Biochemically validated structural model of the 15-subunit intraflagellar transport complex IFT-B.

Cilia are ubiquitous eukaryotic organelles impotant for cellular motility, signaling, and sensory reception. Cilium formation requires intraflagellar transport of structural and signaling components and involves 22 different proteins organized into intraflagellar transport (IFT) complexes IFT-A and IFT-B that are transported by molecular motors. The IFT-B complex constitutes the backbone of polymeric IFT trains carrying cargo between the cilium and the cell body. Currently, high-resolution structures are only available for smaller IFT-B subcomplexes leaving > 50% structurally uncharacterized. Here, we used Alphafold to structurally model the 15-subunit IFT-B complex. The model was validated using cross-linking/mass-spectrometry data on reconstituted IFT-B complexes, X-ray scattering in solution, diffraction from crystals as well as site-directed mutagenesis and protein-binding assays. The IFT-B structure reveals an elongated and highly flexible complex consistent with cryo-electron tomographic reconstructions of IFT trains. The IFT-B complex organizes into IFT-B1 and IFT-B2 parts with binding sites for ciliary cargo and the inactive IFT dynein motor, respectively. Interestingly, our results are consistent with two different binding sites for IFT81/74 on IFT88/70/52/46 suggesting the possibility of different structural architectures for the IFT-B1 complex. Our data present a structural framework to understand IFT-B complex assembly, function, and ciliopathy variants.

Adapting the Ages and Stages Questionnaire to Identify and Quantify Development Among Children With Evidence of Zika Infection.

This article describes novel methods of applying the Ages and Stages Questionnaire-3rd edition (ASQ-3) to assess and quantify developmental delay among children following the 2015-2016 Zika virus outbreak in Brazil. Many of the children with Zika virus infection were expected to have severe developmental delay. However, administering the ASQ-3 to caregivers of these children according to standard protocol would have screened for the overall presence of delay but not the severity of delay. We adopted an amended protocol for administration of the ASQ-3 to quantify the developmental functioning of children severely affected by Zika virus infection in this investigation. Protocols for administering the ASQ-3 among this population were drafted in consultation with developmental measurement experts and are presented here. Specific developmental estimates are discussed, including developmental age equivalents, developmental quotients, and developmental quotient z scores. The calculations of these estimates are presented with examples in the context of the 2015-2016 Zika virus outbreak and associated microcephaly among prenatally infected children from 2 states in northeastern Brazil. Potential applications of these methods for estimating developmental ability among similar pediatric populations are discussed.

The role of the oncostatin M/OSM receptor ß axis in activating dermal microvascular endothelial cells in systemic sclerosis.

BACKGROUND: Scleroderma (SSc) is a rare autoimmune disease characterized by vascular impairment and progressive fibrosis of the skin and other organs. Oncostatin M, a member of the IL-6 family, is elevated in SSc serum and was recognized as a significant player in various stages of fibrosis. The goal of this study was to assess the contribution of the OSM/OSMRß pathway to endothelial cell (EC) injury and activation in SSc. METHODS: IHC and IF were used to assess the distribution of OSM and OSMRß in SSc (n = 14) and healthy control (n = 7) skin biopsies. Cell culture experiments were performed in human dermal microvascular endothelial cells (HDMECs) and included mRNA and protein analysis, and cell migration and proliferation assays. Ex vivo skin organoid culture was used to evaluate the effect of OSM on perivascular fibrosis. RESULTS: OSMRß protein was elevated in dermal ECs and in fibroblasts of SSc patients. Treatments of HDMECs with OSM or IL-6+sIL-6R have demonstrated that both cytokines similarly stimulated proinflammatory genes and genes related to endothelial to mesenchymal transition (EndMT). OSM was more effective than IL-6+sIL-6R in inducing cell migration, while both treatments similarly induced cell proliferation. The effects of OSM were mediated via OSMRß and STAT3, while the LIFR did not contribute to these responses. Both OSM and IL-6+sIL-6R induced profibrotic gene expression in HDMECs, as well as expansion of the perivascular PDGFRß+ cells in the ex vivo human skin culture system. Additional studies in HDMECs showed that siRNA-mediated downregulation of FLI1 and its close homolog ERG resulted in increased expression of OSMRß in HDMECs. CONCLUSIONS: This work provides new insights into the role of the OSM/OSMRß axis in activation/injury of dermal ECs and supports the involvement of this pathway in SSc vascular disease.

Utilization of Ficus carica leaves as a heterogeneous catalyst for production of biodiesel from waste cooking oil.

Biodiesel appears to be a possible substitute for non-renewable fossil fuels; however, its production requires the presence of a catalyst to accelerate the reaction. Serving the purpose of finding effective, cheap and environmentally safe, heterogeneous catalysts, this research used the fig leaves in three different forms, calcined, activated by KOH, and activated by both K2CO3 and CaCO3. Their efficiency in biodiesel synthesis, from spent cooking oil, was examined and compared with that of activated carbon which has been previously investigated. The properties of different catalyst forms were specified using X-ray diffraction, scanning electron microscope and Fourier transform infrared spectroscopy. Operating parameters studied for the three catalysts were reaction time (from 30 to 180 min), alcohol-to-oil molar ratio (from 4:1 to 10:1), catalyst loading (from 0.5 to 5% by wt.), and stirring speed (from 100 to 400 rpm). The increase in reaction time, molar ratio, and catalyst loading proved to have a favorable effect on % conversion to biodiesel but to a certain degree; increasing the stirring speed augmented the conversion. At optimum conditions (2 h of heating, 6:1 alcohol-to-oil molar ratio, 1% by wt. catalyst loading, and 400 rpm stirring), fig leaves activated by KOH provided the highest conversion to biodiesel (92.73%). The measured properties of the produced biodiesel (density, viscosity, flash point, cloud point, and pour point) yielded encouraging results. Graphical Abstract.

Enhancing syntrophic metabolism in up-flow anaerobic sludge blanket reactors with conductive carbon materials.

Syntrophic metabolism of alcohols and fatty acids is a critical step in anaerobic digestion, which if enhanced can better stabilize the process and enable shorter retention times. Direct interspecies electron transfer (DIET) has recently been recognized as an alternative route to hydrogen interspecies transfer as a mechanism for interspecies syntrophic electron exchange. Therefore, the possibility of accelerating syntrophic metabolism of ethanol in up-flow anaerobic sludge blanket (UASB) reactors by incorporating conductive materials in reactor design was investigated. Graphite, biochar, and carbon cloth all immediately enhanced methane production and COD removal. As the hydraulic retention time was decreased the increased effectiveness of treatment in reactors with conductive materials increased versus the control reactor. When these conductive materials were removed from the reactors rates of syntrophic metabolism declined to rates comparable to the control reactor. These results suggest that incorporating conductive materials in the design of UASB reactors may enhance digester effectiveness.

Cognitive functions, fatigue, depression, anxiety, and sleep disturbances: assessment of nonmotor features in young patients with essential tremor.

There is a growing amount of evidence to suggest that besides motor features, patients with essential tremor (ET) may exhibit significant nonmotor features, such as mild cognitive deficits, fatigue, neuropsychiatric symptoms, and sleep disturbances. The goal of this study was to examine nonmotor features in young patients with ET and their impact on quality of life. 45 patients (24.55 ± 7.16 years old) with ET and 35 controls were evaluated using the Pittsburgh Sleep Quality Index, Epworth Sleepiness Scale, Beck Depression Inventory, Beck Anxiety Scale, Fatigue Severity Scale, and Short Form-36. Cognitive functions were evaluated using the Turkish version of the Montreal Cognitive Assessment Battery (MoCA). We ruled out other possible causes of the tremor. The tremor rate was evaluated using the Fahn-Tolosa-Marin Tremor Rating Scale. Poor sleep quality, fatigue, anxiety, and depressive symptoms were more common, and MoCA total scores were lower in the patient group. Fatigue, depressive symptoms, and higher anxiety levels were seen to have a negative effect on physical and mental health. Excessive daytime sleepiness had a negative effect on physical health. There is an emerging interest in nonmotor features of ET. This study showed that even young patients have nonmotor features that decrease their quality of life. This might tell us that nonmotor symptoms could be a part of the disease in the early stages.

Dietary coconut kernel protein beneficially modulates NFκB and RAGE expression in streptozotocin induced diabetes in rats.

Previous studies showed that arginine rich coconut kernel protein (CKP) maintains glucose homeostasis in experimental diabetic rats. But the mechanism of this effect was not clear. This study investigated the effect of CKP on the expression of liver receptor for advance glycated end products (RAGE), inducible nitric oxide synthase (iNOS) and NFkB. Diabetes was induced by injecting a single dose of streptozotocin (75 mg/kg body weight) intraperitoneally. After inducing diabetes, CKP was administered to rats orally for 45 days. After the experimental period, serum glucose, insulin, liver glycogen, glucose metabolizing enzyme activities and the expression of liver RAGE, iNOS and NFkB was evaluated. The results showed that CKP beneficially modulated the levels of glucose and insulin as well as the metabolizing enzyme activities. Expression of RAGE and NFkB was found to be over expressed in diabetic rats but was found to be down regulated in CKP fed diabetic rats. iNOS expression was down regulated in diabetic rats, which was expressed normally in CKP fed diabetic rats. These results clearly demonstrated that anti diabetic activity of CKP is mediated through NFkB pathway.

Health care use and opportunities for human papillomavirus vaccination among young men who have sex with men.

We studied 2941 young gay, bisexual, and other men who have sex with men using National HIV Behavioral Surveillance System data. Within the past 12 months, 88.9% used health care, suggesting many opportunities for recommended care including human papillomavirus vaccination. However, only 61.3% disclosed male-male sexual attraction/behavior to a provider, which may result in some opportunities being missed.

Arginine rich coconut kernel protein modulates diabetes in alloxan treated rats.

Diabetes mellitus is a syndrome characterized by the loss of glucose homeostasis due to several reasons. In spite of the presence of known anti-diabetic medicines in the pharmaceutical market, remedies from natural resources are used with success to treat this disease. The present study was undertaken to investigate the effect of coconut kernel protein (CKP) on alloxan induced diabetes in Sprague-Dawley rats. Diabetes was induced by injecting a single dose of alloxan (150mg/kg body weight) intraperitoneally. After inducing diabetes, purified CKP isolated from dried coconut kernel was administered to rats along with a semi synthetic diet for 45 days. After the experimental period, serum glucose, insulin, activities of different key enzymes involved in glucose metabolism, liver glycogen levels and the histopathology of the pancreas were evaluated. The amount of individual amino acids of CKP was also determined using HPLC. Results showed that CKP has significant amount of arginine. CKP feeding attenuated the increase in the glucose and insulin levels in diabetic rats. Glycogen levels in the liver and the activities of carbohydrate metabolizing enzymes in the serum of treated diabetic rats were reverted back to the normal levels compared to that of control. Histopathology revealed that CKP feeding reduced the diabetes related pancreatic damage in treated rats compared to the control. These results clearly demonstrated the potent anti-diabetic activity of CKP which may be probably due to its effect on pancreatic ß cell regeneration through arginine.

Effect of topical application of virgin coconut oil on skin components and antioxidant status during dermal wound healing in young rats.

OBJECTIVES: The present study was undertaken to evaluate the effect of a topical application of virgin coconut oil (VCO) on excision wounds in young rats. METHODS: Three sets of experiments with 3 groups of female Sprague-Dawley rats each consisting of 6 animals were used for studying wound closure time, antioxidant status and biochemical parameters. Group 1 was the control; groups 2 and 3 were treated with 0.5 and 1.0 ml VCO, respectively, 24 h after wound creation for 10 days. After the experimental period, the healing property of VCO was evaluated by monitoring the time taken for complete epithelization as well as levels of various parameters of the wound's granulation tissue. The collagen solubility pattern, glycohydrolase activity, and histopathology of the granulation tissue were also analyzed. The antioxidant status during wound healing was monitored continuously for 14 days. RESULTS: VCO-treated wounds healed much faster, as indicated by a decreased time of complete epithelization and higher levels of various skin components. Pepsin-soluble collagen showed a significant increase in VCO- treated wounds, indicating a higher collagen cross-linking. Glycohydrolase activities were also found to be increased due to a higher turnover of collagen. Antioxidant enzyme activities, and reduced glutathione and malondialdehyde levels were found to be increased on the 10th day after wounding, which were found to have returned to normal levels on day 14 in the treated wounds. The lipid peroxide levels were found to be lower in the treated wounds. A histopathological study showed an increase in fibroblast proliferation and neovascularization in VCO-treated wounds compared to controls. CONCLUSION: The beneficial effect of VCO can be attributed to the cumulative effect of various biologically active minor components present in it.

Wet and dry extraction of coconut oil: impact on lipid metabolic and antioxidant status in cholesterol coadministered rats.

Because coconut oil extracted by wet process (virgin coconut oil, VCO) is gaining popularity among consumers, this study was conducted to evaluate VCO compared with coconut oil extracted by dry process (copra oil, CO) for their influence on lipid parameters, lipid peroxidation, and antioxidant status in rats coadministered with cholesterol. VCO, CO, and cholesterol were fed in a semi-synthetic diet to 24 male Sprague-Dawley rats for 45 days. After the experimental period, lipid and lipid peroxide levels and antioxidant enzyme activities were observed. Chemical composition and antioxidant properties of the polyphenolic fraction from VCO and CO were also analyzed. The results showed that lipid and lipid peroxide levels were lower in VCO-fed animals than in animals fed either CO or cholesterol alone. Antioxidant enzyme activities in VCO-fed animals were comparable with those in control animals. Although the fatty acid profiles of both oils were similar, a significantly higher level of unsaponifiable components was observed in VCO. Polyphenols from VCO also showed significant radical-scavenging activity compared with those from CO. This study clearly indicates the potential benefits of VCO over CO in maintaining lipid metabolism and antioxidant status. These effects may be attributed in part to the presence of biologically active minor unsaponifiable components.

Polar lipid fatty acids, LPS-hydroxy fatty acids, and respiratory quinones of three Geobacter strains, and variation with electron acceptor.

The polar lipid fatty acids, lipopolysaccharide hydroxy-fatty acids, and respiratory quinones of Geobacter metallireducens str. GS-15, Geobacter sulfurreducens str. PCA, and Geobacter bemidjiensis str. Bem are reported. Also, the lipids of G. metallireducens were compared when grown with Fe(3+) or nitrate as electron acceptors and G. sulfurreducens with Fe(3+) or fumarate. In all experiments, the most abundant polar lipid fatty acids were 14:0, i15:0, 16:1 omega 7c, 16:1 omega 5c, and 16:0; lipopolysaccharide hydroxy-fatty acids were dominated by 3oh16:0, 3oh14:0, 9oh16:0, and 10oh16:0; and menaquinone-8 was the most abundant respiratory quinone. Some variation in lipid profiles with strain were observed, but not with electron acceptor.

Power output and columbic efficiencies from biofilms of Geobacter sulfurreducens comparable to mixed community microbial fuel cells.

It has been previously noted that mixed communities typically produce more power in microbial fuel cells than pure cultures. If true, this has important implications for the design of microbial fuel cells and for studying the process of electron transfer on anode biofilms. To further evaluate this, Geobacter sulfurreducens was grown with acetate as fuel in a continuous flow 'ministack' system in which the carbon cloth anode and cathode were positioned in close proximity, and the cation-selective membrane surface area was maximized in order to overcome some of the electrochemical limitations that were inherent in fuel cells previously employed for the study of pure cultures. Reducing the size of the anode in order to eliminate cathode limitation resulted in maximum current and power densities per m(2) of anode surface of 4.56 A m(-2) and 1.88 W m(-2) respectively. Electron recovery as current from acetate oxidation was c. 100% when oxygen diffusion into the system was minimized. This performance is comparable to the highest levels previously reported for mixed communities in similar microbial fuel cells and slightly higher than the power output of an anaerobic sludge inoculum in the same ministack system. Minimizing the volume of the anode chamber yielded a volumetric power density of 2.15 kW m(-3), which is the highest power density per volume yet reported for a microbial fuel cell. Geobacter sulfurreducens formed relatively uniform biofilms 3-18 mum thick on the carbon cloth anodes. When graphite sticks served as the anode, the current density (3.10 A m(-2)) was somewhat less than with the carbon cloth anodes, but the biofilms were thicker (c. 50 mum) with a more complex pillar and channel structure. These results suggest that the previously observed disparity in power production in pure and mixed culture microbial fuel cell systems can be attributed more to differences in the fuel cell designs than to any inherent superior capability of mixed cultures to produce more power than pure cultures.

Severe pneumonia with pneumatoceles and patent foramen ovale in an infant: optimal ventilation strategy?

We discuss a case of severe pneumonia with pneumatoceles in a 2-month old infant. Despite peak and plateau airway pressures kept below 30 and 25 cmH(2)O, respectively, the infant developed bilateral pneumothorax and pneumomediastinum caused by pneumatocele rupture, necessitating bilateral intercostal drainage. Application of positive end-expiratory pressure (PEEP) of 10 cmH(2)O at FIO(2) of 0.7 worsened oxygenation, and transthoracic echocardiography showed right-to-left shunting of blood through the patent foramen ovale. Therefore, PEEP had to be tapered to 6 cmH(2)O, and ventilation carried out at FIO(2) of 1 until the lung condition improved. The open lung strategy of higher increments of PEEP and lower FIO(2) for lung protective ventilation actually proved deleterious to our patient. We conclude that a subset of patients with acute respiratory distress syndrome with refractory hypoxaemia may have right-to-left shunting of blood through a patent foramen ovale. This can be detected by echocardiography, and these patients may benefit from a ventilation strategy aimed at lowering pulmonary vascular resistance.

Validation of a 7-point Global Overall Symptom scale to measure the severity of dyspepsia symptoms in clinical trials.

BACKGROUND: Currently there is no consensus on the optimal method to measure the severity of dyspepsia symptoms in clinical trials. AIM: To validate the 7-point Global Overall Symptom scale. METHODS: The Global Overall Symptom scale uses a 7-point Likert scale ranging from 1 = no problem to 7 = a very severe problem. Validation was performed in two randomized-controlled trials (n = 1121 and 512). Construct validity: Global Overall Symptom was compared with the Quality of Life in Reflux And Dyspepsia, Gastrointestinal Symptom Rating Scale, Reflux Disease Questionnaire and 10 specific symptoms using Spearman correlation coefficients. Test-retest reliability: The Intraclass Correlation Coefficient was calculated for patients with stable dyspepsia defined by no change in Overall Treatment Effect score over two visits. Responsiveness: effect size and standardized response mean were also calculated. RESULTS: Construct validity: Change in Global Overall Symptom score correlated significantly with Quality of Life for Reflux And Dyspepsia, Gastrointestinal Symptom Rating Scale, Reflux Disease Questionnaire and specific symptoms (all P < 0.0002). Reliability: The Intraclass Correlation Coefficient was 0.62 (n = 205) and 0.42 (n = 270). Responsiveness: There was a positive correlation between change in Global Overall Symptom and change in symptom severity. The effect size and standardized response mean were 1.1 and 2.1, respectively. CONCLUSION: The Global Overall Symptom scale is a simple, valid outcome measure for dyspepsia treatment trials.

Adaptation to disruption of the electron transfer pathway for Fe(III) reduction in Geobacter sulfurreducens.

Previous studies demonstrated that an outer membrane c-type cytochrome, OmcB, was involved in Fe(III) reduction in Geobacter sulfurreducens. An OmcB-deficient mutant was greatly impaired in its ability to reduce both soluble and insoluble Fe(III). Reintroducing omcB restored the capacity for Fe(III) reduction at a level proportional to the level of OmcB production. Here, we report that the OmcB-deficient mutant gradually adapted to grow on soluble Fe(III) but not insoluble Fe(III). The adapted OmcB-deficient mutant reduced soluble Fe(III) at a rate comparable to that of the wild type, but the cell yield of the mutant was only ca. 60% of that of the wild type under steady-state culturing conditions. Analysis of proteins and transcript levels demonstrated that expression of several membrane-associated cytochromes was higher in the adapted mutant than in the wild type. Further comparison of transcript levels during steady-state growth on Fe(III) citrate with a whole-genome DNA microarray revealed a significant shift in gene expression in an apparent attempt to adapt metabolism to the impaired electron transport to Fe(III). These results demonstrate that, although there are many other membrane-bound c-type cytochromes in G. sulfurreducens, increased expression of these cytochromes cannot completely compensate for the loss of OmcB. The concept that outer membrane cytochromes are promiscuous reductases that are interchangeable in function appears to be incorrect. Furthermore, the results indicate that there may be different mechanisms for electron transfer to soluble Fe(III) and insoluble Fe(III) oxides in G. sulfurreducens, which emphasizes the importance of studying electron transport to the environmentally relevant Fe(III) oxides.

Behavioral responses during the forced swim test are not affected by anti-inflammatory agents or acute illness induced by lipopolysaccharide.

Pro-inflammatory cytokines and other molecules traditionally associated with immune function have been implicated in mediating behavioral and physiological consequences of stressor exposure. There is also evidence that cytokines are aberrantly expressed in depressive populations, suggesting they may play an etiological role in the development of depression/despair-related processes. Thus, we conducted a series of experiments to determine whether agents known to suppress cytokine activity or inflammatory responses in the CNS would alter the normal progression of behavioral responses during the forced swim test (FST, an animal model of depression/behavioral despair). Adult male Sprague-Dawley rats were injected with indomethacin (1 or 10 mg/kg intraperitoneally (i.p.)), alpha-MSH (0.25 or 0.5 microg icv), or minocycline (20 or 40 mg/kg i.p.) prior to each day of the FST and behavioral assessments were performed. Injection of indomethacin, alpha-MSH, or minocycline had no effect on the development of the immobility response during the FST on either day of testing. In a second series of experiments, we examined whether behavioral responses during forced swim would be affected by acute illness induced by a single injection of lipopolysaccharide (LPS). Acute injection of LPS (10 or 100 microg/kg i.p.) had no effect on behavioral responding during the FST irrespective of when it was injected, despite pronounced reductions in social behavior following these same doses of LPS. From these studies, we conclude that (a) endogenous inflammatory mediators do not appear to be involved in the normal progression of behavioral responses during the FST, and (b) behavioral responses during the FST are not affected by acute systemic injection of LPS.

Stress-induced increases in hypothalamic IL-1: a systematic analysis of multiple stressor paradigms.

Exposure to stressors such as footshock, tailshock, and immobilization have been shown to induce hypothalamic IL-1 production, while other stressors such as restraint, maternal separation, social isolation, and predator exposure have no effect on hypothalamic IL-1 levels. This disparity of findings has led to considerable controversy regarding the ability of stressors to induce hypothalamic IL-1 expression. Thus, the goal of the following experiments was to examine hypothalamic IL-1 responses in adult male Sprague-Dawley rats following exposure to a diverse set of stressors. Our data indicate that exposure to 2h of restraint in a Plexiglas tube, glucoprivic challenge induced by administration of 2-deoxyglucose (2-DG), or insulin-induced hypoglycemia all fail to alter hypothalamic IL-1 levels despite robust activation of the pituitary-adrenal response. However, when restraint was administered on an orbital shaker or in combination with insulin-induced hypoglycemia, robust increases in hypothalamic IL-1 were observed. No effects of glucoprivic (2-DG) challenge were observed when combined with restraint, indicating some specificity in the hypothalamic IL-1 response to stress. We also provide a preliminary validation of the ELISA detection method for IL-1, showing that (a) Western blot analyses confirmed strong immunopositive banding at the apparent molecular weight of both mature IL-1beta and the IL-1beta prohormone, and (b) footshock led to a two-fold increase in mRNA for IL-1 in the hypothalamus as detected by RT-PCR. These data provide novel insight into the characteristics of a stressor that may be necessary for the observation of stress-induced increases in hypothalamic IL-1.

Effect of Aerva lanata against hepatotoxicity of carbon tetrachloride in rats.

The partially purified petroleum ether extractable fraction of the whole plant Aerva lanata (PF) was evaluated for the protective effect against liver damage induced by carbon tetra chloride (CCl(4)) in Sprague Dawley rats. Rats were orally administered with PF (50 and 100mg/kg body weight) for 14 days before CCl(4) challenge and 100mg of PF alone for toxicity analysis without CCl(4) administration. The results showed that CCl(4) administration significantly damaged the liver as evident from histopathology and very high activity of serum and liver marker enzymes. It also reduced the antioxidant enzyme status of the animals. PF administration significantly reversed the histopathological changes and restored the elevated activities of liver marker enzymes and also enhanced the antioxidant enzyme activities. The extract also reduced hepatic lipid peroxidation and increased the serum total protein and albumin/globulin (A/G) ratio. Preliminary phytochemical analysis of PF showed the presence of alkaloids. These observations clearly indicate that PF contains antioxidant alkaloids capable of ameliorating the CCl(4)-induced hepatic injury by virtue of its antioxidant activity.