Tracking and visualizing the circadian ticking of the cyanobacterial clock protein KaiC in solution.

The circadian clock in cyanobacteria persists even without the transcription/translation feedbacks proposed for eukaryotic systems. The period of the cyanobacterial clock is tuned to the circadian range by the ATPase activity of a clock protein known as KaiC. Here, we provide structural evidence on how KaiC ticks away 24 h while coupling the ATPase activity in its N-terminal ring to the phosphorylation state in its C-terminal ring. During the phosphorylation cycle, the C-terminal domains of KaiC are repositioned in a stepwise manner to affect global expansion and contraction motions of the C-terminal ring. Arg393 of KaiC has a critical function in expanding the C-terminal ring and its replacement with Cys affects the temperature compensation of the period--a fundamental property of circadian clocks. The conformational ticking of KaiC observed here in solution serves as a timing cue for assembly/disassembly of other clock proteins (KaiA and KaiB), and is interlocked with its auto-inhibitory ATPase underlying circadian periodicity of cyanobacteria.

Evaluating the detailed position of the subclavian artery to avoid inadvertent subclavian artery puncture during right internal jugular vein catheterization.

PURPOSE: Central venous catheter placement is useful but is associated with complications. Inadvertent subclavian artery (SCA) puncture is a rare complication associated with internal jugular vein (IJV) catheterization. We determined the position of the SCA by ultrasonography, and propose a needle-insertion position for avoiding inadvertent SCA puncture. METHODS: We positioned an ultrasound probe at an angle of 60° to the skin to mimic a puncture needle halfway between the clavicle and the angle of the mandible (center) and moved the probe parallel to the right IJV (RIJV) toward the clavicle until locating the SCA. We measured the distance from the clavicle to the probe 60 and from the probe to the SCA (P60-SCA) where the SCA was visible by ultrasonography. RESULTS: For 50 volunteers with a mean age of 27.3 years, the center position was, on average, 67 mm from the clavicle. The image of the SCA appeared within 65 mm of the clavicle. P60-SCA differed significantly between men and women (p = 0.0058). For 45 volunteers, P60-SCA was <25 mm with the probe 65 mm from the clavicle on the skin. RIJVP-SCA averaged 4.4 mm. Only P60-SCA correlated well with BMI for men (r = 0.732, p = 0.0068). CONCLUSION: Puncturing the RIJV away from the center should avoid SCA puncture; puncturing it while approaching the clavicle is more dangerous. The exact location of the SCA varies from person to person; thus, confirming SCA position by ultrasonography is recommended every time before puncturing.

Elucidation of the role of clp protease components in circadian rhythm by genetic deletion and overexpression in cyanobacteria.

In the cyanobacterium Synechococcus elongatus PCC7942, KaiA, KaiB, and KaiC are essential elements of the circadian clock, and Kai-based oscillation is thought to be the basic circadian timing mechanism. The Kai-based oscillator coupled with transcription/translation feedback and other intercellular factors maintains the stability of the 24-hour period in vivo. In this study, we showed that disruption of the Clp protease family genes clpP1, clpP2, and clpX and the overexpression of clpP3 cause long-period phenotypes. There were no significant changes in the levels of the clock proteins in these mutants. The overexpression of clpX led to a decrease in kaiBC promoter activity, the disruption of the circadian rhythm, and eventually cell death. However, after the transient overexpression of clpX, the kaiBC gene expression rhythm recovered after a few days. The rhythm phase after recovery was almost the same as the phase before clpX overexpression. These results suggest that the core Kai-based oscillation was not affected by clpX overexpression. Moreover, we showed that the overexpression of clpX sequentially upregulated ribosomal protein subunit mRNA levels, followed by upregulation of other genes, including the clock genes. Additionally, we found that the disruption of clpX decreased the expression of the ribosomal protein subunits. Finally, we showed that the circadian period was prolonged following the addition of a translation inhibitor at a low concentration. These results suggest that translational efficiency affects the circadian period and that clpX participates in the control of translation efficiency by regulating the transcription of ribosomal protein genes.

SWAP70 functions as a Rac/Rop guanine nucleotide-exchange factor in rice.

Rho family small GTPases are involved in diverse signaling processes including immunity, growth, and development. The activity of Rho GTPases is regulated by cycling between guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active forms, in which guanine nucleotide exchange factors (GEFs) predominantly function to promote activation of the GTPases. In animals, most Rho GEFs possess a Dbl (diffuse B-cell lymphoma) homology (DH) domain which functions as a GEF-catalytic domain. However, no proteins with the DH domain have been identified in plants so far. Instead, plant-specific Rho GEFs with the PRONE domain responsible for GEF activity have been found to constitute a large family in plants. In this study, we found rice homologs of human SWAP70, Oryza sativa (Os) SWAP70A and SWAP70B, containing the DH domain. OsSWAP70A interacted with rice Rho GTPase OsRac1, an important signaling factor for immune responses. The DH domain of OsSWAP70A exhibited the GEF-catalytic activity toward OsRac1 as found in animal Rho GEFs, indicating that plants have the functional DH domains. Transient expression of OsSWAP70A enhanced OsRac1-mediated production of reactive oxygen species in planta. Reduction of OsSWAP70A and OsSWAP70B mRNA levels by RNA interference resulted in the suppression of chitin elicitor-induced defense gene expression and ROS production. Thus, it is likely that OsSWAP70 regulates immune responses through activation of OsRac1.

Ultrasound detection of guidewires in-plane during pediatric central venous catheterization.

OBJECTIVE/AIM: To assess the usefulness of longitudinal ultrasound images of guidewires for pediatric central venous catheter (CVC) placement. BACKGROUND: To avoid arterial placement of a CVC, it is important to ensure that the guidewires are in the internal jugular vein (IJV) before inserting the dilators. Ultrasound confirmation of the guidewire position may eliminate accidental arterial dilation during CVC cannulation. METHODS: Fifty pediatric patients undergoing general endotracheal anesthesia for cardiovascular surgery were enrolled. The mean ± sd value for age was 14.8 ± 16.8 months; height, 69.1 ± 14.6 cm; and weight, 7.1 ± 3.2 kg. Longitudinal ultrasound images of guidewires in or around the right IJV during pediatric CVC placement were acquired using an L10-5 MHz probe (TiTAN(®); SonoSite Co., Tokyo, Japan), with the neck of the patient fully extended and the head turned 15-30 degree to the left. RESULTS: In 42 (84%) patients, guidewires were visible in the lumen of the IJV. However, in 8 (16%) patients, guidewires were not visible. Longitudinal imaging of guidewires was achieved with high probability (100%) in children in whom the distance from the clavicle to the angle of the mandible was more than 59 mm, in whom the distance from the clavicle to the puncture site was more than 30 mm, or in children taller than 65 cm. All cannulations were confirmed to be CVCs by chest radiography. CONCLUSIONS: To confirm the insertion of dilators into veins in children, we recommend that longitudinal images of the guidewires should be initially acquired.

[Successful internal jugular venipuncture in an infant with a high risk of puncturing the vertebral artery].

A five-month-old baby girl, 61.8 cm in height and weighing 4.9 kg, underwent ventricular septal defect repair under general anesthesia. Authors checked for small vessels around the IJV using an ultrasound echo apparatus with a 5/10-MHz probe (TiTAN, SonoSite Co., Tokyo, Japan) at a point 28 mm from the clavicle over the IJV. We observed a 3.8-mm-wide and 8.7-mm-deep vertebral artery 1.3 mm behind the 5.5-mm-wide IJV with color Doppler flow imaging for CVC. It seemed difficult to avoid puncturing the vertebral artery. We checked for small arteries again at a point 40 mm from the clavicle. We also confirmed the 3.2-mm-wide and 13.4-mm-deep vertebral artery 5.3 mm behind the IJV. It thus seemed possible to avoid puncturing the vertebral artery at this point. The operator punctured the IJV carefully using real-time ultrasound guidance with a 24 G plastic puncture needle (Jelco Plus, Smith Medical, Tokyo, Japan), and avoided puncturing the vertebral artery. The CVC was successful, and no after-effect was observed following the operation. Before inserting the puncture needle each time, one should check for small arteries behind the IJV with color Doppler flow imaging.

[A case report of a failure to advance a guidewire in an infant from the internal jugular vein to the brachiocephalic vein].

A 2-month-old baby boy, 52 cm in height and weighing 4.6 kg, underwent a Blalock-Taussig shunt operation under general anesthesia. The authors checked the internal jugular vein (IJV) using an ultrasound apparatus with a 5/10-MHz probe (TiTAN, SonoSite Co., Tokyo, Japan) at a mid-portion of the neck. We observed a 3.9-mm-wide and 7.6-mm-deep IJV for central venous catheter (CVC) placement. We started to videotape the procedure. The operator punctured the IJV using real-time ultrasound guidance with a 24 G plastic puncture needle (Jelco Plus, Smith Medical, Tokyo, Japan), and obtained the back flow of the vein. The operator could not advance a guidewire more than 5 cm into the IJV. We suspected that the straight type guidewire had advanced outside of the IJV, and removed the guidewire. We placed the CVC into the left IJV instead of the right IJV. We speculated that the guidewire had advanced into the IJV; however, we could not advance the tip of the guidewire from the IJV to the brachiocephalic vein because the angle between the IJV and the brachiocephalic vein was 90 degrees. We could have advanced a J-type guidewire from the IJV into the brachiocephalic vein.

[Ultrasound evaluation of the anatomical relationships between common carotid arteries and internal jugular veins in children].

BACKGROUND: Relationships between common carotid arteries (CCA) and internal jugular veins (IJV) have been investigated both in adults and children. Overlapping of CCA and IJV are reported in 70 to 90% of cases; however we seldom observe overlapping cases in children. We evaluate the relationships between CCA and IJV in children. METHODS: In this study, 100 children below 60 months undergoing tracheal intubation for general anesthesia were enrolled. The width of the CCA (AW), IJV (VW), and the distance from the internal edge of the CCA to the outer edge of the IJV (DAV) were measured using an ultrasound apparatus. The overlapping ratio was calculated as (AW + VW--DAV)/DAV. RESULTS: Mean (S. D) age was 15.5 (16.0) months, height was 70.8 (16.4) cm, and weight was 7.6 (3.8) kg; the AW was 4.4 (0.8) mm, and the VW was 7.4 (2.2) mm. The percentage of non-overlapping of CCA and IJV was 66.2% and overlapping less than 10% was 92%. The overlapping more than 20% was only in 3%. Puncturing the CCA did not occur. CONCLUSIONS: There appears to be little danger of puncturing the CCA during internal jugular venipuncture from the viewpoint of anatomical relationships between the CCA and IJV

[Pediatric use of cuffed tracheal tubes under general anesthesia].

BACKGROUND: Although the use of cuffed tracheal tubes (CTT) in children less than 8 years of age has increased, the criteria for selecting CTTs of appropriate sizes have not been determined. METHODS: To study the criteria for choosing tubes of appropriate sizes (internal diameter: 4.0, 4.5, 5.0, or 5.5mm), we intubated 51 children aged 2-8 years under general anesthesia using an original protocol. We excluded 2 patients who received 4.0-mm tubes; thus, the outcomes in 48 patients were analyzed statistically. RESULTS: A larger CTT was replaced with a smaller one in 1 patient, and a CTT was replaced with an uncuffed tracheal tube (UTT) in another patient (excluded from analysis). No tube changes were required in 49 patients. Post-extubation stridor was observed in 3 patients. The 4.5-mm tubes seemed appropriate in 12 cases because average airway leakage in these cases was > 10%; 5.0- and 5.5-mm tubes did not seem appropriate in 17 and 19 cases, respectively, because the leakage was < 10% of the tidal volume. CONCLUSIONS: We recommend the pediatric use of CTTs because of the very low tube-exchange ratio and the acceptable incidence of stridor. However, the protocol may require modification.

[Four cases of cardiac arrest during spinal anesthesia].

Incident rates of cardiac arrest are reported as being from 0.54 to 17.5 per 10,000 (0.54-17.5/10,000) spinal anesthesia patients. In our hospital, four cases of cardiac arrest occurred in 903 spinal and combined spinal and epidural anesthesia patients over 15 months (44.3/ 10,000). Common denominators were ages below 60 years, ASA-PS 1, rapid onset of T10 blockade within 2 minutes and leg raising from 3 to 6 minutes after administration of hyperbaric bupivacaine and T4 blockade. All patients recovered without any side effects. The conditions suspected of causing strong bradycardia leading to cardiac arrest are thought to be maintained parasympathetic activity, lack of blood volume before anesthesia, rapid progress of spinal blockade, decreased blood return to the heart by high spinal blockade (T4) and protective cardiac parasympathetic reflex (Bezold-Jarisch reflex).

[Left internal jugular venipuncture in real-time ultrasound-guided pediatric central venous cannulation].

The right internal jugular vein (IJV) is usually selected for pediatric central venous cannulation (CVC); however, the left is rarely selected. We investigated cases of CVC through left IJVs from Apr to Nov of 2011. In all 82 patients with the median age of 11 months, height of 72.2 cm, and weight of 7.4 kg, right IJVs were selected first for real-time ultrasound-guided CVC. Right IJVs were with median width of 7.3 mm, length of 5.0 mm, and depth of 6.8 mm. After three failed attempts through the right IJVs in three patients (3.7%), of 2 to 9 months of age out of the 82 patients, 24 G puncture needles were successfully inserted through left IJVs with the first attempt. One of the three patients, of 9 months of age with a persistent left superior vena cava, had a thinner 2.6-mm-wide right IJV and a thicker 8.3-mm-wide left IJV It was difficult but successful to insert a 0.018-inch guidewire in another patient due to a steep 90 degree angle between the left IJV and the brachiocephalic vein.

Deficiency of protocadherin 9 leads to reduction in positive emotional behaviour.

Protocadherin 9 (Pcdh9) is a member of the cadherin superfamily and is uniquely expressed in the vestibular and limbic systems; however, its physiological role remains unclear. Here, we studied the expression of Pcdh9 in the limbic system and phenotypes of Pcdh9-knock-out mice (Pcdh9 KO mice). Pcdh9 mRNA was expressed in the fear extinction neurons that express protein phosphatase 1 regulatory subunit 1 B (Ppp1r1b) in the posterior part of the basolateral amygdala (pBLA), as well as in the Cornu Ammonis (CA) and Dentate Gyrus (DG) neurons of the hippocampus. We show that the Pcdh9 protein was often localised at synapses. Phenotypic analysis of Pcdh9 KO mice revealed no apparent morphological abnormalities in the pBLA but a decrease in the spine number of CA neurons. Further, the Pcdh9 KO mice were related to features such as the abnormal optokinetic response, less approach to novel objects, and reduced fear extinction during recovery from the fear. These results suggest that Pcdh9 is involved in eliciting positive emotional behaviours, possibly via fear extinction neurons in the pBLA and/or synaptic activity in the hippocampal neurons, and normal optokinetic eye movement in brainstem optokinetic system-related neurons.

Functional polymorphism in the GPR55 gene is associated with anorexia nervosa.

Endocannabinoids, anandamide, and 2-arachidonoyl glycerol are involved in food intake and appetite. Although anandamide is now thought to be a ligand for vanilloid receptor, receptors that are targets of anandamide could play a similar role in eating behaviors and related disorders. This study therefore focused on the receptor, which is called G-protein-coupled receptor 55 (GPR55) that had recently been reported to have binding affinity for endocannabinoids. Functional analysis of the sole missense polymorphism, rs3749073 (Gly195Val) in the GPR55 gene was performed by detecting the phosphorylation level of extracellular signal-regulated kinase (ERK) in Chinese-Hamster-Ovary (CHO) cells engineered to express human GPR55. Val195 type GPR55 appeared to induce less phosphorylated ERK than Gly195 type GPR55 when CHO cells were treated with anandamide and lysophosphatidylinositol (LPI). An association between the functional Gly195Val polymorphism and anorexia nervosa was tested in a female Japanese population comprising 235 patients and 1244 controls. The Val195 allele and homozygote of the Val195 allele were more abundant in the group of patients diagnosed with anorexia nervosa (P = 0.023, Odds ratio = 1.31 (95% Cl = 1.03-1.37), P = 0.0048, OR = 2.41 (95% Cl = 1.34-4.34), respectively). In conclusion, the low-functioning Val195 allele of GPR55 appears to be a risk factor for anorexia nervosa.

The Effects of Paliperidone Palmitate 1 Month on the Employment Status and Social Functioning of Patients with Schizophrenia.

Objective: We sought to evaluate the effects of a one-month paliperidone palmitate formulation (PP1M) on employment status, social function, symptomatology, and safety and conducted a two-year postmarketing surveillance study of Paliperidone Palmitate 1 Month (PP1M). Methods: Patients diagnosed with schizophrenia participated in the study. Employment status was recorded at baseline and changes were measured at one and two years. Social functioning and symptomatology were assessed using the Social and Occupational Functioning Assessment Scale (SOFAS) and the Clinical Global Impression-Schizophrenia (CGI-SCH). Data on adverse events were also collected. Results: A total of 1,319 patients were enrolled in this investigation, including 1,306 who were evaluable for safety and 1,279 who were evaluable for efficacy. The maintenance rate during the observation period was 49.4 percent. During the observation period, the percentages of patients reporting employment significantly increased: 24.3 percent of patients were employed in some capacity at baseline, 32.5 percent of patients were employed at one year, and 34.6 percent of patients were employed at two years. Significant improvements were observed in both SOFAS and CGI-SCH scores during the observation period. The percentage of patients with socially functional remission also significantly increased. A strong association between the improvement of social function, gender, and monotherapy versus polypharmacy and the improvement of employment status was observed. A total of 29.3 percent of patients experienced at least one adverse event. There were no unexpected findings from long-term treatment and a safety profile, including mortality. Conclusion: PP1M treatment appears to improve not only schizophrenic symptoms but also functional outcomes.

Design, synthesis, enzyme inhibition, and tumor cell growth inhibition of 2-anilinobenzamide derivatives as SIRT1 inhibitors.

A series of 2-anilinobenzamide derivatives were designed, synthesized and evaluated for their SIRT1-inhibitory activity. Among these, compounds 3 and 5 inhibited SIRT1 activity in enzyme assays and suppressed the growth of Daudi and HCT116 cells.