Chemo-Enzymatic Synthesis of Non-ribosomal Macrolactams by a Penicillin-Binding Protein-Type Thioesterase.

Penicillin-binding protein-type thioesterases (PBP-type TEs) are an emerging family of non-ribosomal peptide cyclases. PBP-type TEs exhibit distinct substrate scopes from the well-exploited ribosomal peptide cyclases and traditional non-ribosomal peptide cyclases. Their unique properties, as well as their stand-alone nature, highlight PBP-type TEs as valuable candidates for development as biocatalysts for peptide macrocyclization. Here in this chapter, we describe the scheme for the chemoenzymatic synthesis of non-ribosomal macrolactam by SurE, a representative member of PBP-type TEs.

Streamlined Chemoenzymatic Synthesis of Cyclic Peptides by Non-ribosomal Peptide Cyclases.

Macrocyclization improves the pharmaceutical properties of peptides; however, regio- and chemoselective intramolecular cyclizations remain challenging. Here we developed a streamlined chemoenzymatic approach to synthesize cyclic peptides by exploiting non-ribosomal peptide (NRP) cyclases. Linear peptides linked to the resin through a C-terminal diol ester functionality are synthesized on a solid support, to circumvent the installation of leaving groups to the peptidic substrates in the liquid phase which often triggers undesirable epimerization. Cleavage of the resin-bound peptides yielded the diol esters with sufficient purity to be readily cyclized in a head-to-tail manner by SurE, a representative penicillin-binding protein-type thioesterase (PBP-type TE). Explorations of homologous wild-type enzymes as well as rational protein engineering have broadened the scope of the enzymatic macrolactamization. This method will potentially accelerate the exploitation of NRP cyclases as biocatalysts.

New azodyrecins identified by a genome mining-directed reactivity-based screening.

Only a few azoxy natural products have been identified despite their intriguing biological activities. Azodyrecins D-G, four new analogs of aliphatic azoxides, were identified from two Streptomyces species by a reactivity-based screening that targets azoxy bonds. A biological activity evaluation demonstrated that the double bond in the alkyl side chain is important for the cytotoxicity of azodyrecins. An in vitro assay elucidated the tailoring step of azodyrecin biosynthesis, which is mediated by the S-adenosylmethionine (SAM)-dependent methyltransferase Ady1. This study paves the way for the targeted isolation of aliphatic azoxy natural products through a genome-mining approach and further investigations of their biosynthetic mechanisms.

Molecular Basis for Enzymatic Aziridine Formation via Sulfate Elimination.

Natural products containing an aziridine ring, such as mitomycin C and azinomycin B, exhibit antitumor activities by alkylating DNA via their aziridine rings; however, the biosynthetic mechanisms underlying the formation of these rings have not yet been elucidated. We herein investigated the biosynthesis of vazabitide A, the structure of which is similar to that of azinomycin B, and demonstrated that Vzb10/11, with no similarities to known enzymes, catalyzed the formation of the aziridine ring via sulfate elimination. To elucidate the detailed reaction mechanism, crystallization of Vzb10/11 and the homologous enzyme, AziU3/U2, in the biosynthesis of azinomycin B was attempted, and the structure of AziU3/U2, which had a new protein fold overall, was successfully determined. The structural analysis revealed that these enzymes adjusted the dihedral angle between the amino group and the adjacent sulfate group of the substrate to almost 180° and enhanced the nucleophilicity of the C6-amino group temporarily, facilitating the SN2-like reaction to form the aziridine ring. The present study reports for the first time the molecular basis for aziridine ring formation.

Risk prediction of biomarkers for early multiple organ dysfunction in critically ill patients.

BACKGROUND: Shock and organ damage occur in critically ill patients in the emergency department because of biological responses to invasion, and cytokines play an important role in their development. It is important to predict early multiple organ dysfunction (MOD) because it is useful in predicting patient outcomes and selecting treatment strategies. This study examined the accuracy of biomarkers, including interleukin (IL)-6, in predicting early MOD in critically ill patients compared with that of quick sequential organ failure assessment (qSOFA). METHODS: This was a multicenter observational sub-study. Five universities from 2016 to 2018. Data of adult patients with systemic inflammatory response syndrome who presented to the emergency department or were admitted to the intensive care unit were prospectively evaluated. qSOFA score and each biomarker (IL-6, IL-8, IL-10, tumor necrosis factor-α, C-reactive protein, and procalcitonin [PCT]) level were assessed on Days 0, 1, and 2. The primary outcome was set as MOD on Day 2, and the area under the curve (AUC) was analyzed to evaluate qSOFA scores and biomarker levels. RESULTS: Of 199 patients, 38 were excluded and 161 were included. Patients with MOD on Day 2 had significantly higher qSOFA, SOFA, and Acute Physiology and Chronic Health Evaluation II scores and a trend toward worse prognosis, including mortality. The AUC for qSOFA score (Day 0) that predicted MOD (Day 2) was 0.728 (95% confidence interval [CI]: 0.651-0.794). IL-6 (Day 1) showed the highest AUC among all biomarkers (0.790 [95% CI: 0.711-852]). The combination of qSOFA (Day 0) and IL-6 (Day 1) showed improved prediction accuracy (0.842 [95% CI: 0.771-0.893]). The combination model using qSOFA (Day 1) and IL-6 (Day 1) also showed a higher AUC (0.868 [95% CI: 0.799-0.915]). The combination model of IL-8 and PCT also showed a significant improvement in AUC. CONCLUSIONS: The addition of IL-6, IL-8 and PCT to qSOFA scores improved the accuracy of early MOD prediction.

Real-time bowel sound analysis using newly developed device in patients undergoing gastric surgery for gastric tumor.

BACKGROUND AND AIM: Objective measurements are not available for determining bowel sounds. The present study sought to evaluate the efficacy of a novel bowel sound monitoring system for perioperative use in patients undergoing gastric surgery. METHODS: The study enrolled 14 patients who underwent surgery for gastric cancer at Kochi Medical School from 2017 to 2018. Preoperative and postoperative bowel sounds were recorded using a newly developed real-time analysis system in the operating theater and recovery room. Clinical information and bowel sound count data were obtained to compare preoperative and postoperative measures. RESULTS: The median preoperative and postoperative bowel sound counts across all patients were 1.4 and 2.5 counts per minute (cpm), respectively. In patients who underwent laparoscopic gastrectomy, the postoperative bowel sound count was significantly higher than that recorded preoperatively (2.3 vs. 1.6 cpm, P = 0.005). The findings also revealed a significant negative correlation between postoperative bowel sound count and operation time (r = -0.714, P = 0.003). CONCLUSIONS: The real-time bowel sound analysis system tested herein presents a promising diagnostic tool to quantitatively evaluate bowel movements associated with surgery. Our results suggested a need for shorter operation times for gastric procedures with respect to peristalsis recovery and supported the use of minimally invasive surgery.

Accuracy for Mortality Prediction With Additive Biomarkers Including Interleukin-6 in Critically Ill Patients: A Multicenter Prospective Observational Study.

OBJECTIVES: Several inflammation markers have been reported to be associated with unfavorable clinical outcomes in critically ill patients. We aimed to elucidate whether serum interleukin-6 concentration considered with Sequential Organ Failure Assessment score can better predict mortality in critically ill patients. DESIGN: A prospective observational study. SETTING: Five university hospitals in 2016-2018. PATIENTS: Critically ill adult patients who met greater than or equal to two systemic inflammatory response syndrome criteria at admission were included, and those who died or were discharged within 48 hours were excluded. INTERVENTIONS: Inflammatory biomarkers including interleukin (interleukin)-6, -8, and -10; tumor necrosis factor-α; C-reactive protein; and procalcitonin were blindly measured daily for 3 days. Area under the receiver operating characteristic curve for Sequential Organ Failure Assessment score at day 2 according to 28-day mortality was calculated as baseline. Combination models of Sequential Organ Failure Assessment score and additional biomarkers were developed using logistic regression, and area under the receiver operating characteristic curve calculated in each model was compared with the baseline. MEASUREMENTS AND MAIN RESULTS: Among 161 patients included in the study, 18 (11.2%) did not survive at day 28. Univariate analysis for each biomarker identified that the interleukin-6 (days 1-3), interleukin-8 (days 0-3), and interleukin-10 (days 1-3) were higher in nonsurvivors than in survivors. Analyses of 28-day mortality prediction by a single biomarker showed interleukin-6, -8, and -10 at days 1-3 had a significant discrimination power, and the interleukin-6 at day 3 had the highest area under the receiver operating characteristic curve (0.766 [0.656-0.876]). The baseline area under the receiver operating characteristic curve for Sequential Organ Failure Assessment score predicting 28-day mortality was 0.776 (0.672-0.880). The combination model using additional interleukin-6 at day 3 had higher area under the receiver operating characteristic curve than baseline (area under the receiver operating characteristic curve = 0.844, area under the receiver operating characteristic curve improvement = 0.068 [0.002-0.133]), whereas other biomarkers did not improve accuracy in predicting 28-day mortality. CONCLUSIONS: Accuracy for 28-day mortality prediction was improved by adding serum interleukin-6 concentration to Sequential Organ Failure Assessment score.

Hepatocyte growth factor regulates HIF-1α-induced nucleus pulposus cell proliferation through MAPK-, PI3K/Akt-, and STAT3-mediated signaling.

Intervertebral discs are important for maintaining mobility and offer support to the body trunk. If these discs lose their biomechanical features, lower back pain can occur. We previously reported that hepatocyte growth factor (HGF) promotes cell proliferation and suppresses apoptosis, inflammation, and matrix degradation in nucleus pulposus (NP) cells. In the present study, we investigated the molecular mechanisms of how HGF promotes the proliferation of NP cells in hypoxic conditions. Hypoxic stimulation promoted modest cell proliferation, which was further upregulated by HGF. Expression of hypoxia-inducible factor (HIF-1α) protein, which contributes to the maintenance of homeostasis in NP cells, was also upregulated in hypoxia-treated cell groups; HGF further increased HIF-1α expression in NP cells. Additionally, knockdown of HIF-1α expression significantly reduced the proliferation of NP cells. An MAPK inhibitor inhibited the expression of HIF-1α and pERK, as well as cell proliferation in a dose-dependent manner. Similarly, inhibiting the PI3K/Akt and STAT3 pathways also decreased the expression of HIF-1α and cell proliferation. These results show that under hypoxic conditions, HGF promotes NP cell proliferation via HIF-1α-, MAPK-, PI3K/Akt-, and STAT3-mediated signaling which is involved in this pathway. The control of these signaling pathways may be a target for potential therapeutic strategies for the treatment of disc degeneration in hypoxic conditions.

Blood Interleukin-6 Levels Predict Multiple Organ Dysfunction in Critically Ill Patients.

BACKGROUND: Predicting multiple organ dysfunction (MOD) in the late phase of critical illnesses is essential. Cytokines are considered biomarkers that can predict clinical outcomes; however, their predictive value for late-phase MOD is unknown. This study aimed to identify the biomarker with the highest predictive value for late-phase MOD. METHODS: This observational study prospectively evaluated data on adult patients with systemic inflammatory response syndrome, those who presented to the emergency department or were admitted to intensive care units in five tertiary hospitals (n = 174). Seven blood biomarkers levels (interleukin-6 [IL-6], IL-8, IL-10, tumor-necrosis factor-α, white blood cells, C-reactive protein, and procalcitonin) were measured at three timepoints (days 0, 1, and 2). The area under the receiver operating characteristic curve (AUC) was analyzed to evaluate predictive values for MOD (primary outcome, MOD on day 7 [late-phase]; secondary outcome, MOD on day 3 [early-phase]). RESULTS: Of the measured 7 biomarkers, blood IL-6 levels on day 2 had the highest predictive value for MOD on day 7 using single timepoint data (AUC 0.825, 95% confidence interval [CI] 0.754-0.879). Using three timepoint biomarkers, blood IL-6 levels had the highest predictive value of MOD on day 7 (AUC 0.838, 95% CI 0.768-0.890). Blood IL-6 levels using three timepoint biomarkers had also the highest predictive value for MOD on day 3 (AUC 0.836, 95% CI 0.766-0.888). CONCLUSION: Of the measured biomarkers, blood IL-6 levels had the highest predictive value for MOD on days 3 and 7. Blood IL-6 levels predict early- and late-phase MOD in critically ill patients.

Evaluation of Perioperative Intestinal Motility Using a Newly Developed Real-Time Monitoring System During Surgery.

BACKGROUND: This study aimed to investigate perioperative intestinal motility using a novel bowel sound monitoring system in patients undergoing breast and neck surgery. MATERIALS AND METHODS: This study enrolled 52 patients who underwent surgery for breast cancer, thyroid tumor, and parathyroid tumor at Kochi Medical School from May 2019 to June 2020. Perioperative bowel sound counts (BSCs) were recorded using a newly developed real-time analysis system in the operating theater. Clinical information and BSC per minute (cpm) data during the preanesthetic, preoperative, operative, postoperative periods, and period in recovery room were obtained to compare between each period. The Mann-Whitney U and Pearson Chi-square tests were used in data analysis. RESULTS: The BSCs during the intraoperative period and postoperative period were significantly decreased compared to those during the preanesthetic period (0.07 cpm versus [vs.]. 1.4 cpm, P = 0.002 and 0.1 cpm vs. 1.4 cpm, P = 0.025, respectively). The preoperative BSC with a preanesthetic BSC < 1.4 was significantly lower than that with a preanesthetic BSC ≥ 1.4 (0.40 cpm vs. 1.78 cpm, P = 0.006). The preanesthetic, preoperative, and postoperative BSCs with an intraoperative BSC < 0.07 were significantly lower than those with an intraoperative BSC ≥ 0.07 (0.48 cpm vs. 2.83 cpm, P = 0.007; 0.40 cpm vs. 1.81 cpm, P = 0.008; and 0.07 cpm vs. 0.42 cpm, P = 0.006, respectively). CONCLUSION: The real-time bowel sound analysis system demonstrated an inhibitory effect associated with anesthetic and surgical stress on intestinal motility as the BSC sequentially.

Guanidyl modification of the 1-azabicyclo[3.1.0]hexane ring in ficellomycin essential for its biological activity.

The 1-azabicyclo[3.1.0]hexane ring is a key moiety in natural products for biological activities against bacteria, fungi, and tumor through DNA alkylation. Ficellomycin is a dipeptide that consists of l-valine and a non-proteinogenic amino acid with the 1-azabicyclo[3.1.0]hexane ring structure. Although the biosynthetic gene cluster of ficellomycin has been identified, the biosynthetic pathway currently remains unclear. We herein report the final stage of ficellomycin biosynthesis involving ring modifications and successive dipeptide formation. After the ring is formed, the hydroxy group of the ring is converted into the guanidyl unit by three enzymes, which include an aminotransferase with a novel inter ω-ω amino-transferring activity. In the last step, the resulting 1-azabicyclo[3.1.0]hexane ring-containing amino acid is connected with l-valine by an amino acid ligase to yield ficellomycin. The present study revealed a new machinery that expands the structural and biological diversities of natural products.

Scaffold attachment factor B: distribution and interaction with ERα in the rat brain.

Scaffold attachment factor (SAFB) 1 and its homologue SAFB2 are multifunctional proteins that are involved in various cellular mechanisms, including chromatin organization and transcriptional regulation, and are also corepressors of estrogen receptor alpha (ERα). Both SAFBs are expressed at high levels in the brain. However, the distributions of SAFB1 and SAFB2 have yet to be characterized in detail and it is unclear whether both proteins interact with ERα in the brain. In this study, we investigated the expression and distribution of both SAFBs and their interaction with ERα in adult male rat brain. Immunohistochemical staining showed that SAFB1 and SAFB2 have a similar distribution pattern and are widely expressed throughout the brain. Double-fluorescence immunohistochemical and immunocytochemical analyses in primary cultures showed that the two SAFB proteins are localized in nuclei of neurons, astrocytes, and oligodendrocytes. Of note, SAFB2 was also found in cytoplasmic regions in these cell lineages. Both SAFB proteins were also expressed in ERα-positive cells in the medial preoptic area (MPOA) and arcuate and ventromedial hypothalamic nuclei. Co-immunoprecipitation experiments revealed that both SAFB proteins from the MPOA reciprocally interact with endogenous ERα. These results indicate that, in addition to a role in basal cellular function in the brain, the SAFB proteins may serve as ERα corepressors in hormone-sensitive regions.

Occlusal force predicted cognitive decline among 70- and 80-year-old Japanese: A 3-year prospective cohort study.

PURPOSE: Dementia is a growing health problem for countries with aging populations, but few effective dementia treatments are available. However, there is increasing interest in oral health as a modifiable risk factor in interventions to prevent cognitive decline. This study aimed to investigate the impact of oral health on the decline of cognitive function over 3 years among Japanese people aged 70 and 80 years. METHODS: Participants (n = 860) were community-dwelling older adults who participated in baseline and follow-up surveys (at baseline: 69-71 years n = 423; 79-81 years, n = 437). Registered dentists examined the number of teeth, number of functional teeth, number of periodontal teeth, and occlusal force. The Japanese version of the Montreal Cognitive Assessment was used to evaluate cognitive function. We also evaluated socioeconomic factors, medical history, drinking and smoking habits, physical performance, genetic factors, and C-reactive protein concentration in blood. A generalized estimating equation (GEE) was used to examine how oral health at baseline influenced cognitive decline over 3 years. RESULTS: The GEE showed that the number of teeth (non-standardized coefficient: B = 0.031, p = 0.022) and occlusal force (B = 0.103, p = 0.004) at baseline were associated with cognitive function at follow-up, even after adjusting for other risk factors. Furthermore, maintaining more teeth (B = 0.009, p = 0.004) and a stronger occlusal force (B = 0.020, p = 0.040) buffered cognitive decline. CONCLUSIONS: Number of teeth and occlusal force predict cognitive decline over 3 subsequent years in Japanese older adults aged 70 and 80 years.

Occlusal support predicts tooth loss in older Japanese people.

OBJECTIVES: Many older people lose their teeth. However, few studies have examined whether the number of remaining teeth or the amount of occlusal support is more important for tooth loss that occurs in older age after adjusting for systemic factors. The purpose of this study was to investigate comprehensively the factors related to tooth loss over a period of 6 years, including occlusal support in 70- and 80-year-old community-dwelling Japanese people. METHODS: This cohort study included 296 participants in the 70-year-old group and 232 in the 80-year-old group. The number of teeth of all participants was recorded at baseline and after 6 years, and the participants were divided into two groups according to the number of teeth lost (0 or 4 or more). Occlusal support was classified into three groups based on posterior occlusal support. Mean probing pocket depth, tooth brushing habits, frequency of dental check-ups, education level, economic status and systemic factors (carotid atherosclerosis, hypertension, diabetes mellitus, dyslipidemia, cognitive function and smoking habits) were evaluated. Multiple logistic regression analysis was performed to investigate the relationship of tooth loss with occlusal support and systemic factors. RESULTS: Logistic regression analysis revealed that mean probing pocket depth (odds ratio [OR] = 5.70, 95% confidence interval [CI] = 2.70-12.04, P < .01) and posterior occlusal support (reference = Eichner class A; Eichner B1-3, OR = 4.33, 95% CI = 1.54-12.17, P < .01; Eichner B4 or C, OR = 6.16, 95% CI = 1.17-32.44, P = .03) were associated significantly with the loss of four or more teeth. CONCLUSIONS: This study revealed that age itself is not a predictor of multiple tooth loss in community-dwelling older Japanese people. Deep mean probing pocket depth and a lack of posterior occlusal support were significantly associated with the loss of four or more teeth.

Scrobiculosides A and B from the deep-sea sponge Pachastrella scrobiculosa.

Two new steroidal saponins, scrobiculosides A and B, were isolated from the deep-sea sponge Pachastrella scrobiculosa, collected at a depth of 200 m off Miura Peninsula, Japan. The aglycones of scrobiculosides A and B feature a vinylic cyclopropane and a ∆24,25 exomethylene on the side chains, respectively. Both saponins have a common sugar moiety composed of ß-D-galactopyranosyl-(1 → 2)-6-acetyl-ß-D-glucopyranoside, with the exception of an acetyl group on C6″ in scrobiculoside A. Scrobiculoside A exhibited cytotoxicity against HL-60 and P388 cells, with IC50 values of 52 and 61 µM, respectively.

Autologous Stem Cell Rescue for Graft Failure of Second Allogeneic Stem Cell Transplant After Engraftment of Primary Allogeneic Transplant.

Here, we describe a case of primary graft failure with severe sepsis in a boy who experienced frequent relapses of osteosarcoma. The patient had undergone haploidentical bone marrow transplant after engraftment of unrelated cord blood transplant performed 10 months earlier. Considering his severe condition, we transfused autologous peripheral stem cells along with a single dose of etoposide (50 mg/m2). Granulocyte engraftment was confirmed on human leukocyte antigen-microsatellite analysis of bone marrow on day 14. Although the patient died due to respiratory failure, transfusion of autologous hematopoietic stem cells is a reasonable rescue option for graft failure even in patients whose background hematopoiesis is reconstituted by a first donor.

Efficacy of hemofiltration with PEPA membrane for IL-6 removal in a rat sepsis model.

Recently, intensive care physicians have focused on continuous hemodiafiltration with a cytokine-adsorbing hemofilter in the treatment of sepsis. We aimed to establish extracorporeal circulation in a rat sepsis model to evaluate the cytokine removal properties of mini-modules using two types of membrane materials. Rats were divided into polyester polymer alloy (PEPA) and cellulose triacetate (CTA) groups as membrane materials of mini-modules. One hour after 0.1 mg/kg of lipopolysaccharide administration, continuous hemofiltration (CHF) was started in each group. Plasma interleukin-6 (IL-6), an important mediator of sepsis, was measured over time during hemofiltration. The peak IL-6 concentration in PEPA group was approximately 13,000 pg/mL, in comparison to approximately 31,000 pg/mL in CTA group. IL-6 clearance in PEPA group was much more than CTA group. Since IL-6 was not detected in the filtrate in PEPA group, it was considered that IL-6 was adsorbed to the membrane. In conclusion, our results suggest that CHF with PEPA hemofilter can be suitable for removing IL-6 from the blood stream efficiently.

Effect of Footprint Preparation on Tendon-to-Bone Healing: A Histologic and Biomechanical Study in a Rat Rotator Cuff Repair Model.

PURPOSE: To compare the histologic and biomechanical effects of 3 different footprint preparations for repair of tendon-to-bone insertions and to assess the behavior of bone marrow-derived cells in each method of insertion repair. METHODS: We randomized 81 male Sprague-Dawley rats and green fluorescent protein-bone marrow chimeric rats into 3 groups. In group A, we performed rotator cuff repair after separating the supraspinatus tendon from the greater tuberosity and removing the residual tendon tissue. In group B, we also drilled 3 holes into the footprint. The native fibrocartilage was preserved in groups A and B. In group C, we excavated the footprint until the cancellous bone was exposed. Histologic repair of the tendon-to-bone insertion, behavior of the bone marrow-derived cells, and ultimate force to failure were examined postoperatively. RESULTS: The areas of metachromasia in groups A, B, and C were 0.033 ± 0.019, 0.089 ± 0.022, and 0.002 ± 0.001 mm2/mm2, respectively, at 4 weeks and 0.029 ± 0.022, 0.090 ± 0.039, and 0.003 ± 0.001 mm2/mm2, respectively, at 8 weeks. At 4 and 8 weeks postoperatively, significantly higher cartilage matrix production was observed in group B than in group C (4 weeks, P = .002; 8 weeks, P < .001). In green fluorescent protein-bone marrow chimeric rats in group B, bone marrow-derived chondrogenic cells infiltrated the fibrocartilage layer. Ultimate force to failure was significantly higher in group B (19.7 ± 3.4 N) than in group C (16.7 ± 2.0 N) at 8 weeks (P = .031). CONCLUSIONS: Drilling into the footprint and preserving the fibrocartilage improved the quality of repair tissue and biomechanical strength at the tendon-to-bone insertion after rotator cuff repair in an animal model. CLINICAL RELEVANCE: Drilling into the footprint and preserving the fibrocartilage can enhance repair of tendon-to-bone insertions. This method may be clinically useful in rotator cuff repair.

Development of a quick serum IL-6 measuring system in rheumatoid arthritis.

OBJECTIVE: Interleukin-6 (IL-6) plays a crucial role in the pathogenesis of rheumatoid arthritis (RA). Both fulfillment of remission criteria and assessment of other methods of evaluation of RA are important for preventing joint damage progression. Measurement of serum IL-6 concentrations has been reported to be useful for monitoring RA disease activity. However, it takes at least 4-5h to measure serum IL-6 concentrations using traditional methods, which limits its utility during routine assessment in daily clinical practice settings. We established a novel method that enables measurement of serum IL-6 within 24min and requires a very small blood volume. We investigated the accuracy and efficacy of this system in RA patients. METHODS: One hundred fifty blood samples collected from 76 patients were measured using the two systems. We first developed the prototype of the Human IL-6 RAYFAST. Then, we examined the correlation between the prototype RAYFAST and chemiluminescent enzyme immunoassay (CLEIA) methods. Finally, we compared IL-6 concentrations and clinical parameters using both systems. RESULTS: The correlation between RAYFAST (x) and CLEIA (y) for IL-6 was y=0.895x-5.94, r=0.941 (p<0.0001). Serum IL-6 concentrations in RAYFAST correlated with DAS28-CRP (r=0.372, p<0.05) and DAS28-ESR (r=0.397, p<0.01). Serum IL-6 concentrations in CLEIA correlated with DAS28-CRP (r=0.313, p<0.001) and DAS28-ESR (r=0.353, p<0.001). CONCLUSION: This new cytokine quick measure system is as accurate as CLEIA methods. Serum IL-6 concentrations can be measured in 24min using the prototype RAYFAST. It might be usable in the daily clinical practice setting, thereby contributing to improved RA management.

A Sexually Dimorphic Area of the Dorsal Hypothalamus in Mice and Common Marmosets.

We found a novel sexually dimorphic area (SDA) in the dorsal hypothalamus (DH) of mice. The SDA-DH was sandwiched between 2 known male-biased sexually dimorphic nuclei, the principal nucleus of the bed nucleus of the stria terminalis and the calbindin-sexually dimorphic nucleus, and exhibited a female-biased sex difference in neuronal cell density. The density of neurons in the SDA-DH was increased in male mice by orchidectomy on the day of birth and decreased in female mice by treatment with testosterone, dihydrotestosterone, or estradiol within 5 days after birth. These findings indicate that the SDA-DH is defeminized under the influence of testicular testosterone, which acts via both directly by binding to the androgen receptor, and indirectly by binding to the estrogen receptor after aromatization. We measured the activity of SDA-DH neurons with c-Fos, a neuronal activity marker, in female mice during maternal and sexual behaviors. The number of c-Fos-expressing neurons in the SDA-DH of female mice was negatively correlated with maternal behavior performance. However, the number of c-Fos-expressing neurons did not change during female sexual behavior. These findings suggest that the SDA-DH contains a neuronal cell population, the activity of which decreases in females exhibiting higher performance of maternal behavior, but it may contribute less to female sexual behavior. Additionally, we examined the brain of common marmosets and found an area that appears to be homologous with the mouse SDA-DH. The sexually dimorphic structure identified in this study is not specific to mice and may be found in other species.