Flow‐injection chemiluminescence method to detect a β2 adrenergic agonist

A new method for the detection of β₂ adrenergic agonists was developed based on the chemiluminescence (CL) reaction of β₂ adrenergic agonist with potassium ferricyanide–luminol CL. The effect of β₂ adrenergic agonists including isoprenaline hydrochloride, salbutamol sulfate, terbutaline sulfate and ractopamine on the CL intensity of potassium ferricyanide–luminol was discovered. Detection of the β₂ adrenergic agonist was carried out in a flow system. Using uniform design experimentation, the influence factors of CL were optimized. The optimal experimental conditions were 1 mmol/L of potassium ferricyanide, 10 µmol/L of luminol, 1.2 mmol/L of sodium hydroxide, a flow speed of 2.6 mL/min and a distance of 1.2 cm from ‘Y₂’ to the flow cell. The linear ranges and limit of detection were 10–100 and 5 ng/mL for isoprenaline hydrochloride, 20–100 and 5 ng/mL for salbutamol sulfate, 8–200 and 1 ng/mL for terbutaline sulfate, 20–100 and 4 ng/mL for ractopamine, respectively. The proposed method allowed 200 injections/h with excellent repeatability and precision. It was successfully applied to the determination of three β₂ adrenergic agonists in commercial pharmaceutical formulations with recoveries in the range of 96.8–98.5%. The possible CL reaction mechanism of potassium ferricyanide–luminol–β₂ adrenergic agonist was discussed from the UV/vis spectra. Copyright © 2014 John Wiley & Sons, Ltd.     

A Voltage-Gated Calcium Channel Regulates Lysosomal Fusion with Endosomes and Autophagosomes and Is Required for Neuronal Homeostasis

Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis.     

Complete Blood Count Reference Intervals for Healthy Han Chinese Adults

Background

Complete blood count (CBC) reference intervals are important to diagnose diseases, screen blood donors, and assess overall health. However, current reference intervals established by older instruments and technologies and those from American and European populations are not suitable for Chinese samples due to ethnic, dietary, and lifestyle differences. The aim of this multicenter collaborative study was to establish CBC reference intervals for healthy Han Chinese adults.

Methods

A total of 4,642 healthy individuals (2,136 males and 2,506 females) were recruited from six clinical centers in China (Shenyang, Beijing, Shanghai, Guangzhou, Chengdu, and Xi’an). Blood samples collected in K2EDTA anticoagulant tubes were analyzed. Analysis of variance was performed to determine differences in consensus intervals according to the use of data from the combined sample and selected samples.

Results

Median and mean platelet counts from the Chengdu center were significantly lower than those from other centers. Red blood cell count (RBC), hemoglobin (HGB), and hematocrit (HCT) values were higher in males than in females at all ages. Other CBC parameters showed no significant instrument-, region-, age-, or sex-dependent difference. Thalassemia carriers were found to affect the lower or upper limit of different RBC profiles.

Conclusion

We were able to establish consensus intervals for CBC parameters in healthy Han Chinese adults. RBC, HGB, and HCT intervals were established for each sex. The reference interval for platelets for the Chengdu center should be established independently.     

24h Urinary Protein Levels and Urine Protein/Creatinine Ratios Could Probably Forecast the Pathological Classification of HSPN

This study aimed to assess the relevance of laboratory tests in Henoch-Schönlein purpura nephritis (HSPN) classification, and determine accurate classification factors. This prospective study included 694 HSPN patients who underwent ultrasound-guided percutaneous renal biopsy (PRB). Renal specimens were scored according to International Study of Kidney Disease in Children (ISKDC) classification. Meanwhile, blood samples were immediately collected for laboratory examination. The associations between laboratory parameters and HSPN classification were assessed. Significant differences in levels of serum Th1/Th2 cytokines, immunoglobulins, T-lymphocyte subsets, complement, and coagulation markers were obtained between HSPN patients and healthy children. Interestingly, 24h urinary protein (24h-UPRO) levels and urine protein/urine creatinine ratios could determine HPSN grade IIb, IIIa, and IIIb incidences, with areas under ROC curve of 0.767 and 0.731, respectively. At 24h-UPRO >580.35mg/L, prediction sensitivity and specificity were 75.2% and 70.0%, respectively. These values became 53.0% and 82.3%, respectively, with 24h-UPRO exceeding 1006.25mg/L. At urine protein/urine creatinine > 0.97, prediction sensitivity and specificity were 65.5% and 67.2%, respectively, values that became 57.4% and 80.0%, respectively, at ratios exceeding 1.2. Cell and humoral immunity, coagulation and fibrinolytic systems are all involved in the pathogenesis of HSPN, and type I hypersensitivity may be the disease trigger of HSPN. 24h-UPRO levels and urine protein/creatinine ratios could probably forecast the pathological classification of HSPN.     

Low Goiter Rate Associated with Small Average Thyroid Volume in Schoolchildren after the Elimination of Iodine Deficiency Disorders

Background

After the implementation of the universal salt iodization (USI) program in 1996, seven cross-sectional school-based surveys have been conducted to monitor iodine deficiency disorders (IDD) among children in eastern China.

Objectives

This study aimed to examine the correlation of total goiter rate (TGR) with average thyroid volume (Tvol) and urinary iodine concentration (UIC) in Jiangsu province after IDD elimination.

Design

Probability-proportional-to-size sampling was applied to select 1,200 children aged 8–10 years old in 30 clusters for each survey in 1995, 1997, 1999, 2001, 2002, 2005, 2009 and 2011. We measured Tvol using ultrasonography in 8,314 children and measured UIC (4,767 subjects) and salt iodine (10,184 samples) using methods recommended by the World Health Organization. Tvol was used to calculate TGR based on the reference criteria specified for sex and body surface area (BSA).

Results

TGR decreased from 55.2% in 1997 to 1.0% in 2009, and geometric means of Tvol decreased from 3.63 mL to 1.33 mL, along with the UIC increasing from 83 μg/L in 1995 to 407 μg/L in 1999, then decreasing to 243 μg/L in 2005, and then increasing to 345 μg/L in 2011. In the low goiter population (TGR < 3.9%), TGR was positively associated with average Tvol (r = 0.99); UIC showed a non-linear association with average Tvol, and UIC > 300 μg/L was associated with a smaller average Tvol in children.

Conclusions

After IDD elimination in Jiangsu province in 2001, lower TGR was associated with smaller average Tvol. Average Tvol was more sensitive than TGR in detecting the fluctuation of UIC. A UIC of 300 μg/L may be defined as a critical value for population level iodine status monitoring.     

Finite Element Analysis of a New Pedicle Screw-Plate System for Minimally Invasive Transforaminal Lumbar Interbody Fusion

Purpose

Minimally invasive transforaminal lumbar interbody fusion (MI-TLIF) is increasingly popular for the surgical treatment of degenerative lumbar disc diseases. The constructs intended for segmental stability are varied in MI-TLIF. We adopted finite element (FE) analysis to compare the stability after different construct fixations using interbody cage with posterior pedicle screw-rod or pedicle screw-plate instrumentation system.

Methods

A L3–S1 FE model was modified to simulate decompression and fusion at L4–L5 segment. Fixation modes included unilateral plate (UP), unilateral rod (UR), bilateral plate (BP), bilateral rod (BR) and UP+UR fixation. The inferior surface of the S1 vertebra remained immobilized throughout the load simulation, and a bending moment of 7.5 Nm with 400N pre-load was applied on the L3 vertebra to recreate flexion, extension, lateral bending, and axial rotation. Range of motion (ROM) and Von Mises stress were evaluated for intact and instrumentation models in all loading planes.

Results

All reconstructive conditions displayed decreased motion at L4–L5. The pedicle screw-plate system offered equal ROM to pedicle screw-rod system in unilateral or bilateral fixation modes respectively. Pedicle screw stresses for plate system were 2.2 times greater than those for rod system in left lateral bending under unilateral fixation. Stresses for plate were 3.1 times greater than those for rod in right axial rotation under bilateral fixation. Stresses on intervertebral graft for plate system were similar to rod system in unilateral and bilateral fixation modes respectively. Increased ROM and posterior instrumentation stresses were observed in all loading modes with unilateral fixation compared with bilateral fixation in both systems.

Conclusions

Transforaminal lumbar interbody fusion augmentation with pedicle screw-plate system fixation increases fusion construct stability equally to the pedicle screw-rod system. Increased posterior instrumentation stresses are observed in all loading modes with plate fixation, and bilateral fixation could reduce stress concentration.     

Expression of PprI from Deinococcus radiodurans Improves Lactic Acid Production and Stress Tolerance in Lactococcus lactis

PprI is a general switch protein that regulates the expression of certain proteins involved in pathways of cellular resistance in the extremophilic bacterium Deinococcus radiodurans. In this study, we transformed pprI into Lactococcus lactis strain MG1363 using the lactococcal shuttle vector pMG36e and investigated its effects on the tolerance and lactic acid production of L. lactis while under stress. PprI was stably expressed in L. lactis as confirmed by western blot assays. L. lactis expressing PprI exhibited significantly improved resistance to oxidative stress and high osmotic pressure. This enhanced cellular tolerance to stressors might be due to the regulation of resistance-related genes (e.g., recA, recO, sodA, and nah) by pprI. Moreover, transformed L. lactis demonstrated increased lactic acid production, attributed to enhanced lactate dehydrogenase activity. These results suggest that pprI can improve the tolerance of L. lactis to environmental stresses, and this transformed bacterial strain is a promising candidate for industrial applications of lactic acid production.     

Germ cell-specific Atg7 knockout results in primary ovarian insufficiency in female mice

Primary ovarian insufficiency (POI) is a common cause of infertility in around 1–2% of women aged <40 years. However, the mechanisms that cause POI are still poorly understood. Here we showed that germ cell-specific knockout of an essential autophagy induction gene Atg7 led to subfertility in female mice. The subfertility of Atg7 deletion females was caused by severe ovarian follicle loss, which is very similar to human POI patients. Further investigation revealed that germ cell-specific Atg7 knockout resulted in germ cell over-loss at the neonatal transition period. In addition, our in vitro studies also demonstrated that autophagy could protect oocytes from over-loss by apoptosis in neonatal ovaries under the starvation condition. Taken together, our results uncover a new role for autophagy in the regulation of ovarian primordial follicle reservation and hint that autophagy-related genes might be potential pathogenic genes to POI of women.Primary ovarian insufficiency (POI), also known as premature ovarian failure (POF), is an ovarian defect characterized by the premature depletion of ovarian follicles before the age of 40 years. POI is a common cause of infertility in women, affecting 1–2% of individuals aged <40 years and 0.1% of individuals aged <30 years.¹ Potential etiologies for POI are highly heterogeneous, which include iatrogenic, infectious, autoimmune, metabolic, chromosomal and genetic factors.² At present, about 25% of all forms of POF can be classified as iatrogenic and are related to cancer treatment, but >50% of the cases remain idiopathic. Though the pathogenic mechanism remains unexplained in the majority of the cases, several observations support a prevalent role of genetic mechanisms in the pathogenesis of idiopathic POI. It has been reported that mutations in FMR1, BMP-15, GDF-9, FOCL2, FSHR, LHR, INHA, GALT and AIRE are associated with POI.^{3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13} The genetic information of POI is very useful for family counseling, because it can predict the female relatives who may be at higher risk for POI and fertility loss in young age. The female carriers will be able to plan their conception before ovarian failure occurs. This requirement is becoming more and more important, because women nowadays tend to conceive ever more frequently in their thirties and forties,¹⁰ when the risk of POI in the general population is about 1–2%. However, still few genes could be identified that can explain a substantial proportion of the cases of POI.An important phenotype of POI is infertility, thus POI patients do not have large family histories, and therefore are difficult to study using traditional genetic methods, such as linkage analysis. Animal models of POI have been successfully used to identify candidate genes in this disease. The disruption of meiosis-specific genes, Bcl-2 family apoptotic-related genes, Pten-PI3K-Akt-Foxo3 pathway and Tsc1/2-mTOR signaling pathway result in POI-like phenotype in mice.^{14, 15, 16, 17} However, as a complex disorder, the genetic etiologies of POI still need to be further investigated to better understand the underlying molecular mechanisms.Macroautophagy (hereafter referred to as autophagy) is the primary intracellular catabolic mechanism for degrading and recycling long-lived proteins and organelles, which is evolutionarily conserved from yeast to mammals.¹⁸ During autophagy, isolation membrane enwraps parts of the cytoplasm and intracellular organelles, and fuse with each other forming a double membrane structure, known as the autophagosome. Then the outer membrane of the autophagosome fuses with the lysosome to form autolysosome, in which the cytoplasm-derived materials are degraded by resident hydrolases.¹⁹ The primary function of autophagy is to allow cells or organisms to survive nutrient starvation conditions by recycling either proteins or other cellular components. This process is important for cells to adapt their metabolism to starvation caused by decreased extracellular nutrients or by decreased intracellular metabolite concentrations. In addition to nutrient supply and adaptation to stress conditions, a number of observations have revealed that autophagy also functions in many physiological processes in mammalian systems, such as cell death, antiaging mechanisms, innate immunity, development and tumor suppression.^{20, 21, 22, 23, 24, 25}From the discovery of the molecular mechanism underlying autophagy, it was found that autophagy is required for the reproductive process in budding yeast.²⁶ In mammals, fertilization induces massive autophagy to degrade maternal proteins and messenger RNAs, and autophagy functions as a major nutrient-providing system for embryos before their implantation.²⁷ Our recent work indicates that autophagy is required for acrosome biogenesis during spermatogenesis in mice, thus essential to male fertility.²⁴ However, whether autophagy is involved in female gametogenesis or not is still unknown. Here, we showed that germ cell-specific knockout of an essential autophagy induction gene Atg7 led to POI in female mice, and the numbers of the oocytes and follicles were significantly declined in the adult mutant mice. Further investigation revealed that autophagy protected oocytes over-loss during the neonatal transition period. Our results suggest that autophagy-related genes might be pathogenic genes to POI.     

Tea Consumption and Cognitive Impairment: A Cross-Sectional Study among Chinese Elderly

Background

Laboratorial and epidemiological researches suggested that tea exhibited potential neuroprotective effect which may prevent cognitive impairment, but there were few data among the elderly aged 60 years and above in China.

Objective

The objective was to explore the relationship between characteristics of tea consumption and cognitive impairment.

Design

We analyzed the baseline data from Zhejiang Major Public Health Surveillance Program (ZPHS) which was conducted in 2014. Totally 9,375 residents aged 60 years and above were recruited in this study. Face-to-face interview based on a self-developed questionnaire was performed for each participant. Detailed tea consumption habits were included in the questionnaire. Cognitive impairment screening was performed by using Mini-Mental State Examination (MMSE). Education-specific cut-off points for Chinese were applied to determine the status of cognitive impairment. Logistic regression analysis was used to calculate odds ratios (ORs) of cognitive impairment associated with tea consumption.

Results

The means (SD) of MMSE scores for the subjects who did not consume tea and consumed <2 cups/d, 2–4 cups/d, ≥4 cups/d were 23.3 (SD = 5.61), 23.8 (SD = 5.60), 24.5 (SD = 5.63) and 25.0 (SD = 5.08), respectively. An inverse correlation was found between tea consumption (of all types) and prevalence of cognitive impairment. Volume of tea consumption was significantly associated with cognitive impairment: compared with non-consumption participants, those who consumed < 2 cups/d, 2–4 cups/d, and ≥4 cups/d were observed ORs of 0.77 (95% CI: 0.56, 1.07), 0.62 (95% CI: 0.47, 0.81), and 0.49 (95% CI: 0.36, 0.66), respectively. Compared with non-consumption, black tea presented a positive correlation with cognitive function after controlling for potential confounders (OR = 0.52, 95% CI: 0.28, 0.95), while green tea showed no significant difference (OR = 1.04, 95% CI: 0.72, 1.51). Participants who consumed weak tea, moderate tea or strong tea more often were observed a better cognitive status when compared with those who did not have tea, with an OR of 0.51 (95% CI: 0.28, 0.92), 0.32 (95% CI: 0.19, 0.56) and 0.42 (95% CI: 0.22, 0.78) after adjusting for the potential confounders. But there was no statistically significant difference between any two of these ORs.

Conclusion

Black tea consumption was association with better cognitive performance among the elderly aged 60 years and above in China, while green tea presented no correlation. The positive association of cognitive status with tea consumption was not limited to particular type of concentration.