Epidemiological Characterization of Influenza A(H1N1)pdm09 Cases from 2009 to 2010 in Baguio City,the Philippines

Background

Baguio City, Philippines experienced its first influenza A(H1N1)pdm09 [A(H1)pdm09] case in May 2009. In spite of numerous reports describing the epidemiological and clinical features of A(H1)pdm09 cases, there are no studies about A(H1)pdm09 epidemiology in the Philippines, where year-round influenza activity was observed.

Objectives

We aimed to investigate the epidemiological and clinical features of A(H1)pdm09 in pandemic and post-pandemic periods.

Methods

Data were collected under enhanced surveillance of influenza-like illness (ILI) and severe acute respiratory infection (SARI) from January 2009 to December 2010. RT-PCR was used to detect A(H1)pdm09, following the protocol of the United States Centers for Disease Control and Prevention. The reproduction number was computed as a simple exponential growth rate. Differences in proportional and categorical data were examined using chi-square test or Fishers’ exact test.

Results and Conclusions

The outbreak was observed from week 25 to 35 in 2009 and from week 24 to 37 in 2010. The highest proportion of cases was among children aged 5–14 years. The number of ILI outpatients was 2.3-fold higher in 2009 than in 2010, while the number of inpatients was 1.8-fold higher in 2009. No significant difference in gender was observed during the two periods. The clinical condition of all patients was generally mild and self-limiting, with only 2 mortalities among inpatients in 2009. The basic reproduction number was estimated as 1.16 in 2009 and 1.05 in 2010 in the assumption of mean generation time as 2.6 days. School children played a significant role in facilitating influenza transmission.     

Synthesis of Very-Long-Chain Fatty Acids in the Epidermis Controls Plant Organ Growth by Restricting Cell Proliferation

Plant organ growth is controlled by inter-cell-layer communication, which thus determines the overall size of the organism. The epidermal layer interfaces with the environment and participates in both driving and restricting growth via inter-cell-layer communication. However, it remains unknown whether the epidermis can send signals to internal tissue to limit cell proliferation in determinate growth. Very-long-chain fatty acids (VLCFAs) are synthesized in the epidermis and used in the formation of cuticular wax. Here we found that VLCFA synthesis in the epidermis is essential for proper development of Arabidopsis thaliana. Wild-type plants treated with a VLCFA synthesis inhibitor and pasticcino mutants with defects in VLCFA synthesis exhibited overproliferation of cells in the vasculature or in the rib zone of shoot apices. The decrease of VLCFA content increased the expression of IPT3, a key determinant of cytokinin biosynthesis in the vasculature, and, indeed, elevated cytokinin levels. These phenotypes were suppressed in ipt3;5;7 triple mutants, and also by vasculature-specific expression of cytokinin oxidase, which degrades active forms of cytokinin. Our results imply that VLCFA synthesis in the epidermis is required to suppress cytokinin biosynthesis in the vasculature, thus fine-tuning cell division activity in internal tissue, and therefore that shoot growth is controlled by the interaction between the surface (epidermis) and the axis (vasculature) of the plant body.     

Vertical Distribution and Estimated Doses from Artificial Radionuclides in Soil Samples around the Chernobyl Nuclear Power Plant and the Semipalatinsk Nuclear Testing Site

For the current on-site evaluation of the environmental contamination and contributory external exposure after the accident at the Chernobyl Nuclear Power Plant (CNPP) and the nuclear tests at the Semipalatinsk Nuclear Testing Site (SNTS), the concentrations of artificial radionuclides in soil samples from each area were analyzed by gamma spectrometry. Four artificial radionuclides (²⁴¹Am, ¹³⁴Cs, ¹³⁷Cs, and ⁶⁰Co) were detected in surface soil around CNPP, whereas seven artificial radionuclides (²⁴¹Am, ⁵⁷Co, ¹³⁷Cs, ⁹⁵Zr, ⁹⁵Nb, ⁵⁸Co, and ⁶⁰Co) were detected in surface soil around SNTS. Effective doses around CNPP were over the public dose limit of 1 mSv/y (International Commission on Radiological Protection, 1991). These levels in a contaminated area 12 km from Unit 4 were high, whereas levels in a decontaminated area 12 km from Unit 4 and another contaminated area 15 km from Unit 4 were comparatively low. On the other hand, the effective doses around SNTS were below the public dose limit. These findings suggest that the environmental contamination and effective doses on the ground definitely decrease with decontamination such as removing surface soil, although the effective doses of the sampling points around CNPP in the present study were all over the public dose limit. Thus, the remediation of soil as a countermeasure could be an extremely effective method not only for areas around CNPP and SNTS but also for areas around the Fukushima Dai-ichi Nuclear Power Plant (FNPP), and external exposure levels will be certainly reduced. Long-term follow-up of environmental monitoring around CNPP, SNTS, and FNPP, as well as evaluation of the health effects in the population residing around these areas, could contribute to radiation safety and reduce unnecessary exposure to the public.     

Basal and Ischemia-Induced Transcardiac Troponin Release into the Coronary Circulation in Patients with Suspected Coronary Artery Disease

Background

Cardiac troponin is a specific biomarker for cardiomyocyte necrosis in acute coronary syndromes. Troponin release from the coronary circulation remains to be determined because of the lower sensitivity of the conventional assay. We sought to determine basal and angina-induced troponin release using a highly sensitive troponin assay.

Methods and Results

The cardiac troponin T levels in serum sampled from the peripheral vein (PV), the aortic root (AO), and the coronary sinus (CS) were measured in 105 consecutive stable patients with coronary risk factor(s) and suspected coronary artery disease (CAD) and in 33 patients without CAD who underwent an acetylcholine provocation test. At baseline, there was a significant increase in the troponin levels from AO [9.0 (6.4, 13.1) pg/mL for median (25^th, 75^th percentiles)] to CS [10.3 (7.3, 15.5) pg/mL, p<0.001] in 96 (91.4%) patients and the difference was 1.1 (0.4, 2.1) pg/mL, which reflected basal transcardiac troponin release (TTR). TTR was positively correlated with PV levels (r = 0.22, p = 0.03). Male sex, left ventricular hypertrophy determined by echocardiography, T-wave inversion, and CAD correlated with elevated TTR defined as above: median, 1.1 pg/mL. A significant increase in TTR was noted in 17 patients with coronary spasms [0.6 (0.2, 1.2) pg/mL, p<0.01] but not in 16 patients without spasms [0.0 (−0.5, 0.9) pg/mL, p = 0.73] after the acetylcholine provocation.

Conclusion

Basal TTR in the coronary circulation was observed in most of the patients with suspected CAD and risk factor(s). This sensitive assay detected myocardial ischemia-induced increases in TTR caused by coronary spasms.     

A Decreased Level of Serum Soluble Klotho Is an Independent Biomarker Associated with Arterial Stiffness in Patients with Chronic Kidney Disease

Background

Klotho was originally identified in a mutant mouse strain unable to express the gene that consequently showed shortened life spans. In humans, low serum Klotho levels are related to the prevalence of cardiovascular diseases in community-dwelling adults. However, it is unclear whether the serum Klotho levels are associated with signs of vascular dysfunction such as arterial stiffness, a major determinant of prognosis, in human subjects with chronic kidney disease (CKD).

Methods

We determined the levels of serum soluble Klotho in 114 patients with CKD using ELISA and investigated the relationship between the level of Klotho and markers of CKD-mineral and bone disorder (CKD-MBD) and various types of vascular dysfunction, including flow-mediated dilatation, a marker of endothelial dysfunction, ankle-brachial pulse wave velocity (baPWV), a marker of arterial stiffness, intima-media thickness (IMT), a marker of atherosclerosis, and the aortic calcification index (ACI), a marker of vascular calcification.

Results

The serum Klotho level significantly correlated with the 1,25-dihydroxyvitamin D level and inversely correlated with the parathyroid hormone level and the fractional excretion of phosphate. There were significant decreases in serum Klotho in patients with arterial stiffness defined as baPWV≥1400 cm/sec, atherosclerosis defined as maximum IMT≥1.1 mm and vascular calcification scores of ACI>0%. The serum Klotho level was a significant determinant of arterial stiffness, but not endothelial dysfunction, atherosclerosis or vascular calcification, in the multivariate analysis in either metabolic model, the CKD model or the CKD-MBD model. The adjusted odds ratio of serum Klotho for the baPWV was 0.60 (p = 0.0075).

Conclusions

Decreases in the serum soluble Klotho levels are independently associated with signs of vascular dysfunction such as arterial stiffness in patients with CKD. Further research exploring whether therapeutic approaches to maintain or elevate the Klotho level could improve arterial stiffness in CKD patients is warranted.     

An In Vitro ES Cell-Based Clock Recapitulation Assay Model Identifies CK2α as an Endogenous Clock Regulator

We previously reported emergence and disappearance of circadian molecular oscillations during differentiation of mouse embryonic stem (ES) cells and reprogramming of differentiated cells, respectively. Here we present a robust and stringent in vitro circadian clock formation assay that recapitulates in vivo circadian phenotypes. This assay system first confirmed that a mutant ES cell line lacking Casein Kinase I delta (CKIδ) induced ∼3 hours longer period-length of circadian rhythm than the wild type, which was compatible with recently reported results using CKIδ null mice. In addition, this assay system also revealed that a Casein Kinase 2 alpha subunit (CK2α) homozygous mutant ES cell line developed significantly longer (about 2.5 hours) periods of circadian clock oscillations after in vitro or in vivo differentiation. Moreover, revertant ES cell lines in which mutagenic vector sequences were deleted showed nearly wild type periods after differentiation, indicating that the abnormal circadian period of the mutant ES cell line originated from the mutation in the CK2α gene. Since CK2α deficient mice are embryonic lethal, this in vitro assay system represents the genetic evidence showing an essential role of CK2α in the mammalian circadian clock. This assay was successfully applied for the phenotype analysis of homozygous mutant ES cells, demonstrating that an ES cell-based in vitro assay is available for circadian genetic screening.     

Focus Issue on Calcium Signaling: Analyses of a Gravistimulation-Specific Ca2+ Signature in Arabidopsis using Parabolic Flights

Gravity is a critical environmental factor affecting the morphology and functions of organisms on the Earth. Plants sense changes in the gravity vector (gravistimulation) and regulate their growth direction accordingly. In Arabidopsis (Arabidopsis thaliana) seedlings, gravistimulation, achieved by rotating the specimens under the ambient 1g of the Earth, is known to induce a biphasic (transient and sustained) increase in cytoplasmic calcium concentration ([Ca2+]_c). However, the [Ca2+]_c increase genuinely caused by gravistimulation has not been identified because gravistimulation is generally accompanied by rotation of specimens on the ground (1g), adding an additional mechanical signal to the treatment. Here, we demonstrate a gravistimulation-specific Ca²⁺ response in Arabidopsis seedlings by separating rotation from gravistimulation by using the microgravity (less than 10⁻⁴g) conditions provided by parabolic flights. Gravistimulation without rotating the specimen caused a sustained [Ca2+]_c increase, which corresponds closely to the second sustained [Ca2+]_c increase observed in ground experiments. The [Ca2+]_c increases were analyzed under a variety of gravity intensities (e.g. 0.5g, 1.5g, or 2g) combined with rapid switching between hypergravity and microgravity, demonstrating that Arabidopsis seedlings possess a very rapid gravity-sensing mechanism linearly transducing a wide range of gravitational changes (0.5g–2g) into Ca²⁺ signals on a subsecond time scale.Calcium ion (Ca²⁺) functions as an intracellular second messenger in many signaling pathways in plants (White and Broadley, 2003; Hetherington and Brownlee, 2004; McAinsh and Pittman, 2009; Spalding and Harper, 2011). Endogenous and exogenous signals are spatiotemporally encoded by changing the free cytoplasmic concentration of Ca²⁺ ([Ca2+]_c), which in turn triggers [Ca2+]_c-dependent downstream signaling (Sanders et al., 2002; Dodd et al., 2010). A variety of [Ca2+]_c increases induced by diverse environmental and developmental stimuli are reported, such as phytohormones (Allen et al., 2000), temperature (Plieth et al., 1999; Dodd et al., 2006), and touch (Knight et al., 1991; Monshausen et al., 2009). The [Ca2+]_c increase couples each stimulus and appropriate physiological responses. In the Ca²⁺ signaling pathways, the stimulus-specific [Ca2+]_c pattern (e.g. amplitude and oscillation) provide the critical information for cellular signaling (Scrase-Field and Knight, 2003; Dodd et al., 2010). Therefore, identification of the stimulus-specific [Ca2+]_c signature is crucial for an understanding of the intracellular signaling pathways and physiological responses triggered by each stimulus, as shown in the case of cold acclimation (Knight et al., 1996; Knight and Knight, 2000).Plants often exhibit biphasic [Ca2+]_c increases in response to environmental stimuli. Thus, slow cooling causes a fast [Ca2+]_c transient followed by a second, extended [Ca2+]_c increase in Arabidopsis (Arabidopsis thaliana; Plieth et al., 1999; Knight and Knight, 2000). The Ca²⁺ channel blocker lanthanum (La³⁺) attenuated the fast transient but not the following increase (Knight and Knight, 2000), suggesting that these two [Ca2+]_c peaks have different origins. Similarly, hypoosmotic shock caused a biphasic [Ca2+]_c increase in tobacco (Nicotiana tabacum) suspension-culture cells (Takahashi et al., 1997; Cessna et al., 1998). The first [Ca2+]_c peak was inhibited by gadolinium (Gd³⁺), La³⁺, and the Ca²⁺ chelator EGTA (Takahashi et al., 1997; Cessna et al., 1998), whereas the second [Ca2+]_c increase was inhibited by the intracellular Ca²⁺ store-depleting agent caffeine but not by EGTA (Cessna et al., 1998). The amplitude of the first [Ca2+]_c peak affected the amplitude of the second increase and vice versa (Cessna et al., 1998). These results suggest that even though the two [Ca2+]_c peaks originate from different Ca²⁺ fluxes (e.g. Ca²⁺ influx through the plasma membrane and Ca²⁺ release from subcellular stores, respectively), they are closely interrelated, showing the importance of the kinetic and pharmacological analyses of these [Ca2+]_c increases.Changes in the gravity vector (gravistimulation) could work as crucial environmental stimuli in plants and are generally achieved by rotating the specimens (e.g. +180°) in ground experiments. Use of Arabidopsis seedlings expressing apoaequorin, a Ca²⁺-reporting photoprotein (Plieth and Trewavas, 2002; Toyota et al., 2008a), has revealed that gravistimulation induces a biphasic [Ca2+]_c increase that may be involved in the sensory pathway for gravity perception/response (Pickard, 2007; Toyota and Gilroy, 2013) and the intracellular distribution of auxin transporters (Benjamins et al., 2003; Zhang et al., 2011). These two Ca²⁺ changes have different characteristics. The first transient [Ca2+]_c increase depends on the rotational velocity but not angle, whereas the second sustained [Ca2+]_c increase depends on the rotational angle but not velocity. The first [Ca2+]_c transient was inhibited by Gd³⁺, La³⁺, and the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid but not by ruthenium red (RR), whereas the second sustained [Ca2+]_c increase was inhibited by all these chemicals. These results suggest that the first transient and second sustained [Ca2+]_c increases are related to the rotational stimulation and the gravistimulation, respectively, and are mediated by distinct molecular mechanisms (Toyota et al., 2008a). However, it has not been demonstrated directly that the second sustained [Ca2+]_c increase is induced solely by gravistimulation; it could be influenced by other factors, such as an interaction with the first transient [Ca2+]_c increase (Cessna et al., 1998), vibration, and/or deformation of plants during the rotation.To elucidate the genuine Ca²⁺ signature in response to gravistimulation in plants, we separated rotation and gravistimulation under microgravity (μg; less than 10⁻⁴g) conditions provided by parabolic flight (PF). Using this approach, we were able to apply rotation and gravistimulation to plants separately (Fig. 1). When Arabidopsis seedlings were rotated +180° under μg conditions, the [Ca2+]_c response to the rotation was transient and almost totally attenuated in a few seconds. Gravistimulation (transition from μg to 1.5g) was then applied to these prerotated specimens at the terminating phase of the PF. This gravistimulation without simultaneous rotation induced a sustained [Ca2+]_c increase. The kinetic properties of this sustained [Ca2+]_c increase were examined under different gravity intensities (0.5g–2g) and sequences of gravity intensity changes (Fig. 2A). This analysis revealed that gravistimulation-specific Ca²⁺ response has an almost linear dependency on gravitational acceleration (0.5g–2g) and an extremely rapid responsiveness of less than 1 s.Open in a separate windowFigure 1.Diagram of the experimental procedures for applying separately rotation and gravistimulation to Arabidopsis seedlings. Rotatory stimulation (green arrow) was applied by rotating the seedlings 180° under μg conditions, and 1.5g 180° rotation gravistimulation (blue arrow) was applied to the prerotated seedlings after μg.Open in a separate windowFigure 2.Acceleration, temperature, humidity, and pressure in an aircraft during flight experiments. A, Accelerations along x, y, and z axes in the aircraft during PF. The direction of flight (FWD) and coordinates (x, y, and z) are indicated in the bottom graph. The inset shows an enlargement of the acceleration along the z axis (gravitational acceleration) during μg conditions lasting for approximately 20 s. B, Temperature, humidity, and pressure in the aircraft during PF. Shaded areas in graphs denote the μg condition.