Emergence and spread patterns of antibiotic resistance genes during two different aerobic granular sludge cultivation processes.

The prevalence and accumulation of antibiotic resistance genes (ARGs) were frequently detected in biological wastewater treatment processes, which might cause potential health crisis to human. In present study, the fates of ARGs during two different aerobic granular sludge (AGS) cultivation processes were investigated. The results showed that traditional AGS (T-AGS) cultivation process and enhanced AGS (E-AGS) cultivation process had significant differences (P < 0.005) in ARGs shift patterns. E-AGS process had higher average relative abundance (0.280 ± 0.079) of ARGs than T-AGS process (0.130 ± 0.041), while the intensity of ARGs enrichment during E-AGS (1.52-5.29 fold) was lower than T-AGS (3.79-75.31 fold) process. TnpA and intI1 as two different types of mobile genetic elements (MGEs) carrying ARGs, were observed to contribute significantly to the horizontal gene transfer (HGT) during T-AGS (r = 0.902, P < 0.050) and E-AGS (r = 0.823, P < 0.001) processes, respectively. Higher HGT level took place and more possible potential hosts (25 hosts) harboring ARGs were detected during E-AGS process comparing with T-AGS process (17 hosts). Meanwhile, over large AGS might increase the propagation of several antibiotic deactivation ARGs, so it was not advised. Overall, whether during T-AGS or during E-AGS process which was applied in a pilot-scale sequencing batch reactor treating municipal wastewater, the accumulation and spread of ARGs were inevitable. It should be valued that some suitable pre-treatments of seed sludge should be executed, meanwhile, advanced treatment for removing of ARGs in AGS should be conducted to maintain the relative abundances of ARGs at relatively low level.

Enhanced granulation process, a more effective way of aerobic granular sludge cultivation in pilot-scale application comparing to normal granulation process: From the perspective of microbial insights.

Aerobic granular sludge (AGS) could be cultivated from only flocs (called normal granulation (NG) process) or mixture of flocs and crushed AGS (called enhanced granulation (EG) process), which might lead to different system performances such as granulation speed and pollutants removal efficiencies. However, the differences of mechanisms between NG and EG processes at microbial community level are still unknown. In this study, the NG and EG processes were implemented successively in a pilot-scale sequencing batch reactor (SBR) with certain amounts of additional carbon sources. Illumina MiSeq sequencing and quantitative PCR were applied to investigate the dynamics of bacterial communities during NG and EG processes and explore the possible explanations for faster EG process. The results showed that significant distinctions in bacterial diversities and community structures were observed between NG and EG processes. The major contributor to NG process was bacterial communities with 32.04% contribution. While EG process was more dependent on the interactions (73.16% contribution) between the bacterial communities and environmental variables (operational parameters and self-adaptive variable). EG process had higher relative abundances of functional bacteria than NG process. Glycogen accumulating organisms (GAOs) related bacteria with a total relative abundance of maximum 65.43% might be mainly responsible for the faster EG process. This study provided microbial insights for practical application of AGS technology that inoculating crushed AGS might be an effective way to cultivate AGS.

Hippocampal atrophy and functional connectivity disruption in cirrhotic patients with minimal hepatic encephalopathy.

The hippocampus is a crucial pathological node for minimal hepatic encephalopathy (MHE) and it is associated with various cognitive impairments. Investigations on alterations involving hippocampal morphology and functional connectivity (FC) in MHE are limited. This study aimed to simultaneously evaluate hippocampal volume and FC alterations and their association with cognitive decline in MHE. Twenty-two cirrhotic patients with MHE, 31 cirrhotic patients without MHE (NHE), and 43 healthy controls underwent high-resolution T1-weighted imaging, resting-state functional magnetic resonance imaging, and cognition assessment based on Psychometric Hepatic Encephalopathy Score (PHES). The structural images were preprocessed using a voxel-based morphometry method, during which hippocampal volume was measured. The hippocampal connectivity network was identified using seed-based correlation analysis. Hippocampal volume and FC strength were compared across the three groups and correlated against the PHES results of the cirrhotic patients. Compared to the controls, MHE patients exhibited a significantly lower bilateral hippocampal volume. A slight decrease in hippocampal volume was obtained from NHE to MHE, but it did not reach statistically significance. In addition, the average FC strength of the bilateral hippocampal connectivity network was significantly lower in the MHE patients. In particular, the MHE patients showed a decrease in FC involving the left hippocampus to bilateral posterior cingulate gyrus and left angular gyrus. The MHE patients also showed FC reduction between the right hippocampus and bilateral medial frontal cortex. A progressive reduction in hippocampal FC from NHE to MHE was also observed. The bilateral hippocampal FC strength (but not hippocampal volume) was positively correlated with the PHES results of the cirrhotic patients. Our assessment of MHE patients revealed decreased hippocampal volume, which suggests regional atrophy, and reduced hippocampal connectivity with regions that are primarily involved in the default-mode network, thereby suggesting a functional disconnection syndrome. These alterations reveal the mechanisms underlying cognitive deterioration with disease progression.

Functional Network-Based Statistics Reveal Abnormal Resting-State Functional Connectivity in Minimal Hepatic Encephalopathy.

Purpose: Whole-brain functional network analysis is an emerging methodology for exploring the mechanisms underlying hepatic encephalopathy (HE). This study aimed to identify the brain subnetwork that is significantly altered within the functional connectome in minimal HE (MHE), the earliest stage of HE. Materials and Methods: The study enrolled 19 cirrhotic patients with MHE and 19 controls who underwent the resting-state functional magnetic resonance imaging and cognitive assessment based on the Psychometric Hepatic Encephalopathy Score (PHES). A whole-brain functional connectivity (FC) matrix was calculated for each subject. Then, network-based statistical analyses of the functional connectome were used to perform group comparisons, and correlation analyses were conducted to identify the relationships between FC alterations and cognitive performance. Results: MHE patients showed significant reduction of positive FC within a subnetwork that predominantly involved the regions of the default-mode network, such as the bilateral posterior cingulate gyrus, bilateral medial prefrontal cortex, bilateral hippocampus and parahippocampal gyrus, bilateral angular gyrus, and left lateral temporal cortex. Meanwhile, MHE patients showed significant reduction of negative FC between default-mode network regions (such as the bilateral posterior cingulate gyrus, medial prefrontal cortex, and angular gyrus) and the regions involved in the somatosensory network (i.e., bilateral precentral and postcentral gyri) and the language network (i.e., the bilateral Rolandic operculum). The correlations of FC within the default-mode subnetwork and PHES results were noted. Conclusion: Default-mode network dysfunction may be one of the core issues in the pathophysiology of MHE. Our findings support the notion that HE is a neurological disease related to intrinsic brain network disruption.

Altered Topological Properties of Gray Matter Structural Covariance Networks in Minimal Hepatic Encephalopathy.

Background and Aims: Liver cirrhosis commonly induces brain structural impairments that are associated with neurological complications (e.g., minimal hepatic encephalopathy (MHE)), but the topological characteristics of the brain structural network are still less well understood in cirrhotic patients with MHE. This study aimed to conduct the first investigation on the topological alterations of brain structural covariance networks in MHE. Methods: This study included 22 healthy controls (HCs) and 22 cirrhotic patients with MHE. We calculated the gray matter volume of 90 brain regions using an automated anatomical labeling (AAL) template, followed by construction of gray matter structural covariance networks by thresholding interregional structural correlation matrices as well as graph theoretical analysis. Results: MHE patients showed abnormal small-world properties of the brain structural covariance network, i.e., decreased clustering coefficient and characteristic path length and lower small-worldness parameters, which indicated a tendency toward more random architecture. In addition, MHE patients lost hubs in the prefrontal and parietal regions, although they had new hubs in the temporal and occipital regions. Compared to HC, MHE patients had decreased regional degree/betweenness involving several regions, primarily the prefrontal and parietal lobes, motor region, insula and thalamus. In addition, the MHE group also showed increased degree/betweenness in the occipital lobe and hippocampus. Conclusion: These results suggest that MHE leads to altered coordination patterns of gray matter morphology and provide structural evidence supporting the idea that MHE is a neurological complication related to disrupted neural networks.

Diffusion Tensor Imaging With Tract-Based Spatial Statistics Reveals White Matter Abnormalities in Patients With Vascular Cognitive Impairment.

Purpose: The aim of this study was to evaluate microstructural changes of major white matter (WM) tracts in patients with vascular cognitive impairment (VCI). Method: Diffusion tensor imaging (DTI) data were obtained from 24 subjects with subcortical ischemic vascular disease (SIVD), including 13 subjects with VCI-no dementia (VCIND) and 11 subjects with normal cognition (as a control group). A tract-based spatial statistics approach was performed to investigate WM microstructure in VCIND by integrating multiple indices including fractional anisotropy (FA) and mean diffusivity (MD), which are intra-voxel metrics, and local diffusion homogeneity (LDH), which is an inter-voxel metric. Results: The VCIND group had decreased FA and increased MD values throughout widespread WM areas predominately in the corpus callosum, bilateral internal capsule/corona radiata/posterior thalamic radiation/inferior fronto-occipital fasciculus and right inferior/superior longitudinal fasciculus. There was a slight discrepancy between the distribution of areas with decreased FA and LDH. The FA, MD and LDH values were significantly correlated with cognitive test results. According to a WM tract atlas, 10 major tracts were identified as tracts of interest in which three diffusion metrics simultaneously differed between groups, including bilateral anterior thalamic radiation, forceps minor, right corticospinal tract, bilateral inferior fronto-occipital fasciculus, left inferior and superior longitudinal fasciculus, and bilateral uncinate fasciculus. Receiver operating characteristic (ROC) analysis demonstrated the feasibility of using diffusion metrics along the forceps minor and left anterior thalamic radiation for separating two groups. Conclusion: The results suggest WM microstructural abnormalities contribute to cognitive impairments in SIVD patients. DTI parameters may be potential biomarkers for detecting VCIND from SIVD.

[Microbial Community Dynamics During Two Sludge Granulation Processes].

Aerobic granular sludge (AGS) was cultivated in a sequencing batch reactor (SBR). In this study, AGS was broken during the formation process and then mature AGS formed again. The microbial community dynamics during two sludge granulation processes were investigated using high-throughput sequencing to reveal the dominant bacteria beneficial to AGS formation. The abundance dynamics of nitrifying microorganisms were analyzed by a quantitative polymerase chain reaction (qPCR). The results showed that the amount of extracellular protein and polysaccharides increased during two sludge granulation processes. The abundance of ammonia oxidizing archaea (AOA) increased during the first AGS formation process and during the process of AGS maturation. The abundance of ammonia oxidizing bacteria (AOB) decreased during the first AGS formation process, while it maintained a higher abundance than AOA during AGS cultivation. Microbial diversity decreased with AGS formation. The relative abundance of Proteobacteria increased by 12.29% and 5.90% during two sludge granulation processes, respectively. Candidatus Competibacter belonging to Proteobacteria was enriched during two sludge granulation processes, accounting for 14.20% in mature AGS. Overall, extracellular protein and polysaccharides may have contributed to the sludge granulation. Both AOA and AOB might have been involved in ammonia oxidation. This study indicated that Ca. Competibacter might contribute to AGS formation.

Comparative effects of EtOH consumption and thiamine deficiency on cognitive impairment, oxidative damage, and ß-amyloid peptide overproduction in the brain.

The effects of chronic EtOH consumption, associated or not with thiamine deficiency (TD), on cognitive impairment, oxidative damage, and ß-amyloid (Aß) peptide accumulation in the brain were investigated in male C57BL/6 mice. We established an alcoholic mouse model by feeding an EtOH liquid diet, a TD mouse model by feeding a thiamine-depleted liquid diet, and an EtOH treatment associated with TD mouse model by feeding a thiamine-depleted EtOH liquid diet for 7 weeks. The learning and memory functions of the mice were detected through the Y-maze test. Biochemical parameters were measured using corresponding commercial kits. The Aß expression in the hippocampus was observed by immunohistochemical staining. Several results were obtained. First, EtOH significantly reduced cognitive function by significantly decreasing the Glu content in the hippocampus; increasing the AChE activity in the cortex; and reducing the thiamine level, and superoxide dismutase (SOD), glutathione peroxidase (GPx), and choline acetyltransferase (ChAT) activities in both the hippocampus and cortex. The treatment also increased the levels of malondialdehyde (MDA), protein carbonyl, 8-hydroxydeoxyguanosine (8-OHdG), and nitric oxide (NO) and the activities of total nitric oxide synthase (tNOS), inducible nitric oxide synthase (iNOS), and monoamine oxidase B (MAO-B). Furthermore, EtOH enhanced the expression levels of Aß1-42 and Aß1-40 in the hippocampus. Second, TD induced the same dysfunctions caused by EtOH in the biochemical parameters, except for learning ability, 8-OHdG content, and GPx, tNOS, and AChE activities in the cortex. Third, the modification of MDA, protein carbonyl and NO levels, and GPx, iNOS, ChAT, and MAO-B activities in the brain induced by chronic EtOH treatment associated with TD was greater than that induced by EtOH or TD alone. The synergistic effects of EtOH and TD on Aß1-40 and Glu release, as well as on SOD activity, depended on their actions on the hippocampus or cortex. These findings suggest that chronic EtOH consumption can induce TD, cognitive impairment, Aß accumulation, oxidative stress injury, and neurotransmitter metabolic abnormalities. Furthermore, the association of chronic EtOH consumption with TD causes dramatic brain dysfunctions with a severe effect on the brain.

[Microbial Population Dynamics During Sludge Granulation in a Simultaneous Nitrogen and Phosphorus Removal System].

In this study, domestic sewage was utilized to cultivate aerobic granular sludge (AGS) in a simultaneous nitrogen and phosphorus removal (SNPR) system. The bacterial population dynamics during the aerobic sludge granulation were investigated to reveal the granulation mechanisms using Illumina MiSeq PE300 high-throughput sequencing. Quantitative real time polymerase chain reactions (PCR) were used to investigate shifts in the abundance of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), nitrite-oxidizing bacteria (NOB) and polyphosphate accumulating organisms (PAOs). After cultivation for 100 d, the AGS was compact and demonstrated good SNPR performance. During the AGS formation process, extracellular polysaccharides obviously increased, while extracellular proteins kept relatively stable. The abundance of AOA significantly decreased during the formation of AGS process, while the abundance of PAOs increased. The bacterial diversity increased at first and then decreased during the formation of AGS. The bacterial community changed dramatically during aerobic sludge granulation. Persistent operational taxonomic units (OTUs) accounted for 92.70% of the total sequences. Proteobacteria (31.07%-53.67%), Bacteroidetes (6.70%-16.50%) and Chloroflexi (7.84%-13.36%) were the dominant phyla. Candidatus competibacter was obviously enriched in the AGS formation process (increased from 0.11% in the seed sludge to 35.33% in the AGS) and may play an important role in the formation of AGS.

Ameliorative effect of lotus seedpod proanthocyanidins on cognitive impairment and brain aging induced by D-galactose.

This study mainly investigated the ameliorative effect of lotus seedpod proanthocyanidins (LSPC) and the mechanism underlying such effect on cognitive impairment and brain aging induced by d-galactose. Aging mice induced by d-galactose (150 mg/kg, sc injection daily for 6 weeks) were chosen for the experiment. LSPCs (30, 60, and 90 mg/kg, ig) were provided after d-galactose injection. Learning and memory functions were detected by Y-maze and step-down avoidance tests. Then, some biochemical indexes related to cognitive ability and aging were measured. Histopathological feature and P53 protein expression in the hippocampus were observed. Results showed that the three different doses of LSPC could significantly ameliorate the learning and memory abilities impaired by d-galactose. LSPC significantly reduced the levels of malondialdehyde and nitric oxide (i.e. 90 mg/kg LSPC group vs. model group, P=0.008), reduced the content of ß-amyloid peptide 1-42 (i.e. 90 mg/kg LSPC group vs. model group, P=0.009), decreased the activities of acetylcholinesterase, monoamine oxidase B, total nitric oxide synthase (i.e. 90 mg/kg LSPC group vs. model group, P=0.006), and neuronal nitric oxide synthase and synchronously increased the activities of superoxide dismutase and glutathione peroxidase in the brain. Furthermore, LSPC could prevent neuron damage and could lessen the expression of P53 protein in the hippocampus. These findings demonstrated that LSPC effectively attenuated cognitive damage and improved parameters related to brain aging in senescent mice induced by d-galactose, and may be used to treat Alzheimer's disease.

Modification of a smoking motivation questionnaire for Chinese medical students.

INTRODUCTION: Smoking prevalence among the medical students is high in China. Therefore, understanding the smoking motivations of medical students is crucial for smoking control, but currently there are no scales questionnaires customized for probing the smoking motivations of medical students. This aim of study was to test and modify a questionnaire for investigating smoking motivations among medical students. METHODS: A cross-sectional survey was conducted among 1,125 medical students at Xuzhou Medical College in China in 2012.The model fit and validity was assessed by confirmatory factor analysis (CFA) and the reliability was tested by single-item reliability, composite reliability, and item-total correlation. RESULTS: The prevalence of smoking was 9.84 % among study population. In the modified scales, the global fit indices identified a CFI value of 0.96, TLI was 0.96, and the RMSEA was 0.063. CFA supported the two dimensional structure of the instrument. The average variance extracted ranged from 0.45 to 0.62. All single-item reliability scores were greater than 0.20, and the composite reliability ranged from 0.74 to 0.91. CONCLUSION: Modified scales could be the preliminary instrument used in evaluating the smoking motivations of medical students. However, it should be further assessed using other forms and methods of validity and reliability, additional motivations of smoking, and the survey of other medical colleges in China.

Molecular diagnosis of putative Stargardt Disease probands by exome sequencing.

BACKGROUND: The commonest genetic form of juvenile or early adult onset macular degeneration is Stargardt Disease (STGD) caused by recessive mutations in the gene ABCA4. However, high phenotypic and allelic heterogeneity and a small but non-trivial amount of locus heterogeneity currently impede conclusive molecular diagnosis in a significant proportion of cases. METHODS: We performed whole exome sequencing (WES) of nine putative Stargardt Disease probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Follow-up dideoxy sequencing was performed for confirmation and to screen for mutations in an additional set of affected individuals lacking a definitive molecular diagnosis. RESULTS: Whole exome sequencing revealed seven likely disease-causing variants across four genes, providing a confident genetic diagnosis in six previously uncharacterized participants. We identified four previously missed mutations in ABCA4 across three individuals. Likely disease-causing mutations in RDS/PRPH2, ELOVL, and CRB1 were also identified. CONCLUSIONS: Our findings highlight the enormous potential of whole exome sequencing in Stargardt Disease molecular diagnosis and research. WES adequately assayed all coding sequences and canonical splice sites of ABCA4 in this study. Additionally, WES enables the identification of disease-related alleles in other genes. This work highlights the importance of collecting parental genetic material for WES testing as the current knowledge of human genome variation limits the determination of causality between identified variants and disease. While larger sample sizes are required to establish the precision and accuracy of this type of testing, this study supports WES for inherited early onset macular degeneration disorders as an alternative to standard mutation screening techniques.

Using excess sludge as carbon source for enhanced nitrogen removal and sludge reduction with hydrolysis technology.

In order to improve the nitrogen removal efficiency and to achieve the sludge reduction in traditional wastewater treatment plants, a combined hydrolysis-anoxic-oxic (H-A-O) pilot-scaled reactor was used in this study to investigate the possibility and validity of using excess activated sludge (EAS) fermentation liquids to enhance the nitrogen removal. The results clearly showed that sludge acidification rate in fermentation reactor can reach to 43.2%. The percentages of acetic acid, propionic acid and butyric acid in the fermentation liquids were 68.4, 25.3 and 6.3%, respectively, while those in domestic wastewater were 73.0, 12.2 and 13.8%, respectively. Bioavailability of soluble chemical oxygen demand (SCOD) from fermentation liquids and domestic wastewater were investigated in batch reactors with nitrate as the electron accepter as well. The corresponding specific denitrification rates were 0.15 g NO3⁻-N/g VSS d⁻¹ and 0.09 g NO3⁻-N/g VSS d⁻¹. When the substances were enough, the denitrification reaction appeared to follow the zero-order kinetics. The results also showed that, when the H-A-O pilot-scaled reactor was operated continuously and sludge fermentation liquids were applied as additional carbon source in the A-O reactor, the removal efficiencies of SCOD, NH4+-N and total nitrogen (TN) were higher than 90, 95 and 79%, respectively. EAS reduction rate in this system was able to reach 40.4%, and the sludge VSS/SS ratio decreased from 0.82 to 0.59 after hydrolysis step.

[Inhibitory effect of free nitrous acid and cross inhibition in denitrification].

Nitrite and nitrate have been generally recognized to have an inhibitory effect on metabolism of denitrifiers. The nitrite inhibition on nitrate reduction under various pH conditions and the cross effect of the both electron acceptors on each other were investigated through a series of batch tests by using a biological nutrient removal (BNR) sludge. The results showed that the nitrate reduction activity had a much stronger relationship with the free nitrous acid (FNA) than that of nitrite concentration, suggesting that FNA, rather than nitrite is likely the actual inhibitor on nitrate reduction. Sixty percent inhibition of nitrate reduction was observed at an FNA concentration of 0.01-0.025 mg x L(-1), while total inhibition occurred when the FNA concentration was greater than 0.2 mg x L(-1). Furthermore, nitrite reduction by the BNR sludge was also found to be inhibited by HNO2. The reduction rate decreased by approximately 80% when the FNA concentration was increased from 0.01 mg x L(-1) to 0.2 mg x L(-1). The inhibitory effect of nitrate on nitrite reduction was found to be insignificant, with the most recovery rates under different nitrate concentrations larger than 90%. However, the nitrate reduction rate was observed to recover only 3.04%-72.54%. The recovery rate from inhibition was independent of the duration of the inhibition and the feeding mode of inhibitor, but strongly dependent on the concentration of inhibitor the biomass was exposed to during the inhibition period.

[Simultaneous nitrogen and phosphorous removal under low DO in a hybrid biological reactor].

Study was conducted by use of the mixed organism of activated sludge and suspended biofilm coexisting in a hybrid biological reactor (HBR) system for simultaneous nitrogen and phosphorous removal. The experimental results showed that, DO and COD/TN were the main factors affected the effluent. The maximum phosphorous releasing rate (the rate of PO4(3-)-P concentration at releasing end to the initial PO4(3-)-P concentration) was 249%, TN removal efficiency was 80.0%, and PO4(3-)-P removal efficiency was 92.2% by controlling the aeration rate (Qair) at 0.07 m3/h. TN and PO4(3-)-P removal efficiency will be decreased, whenever aeration rate increasing or decreasing. With the increasing of COD/TN, the TN and PO4(3-)-P removal efficiency was increased. TN removal efficiency was increased from 70.3% to 84.9%, and PO4(3- -P removal efficiency was increased from 82.2% to 96.0%, when COD/TN was increased from 3.2 to 10.5. The SVI of the activated sludge was less than 90 mL/g during the experiments. It was found during the research process that high phosphorus and nitrogen removal efficiency can be simultaneous achieved without anaerobic and anoxic phase, which was conventionally considered as a key phase for phosphorus and nitrogen removal.

[Nitrogen removal by enhanced endogenous denitrification with excess activated sludge reduction technology].

In order to increase the efficiency of nitrogen removal by endogenous denitrification, and to realize the excess activated sludge reduction in the traditional wastewater treatment process, this investigation proposes a hydrolysis/anoxic/oxic (H/A/O) process utilizing nitrogen removal with excess activated sludge reduction. The experimental equipment for continuous treatment of domestic wastewater was set up and the pilot experiment was conducted. The results showed that, without adding external carbon sources and alkalinity, the removal efficiency of COD, NH4+-N and TN were higher than 90%, 95% and 75%, respectively, under the condition that the hydraulic retention time was 10 h and the backflow ratio of nitrification liquor was 300%, the COD of influent between 220-410 mg/L, the NH4+-N of influent between 36-58 mg/L. When the carbon source was not the limiting condition of the denitrification, with the increasing of the return nitrification liquor, the nitrogen removal efficiency increased. On the contrary, with the increasing of the return nitrification liquor, the nitrogen removal efficiency decreased. It has been tested that the reduction rate of excess activated sludge in the system reached 56.2%. The nitrogen removal efficiency was improved greatly by the wastewater and excess activated sludge hydrolysis. It's proved that using hydrolysis as pre-treatment of wastewater and excess activated sludge is feasible, thas it not only improved the biodegaradability of wastewater and the nutrient removal efficiency, but also enhanced the stability for the operation of the wastewater treatment system, and then the wastewater and excess activated sludge were treatment simultaneously.

Association of the Asn306Ser variant of the SP4 transcription factor and an intronic variant in the beta-subunit of transducin with digenic disease.

PURPOSE: SP4 is a transcription factor abundantly expressed in retina that binds to the GC promoter region of photoreceptor signal transduction genes. We have previously shown that SP4 may be involved in the transcriptional activation of these genes alone or together with other transcription factors such as SP1, neural retina leucine zipper protein (NRL), and cone-rod homeobox gene (CRX). Since mutations in NRL and CRX are involved in inherited retinal degenerations, SP4 was considered a good candidate for mutation screening in patients with this type of diseases. The purpose of this work, therefore, was to investigate possible mutations in SP4 in a cohort of patients affected with different forms of retinal degenerations. METHODS: 270 unrelated probands with various forms of retinal degeneration including autosomal dominant and autosomal recessive retinitis pigmentosa (RP), autosomal dominant and autosomal recessive cone-rod dystrophy (CRD), and Leber's congenital amaurosis (LCA), were screened for mutations in the SP4 gene. Single strand conformation polymorphism (SSCP) analysis was performed on the six SP4 gene exons including flanking regions followed by direct sequencing of SSCP variants. RESULTS: Nine different sequence variants were found in 29 patients, four in introns and five in exons. Many of the probands were previously screened for mutations in the genes encoding the alpha-, beta- and gamma-subunits of rod-specific cGMP phosphodiesterase (PDE6A, PDE6B, PDE6G), the beta-subunit of rod-specific transducin (GNB1), and peripherin/rds (RDS). One group of seven probands of Hispanic background that included five with arRP, one with RP of unknown inheritance (isolate) and 1 with arCRD carried an Asn306Ser mutation in SP4. Of the seven, the isolate case was homozygous and the other 6 heterozygous for the variant. Two arRP and the arCRD probands carried an additional intronic GNB1 variant. DNA from the family members of the arCRD proband could not be obtained, but for the other two families, all affected members and none of the unaffected carried both the SP4 Asn306Ser allele and the GNB1 intronic variant. CONCLUSIONS: If mutations in SP4 do cause retinal degenerative disease, their frequency would be low. While digenic disease with the SP4 Asn306Ser and the GNB1 intronic variant alleles has not been established, neither has it been ruled out. This leaves open the possibility of a cooperative involvement of SP4 and GNB1 in the normal function of the retina.

[Factors influencing snack behaviours of young children in Hefei and evaluation of the effection of interventions].

OBJECTIVE: To evaluate the effect of the parent-to-child and teacher-to-child models used in the Snack Risk Factor Intervention Project for Children (SRFIPC) to change the snack pattern of children. METHODS: Using the community-based intervention trail, nutrition education was implemented based on the parent-to-child and teacher-to-child models among 2102 pre-school children aged 4-6 years in 8 day-care centres of Hefei City, Anhui Province. Data on snack consumption of young children was obtained using a snack behaviour questionnaire. RESULTS: (1) The rate of snacking was high in young children of Hefei. The attitude and diet behavior of the parent are the main influence of the young children snack behavior. (2) The snack frequency of children in intervention group decreased as compared with the control group (P < 0.001). However, children who have higher frequent snacking in the intervention group did not have significant change in their snack behaviours compared with children in the control group. (3) The percentage of children who consumed snacks between meals increased, while the percentage of children who consumed snacks ad arbitrium was greatly decreased in the intervention group (both P < 0.001). (4) The percentages of snacks low in nutrition density, such as the biscuit, cake, confectionery, ham sausage, beverages were lower in intervention group than in the control group. CONCLUSION: It is useful to intervention the children's snack behaviours parent-to-child and teacher-to-child models.

A substitution of G to C in the cone cGMP-phosphodiesterase gamma subunit gene found in a distinctive form of cone dystrophy.

OBJECTIVE: To identify genes responsible for cone dystrophies and determine the functional consequences of their underlying mutations. DESIGN: Case-control study. PARTICIPANTS: Two hundred forty unrelated patients diagnosed with cone dystrophy, cone-rod dystrophy, macular dystrophy, macular degeneration, or Stargardt disease, 95 control individuals, and 2 unrelated families with a distinctive type of cone dystrophy. METHODS: The DNAs of the 240 probands were screened for sequence variants in the PDE6H gene (that encodes the inhibitory gamma-subunit of cone cyclic guanosine monophosphate [cGMP]-phosphodiesterase [PDE]) by single-strand conformation polymorphism electrophoresis. The effect of a nucleotide substitution in the DNA of a patient on gene expression efficiency was analyzed by in vitro transcription/translation. MAIN OUTCOME MEASURES: Cone-specific gene variants, fundus, visual field and electroretinogram (ERG) findings, and protein synthesis efficiency. RESULTS: We found a heterozygous G to C substitution in the 5' untranslated region (UTR) of the PDE6H gene in the DNA of a patient with a distinctive form of cone dystrophy, her sibling, and their father. This rare form of disease is very different in manifestation from other cone dystrophies and has been described as "cone dystrophy with nyctalopia and supernormal rod responses," "cone dystrophy with supernormal scotopic ERGs" and "supernormal and delayed rod ERG syndrome." Among the 240 patients that we studied, only 1 proband had the G to C variant. Furthermore, none of the 95 controls used in this study had this nucleotide change. We also determined that the PDE6H variant was not present in another family affected with this particular type of cone dystrophy. Because the 5' UTR of mRNAs plays a critical role in the regulation of protein synthesis, we determined the effect of the G to C change in this process. By use of in vitro transcription/translation experiments, we demonstrated that this substitution could lead to an increase in PDE6H gene expression. CONCLUSIONS: Our results indicate that mutations in the PDE6H gene are not common, because only 1 of 240 patients with cone dystrophy showed a single nucleotide substitution in the 5' UTR of PDE6H mRNA that could be associated with the disease. If the effect of the G to C substitution we observed in vitro also occurs in vivo, it will lead to PDE6H overexpression in the photoreceptors. Excess of PDEgamma may affect normal cone cGMP-PDE function by inhibiting the catalytic PDEalpha,beta activity and lead to pathogenic elevation of cGMP and eventual degeneration of cone photoreceptors.