADCs within tumour ROIs. Tumour volume, SUVmax, ADCminimum, the heterogeneity and the correlation coefficients of SUV and ADC were recorded. The parameters of high- and low/intermediate-grade groups were compared, and receiver operating characteristic (ROC) analysis was also performed. RESULTS: The mean correlation coefficient for SUV and ADC in high-grade sarcomas was lower than that of low/intermediate-grade tumours (-0.41 ± 0.25 vs. -0.08 ± 0.34, P < 0.01). Other parameters did not differ significantly. ROC analysis demonstrated that correlation coefficient showed the best diagnostic performance for differentiating the two groups (AUC 0.79, sensitivity 96.0%, specificity 60%, accuracy 85.7%). CONCLUSIONS: SUV and ADC determined via PET/MR may be useful for differentiating between high-grade and low/intermediate-grade soft tissue tumours. KEY POINTS: • PET/MR allows voxel-based comparison of SUVs and ADCs in soft-tissue tumours. • A comprehensive assessment of internal heterogeneity was performed with scatter plots. • SUVmax or ADCminimum could not differentiate high-grade sarcoma from low/intermediate-grade tumours. • Only the correlation coefficient between SUV and ADC differentiated the two groups. • The correlation coefficient showed the best diagnostic performance by ROC analysis.

Effects of hypnotic bromovalerylurea on microglial BV2 cells.

An old sedative and hypnotic bromovalerylurea (BU) has anti-inflammatory effects. BU suppressed nitric oxide (NO) release and proinflammatory cytokine expression by lipopolysaccharide (LPS)-treated BV2 cells, a murine microglial cell line. However, BU did not inhibit LPS-induced nuclear translocation of nuclear factor-κB and subsequent transcription. BU suppressed LPS-induced phosphorylation of signal transducer and activator of transcription 1 (STAT1) and expression of interferon regulatory factor 1 (IRF1). The Janus kinase 1 (JAK1) inhibitor filgotinib suppressed the NO release much more weakly than that of BU, although filgotinib almost completely prevented LPS-induced STAT1 phosphorylation. Knockdown of JAK1, STAT1, or IRF1 did not affect the suppressive effects of BU on LPS-induced NO release by BV2 cells. A combination of BU and filgotinib synergistically suppressed the NO release. The mitochondrial complex I inhibitor rotenone, which did not prevent STAT1 phosphorylation or IRF1 expression, suppressed proinflammatory mediator expression less significantly than BU. BU and rotenone reduced intracellular ATP (iATP) levels to a similar extent. A combination of rotenone and filgotinib suppressed NO release by LPS-treated BV2 cells as strongly as BU. These results suggest that anti-inflammatory actions of BU may be attributable to the synergism of inhibition of JAK1/STAT1-dependent pathways and reduction in iATP level.

Laparoscopy-assisted distal gastrectomy is feasible also for elderly patients aged 80 years and over: effectiveness and long-term prognosis.

BACKGROUND: Elderly patients usually have concurrent ailments, and the safety and effectiveness of laparoscopy-assisted distal gastrectomy (LADG) for these patients have been controversial. This study aimed to evaluate whether laparoscopy-assisted distal gastrectomy is safe and effective for elderly patients aged 80 years and over, as well as to clarify their long-term prognosis. METHODS: A total of 31 patients aged 80 years and over who underwent LADG in our hospital were retrospectively reviewed. Peri- and postoperative data were compared with those of 38 patients aged 65 years and younger. The median follow-up period of the elderly and younger group was 56.0 and 63.0 months, respectively, and their prognosis was examined. RESULTS: There were significant differences between the two groups in preoperative respiratory and renal functions, hemoglobin, and nutritional index. Significant differences in postoperative complications were seen only in pneumonia and delirium. There were no hospital deaths, but the 3-year and 5-year overall survival rates were significantly lower in the elderly group than in the non-elderly group. However, in the elderly group, only one patient died of gastric cancer recurrence, whereas four died of cardiovascular disease and three died of pneumonia during follow-up. Therefore, the recurrence-free survival rate was not significantly different between the groups. CONCLUSIONS: LADG seems to be safe and effective even for elderly patients, and their prognosis was satisfactory. However, careful monitoring for cardiovascular and pulmonary disease should be observed during the follow-up period.

Structure of a dominant-negative helix-loop-helix transcriptional regulator suggests mechanisms of autoinhibition.

Helix-loop-helix (HLH) family transcription factors regulate numerous developmental and homeostatic processes. Dominant-negative HLH (dnHLH) proteins lack DNA-binding ability and capture basic HLH (bHLH) transcription factors to inhibit cellular differentiation and enhance cell proliferation and motility, thus participating in patho-physiological processes. We report the first structure of a free-standing human dnHLH protein, HHM (Human homologue of murine maternal Id-like molecule). HHM adopts a V-shaped conformation, with N-terminal and C-terminal five-helix bundles connected by the HLH region. In striking contrast to the common HLH, the HLH region in HHM is extended, with its hydrophobic dimerization interfaces embedded in the N- and C-terminal helix bundles. Biochemical and physicochemical analyses revealed that HHM exists in slow equilibrium between this V-shaped form and the partially unfolded, relaxed form. The latter form is readily available for interactions with its target bHLH transcription factors. Mutations disrupting the interactions in the V-shaped form compromised the target transcription factor specificity and accelerated myogenic cell differentiation. Therefore, the V-shaped form of HHM may represent an autoinhibited state, and the dynamic conformational equilibrium may control the target specificity.

Generation of a neutralization-resistant CCR5 tropic simian/human immunodeficiency virus (SHIV-MK38) molecular clone, a derivative of SHIV-89.6.

Previously, we reported that a new genetically diverse CCR5 (R5) tropic simian/human immunodeficiency virus (SHIV-MK38) adapted to rhesus monkeys became more neutralization resistant to SHIV-infected plasma than did the parental SHIV-KS661 clone. Here, to clarify the significance of the neutralization-resistant phenotype of SHIV in a macaque model, we initially investigated the precise neutralization phenotype of the SHIVs, including SHIV-MK38 molecular clones, using SHIV-MK38-infected plasma, a pooled plasma of human immunodeficiency virus (HIV)-infected individuals, soluble CD4 and anti-HIV-1 neutralizing mAbs, the epitopes of which were known. The results show that SHIV-KS661 had tier 1 neutralization sensitivity, but monkey-adapted R5 tropic SHIV-MK38 acquired neutralization resistance similar to that of tier 2 or 3 as a clone virus. Sequence analysis of the env gene suggested that the neutralization-resistant phenotype of SHIV-MK38 was acquired by conformational changes in Env associated with the net charge and potential N-linked glycosylation sites. To examine the relationship between neutralization phenotype and stably persistent infection in monkeys, we performed in vivo rectal inoculation experiments using a SHIV-MK38 molecular clone. The results showed that one of three rhesus monkeys exhibited durable infection with a plasma viral load of 105 copies ml- 1 despite the high antibody responses that occurred in the host. Whilst further improvements are required in the development of a challenge virus, it will be useful to generate a neutralization-resistant R5 tropic molecular clone of the SHIV-89.6 lineage commonly used for vaccine development - a result that can be used to explore the foundation of AIDS pathogenesis.

Statistical approach for the retrieval of phytoplankton community structures from in situ fluorescence measurements.

Knowledge of phytoplankton community structures is important to the understanding of various marine biogeochemical processes and ecosystem. Fluorescence excitation spectra (F(λ)) provide great potential for studying phytoplankton communities because their spectral variability depends on changes in the pigment compositions related to distinct phytoplankton groups. Commercial spectrofluorometers have been developed to analyze phytoplankton communities by measuring the field F(λ), but estimations using the default methods are not always accurate because of their strong dependence on norm spectra, which are obtained by culturing pure algae of a given group and are assumed to be constant. In this study, we proposed a novel approach for estimating the chlorophyll a (Chl a) fractions of brown algae, cyanobacteria, green algae and cryptophytes based on a data set collected in the East China Sea (ECS) and the Tsushima Strait (TS), with concurrent measurements of in vivo F(λ) and phytoplankton communities derived from pigments analysis. The new approach blends various statistical features by computing the band ratios and continuum-removed spectra of F(λ) without requiring a priori knowledge of the norm spectra. The model evaluations indicate that our approach yields good estimations of the Chl a fractions, with root-mean-square errors of 0.117, 0.078, 0.072 and 0.060 for brown algae, cyanobacteria, green algae and cryptophytes, respectively. The statistical analysis shows that the models are generally robust to uncertainty in F(λ). We recommend using a site-specific model for more accurate estimations. To develop a site-specific model in the ECS and TS, approximately 26 samples are sufficient for using our approach, but this conclusion needs to be validated in additional regions. Overall, our approach provides a useful technical basis for estimating phytoplankton communities from measurements of F(λ).