diff --git "a/deduped/dedup_0212.jsonl" "b/deduped/dedup_0212.jsonl" new file mode 100644--- /dev/null +++ "b/deduped/dedup_0212.jsonl" @@ -0,0 +1,46 @@ +{"text": "Arabidopsis in vitro transcripts and arrayed serial dilutions of homologous probes; 2) how curing time of in-house poly-L-lysine coated slides impacts probe retention capacity; and 3) the retention characteristics of 13 commercially available surfaces.Global gene expression studies with microarrays can offer biological insights never before possible. However, the technology possesses many sources of technical variability that are an obstacle to obtaining high quality data sets. Since spotted microarrays offer design/content flexibility and potential cost savings over commercial systems, we have developed prehybridization quality control strategies for spotted cDNA and oligonucleotide arrays. These approaches utilize a third fluorescent dye (fluorescein) to monitor key fabrication variables, such as print/spot morphology, DNA retention, and background arising from probe redistributed during blocking. Here, our labeled cDNA array platform is used to study, 1) compression of array data using known input ratios of When array element fluorescein intensity drops below 5,000 RFU/pixel, gene expression measurements become increasingly compressed, thereby validating this value as a prehybridization quality control threshold. We observe that the DNA retention capacity of in-house poly-L-lysine slides decreases rapidly over time and that there are considerable differences in retention characteristics among commercially available poly-L-lysine and amino silane-coated slides.High DNA retention rates are necessary for accurate gene expression measurements. Therefore, an understanding of the characteristics and optimization of protocols to an array surface are prerequisites to fabrication of high quality arrays. The generation of reliable gene expression data with cDNA microarrays requires fabrication of quality arrays. This task encompasses the amplification of adequate amounts of concentrated PCR product for use as probe from the cDNA clone, followed by ordered arraying of the probes onto coated glass slides. The glass slide is a key variable in either spotted cDNA or oligonucleotide array fabrication since it must possess: 1) a uniform surface that yields spots of consistent shape and size, 2) low background fluorescence, and 3) high DNA retention capacity. Since the array is clearly a source of experimental variability, we have developed a novel three-color array approach where it is possible to directly visualize either cDNA or oligonucleotide arrays prior to hybridization [in vitro transcript we experimentally correlate the quantity of support bound probe to measured expression ratios, in order to validate our quality control threshold for array acceptance. We then utilize our three-color array platform to evaluate the characteristics of in-house prepared poly-L-lysine coated slides and 13 additional commercially available coating surfaces, in terms of background auto-fluorescence, spot morphology, and DNA retention.By labeling the array itself with a third color, we have observed that arrays fabricated together are not equivalent in terms of a number of measurable physical parameters, including the amount of DNA probe deposited and retained and the amount of background arising from probe solublized and re-deposited during post-processing. In prior studies, we observed that these pre-hybridization array-based variables play a direct and significant relationship in replicate consistency, and that microarray data quality can be improved through prehybridization slide selection based upon these quality parameters ,2. As a Saccharomyces cerevisiae probes and complementary Cy5 and Cy3 labeled cDNA targets derived from in vitro transcripts, indirectly demonstrated this by printing yeast probes at increasingly dilute concentrations (<50 ng/ul) and observed elimination of the measured dynamic range to where input transcript ratios of 30:1 or 1:30 were both detected as output ratios close to 1:1, illustrating that limiting bound probe results in an underestimation or failure to detect differential gene expression [It has been assumed that the amount of cDNA probe deposited and retained on the array surface would have a nominal effect on observed differential expression ratios due to the competitive nature of two channel fluorescent hybridizations ; howeverArabidopsis probes and transcripts. Total thymus RNA extracted from the DR+/+ and DRlyp/lyp [Arabidopsis gene in vitro transcript and hybridized to 18,000 probe rat cDNA arrays possessing serially diluted fluorescein-labeled Arabidopsis probes . This approach allowed comparison of known RNA input ratio to measured output ratio, enabling a direct and quantitative measure of the relationship between the amount of support-bound probe and ratio data compression were filtered; these spots were either saturated or possessed high background. Spots with low hybridization intensities, which would normally be flagged by Matarray, were intentionally retained to study ratio compression due to low amounts of support-bound probe. After filtering, 896/1536 data points from 16 different arrays were available for analysis. Plotted on the y-axis of Figure Arabidopsis in vitro transcript output log ratio divided by the log ratio of transcript actually introduced into the Cy3 and Cy5 labeling reactions. In this analysis, a perfect measurement is represented by a value of \"1\". On the x-axis, is plotted the spot fluorescein intensity. When the spot fluorescein intensity falls below 5000 RFU/pixel, the data variability and data compression dramatically increase. These results recapitulate our previous observations where replicate consistency was found to decrease when the array average spot fluorescein intensity dropped below 5000 RFU/pixel, whereas arrays possessing average fluorescein intensities above 5000 RFU/pixel were found to generate equally good data. These results further demonstrate that use of measurable array characteristics are effective quality markers for printed arrays (judged by their effect on the hybridization data) and serve to validate our array intensity quality control threshold of >5000 RFU/pixel.To expand upon these observations and place them in context with the quality control standards of our three-color array platform, we conducted similar experiments using Rlyp/lyp BioBreedn Figure and 1B. ; ; ). Although most available protocols are quite similar, some recommend the curing of slides for two weeks prior to spotting, while others state that coated slides are not stable for extended periods of time and recommend not printing onto slides that are greater than 4 months old. To investigate slide coating age as a potential variable in retention capacity, we fabricated more than 1,000 rat cDNA arrays using in-house poly-L-lysine coated slides ranging in age from 3 to 12 weeks. These slides were coated in 26 independent sessions and utilized over 12 different print runs. After printing all arrays were post-processed [Clearly, the amount of immobilized probe on the coated glass surface is a critical array fabrication variable, therefore factors that affect the amount of retention characteristics, such as surface chemistry, probe concentration, spotting buffer, spotting conditions, cross-linking and blocking conditions are important to understand. Protocols for coating glass microscope slides with poly-L-lysine are readily available on-line and reasonably simple to perform . An average array fluorescein intensity of >15,000 RFU/pixel was observed when printing on slides 4 weeks old or less, however a nearly 50% reduction in retention capacity is observed when printing on poly-L-lysine coating greater than 10 weeks old. We speculate that the poly-L-lysine may become oxidized thereby losing its positive charge and ability to initially electrostatically interact with the negatively charged DNA. Irregardless of the type of degradation occurring to the surface coating over time, these results indicate that, at least for in house fabricated poly-L-lysine coated slides, shelf-life is a significant variable in the fabrication of quality arrays capable of yielding reliable gene expression measurements.A significant loss of DNA retention capacity is observed Figure when theGiven the potential time-dependent variability of in-house prepared poly-L-lysine coated slides, we investigated the retention characteristics of commercially available coated slides. Our objective was to identify a surface with consistently higher retention characteristics than our \"fresh\" in-house slides without having to change the spotting buffer (1.5 M betaine/5% DMSO) or the nonaqueous post-processing protocol ,9, sinceTo study retention characteristics, a single 9600 element human cDNA array was spotted onto each slide in 1.5 M betaine/3%DMSO. The in-house poly-L-lysine slides were less than 6 weeks old and all vendor-supplied slides were unpacked from any special packaging immediately before printing. Five replicate arrays for each slide type were generated. The five replicates were evenly distributed over the arrayer deck (capacity 100 slides) by arranging the slides into 5 groups of 18 to account for any variance introduced through print rank order (ie first versus last), since we previously identified this as a variable that influences array average array fluorescein intensity . The aveIt has been reported that the amount of UV irradiation may be an important array fabrication variable since the amount of hybridization signal from spotted 70-mer oligonucleotides has been found to be dependent on the amount of cross-linking . In thisFabrication of high quality spotted arrays is a daunting task possessing a high number of variables. The vendor supplied slides tested here were done so under conditions that have been optimized for our in-house prepared poly-L-lysine coated slides, although our optimized protocol is not drastically different than those used by other laboratories nor drastically different from any of the vendor provided protocols. Our observations, as well as the observations of others, suggest that optimization of ones protocol to a surface chemistry is an essential first step to generating reliable global gene expression measurements using in-house spotted microarrays.2, 50 mM KCl, 0.2 mM each dNTP , 1 M betaine [Taq polymerase . Reactions were amplified with a touchdown thermal profile consisting of 94\u00b0C for 5 min; 20 cycles of 94\u00b0C for 1 min, 60\u00b0C for 1 min (minus 0.5\u00b0 per cycle), 72\u00b0C for 1 min; and 15 cycles of 94\u00b0C for 5 min; 20 cycles 94\u00b0C for 1 min, 55\u00b0C for 1 min, 72\u00b0C for 1 min; terminated with a 7 min hold at 72\u00b0 [A sequence-verified human library , consisting of 41,472 clones or a 36,000 clone rat cDNA library obtained from the University of Iowa was used as a source of probe DNA. Cultures were grown in 150 ul Terrific Broth supplemented with 100 mg/ml ampicillin in 384 deep-well plates sealed with air pore tape sheets and incubated with agitation for 14\u201316 hr. Clone inserts were amplified in duplicate in 384-well format from 0.5 ul bacterial culture or from 0.5 ul purified plasmid (controls only) using 0.26 \u03bcM of each vector primer as part of the SpotReport\u00ae-10 Array Validation System. Arabidopsis thaliana PCR product was cloned into the pCRII vector using the TA cloning kit and fluorescein-labeled PCR products for photosystem I chlorophyll a/b-binding protein, RUBISCO activase, ribulose-1,5-biphosphate carboxylase/oxygenase, lipid transfer protein 6 lipid transfer protein 5, papain-type cysteine endopeptidase, root cap 1, and triosphophate isomerase were generated using vector-specific primers essentially as described above. Products were purified, quantified, and a 1:2 dilution series (200 ng/ul to 12.5 ng/ul) was prepared and printed in duplicate onto each array.2, deceleration: 100 cm/sec2. All slides were post-processed using the previously described non-aqueous protocol [2 UV cross-linking energy. This protocol has yielded more favorable fluorescein post-blocking signal-to-noise values as compared to blocking in aqueous solutions[Poly-L-lysine coated slides were prepared in-house as previously described on Corniprotocol using 60solutions,8,17.. Known input ratios of photosystem I chlorophyll a/b-binding protein (30:1); RUBISCO activase (10:1); ribulose-1,5-biphosphate carboxylase/oxygenase (5:1); lipid transfer protein 6 (1:1); 0.7 lipid transfer protein 5 (1:1); papain-type cysteine endopeptidase (1:5); root cap 1 (1:10); and triosphophate isomerase (1:30) were spiked into Cy3 and Cy5 RNA labeling reactions, respectively. After hybridization, arrays were scanned with a ScanArray 5000 and image files were obtained. Again, array image files were analyzed with the Matarray software [Isolation of mRNA, labeling, and hybridization were performed as described previously software ,17.MJH and XW conceived of the study, its design and coordination, and drafted the manuscript. LM, JT, and SM carried out the array fabrication and gene expression studies. XW performed the statistical analysis. All authors read and approved the final manuscript."} +{"text": "Helicoverpa zea moths is manifested as insects that are either sterile \"agonadal\" individuals with malformed reproductive tissues or fertile asymptomatic carriers which are capable of transmitting virus on to their progeny. Virus infected progeny arising from eggs laid by asymptomatic carrier females may themselves be either sterile agonadals or asymptomatic carriers.Hz-2V infection of female By injecting virus into female moths, a correlation was established between virus doses administered to the females and the levels of resulting asymptomatic and sterile progeny.The results of these experiments indicate that high virus doses produced a higher level of agonadal progeny and lower doses produced higher levels of asymptomatic carriers. Helicoverpa zea originating at the USDA-ARS in Stoneville, MS [in vitro and in genome structure and size [The insect virus, Hz-2V originally named gonad-specific virus (GSV) was firsille, MS ,2. Insecand size -5.et al. [et al. [While examining progeny from eggs laid by infected female moths, Hamm et al. identifi [et al. , were abet al. [et al. [Hamm et al. presente [et al. were unaet al. [et al. [Raina et al. showed t [et al. .In this study we have used the approach of injecting virus into healthy female moths to examine the relationship between virus dose and the level of infected, agonadal and asymptomatic carrier progeny insects hatching from eggs laid on successive ovipostion days. The results presented here demonstrate that virus dose affects both the level of infected progeny and the kind of infection found in insects hatching from eggs laid by virus infected females, indicating a direct correlation between virus dose received by females and the level of infected progeny they produce. Also demonstrated here is the fact that for each virus dose, as the level of agonadal insects hatching from eggs laid on successive oviposition days increase, the level of asymptomatic carrier progeny decreases.5, 2 \u00d7 106, 2 \u00d7 107, or 2 \u00d7 108 TCID50 units of Hz-2V were dissected and the reproductive tissues examined for signs of virus pathology. The PCR products of DNA samples from reproductive tissues of all apparently healthy progeny moths were examined for the presence of Hz-2V DNA via slot blot hybridization , and uninfected progeny moths hatching from eggs laid on each oviposition day was recorded.5 to 2 \u00d7 108 TCID50 units (figure 7 and 2 \u00d7 108), whereas the lowest percentage (about 60%) was produced by females infected with the lowest doses of virus (2 \u00d7 105 and 2 \u00d7 106 TCID50).The analysis of these results showed that the percentage of total infected progeny at all doses tested increased with each successive oviposition day, and the level of infected progeny increased as virus dose increased from 2 \u00d7 10s figure . For indVirus infected progeny moths arising from eggs laid on each oviposition day by females infected with different virus doses were divided into agonadal and asymptomatic carriers and these results are presented in figure For all F1 insects hatching from eggs laid on day two, figure the percIn order to better illustrate the relationship between the two types of infections and to emphasize the effects of virus dose upon the proportions of asymptomatic and agonadal infections, percentages of asymptomatic carriers and agonadal progeny for only the highest and lowest dose are presented in figure et al. [Injecting Hz-2V into female moths results in experimentally infected insects that resemble asymptomatic females and females that have become infected with the virus during copulation, not unlike the females infected during mass-matings by infected males in transmission experiments conducted by Hamm The results presented in figure The percentage of agonadal progeny also increases with each successive oviposition day, approaching 100% agonadal progeny by day three at all virus doses tested, and all progeny moths arising from eggs laid on oviposition day four in all groups were agonadal. Based on the correlation between virus titer and percent agonadal progeny observed in these experiments, the increase in agonadal progeny per oviposition day is likely due to an increase in the titer of virus transmitted to the eggs, suggesting that the titer of virus increases in the parent female moths with each successive oviposition day.in vitro revealed a rapid increase in virus titer by 24 hours post infection in Tn-368 and Ld-652Y cells [in vivo in the epithelial cells of agonadal female oviduct tissue has been described previously by Rallis and Burand [in vitro also occurs in vivo, resulting in a significant daily increase in the titer of Hz-2V in these experimentally infected female moths. Although the precise site of virus replication in these experimentally infected females is not known, the increase in virus titer in these individuals almost certainly results in an increase in virus being transmitted to the progeny with each successive oviposition day and ultimately in the patterns of infection reported here.Studies of Hz-2V replication If, as we have proposed, low virus doses result in asymptomatic carrier moths, and high virus doses produce agonadal progeny, then asymptomatic carrier progeny would likely arise from eggs produced on the earliest oviposition days and decrease with each day, as the virus titer in the egg-laying female moth increases. In fact, the percentage of asymptomatic carrier progeny in these experiments does decrease with each successive oviposition day to 0% by day four. The percent asymptomatic carriers is highest in progeny that receive the lowest virus dose, specifically progeny from oviposition day one and progeny arising from the parent female moths that were experimentally infected with the lowest dose of virus. This is directly opposite of what is observed for agonadal progeny, which is at its highest level at the highest virus dose, specifically on the later oviposition days (days three or greater) and in progeny arising from parent female moths that were infected with the highest virus dose. Interestingly, the lowest percentage of asymptomatic carrier progeny arose from eggs laid by the group of female moths that received the highest virus dose of Hz-2V figure. . These dThe results presented here clearly show that there is a direct correlation between virus dose and the relative percentage of agonadal and asymptomatic progeny. That is, increasing the virus dose causes an increase in the percentage of agonadal progeny, but a decrease in the percentage of asymptomatic progeny. At the present time, it is unknown how the development of an infected individual into an agonadal adult or an asymptomatic carrier is regulated. It is likely that a minimum titer of Hz-2V is needed at a key point in larval development to produce a viral factor(s) within the larval tissues at a threshold level required to reprogram the development and differentiation of the reproductive tissues into the agonadal structures. If this threshold is equaled or exceeded at this point in development, the progeny will exhibit the agonadal condition. However, if this threshold level is not attained, then the reproductive tissues are not reprogrammed and the infected insect becomes an apparently healthy, fertile, asymptomatic carrier of Hz-2V.H. zea has resulted in the ability of the virus to produce two different types of infections in the insects that enable the virus to replicate to high titers in the reprogrammed reproductive tissues in sterile agonadal moths, while maintaining itself in a population in asymptomatic carrier moths. This replication strategy appears to be essential for the continued existence of Hz-2V, since the development of the sterile, agonadal condition in all infected moths would lead to the extinction of the insect host, and the possibly the virus as well. The production of asymptomatic carrier moths ensures that some fertile, infected moths exist that can mate and produce infected progeny, enabling an Hz-2V-infected population to sustain itself, as in the case of the Stoneville colony.The evolution of Hz-2V infection in H. zea were obtained from the USDA-ARS in Stoneville, MS. Insects were reared on artificial diet and maintained as outlined previously [Corn earworm larvae used to start a laboratory colony of healthy eviously .Hz-2V for infecting female moths was prepared as described previously and purified via sucrose gradient centrifugation .5, 2 \u00d7 106, 2 \u00d7 107, and 2 \u00d7 108 TCID50 units.Newly emerged adult female moths were prepared and injected with Hz-2V as outlined by Rallis and Burand . The fem4 Tn368 cells were seeded into each well of a 96-well plate. Between 6 and 13 serial dilutions were made from each virus sample assayed and 10 or 20 wells were plated with 10 ul for each dilution. Plates were incubated at 27\u00b0C for 3 to 4 days and examined for the appearance of cytopathic effect (CPE). The numbers of wells with CPE were counted and the TCID50 calculated [Tn368 cells were cultured as per Burand & Lu and 100 DNA was extracted from the reproductive tissues of adult moths by first homogenizing dissected tissues in 200 ul of TE buffer followed by a 2-minute incubation in a boiling water bath. The homogenate was then chilled on ice, after which Ribonuclease A (10 ug/ul) was added to each sample, which was then incubated at room temperature for 15 min. The samples were then clarified by centrifugation at 15,600 \u00d7 g for 2 min.Viral DNA used as template for PCR reactions was extracted from purified virus using 1% SDS in TE containing 1 mg/ml Protease K as outlined by Burand and Lu .\u00ae PCR reagent premix kit with 1 unit of Taq DNA polymerase. Each reaction was carried out in10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl2 and 40 mM KCl, containing 250 uM of each of the four dNTP's, with 100 pM of P4-1 forward and P4-2 reverse primers, and 10 ng of purified viral DNA as template. These primer set and reaction conditions were also used to amplify viral DNA sequences in approximately 100 ng of DNA from reproductive tissues of moths thought to be asymptomatic carriers of Hz-2V.Two sets of primers were used to amplify Hz-2V genomic DNA to prepare a probe for use in slot blot analysis of insect reproductive tissues. The first set was designed to amplify a 434 bp sequence of the Hz-2V genome . PCR rea2), 100 pM of both P4-3 and P4-4 primers, a hexanucleotide mixture containing DIG-labeled dUTP , and 100 pM of Hz-2V genomic DNA. The DIG-labeled PCR product was purified on a 0.8% agarose gel using the Qiagen gel electrophoresis purification kit.The second set of primers was designed to amplify a 350 bp region directly interior to that of the P4-1 and P4-2 amplified sequence. These primers were used to generate a DIG-labeled probe for Hz-2V to be used in slot blot hybridization assays. PCR reactions for production of the DIG-labeled probe were carried out in a final volume of 50 ul using the Boehringer Manheim DIG High Prime DNA Labeling and Detection Kit, with 1X concentrations of Taq Polymerase buffer , and 50% Formamide). Slot blots were hybridized with 150 ng DIG-labeled Hz-2V probe at 37\u00b0C for 12\u201314 hrs. Following washing, chemiluminescent detection was carried out as recommended by the DIG High Prime Labeling and Detection Kit Manual for DNA Hybridization (Boehringer Mannheim).To prepare the DNA for slot blot analysis, 15 ul of the P4-1 and P4-2 PCR amplified DNA from insect samples was denatured by incubating with NaOH (0.4 M)/ EDTA at 100\u00b0C for 10 min., then applied to a Hybon-N+ membrane prewashed with 500 ul 5X SSC buffer in a Manifold II slot blotter (Schleicher & Schuell). After applying the DNA, the membrane was baked at 88\u00b0C for 2 hrs under vacuum and prehybridized for 6 hrs. at 42\u00b0C in 50% formamide prehybridization buffer N-laurylsarcosine, 1% (w/v) NaIn order to confirm that the PCR products that hybridized to the viral DNA probe contained an amplified DNA fragment of the appropriate size (434 bp), representative samples were analyzed by electrophoresis on 0.8% agarose gels with 0.5X TBE buffer at 100 volts for approximately 1 hr, then stained with EtBr to visualize DNA bands under ultraviolet light.The author(s) declare that they have no competing interests.CPR participated in the design of the study, carried out the work with the insects, coordinated the project and assisted in the molecular analysis and drafting of the manuscript. JPB conceived the study, designed and supervised the experimental work and drafted the manuscript. All authors read and approved the final manuscript."} +{"text": "Array Comparative Genomic Hybridization (aCGH) is a rapidly evolving technology that still lacks complete standardization. Yet, it is of great importance to obtain robust and reproducible data to enable meaningful multiple hybridization comparisons. Special difficulties arise when aCGH is performed on archival formalin-fixed, paraffin-embedded (FFPE) tissue due to its variable DNA quality. Recently, we have developed an effective DNA quality test that predicts suitability of archival samples for BAC aCGH.In this report, we first used DNA from a cancer cell-line (SKBR3) to optimize the aCGH protocol for automated hybridization, and subsequently optimized and validated the procedure for FFPE breast cancer samples. We aimed for highest throughput, accuracy, and reproducibility applicable to FFPE samples, which can also be important in future diagnostic use.Our protocol of automated array-CGH on archival FFPE ULS-labeled DNA showed very similar results compared with published data and our previous manual hybridization method.This report combines automated aCGH on unamplified archival FFPE DNA using non-enzymatic ULS labeling, and describes an optimized protocol for this combination resulting in improved quality and reproducibility. DNA fragmentation and cross-links) and sample heterogeneity. Therefore, selection of DNA of sufficient quality, especially when using FFPE material, is of great importance for aCGH [Array CGH has become a successful and valuable tool for the analysis of chromosome copy-number alterations including the detection of sub-megabase alterations and has been applied to phocytes -5. The pfor aCGH . Furtherfor aCGH -9. In adfor aCGH , especiafor aCGH . As an afor aCGH , also fofor aCGH . One of There have been earlier reports on array CGH of FFPE material -8,12-14 \u00ae slides containing the human 3.5 k BAC/PAC genomic clone set in triplicate were used as before [DNA was isolated from the breast cancer cell-line SKBR3 (obtained from ATCC) or from FFPE tumor tissue with at least 70% tumor cells as described before . Two mics before . As optis before . Automat0t-1 DNA and precipitated. The pellet was dissolved in 140 \u03bcl 0.22 \u03bcm filtered hybridization buffer , 0.1% Tween20, 2 \u00d7 SSC, 10 mM Tris pH 7.4, and 25 mM EDTA) and 10 \u03bcl (100 \u03bcg/\u03bcl) yeast tRNA . The pre-hybridization solution consisted of 400 \u03bcg single stranded sheared herring sperm DNA and 125 \u03bcg C0t-1 DNA dissolved in 150 \u03bcl hybridization buffer. Both hybridization and pre-hybridization mixtures were dissolved at 37\u00b0C continuously shaking at 650 rpm (Eppendorf Thermomixer) for at least one hour, denatured for 10 min at 95\u00b0C and spun for 1 min at 14000 rpm (Eppendorf centrifuge) to pellet potential particles prior to injecting 120 \u03bcl pre-hybridization mixture followed by 120 \u03bcl sample mixture into the hybridization chamber.Labeled sample and reference DNA were pooled with 125 \u03bcg Cstep 1; wet the array with 2 \u00d7 SSC for 30s at 37\u00b0C, no soak. Step 2; 120 \u03bcl pre-hybridization solution was slowly injected and incubated for 1 hour at 37\u00b0C, agitation set at 'high'. Step 3; 15s wash at 37\u00b0C with 2 \u00d7 SSC, no soak. Step 4; 120 \u03bcl sample mixture was injected and hybridized for 72 hours at 37\u00b0C, agitation set at 'high'. Step 5; 12 \u00d7 with 2 \u00d7 SSC + 0.1% SDS at 37\u00b0C. Step 6; 6 \u00d7 (1 min wash + 1 min soak) with 2 \u00d7 SSC + 0.1% SDS at 68\u00b0C. Step 7; 2 \u00d7 (1.5 min wash + 1 min soak) with 2 \u00d7 SSC at 68\u00b0C. Step 8; 1,5 min wash with 0.1 \u00d7 SSC at 23\u00b0C, no soak. Step 9; 2 min with nitrogen gas at 23\u00b0C. Slides were scanned with an Agilent DNA Microarray Scanner BA on the same day. Data processing included signal intensity measurement in ImaGene Software followed by median pintip (c.q. subarray) normalization and plotting in custom Matlab code as before [Optimal hybridization parameters for the hybridization station: Three statistics were used to determine the quality of the hybridization, the CGH profile, and to compare experiments with each other. For each CGH profile, we calculated the variance across all log2 ratios relative to the ratios of the underlying true ploidy levels as estimated by CGH-segmentation , secondlMicroarray data have been deposited in NCBIs Gene Expression Omnibus and are accessible through GEO Series accession number GSE7122.Further automation of CGH is indispensable to meet the demand for higher quality, higher throughput, and improved reproducibility. We here describe automated and reproducible array-CGH on FFPE material. As optimization goals we aimed to reproduce results from manual hybridizations and published results, to minimize the variance, to maximize dynamic range, to minimize standard deviation of the triplicate spot measurements, and to maximize signal-to-noise.It is difficult to determine the quality of aCGH profiles without an independent methodology to verify gains and losses. Therefore, we chose to use the widely studied cell-line SKBR3 as a model for which chromosomal aberrations have been well documented ,3,17, al0t-1 DNA for 1 hour at 37\u00b0C. The optimal hybridization buffer contained 15% 50 kDa dextran sulphate. Washing was performed as described in Material and Methods. The steps that led to this protocol are described in detail below.Using 2 \u03bcg unamplified sample DNA from FFPE tissue and 2 \u03bcg reference DNA both CyDye labeled, incubation duration was optimal at 72 hours at 37\u00b0C after pre-hybridization with a mixture of herring sperm and Cet al., hybridized to a human cDNA micro array containing 6,691 different mapped human genes [et al., hybridized to a human oligonucleotide array containing 28,830 unique genes [We used SKBR3 as a model cell-line and compared its CGH profile with published ,3,17 andan genes . Figure ue genes . BreakpoThe effects of hybridization duration and temperature were measured in two experiments using SKBR3 DNA and reference DNA hybridized for 24 or 48 hour at 37\u00b0C. Hybridization mixture containing 7% 50 kDa dextran sulphate was used. Figure A major further improvement of the hybridization was obtained by increasing the 50 kDa dextran sulphate concentration from 7 to 15%. Here we describe our results for 10 and 15%. Four hybridizations were done at 37 and 42\u00b0C each with 10 or 15% dextran sulphate. The resulting profiles were very similar to each other and to our manual hybridized aCGH. A slight systematic difference was detected in the variance, standard deviation of the triplicates and dynamic range. Hybridizing at 37\u00b0C, standard deviations were 0.06 and 0.07 at 10 and 15% dextran sulphate respectively and at both concentrations the variances were 0.11. At 42\u00b0C, both the variances increased to 0.14 and 0.12 and the mean standard deviations to 0.10 and 0.09 at 10 and 15% dextran sulphate respectively (table i.e. lower viscosity at the same concentration). We used 5 kDa (Sigma) and 10 kDa dextran sulphate at 15, 17.5 or 20% for SKBR3 profiling. All six hybridizations showed inferior dynamic ranges compared with the 50 kDa dextran sulphate experiments, shown in table Increasing the concentration of 50 kDa dextran sulphate from 15% to 17.5 or 20% did not further improve the array results. At these concentrations the variance was 0.11 and 0.09 at 17.5% and 20% dextran sulphate respectively, notably at 20% dextran sulphate the dynamic range decreased below 3.5. Elevated concentrations of dextran sulphate render the hybridization mixture viscosity beyond the mixing capability of the hybridization station. This prompted us to evaluate the effect of lower molecular weight dextran sulphate with 2 \u00d7 SSC + 0.1% SDS at the hybridization temperature of 37, 42 or 45\u00b0C (previously discussed), step 6; 6 \u00d7 with 2 \u00d7 SSC + 0.1% SDS at 37, 46 or 65\u00b0C, step 7; 2 \u00d7 with 2 \u00d7 SSC at 37, 46 or 65\u00b0C, step 8; 15 sec wash with 0.1 \u00d7 SSC at 23\u00b0C, step 9; dry slides for 2 minutes with nitrogen gas at 23\u00b0C.Most wash protocols use large amounts of formamide to wash off non-specifically bound probe. Formamide is a toxic that we wished to exclude from all washes. The wash procedure now consists of: As described before, hybridization was performed at 37, 42 or 45\u00b0C. Step 5 was done at these temperatures and results are discussed above. Step 6 and 7 were done at 37 or 46\u00b0C, both resulting in inferior profiles compared with the manual hybridization. A large proportion of the deletions and amplifications in the CGH profile could not be detected and the data were essentially as in figure i.e. mix of cells of different genomic composition), fragmented, and rarely composed of 100% tumor cells. Therefore, aCGH profiles of FFPE material generally have larger variances (defined as the spread around the common levels between adjacent chromosome breakpoints), lower intensities and lower dynamic range compared with hybridizations of cell-line DNA.To develop aCGH also as a diagnostic tool, it will be essential to validate its applicability on patient tumor samples and especially on archival FFPE tissue . ExtractTo validate our automated hybridization method for FFPE material we compared CGH profiles from unfixed and formalin fixed paraffin embedded SKBR3 cells. Figure step 1: wetting or chamber filling), for 1 hour at 37\u00b0C. The pre-hybridization mixture consisted of 400 \u03bcg single stranded sheared herring sperm DNA and 125 \u03bcg C0t-1 DNA dissolved in 150 \u03bcl hybridization buffer. With pre-hybridization, signal intensities were almost 50% higher and the mean standard deviation of the triplicate spots 15% lower compared to the protocol without pre-hybridization resulting in good CGH profiles of FFPE material (data not shown). Although CGH profiles of SKBR3 did not improve upon adding pre-hybridization, it clearly benefited CGH profiles of DNA extracted from FFPE patient tissue (data not shown).Therefore, we validated our method on archival material. The first hybridization was done with tumor #1 DNA with or without pre-hybridization after the first wash step both in duplicate on FFPE extracted material tumor #2. Figure To validate the optimal automated hybridization described above, we hybridized four FFPE samples that were previously hybridized using our manual method. Figure e.g. chromosome 7p), more then one copy number gain (chromosome 1q) and unchanged chromosome copy numbers (e.g. chromosome 10) in FFPE tumor tissue.So far, we performed over one hundred automated array-CGH experiments, the oldest archival material used was fixed and embedded in 1971, all with reproducible and high quality results. Figure To develop an automated hybridization method, we first used the breast cancer cell-line SKBR3 as a model-genome and subsequently optimized and validated the protocol for FFPE breast tumors. Reproducible hybridization results for FFPE tumor tissue were obtained using ULS-labeled unamplified tumor DNA with pre-hybridization, hybridized on a hybridization station at 37\u00b0C for 72 hours with a hybridization mixture containing 15% 50 kDa dextran sulphate and post-hybridization washing steps without using formamide. Pre-hybridization did not have a detectable effect on the CGH profile of the cell-line SKBR3 but did improve CGH profiles of FFPE tissue samples. All hybridization parameters studied are optimized for the 3.5 k BAC array-CGH platform but may be different for other platforms. This protocol of automated array-CGH on archival FFPE ULS-labeled DNA outperformed all our manual methods with respect to accuracy, reproducibility, easy of handling, and speed.aCGH = array-Comparative Genomic Hybridization; BAC = Bacterial Artificial Chromosome; bp = base pairs; FFPE = Formalin-Fixed, Paraffin-Embedded; ULS = Universal Linkage SystemThe author(s) declare that they have no competing interests.SJ performed all experiments, data analyses, participated in the study design and wrote the manuscript. EvB helped in writing and data analyses, and participated in the study design. PN participated in the study design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:et al., Shadeo and Lam, Jong et al., manually, automatically, and hybridized FFPE SKBR3. Calling copy number changes by setting thresholds allowed comparison of different hybridization platforms and methods.This file includes the aberrations calculated by CGH-segmentation for the SKBR3 CGH profiles hybridized by Pollack Click here for fileY-axis) from SKBR3 CGH profiles per chromosome (X-axis), hybridized by Pollack et al., Shadeo and Lam, Jong et al., manually, automatically, and hybridized FFPE SKBR3. Gain at 1, unchanged at 0, heterozygous loss at -1, and homozygous loss at -2.This picture shows the copy number calls (Click here for file"} +{"text": "The evolution of viral quasispecies can influence viral pathogenesis and the response to antiviral treatments. Mutant clouds in infected organisms represent the first stage in the genetic and antigenic diversification of RNA viruses, such as foot and mouth disease virus (FMDV), an important animal pathogen. Antigenic variants of FMDV have been classically diagnosed by immunological or RT-PCR-based methods. DNA microarrays are becoming increasingly useful for the analysis of gene expression and single nucleotide polymorphisms (SNPs). Recently, a FMDV microarray was described to detect simultaneously the seven FMDV serotypes. These results encourage the development of new oligonucleotide microarrays to probe the fine genetic and antigenic composition of FMDV for diagnosis, vaccine design, and to gain insight into the molecular epidemiology of this pathogen.A FMDV microarray was designed and optimized to detect SNPs at a major antigenic site of the virus. A screening of point mutants of the genomic region encoding antigenic site A of FMDV C-S8c1 was achieved. The hybridization pattern of a mutant includes specific positive and negative signals as well as crosshybridization signals, which are of different intensity depending on the thermodynamic stability of each probe-target pair. Moreover, an array bioinformatic classification method was developed to evaluate the hybridization signals. This statistical analysis shows that the procedure allows a very accurate classification per variant genome.A specific approach based on a microarray platform aimed at distinguishing point mutants within an important determinant of antigenicity and host cell tropism, namely the G-H loop of capsid protein VP1, was developed. The procedure is of general applicability as a test for specificity and discriminatory power of microarray-based diagnostic procedures using multiple oligonucleotide probes. Indeede copied ,3. As a e copied ,5. Virale copied -8, and te copied . Mutant Picornaviridae, whose genome is a single stranded RNA of about 8200 nucleotides, of positive polarity, replicated by a virus-coded RNA-dependent RNA polymerase, devoid of a proofreading-repair activity . FMDV iactivity . The ant culture ,13. Inac culture . The ant culture . In vacc culture . Therefo culture .Antigenic variants of FMDV have been classically diagnosed by immunological methods [review in ]. RecentA major antigenic site of FMDV (termed site A) is located at the mobile, exposed G-H loop of capsid protein VP1 ,20,21. T15 spacers, and the oligonucleotide concentration. A number of conclusions were drawn from the results (not shown). First, the hybridization signals were weaker with oligonucleotides of 11 residues than with oligonucleotides of 15 residues. We have not assessed oligonucleotides longer than 15 residues because they are more likely to accommodate, without destabilization of the helical duplex, a single nucleotide mismatch at a central position at their 5'-end, followed by an oligo (dT)15 spacer and the specific 15-mer sequence; the oligonucleotides were purified by HPLC. The oligonucleotides [the numbering of FMDV genomic residues is according to [15CAATACATGGATGATT-3' and 5'-C6-T15GATGCATATTTTTCAG-3', corresponding to the HIV reverse transcriptase coding region and termed HIVa and HIVb respectively) and spots with spotting solution with no nucleotide defining four grids per slide. Each oligonucleotide was spotted in duplicate dots 150 \u03bcm in diameter, with a center-to-center distance of 250 \u03bcm RNA from mAb SD6-escape mutants was extrIn a number of preliminary assays, a streptavidin-biotin system was assessed to obtain single-strand DNA target . Additionally, Cy3 and Cy5 fluorescence dyes (Amersham) were used as a direct labeling system. The final protocol includes the reagents showing in our hands the highest sensitivity and reproducibility.Immediately before hybridization, slides were processed as follows: They were washed for 2 min. at room temperature with 2X sodium saline citrate (SSC), 0.1% lauroylsarcosine, and for an additional 2 min. wash with 2 \u00d7 SSC at room temperature, to remove unbound DNA and components of the printing buffer. The oligonucleotides were denatured by placing the slides 2 min. in distilled water at 100\u00b0C, cooled 10 sec. at room temperature, and then the oligonucleotides were fixed by plunging the slides into ice-cold 100% ethanol for 2 min., finally the slides were centrifuged 1 min. at 500 \u00d7 g (Minicentrifuge Arrayit). Microarrays were incubated in a hybridization chamber (Genetix) with 20 \u03bcl of hybridization buffer under a 24 \u00d7 24 mm cover slip, and bathed at 42\u00b0C for 45 min. Then the microarrays were washed with distilled water, and dried by a brief centrifugation.The hybridization with the labeled DNA was carried out in hybridization buffer at the appropriate temperature (58\u201360\u00b0C) and with the required amount of target . After a 3 hours incubation in the hybridization chamber, the slides were washed for 5 min. in 2 \u00d7 SSC, 0.1% lauroylsarcosine, followed by 5 min. in 2 \u00d7 SSC, and finally rinsed 10 sec. in 0.2 \u00d7 SSC, and 5 min. in distilled water, at 45\u00b0C. The slides were dried by spinning 1 min. at 500 \u00d7 g and, finally, scanned using a G2565AA/G2565AB Scanner (Agilent). The Agilent and Scan Array Express (Perkin Elmer Life Sciences) analysis software was used for reading and quantifying the hybridization images. The reproducibility of the method was assessed by comparing the results of at least five different hybridization experiments for each mutant.a), the log2 of the average of its replicated spots mean foreground intensities, subtracted from its background:Array quantification was performed with the program Scan Array Express. Each probe was duplicated in the array. For each spot in the array, measures for the mean and median foreground intensity and for the mean and median background intensities were available. Visual inspection of scanned hybridized arrays revealed some noise due to the presence of dust and scratches, introducing an uneven increase in the mean foreground signal for some spots. We have tried to detect the affected spots by calculating a Z-score of their mean foreground intensity per pixel, using the four measurements available for each probe in every hybridization experiment. For this, we have used the additional measures available for the same probe (median foreground intensity and replicated spot mean and median foreground intensity) and computed their average and standard deviation. Then we calculated the Z-score in the usual way, subtracting the average from the mean foreground intensity and then dividing it by the standard deviation. After testing several absolute Z-score thresholds for discarding spots, we have found that a Z-score of 7 provides optimal results. If neither of the spots is discarded, we take as a measure for the presence of each mutant in the sample , discarding those arrays for which the logn-dimensional vector space. The SVM algorithm operates by first mapping the training set into a high-dimensional feature space and then attempting to locate in that space a hyperplane that separates positive from negative examples. Having found such a hyperplane, the SVM can then predict the classification of an unlabeled example by mapping it into the feature space and asking on which side of the separating plane the example is found. The multiclass SVM is an extension of the classification problem to multiple classes, instead of just a binary classification.Data classification was carried out with a multiple class support vector machine tool (mcSVM) ,50. Brie-2 and \u03b1 = 103 as optimal parameters.In our case, we have used 39 probes in the array for classification purposes, one for quality control ICF and 38 for detecting different genotypes, including mutants and wild type. Therefore, each sample was encoded by a 39 dimensional vector, each dimension corresponding to a variable computed in equation 1. We analyzed 202 samples distributed among 17 phenotype classes to classify analysis software for reading and quantifying the hybridization images.Click here for file"} +{"text": "Limb length discrepancy and its effects on patient function have been discussed in depth in the literature with respect to hip arthroplasty but there are few studies that have examined the effect on function of limb length discrepency following total knee arthroplasty (TKA). The aim of this study was to determine whether limb length discrepancy after TKA in patients with bilateral osteoarthritis of knee with varus deformity affects functional outcome.Fifty-four patients with bilateral osteoarthritis of knee with varus deformity, who were operated for total knee arthroplasty from 1996 to 2008, were reviewed retrospectively. The patients were divided into two groups. Thirty patients (mean age 64 years) were operated for unilateral TKA and thirty patients (mean age 65.8 years) were operated for bilateral total knee arthroplasty. Six patients underwent staged surgery and were included in both groups as the time interval between the two surgeries was more than the minimum 6-month follow-up period specified for inclusion in the study. The limb length discrepancy was measured and statistically correlated with the functional component of the Knee Society Score.P=0.006), while no statistically significant correlation was found in the bilateral group .In the unilateral group (n=30), the mean limb length discrepancy was 1.53 cm (range: 0-3 cm) and the mean functional score was 73 (range: 45-100). In the bilateral group (n=30), the mean limb length discrepancy was 0.5 cm (range: 0-2 cm) and the mean functional score was 80.67 (range: 0-100). A statistically significant negative correlation was found between limb length discrepancy and functional score in the unilateral group (Spearman correlation coefficient, r =\u22120.52, Limb length discrepancy affects functional outcome after total knee arthroplasty, especially so in patients of bilateral osteoarthritis with varus deformity undergoing surgery of only one knee. Improved surgical techniques and rehabilitation protocols have resulted in excellent knee function and range of motion following total knee arthroplasty. Nevertheless, there remain 15-20% of patients with persistent dysfunction that is difficult to treat.et al.Ulrich Functional problems following total knee arthroplasty may be incapacitating as a result of persistent pain,79The aim of this study was to determine whether limb length discrepancy after TKA in patients with bilateral osteoarthritis of knee with varus deformity affects functional outcome.The aim of this study was to investigate for the presence of limb length discrepancy after total knee arthroplasty, the amount of discrepancy, patient perception of the limb length discrepancy, its effects on the patient function, and any difference between patients undergoing unilateral and bilateral total knee arthroplasty with regard to function due to limb length discrepancy.This was a retrospective study conducted in a tertiary level hospital by a specialist arthroplasty unit. Fifty-four patients of bilateral osteoarthritis of the knee with varus deformity who were operated for unilateral or bilateral total knee arthroplasty consecutively and had minimum 6 months follow up were enrolled into this study. The surgeries were performed over the time period from 1996 to 2008. Cases of osteoarthritis of the knee with valgus deformity and of rheumatoid arthritis were excluded from the study.Thirteen patients were males and 41 were females. The mean age of patient was of 64.9 years (range 48 to 83 years). The 54 patients were divided into two groups. Group A (30 patients) were operated for unilateral total knee arthroplasty and Group B (30 patients) for bilateral total knee arthroplasty. Six patients were included in both the unilateral and the bilateral group; these six patients underwent staged surgeries, during the time interval of this study, with a gap more than 6 months, hence were available for evaluation in both groups. They were thus included in the unilateral group after being operated for one knee and then in the bilateral group following the second knee arthroplasty. The 24 patients who underwent unilateral total knee arthroplasty did not get operated on the second knee for reasons that were personal or financial.All the surgeries were performed by the same arthroplasty surgeon using the midvastus approach10The unilateral TKA group consisted of 30 patients, with 7 males and 23 females. The mean age in this group was 64 years (range 48 to 80 years) (mean of 64 years). The bilateral TKA group consisted of 30 patients, with 6 males and 24 females. The mean age was 65.8 years (range 54-83 years). Of the 30 patients who had bilateral total knee arthroplasty, 10 were operated for both the knees simultaneously, while the remaining 20 underwent staged procedures, with the time interval between the two surgeries ranging from 1 week to 12 years. The time interval between the staged surgeries was less than six months for 13 patients and more than six months for the remaining 7 patients. The evaluation was done at recent follow up for all the cases.Limb length measurement was done in the supine position. The pelvis was squared, i.e., the line joining the anterior superior iliac spines was kept perpendicular to the long axis of the body (xiphisternum to pubic symphysis). The lower limbs were placed parallel to the long axis of the body, and limb length measurement (in centimeters) was done from the anterior superior iliac spine to the medial malleolus using a measuring tape. The measurement was taken twice by two different observers and the mean of the two values was recorded as the limb length.An anteroposterior (AP) roentgenogram of the operated limb was obtained in the supine position with the patella facing the ceiling. Similarly, an AP standing roentgenogram of the operated limb was obtained in the standing position with the patella facing forward. The x-ray beam was directed at the joint line. A lateral view was obtained in the supine position, with the beam directed perpendicular to that in the AP view. A skyline view was obtained to study the patellar component and tracking. Alignment, component positioning, and loosening were evaluated from the above roentgenograms by an independent observer as per the Knee Society total knee arthroplasty roentgenographic evaluation and scoring system.Only the functional component of the Knee Society Clinical Rating SystemIn the Group A , mean limb length discrepancy was 1.53 cm (range 0 to 3 cm). Five patients (16.67%) had no limb length discrepancy, five (16.67%) had limb length discrepancy of 1 cm, nineteen (63.33%) had limb length discrepancy of 2 cm, and one (3.33%) had a limb length discrepancy of 3 cm.P=.006).The mean functional score in this group, was 73 (95% CI: 66.73 to 79.27) with range from 45 to 100. The standard deviation was 16.79. The mean functional score of patients with no limb length discrepancy was 85. Those with a limb length discrepancy of 1 cm had a mean functional score of 83, those with limb length discrepancy of 2 cm had a mean score of 68.16, and those with limb length discrepancy of 3 cm had a mean functional score of 55 . A statiA total of eight patients 26.7%) perceived the limb length discrepancy, while the remaining 22 patients 73.3%) did not. Of the eight patients who perceived limb length discrepancy, seven (87.5%) had limb length discrepancy of 2 cm and the remaining one (12.5%) patient had a limb length discrepancy of 3 cm. None of the patients with limb length discrepancy of 1 cm perceived it. Of the 19 patients with limb length discrepancy of 2 cm, seven (36.8%) perceived the limb length discrepancy. The single patient with a 3 cm limb length discrepancy also perceived the same [Tables 6.7% perc.3% did nP=.458). None of the patients in the bilateral group perceived any limb length discrepancy.In the group B mean limb length discrepancy was 0.5 cm (range 0 cm to 2 cm). Sixteen patients (53.33%) had no limb length discrepancy, thirteen (43.33%) had limb length discrepancy of 1 cm, and 1 (3.33%) had a limb length discrepancy of 2 cm. In the bilateral group the functional score ranged from 0 to 100, with a mean of 80.67 (95% CI: 72.75 to 88.58); the standard deviation was 21.2. The mean functional score of patients with no limb length discrepancy was 81.25, that of those with 1-cm limb length discrepancy was 80.00, and that of those with 2 cm limb length discrepancy was 80.00 . No statt test, P=.005 at 95% confidence interval) [Tables Six patients in the study were included in both the groups. After unilateral total knee arthroplasty, five patients had limb length discrepancy of 2 cm and one had limb length discrepancy of 3 cm. Two patients perceived the limb length discrepancy. After bilateral total knee arthroplasty, only one patient had limb length discrepancy of 1 cm; the remaining five patients had no limb length discrepancy. The one patient with limb length discrepancy did not perceive it. The mean functional score after unilateral total knee arthroplasty was 64.17 and that after bilateral total knee arthroplasty was 80.00. The difference in functional score was statistically significant did not perceive any limb length discrepancy.Patients with unilateral total knee arthroplasty had a significant increase in functional scores after being operated for the other knee. The limb length discrepancy present after the unilateral surgery got corrected after the second surgery. The two patients who perceived limb length discrepancy after unilateral total knee arthroplasty did not do so after the second surgery.A number of factors may be responsible for the limb length discrepancy, including correction of the varus alignment after surgery, the amount of preoperative flexion deformity, and the postoperative flexion deformity. Investigation for these factors was not the aim of this study and hence we have not considered these variables. None of the patients in the study had any malalignment, component malposition, patellar maltracking, or instability. Thus, these factors did not act as confounders in this study.The mean limb length discrepancy after total hip arthroplasty varies from 1 to 15.9 mm.2218Limb length discrepancy is more common after unilateral than after bilateral total knee arthroplasty (83.33% vs 46.66%). Limb length discrepancy of 2 cm or more is perceived by patients operated for unilateral total knee arthroplasty. Limb length discrepancy of 2 cm is not perceived by patients operated for bilateral total knee arthroplasty. Limb length discrepancy after unilateral total knee arthroplasty for bilateral varus osteoarthritis significantly affects the functional outcome, but the same is not true for patients operated for bilateral total knee arthroplasty. The functional outcome of patients of bilateral knee osteoarthritis with varus deformity operated for unilateral total knee arthroplasty improves significantly after being operated for the other side."} +{"text": "Brassica juncea seedlings. Treatment of seedlings with substrate, cofactor and inhibitor of Nitric-oxide synthase and nitrate reductase (NR), indicated NR mediated NO biosynthesis in cold. Analysis of the in vivo thiols showed depletion of low molecular weight thiols and enhancement of available protein thiols, suggesting redox changes. To have a detailed view, S-nitrosylation analysis was done using biotin switch technique (BST) and avidin-affinity chromatography. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is S-nitrosylated and therefore, is identified as target repeatedly due to its abundance. It also competes out low abundant proteins which are important NO signaling components. Therefore, RuBisCO was removed (over 80%) using immunoaffinity purification. Purified S-nitrosylated RuBisCO depleted proteins were resolved on 2-D gel as 110 spots, including 13 new, which were absent in the crude S-nitrosoproteome. These were identified by nLC-MS/MS as thioredoxin, fructose biphosphate aldolase class I, myrosinase, salt responsive proteins, peptidyl-prolyl cis-trans isomerase and malate dehydrogenase. Cold showed differential S-nitrosylation of 15 spots, enhanced superoxide dismutase activity (via S-nitrosylation) and promoted the detoxification of superoxide radicals. Increased S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase sedoheptulose-biphosphatase, and fructose biphosphate aldolase, indicated regulation of Calvin cycle by S-nitrosylation. The results showed that RuBisCO depletion improved proteome coverage and provided clues for NO signaling in cold.Although in the last few years good number of S-nitrosylated proteins are identified but information on endogenous targets is still limiting. Therefore, an attempt is made to decipher NO signaling in cold treated Research in the last two decades has proved beyond doubt, the versatility of nitric oxide (NO) as an important signaling molecule in plants. It regulates numerous biological processes for 7 days.For cold stress, 7 days old seedlings were kept in a cold chamber at 4\u00b0C for 2\u201396 h under the same conditions as mentioned in the above section. Control seedlings were kept at 25\u00b0C. Seedlings were treated with SNP or cPTIO . Following the treatment, the seedlings were rinsed with the double distilled water and blotted onto a filter paper and were immediately frozen in the liquid nitrogen.NO was measured using the NO measuring system following manufacturer's instructions. inNO consist of a nitric oxide meter, a sensor and a data acquisition system which measure free NO in the sample. NO measurement experiments were performed following in 20 mM Tris-HCl (pH 7.0) containing 20% glycerol and 5 mM PMSF. The homogenate was centrifuged at 12,000 rpm for 20 min at 4\u00b0C. Protein was estimated by Bradford assay was used following the manufacturer's instructions. Briefly, the column was washed thrice before use with 500 \u03bcL tris buffered saline to remove the suspension buffer. Immuno-capture of RuBisCO was performed by incubating the supernatant (90 \u03bcg protein) with the matrix for 15 min at 25\u00b0C with gentle shaking. After 15 min, the flow through was collected by centrifugation at 2000 rpm for 30 s. Unbound protein were removed by washing with TBS. Elution was done with the stripping buffer and the fractions were immediately neutralized with 1M Tris-HCl, pH 8.0.The S-nitrosylated proteins were detected and purified from RuBisCO depleted fractions by BST and neutravidin-agarose column chromatography following Abat and Deswal except tTwo dimensional electrophoresis was performed following Abat and Deswal with mint-test (p < 0.05) was applied to determine any significant quantitative change.The gels were scanned using Alpha Imager . ImageMaster 2-D Platinum software was used for the spot detection in the 2-D gels. Protein spot pattern from the gels of three independent biological replicates were used to create a master gel in the first level match set. The gels were normalized in the percentage spot volume mode to reduce the differences in the protein loading and gel staining. This was followed by the formation of a second level match set where master gel of different samples was compared. Intensity of each spot is defined as the sum of the intensities of the pixels constituting that spot and is represented in the spot volume. Students's http://www.matrixsciences.com) and was searched against the NCBInr in Viridiplantae. The search parameters were same as described in with mowse score 50 and above were selected. The unidentified/hypothetical proteins were subjected to BLASTP search against the NCBInr protein database to assign function to the unnamed or unknown proteins.For the MS identification, polypeptides/spots were manually excised from silver stained 1-D or 2-D gel. Identification was done at Proteomics International by Electrospray mass spectrometry on a 4000 Q TRAP mass spectrometer (Applied Biosystems). Utimate 3000 nanoflow LC system was used for sample introduction as described in Bringans et al. . The peaFor the enzyme assays, the seedlings were extracted in the HEN buffer and the homogenate was centrifuged at 14,000 g for 25 min at 4\u00b0C. The supernatant was passed through two layers of the cheese cloth and was incubated without or with GSNO (100-500 \u03bcM) or GSH (250 \u03bcM) in the dark for 20 min at 25\u00b0C. For the DTT treatment, after incubation with GSNO (100 \u03bcM), the samples were incubated with DTT (10 mM) in dark for 40 min. GSNO, GSH and DTT were removed using Micro Bio-Spin 6 columns (Bio-Rad).The total SOD (EC 1.15.1.1) activity was assayed by monitoring the inhibition of photochemical reduction of nitroblue tetrazolium [NBT, with three repeats. The data shown in the NO measurement, thiol pool analysis and the enzymatic assay represents mean \u00b1 SD from three independent experiments performed in triplicates and significant differences were calculated by Student's in vivo NO generating capacity of the plant, could be due to the higher concentrations of the substrate and cofactors being provided from the outside. A decrease in NO to the basal level by tungstate in cold confirmed the results. The control (RT) sets showed a similar trend.NO content was measured in the cold (4\u00b0C) treated seedlings using NO measuring system. The sensing element of the iNO sensor has a NO selective permeable membrane. Cold stress led to NO evolution right from 2 h with maximum NO accumulation (2 fold) at 6 h Figure . In contAs NO modulates the cellular thiols, these were quantified in cold stress. Thiols are broadly categorized into protein-based (high molecular weight), non-protein based (low molecular weight) and pellet bound thiols. Protein-based thiols are further categorized as available and buried thiols. Low molecular weight thiols include GSH and free cysteines. Pellet bound thiols are the thiols present in the broken organelles and cell membranes. Cold stress increased available thiols and pellet bound thiols by 54.5% and 14.2% respectively, while decreased the buried thiols and low molecular weight thiols by 53.8% and 24.1% respectively Figure . Overall2+/phytate fractionation . Seppro RuBisCO spin columns (IgY affinity purification) removed 83% and 87.5% of large and small subunit of RuBisCO respectively as shown by the densitometric quantification Figures . For thein vivo S-nitrosylation. Immunoblot of GSNO (250 and 500 \u03bcM) treated fractions showed 17 immunopositive polypeptides were dissolved in the HEN buffer and GSNO was used for mimicking the Affinity purified S-nitrosylated proteins showed 16 polypeptides on a 12% gel Figure , includip<0.05) change in the abundance , epithiospecifier protein, vacuolar calcium binding protein, inorganic pyrophosphatase I, unnamed protein products and unknown proteins are identified as S-nitrosylated proteins for the first time in plants.A comparison of S-nitrosylation of the crude with RuBisCO depleted fractions showed that RuBisCO depletion increased polypeptide/spot number on the 1-D/2-D gels, indicating its effectiveness in S-nitrosylation analysis Figure . MS idenIn the present study, Fe-SOD showed an increase in S-nitrosylation in cold was removed from To test, if RuBisCO removal improves efficacy of S-nitrosylation analysis, BST of RuBisCO depleted fractions (using GSNO) was performed. It improved the protein resolution as 7 new polypeptides (on the 1-D gel) and 13 new spots (on the 2-D gel) were observed. Increased polypeptide/spot number also suggests improved efficacy of the BST and neutravidin affinity chromatography. Moreover, it also enhanced the identification of the regulatory targets , which earlier escaped detection in the crude and inorganic pyrophosphatase 1. Although, tyrosine nitration of Gly I in salt stress was shown in citrus , involved in the glucosinolates hydrolysis as targets, suggest the role of S-nitrosylation in regulating \u201cglucosinolate hydrolysis pathway.\u201d This pathway is specific to Brassicaceae and is involved in protection against abiotic stress , a database of protein oxidative modifications. No other redox modification was identified supporting that these targets are not yet reported and are novel in plants.The novel targets were searched to detect other redox based PTMs using RedoxDB (B. juncea. Cold induced NO reacts with low molecular weight thiols and promotes SNOs formation leading to S-nitrosylation. The fact that 17 new S-nitrosylated targets (4 GSNO treated and 13 cold responsive) were identified, which were not detected in crude (Abat and Deswal, To conclude, an increase in the NO production in cold suggested its role in maintaining cellular redox homeostasis in The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "We investigated the independent effects of HIV-1 \u201dtarget not detected\u201d measurements versus those that were detectable but below the limit of quantification by Taqman RT-PCR assay on subsequent viral rebound as there are conflicting data regarding the clinical implications of arbitrary or isolated low-level viremia. Cox proportional hazard regression modeling was used to investigate the independent effects of the first HIV-1 load measurement after introduction of the Taqman RT-PCR assay (time-point 0 [T0]), pre-T0 viral loads, CD4 T cell count, race/ethnicity, gender, age and NNRTI use on risk of a confirmed VL >50, >200, >400 and >1000 copies/mL at 22 months follow-up in analyses of all patients and propensity-matched baseline cohorts. 778 patients had a viral load that was either not detected by RT-PCR (N\u200a=\u200a596) or detectable, but below the limit of quantification (N\u200a=\u200a182) at T0. Detectable viremia, lower T0 CD4 count, decreased age, and having detectable or unknown VL within a year prior to T0 were each associated with viral rebound to >50, >200 and >400 copies/mL. Overall failure rates were low and <5.5% of all patients had confirmed VL >1000 copies/mL. A majority of patients with rebound >200 copies/mL subsequently re-suppressed (28 of 53). A detectable VL <48 copies/mL was independently and significantly associated with subsequent viral rebound, and is cause for clinical concern. Monitoring the response to antiretroviral therapy relies upon measurements of HIV-1 RNA, with the goal to achieve virologic suppression, defined as a level below the limit of detection of the assay These mixed findings leave clinicians with a conundrum when faced with plasma HIV-1 RNA results that fall into the detectable but not quantifiable range: should such a finding prompt a change of therapy, closer monitoring, or no action at all? Further study is warranted to understand fully the clinical implications of VLLV in various populations and in individuals who rebound with higher viral loads. We investigated the independent effects of \u201dtarget not detected\u201d measurements versus those that were detectable but below the limit of quantification using the Roche Cobas Taqman RT-PCR assay on risk of virologic rebound in patients followed at two academic medical centers, and described virologic outcomes of patients experiencing rebound.The Partners Healthcare Human Research Committee reviewed and approved this study. The requirement to obtain informed consent from each individual was waived by the institutional review board as the study was limited to review of existing medical records.Data from electronic medical records of HIV-1-infected patients on treatment at or after the time the Roche Cobas Taqman RT-PCR assay v.1 was introduced into use were collected at two academic medical centers in Boston, Massachusetts. One institution changed from the Versant bDNA assay (limit of detection \u200a=\u200a75 copies/mL) to the Taqman assay in July 2008, and the second institution changed from the Cobas Amplicor assay (limit of detection \u200a=\u200a50 copies/mL) to the Cobas Taqman assay in December, 2009. Patient information was collected at all available time-points following the first viral load (VL) result obtained with the new Taqman assay (time-point 0 [T0]). Information collected included patient demographics, CD4 T-cell count, VL, and antiretroviral regimen, and if all known pre-Taqman VL assays were below the limit of detection (50, >200, >400 or >1000 copies/mL with follow-up censored at 22 months. A confirmed VL was defined as a result with at least one consecutive measurement greater than the VL threshold value. The study period was determined by the mean length of follow-up and availability of laboratory data. Patient demographics, CD4 T-cell count at T0, NNRTI use, VL group at T0, and known VL < assay threshold 1 year prior to T0 were included in the Cox regression model. The regression was repeated using a viral rebound definition excluding a single VL above the cutoff value at the last available determination. A second hazard analysis was performed to determine the effects of T0 viral load group using a propensity score matched TND comparator cohort (1\u22362 BLQ to TND). Propensity scores were calculated by the nearest neighbor method including all study variables as covariates.Baseline characteristics for the TND and BLQ groups were compared using Fisher's exact test for gender and use of non-nucleoside reverse transcriptase inhibitor (NNRTI) therapy, \u03c7Additional clinical information was obtained from the records of patients with confirmed or last VL >200 copies/mL, including whether or not resistance testing was requested because of virologic rebound, resistance test results, changes in antiretroviral drugs at the time of rebound, and whether virologic suppression was re-established (defined as a confirmed or last VL of <50 copies/mL) after initial rebound. All statistical analyses were performed with SPSS vs. 20 and R Essentials.A total of 778 patients had a VL that was TND (N\u200a=\u200a596) or BLQ (N\u200a=\u200a182) at T0. Baseline CD4 cell counts were significantly lower in the BLQ group . The proDuring 22 months of follow-up, 66 patients in the TND group (11.4%) and 60 in the BLQ group (33%) had a confirmed or last VL >50 copies/mL. Thirty-two (5.4%) and 21 (11.5%) of patients in the TND and BLQ groups experienced rebound to a VL >200 copies/mL, whereas 26 (4.4%) and 18 (9.9%), respectively, rebounded to >400 copies/mL. Only 20 of patients in the TND group (3.4%) and 10 in the BLQ group (5.5%) experienced viral rebound >1000 copies/mL. A similar percentage of patients experienced viral rebound >50, >200, >400, and >1000 copies/mL from the propensity-matched TND cohort when compared to the unmatched TND population .Factors including a VL BLQ at T0, lower CD4 count, and known detectable viral load prior to T0 by the older VL assays were independently associated with a subsequent confirmed VL >50, >200 and >400 copies/mL from Cox regression analysis of all patients defining viral rebound as only a confirmed, consecutive VL above the cutoff value. Younger age was also associated with VL>200 and >400 copies/mL . Only lower CD4 count and known detectable viral load prior to T0 were independently associated with viral rebound >1000 copies/mL.Of the 53 patients from the entire population who experienced viral rebound to >200 copies/mL, 55% re-suppressed to <50 copies/mL. Of the 24 patients that did not resuppress, 10 had persistent VL >200, 5 had persistent LLVL >50 but <200 and 9 had no further VL info available. Of note, 26% of patients were receiving NNRTI based therapy at the time of viral rebound, and 30% of patients experiencing rebound to >200 copies/mL underwent a change in ART. Interpretable results were obtained from 22 of 34 genotypic resistance tests attempted. Nine of the 22 viral genotypes showed evidence of drug resistance, including 6 with NNRTI resistance. The median viral load at the time of failure for the 9 patients with resistance was higher than for the 13 patients without measurable resistance . All 9 patients with documented drug resistance changed antiretroviral regimens, of whom 7 subsequently re-suppressed.A better understanding of the mechanisms and clinical importance of residual viremia is needed as newer, more sensitive assays are implemented. We show that viral load measurements that are detectable but <48 copies/mL independently predicted a higher risk of virologic rebound to >50, >200 and >400 copies/mL but <1000 copies/mL when compared to undetectable viremia .The strongest predictor of viral rebound was having quantifiable or unknown viral loads within the year prior to rollout of the ultrasensitive Taqman assay; lower CD4 count at baseline was also a strong predictor of rebound. Although power to detect small differences between viral rebound at higher VL copy numbers was lacking, a higher proportion of patients within the T0 VL BLQ group experienced rebound >1000 copies/mL. As a result, detectable but unquantifiable viral load measurements are cause for concern.Prior studies that examined the relationship between VLLV and subsequent virologic rebound or failure have given mixed results. For example, a single VLLV was associated with subsequent viral rebound to >50 or 400 copies/mL at 12 to18 months in one study Previous studies that identified significant positive associations between VLLV and virologic rebound utilized the Abbot RealTime PCR assay for VL testing. We identified a similar association when VL testing was performed using the Roche Cobas Taqman assay v.1. A higher frequency of low-level viral blips using the Cobas Taqman v.1 assay compared to other quantification platforms has been described A majority of patients (53%) with viral rebound to >200 copies/mL in our study resuppressed, even though less than a third of patients changed ART regimens. A previous study showed that low-level viremia appeared to be transient, with 40% of patients reverting to complete suppression over time This study was limited by information available from medical record review. Reliable information regarding CD4 T cell count nadirs, duration of ART and suppressed viremia prior to T0 were not available from the medical records, as patients either presented to care already on therapy or had been followed intermittently at facilities outside of our hospital network. As a result, survivor or other bias may have been introduced into the analyses. Our study incorporated the longest follow-up time used in recent studies of VLLV and was unique in the use of propensity score-matching to reduce standardized differences between baseline variables and investigation of viral rebound at higher viral loads (e.g. >1000 copies/mL) nd and 3rd line regimens that may result from lowering VL cutoffs that define full viral suppression In conclusion, our finding that VLLV is associated with a greater risk of subsequent viral rebound is a cause for concern, but the implications for clinical management are uncertain. Studies in larger cohorts are needed to answer important clinical questions regarding the potential increased costs of more frequent VL monitoring or switching to more expensive or less well-tolerated 2"} +{"text": "High-sensitive real-time PCR assays are routinely used to monitor HIV-1 infected subjects. Inter-assay discrepancies have been described at the low viral load (VL) end, where clinical decisions regarding possible virological rebound are based.A retrospective study was performed to analyze frequencies of viral blips after transition to the COBAS Ampliprep/COBAS TaqMan v2.0 HIV-1 assay (Taqman v2.0) in patients with prior undetectable VLs as measured with the Roche Cobas Ampliprep Amplicor HIV-1 Monitor Test, v1.5 (Amplicor) and was evaluated in comparison to a group of patients monitored with the Abbott Real-time HIV-1 assay (Abbott RT) during the same period of time.85 of 373 patients with VLs below the limit of quantification with Amplicor had VLs >50 copies/mL after transition to the TaqMan v2.0 assay. Among these 74.1% had VLs ranging from 50\u2013499 copies/mL, 22.9% had VLs >500 copies/mL. From 22 patients with initial Taqman v2.0 based VLs exceeding 500 copies/mL, 6 patients had VLs <20 copies/mL after novel VL measurement on a next visit. In our control group with VL quantification using the Abbott RT assay, only 1 patient became detectable and showed a VL of <40 copies/mL after new measurement.Transition to the Taqman v2.0 assay was accompanied by an increase of quantifiable HIV-1 VLs in patients with long term viral suppression under antiretroviral therapy that might be attributed to technical shortcomings of the Taqman v2.0 assay. A high test variability at the low VL end but also beyond was observed, making meaningful clinical interpretation of viral blips derived from different assays difficult. Viral load (VL) quantification constitutes a fundamental cornerstone of antiretroviral therapy management in HIV-1 infected subjects gag region, was released, numerous studies reported unusual findings including false negative or underestimated VL measurements in patients monitored with the TaqMan v1.0 assay Shortly after the first version of the TaqMan assay (TaqMan v1.0), which targets the Although the overall reliability and accuracy of the TaqMan v2.0 and the Abbott RT assay, targeting the integrase region, were found to be similar Plasma samples were recruited from HIV-1 infected individuals from two Austrian HIV outpatient clinics in Salzburg , and in Vienna (Vienna Center). Both HIV centers are part of the Austrian HIV Cohort (AHIVCOS) and the study has been approved by the ethics committee of the Vienna Medical University (No. 898/2010) and the ethics committee of the Salzburg Federal Government (No. 1159/2010), respectively. Written informed consent was given by the patients for their information to be stored in the hospital database and used for research.Both centers followed similar hospital procedures for routine venipuncture and blood collection in ethylene diamine tetra acetic acid (EDTA) anticoagulated tubes. Blood was collected in 10 mL BD Vacutainer EDTA tubes and transferred within 2\u20134 h post collection from the clinic site to the laboratory for further processing. The EDTA tubes were centrifuged at arrival in the diagnostic laboratory for 10 min at 1,100 g for plasma separation. After centrifugation, 1 mL of plasma was transferred into secondary tubes and stored at \u221220\u00b0C. On the day of analysis, the plasma aliquots were thawed, vortexed and analyzed for viral load.We performed a retrospective analysis comparing both centers in order to evaluate whether significant differences in the number of elevated VL measurements in formerly virologically suppressed patients after implementation of the TaqMan v2.0 assay could be found.On August 6, 2009 the Vienna Center switched from the Cobas Ampliprep/ Amplicor HIV-1 Monitor Test v1.5 (Amplicor) to real-time PCR technology using TaqMan v2.0. Throughout the same period of time, the Salzburg Center used Abbott RT testing for HIV-1 quantification, which had formerly been introduced in August 2008. Inclusion criteria were chosen as follows: 1) Initiation of ART prior to August 6, 2008, 2) \u22651 VL measurement in the pre TaqMan v2.0 period from August 6, 2008 to August 5, 2009 (designated as time point 1) and all VL measurements below the LOQ and no ART changes; 3) \u22651 VL measurement during the TaqMan v2.0 period from August 6, 2009 to August 5, 2010 (designated as time point 2) and no ART changes. Furthermore, patients who reached VLs above 20 copies/mL at time point 2, were reassessed with a new VL measurement on their next clinical visit (designated as time point 3). For the Amplicor and the TaqMan v2.0 assay, automated RNA extraction was performed using the COBAS Ampliprep system. The Abbott m24 sp automated sample preparation system was used for RNA isolation for the Abbott RT HIV-1 assay. PCR amplification was then performed either on the COBAS Amplicor system using the Amplicor assay with a dynamic range from 50 to 750.000 copies/mL, on the COBAS TaqMan 48 system using the TaqMan v2.0 assay with a dynamic range from 20 to 10.000.000 copies/mL or on the m2000 rt Real- Time PCR system using the Abbott RT HIV-1 assay with a dynamic range of 40 to 10.000.000 copies/mL. Genotypic drug resistance testing was performed using the ViroSeq\u00ae HIV-1 Genotyping System (Abbott Diagnostics).Following the switch to the TaqMan v2.0 assay in the Vienna Center, multiple internal reports accumulated concerning an increase of detectable VLs in patients with previous long-term virological suppression.In the Vienna Center, 373 of 2078 recruited HIV-1 infected individuals met the inclusion criteria. 288 (77.2%) patients remained with a HIV-1 VL below 50 copies/mL after implementation of the TaqMan v2.0 assay. Due to increased dynamic range of the TaqMan v2.0 assay 67 patients now had quantifiable VL levels ranging from 20 to 49 copies/ml and 221 patients had VL measurements <20 copies/ml. Interestingly for 85 subjects (22.8%) VLs >50 copies/mL were reported. Among these patients, 63 had VLs ranging from 50\u2013499 copies/mL while 22 patients showed VLs >500 copies/mL . As a coDuring the same time period, 288 HIV-1 infected individuals were recruited in the Salzburg Center and 52 patients met our inclusion criteria. For these patients, VL quantification was performed continuously using the ABB assay and only 1 patient became detectable (137 copies/mL) from August 2008 to August 2010. A new measurement resulted in a VL of <40 copies/mL at the next clinical visit. In addition a direct comparison of the TaqMan v2.0 and the Abbott RT assay was performed at the Salzburg Center using 77 samples originating from HIV-1 infected subjects under ART with previous long-term virological suppression and with HIV-1RNA levels below the LOQ with at least one of the two assays. One sample had a VL of 76 copies/ml as measured with the Abbott RT assay but was undetectable with the TaqMan v2.0 assay whereas 12 had a VL of >50 copies/ml with the TaqMan v2.0 assay but were below the limit of detection with the Abbott RT assay (data not shown).Transition from the Amplicor assay to the TaqMan v2.0 assay in the Vienna Center was followed by an increase of quantifiable VLs in patients with stable ART and prior successful viral suppression, which at least in part, could not be reproduced in subsequent VL measurements. We are aware that our findings are not derived from repeat measurements of the same samples but from sequential clinical visits.The implementation of newer high-sensitive RT-PCR assays with lower detection limits for HIV-RNA VL monitoring has been shown to lead to increased frequency of blips LTR and gag region, respectively. Although we are aware that our study does not provide any mechanistically insight this technical issue might be addressed in the future by using different fluorescent dyes for each target and calculating VLs for each target from separate calibration curves allowing comparison of two VL measurements in the same sample within the same run.A study by Garrett et al compared the size and rate of blips between TaqMan v1.0 and 2.0 in a cohort of virologically suppressed HIV-1 patients and found similar blip rates for both test systems but a higher amplitude of blips with v2.0. Also a higher proportion of blips on TaqMan v2.0 exceeding a cut-off of 200 copies/mL was observed The clinical significance of viral \u201cblips\u201d in previously stable patients is still a matter of debate and remains a source of dilemma for many clinicians. Recently, it was shown that virological blips of >500 copies/mL were associated with increased rebound risk, whereas no significant association was observed for blips between 50\u2013500 copies/mL In an attempt to address the socio-economic impact of our findings, we noted that after introduction of the TaqMan v2.0 assay in the Vienna Center 132 additional VL repeat measurements as well as 3 genotypic testings were required. Considering 58\u20ac per VL test, 16\u20ac per CD4 cell count, 5\u20ac per venipuncture and 171\u20ac per genotypic resistance test, the overall cost for additional laboratory testing reached 11000\u20ac not including the supplementary financial burden due to ART modification. Our very conservative estimate does also not take into account the increased workload and time required by clinic staff as well as the psychological stress for patients confronted with the possibility of an emerging drug resistance and numerous adherence discussions between patients and physicians.In summary, we show that transition from the Amplicor assay to a high-sensitive, dual-target RT-PCR assay (TaqMan v2.0) led to an increased frequency of reported quantifiable VLs in patients with long term viral suppression under ART. Although the control group was considerably smaller than our group of interest in Vienna, the comparison with a second center with the same clinical standards of care using the Abbott RT assay for VL monitoring questions the reliability of the Taqman v2.0 assay at the lower VL end. Furthermore, the variability of VL measurements in patients with VL above 500 copies/mL as determined by TaqMan v2.0 was surprising and constitutes an additional matter of concern. For clinicians interpretation of single detectable HIV VLs especially when longitudinal data are derived from different assays is very difficult and should not lead to immediate genotypic testing or therapy intensification."} +{"text": "Mechanistically, CDK1 inhibition caused a reduction in the S-phase fraction of KRAS mutant cells, an effect also characterised by modulation of Rb, a master control of the G1/S checkpoint. Taken together, these observations suggest that the KRAS/CDK1 interaction is a robust synthetic lethal effect worthy of further investigation.Activating KRAS mutations are found in approximately 20% of human cancers but no RAS-directed therapies are currently available. Here we describe a novel, robust, KRAS synthetic lethal interaction with the cyclin dependent kinase, CDK1. This was discovered using parallel siRNA screens in KRAS mutant and wild type colorectal isogenic tumour cells and subsequently validated in a genetically diverse panel of 26 colorectal and pancreatic tumour cell models. This established that the KRAS/CDK1 synthetic lethality applies in tumour cells with either amino acid position 12 or amino acid position 13 (p.G13D) KRAS mutations and can also be replicated KRAS oncogene is one of the most frequently mutated genes in human cancer [KRAS have profound effects on signalling, which can result in a hyper-proliferative and anti-apoptotic phenotype [KRAS mutations affecting amino acid position p.G12, cause resistance to EGFR targeted therapy in colorectal cancer (CRC) [KRAS, also known as the Kirsten rat sarcoma viral oncogene homolog protein V-Ki-ras2), is a member of the RAS superfamily , 2. RAS , is a men cancer , being an cancer . Oncogenhenotype , 8\u201310. Ier (CRC) , 12.KRAS mutations in human cancers considerable attention has been paid to targeting this oncogene. These efforts include; (i) approaches that are based on inhibiting signal transduction pathways that act downstream of KRAS, such as the use of MEK inhibitors [Because of the frequency of hibitors , (ii) thhibitors \u201321 and, hibitors . In the hibitors .in vitro and in vivo.Here, we describe the identification of a novel KRAS SL interaction involving the cyclin dependent kinase, CDK1. This was identified using siRNA screens, was shown to operate in a genetically diverse set of colorectal and pancreatic tumour cell models and was replicated with small molecule inhibitors of CDK1, both The LIM1215 and SW48 KRAS isogenic cell lines were generated and provided by Horizon Discovery. The LIM1215 cell lines were cultured in RPMI supplemented with 10% FBS and SW48 cell lines were grown in DMEM with 10% FBS. The C2BBe1, HT115, HT29, HT55, LS411N, RKO, SNU-C1, SW1417, WiDr, DLD1, GP2D, HCT116, LOVO, LS513, SKCO1, SW1116, SW480, SW620, T84, BxPC-3, HPAC, MIAPACA2, PANC1, PL45 and PL5 were obtained from the American Type Tissue Collection and cultured according to the suppliers\u2019 instructions. All cell lines were routinely confirmed as being mycoplasma negative using the MycoAlert Kit (Lonza) throughout experimentation.AZD5438, RO-3306, AT7519, Dinaciclib and PD023309 were purchased from Selleck chemicals. Antibodies targeting pan-RAS (Millipore), KRAS (Sigma), \u03b2-Actin, PARP-1 (F-2) (Santa Cruz), CDK1, Phospho-CDK1 (Thr161), Rb and Phospho-Rb (Ser 807/811) , were employed in western blot.2 transformed. To account for plate-to-plate variation, we calculated the median effect in each plate and then normalized each well value according to the plate median. Plate normalized values from triplicate screens were then combined by the calculation of median values. To allow cell inhibitory effects to be compared between cell lines and screens, we converted median plate normalized values to Z, or standardized, scores, using the calculation Za = (xa\u2212screen median) / variance of screen, where Za = Z score for gene a, xa = plate normalized value for gene a. The screen median was calculated according to the median value for all 784 siRNAs in the screen and the variance of the screen was estimated by calculation of the Median Absolute Deviation (MAD).A 384 well plate arrayed siRNA library targeting 784 genes (Dharmacon) was used ethanol. Cells were then washed twice in PBS and resuspended in 1 mL of PBS containing 100 \u03bcL RNase A (Sigma) and 20 \u03bcL propidium iodide (PI) (Sigma). Total DNA content was quantified and analyzed by flow cytometry on a Becton Dickinson fluorescence-activated cell scan cytometer and data was analysed using BD FACSDiva Software (BD Biosciences).The Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit was used for analyzing DNA replication in proliferating cells, according to the manufacturer\u2019s instructions. EdU (5-ethynyl-2 \u2032-deoxyuridine) is a thymidine analog, which is incorporated into DNA during active DNA synthesis.Cells were incubated with 10 \u03bcL of a 10 mM solution of EdU in a 10 cm dish for 1h at 37\u00b0C, 5% CO2. After the incubation the cells were harvested and washed once with 3 mL of 1% (w/v) BSA in PBS. The cells were then centrifuged and incubated with 100 \u03bcL of Click-iT fixative for 15 minutes at room temperature, protected from light. The cells were washed with 3 mL of 1% BSA (w/v) in PBS, centrifuged, then resuspended in 100 \u03bcL of 1X Click-iT saponin-based permeabilization and wash reagent and incubated for 15 minutes. 0.5 mL of Click-iT reaction cocktail was added to each tube and incubated for 30 minutes at room temperature, protected from light. The cells were washed once with 3 mL of 1X Click-iT saponin-based permeabilization and wash reagent, centrifuged and resuspended in 500 \u03bcL of 1X Click-iT saponin-based permeabilization and wash reagent. DAPI staining was added for DNA staining. Standard flow cytometry methods were used for determining the percentage of S- phase cells in the population and DNA content.Genomic DNA was isolated from the cell lines using the Puregene\u2014blood, cell and tissue kit (Qiagen), according to manufacturer\u2019s instructions, eluted in 20 \u03bcL H2O and stored at -20\u00b0C. DNA concentration was measured using a spectrophotometer measuring the UV absorbance at 260 nm. Typically PCR reactions contained 10\u2013100 ng DNA, and were done in 50 \u03bcL reaction volume, using Expand High Fidelity PCR system (Roche). PCR conditions were as follows: 95\u00b0C for 5 min; thirty cycles of 94\u00b0C for 1 min, 55\u00b0C for 1 min, 72\u00b0C for 45 sec, followed by a single cycle at 72\u00b0C for 5 min and 4\u00b0C thereafter. PCR products were purified from agarose gel using QIAquick Gel extraction Kit (Qiagen). Cycle sequencing was carried out using BigDye Terminator v3.1 Cycle Sequencing kit with an initial denaturation at 95\u00b0C for 1 min, then 25 cycles at 96\u00b0C for 10 sec, 50\u00b0C for 5 sec, and single cycle at 60\u00b0C for 4 min. Sequencing products were purified using DyeEx 2.0 spin protocol for Dye-Terminator removal (Qiagen), and analysed using the ABI PRISM 377 DNA sequencer (Perkin Elmer). The data was analysed using Sequencher 4.8 software.Cells were plated in 96-well (250\u20131000 cells/well (depending on the cell line)) plates in 80 \u03bcL. After 24 hours, drug or vehicle (DMSO) dilutions in media were added to the cells to make a total volume of 100 \u03bcL. Cells were left exposed to drug for five days. Cell viability was assessed using the luminescent CellTiter-Glo reagent (Promega) whereby 50 \u03bcL of reagent diluted 1:4 in PBS was added to each well, which was shaken for 10 minutes at room temperature according to the manufacturer\u2019s protocol. Luminescence was measured using the Victor X5 Multilabel plate reader (Perkin Elmer).Activated RAS was measured using a RAS activation assay kit according to manufacturer\u2019s instructions (Millipore). RAS alternates between an active, GTP-bound state and an inactive, GDP-bound state. RAS family effector proteins specifically recognize the GTP-bound form of RAS. This is exploited experimentally to determine the levels of RAS protein activation. The assay uses the RAS-binding domain (RBD) of the RAS effector kinase c-RAF, which binds specifically to the GTP-bound form of RAS proteins. The RAF-RBD is conjugated with agarose beads, which allows the precipitation of the RAF-RBD/GTP-RAS complex. For the RAS pull-down assay, 500 \u03bcg of whole cell lysates (freshly collected) were incubated with the RAF-RBD-agarose beads for 45 min at 4oC with gentle agitation. After washing the agarose beads, the amount of GTP-RAS was quantified by western blotting of purified samples with a mouse monoclonal antibody recognizing all three isoforms of RAS. MCF7 and HeLa cells were used as controls of the experiment. HeLa cells were also stimulated with 50 nM EGF as a positive control.All mouse work was carried out in accordance with the Institute of Cancer Research (ICR) guidelines and with the UK Animals (Scientific Procedures) Act 1986 and approved by the ICR Animal Welfare and Ethical Review Body. Animals were housed in IVC type cages , which were maintained under negative airflow. Mice were companion held and a density commensurate with the UK Home Office Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes. Animals were provided with Corncob bedding, nesting material and environment enrichment. All animals were fed Ad-libitum with Lab diet 5002 rodent diet. Water was filtered and chlorinated. Animal holding rooms were maintained within the parameters recommended in the Home Office Code of Practice with temperatures being 21\u00b0C +/- 2 degrees, Humidity 55% +/- 10% and a light cycle of 12 hours dark/light. Animals were monitored daily by facility staff for basic husbandry requirements and signs of ill health. Study animals were also monitored by AK and SCC.in vivo efficacy of AZD5438, 5x106 of SW620 cells, or SW48 KRAS WT or p.G12V isogenic cells were injected into the flank regions of female athymic Balb/C mice, twenty mice per cell line (Harlan Laboratories). In the drug arm ten mice were treated once daily with AZD5438 by oral administration starting immediately after tumour establishment at a dose of 20mg/kg and ten mice were treated once daily with vehicle (0.5% methylcellulose) in the control arm.For assessment of the 3) or if there was more than 20% weight loss. Tumour volume was calculated using the formula (L*W)*(SQRT(L*W))*(\u03c0/6) , where length represented the longer diameter of an asymmetrical tumour.Tumour growth was monitored at least twice a week, taking two-dimensional measurements with callipers. The mice were monitored daily for toxicity, and culled when the maximum tumour volume was reached Act 1986. Mice were sacrificed by the Schedule 1 methods of cervical dislocation or decapitation according to the UK Animals Act 1986.KRAS mutations. We selected the KRAS wild type (WT) colorectal tumour cell line, LIM1215 as a model [KRAS alleles into the endogenous KRAS gene at position glycine 12 [KRAS alleles with a mutant allele caused constitutive KRAS activation in all three cases, as assessed by measuring levels of active GTP-RAS cells and of KRASWT/G12D, KRASWT/G12S and KRASWT/G12V models in parallel siRNA screens to identify candidate synthetic lethal effects. For each genotype, we siRNA screened two independently derived clones. A LIM1215 clone encompassing a control targeting vector (LIM1215neo) was also screened in parallel. As a screening library, we used a 384 well plate arrayed siRNA library targeting 853 genes, predominantly protein kinase encoding genes. We focused on protein kinases given their inherent tractability as drug targets as well as their involvement in a wide range of intracellular signalling processes. Cells were reverse transfected with siRNA on day 0, and cell viability was estimated six days later using Cell Titre Glo reagent. In total we carried out two biological replicate screens for each clone, with each biological replicate encompassing three technical replica screens of \u2264 -2 and a median Z score in the control, non-KRAS mutant lines of \u2265 -1. Using these thresholds we identified six candidate KRAS SL genes; Cyclin-Dependent Kinase 11B (CDK11B), Integrin-Linked Kinase (ILK), Casein Kinase 1, Gamma 1 (CSNK1G1), Cell Division Cycle 73 (CDC73), Cyclin Dependent Kinase 1 (CDK1) and General Transcription Factor IIH, Polypeptide 1, 62kDa (GTF2H1) .WT/WT) clone, we generated isogenic KRASWT/G12D, KRASWT/G12S, KRASWT/G12V and KRASWT/G13D clones and assessed RAS activation in these clones validated, as the CDK1 siRNA gave a more profound effect in the SW48 KRAS mutant cells than WT cells . We did mitosis , 29. Whiansition \u201332. CDK1ansition \u201333.KRAS mutant (n = 10) and WT cohorts (n = 10). We found that although CDK1 siRNA did inhibit multiple KRAS mutant models it also had inhibitory effects in KRAS WT models, HT29, WiDr, LS411N, SW1417 and RKO , a disease where where somatic of cases . We founAC model .KRAS WT/BRAF mutant models RKO and LS411N showed sensitivity to as well as sensitising to CDK1 siRNA results previously discussed . This difference was enhanced when cells were exposed to AZD5438 (G1 fraction in p.G12V KRAS mutant cells exposed to 0.3 \u03bcM = 87.7% compared to 68.7% for similarly treated WT cells). Concomitantly, we noticed a significant reduction in the proportions of KRAS p.G12V mutant cells in the S- and G2/M phases when compared to WT cells after cells were exposed to AZD5438 , S8 Fig.KRAS p.G12V cells than in the WT cells, we examined S phase progression by measuring DNA synthesis using the Click-iT Plus 5-ethynyl-2'-deoxyuridine (EDU) assay. In this assay, the fluorescently labelled thymidine analogue EDU is incorporated into newly synthesized DNA, an event that can be monitored by measuring fluorescence [KRAS mutant cells when compared to both untreated mutant and WT cells is phosphorylated by either CDK1 or CDK2 at Ser807 and Ser811. This post translational modification inactivates Rb and facilitates cell cycle progression . We therrylation . In KRASexposure . Taken t1 arrest followed by apoptosis.We further assessed the mechanism of cell inhibition caused by AZD5438. We found that a high, non KRAS selective concentration of AZD5438 caused PARP cleavage in both KRAS WT and mutant cells, but a KRAS selective concentration of AZD5438 caused apoptosis in the KRAS mutant cell line but not in the KRAS model . This suThe druggable nature of CDKs, along with the recurrent dysregulation of CDK activity in human cancer, has led to intensive efforts to develop selective CDK inhibitors . CDK4 haIn order to identify whether other small molecule CDK inhibitors elicited the same KRAS selective effects as AZD5438, we assessed the tumour cell inhibitory effects of a range of CDK inhibitors: (i) AT7519, a CDK1/2/4/5/9 inhibitor ; (ii) diin vitro selective effects of AZD5438 on KRAS mutant cells, we assessed in vivo efficacy of AZD5438. We xenografted SW620 (KRAS p.G12V) colorectal tumour cells into immunocompromised mice and once tumours had established, we treated animals with AZD5438. Specifically, animals with established SW620 tumours (80 mm3) were randomized into one of two cohorts and treated with either 20 mg/kg/day AZD5438 or vehicle (n = 10 in each cohort) . The gro cohort) . SW620 x cohort) . Moreove cohort) , suggest48 cells . Whilst 2/M phases of the cell cycle in KRAS mutant cells. This was accompanied by an increased in the proportion of cells in G1. Moreover, western blotting revealed that AZD5438 decreased Rb phosphorylation levels in KRAS mutant cells compared to KRAS WT cells. As cells require hyperphosphorylation of Rb to pass the restriction point in G1 prior to entering S-phase, the reduction in Rb phosphorylation could provide an explanation as to the cell cycle arrest, which could be a possible cause for the reduced proportion of KRAS mutant cells in S-phase [in vivo setting. These experiments were performed in immunocompromised mice with SW620 cell together with SW48 KRAS isogenic p.G12V and WT cells xenografts, and have confirmed the KRAS selective inhibitory effect of AZD5438, supporting the in vitro results. Overall the results from this experiment suggested disease stabilisation after AZD5438 treatment, but in some cases complete tumour inhibition was observed. Already a number of other synthetic lethal interactions involving cyclin dependent kinases have been proposed. Perhaps the most notable synthetic lethal interaction involving CDK1 described to date is between CDK1 and the oncogenic transcription factor, MYC [Here, we describe a series of experiments aimed at identifying novel dependencies in KRAS mutant tumour cells. Using genetic screens in LIM1215 KRAS isogenic cell lines we identified novel KRAS synthetic lethal effects, including CDK1. One weakness of the HT screen might be the use of engineered models of KRAS mutation, rather than the use of models with naturally occurring KRAS mutations. It is possible that these engineered models do not fully replicate all of the KRAS synthetic lethal effects found in real human tumours. However, we do note that we also observed the KRAS selective effect of CDK1 inhibition in tumour cell lines with naturally occurring KRAS mutations . CDK1 wa S-phase . We detetor, MYC . In thistor, MYC . This pator, MYC . As welltor, MYC as well tor, MYC . Here weS1 FigKRAS transcript. The different introduced mutations are indicated for each cell line with a black arrow in the chromatograms.The introduced genetic alterations in the targeted cells was determined by RT-PCR and sequencing of the (PDF)Click here for additional data file.S2 Fig(A) (PDF)Click here for additional data file.S3 FigCell viability was assessed at day six by CellTiter-Glo (Promega) luminescence reading. Only screens fulfilling the pre-established quality criteria (Z\u2019 factor > 0 and Spearman rank correlation between replicates > 0.7) were considered further. After data processing, potentially interesting siRNAs were selected based on a Z score > -1 in the KRAS WT and < -2 in the KRAS mutant cells.(PDF)Click here for additional data file.S4 Fig(A) (PDF)Click here for additional data file.S5 FigKRAS WT/BRAF mutant (green) and KRAS WT/BRAF WT (black) cells after AZD5438 exposure in a five-day survival assay. ****P<0.0001, Two-way ANOVA. Error bars represent SEM of three technical replicates.(A) Drug-dose response curves of PDAC cells after AZD5438 exposure in a fifteen-day colony formation assay. **P<0.01, ***P<0.001, ***P<0.0001, Two-way ANOVA. (B) Drug-dose response curves of CRC cells, (PDF)Click here for additional data file.S6 Fig(A) (PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig2/M-phase cells after exposure with AZD5438 when compared to the control (DMSO) and to KRAS WT cells (AZD5438 and DMSO).Propidium iodide (PI) flow cytometry plots. SW48 KRAS WT (A) and p.G12V (B) were exposed to 0.3 \u03bcM AZD5438 or DMSO for 16, 24 and 48 hours after which cell cycle profiles were assessed by flow cytometry. The KRAS p.G12V mutant cells showed a decrease in S and G(PDF)Click here for additional data file.S9 Fig(A) (PDF)Click here for additional data file.S10 Fig(A) AT7519, (B) dinaciclib and (C) PD023309 survival curves from a five-day cell viability assay to assess the KRAS selectivity of the CDK inhibitors in ten colorectal cell lines, four KRAS WT (black) and six mutant (pink) cell lines.(PDF)Click here for additional data file.S11 Fig(A and B) Average increase in tumour volume of KRAS WT and mutant xenografts. In the KRAS WT xenografts there is no significant difference between the treatment arm and the non-treatment. As in the KRAS mutant xenografts, the drugged arm shows significantly reduced tumour growth compared to the vehicle. Error bars represent SEM. . (C) Average final tumour weight. There is no significant difference between the vehicle and treatment arms, however the difference in weight between the WT and mutant treated with AZD5438 is significant .(PDF)Click here for additional data file.S1 TableThis table lists the genes included in the siRNA library alongside the gene accession number and the median Z scores from three replicate screens for each cell line.(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table50, and the area under the curve (AUC) of CDK1 inhibitors, RO-3306 and AZD5438, for SW48 KRAS isogenic cell lines (A), Colorectal (B) and Pancreatic Adenocarcinoma Cancer cell lines (C).Tables presenting SF(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file."} +{"text": "In non-small-cell lung cancer (NSCLC), one-fifth of patients have KRAS mutations, which are considered a negative predictive factor to first-line therapy. Evidence is emerging that not all KRAS mutations have the same biological activities and possible remodeling of cell metabolism by KRAS activation might complicate the scenario. An open question is whether different KRAS mutations at codon-12 affect cellular metabolism differently with possible implications for different responses to cancer treatments.We applied an explorative mass spectrometry-based untargeted metabolomics strategy to characterize the largest possible number of metabolites that might distinguish isogenic NSCLC cells overexpressing mutated forms of KRAS at codon-12 and the wild-type. The glutamine deprivation assay and real-time PCR were used to confirm the involvement of some of the metabolic pathways highlighted.Cell clones indicated distinct metabolomic profiles in KRAS wild-type and mutants. Clones harboring different KRAS mutations at codon-12 also had different metabolic remodeling, such as a different redox buffering system and different glutamine-dependency not driven by the transcriptional state of enzymes involved in glutaminolysis.These findings indicate that KRAS mutations at codon-12 are associated with different metabolomic profiles that might affect the responses to cancer treatments. Lung cancer is the leading cause of cancer-related mortality worldwide , closelyKRAS is frequently mutated in largely diffused tumors, such as pancreatic, colon and NSCLC , 5. ThesApproximately 20-25% NSCLC patients present KRAS mutations, which are a negative predictive factor of response to first-line therapy . The majKRAS activation supports the decoupling of glycolysis and TCA metabolism, with glutamine supplying increased carbon to drive the TCA cycle. These results provide evidence that oncogenic KRAS is involved in the metabolic reprogramming of cancer cells -13. It hStudies of KRAS-mediated metabolism have mainly examined transformed fibroblasts -11 or siWe applied an explorative, untargeted metabolomics approach with liquid chromatography/tandem mass spectrometry (LC-MS/MS) to characterize the largest possible number of metabolites from relevant or potentially affected metabolic pathways in isogenic NSCLC cells overexpressing mutated forms of KRAS at codon-12 . This enabled us to draw metabolic portraits characterizing KRAS mutant clones in lung cancer cells. The different metabolic states associated with different KRAS mutations will help in designing more efficacy cancer therapy, aimed at exploiting metabolic differences among KRAS mutations in NSCLC.We applied unsupervised mass spectrometry-based metabolomics to discover unbiased small-molecule metabolic profiles that might distinguish human NSCLC cell line NCI-H1299 clones overexpressing KRAS mutations from the wild-type (WT). Two independent clones were screened for each KRAS mutant. All the clones had comparable KRAS expression levels and actiMass-spectral data were subjected to peak alignment and data pre-proccesing by SIEVE 1.3. Then data were analyzed for global changes, using multivariate statistics to determine group separation and to assess the number and percentage of molecular features that differed significantly in the four sample sets. OPLS-DA analysis indicated good separation of all the clones under both positive and negative ionization. Each KRAS mutant clone has its own metabolomics signature, distinct from KRAS WT. Each mutant was separated from KRAS WT, as shown by the score plots supportihttp://metlin.scripps.edu; HMBD, http://www.hmdb.ca/; LipidMaps, http://www.lipidmaps.org/) in positive and negative ionization. KRAS G12C, G12D and G12V were significantly separated from KRAS WT by respectively 74, 58 and 48 singular metabolic species, identified by database searches . The metabolic class distribution was similar for the different KRAS mutations, where glycerophospholipids and amino acids were the most abundant classes Figure . The G12To further interpret the biological significance of the metabolite changes in the three KRAS mutant clones, we used MetaboAnalyst tools to link metabolites to metabolic pathways. For the reasons mentioned, lipid classes were not included in the list uploaded on MetaboAnalyst.+, an essential coenzyme regulating numerous cellular metabolic pathways . KRAS mutant clones had a redox state comparable to the WT and small, but significant, differences were mainly observed among mutations at codon-12. Altered detoxification status in KRAS-transformed fibroblasts has been reported, with better detoxification potential in G12D and G12V mutants than their control .We characterized the antioxidant profile of our clones by measuring ophthalmate (OPA) which is an endogenous analog of GSH, indicated as a potential biomarker for GSH depletion . The G12Overall our data further support the notion that KRAS mutational status involves a general metabolic reprogramming to fuel growth and counter stress, including enhanced amino acids catabolism, alterations in lipid biochemistry and antioxidant program.Our evidence was obtained using a robust isogenic system generated by stable transfection of WT or mutated KRAS. Although the system has some limitations, as it was generated by overexpressing KRAS (WT or mutated) in a WT KRAS-expressing cell line, it has several advantages over cell lines with different KRAS status. In fact it allows us to determine, in a similar genetic background, the role of a single point mutation in KRAS. The system was generated to express similar levels of wild-type or mutant protein and relies on the evidence that two independent clones, generated for each mutation, have similar growth rates, protein expression and GTPase activity, thus reducing the risks of clonal selection possible with stable transfectants.The particular metabolic adaptations of KRAS mutational status might contribute to their different responses to anticancer treatments and migh2 in air. For growth curve experiments, cells were cultured in six-well plates with or without glutamine and were counted 24, 48, 72, 96 and 168 hours after seeding, using a cell counter (Beckman). The growth curves were plotted as absolute numbers of cells. Each experiment consisted of three replicates for each point and the plotted data are the mean and SD of three independent experiments. Statistical analyses (two-way ANOVA and Bonferroni post-test for multiple comparison) were done using GraphPad Prism software .Human non-small-cell lung carcinoma NCI-H1299 KRAS overexpressing clones were grown in RPMI1640 medium with 500 microG/mL of G418 (Gibco) added. Cells were maintained at 37\u00b0C in a humidified atmosphere of 5% (v/v) COg for 10 min at 4\u00b0C and the protein concentration was determined using a BioRad assay kit (BioRad). A 50-microG sample of total cellular proteins was separated on SDS-PAGE and electrotransferred to PVDF membrane (Millipore). Antibodies were diluted following the manufacturer\u2019s instructions in 5% non-fat dry milk in TBS-Tween 20 0.05% (TBS-T). Immunoblotting was carried out with the following antibodies: anti-KRAS and anti-actin, obtained from Santa Cruz Biotechnology. Antibody binding was detected using peroxidase secondary conjugated antibodies (Santa Cruz Biotechnology) and visualized by enhanced chemiluminescence .KRAS expression level in all clones was evaluated by Western blotting analysis. Briefly, cells were plated at different concentrations in 100-mm tissue culture dishes (Sterilin). Forty-eight hours after seeding, extracts were prepared by lysing cells for 30 minutes on ice in protein lysis buffer in the presence of protease inhibitors. Insoluble material was pelleted at 13,000xActive (GTP-bound) KRAS was measured in mutated and WT expressing cells by the KRAS Activation assay Kit (Cell Biolabs) according to the manufacturer\u2019s instructions. 1000 microG of whole-cell cleared lysate was incubated with RAS-RAF binding domain for 60 min at 4\u00b0C. The complexes were collected by centrifugation and washed. Proteins were separated by SDS-PAGE, followed by Western blot. The KRAS protein was detected with anti-KRAS antibody (Santa Cruz Biotechnology). Actin was detected as a loading control.2 to the dish. The plates were then stored at \u221280\u00b0C, and extracted and analyzed within seven days. Extraction was done by adding 1 mL of ice-cold 90% 9:1 MeOH:CHCl3 to each plate and cells were scraped. The extraction solvent contained tryptophan-D8, 3-hydroxyindoleacetic acid-D5, methionine-D3, 17-alpha-methyltestosterone-D3, fludrocortisone and desoximetasone as internal standards (1 microM/each) to ensure metabolite extraction, injection and chromatographic consistency for positive and negative ionization modes. Extracts were transferred to 1.5 mL micro-centrifuge tubes and pelleted at 4\u00b0C for 15 min at 10000xg. Supernatants were divided into two aliquots, dried, then reconstituted in acidic or basic LC-compatible solvent.For metabolomics analysis, NCI-H1299 KRAS overexpressing clones were grown for 48 hours in biological triplicate. At 48 hours, all four clones have the same proliferation rate. Metabolites were extracted from clones as reported , with miA portion (2 microL) of metabolite extract from all the KRAS clones was directly analyzed by LC-MS/MS, using an LTQ Orbitrap XL\u2122 (Thermo Scientific), interfaced with a 1200 series capillary pump (Agilent). The MS instrument was operated in positive (POS) and negative (NEG) ionization modes. Chromatographic and MS conditions were as reported . UntargeThe normalized ion intensity data for each clone were submitted to the SIMCA-P13 software package (Umetrics) for multivariate data analysis. The variables were scaled using Pareto scaling to increase the low abundance ions without significantly amplifying the noise. To maximize class discrimination, the data were analyzed by orthogonal partial least-squares discriminant analysis (OPLS-DA). S-plots were calculated to visualize the relationship between covariance and correlation within the OPLS-DA results. Variables that significantly contributed to discrimination between groups were identified.http://metlin.scrpss.edu) and Human Metabolome Database . Accurate mass data and isotopic distribution for the precursor and product ion were compared to spectral data of the reference compounds in the databases. Lipids were tentatively identified by high mass accuracy and MS/MS fragment ions using the LIPID Mass database without authentic standards. Identifications were reported only for metabolites with accurate mass match < 5 ppm.For metabolite identification, the frame m/z values were used for batch searches on the METLIN database (www.genome.jp/kegg/), using MetaboAnalyst 2.0, a comprehensive online tool suite for metabolomic data analysis and interpretation .For biological interpretation of the metabolite dataset by our untargeted strategy, we mapped the identified metabolites to the KEGG pathway database and OPA were quantified by LC-MRM-MS , 31, witOne-hundred microL of conditioned cell culture media from KRAS-overexpressing clones were filtered through a 10 kDa MW spin filter (Millipore) to remove proteins and used to determine lactate secretion. Lactate was measured using the Lactate Colorimetric Assay Kit (Abcam). Statistical analysis (one-way Anova) was done using GraphPad Prism software.http://bioinfo.ut.ee/primer3-0.4.0/primer3/input.htm) and the specificity was verified by detecting single-band amplicons of the PCR products. cDNA was amplified by real time RT-PCR with the SYBR Green technique. Relative quantification of mRNA was done using the \u0394\u0394Ct method. Actin was used as reference gene and H1299 KRAS WT clone was arbitrarily set to 1.RNA from H1299 KRAS-overexpressing clones grown for 48 hours with or without glutamine was purified using the Simply RNA Maxwell Total RNA Purification Kit (Promega). Retrotranscription to cDNA was done using the High Capacity cDNA Retrotranscription Kit (Applied Biosystems). Optimal primer pairs for selected genes , spannin"} +{"text": "KRAS mutations in non-small-cell lung cancer (NSCLC) patients are considered a negative predictive factor and indicate poor response to anticancer treatments. KRAS mutations lead to activation of the PI3K/akt/mTOR pathway, whose inhibition remains a challenging clinical target. Since the PI3K/akt/mTOR pathway and KRAS oncogene mutations all have roles in cancer cell metabolism, we investigated whether the activity of PI3K/akt/mTOR inhibitors (BEZ235 and BKM120) in cells harboring different KRAS status is related to their metabolic effect. Isogenic NSCLC cell clones expressing wild-type (WT) and mutated (G12C) KRAS were used to determine the response to BEZ235 and BKM120. Metabolomics analysis indicated the impairment of glutamine in KRAS-G12C and serine metabolism in KRAS-WT, after pharmacological blockade of the PI3K signaling, although the net effect on cell growth, cell cycle distribution and caspase activation was similar. PI3K inhibitors caused autophagy in KRAS-WT, but not in KRAS-G12C, where there was a striking decrease in ammonia production, probably a consequence of glutamine metabolism impairment.These findings lay the grounds for more effective therapeutic combinations possibly distinguishing wild-type and mutated KRAS cancer cells in NSCLC, exploiting their different metabolic responses to PI3K/akt/mTOR inhibitors. As shown in Figure PI3K inhibitors were able to induce authophagy only in KRAS-WT clone.Since autophagy and apoptosis can be triggered by common upstream signals, and sometimes this results in combined induction of autophagy and apoptosis, we investigated the activation of caspase 3/7 in our system after treatment. Neither of the drugs at the doses used for autophagy evaluation had any relevant effect on the central step of apoptosis in the two KRAS clones Figure and 5B. To further clarify the behavior of clones, cell cycle perturbation was evaluated by flow cytometric analysis at different times after BEZ235 or BKM120. Cell cycle distribution after the PI3K inhibitors at low doses was similar for both KRAS isoforms. BEZ235 induced a partial accumulation of cells in G1 phase starting at 6h and up to 48h of treatment, while a small amount of cells treated with BKM120 was intercepted by the G2/M checkpoint 6h after the start of treatment Figure . The perKRAS mutations are genetic events that occur early in tumor progression and are associated with more aggressive tumor phenotypes and/or resistance to treatment. KRAS mutations lead to the activation of several signaling pathways including PI3K/akt/mTOR . In NSCLHere we show that the dual PI3K/mTOR inhibitor BEZ235 and the pan PI3K inhibitor BKM120 have similar activity in terms of cell growth inhibition in KRAS-WT and KRAS-G12C mutated clones. In view of this similar growth inhibition, and since they induce a comparable block in the PI3K signaling pathway, we wondered whether this final effect was reached through different mechanisms in KRAS-WT and -G12C cells, considering their different basal metabolic profiles recently reported by our group \u201333.More is known about the mechanism of action of PI3K inhibitors but nothing about the metabolic rewiring they induce on NSCLC harboring different KRAS isoforms. Identification of mutant KRAS specific metabolic alterations in response to treatment would open up the possibility of combining these drugs with specific agents on metabolism to maximize their anticancer activity. We found that the presence of different KRAS isoforms in NSCLC cell clones did indeed induce different metabolic responses after pharmacological impairment of the PI3K signaling. In particular, the KRAS-G12C mutant clone presented alterations of the glutamine metabolism supported by the accumulation of glutamine, glutamate and aspartic acid, all utilized to generate \u03b1-ketoglutarate and ammonia to sustain cell proliferation . We haveIn contrast, the KRAS-WT clone did not show any significant accumulation of metabolites involved in the glutamine metabolism after BEZ235 and BKM120 treatment, again supporting our previous evidence that the KRAS-G12C mutation affects the cell responses , 23. StrThe impairment of glutamine and serine metabolism respectively in KRAS-G12C and KRAS-WT clones, was also confirmed by the significantly lower uptake of glutamine and serine in their conditioned media. These altered metabolic pathways represent attractive therapeutic targets. For instance agents able to interfere with central metabolic pathways already exist and some have been shown to be effective in preclinical cancer models \u201342. DespThis again indicates the presence of different metabolic responses induced by KRAS-G12C and KRAS-WT in NSCLC cells after pharmacological impairment of PI3K signaling, although the net effect on cell growth, cell cycle distribution and caspase activation are similar. It is likely that KRAS-WT cells activate ammonia-induced autophagy after drug treatment probably to cope with energy imbalance stress (derangement in serine metabolism), whereas the KRAS-G12C cells do not display this compensatory mechanism triggered by ammonia because of their glutamine metabolism impairment. Figure In conclusion, these findings may have important implications in NSCLC through the development of combination strategies (e.g. nutrient modification) to increase the killing of cancer cells expressing mutant KRAS, enhancing the selectivity and finally improving the therapeutic index.The NCI-H1299 derived clones were grown in RPMI-1640 medium including 500 \u03bcg/ml of G418 (Gibco). Clones were obtained by transfecting the NCI-H1299 cell line with the expression plasmids encoding for the mutant KRAS-G12C and KRAS-WT as a control. Details of transfection, KRAS protein expression and activation are reported in our previous works [Cells are routinely tested for mycoplasma contamination by PCR and authenticated with the PowerPlex 16 HS System (Promega) every six months by comparing the STR profiles with those deposited in ATCC and/or DSMZ databases. All drugs were dissolved in medium just before use. Treatments were performed with BEZ235 at 25 nM or with BKM120 at 1 \u03bcM for 6h, 24h and 48h unless otherwise specified. The MTS assays (Promega) were done as described in [NCI-H1299 cell lines harboring KRAS-G12C or KRAS-WT isoforms treated with BEZ235 (25 nM) or BKM (1 \u03bcM) and their untreated counterparts were collected after 6, 24 and 48h of drug treatment. Metabolites were extracted as reported [Conditioned cell culture medium was collected 48 h after inhibitors treatment, 1mL of each culture medium was filtered and thirty microliters of filtered medium were used for metabolomic analysis.A targeted quantitative approach using a combined direct flow injection and liquid chromatography (LC) tandem mass spectrometry (MS/MS) assay was applied for the metabolomics analysis. The method combines derivatization and extraction of analytes with the selective mass-spectrometric detection using multiple reaction monitoring (MRM) pairs. Isotope-labeled internal standards are integrated into the platform for metabolite absolute quantification. This targeted strategy allows for simultaneous detection and quantification of up to 186 metabolites from five analyte groups: acylcarnitines, amino acids, biogenic amines, hexoses (sum of hexoses), phosphatidylcholines (PCs), and sphingomyelins (SMs) . SamplesThe method of AbsoluteIDQ p180 kit has been proven to be in conformance with FDA Guideline Guidance for Industry\u2014Bioanalytical Method Validation (May 2001), which implies proof of reproducibility within a given error range. Data evaluation for quantification of metabolite concentrations and quality assessment have been performed with the MetIDQ software package, which is an integral part of the AbsoluteIDQ kit. The metabolite concentration of each metabolite in each experimental condition were compared with the measurement detection limit specifications as reported by the manufacturer of the AbsoluteIDQ p180 kit (Biocrates). A metabolite was excluded from further analyses if its concentration measurement data did not meet all of the following criteria: (1) less than 20% of missing values (non-detectable peak) for each quantified metabolite in each experimental group; (2) 50% of all sample concentrations for the metabolite had to be above the limit of detection (LOD).http://www.TM4.org).Metabolite data expressed as micromolar concentration (\u03bcM), from each experimental condition (cell clones/treatments), were examined using the SIMCA-P13 software package (Umetrics) for multivariate analysis. The variables were scaled using Pareto. To maximize class discrimination, the data were analyzed by orthogonal partial least-squares discriminant analysis (OPLS-DA). S-plots were calculated to visualize the relationship between covariance and correlation within the OPLS-DA results. Heat map and Hierarchical clustering were done using the MeV module of the TM4 package to identify metabolites differing significantly between treated and untreated KRAS isoforms.Total RNA was reverse-transcribed with a High-Capacity cDNA Kit (Life Technologies) and amplified by 7900HT Sequence Detection System (Life Technologies). Actin was used as internal control. Primers and TaqMan probes were purchased for all genes as ready-to-use solutions (Life Technologies). Two samples that showed at least two-fold differences were considered differently expressed.Proteins were extracted and visualized as reported in . ImmunobTwenty-four hours after cell plating, BEZ235 (25 and 50 nM) or BKM (1 and 2 \u03bcM) was added. Twenty-four or 48h later, caspase activity was assessed using the Caspase-Glo 3/7 Assay (Promega) according to the manufacturer's instructions.Sample preparation and monoparametric DNA histograms analysis were done in untreated or treated BEZ235 (25 nM) or BKM (1 \u03bcM) as described in .Ammonia levels were measured in cell-conditioned medium in NSCLC harboring KRAS-G12C or KRAS-WT isoforms 48 hours after BEZ235 (25 nM) or BKM120 (1 \u03bcM) treatment, following the manufacturer's instruction .Statistical analyses were done using GraphpadPrism version 6.05. Tests to analyze specific experiments are indicated in the legends to the figures. Differences between groups were considered statistically significant when the p-values were \u22640.05."} +{"text": "Gadus morhua) may instead increase. Arctic aquaculture that constitutes about 2% of global farming is mainly made up of Norwegian salmon farming. The sector will face many challenges in a warmer future and some of these are already a reality impacting negatively on salmon growth. Other more indirect effects from climate change are more uncertain with respect to impacts on the economic conditions of Arctic aquaculture.We review current knowledge about climate change impacts on Arctic seafood production. Large-scale changes in the Arctic marine food web can be expected for the next 40\u2013100\u00a0years. Possible future trajectories under climate change for Arctic capture fisheries anticipate the movement of aquatic species into new waters and changed the dynamics of existing species. Negative consequences are expected for some fish stocks but others like the Barents Sea cod ( Gadus morhua) in the Barents Sea environmental carrying capacity may instead increase farmed in Norway. The sector may face many challenges in a warmer future and some of these, being directly related to temperature increase, are already a reality for the industry. Other more indirect impacts will have more uncertain influence on the economic conditions of Arctic aquaculture production fishery takes place in this area . Catches from East and West of Greenland (including the Arctic Sea) are marginal in comparison; however, fishing in these areas constitutes important livelihoods for many small-scale operators. North-eastern Pacific fisheries take place south of the Arctic Circle and their numbers are included just as a reference as important Alaska Pollock (ea Table\u00a0.Fishery is one of the most important industries in the Arctic representing large shares of gross domestic product (GDP) in some countries. For local communities fishing, fish processing and/or fish farming can be even more important. Thus, local communities, regions and nations\u2019 degree of dependency on the fishing or associated activities are important, and since fish production volumes are the most available (and reliable) data at hand, this is usually used as a proxy for importance.Fisheries have historically been the main reason for settlement in many peripheral Arctic coastal areas. Arctic aquaculture has the last two decades grown significantly and is today dominated by Norwegian Atlantic salmon farming. Arctic aquaculture only constitutes a small share of world aquaculture production volumes (~\u00a02%) but its specific contribution to global marine aquaculture production is important (ca. 25%) FAO and its Volumes of catches and targeted species from Arctic fisheries are presented in Table\u00a0Mallotus villosus), Atlantic herring (Clupea harengus), Atlantic cod (Gadus morhua), blue whiting (Micromesistius poutassou), Greenland halibut (Reinhardtius hippoglossoides), queen crab (Chionoecetes opilio), deepwater redfish (Sebastes mentella) and northern prawn are capelin (Boreogadus saida) spawning stock was particularly large in 2008 (FAO Eleginus nawaga) and Arctic flounder according to Christensen et al. mostly [for] freshwater and diadromous fishes\u201d are very low, with only 4 tonnes reported in 2014 , together contributing to almost 60% of total catch. The capelin stock was not exploited in 2014 while it represented the largest catch 2\u00a0years before (FAO Norway is the dominant fishing nation in the Northeast Atlantic but many countries have active fisheries in this area Table\u00a0. The maifore FAO . This lafore FAO .Scomber scombrus) has a larger (and increasing) distribution area, and coastal states have not reached a final agreement on the allocation of quotas since 2007. For that stock, the advice went from 349 000\u2013456 000 tonnes in 2008 to 927 000\u20131 011 000 tonnes in 2014. ICES estimated that 1 396 000 tonnes were landed in 2014 advises on total allowable catch (TAC), which are then set by governments. The TAC advice for Norwegian spring spawning herring in 2015 was 283 000 tonnes compared to 1 687 000 tonnes in 2009 ICES . The Nor014 ICES .While the fleets from the different nations all target the same common resource, they typically have different structures, with different boat sizes and gear types\u2014often resulting from national institutional constraints. The industry is rather dynamic; for example, in 2013 a new fishery developed along the Eastern Greenland coast targeting mackerel that had recently reached further north in this region and all the way to the Spitsbergen fjords flow into the Barents Sea basin, significantly affecting cod\u2013capelin interaction and haddock (8%). Between 2000 and 2014, the number of registered fishermen in Norway decreased from 20 000 to 11 300 (\u2212 44%).Norwegian fisheries are diverse and like in other countries they have experienced considerable changes recently. From 2000 to 2014, the number of registered fishing vessels decreased from 13 000 to 6000. About 80% of the fleet in 2014 consisted of small vessels below 11 metres . The industrial fleet is located mainly in the Murmansk and also in Arkhangelsk Oblast. Murmansk Regional Government MRG reports Russian official catch statisticsThe Murmansk fishery sector\u2019s share of regional GDP is 7%, and the sector employs roughly 7800 persons MRG . MurmansPandalus borealis) are the most important species. Greenland fisheries\u2019 share of GDP was 13.6 and 90% of total export in 2013http://www.stat.gl/publ/da/IE/201401/pdf/Udenrigshandel2013.pdf ). In 2013, the fishing fleet consisted of 384 vessels, of which 193 were below 10\u00a0m, 149 were 10\u201320\u00a0m, 19 were 20\u201330\u00a0m and 23 were larger than 30\u00a0m in length. In addition, Greenland fisheries sector also had 185 snowmobiles, 602 dog sledges and 1422 jolly boats, mainly located in Northwest Greenland (Qaasuitsup) and with permit to fish and land fish . These \u201cvessels\u201d had a share of 29% of total Greenland landings value (about 117 million \u20ac in 2013). Greenland\u2019s total catch in 2013 amounted to 170 000 tonnes, 70% from within their own exclusive economic zone (EEZ). The most important species (in volume) were mackerel (31%) caught in East Greenland waters and ICES areas XIV a/b, shrimp (25%) and capelin (16%) caught in Icelandic waters and Greenland halibut (6%). In value terms, shrimp was by far the most important species , then Greenland halibut (12%) and cod (7%). Shrimp trawlers were either larger offshore trawlers or inshore trawlers. The former operated outside three nautical miles from the baseline and in open waters and had an obligation to land 25% of its catch to land-based production (leaving 75% to be on-board processed and exported). The inshore trawlers had an obligation to land 100% for land-based production. Greenlandic shrimp quotas were divided between offshore and inshore trawlers in a 57/43 percentage distribution. In addition to fish and crustacean, Greenlandic hunting landed 51 000 sealskins and 3300 whales in 2013.Fishing is Greenland\u2019s primary industry and shrimps , approximately 116 000 tonnes were caught in NAFO areas 1a (Baffin Bay) and 1b (Davies\u2019 Strait), while 8 000 tonnes were caught in ICES area XIV (a and b) and other ICES areas. Overall, approximately 125 000 tonnes of Greenland\u2019s catch were caught in Arctic waters (55%). Catches in Arctic Northeast Atlantic waters are unclear (ICES catch statistics for 2012 report 39 000 tonnes but EuroStat has no records). Total employment in the Greenland fisheries sector (fish processing industry excl.) was roughly 3550 in 2013\u2014approximately 13% of Greenland\u2019s labour force.Oncorhynchus mykiss). Iceland mainly produces Atlantic salmon, rainbow trout and Arctic char , while Russia produces primarily salmon. In Finland and Sweden, small volumes of freshwater species dominate the production. Some mussels (Mytilus edulis) are produced in areas close to the Arctic such as Newfoundland and in the southern parts of Alaska. Fish farming is currently prohibited in Alaska. In the Canadian provinces south of Newfoundland, both salmon and mussels are farmed.Aquaculture is an important economic activity in some concentrated parts of the Arctic region Fig.\u00a0. Total ANorway is the world\u2019s largest producer of Atlantic salmon and also has a significant production of rainbow trout and a smaller production of several other marine and freshwater species. A considerable part of this production takes place in the Arctic region. Table\u00a0Salmon farming was introduced in the Norwegian Arctic around 1970 and grew rapidly after 1994. Salmon dominated during the whole period and the value of production accelerated after 2000. Other species were introduced, but have not experienced the same growth as salmon despite considerable research and development investments for farming halibut and cod for example. Halibut was primarily in focus during the early 1990s and cod in focus during the latest part of 1990s. Arctic charr was early in 1900 a pioneering aquaculture species in Norway, but farming north of the Arctic Circle developed first during the 1980s , turbot , trout and blue mussels constitute the rest of the production. A large share of the overall production originates from land-based systems.Only a limited part of Iceland\u2019s coastline is protected against waves and suitable for aquaculture, and hence production is relatively small. Icelandic aquaculture has gone through several phases, with rapid growth in the 1980s followed by a period of stable production in the 1990s and a rapid decline in the mid-2000s when salmon production declined from about 7000 to about 500 tonnes. Arctic charr have had a relatively steady growth during the whole period and cod aquaculture started to develop first in early 2000. Arctic charr constitute about 50% of total value Table\u00a0. AtlantiArctic charr constitute over 50% of total production Table\u00a0, with AtClimate change can affect the main drivers causing distributional changes in the productivity of an ecosystem Brander . NaturalHistorically (17th and 18th century) claimed increased catches were associated with increased temperatures . Price premiums have been observed on labelled seafood , the most common salmon parasite that dominates global fishmeal and fish oil production. Most aquaculture species (fish and crustaceans) increasingly feed on terrestrial crops instead of feed from the sea . Small farming volumes could expand into the current Arctic. However, Alaska has banned finfish farming. Lifting this ban could trigger the introduction of fish farms in the current farming areas and into the Arctic.In Canada, Atlantic salmon is the main species at USD 192 million in 2014 FAO , but somOstrea edulis) and scallops (Pecten maximus) are primarily grown in warmer waters and the anticipated warming may not be sufficient to bring temperatures in the Arctic to comparable levels.Iceland has no close \u201cneighbours\u201d that can inform future potential aquaculture species expansion. The Norwegian and Russian Arctic are likely to follow the current activities in the remaining part of Norway, focusing mainly on salmon. Norway hosts a considerably higher production of rainbow trout in the south and more Atlantic halibut Table\u00a0. Both thSocioeconomic impacts from climate change could be significant for the aquaculture sector. However, these effects are also linked with changes in other economic sectors in the Arctic, and in the rest of the world so it is difficult to foresee how the overall effects will play out (Cr\u00e9pin et al. Nations at higher latitudes may benefit from climate change effects on ocean ecosystems, at least initially\u201d (IPCC Increased variability could increase tensions among fishing nations creating climate change-related conflicts like the recent conflict over Atlantic mackerel stocks, previously shared between EU and Norway but now also targeted by Icelandic fishermen \u2013 a response to mackerel stocks migrating into the Icelandic economic exclusive zone during summertime\u201d (IPCC Current Arctic fisheries mainly operate in the Northeast Atlantic, with some fishing, whaling and sealing activities in the Northwest Atlantic. This review indicates that substantial spatial and temporal variability already characterise these fisheries and climate change will likely exacerbate these. According to IPPC, \u201cly\u201d IPCC . Also, \u201cme\u201d IPCC . The manThe climate-induced temperature rise on the Norwegian coasts is likely to range between 0.5\u00b0 and 2.5\u00b0 and play out differently during different seasons. Despite large uncertainties, and just a few detailed studies that specifically target climate change impacts on Arctic aquaculture, the direct effects of a temperature change on the aquaculture industry can be modelled with fairly good accuracy, including effects on fish growth and impacts on the whole industry. These models indicate positive effects from warming water temperatures on Arctic aquaculture. Direct effects related to storm frequencies and intensities can be relatively well anticipated, but with high uncertainty. Other indirect effects, such as diseases and pest species and freshwater runoff, are much harder to predict. However, it is certain that the environmental conditions will change and that the industry will have to adapt to these changes. For enabling the industry to do so, there is a need to look over existing regulatory frameworks and start a multi-stakeholder dialogue to find out where and how aquaculture operations can move or change their operations. As the Arctic Region is undergoing multiple changes, involving changes in economic conditions and large-scale environmental changes, the different ways that aquaculture in the Arctic can adapt will be linked to the overall changes occurring in the region. Thus, a broader integrative approach is needed for successful governance of the Arctic system (Cr\u00e9pin et al."} +{"text": "However, the rigid and compact structure of the plant cell walls significantly blocks the separation of lignin. In this study, wheat straw was hydrothermally pretreated at different temperatures (120\u2013200\u2009\u00b0C) followed by post-treatment with 70% ethanol containing 1% NaOH to improve the isolation of lignin. Results demonstrated that the content of associated carbohydrates of the lignin fractions was gradually reduced with the increment of the hydrothermal severity. The structure of the lignins changed regularly with the increase of the pretreatment temperature from 120 to 200\u2009\u00b0C. In particular, the contents of As we know, fossil resources have been widely used as fuels, chemicals, and materials for decades. However, the excessive consumption of the non-renewable fossil fuels has caused serious energy crisis and environmental problem. As a renewable and unique carbon-containing resource, biomass is considered as the most important alternative to fossil resources for providing chemicals and materials in the future23Lignocellulosic materials are mainly composed of cellulose, hemicelluloses, and lignin. Among the three components, lignin is the most abundant aromatic compound in nature that consists of phenylpropane units with various types of linkages. The aromatic nature of lignin presents great potential for the production of industrially useful fuels, chemicals, and materials2, alkaline hydrolysis, hydrothermal pretreatment (HTP), organosolv process, have been extensively explored to process different biomasses for cellulosic ethanol production12Generally, pretreatments for lignocellulosic materials can be roughly divided into chemical, physical, physical-chemical and biological methods13C NMR), quantitative two-dimensional heteronuclear single quantum coherence (2D-HSQC) and 31P NMR spectroscopy. In addition, thermogravimetric analysis (TGA) was also performed to unveil the relationship between structural features and thermal behaviors. The relationships between physicochemical properties of the isolated lignins and hydrothermal pretreatment conditions were also discussed.Wheat straw is an abundant agriculture residue worldwide. However, it is often burned or discarded in a low value-added utilization method due to the lack of effective use pattern. As a kind of grass, wheat straw possesses much loose structure than wood, which shows great potential for the isolation of lignin and the production of bioethanol. In this work, wheat straw was firstly hydrothermally pretreated under selected conditions and then subsequently post-treated with alkaline ethanol solution to improve the separation of lignin fractions. The structures of the obtained lignins were thoroughly characterized by high performance anion exchange chromatography (HPAEC), gel permeation chromatography (GPC), Fourier transform infrared (FT-IR), carbon-13 nuclear magnetic resonance spectroscopy and the content of associated carbohydrates (based on the lignin content in the dewaxed wheat straw) are listed in Mw) and number average (Mn) molecular weights and the polydispersity (Mw/Mn) of the isolated lignins were analyzed by GPC. The results are shown in Mw value of the lignin fraction obtained from the HTP samples gradually decreased from 2770\u2009g/mol to 1560\u2009g/mol as the HTP temperature increased from 120 to 200\u2009\u00b0C. Meanwhile, the Mw values of the lignin fractions obtained from the wheat straw with mild HTP were slightly higher than that of AL (2180\u2009g/mol). It suggested that the mild HTP process was helpful to the decomposion of the cell walls and the release of the lignin fractions with high molecular weights in the following alkaline ethanol extraction. However, the Mw values of the L180 and L200 were obviously lower than that of AL, implying that partial linkages between lignin units might be broken and the lignin was degraded into smaller molecules under such severe HTP conditions. Additionally, all the lignin fractions showed relatively narrow molecular distributions (1.54\u20131.73).To study the effect of the HTP of wheat straw on the molecular weight of the isolated lignin fractions, the weight-average decreased from L120 to L200, suggesting that the content of methoxy groups was decreased with increasing HTP severity as a result of demethoxylation reactions. The absorption at 1342\u2009cm\u22121 (C-O stretching of the S ring breathing) presented a higher intensity than that of the reference band at 1265\u2009cm\u22121 (C-O vibration of the G ring) in L120, but the intensity of the band at 1342\u2009cm\u22121 was lower than that at 1265\u2009cm\u22121 in L160 and L200. It might be related to the demethoxylation reactions of S-type lignin units under the harsh pretreatment conditions, which led to the decrease of the S-type lignin units and the elevation the content of G-type lignin units16\u22121 observed in all of the lignin spectra is assigned to the antisymmetric C-O stretching of ester groups, suggesting that a portion of ester bonds were remained in the isolated lignins. A band at 1119\u2009cm\u22121 derived from the aromatic in-plane C-H bending deformation of S-type lignin was also observed in all spectra. The intensity of this band decreased with the increment of the HTP temperature from 120 to 200\u2009\u00b0C, indicating the reduction of S-type lignin caused by demethoxylation. This result was also supported by the NMR measurements presented afterwards. In addition, it was observed that the intensity of the peak at 1705\u2009cm\u22121 significantly increased with the increment of the HTP severity. It was probably due to the fact that the elimination of water from the benzylic position led to the formation of a carbocation, and then the cleavage of \u03b2-O-4\u2032 linkages gave rise to the formation of Hibbert ketones under severe pretreatment condition141819The FT-IR spectra of the lignin fractions obtained from the wheat straw with and without HTP are shown in 13C NMR spectra of AL, L120, L160, and L200 are shown in The quantitative Signals between 104.4 and 168.2\u2009ppm are attributed to the aromatic region of lignin. The S units signals were observed at 152.2 , 147.1 , 138.2 , 134.3 , and 104.4\u2009ppm (C-2/C-6), while the G units gave signals at 149.7 , 145.5 , 134.3 , 119.4 , 114.8 , and 111.1\u2009ppm . The signals at 128.2 (C-2/C-6) and 115.3\u2009ppm are related to the H units in lignins. These signals indicated that the lignins obtained from wheat straw can be classified as SGH-lignin, which is a typical grass lignin. Additionally, the content of aromatic C-O and aromatic C-H structures in lignins was reduced, while the amount of aromatic C-C structures increased with the increment of HTP intensity. This is likely attributed to the replacement of C-H bonds with C-C bonds during the condensation reaction. Generally, the condensation of lignin occurs simultaneously with its depolymerization during the fractional isolation of lignin. The condensation of lignin tends to form more C-C linkages with the elimination of partial C-H and C-O linkages\u03b2, C-\u03b1, and C-\u03b3 position of \u03b2-O-4\u2032 linkages, respectively. The signal at 56.0\u2009ppm is assigned to the \u2212OCH3 groups in G and S units. The signals of \u03b2-O-4\u2032 linkages and \u2212OCH3 groups gradually decreased with the increase of HTP temperature, suggesting that both de-etherification and demethoxylation occurred during the HTP process. The signals between 57.0 and 103.0\u2009ppm, related to the associated polysaccharides, were almost absent in the spectra of the extracted lignins, indicating that a large proportion of linkages between lignin and polysaccharides have been broken as confirmed by the sugar analysis. Additionally, the signal intensity of esterified p-coumaric acid decreased with the increase of hydrothermal temperature. In particular, the PCA signals were hardly observed as the HTP temperature increased to 200\u2009\u00b0C. Meanwhile, the etherified ferulic acid (FA) was detected with a weak signal at 111.1\u2009ppm corresponding to its C-2 position. The variations of these signals suggested that the cleavage of both PCA and FA occurred under the given HTP conditions.Signals between 50 and 86\u2009ppm are attributed to the interunit linkages of lignin. Signals detected at 86.1 (S units)/84.6 (G and H units), 72.3, and 60.1\u2009ppm are corresponding to the C-\u03b2-O-4\u2032 aryl ether (substructure A) signals appeared at \u03b4C/\u03b4H 71.7/4.82 and 59.6/3.61 are corresponding to its C\u03b1-H\u03b1 and C\u03b3-H\u03b3 correlations, respectively. The C\u03b2-H\u03b2 correlations observed at \u03b4C/\u03b4H 85.8/4.09 and 83.7/4.28 are assigned to the \u03b2-O-4\u2032 structure linked to S and G/H lignin units, respectively. The interunit \u03b2-O-4\u2032 linkages appeared to be the most abundant linkages of the wheat straw lignin. In addition to the abundant \u03b2-O-4\u2032 linkages, signals for resinol substructures also appeared in the spectra in noticeable amounts with their C\u03b1-H\u03b1, C\u03b2-H\u03b2, and double C\u03b3-H\u03b3 correlations at \u03b4C/\u03b4H 84.8/4.64, 53.6/3.06, and 71.0/3.79 and 4.16, respectively. Phenylcoumaran substructures were present in low amounts, and their signals for C\u03b1-H\u03b1, C\u03b2-H\u03b2 and C\u03b3-H\u03b3 correlations occurred at \u03b4C/\u03b4H 87.4/5.58, 53.5/3.45 and 62.2/3.70, respectively. In addition, \u03b1-O-4\u2032 aryl ether and p-hydroxycinnamyl alcohol end groups were also detected in the side-chain regions.To obtain the detailed structural information of the lignin samples, 2D-HSQC NMR technique was applied to analyze the lignins. The HSQC spectra of the lignins are presented in 2223242,6-H2,6 correlations of S and C\u03b1-oxidized S units (S\u2032) were observed at \u03b4C/\u03b4H 104.0/6.69 and 104.1/7.31, respectively. The C2,6-H2,6 signal of H units was observed at \u03b4C/\u03b4H 128.3/7.21, while the G units presented various correlations for C2-H2 (\u03b4C/\u03b4H 111.0/6.97), C5-H5 (\u03b4C/\u03b4H 114.6/6.71), and C6-H6 (\u03b4C/\u03b4H 118.9/6.77). In addition, the signals for PCA substructures were observed at \u03b4C/\u03b4H 129.7/7.50, 115.4/6.81, 144.0/7.48 and 115.1/6.30 corresponding to their C2,6-H2,6, C3,5-H3,5, C7-H7 and C8-H8 correlations, while the signals for FA substructures were detected at \u03b4C/\u03b4H 111.0/7.26, 122.1/7.08, 144.0/7.48 and 116.5/6.40 corresponding to C2-H2, C6-H6, C7-H7 and C8-H8 correlations.In the aromatic region, signals from S, G, and H units could be observed clearly. The C\u03b2-O-4\u2032 aryl ether linkages, followed by low amounts of phenylcoumaran (\u03b2-5\u2032), resinol (\u03b2-\u03b2\u2032), and \u03b1-O-4\u2032 aryl ether linkages. The content of \u03b2-O-4\u2032 linkages in AL (39.35/100Ar) was slightly lower than that of L120 (43.10/100Ar). The phenomenon was probably due to the fact that mild hydrothermal pretreatment condition was conducive to the subsequence fractionation of wheat straw lignin to some extent, resulting in the decrease of cleavage of ether linkages during the alkaline ethanol extraction process. However, the content of \u03b2-O-4\u2032 linkages in AL (39.35/100Ar) was much higher than those of L160 (19.54/100Ar) and L200 (1.50/100Ar). It indicated that extensive depolymerization reactions occurred under such severe pretreatment condition. Except for the cleavage of \u03b2-O-4\u2032 linkages, comprehensive degradation of other aryl ether linkages (\u03b1-O-4\u2032 linkages) and carbon-carbon linkages (\u03b2-\u03b2\u2032 and \u03b2-5\u2032) was also observed.The relative abundances of the interunit linkages presented in lignins as well as PCA, FA and S/G/H molar ratios were calculated from the 2D HSQC spectra based on a previous computing method26120, L160 and L200 were lower than those in AL, suggesting that partial etherified FA and esterified PCA in lignin were disrupted during the HTP process22Besides the interunit linkages difference among these lignins obtained under different HTP conditions, a notable lignin units difference among the lignins were also found. As shown in 120, L160, and L200) were analyzed using the quantitative 31P NMR technique. The 31P NMR spectra of the lignins are illustrated in et al.13C NMR analysis.To further investigate the functional groups of the obtained lignins, the lignin samples is in line with the temperature of the maximum decomposition rate (VM) of substrateM of the lignin obtained with HTP was higher than that of the lignin extracted without HTP, and the TM of the isolated lignin increased with the raising HTP temperature. It has been reported that the TM of lignin was positively related to its molecular weightM and Mw was not found in the present work. The shift of TM to higher temperature might be explained by the formation of more stable lignin structure, such as condensed lignin, which was confirmed by the NMR results. Additionally, a large proportion of residual char was observed after calcination at 700\u2009\u00b0C. The residual chars were 37.06% and 41.24\u201350.04% for AL and L120-L200, respectively. Moreover, the residual char percentage showed an increased trend with the raising HTP severity. The char percentage was positively correlative with the content of the condensed lignin units in lignin uncovered by the 31P NMR analysis, indicating the high thermal stability of condensed lignin.The main degradation temperature range of the lignins was observed between 200 and 700\u2009\u00b0C. Generally, the maximum decomposition temperature obtained from the hydrothermally pretreated wheat straw was much higher than that of AL (3.87%) isolated from the wheat straw without hydrothermal pretreatment. The structures and physicochemical properties of the lignins isolated from the hydrothermally pretreated wheat straw changed regularly with the increase of pretreatment temperature from 120 to 200\u2009\u00b0C. In particular, the contents of Wheat straw was obtained from Shaanxi province, China. The dried wheat straw was grounded, and the fraction between 40\u201360 mesh was collected for further experiments. The straw powder was further dewaxed with toluene-ethanol in a Soxhlet apparatus for 6\u2009h and then dried in an oven at 60\u2009\u00b0C for 16\u2009h. The dewaxed straw was mainly composed of 41.2% cellulose, 27.7% hemicelluloses, 18.5% lignin (16.6% Klason lignin and 1.9% acid-soluble lignin), and 6.9% ash, according to National Renewable Energy Laboratory\u2019s (NREL) standard analytical procedure120, L140, L160, L180, and L200 according to the HTP temperature. A lignin sample (AL) fractionated from the wheat straw without hydrothermal pretreatment was also prepared for comparison. All experiments were performed in duplicate, and the average values were given. The schematic diagram of the experimental procedure is shown in The HTP of wheat straw was conducted in a 100\u2009mL batch reactor . The feedstock 5.0\u2009g was immersed in 50\u2009mL deionized water. Then, the mixture was heated up to desired temperature and maintained at this temperature for 0.5\u2009h under continuous mechanical stirring at 800\u2009rpm. After completion of the reaction, the reactor was cooled down to room temperature by ice water. The hydrothermally pretreated residue was collected by filtration, washed thoroughly with deionized water and freeze-dried. Then, the hydrothermally pretreated residue (2.0\u2009g) was subsequently treated with 70% ethanol solution containing 1% NaOH at 90\u2009\u00b0C for 2\u2009h at a solid to liquid ratio of 1:20 (g/mL). The solid residue and liquid stream were separated on a Buchner funnel, washed with deionized water until the filtrate was neutral. The filtrate was collected and adjusted to pH 5.5\u20136.0 with 6\u2009M HCl and further concentrated to 40\u2009mL on a rotary evaporator under reduced pressure. The concentrated liquid was poured into three volumes of 95% ethanol and further filtrated. The supernatant was further concentrated to about 10\u2009mL and poured into 100\u2009mL of acidic water (pH 2.0 adjusted by HCl) to precipitate lignin. The obtained lignin fractions were freeze-dried and labeled as LMw) and number-average (Mn) molecular weights of the obtained lignin fractions were determined by GPC with an ultraviolet detector (UV) at 240\u2009nm according to a previous literature procedure\u22121 with a resolution of 4\u2009cm\u22121.The weight-average acetylacetonate (0.01\u2009M) was added as a relaxation agent for the quantitative 13C NMR spectrum to reduce the relaxation delay according to previous reports3513C NMR spectra were recorded in the FT mode at 100.6\u2009MHz. The inverse gated decoupling sequence (C13IG sequence from Bruker Standard Library) allows quantitative analysis and comparison of signal intensities, which was used with the following parameters: 30\u00b0 pulse angle; 1.4\u2009s acquisition time; 2\u2009s relaxation delay; 64\u2009K data points, and 30,000 scans. For 2D HSQC NMR spectra, 50\u2009mg lignin was dissolved in 0.5\u2009mL DMSO-d6. A semiquantitative analysis of the 2D-HSQC contour intensities was performed according to literature methods3731P NMR spectra39NMR spectra were recorded on a Bruker AVIII 400\u2009MHz spectrometer at 25\u2009\u00b0C in DMSO-oC/min under nitrogen atmosphere.Thermal analysis of the lignin fractions was measured by using TGA and differential thermogravimetric (DTG) analyses on a simultaneous thermal analyzer . About 3\u20135\u2009mg of the lignin sample was heated in an alumina crucible from room temperature to 700\u2009\u00b0C at a heating rate of 20\u2009How to cite this article: Chen, X. et al. Effect of hydrothermal pretreatment on the structural changes of alkaline ethanol lignin from wheat straw. Sci. Rep.6, 39354; doi: 10.1038/srep39354 (2016).Publisher\u2019s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."} +{"text": "G93A mouse model of ALS, and have been proposed to play a role in motor neuron degeneration as well as in other pathologies of the nervous system, such as Alzheimer\u2019s disease and hereditary neuropathies. In this study, we screen a library of small-molecule kinase inhibitors towards the identification of pharmacological enhancers of the axonal retrograde transport of signalling endosomes, which might be used to normalise the rate of this process in diseased neurons. Inhibitors of p38 mitogen-activated protein kinases (p38 MAPK) were identified in this screen and were found to correct deficits in axonal retrograde transport of signalling endosomes in cultured primary SOD1G93A motor neurons. In vitro knockdown experiments revealed that the alpha isoform of p38 MAPK (p38 MAPK\u03b1) was the sole isoform responsible for SOD1G93A-induced transport deficits. Furthermore, we found that acute treatment with p38 MAPK\u03b1 inhibitors restored the physiological rate of axonal retrograde transport in vivo in early symptomatic SOD1G93A mice. Our findings demonstrate the pathogenic effect of p38 MAPK\u03b1 on axonal retrograde transport and identify a potential therapeutic strategy for ALS.Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by the degeneration of upper and lower motor neurons. Defects in axonal transport have been observed pre-symptomatically in the SOD1 However, which of these mechanisms play a causative role in ALS pathogenesis is currently unknown2.Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by the degeneration of both upper and lower motor neurons, resulting in progressive muscle paralysis and ultimately death. Although the precise cause of motor neuron degeneration in ALS is not yet fully understood, several mechanisms have been proposed to play a role in this process, including mitochondrial dysfunction, excitotoxicity and axonal transport deficits2. In mice overexpressing the ALS-associated human superoxide dismutase 1 G93A (SOD1G93A) mutant, intravital imaging in the sciatic nerve has revealed abnormalities in the axonal retrograde transport of signalling endosomes and mitochondria in pre-symptomatic mice3. The deficit in endosome motility was demonstrated using two independent probes: the binding fragment of tetanus toxin (HCT)4 and an antibody specific for the p75 neurotrophin receptor (\u03b1p75NTR)5. The early appearance of transport impairments in the SOD1G93A mouse model3 suggests that these deficits play a crucial role in triggering motor neuron dysfunction, leading to the motor neuron degeneration observed in ALS.Deficits in axonal transport have been inferred from patient data and observed in ALS mouse models2 and other neurodegenerative conditions7 a causal relationship between these transport impairments and neurodegeneration has not yet been shown. Indeed, the role of axonal transport defects in ALS pathogenesis remains a matter of some debate. Work using an ALS mouse model expressing the SOD1G85R mutant has shown that motor neuron degeneration can also occur in the absence of overt axonal transport deficits8, although it should be noted that these results have been obtained using explants rather than intravital microscopy, and disease progression is much more variable in the SOD1G85R mouse model than in the SOD1G93A mice used in our study3. Hence, the identification of compounds able to specifically enhance axonal transport and thereby rescue the deficits observed in SOD1G93A mice would conclusively prove the role of axonal transport defects in ALS pathogenesis.Despite the strength of evidence demonstrating the presence of axonal transport defects in ALS9. It has been proposed that disease-associated pathological proteins, such as amyloid beta (A\u03b2) and SOD1G93A, mediate their toxic effects through the activation of specific kinase cascades10, such as\u00a0p38 mitogen-activated protein kinase\u00a0(MAPK)16. In this study, we demonstrate that p38 MAPK is responsible for SOD1G93A-induced deficits in axonal retrograde transport in motor neurons and establish that specific inhibition of p38 MAPK alpha (p38 MAPK\u03b1) or its down-regulation corrects axonal transport deficits both in vitro and in vivo in SOD1G93A mice. Inhibitors of p38 MAPK\u03b1 are thus powerful tools to determine the role of axonal retrograde transport deficits in ALS pathogenesis and could be explored for future therapeutic intervention.Protein kinases have been suggested to be key players in several neurodegenerative diseasesCT and \u03b1-p75NTR in mouse embryonic stem (ES) cell-derived motor neurons has been previously validated in our laboratory as a biological read-out capable of identifying novel axonal trafficking effectors when combined with a siRNA screen18. In this study, we adapted this assay to screen a library of kinase inhibitors to identify novel regulators of axonal retrograde transport. As before18, transgenic HB9-GFP ES cells (HBG3) differentiated into motor neurons were used to overcome the intrinsic cellular heterogeneity of primary motor neuron cultures and obtain the large amount of neurons required for the screen. The expression of green fluorescent protein (GFP) driven by the Hb9 homeobox gene enhancer facilitated the identification of motor neurons and enabled the implementation of a reliable automatic quantification protocol17.The accumulation of HCT-containing signalling endosomes19. Treatment of ES cell-derived motor neurons with 1\u2009mM EHNA resulted in a significant decrease in HCT or \u03b1-p75NTR accumulation in the soma and \u03b1-p75NTR adenine (EHNA) is an established inhibitor of cytoplasmic dynein, and blocks the axonal retrograde transport of Hles Fig.\u00a0, whilst \u00a0CT Fig.\u00a0 and \u03b1-p7NTR Fig.\u00a0 accumula23. Accordingly, treatment of ES cell-derived motor neurons with 1\u2009mM ALCAR increased the retrograde transport speed of HCT-labelled organelles in cultured motor neurons was previously reported to enhance axonal retrograde transport in diabetic ratsons Fig.\u00a0. We were\u00a0CT Fig.\u00a0 and \u03b1-p7NTR Fig.\u00a0 in ALCARG93A motor neurons24. Using primary motor neurons cultures, we found a significant decrease in HCT accumulation in SOD1G93A motor neurons compared to wild-type controls , representing a primary hit rate of 4%. We also identified three inhibitory molecules is an inhibitor of p38 MAPK. Interestingly, abnormal activation of p38 MAPK has been previously implicated in ALS pathogenesisons Fig.\u00a0. In contons Fig.\u00a0.Fig. 3Im26 . Four chemically diverse p38 MAPK inhibitors were found to increase HCT and \u03b1-p75NTR accumulation in ES cell-derived motor neurons Fig.\u00a0. We alsoG93A motor neurons. This in turn would provide a more specific therapeutic target reducing undesirable side effects, a problem which has severely hampered the development of new therapeutic approaches directed towards p38 MAPK27.The four isoforms of p38 MAPK are all expressed in primary motor neurons Fig.\u00a0. Because12, we sought to test whether this isoform is responsible for the defects in axonal retrograde transport observed in SOD1G93A mice. Lentiviral shRNA vectors expressing GFP as a reporter were used to knockdown p38 MAPK\u03b1 in SOD1G93A motor neurons -derived motor neurons29. Therefore, to demonstrate that inhibition of JNK2/3 is not involved in the restoration of axonal retrograde transport, we tested SB-239063, a second generation p38 MAPK\u03b1 and \u03b2 inhibitor with enhanced specificity30. Similarly to SB-203580, SB-239063 was able to rescue axonal transport in vitro in SOD1G93A motor neuron cultures and axonal retrograde transport was assessed by imaging the sciatic nerve 4\u2009h later in live, anaesthetised mice31 dose of 100\u2009mg/kg allowed for circulating concentrations in the brain, spinal cord and muscle to reach, albeit transiently, levels similar to those used in our in vitro axonal transport assays i.p. twice daily until termination at 90 (symptomatic) and 120 d (end-stage of disease) Fig.\u00a0. Hindlimely Fig.\u00a0. Treatmeely Fig.\u00a0.Fig. 7EfG93A mice is significantly reduced by 90 d , a significant protection was observed at 120 d in SOD1G93A mice treated with SB-239063 in lumbrical muscles. These muscles innervate the digits of the hindlimbs and are one of the most distally affected muscle groups in SOD1G93A mice32. Endplates were scored according to whether they were fully innervated (arrows), partially innervated (arrowheads), or denervated than wild-type mice . By 120 d, 42.8\u2009\u00b1\u20092.8% of endplates were denervated in SOD1G93A mice and late symptomatic (120 d) SOD1G93A mice display subtle deficits in axonal transport. This is likely due to the decrease in ALS-susceptible motor neuron axons observed at late time points of disease progression, and the increase in the contribution of ALS-resistant neurons to overall axonal transport rates. We therefore assessed the effects of long-term treatment with SB-239063 in pre-symptomatic 70 d old SOD1G93A mice, the same age at which we found acute treatment to completely revert transport deficits for 20 d , and spleen and liver toxicity . These results suggest that SOD1G93A causes the sustained activation of the p38 MAPK signalling cascade , and colocalises with phosphorylated neurofilaments in SOD144, a large body of evidence indicates that restoring axonal transport could be a promising therapeutic strategy. For example, pharmacological inhibition or knockout of histone deacetylase 6 (HDAC6) accelerates axonal transport in motor and sensory neurons47, causing an improvement in motor behaviour in a peripheral neuropathy model47, and increasing motor neuron survival and lifespan in SOD1G93A mice48. In addition, the p38 MAPK\u03b1 and \u03b2 inhibitor SB-203580, shown in this study to restore axonal transport, reduces SOD1G93A motor neuron death in vitro13.Although some controversy exists regarding the role of axonal transport deficits in ALSG93A mice and hereditary neuropathies. Henceforth, the identification of novel enhancers of axonal transport could yield novel therapeutic approaches for these presently untreatable pathologies. Interestingly, the p38 MAPK\u03b1 selective inhibitor MW01-18-150SRM has been found to attenuate disease progression in two AD mouse models53, suggesting that pathological mechanisms may be shared among distinct neurodegenerative diseases.Deficits in axonal transport have been shown to be a hallmark of other neurodegenerative diseasesG93A (TgN[SOD1-G93A]1Gur) gene were used in these experiments. Colonies were maintained by breeding male heterozygous carriers with female (C57BL/6\u2009\u00d7\u2009SJL) F1 hybrids. Mice were genotyped for the human SOD1 transgene from ear or tail genomic DNA. Spinal cords were harvested from terminally anaesthetized mice at different stages of disease progression, snap-frozen in liquid nitrogen and stored at \u221280\u2009\u00b0C.All experiments were carried out following the guidelines of the UCL-Institute of Neurology Genetic Manipulation and Ethic Committees and in accordance with the European Community Council Directive of November 24, 1986 (86/609/EEC). Animal experiments were undertaken under license from the UK Home Office in accordance with the Animals (Scientific Procedures) Act 1986 (Amended Regulations 2012) and the GSK Policy on the Care, Welfare and Treatment of Animals. Female transgenic mice carrying a human wild-type SOD1 (B6SJLTg[SOD1]2Gur/J) or mutant SOD1G93A mice at different ages (25 or 70 d) were randomly allocated into vehicle treated and SB-239063 treated groups. A separate group of wild-type mice was also included, which was treated with vehicle. Mice were injected intraperitoneally twice daily with 10\u2009mg/kg SB-239063 until the day of experiment. In each experimental group and assessment, 5\u20138 mice were included.Female mutant SOD117. Primary motor neurons were isolated from spinal cords of E12.5\u201313.5 mouse embryos54. Briefly, embryos were sacrificed, the spinal cords dissected and the ventral horns isolated and dissociated by incubation with trypsin, followed by incubation with DNase and centrifugation through a BSA cushion. The resulting cell pellet was resuspended in complete motor neuron medium . Cells were immediately plated on poly-DL-ornithine/laminin coated plates and maintained in culture for 5\u20138 d (5\u20138 DIV).ES cell-derived motor neurons were cultured as previously describedCT and \u03b1-p75NTR in the cell body of motor neurons was performed as previously described17, except that the HCT and \u03b1-p75NTR were applied in complete medium. Internalised probes were quantified using the Cell Profiler software18. For drug treatments, compounds were dissolved in dimethylsulphoxide (DMSO) and were applied at the same time as the HCT and \u03b1-p75NTR.The accumulation of AlexaFluor 555-conjugated HCT and \u03b1-p75NTR were performed as previously described55 and quantified using Motion Analysis software . Compounds were applied at the same time as HCT or \u03b1-p75NTR, and the speed distribution profiles of their carriers have been obtained using a 0.2 \u03bcm/s binning interval3.Axonal retrograde transport assays with AlexaFluor 647-conjugated HNTR used in this study has been previously described5.Chemicals were from Sigma-Aldrich, unless otherwise stated. SB-239272, SB-239063 and SB-203580 were from GlaxoSmithKline; ciliobrevin A was from InterBioScreen . Tissue culture media and supplements were purchased from Life Technologies. Antibodies for phospho-p38 MAPK , p38 MAPK\u03b1 (#9211), p38 MAPK\u03b3 (#2307), p38 MAPK\u03b4 (#2308), pan-p38 MAPK (#9212) and JNK (#9252) were from Cell Signalling . p38 MAPK\u03b2 (PA1-41154) was from ThermoFisher and phospho-JNK was from Santa Cruz Biotechnology . The GAPDH antibody (mab374) was from Merck Millipore . The mouse monoclonal antibody specific for phosphorylated neurofilament heavy chain was from Biolegend . The antibody for GFP (4E12/8) was a kind gift from the Cancer Research UK London Research Institute monoclonal facility. The polyclonal antibody against the extracellular domain of p75shRNA constructs directed against p38 MAPK\u03b1 (MSH030695-LVRU6GP) and \u03b4 (MSH0306954-LVRU6GP) and scrambled controls were purchased from the OmicsLink\u2122 shRNA clone collection . All constructs were in psi-LVRU6GP plasmids with an eGFP reporter gene. Briefly, Lenti-X 293\u2009T cells were co-transfected with shRNA, packaging, and envelope plasmid vectors using Lipofectamine 3000 (ThermoFisher). Medium containing lentiviral particles was collected every day for 3 d after transfection. Medium containing viral particles was concentrated using LentiX Concentrator (ClonTech) and the lentiviral particles resuspended in complete motor neuron medium. The lentivirus titre was determined using the Lenti-X p24 Rapid Titre kit (ClonTech) and lentiviral particles were stored at \u221280\u2009\u00b0C until further use. To transduce motor neurons, lentivirus was added to the culture medium 6\u2009h after plating. The medium was replaced 16\u2009h after transduction, and motor neurons were assayed one week later.Motor neuron and spinal cord lysates were prepared in RIPA buffer containing Halt\u2122 phosphatase and protease inhibitor cocktail and let to incubate on ice for at least 30\u2009min. Lysates were centrifuged at 14,800\u2009rpm for 15\u2009min at 4\u2009\u00b0C and protein concentration determined using Pierce\u2122 BCA Protein Assay (ThermoFisher). Samples (20\u2009\u00b5g) were run on 4\u201312% NuPAGE Bis-Tris gradient gels (ThermoFisher) and proteins blotted onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore). Membranes were blocked in 5% bovine serum albumin (BSA) dissolved in Tris-buffered saline (TBS) containing 0.5% Tween-20 (TBST) for 1\u2009h at 4\u2009\u00b0C and then incubated with primary antibodies overnight at 4\u2009\u00b0C. Blots were washed and incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies . Immunoreactivity was detected using Crescendo ECL substrates (Merck Millipore) and FujiFilm X-ray film (ThermoFisher). Quantification was performed using ImageJ. For the analysis of neurofilament phosphorylation, motor neurons (7 DIV) were treated for either 2\u2009h or 24\u2009h with 2\u2009\u00b5M SB-239063 or vehicle control (DMSO). Five micrograms of protein lysates were run on a 7.5% acrylamide gels, blotted onto PVDF membranes and then stained with a mouse monoclonal specific for phosphorylated NFH\u00a0(SMI34).Motor neurons were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 15\u2009min at room temperature, permeabilized with 0.1% Triton X-100 in 5% BSA in PBS for 60\u2009min and then incubated with the relevant primary antibodies in blocking solution (5% BSA in PBS) overnight at 4\u2009\u00b0C. Motor neurons were washed in blocking solution and then incubated with the appropriate secondary antibodies (1:400 in blocking solution). Cells were washed in PBS, mounted in Mowiol 4\u201388 and imaged using a Zeiss LSM 780 confocal microscope equipped with a 63\u2009\u00d7\u2009oil-immersion objective .31. Briefly, mice were anesthetized with isoflurane , and AlexaFluor 555-conjugated HCT (13\u2009\u03bcg) and BDNF (50\u2009ng) were injected intramuscularly into the exposed TA and gastrocnemius (GC) muscles of one hind leg, and the wound sutured. Mice were then left to recover and kept under standard conditions with unlimited food and water supply. Four hours later, animals were re-anesthetized and the sciatic nerve exposed. Mice were placed on a heated stage in an environmental chamber (both kept at 37\u2009\u00b0C) and axonal transport was imaged in the intact sciatic nerve by time-lapse confocal microscopy. Images of axons were acquired every 3\u20134\u2009s with an inverted Zeiss LSM 780 equipped with a 63\u2009\u00d7\u2009oil-immersion objective . SB-239063 was suspended in a solution of 1% methylcellulose and delivered by intraperitoneal injection at the same time as the AlexaFluor555-conjugated HCT was injected intramuscularly.In vivo axonal transport assays were performed as previously described56.Experimental animals were anesthetised using isoflurane on the day of assessment. The distal tendon of the TA and EDL muscles was dissected and connected to a force transducer, and the exposed sciatic nerve was attached to a stimulating electrode. Maximum tetanic force as well as the number of functional motor units were assessed as previously described57. Endplate occupancy was scored according to the level of co-localisation between the endplate and terminal motor axon (combined neurofilament and SV2 labelling). Endplates were classified into three categories: fully innervated, partially innervated or denervated, according to the overlap of endplate labelling with the neurofilament/SV2 staining. For each animal a total of minimum 100 endplates were analysed from different regions of the muscle to include endplates innervated by multiple terminal motor axons. Innervation was then expressed as a percentage of total number of endplates assessed.At the end of the axonal transport experiments, animals were euthanized and the lumbrical muscles were removed from the hind paws, fixed in 4 % paraformaldehyde for 5\u2009min and then stained with a combination of antibodies against neurofilament (2HG3) and SV2 as well as with AlexaFluor 568-labelled bungarotoxin n\u2009=\u20092 groups to compare) or one-way/two-way analysis of variance (ANOVA) (n\u2009>\u20092 groups to compare), followed by Dunnett\u2019s or Sidak\u2019s multiple comparisons test, was used. Muscle physiology data were analysed using a two-way ANOVA with Tukey\u2019s multiple comparisons test. If the data were not found to be normally distributed, a Kruskal\u2013Wallis test was used, followed by Dunn\u2019s multiple comparison test (n\u2009>\u20092 groups to compare). If there were too few data points to accurately test for normality, data were assumed to be normally distributed. The test used and associated p values are indicated in the figure legends.Statistical analysis was performed using Graphpad Prism software . Unless otherwise stated, data is expressed as mean\u2009\u00b1\u2009SEM. To determine the most appropriate statistical test to use, data were tested for normality using three tests: D\u2019Agostino & Person omnibus normality test, Shapiro-Wilk normality test and KS normality test. If the data were found to be normally distributed, either a Student\u2019s t-test (Supplemental Material"} +{"text": "SOD1, TARDBP (TDP-43), FUS and C9orf72. Functional analyses of these genes and their pathogenic mutations have provided great insights into the underlying disease mechanisms. Defective axonal transport is hypothesized to be a key factor in the selective vulnerability of motor nerves due to their extraordinary length and evidence that ALS occurs as a distal axonopathy. Axonal transport is seen as an early pathogenic event that precedes cell loss and clinical symptoms and so represents an upstream mechanism for therapeutic targeting. Studies have begun to describe the impact of a few pathogenic mutations on axonal transport but a broad survey across a range of models and cargos is warranted. Here, we assessed the axonal transport of different cargos in multiple Drosophila models of ALS. We found that axonal transport defects are common across all models tested, although they often showed a differential effect between mitochondria and vesicle cargos. Motor deficits were also common across the models and generally worsened with age, though surprisingly there was not a clear correlation between the severity of axonal transport defects and motor ability. These results further support defects in axonal transport as a common factor in models of ALS that may contribute to the pathogenic process.Amyotrophic lateral sclerosis (ALS) is characterized by the degeneration of motor neurons resulting in a catastrophic loss of motor function. Current therapies are severely limited owing to a poor mechanistic understanding of the pathobiology. Mutations in a large number of genes have now been linked to ALS, including SOD1, TARDBP (TDP-43), FUS and C9orf72. Functional analyses of these genes and pathogenic mutations have provided great insight into the underlying disease mechanisms is a typically adult onset progressive neurodegenerative disorder and the most common form of motor neuron disease. It is characterized by the loss of both the upper and lower motor neurons representing a catastrophic loss of motor function. The condition is fatal, usually due to respiratory failure, with an average life expectancy of 3\u20135 years from diagnosis . There iTDP-43 is a multi-functional DNA/RNA-binding protein that shuttles between the nucleus and cytoplasm . In the C9orf72 is the most common genetic cause of ALS , FUS and C9orf72. We also analyzed the effect of loss-of-function mutants of the Drosophila orthologs of TDP-43 and FUS, TBPH and caz, respectively. The motor activities of larvae and adults in these models were assessed to correlate potential defects in axonal transport with locomotor deficits. We found that axonal transport defects are common across all models tested, although they often showed a differential effect between mitochondria and vesicle cargos. Motor deficits were also common across the models, though did not always show a clear correlation between the severity of axonal transport defects and motor ability.Here, we analyzed fast axonal transport in larval motor neurons of TDP-43 cause ALS in an autosomal dominant manner and likely impact on neuronal function in a multitude of ways. We first analyzed whether ectopic expression of a pathogenic variant of TDP-43, M337V, caused any disruption of axonal transport in Drosophila motor nerves in comparison to WTTDP-43. To minimize the likelihood of artifacts from different expression levels or from disruption of genomic insertion, we made use of transgenes generated in the same integration site and expressing at equivalent levels . Somewhat surprisingly no significant effect on mitochondrial transport was observed for any of the transgenes . Ectopic expression of WTFUSor a pathogenic variant P525LFUS caused no disruption to mitochondrial transport , animals needed to be raised at 29 \u00b0C, increasing transgene expression levels, to see a complete rescue of the mitochondrial transport. Interestingly, the re-expression of the pathogenic variant, P398Lcaz, had differential rescuing effects. Although it was able to rescue the mitochondrial transport defect, it was unable to rescue the disruption in vesicle transport. Similarly, expression of WTFUS was able to rescue the 1caz mutant transport deficit of mitochondria and vesicles repeats. Transgenic expression of a non-pathogenic repeat length (G4C2-3) had no effect on mitochondrial transport; however, expression of 36 repeats (G4C2-36), previously shown to cause neurotoxicity , and the locomotor capacity of larvae and young adults was modestly affected only by PR-36 C and D. Drosophila models of ALS, we examined the transport of two different cargos and correlated any transport deficiencies with motor ability. Overall, we found that axonal transport deficits are a common feature in all the models analyzed here, but the severity and cargo selectivity differ substantially between models. Moreover, the severity of motor deficits does not always mirror the severity of axonal transport defects.In this study we sought to determine whether disruption of axonal transport is a common feature of ALS pathogenesis, which may underlie the disruption of neuromuscular function and loss of motor ability. The neuromuscular junction is highly energetically demanding and accordingly is dependent on correct distribution and function of mitochondria . Thus, dTDP-43/TBPH exert differential effects on axonal transport. The overexpression of TDP-43 variants did not affect mitochondrial axonal transport but impaired vesicle transport, whereas loss of TBPH caused a decrease in the transport of mitochondria but vesicle transport was unaffected. The relatively modest deficit in vesicle transport in the overexpression models correlated with modest locomotor deficits, whereas the loss of mitochondrial transport in TBPH mutants correlated with severe motor impairment.Gain and loss of FUS/caz affected axonal transport, though the effects were generally more widespread. Although the overexpression of caz variants affected both mitochondria and vesicle transport, overexpression of FUS variants only perturbed vesicle transport. Loss of caz substantially affected both vesicle and mitochondrial transport. The effects on motor ability were similar to TDP-43/TBPH; although the caz mutants exhibit severe developmental and locomotor defects, the overexpression models have only modest effects on adult locomotion that become more pronounced with age.Both gain and loss of function of C9orf72 models. The expanded pure G4C2-36 repeats caused a disruption of mitochondrial transport; however, the effect on the transport of vesicles is more complicated as the G4C2-3 repeats also slightly inhibit vesicles transport. Nevertheless, results from the RO-36 and PR-36 transgenes further support the view that toxicity of these expanded repeats is caused by DPR production and not \u2018RNA toxicity\u2019 as the RO-36 transgene appeared inert in all assays whereas the PR-36 transgene was highly toxic, substantially inhibiting mitochondrial and vesicle transport. Interestingly, expression of this transgene caused only modest motor deficits.Axonal transport was also disrupted in the Thus, our data indicate that axonal transport deficits are a common feature in all the models analyzed here. Disruption of axonal transport has also been noted in several vertebrate models of ALS, most notably in models of SOD1 mutations and TDP-43 models. We chose to not investigate the impact of SOD1 mutations as they have been fairly well characterized in vertebrates, and have been found to affect neurofilaments ,32, mitoDrosophila models of ALS, axonal transport deficits are by no means specific to ALS because they have also been observed in other disease models. Axonal transport has been shown to be disrupted in models of Alzheimer\u2019s disease (AD), polyglutamine (polyQ) diseases such as Huntington\u2019s disease, peripheral neuropathies and Parkinson\u2019s disease (PD). It is notable that the proteins in question in these disease models are all functionally quite different from each other and from the ALS-related disease factors studied here, indicating that diverse mechanisms can disrupt axonal transport. The models of AD implicate Tau-linked disruption of microtubules or dysregulation of kinases and phosphatases to impair transport of axonal transport defects but emerging evidence strongly implicates RNA dysregulation in TDP-43, FUS and G4C2-repeats mechanisms. TDP-43 and FUS function as part of ribonucleoprotein complexes in the regulation of RNA metabolism, and dysregulation leads to widespread effects on transcription and RNA splicing and transport. A growing consensus proposes that these RBPs sequester mRNAs in stress granules . Recent TDP-43 and FUS result in neurodegenerative phenotypes by which are not . Further are not . Thus, tDrosophila were raised under standard conditions at 25 \u00b0C on food consisting of agar, cornmeal, molasses and yeast unless otherwise stated. The UAS transgenes for TBPH, TDP-43, caz and FUS, which are all inserted in the attP2 landing site, as well as the 1caz mutant were a gift from Brian McCabe (Columbia University) (1TBPH line was a gift from Aaron Voigt (RWTH Aachen University) (UAS-NPY.GFP was a gift from Iain Robinson (Plymouth University) .versity) . The TBPversity) and the Trieste) . All C9o London) . UAS-NPYversity) . UAS-lacm NaCl, 1 mm EGTA, 4 mm MgCl2, 2 mm KCl, 5 mm HEPES and 36 mm sucrose, adjusted to pH 7.2. Larvae were cut along the dorsal midline using micro-dissection scissors, the sides pinned back and the internal organs removed. Movies were taken using an Olympus FV1000 fluoview confocal microscope with a 60\u00d7 water immersion lens (NA 0.90 Olympus LUMPFL). Images were captured at a rate of 1 frame per 5 s for 100 frames for mitochondria, or 1 frame per 0.5 s for 100 frames for vesicles. CCAP-GAL4 was used as it expresses in a very sparse population of cells which secrete the neuropeptide CCAP (Crustacean Cardio-Active Peptide). These neurons send out a single axon in the segmental nerve thus facilitating precise imaging of transport in an individual axon.Analysis of axonal transport was performed live on third instar larvae as previously described ,59. BrieMovies were processed and analyzed using the ImageJ software as previously described to produce kymographs . These wPupae were placed in groups of 25 to a vial and the proportion of adult flies that emerged was assessed. Animals from at least three replicate crosses were analyzed.Control and experimental crosses were established at 25 \u00b0C and transferred to fresh vials every 2 days. Vials containing wandering third instar larvae were coded by an independent researcher, placed at 23 \u00b0C and left to acclimatize for 2 h. Individual larvae were taken and rinsed in distilled water to remove any residual food, placed on a 2% agarose plate under a viewing microscope and left to acclimatize for 5 s. The number of peristaltic movements in 2 min was counted by direct observation. Animals from at least three replicate crosses were analyzed.Male flies between 0 and 3 days post-eclosion were placed at 23 \u00b0C to acclimatize for 1 h. The flies were then transferred to the climbing tubes at a maximum number of 25 per tube for a further 1 h. Flies were introduced into a negative geotaxis counter-current apparatus and given 10 s to climb at each position for total of five attempts. A climbing index score was generated based on the number of flies at each of the positions. The score for each genotype within an experiment was normalized to the control. Animals from at least three replicate crosses were analyzed. For the longitudinal study of the effects of age-related neurodegeneration on climbing ability, male flies were aged in cohorts and the climbing assay was performed at set time points as indicated. Between assays, the flies were transferred every 2 days into fresh vials with no more than 25 flies per vial.Calculations were performed using GraphPad Prism 6.0. Adult climbing analysis is not normally distributed so the data were analyzed using Kruskal\u2013Wallis non-parametric test with Dunn\u2019s correction for multiple comparisons. All other quantifications passed the D\u2019Agostino & Pearson omnibus normality test. For axonal transport, eclosion and larval crawling significance was determined by one-way analysis of variance (ANOVA) with the Sidak multiple comparison test (being more powerful than Bonferroni\u2019s correction)."} +{"text": "In communities with high social cohesion, older men who perceived that their communities\u2019 social cohesion was high showed greater functional ability improvement than men who perceived it to be low (pinteraction = 0.02). Community social capital can thus affect functional ability improvements variously, depending on individual psychosocial characteristics and gender. Community interventions aiming to foster social capital should focus on people who are excluded from existing opportunities to participate.We investigated the contextual effects of community social capital on functional ability among older people with functional disability in Japan, and the cross-level interaction effects between community social capital and individual psychosocial characteristics. We used data from the Japan Gerontological Evaluation Study for 1936 men and 2207 women nested within 320 communities and followed for 46 months. We used objective data for functional ability trajectories derived from the national long-term care-insurance system, and a validated measure of health-related community social capital comprising three components: civic participation, social cohesion, and reciprocity. A multilevel survival analysis with a community-level random intercept showed that in communities with high civic participation, women who actively participated in any community group showed greater functional ability improvement than did women who did not participate ( Populations are ageing rapidly worldwide, and over a third of older people in high-income countries have a functional disability . In 2016The World Health Organization and the Japanese government have stressed that to achieve greater improvement in functional ability, enriching local resources that facilitate older adults\u2019 participation in communities is essential ,6. SpeciA number of studies have suggested that community social capital is associated with good perceived health ,10, low There may be cross-level interactions between community social capital and individual psychosocial characteristics. That is, the effects of community social capital may vary depending on the characteristics of subpopulations, making it beneficial for some and harmful for others. In particular, caution is needed when conducting community-empowering interventions, given the potential unfavorable side of social capital: bonding social capital can induce the exclusion of outsiders, excessive demands on community members, restrictions on individual freedoms, and downward-leveling norms . AlthougFurthermore, the association between social capital and health may differ by cultural context. Many studies so far have been conducted in Western countries, but evidence from other parts of the world, including Asia, is scarce. For example, a large-scale study in China revealed that higher community-level civic participation was associated with poor mental health in urban areas . As one Thus, this study examined, by measuring three components, the effect of community social capital on improvements in the functional ability of older people with disabilities, using longitudinal large-scale cohort data linked to a national long-term care system database in Japan. To perform this, we used a validated instrument with multiple indicators that was designed to measure the health-related community social capital (HR-CSC) of older adults at the school district level . Specifin = 85), and missing data for residence (n = 6). Consequently, the final analysis included 1936 men and 2207 women living in 320 communities. To assess community social capital, we used the data of 93,983 people living in 530 communities (school districts), excluding those with missing data for the area of residence (n = 375). The study protocol was approved by the Ethics Committee for Research on Human Subjects at Nihon Fukushi University, Japan (No. 10\u201305), and the Ethics Committee for Medical Research at the University of Tokyo (No. 10555).For this study, we used data from the Japan Gerontological Evaluation Study (JAGES) program and Japan\u2019s Long-term Care Insurance (LTCI) database. The JAGES program was designed to investigate the social determinants of the health of older adults. The study participants were Japanese people aged 65 or older without functional impairment (which was defined as not being certified by the public LTCI system as using care services) at baseline . In thisLevels of disability were objectively assessed at the time of certification for the utilization of Long-term Care (LTC) services, based on nationally standardized criteria 25,26].,26.25,26There are seven levels of disability: Requiring Support-1 and -2 and Requiring LTC-l to LTC-5 . These measurements have frequently been used in previous studies exploring the predictors of functional disability and mortality ,27. The We assessed three components of community social capital using the HR-CSC instrument . SpecifiThe developers of the HR-CSC defined three individual characteristics of psychosocial conditions or social relationships that are closely related to the components of community social capital: (1) participation in community groups, (2) perception of community social cohesion, and (3) social support . ParticiOther covariates included age at initial LTC certification, income , education (\u2264nine or >nine years), marital status, living status, and comorbidities ,29. Missn = 1936 men; n = 2207 women) and the community level (n = 320). We first estimated empty models and then included the individual and community predictors (Model 1). Then, we further included cross-level individual-community interaction (Model 2). Individual psychosocial characteristics were treated as dichotomous variables (0 vs. 1 or more) because these can explicitly model meaningful conditions. We also conducted a sensitivity analysis using continuous values. To avoid multicollinearity, individual psychosocial characteristics were centered around the group mean [To investigate the individual and contextual community characteristics and their cross-level interactions with respect to improvement in functional ability, we used multilevel Weibull survival models, including a community-level random intercept. We conducted analyses separately for community civic participation, social cohesion, and reciprocity. Additionally, we analyzed the data by considering its structure at two levels: the individual level (ty) mean . Stata 1ty) mean .The average follow-up period was 315 days . Among the participants, 17.8% of the men and 21.1% of the women were found to have improved their functional ability at a follow-up . For botp for interaction = 0.70) (p for interaction = 0.05), while the main effect was not : 0.93, 95% confidence interval (CI): 0.78\u20131.12) nor the cross-level interaction effect between community civic participation and individual group participation in the community were observed with regard to improvements in functional ability . For wom75\u20131.04) . Among w75\u20131.04) .p for interaction = 0.02) ; however, the cross-level interaction with individual perception of community social cohesion was observed . Among m = 0.02) .p for interaction = 0.43) was observed nor the cross-level interaction effect (HR: 1.15, 95%CI: 0.81\u20131.63, observed .We did not find either the main effect of community reciprocity or cross-level interactions between community reciprocity and individual social support for men or womenA sensitivity analysis with continuous values of individual psychosocial characteristics supported these results .The results of our multilevel longitudinal study showed diverse patterns\u2014based on individual psychosocial characteristics and gender\u2014in the association between community social capital and improvements in functional ability. The community characteristics that facilitate civic participation may help women who participate in group activities to improve their functional ability. However, for women who do not participate in any group activities, such community characteristics may reduce the possibility of improvement in their functional ability. Similarly, in communities with high social cohesion, men who perceive their communities to be cohesive are more likely to improve their functional ability, whereas, for men who perceive their communities not to be cohesive, the cohesive community characteristics may reduce the possibility of improvement in their functional ability.These results accord with the findings of a recent study conducted in 22 European countries, which found that when a nation has a high overall participation rate, lower self-rated health is only associated with individuals who do not participate in any types of clubs or associations . FurtherOn the other hand, the potential adverse effects of community social capital on socially inactive women may reflect the drawbacks of social capital, as suggested by Portes: groups with rich, bonding social capital may exclude others outside the group . Older wCommunity social cohesion was determined to be inversely associated with improvements in the functional ability of men who did not perceive their residential communities to be cohesive. This result conforms with those of recent studies conducted in the USA and Europe, which found that in communities with high mutual trust, those who did not trust others showed lower self-rated health than those who trusted others ,19,20. TCommunity reciprocity was determined not to be associated with improvements in functional ability, regardless of individual characteristics, including the reception/provision of social support. Almost 90% of the men and women reported that they received or provided social support, and the level of community reciprocity was measured by aggregating the responses to those questions for each community. This means that the measurement of community reciprocity applied in the current study was not suitable for detecting statistical differences in improvements in functional ability across levels of community reciprocity.There are several limitations to the current study. First, there might be self-selection bias regarding where the respondents lived . Second,Despite the above limitations, the current study has important implications for health policy. Community social capital can have differing impacts on the improvements in functional ability experienced by older people living in the same community, depending on individual psychosocial characteristics and gender. Community-empowering policies and actions aiming to strengthen community social capital might have negative impacts for specific vulnerable or socially isolated populations, such as those who do not participate in any group in a community. Given the findings of this study, when implementing those policies, a better understanding and pre- and post-intervention assessments involving multiple stakeholders and subpopulations are critical ,42."} +{"text": "This study aimed to examine the contextual effects of community-level social capital on the onset of depressive symptoms using a longitudinal study design.n = 7,424). Using sex-stratified multilevel logistic regression, we investigated the lagged associations between three scales of baseline community social capital and the development of depressive symptoms.We used questionnaire data from the 2010 and 2013 waves of the Japan Gerontological Evaluation Study that included 14,465 men and 14,600 women aged over 65 years from 295 communities. We also used data of a three-wave panel (2006\u20132010\u20132013) to test the robustness of the findings , while no such association was found in relation to the other two scales on social cohesion and reciprocity. This association was attenuated by the adjustment of individual responses to the civic participation component. Individual-level scores corresponding to all three community social capital components were significantly associated with lower risks for depressive symptoms. The results using the three-wave data set showed statistically less clear but similar associations.Promoting environment and services enhancing to community group participation might help mitigate the impact of late-life depression in an aging society. At a community level, Ehsan and De Silva concluded that higher cognitive social capital was associated with lower risk of CMD in seven cross-sectional studies, but that structural social capital was not associated with CMD.6Mental disorders, including depression, have been linked with social capital.8,9 For example, communities rich in social capital can influence political decisions to make healthcare services readily available in their communities .8,9 Health-related information or positive feelings are known to be transmitted more rapidly in communities with high social capital .9 In addition, living in communities with higher neighborhood safety as a result of informal social control may reduce psychological stress.9Several pathways have been proposed to explain the mechanism linking community social capital and depressive symptoms, including collective efficacy, informal social control, and social influence.10\u201312 This sense of belonging may improve people\u2019s ability to cope with stressors, and thus may have protective effects against the onset or progression of depressive symptoms. Social support also helps people cope with stressors (related to community reciprocity).13 Increased physical activity owing to civic participation has also been shown have such protective effects.14,15These potential contextual effects may directly affect health regardless of individual behaviors, but they may also enhance individual-level pathways. For instance, sense of belonging may improve by participating in community organizations (related to community civic participation) and living in communities whose residents are trustworthy .16,17 few studies have investigated the association between social capital and mental illnesses using a longitudinal design while accounting for both community-level social capital variables and individual-level responses.18 It is essential to include both community- and individual-level variables in a model to evaluate the contextual effects of community social capital .11,19 Second, we examined the effect of community-level social capital on depressive symptoms of the older population in Japan, a country experiencing rapid population aging. Given that depression is a mental disorder common in older people20 and that late-life depression is linked with a variety of negative health outcomes,21 it has become important to identify factors that help mitigate the impact of depression in aging societies. Third, we used study-specific social capital measures that have been validated in relation to depressive symptoms and self-rated health in the current study population of older people living in Japan.22 While these scales were originally developed to measure community-level social capital, we also used individual responses as indicators of individual-level social capital in this study, as they were deemed to reflect individual-level social capital in terms of neighborhood organization participation, individual perception of community cohesion, and availability of mutual support.23,24The present study was designed to extend the findings of previous studies on the association between social capital and depression. First, despite the existence of some cross-sectional studies,Thus, the present study aimed to investigate the contextual effects of community-level social capital on the onset of depression using longitudinal panel data from the Japan Gerontological Evaluation Study (JAGES).25,26 For the present study, we used longitudinal data from the 2010 and 2013 waves; in the 2010 wave, data were collected from 112,123 residents (response rate: 66.3%) aged 65 years or older who did not receive long-term care and resided in 533 school districts in 31 municipalities. The follow-up survey was conducted among the residents who participated in the 2010 wave, and 62,438 participants responded to the 2013 wave. We excluded 555 participants with self-reported difficulties in activities of daily living at the 2010 wave. After excluding those who lived in school districts in which fewer than 50 residents responded ,22 those with missing information on self-reported depressive symptoms , and those who reported to have depression/depressive symptoms in the 2010 wave score \u22655 [described later] or previous diagnosis of depression) , a total of 29,065 subjects nested within 295 school districts were included in the subsequent analyses.Data was obtained from the JAGES, which is a prospective cohort study that examined the influence of social determinants of health among older people in Japan.We also created an alternative three-wave dataset using the 2006, 2010, and 2013 waves. As the 2006 wave was conducted only in 25 districts in Chita Peninsula, Aichi Prefecture, the sample size was 7,424 . We excluded those who had depression/depressive symptoms at an earlier wave to investigate the association at the subsequent waves.The JAGES protocol and informed consent procedure were approved by the Ethics Committee for Research of Human Subjects at Nihon Fukushi University (no. 10-05 and no. 13-14) and the Ethics Committee for Medical Research at the University of Tokyo (no. 10555).22 The scale comprised three dimensions: civic participation (five questions), social cohesion (three questions), and reciprocity (three questions). Civic participation refers to the level of residents\u2019 participation in community organizations and activities.27,28 Social cohesion pertains to the cognitive aspects of interpersonal trust, reciprocity, and attachment to the community.28,29 It was assessed using the questions, \u201cGenerally speaking, would you say that most people can be trusted?\u201d, \u201cDo you think that it is important to be helpful to other people in your local area?\u201d, and \u201cDo you have an attachment to your local area?\u201d. Responses options were presented as a Likert scale . Respondents who chose \u201cstrongly or moderately agree/yes\u201d were coded as 1, and were otherwise coded as 0. These scores were then summed to compute a composite score ranging from 0\u20133. In our three-wave analysis, as information on attachment to the community was not available in the 2006 wave, we calculated the score of social cohesion based on scores on community trust and community contribution (score range: 0\u20132). Reciprocity represents the community characteristics of exchanging support,3 and the following three questions were used in the scale: \u201cDo you have someone who listens to your concerns and complaints?\u201d, \u201cDo you listen to someone\u2019s concerns and complaints?\u201d, and \u201cDo you have someone who looks after you when you are sick and confined to bed for a few days?\u201d. Respondents who chose \u201cstrongly or moderately agree/yes\u201d were coded as 1 and were otherwise coded as 0 to compute a composite score ranging from 0\u20133.We used the study-specific health-related community social capital indicators validated in the JAGES population.22 and other previous literature,11 we aggregated individual-level scores on the three types of social capital to obtain community-level scores by school district (n = 295). Specifically, the average scores of individual-level responses in relation to civic participation, social cohesion, and reciprocity were calculated for each school district. School district is the smallest area unit available in the JAGES. Historically, school districts are likely to represent the unit of former \u201cvillages\u201d before repeated municipality mergers took place in the last few decades in Japan.30 For comparison between the waves, we standardized the community-level and individual-level scores to a mean of 0 and standard deviation (SD) of 1.Following Saito et al22 those who participated in these organizations once a month or more were categorized as having a high level of participation. This civic participation score ranged from 0 to 3. By conducting confirmatory factor analyses using data of the 2013 wave, limited to the questions we used in the present study, we investigated that our social capital measures, lacking two questions in our sample for the two-wave analysis, and an additional question for the three-wave analysis, had factor components similar to those of their original versions provided by Saito et al.22Since the 2006 and 2010 waves lacked data on two questions in relation to for civic participation , the present study used three out of five of the original questions. Consequently, civic participation in the 2006 and 2010 waves was measured by asking if the respondents participated in volunteer groups, hobby groups, and sports groups. Following a previous study,31\u201333 Participants were judged to have depressive symptoms when they had a score of 5 points or above30,31 or when they reported a previous diagnosis of depression. The outcome was defined as the onset of depressive symptom between the baseline (2010) wave and the follow-up (2013) wave.To evaluate depressive symptoms, we used the short form of the GDS, comprising 15 questions .34 If participants had missing information for any of the covariates, they were assigned to a \u201cmissing\u201d category and included in the analysis.As potential confounders, we adjusted for the following covariates in our analysis, measured at the 2010 wave: age , family structure , marital status , equivalent household income , current employment , educational attainment , and comorbidity . We used these four diseases following a previous study on social capital and depression.35\u201337 We modeled the onset of depressive symptoms (GDS \u22655) between 2010 and 2013, incorporating one of the three community-level social capital components at baseline. The models were as follows:i in school district j having depressive symptoms at 2013 was modeled by individual responses to social capital components (ISC) and community-level social capital scores (CSC) at baseline (2010), controlling for their covariates (C) and random intercepts for school districts (ib).To investigate the association between community-level social capital and the onset of depressive symptoms, we used a sex-stratified multilevel logistic regression. After confirming the presence of suggestive interaction between sex and social capital , we straFor the three-wave analysis, we included those who participated in the study at least twice . The models were modified as follows:ij,2006, CSCj,2006, and ijC,2006), assuming that their variables were stable over the 2006\u20132010 period. We first tested a null model to evaluate the community level variability in the onset of depressive symptoms. Next, in model 1, we examined the association between community-level social capital and the onset of depressive symptoms separately, while adjusting for possible covariates at baseline. Then, in model 2, we modeled the community-level social capital scores by further adjusting for ISC scores. We provide the adjusted odds ratios (AORs) and their 95% confidence intervals (CIs). Based on the random-effects variance, we calculated the median odds ratio (MOR) to examine the unexplained heterogeneity between communities. If there was a strong difference between school districts, the MOR would be further away from 1.38 All statistical analyses were performed using Stata 14.0 . The level of statistical significance was set at P < 0.05 (two-tailed).That is, we modeled the discrete hazards of the onset of depressive symptoms by social capital measured in an earlier wave with time-invariant effects and time-specific intercepts. For those who participated at the 2006 and 2013 waves, the predictor variables were substituted by those measured at 2006 for men and 72.4 years for women. Most of the participants lived with their family and were likely to be married, retired, and have a middle socio-economic status according to the household income and years of schooling in men and 2,298 (15.7%) in women . The null model of multilevel logistic regression model and 798 women (21.9%) experiencing the onset of depressive symptoms. Discrete-hazard analyses using the data set showed statistically less clear but similar associations .This prospective longitudinal study found that community-level civic participation was inversely associated with the onset of depressive symptoms among older people in Japan. No such association was found in relation to community-level social cohesion and reciprocity. On the other hand, all individual-level responses to community-level social capital components were inversely associated with the onset of depressive symptoms; a one-SD increase in each of the social capital scores was linked to 12\u201324% lower odds for the onset of depressive symptoms. When we modeled community- and individual-level scores simultaneously, the statistical significance of the association between community-level civic participation and depressive symptoms onset was attenuated.18 and they found that neither an increase in neighborhood-level nor in individual-level social cohesion was significantly associated with a change in depression. The inverse association between community-level social cohesion and depression reported by Mair et al was not adjusted for individual-level social cohesion.39 The discrepancy between the current findings and those of these recent studies might have resulted from measurement issues. Using the community-level social capital measures utilized in the present study, Saito et al reported that civic participation is more closely linked with positive health outcomes than with community-level social cohesion, probably because community-level social cohesion is measured more objectively, and that it might have less measurement bias that is caused by the absence of those who do not trust others in the community.22 An alternative explanation may the variations caused by the country in which the study was conducted.Moore et al simultaneously investigated the effects of individual- and neighborhood-level social cohesion on depression among adults aged 45\u201384 years in the United States,40\u201342 For example, an 18-year longitudinal survey in Taiwan conducted by Chiao et al revealed that social participation was associated with fewer depressive symptoms in an older population.40 Croezen et al reported that, among the population aged 50 years or older in 10 European countries, participation in religious organizations was associated with a decline in depressive symptoms, while participation in political/community organizations was linked with an increase in depressive symptoms, highlighting the differences in the direction of the association by the type of social activity.41 Park et al also reported different effects of the type of activity; participating in leisure activities was associated with fewer depressive symptoms, while participating in volunteer activities was not.42The findings observed in this study in relation to individual group participation were consistent with those reported in previous studies.3,43The non-significant association between community-level social capital and depressive symptoms after adjusting for individual-level social capital suggests that individual participation mediated the association between community-level social capital and the onset of depressive symptoms. This finding is in line with a recent intervention that reported that a municipality project on strategically building community social gathering opportunities increased individuals\u2019 group participation, resulting in better health outcomes.42,44 and the general population.42,44\u201346 For example, Park et al conducted a longitudinal study (2006\u20132013) among older women in South Korea and reported that interpersonal trust and reciprocity were both associated with fewer depressive symptoms.42 In their cross-sectional study, Forsman et al reported that trust in friends and neighbors was associated with less depression among older adults in Sweden and Finland.44 In their prospective study conducted among the United States\u2019 general population, Fujiwara and Kawachi showed that trusting neighbors was inversely associated with the risk of major depression.45 In 2007, Kim et al showed that interpersonal trust in 2006 was inversely associated with developing depression among the general population in South Korea,46 while the significant inverse association observed in relation to reciprocity was attenuated and became non-significant when only healthy people were included in the analysis.Although the present study did not suggest the contextual effects of community-level social cohesion and reciprocity on depressive symptoms, individual responses to them were strongly associated with lower risks for depressive symptoms. Other recent studies have also reported that individual-level social cohesion and reciprocity are associated with fewer depressive symptoms in the older population22 However, our factor analysis identified the same three components as those in their study, suggesting that our measure properly measured the components of community social capital adequately. Fourth, as social capital and depressive symptoms were based on a questionnaire, these responses are subject to common method biases, and those who were depressed might have perceived themselves as having lower social capital irrespective of their actual social capital. Finally, when we created community-level social capital variables, we depended solely on individual responses collected from older individual who participated in the survey. While Saito et al22 confirmed the validity of the scores, excluding younger generations and those who did not participate in the survey when calculating community-level social capital might have resulted in measurement error.There are some limitations to the present study. First, this study was a postal survey and might have been difficult for those with severe depression or other serious health problems to complete. It is possible that our participants were healthier than the general population and they might not represent the older population in Japan adequately. Second, a selection bias might also exist due to attrition: those with lower social capital may have been more likely to drop out in the follow-up surveys. Third, our social capital score lacked a few questions from the original social capital scale used by Saito et al.18,39 while a still larger number of community units might have been necessary to examine the effect of community-level social capital. Second, our finding was robust in that it was also confirmed by the analysis using three-wave data obtained from 25 school districts. Third, we used study-specific social capital scores, which were previously shown to be associated with self-rated health and depression in Japan.Despite these limitations, this study has extended the findings of previous studies on the association between social capital and depressive symptoms in many ways. First, our longitudinal study investigated the association among larger numbers of participants and community units as compared to two previous reports,43This study suggests that the promotion of civic participation might help mitigate the social impact of late-life depressive symptoms. While more research is needed, community programs for older adults, which focus on creating more opportunities for group participation, might increase actual individual participation, resulting in better health."} +{"text": "Food environment policies play a critical role in shaping food choices, diets, and health outcomes. This study endeavored to characterize and evaluate the current food environment policies in Canada using the Healthy Food Environment Policy Index (Food-EPI) to compare policies in place or under development in Canada as of 1 January 2017 to the most promising practices internationally. Evidence of policy implementation from the federal, provincial, and territorial governments was collated and verified by government stakeholders for 47 good practice indicators across 13 policy and infrastructure support domains. Canadian policies were rated by 71 experts from across Canada, and an aggregate score of national and subnational policies was created. Potential policy actions were identified and prioritized. Canadian governments scored \u2018high\u2019 compared to best practices for 3 indicators, \u2018moderate\u2019 for 14 indicators, \u2018low\u2019 for 25 indicators, and \u2018very little or none\u2019 for 4 indicators. Six policy and eight infrastructure support actions were prioritized as the most important and achievable. The Food-EPI identified some progress and considerable gaps in policy implementation in Canada, and highlights a particular need for greater attention to prioritized policies that can help to shift to a health-promoting food environment. It is well established that food choices are heavily influenced by the food environment within which they are made . The fooCanada is no exception to this global trend. Overall diet quality in Canada is poor, with few Canadians meeting recommendations for fruit and vegetable consumption and sodium intake ,7,8. In Governments play an important role in shaping the food environment, as policies have the potential to shift the environment towards one that promotes healthy diets. Globally, many middle and high-income countries are assuming leadership in implementing food environment policies, regulations, and programs at the national, state, and local level . In CanaThis study employed the Healthy Food Environment Policy Index (Food-EPI) tool and process developed by the International Network for Food and Obesity Research, Monitoring and Action Support (INFORMAS) to assess Canadian food environment policies compared to best practice . All parThe Food-EPI tool and process are described in detail in previous publications ,25. BrieGiven the nature of regulatory jurisdiction in Canada, food environment policies and related infrastructure support were evaluated for federal as well as provincial/territorial governments. Out of the 47 good practice indicators under evaluation, 7 were identified as outside of the regulatory authority of provincial and territorial governments in Canada and were only rated at the federal level. The indicator for a systems-based approach with local organizations was erroneously excluded from provincial/territorial ratings as the result of a computer programming error, and was only analyzed at the federal level. One indicator was identified as not relevant at the federal level (support for implementation of nutrition policies in school settings) and was only rated at the provincial/territorial level.http://labbelab.utoronto.ca/food-epi-canada-2017/.Evidence on implementation of food environment policies and actions in Canada was collated up to 1 January 2017. Data were compiled through comprehensive online searches of government websites and databases, policy documents, and non-governmental and advocate websites. The evidence documents were then shared with government stakeholders in relevant ministries to verify that the information collated was correct and comprehensive, to the extent possible, including Health Canada , the Public Health Agency of Canada, Department of Finance, Finances Canada, Global Affairs Canada, and others. The final evidence documents for each of the provinces and territories and the federal government are available at: A panel was convened from the various areas of food and nutrition research, practice, and advocacy in Canada. A total of 111 experts were invited, 75 from academia and 36 from non-governmental or other civil society organizations. The group included academic experts, health and nutrition-related non-governmental organizations, and individuals from other types of organizations with relevant expertise and with no conflict of interest in participating. Potential participants were identified through the authors\u2019 extensive networks, and snowball sampling was conducted among government stakeholders to identify other potential experts to invite to participate in the process. Individuals with government or industry affiliations were purposefully excluded from the sample to avoid conflict of interest in rating policy actions; however, key government stakeholders were invited to be observers and attended all of the rating workshops.n = 2) and Vancouver (n = 1).A two-step process was used to rate federal and provincial/territorial policies in Canada in May and June of 2017. First, each expert was randomized to rate 1 of 12 provinces or territories usinExperts were asked to review the relevant evidence documents for the provincial/territorial jurisdiction to which they were randomized prior to undertaking the online process and for the federal jurisdiction prior to the workshop. The process for online and in-person workshop ratings was similar. For each good practice indicator, the international benchmark examples were presented to identify what types of policies and regulations had been implemented in other jurisdictions. Next, a brief paragraph of context for the Canadian policy environment was provided, and the current evidence of implementation by the federal/provincial/territorial government for the indicator was described in detail. An example of the data that were provided in the online survey is depicted in Participants were asked to rate Canadian federal/provincial/territorial policies in comparison to the international benchmarks that were presented on a 5-point scale . Participants were instructed to rate the level of implementation in Canada compared to the international benchmarks, and not against theoretical or ideal standards. There were instances where the international benchmarks did not cover all aspects of the good practice indicator; however, the process purposefully compared Canadian policies to real-world examples of policy action, despite some examples being limited in scope compared to the policy described in the theoretical good practice statement and some examples of implemented policies being limited in their strength or comprehensiveness.Participants were asked to consider the quality of the policies compared to international best practice and the extent of implementation, bearing in mind the various levels of the policy cycle , and ratings were to be assigned accordingly. For example, policies in the policy development phase would be rated lower than policies in the implementation or evaluation stages. Implementation was considered to include public intentions and plans of the government, as well as government funding for implementation of actions taken by NGOs and working or advisory groups.n = 60) was compiled using the feedback from all three workshops to identify concrete actions that could be recommended to the government to improve the Canadian food environment. Unique provincial and territorial actions were created based on relevant ratings and federal policy discussions. The long list of actions was cross-referenced with proposed policy actions and policy positions from prominent non-governmental organizations and groups that were participating in the Food-EPI Canada process, including Alberta Policy Coalition for Chronic Disease Prevention [After the ratings workshops were conducted, based on implementation gaps identified, a long list of federal policy and infrastructure support actions . Policy actions that were considered more important or achievable scored more points, while lower priority actions scored fewer. Similarly, participants were asked to rate the importance and achievability of the 30 proposed infrastructure support actions, with 150 points for importance and 150 points for achievability. The same exercise was completed for each province and territory for which the expert had been randomized. The total number of points for each province or territory varied by the number of proposed actions. Participants were also given an opportunity to weight the \u2018Importance\u2019 and \u2018Achievability\u2019 criteria, to indicate whether the expert placed greater value on the importance or achievability of a policy. Weighting was initially set at 50%\u201350% for \u2018Importance\u2019 and \u2018Achievability\u2019.A process evaluation was conducted to examine expert evaluations of participation in the Food-EPI process. Participants were asked to rate the comprehensiveness of the Food-EPI tool and the comprehensiveness and completeness of the Federal Evidence document and how easy it was to conduct the ratings in the workshop and the online ratings . Then, participants were asked their level of agreement with the following statements: \u201cMy knowledge of food environments and related food and nutrition policy increased as a result of the project\u201d, \u201cI have increased my knowledge of current best practice/what other governments are doing internationally\u201d, \u201cI made new professional connections or strengthened existing relationships as a result of the project\u201d, \u201cThe project is likely to contribute to beneficial policy change\u201d and \u201cIt is important to repeat the study in order to monitor government progress.\u201d Lastly, participants were asked whether or not they would be willing to participate in the assessment process again if the Food-EPI were to be repeated.Inter-rater reliability was assessed using AgreeStat, estimated as the percentage of agreement between experts using quadratic weights . For the estimation of variance, the sample of subjects was set at 100%, and the sample of raters was set at 64% (response rate for ratings workshops).Any policy indicators for which greater than 20% of participants selected \u2018cannot rate\u2019 were excluded from analysis, as it was assumed that either the policy evidence document or the international benchmark were not well understood by participants. This excluded one indicator (sufficient population nutrition budget) from the ratings, as 51% of participants selected \u2018cannot rate\u2019 due to the lack of public information on funding that is specific to diet-related NCDs, obesity or population nutrition, and a poorly defined international benchmark for this indicator.n = 39). The composite score weighted the federal government and each of the provincial and territorial governments equally . Scores were grouped into four levels of implementation: very little or no implementation (0\u201325%), low implementation (26\u201350%), moderate implementation and high implementation (>75%).Mean ratings were assessed for the federal and provincial/territorial indicators. To assess overall food environment policy implementation incorporating both federal and provincial/territorial governments, a composite score was derived for each indicator that fell within both federal and provincial/territorial jurisdiction were from western provinces, 51% (n = 36) from Ontario, 13% (n = 9) from Quebec, 11% (n = 8) from the Atlantic provinces, and 1% (n = 1) from the territories. Experts came from a variety fields of research and practice, including dietetics, nutrition, public health, health policy, health economics, law, agriculture, sustainability, food security, and epidemiology, among others.A total of 71 experts participated in the ratings, 22 provided feedback on the proposed actions that were considered, including on the province or territory that they rated, any other provinces or territories for which they had policy familiarity and the proposed federal actions, and 74 participated in the prioritization exercise, for an overall response rate of 67%. The number of experts that rated each of the provinces and territories ranged from 5 to 7 experts, and a minimum of 3 experts conducted the prioritization activity for each province and territory. A total of 7 government observers attended the different federal workshops to listen to the expert panel discussion and provide clarity on current policy status, as required. Overall, 62% of the experts were from academia, 32% were from non-governmental organizations, and 6% were from other civil society organizations. There was geographic representation from all areas of Canada: 24% , which is considered relatively good agreement. Simple percent agreement was 89.6% (95% CI 88.9\u201390.3%) between raters across all indicators. The Gwet\u2019s AC2 inter-rater reliability coefficient for provincial and territorial ratings ranged from 0.33 to 0.92, with average inter-rater reliability of 0.64, and 9 of 12 jurisdictions had a coefficient greater than 0.5. In some instances, provinces and territories with similar or identical policies were rated differently, as a result of varied expert interpretation and a small number of experts rating within each province and territory. To address this, manual adjustments were made to two indicators and Public Access to Information (GOVERNANCE4) to ensure that provinces or territories that were clearly meeting the international benchmark were rated as such.Gwet\u2019s ACSee n = 6 for policy actions and n = 8 for infrastructure support actions) were considered highest priority.The full list of prioritized policy action recommendations for the federal government can be found in Prioritized policy and infrastructure support recommendations for each province and territory are listed in detail in summary reports for each province that are publicly available . OverallOverall, participants rated the comprehensiveness of the Food-EPI tool \u2018about right\u2019 , and similarly rated the comprehensiveness of the federal evidence document (3.3 out of 5). Participants found the ratings somewhat challenging, with online ratings being somewhat more challenging (mean = 3.1 for workshop ratings and 3.5 for online ratings). Overall, 84% of participants agreed or strongly agreed that they had increased their knowledge of food environments and related food and nutrition policy, 86% had increased their knowledge of current best practice and international government actions, and 73% had made new connections in the area of food environments. Overall, 77% agreed or strongly agreed that the project was likely to have a policy impact, and 91% agreed that it was important to repeat the same study over time to assess progress.Overall, the results suggest that there are few policy and infrastructure support areas where Canadian governments are reaching international benchmarks for promising practices in food environment policy. The level of implementation varied between domains, and also between national and subnational governmental bodies. Notably, there was one Canadian policy that was an international benchmark, the restrictions on marketing to children in Quebec. Infrastructure support indicators had higher ratings of implementation than policy indicators, particularly for the federal government compared to provincial and territorial governments. The prioritization exercise rated policy and infrastructure supports separately, and did not compare the importance and achievability between these two components; however, the results from the rating exercise suggest that there may be larger gaps in policy implementation in the Canadian setting compared to implementation of infrastructure supports, where ratings were overall higher.The large geographic area and multiple levels of government that bear regulatory jurisdiction over food environment policies in Canada represented a unique challenge in assessing the overall Canadian food policy environment. The Food-EPI process identified challenges with identifying \u2018who\u2019 is responsible for taking action to address some of these areas where significant gaps remain in many or most jurisdictions. The current patchwork of policies across provinces and territories that are implemented in some policy domains represent inherent inequities in exposure to less healthy food environments between Canadians living in different jurisdictions, which are likely contributing to disparities in diet quality and rates of obesity and NCDs between provinces and regions . Given tStrengths of the Food-EPI Canada process include the use of internationally-developed methods created by leading experts in food environment policy that have been implemented in 23 countries to date ,37. WhilThe Food-EPI Canada process supports a call to action for increased and sustained actions to improve food environments in Canada. The results were endorsed by several of the major NGOs involved in the process, including the Canadian Cancer Society, Canadian Nutrition Society, Canadian Obesity Network (now Obesity Canada), Childhood Obesity Foundation, Diabetes Canada, Dietitians of Canada, and several other provincial and territorial organizations. The initial reports publisheSome of the policy and infrastructure support actions that were recommended in this process are captured in the Healthy Eating Strategy and A Food Policy for Canada, including restrictions on marketing to children , and revResearch comparing these results to other countries who have implemented the Food-EPI process using an aggregated, weighted score for policy and infrastructure support actions suggest that Canada performs moderately well, although more policy action is required to achieve the amount of progress demonstrated by some other governments globally . NotablyBringing together experts to systematically assess Canadian food environment policy has the potential to identify areas with broad expert recognition as important contributors to shaping a food environment that is health promoting, and also serves as an opportunity to build capacity among experts working in this field. The current study identified key policy areas and infrastructure support areas that experts project will contribute to healthier food environments at the federal, provincial, and territorial level, some of which have been proposed in Canada and some which have yet to be considered by governments. The results will help to shape priority areas for action by current and future governments in years to come. This study contributes to the efforts of INFORMAS to broadly characterize the global food environment.The Food-EPI Canada process supports a call to action for increased and sustained actions to improve food environments in Canada. The current study identified key policy areas and infrastructure support areas , that experts project will contribute to healthier food environments at the federal, provincial, and territorial level, some of which have been proposed in Canada and some which have yet to be considered by governments. The results will help to shape priority areas for action by current and future governments in years to come. This study contributes to the efforts of INFORMAS to broadly characterize the global food environment."} +{"text": "An accumulation of evidence has revealed the important role of epigenetic factors in explaining the etiopathogenesis of human diseases. Several empirical studies have successfully incorporated methylation data into models for disease prediction. However, it is still a challenge to integrate different types of omics data into prediction models, and the contribution of methylation information to prediction remains to be fully clarified.A stratified drug-response prediction model was built based on an artificial neural network to predict the change in the circulating triglyceride level after fenofibrate intervention. Associated single-nucleotide polymorphisms (SNPs), methylation of selected cytosine-phosphate-guanine (CpG) sites, age, sex, and smoking status, were included as predictors. The model with selected SNPs achieved a mean 5-fold cross-validation prediction error rate of 43.65%. After adding methylation information into the model, the error rate dropped to 41.92%. The combination of significant SNPs, CpG sites, age, sex, and smoking status, achieved the lowest prediction error rate of 41.54%.Compared to using SNP data only, adding methylation data in prediction models slightly improved the error rate; further prediction error reduction is achieved by a combination of genome, methylation genome, and environmental factors. BRCA1 promoter methylation was useful in predicting the response to chemotherapy in epithelial ovarian cancer [Increasing evidence reveals the important role of epigenetic factors in explaining the etiopathogenesis of human diseases, especially in cancer . For exan cancer , and Shin cancer . Diseasen cancer , 5. Consn cancer \u20139. HowevIn this study, a stratified drug-response prediction model is built based on an artificial neural network (ANN) to identify the contribution of methylation information to predicting the change in the circulating triglyceride (TG) level after fenofibrate intervention. Omics data, including genetic, epigenetic, and clinical factors, are used as predictors. The analysis of GAW20 real data demonstrates that the inclusion of the methylation data improves the prediction accuracy marginally, which provides an indication for future prediction research.+ T cells harvested from stored buffy coats and the proportion of sample methylation was quantified at >\u2009450,000 cytosine-phosphate-guanine (CpG) sites [GAW20 real data were used in this study and were provided by the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) study, which aimed to identify the genetic determinants of the responses of circulating lipid levels to fenofibrate treatment interventions. In total, 1053 individuals from families with at least 2 siblings were recruited. They all self-reported as being of white ethnicity [In the quality control process, 39 participant outliers were removed, and only subjects without any missing data for the key variables were used. A total of 523 participants were included in the analysis. For the genotype data, single-nucleotide polymorphisms (SNPs) with a minor allele frequency\u2009<\u20090.01 were excluded. Missing variants were imputed according to the probability distribution of the genotype in all subjects. For the methylation data, cross-reactive probes and probes containing common variants were filtered. Beta-mixture quantile normalization was used to correct for the Infinium Type I/II bias , and parDrug response was used as the dependent variable which could be defined as the percentage change in the TG level.TG pre is the average of TG levels at visits 1 and 2, and TG post is the average of TG levels at visits 3 and 4. It was reported that fenofibrate, which was the intervention drug for the GAW20 real data, usually reduced the plasma TG level by approximately 30 to 60% in hyperlipoproteinemia patients at a dosage of 200\u2013400\u00a0mg daily [Where mg daily . In thisThe features related to drug response were selected in a stratified manner , first wp value threshold of 10\u2212\u20094 was applied to filter the biomarkers for GEE and LMM so that a moderate number of predictors can be used in the prediction model. SNPs were pruned to avoid the strong influence of SNP clusters, by snpgdsLDpruning, and the linkage disequilibrium threshold was set at 0.2 [The generalized estimation equation (GEE) model was used to select significant SNPs and adjust for family relatedness . CpG sitt at 0.2 , 22. ThePredictors were added into the prediction model step-by-step by data types. Afterward, chosen SNPs were inputted into the ANN first, followed by significant CpG sites. Finally, age, sex, and smoking status were included. This stratified method made it easy to identify the respective contribution of each category of information to prediction.a) for the hidden layer, which has the following form:A three-layer ANN was applied with one hidden layer. The hyperbolic tangent sigmoid transfer function was used as the activation function (purelin):A linear function was used as the activation function for the output layer , which implies that the inclusion of methylation information improves the prediction model. When clinical factors were also included, the error rate dropped slightly to 41.54% were included in the ANN model in a stepwise manner to compare their contributions to the prediction ability of the model. The baseline model simulates the null scenario; that is, 100 SNPs were selected from the autosomes at random and used to predict the phenotype with the ANN in 5-group cross-validation. This gave a baseline error rate of 47.15% (SD: 3.79%), representing a random-guess prediction error under the ANN. Next, including the SNP information yielded a mean test prediction error rate of 43.65% (SD: 4.79%). When methylation information was added, the prediction model achieved an error rate of 41.92% and CTNNBL1 have both been strongly associated with obesity risk and related traits [DGAT1 plays a role in catalyzing the committed step in the biosynthesis of TGs [ALDH4A1 is known to catalyze ester hydrolysis, suggesting that it may lead to a change in the TG level [Finally, we report the biological meaning of variables identified using all data. Many of the identified SNP and CpG markers had functions that are related to the regulation of the circulating level of TG, which is a major storage molecule for metabolic energy . To listd traits , 25. Thes of TGs , and ALDTG level .Table 2Sp value\u2009=\u20090.3759). The combination of significant SNPs, CpG sites, age, sex, and smoking status achieved the best prediction error rate of 41.54%.Epigenetic factors are thought to be significantly associated with human diseases, making it plausible to incorporate methylation information for better disease prediction. In this study, we used an ANN to build a stratified drug-response prediction model in which SNPs, methylation, age, sex, and smoking status were considered as predictors. The GAW20 real-data analysis shows that the incorporation of methylation information could reduce the prediction error rate by approximately 4% (+ T cells harvested from stored buffy coats, and the phenotype was the TG level in blood, which has a strong correlation with T-cell functions [In previous studies, Deng et al. used fusing networks to predict schizophrenia from SNPs, methylation, and functional magnetic resonance imaging data . They acunctions . Second,Adding methylation data slightly improved the prediction accuracy for drug response using a neural network based prediction algorithm with GWAS data. The result could be constraint by the source of tissue, the outcome variable and the disorder under study. Further studies in other cohorts are necessary to validate the results."} +{"text": "Glioblastoma (GBM) is an aggressive central nervous system tumor with a poor prognosis. This study was conducted to determine any comorbid medical conditions that are associated with survival in GBM. Data were collected from medical records of all patients who presented to VCU Medical Center with GBM between January 2005 and February 2015. Patients who underwent surgery/biopsy were considered for inclusion. Cox proportional hazards regression modeling was performed to assess the relationship between survival and sex, race, and comorbid medical conditions. 163 patients met inclusion criteria. Comorbidities associated with survival on individual-characteristic analysis included: history of asthma , hypercholesterolemia , and incontinence . History of asthma and hypercholesterolemia were associated with shorter survival on multivariable analysis. Surgical patients with GBM who had a prior history of asthma or hypercholesterolemia had significantly higher relative risk for mortality on individual-characteristic and multivariable analyses. It is the most prevalent glioma (57.3%) and most common primary malignant brain tumor (14.6%) with an annual incidence rate of 3.2 per 100,000 population in the United States2. Survival time from diagnosis to death is short, with the Surveillance, Epidemiology, and End Results (SEER) database showing median overall survival ranging from 12\u201315 months, and the Central Brain Tumor Registry of the United States (CBTRUS) listing a 5-year survival of 6.8%3.Glioblastoma (GBM) is a common and fast-growing central nervous system (CNS) tumor with a poor prognosis. GBM, classified as World Health Organization (WHO) grade IV glioma, is a primary CNS tumor11 have focused on the effects of treatment modalities15 or specific patient populations such as pediatrics16 and geriatrics18. Limited research has been conducted on demographic factors, clinical characteristics, or medical comorbidities as predictors for overall survival19. A few predictors of survival are well-established, including: age, extent of resection (EOR), performance status, and O6-methylguanine-DNA-methyltransferase (MGMT) status21. Other pathologic and molecular tumor biomarkers, such as epidermal growth factor receptor (EGFR) amplification, aldehyde dehydrogenase 1A3 (ALDH1A3), and isocitrate dehydrogenase (IDH1/IDH2) isoforms are current foci of research and have been linked to prognosis24. Tumor location has also been linked to prognosis in GBM patients25.To date, most studies on survivalGiven the short natural history of GBM and its prevalence among malignant brain tumors, there exists a need to elucidate further prognostic factors that can improve patient outcomes. A better understanding of medical comorbidities that affect overall survival will help determine whether or not targeted medical optimization could further improve survival in patients with GBM. Therefore, the objective of this study was to investigate demographic, clinical characteristics, and pre-existing medical comorbidities as predictors of overall survival among patients with GBM.Retrospective data were obtained from the Virginia Commonwealth University (VCU) Brain and Spine Tumor Registry, which contains data from medical records for all patients who presented with a brain tumor between January 2005 and February 2015. Data were extracted in August of 2017. Inclusion criteria for this study were: (1) age greater than 18, (2) pathology-confirmed diagnosis of GBM, (3) documented tumor location, (4) documented EOR, and (5) validated living status at time of censorship.The date of first clinical encounter with VCU was considered a patient\u2019s entry into the study. Date of death was used to define event time. Patients that were either lost to follow up or still alive at the event time were censored based on the time of their last clinic visit. Survival time was calculated as the difference between entry date and event time or censorship in days. Demographic variables included age, sex, race, insurance status, marital status, alcohol use, and tobacco use. Clinical characteristics included EOR, tumor volume, and tumor location. Performance status and MGMT promoter methylation were not included, as these variables were not available for many of the patients in the study. Tumor location was determined from pre-operative radiology reports, with \u201cmultiple locations\u201d chosen if more than one distinct location was indicated by the radiological interpretation. EOR was determined by neurosurgery post-operative notes. EOR was graded as complete or near total if these descriptors were used in the postoperative neurosurgery notes that referenced postoperative imaging, or subtotal if the postoperative note referenced any residual contrast enhancement. Comorbidities included: arthritis, asthma, previous cancer, depression or anxiety, diabetes, heart disease, high blood pressure, hypercholesterolemia, incontinence, migraines, seizures, stomach ulcer or upset stomach, stroke, and thyroid problems. Comorbidities were collected from neurosurgery outpatient clinic and inpatient notes.survival and survminer packages . The Virginia Commonwealth University Institutional Review Board approved this study, and waived the need for informed consent as all data were pre-existing in the medical record with no additional risk to patients. All patient data were collected, stored, and analyzed in a HIPAA-compliant fashion and in accordance with VCU institutional and Collaborative Institutional Training Initiative guidelines.A Cox proportional hazards regression was applied to assess the relationship between each of the predictors and overall survival. First, regressions were performed to determine whether or not demographic characteristics, tumor volume, or past medical conditions were individually associated with overall survival. Predictors that were at least marginally significant (p\u2009<\u20090.15) were entered into a final multivariable model. All analyses controlled for age, EOR, and tumor location, as these have been shown to be prognostic factors as stated above. Estimates of overall survival were reported at six, twelve, eighteen, and twenty-four months. Models were fit using the SAS statistical software version 9.4 with inferences made at the 5% level. A Kaplan-Meier survival plot was created using the R statistical software version 3.5.0 with the A total of 197 patients diagnosed with GBM presented to VCU from January 2005 to February 2015. Of those, 163 patients met criteria for inclusion. The 34 patients who did not meet inclusion criteria were excluded primarily due to missing tumor location, missing extent of resection, or missing both variables. Table\u00a0At the time of data collection, 147 (90%) patients were deceased, 15 (9%) patients were lost to follow-up, and 1 (1%) patient was alive. The overall probability of survival in our sample was 71% at six months, 50% at twelve months, 34% at eighteen months, and 26% at twenty-four months. Figure\u00a0Table\u00a0The final multivariable model included age, tumor location, EOR, asthma, hypercholesterolemia, and urinary incontinence Table\u00a0. MultivaSurgical patients with GBM who had a prior history of asthma or hypercholesterolemia had a significantly higher relative risk of mortality in multivariable analysis. Sex, race, and other medical comorbidities were not significantly associated with survival in this study. The poor prognosis of patients with GBM following diagnosis likely negates the hazardous effects of other medical comorbidities on survival. Many of such other diseases require years to cause fatal complications or contribute otherwise to earlier death.26. It has been shown that there is an inverse relationship between serum IgE levels and risk of glioma, and high amounts of mast cells have been detected in GBM tissues28. Gohar et al.28 linked certain IL-4R\u03b1 and IL-13 alleles to increased glioma susceptibility, and found the IL-4R\u03b1 AA genotype in GBM patients was associated with prolonged survival. A 2011 study by Scheurer et al.29 found that those with a self-reported history of asthma/allergies and regular anti-inflammatory/NSAID use had a protective association with development of GBM, but there was no significant effect of regular antihistamine use on risk of GBM. A later study by Amirian et al.30 revealed that regular antihistamine use in patients with self-reported asthma/allergy history was associated with increased risk of glioma, but without any observed effect on survival. Atopy is a disease of the immune system, and the immunologic properties of GBM have been well-described31. It is reasonable to suspect that systemic diseases affecting the immunologic and inflammatory responses would have an effect on GBM development and prognosis.One hypothesis for these findings is that there is a unique interaction between glioblastoma and these comorbidities. The pathophysiology behind asthma and hypercholesterolemia could influence the progression or development of GBM. Prior studies have found that patients with atopic diseases including allergies, eczema, and asthma have a decreased susceptibility to grade II and III gliomas and improved prognosisPerhaps those individuals with asthma have lesser risk of developing glioma, but are prone to more aggressive tumors or a worse clinical course when the tumor does emerge, which is plausible given the results of this study. It could also be that individuals with atopic diseases are less likely to develop secondary GBM arising from low-grade glioma, due to the previously-reported decreased susceptibility to lower-grade gliomas in these patients. Since primary GBM has a worse prognosis than secondary GBM this could explain the poorer survival seen in this sample. The relationship between atopy and GBM is actively being researched, and the relationship between asthma and overall survival in particular needs additional evidence.32. The effect of statin use on GBM has been better studied with data suggesting statins reduce risk of seizure prior to diagnosis, but do not significantly affect survival following diagnosis33. A study by Gaist et al.34 demonstrated a dose-dependent survival benefit in those taking statins prior to diagnosis of GBM. This suggests that the level of control or treatment of hypercholesterolemia at the time of diagnosis may be an underlying factor explaining our observed relationship between hypercholesterolemia and overall survival.There is less research on the relationship between hypercholesterolemia and GBM. Studies have shown the importance of cholesterol on GBM cell survival35. Beyond the post-operative period, asthma still remains an immediate threat to life due to pneumonia and/or acute respiratory failure36. Hypercholesterolemia was associated with poorer overall survival within this study, but an explanation for these findings remains unclear. Hypercholesterolemia is related to myocardial infarction and other acute vascular events that can be rapidly fatal especially within the acute post-operative period. It would be expected that a history of heart disease and stroke would exhibit a similar relationship as hypercholesterolemia with overall survival, which was not observed within this study.Another hypothesis to explain the association between asthma and hypercholesterolemia and decreased survival is that these diseases could be fatal or lead to organ failure within a short time frame. Asthma is associated with increased post-operative pulmonary complications, including infection, asthma exacerbation, and acute respiratory failure37. Additionally, the usual risk of mortality associated with hypertension, diabetes, stroke, and heart disease may be overcome by the drastically higher rate of mortality of patients within our sample population. It could be that individuals in our study with these diagnoses, which would reasonably be expected to cause many of the same acute vascular events as hypercholesterolemia, were already well-treated following prior diagnoses and thus did not experience these complications after their diagnosis of GBM. The other comorbidities measured in our sample, such as arthritis, incontinence, seizures, migraines, depression/anxiety, and thyroid disease, may increase the comorbid burden and poorer overall health status of patients, but would not be expected to increase the risk of mortality within the shortened life expectancy of patients presenting with GBM.Prior studies evaluating comorbidities and long-term survival in patients with GBM have focused on a limited number of comorbid conditions, primarily diabetes mellitus3. There was a clear male predominance amongst patients who presented to our institution with GBM, which has been demonstrated in nationwide samples7. In controlling for surgical EOR and age, we saw that complete resection and near total resection both were associated with significantly longer survival time when compared to biopsy alone, while subtotal resection was not significantly protective. This finding is consistent with previous studies suggesting the survival benefit of complete resection18. Increasing age was associated with decreased survival, which has been repeatedly demonstrated in the literature10. Tumor location was not associated with overall survival. Prior research has demonstrated that tumor location influences overall survival via surgical approaches, oncological management, and the ability to pursue surgical resection25. Our findings could be explained by not stratifying our tumor locations based on eloquence, or by preselecting surgical candidates based on those whose tumors were more amenable to surgery and underwent resection rather than conservative management alone.In our study, the observed probability of survival at 6-, 12-, 18-, and 24-months was 71%, 50%, 34%, and 26%, respectively, which is consistent with estimates of median survival from previous studies19. This sample has a higher proportion of nonwhite patients compared to other studies on glioblastoma patients in the literature2. This may account for some of the divergence in findings and also may improve the applicability of these findings to medical centers with diverse patient populations. We did not evaluate tumor biomarkers, such as MGMT, IDH1/2, P53, EGFR, and PTEN, that are commonly related to overall survival, as these were not consistently available at the time of presentation to our institution for this study sample (2005\u20132015). VCU is an academic medical center with a large referral base, thus patients in the sample may have been diagnosed with a brain mass at an outside facility before coming to VCU for treatment. In addition, the 9% of patients who were censored at the date of their last clinic visit could have survived for significant amount of time afterwards, which would not be captured in this dataset. Lastly, we did not assess the underlying cause of death for each patient, which could further delineate whether progression of disease or other medical comorbidities were the attributable factor for mortality within this patient population.Limitations of this study include its retrospective nature and power. The sample size is comparable to other single-institution studies, although there was a limited sample size for subgroups for some comorbiditiesFuture research with larger sample sizes using prospective or multi-institutional design would be valuable for investigating comorbid predictors of survival in GBM. Specifically, the underlying pathophysiology for asthma or hypercholesterolemia in relation to GBM should be investigated to understand how each disease process alters the overall length of survival. Properly treated hypercholesterolemia or asthma could potentially alter prognoses when compared to untreated disease in this population. The effects of lipid-lowering and asthma/allergy medications on the natural history, progression, and survival of patients with GBM is one possible avenue of further investigation. An examination of tumor markers present in patients with history of asthma or hypercholesterolemia could be useful in assessing potential mechanisms for differences in prognosis of these patients.GBM is an aggressive and prevalent primary CNS tumor with unfavorable overall survival and few known prognostic factors. This study demonstrates that, in addition to patient age and decreased extent of resection, history of asthma and hypercholesterolemia are each associated with a worse prognosis in GBM. Further research should establish the interactions of the pathophysiology of these diseases with glioblastoma and whether or not optimal medical control of asthma and hypercholesterolemia provides survival benefit. Recognizing and ensuring that comorbid conditions are adequately treated remains essential in the treatment of patients with GBM."} +{"text": "P < 0.05). These DEGs were mainly involved in melanogenesis, the calcium signaling pathway, the Wnt signaling pathway, the mTOR signaling pathway, and vascular smooth muscle contraction. The pathway analysis of the DEGs identified some key genes associated with pigmentation, such as DCT and EDNRB2. In summary, the melanin content of breast muscle could be markedly enhanced by adding an appropriate amount of tyrosine to the diet of Xichuan black-bone chickens, and the EDNRB2-mediated molecular regulatory network could play a key role in the biological process of tyrosine-induced melanin deposition. These results have deepened the understanding of the molecular regulatory mechanism of melanin deposition in black-bone chickens and provide a basis for the regulation of nutrition and genetic breeding associated with melanin deposition in Xichuan black-bone chickens.The Xichuan black-bone chicken, which is a rare local chicken species in China, is an important genetic resource of black-bone chickens. Tyrosine can affect melanin production, but the molecular mechanism underlying tyrosine-induced melanin deposition in Xichuan black-bone chickens is poorly understood. Here, the blackness degree and melanin content of the breast muscle of Xichuan black-bone chickens fed a basic diet with five levels of added tyrosine were assessed, and the results showed that 0.8% tyrosine was the optimal level of added tyrosine. Moreover, the effects of tyrosine supplementation on the proliferation and tyrosinase content of melanocytes in Xichuan black-bone chickens were evaluated. The results revealed a dose-dependent relationship between tyrosine supplementation and melanocyte proliferation. In addition, 417 differentially expressed genes (DEGs), including 160 upregulated genes and 257 downregulated genes, were identified in a comparative analysis of the transcriptome profiles constructed using the pooled total RNA from breast muscle tissues of the control group and test group IV, respectively (fold change \u22652.0, The black-bone chicken is an important resource in poultry production, and the fact that its body contains melanin significantly differentiates it from other chicken breeds . Many stMelanin is a type of high-protein molecule synthesized from melanin bodies in melanocytes , and stuThe Xichuan black-bone chicken, which is a rare local chicken species, is mainly distributed in Xichuan County, Henan Province, China. This variety has five black parts and is an important genetic resource of black-bone chickens. Because its meat is tender and delicious and has high nutritional and medicinal value, this variety has become an important object of production and development in recent years. Increased production performance and the production of high-quality poultry meat with a high content of melanin through nutritional regulation or genetic improvement are currently the main goal of the resource exploitation of Xichuan black-bone chickens. The breast muscle is an important part of skeletal muscle and the main source of poultry meat. Therefore, in this study, the breast muscle of Xichuan black-bone chicken was used as the research material to evaluate the effects of different concentrations of tyrosine on melanin deposition and to analyze the transcriptome profiles of breast muscle under tyrosine supplementation. We also verified the effect of tyrosine on the proliferation and tyrosinase content of melanocytes and determined the key genes and molecular regulatory network associated with tyrosine-induced melanin deposition. The main objectives of this study were to optimize the optimal supplementary amount of tyrosine in feed for the promotion of melanin deposition in the breast muscle of Xichuan black-bone chickens and to clarify the potential molecular mechanism underlying tyrosine-induced melanin deposition in black-bone chickens. The results obtained in this study will provide a better understanding of the molecular regulatory mechanisms of melanin deposition in black-bone chickens and contribute to nutrient regulation and genetic breeding related to melanin deposition in Xichuan black-bone chickens.All sample collections and treatments were conducted strictly in accordance with the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Henan Agricultural University, China (11-0099).A total of 480 one-day-old healthy and weight-matched Xichuan black-bone chickens were randomly divided into six groups (the control group and test groups I-V), with five biological replicates of each group and 16 chickens per biological replicate. The control group was fed a basal diet . For tesAt the end of the feeding trial, all the experimental chickens were fasted for 12 h with continued access to water. Two Xichuan black-bone chickens from each replicate were randomly selected. The live weight and dressing percentage of the 60 chickens were measured. These experimental chickens were euthanized by intravenous injection of KCl (1\u20132 mg/kg) under deep anesthesia, and 1\u20132 g of breast meat was frozen immediately in liquid nitrogen and stored at -80\u00b0C until RNA-seq analysis. The remaining breast meat was stripped from both sides for analysis of the melanin content.\u2217). All color values were obtained from three areas of each breast muscle. A higher reading indicated a lower degree of blackness, and vice versa. Second, the melanin content in the breast muscle of each experimental chicken was analyzed. Briefly, 50-g breast muscle samples from each chicken were weighed, and after the fascia, membrane and fat were removed, the samples were heated in an oven at 65\u00b0C for 24 h. The crushed samples were hydrolyzed with papain under neutral conditions for 3 h during heating at 55\u00b0C in a water bath, and after centrifugation, the precipitates were separated, soaked in 200 mL of 6 mol/L HCl and heated in an electric furnace for 30 min. The residue was collected, washed, wrapped with filter paper and placed in a Soxhlet extraction tube. Subsequently, the residue was degreased with ether in a water bath at 42\u00b0C, and the filter paper was washed repeatedly with distilled water and dried at 80\u00b0C in an oven. The melanin was removed and weighed using a microbalance. The melanin content (as the percentage of fresh tissue weight) was then calculated as follows: melanin weight (g)/sample weight (g) \u00d7 100%.First, the breast muscle color of each experimental chicken was determined with a portable NR10QC colorimeter . The color parameter was measured based on the lightness index . Fourth-generation melanocytes at the logarithmic phase of growth were digested with 0.25% trypsin and seeded into cell culture plates for follow-up testing. On the one hand, the effect of tyrosine on the proliferation of melanocytes was evaluated by the Cell Counting Kit-8 (CCK-8) method on 96-well cell culture plates. Ten replicate wells were used for each group. Ten microliters of CCK-8 solution was added to each well, and the plates were incubated for 2 h at 37\u00b0C in an atmosphere with 5% CO2. The absorbance of each well at 450 nm was measured using a high-throughput multifunctional microplate test system. The proliferation rate was calculated as follows: [(A450 (with tyrosine) \u2013 A450 (medium only)]/[A450 (without tyrosine) \u2013 A450 (medium only)] \u00d7 100%. On the other hand, the effect of tyrosine on the tyrosinase content in melanocytes was detected using a chicken tyrosinase ELISA kit on six-well plates. Each group was tested in six replicate wells in triplicate. The plates were incubated for 72 h in a humidified 5% CO2 incubator at 37\u00b0C, and the intracellular components were then extracted using an ultrasonic cell disrupter. The content of tyrosinase in the cells was detected in strict accordance with the instructions provided with the tyrosinase ELISA kit. Moreover, the effects of tyrosine on endothelin receptor B subtype 2 (EDNRB2) gene expression in the above-treated melanocytes were also determined by qRT-PCR analysis.To evaluate the effect of tyrosine on melanin deposition at the cellular level, we exposed melanocytes from Xichuan black-bone chickens to different concentrations of tyrosine. The initial density of the cells was 1 \u00d7 10\u00ae UltraTM Directional RNA Library Prep Kit for Illumina\u00ae . The processes necessary for library construction, such as mRNA isolation, fragmentation, first-strand cDNA synthesis, second-strand cDNA synthesis, terminal repair, 3\u2019 end A-tailing, ligation, and enrichment, were completed according to the manufacturers\u2019 instructions. Sequencing was performed according to the corresponding requirements with a paired-ending cDNA sequencing program. The sequencing process was controlled using the Illumina HiSeq 2000 platform data collection software program that was used for the real-time data analysis. The raw reads were processed to remove adaptor sequences, low-quality reads and reads containing poly-N sequences with Seqtk1. Using the spliced mapping algorithm in TopHat (version: 2.0.9) method . Therefore, breast muscle samples from the control group and test group IV were selected for transcriptome sequencing. The total RNA from the breast muscle samples was extracted using an RNA extraction kit following the appropriate procedure and were processed using DNase I . To characterize a general overview of genes expressed in the breast muscle tissues of the highest amount of melanin deposition, the pooled total RNA from three individuals of test group IV and the control group was used for cDNA library construction, respectively. The libraries were generated using the NEBNext: 2.0.9) , the cle) method . The fra) method .P-value was determined by controlling the false discovery rate (FDR), and the corrected p-value was the q-value functional enrichment analysis network analysis between the control group and test group IV . The thr q-value . The folanalysis and KOBAanalysis . The GO analysis , and theanalysis .TM RT Reagent Kit and SYBR\u00ae Premix Ex Taq II . The PCRs were conducted with an initial denaturation step at 95\u00b0C for 5 min followed by 34 cycles of 95\u00b0C for 15 s (denaturation), 60\u00b0C for 45 s , and 72\u00b0C for 40 s (extension) and a final extension at 72\u00b0C for 10 min. The reactions for each qRT-PCR were performed in triplicate. The relative expression levels of the genes were calculated using the 2-\u0394\u0394Ct method, and the levels were normalized to those of two housekeeping genes, \u03b2-actin and GAPDH between the test groups and the control group. The results are expressed as the mean \u00b1 standard deviation (SD).All the data were statistically analyzed with SPSS 20.0 software to determine the significance of the differences (P > 0.05). However, the melanin contents of the test groups II-V were significantly higher than those of the control group and test group I (P < 0.05), and the maximal value was obtained with 0.8% tyrosine supplementation (P < 0.05) (The results indicate that dietary supplementation with different doses of tyrosine significantly improved the breast muscle blackness and increntation . Althoug < 0.05) .-9 mol/L tyrosine . These results indicated that supplementation with an appropriate concentration of tyrosine can significantly increase the proliferation and tyrosinase content of melanocytes of Xichuan black-bone chickens.We also evaluated the effect of tyrosine in chicken melanocytes. Melanocytes were cultured with different concentrations of tyrosine for 72 h, and the melanocyte morphology was then observed under an inverted microscope. The test groups treated with different concentrations of tyrosine showed significantly higher numbers of cells and evident cytoplasm enlargement compared with the control group. In particular, the most obvious cytoplasmic change was detected in the group treated with 10tyrosine . The ana < 0.05) . Moreove < 0.05) , and thein vivo. To further reveal the molecular regulatory mechanism underlying this biological process, we used the pooled total RNA from breast muscle tissues of the control group and test group IV (0.8% dietary tyrosine level and the highest melanin content) and performed transcriptome sequencing using the Illumina HiSeq 2000 platform. A total of 47,038,307 (95.34%) and 51,161,382 (95.46%) clean reads were obtained for the control group and test group IV, respectively (GSE128028) of the National Center for Biotechnology Information (NCBI). The mapping rates for the reads that were uniquely mapped to the Galgal5 assembly of the chicken genome were 84.01 and 85.01% for the control group and test group IV, respectively. The comparison of the results aligned to their respective chromosomes showed no significant differences between the samples of 0.974 and four upregulated genes were selected for qRT-PCR verification. The log2 (fold change) values of the seven genes obtained from the qRT-PCR analysis were consistent with the RNA-seq results . It is known that the EDNRB2 gene activates the downstream signaling molecule PKC, upregulates the downstream gene DCT and thereby affects melanin deposition.Based on the results of the KEGG pathway enrichment analysis of the DEGs, some melanin synthesis-related pathways, including melanogenesis, the calcium signaling pathway, the Wnt signaling pathway, the mTOR signaling pathway, vascular smooth muscle contraction and adrenergic signaling in cardiomyocytes, were screened . Some kechickens . In partAnimal phenotypes are influenced by both genetics and the environment. The most obvious difference between black-bone chickens and ordinary chickens is that the skin, muscle, periosteum and other tissues of black-bone chickens contain high levels of melanin . Therefoin vitro. The proliferation of chicken melanocytes increased with increases in the tyrosine concentrations within a certain concentration range, but tyrosine concentrations that exceed a certain range inhibited melanocyte proliferation to a certain degree. We also found that tyrosine dose-dependently affects the tyrosinase content of chicken melanocytes. Specifically, the tyrosinase content of melanocytes was significantly upregulated in the tyrosine-treated groups compared with the control group, and higher levels of tyrosine supplementation increased the intracellular tyrosinase content. Analogously, previous studies have also demonstrated that increases in the tyrosine concentrations can promote melanin synthesis in hamster melanoma cells, and a certain dose-dependent relationship has been found between tyrosine and tyrosinase (Melanin is a type of high-protein molecule synthesized from melanin bodies in melanocytes . Black-brosinase . These sPMEL), melanophilin (MLPH), EDNRB2, DCT, and other pigment-related genes, were identified based on the transcriptome profiles constructed using the pooled total RNA from the breast muscle tissue of the control group and experimental group treated with 0.8% tyrosine supplementation. Therefore, PMEL, which is also known as gp100, ME20, PMEL17, and silver, is a melanoma-specific glycoprotein that plays an important role in the development of melanin bodies by forming a proteolytic fibrous matrix for melanin deposition and myosin-Va form ternary complexes that play a key role in the transport of melanosomes gene and encodes a G protein-coupled receptor (GPCR) with seven transmembrane domains (TYR) (TYRP1) , tyrosinT) (TYR) , and tyr (TYRP1) , in melain vivo. For Xichuan black-bone chickens, the optimal tyrosine dietary supplementation level was found to be 0.8%. We also analyzed the characteristics of the transcriptome profiles of breast muscle tissue from the control group and test group IV (0.8% dietary tyrosine level and the highest melanin content). The EDNRB2-mediated regulatory network might play a key role in tyrosine-induced melanin deposition in the breast muscle of Xichuan black-bone chickens. These results provide insights into the molecular regulatory mechanisms for melanin deposition in black-bone chickens and are important for the nutrition regulation and the genetic improvement of the meat quality of Xichuan black-bone chickens.In summary, this study provides the first demonstration that tyrosine can promote melanin deposition in the breast muscle of Xichuan black-bone chickens at the cellular level and The whole sample collection and treatment were conducted in strict accordance with the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Henan Agricultural University, China (11-0099).XK and GL conceived and designed the experiments. YC, JW, and YC contributed to animal husbandry. YZ, YL, and YF performed the experiments. YT and XW analyzed the data. CZ, RJ, and ZL contributed reagents and materials. GS and WL contributed analysis tools. DL wrote the manuscript. All authors reviewed and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Aspergillus species, like Aspergillus niger. Compared to the traditional ways of process optimization, the metabolic engineering strategies to improve glucoamylase production are relatively scarce.Glucoamylase is one of the most industrially applied enzymes, produced by A. niger. To further verify this, five mutants namely OE-ala, OE-glu, OE-gly, OE-asp1 and OE-asp2, derived from the parental strain A. niger CBS 513.88, were constructed respectively for the overexpression of five genes responsible for the biosynthesis of the four kinds of amino acids . Real-time quantitative PCR revealed that all these genes were successfully overexpressed at the mRNA level while the five mutants exhibited different performance in glucoamylase production in shake flask cultivation. Notably, the results demonstrated that mutant OE-asp2 which was constructed for reinforcing cytosolic aspartate synthetic pathway, exhibited significantly increased glucoamylase activity by 23.5% and 60.3% compared to CBS 513.88 in the cultivation of shake flask and the 5\u00a0L fermentor, respectively. Compared to A. niger CBS 513.88, mutant OE-asp2 has a higher intracellular amino acid pool, in particular, alanine, leucine, glycine and glutamine, while the pool of glutamate was decreased.In the previous study combined multi-omics integrative analysis and amino acid supplementation experiment, we predicted four amino acids as the limited precursors for glucoamylase production in Our study combines the target prediction from multi-omics analysis with the experimental validation and proves the possibility of increasing glucoamylase production by enhancing limited amino acid biosynthesis. In short, this systematically conducted study will surely deepen the understanding of resources allocation in cell factory and provide new strategies for the rational design of enzyme production strains. Aspergillus niger, as one of the most efficient cell factories for enzymes, organic acids and food additives production, is widely applied in biochemistry industry. Glucoamylase, especially fungal glucoamylases, has gained great importance because of its wide application in different industries. Therefore, it has been extensively studied during the past few decades [A. niger include fermentation process optimization and genetic engineering. Fermentation process optimization mainly include mycelial morphology control [ decades . Common control , ferment control , culture control , 5 and t control .In industrial glucoamylase production, oxygen limitation strategy is widely applied because high glucoamylase yield to carbon source can be achieved and the byproducts are more efficiently reused . In the P. pastoris, P. stipitis and S. cerevisiae, exogenous amino acids are supplemented in the culture media to improve the heterologous protein production [E. coli and yeast. Xia et al. [E. coli by metabolic engineering to enhance the production of a 284.9\u00a0kDa recombinant spider silk protein which is extremely rich in glycine. Another similar study was reported by Cao et al. [E. coli to achieve high expression of recombinant protein with high molecular weight and Gly-Ala repeated sequence. In P. pastoris, Zahrl et al. [Addition of amino acids in the medium is a common way of increasing the supply of limited amino acids to enhance cell growth as well as production of protein products. In several yeast species such as oduction \u201310. Howea et al. elevatedo et al. . Glycyl/l et al. concludeNeurospora crassa [S. cerevisiae [N. crassa, Carsiotis et al. [S. cerevisiae affects biosynthetic pathways of all branched amino acids (aromatic amino acids and the aspartate family) and the pathways for the basic amino acids. For these amino acids, allosteric feedback inhibition, repression of enzymes in common for amino acids of branched pathways and active sequestration of amino acids [Metabolic engineering of amino acid biosynthesis is rather challenging because there exists a so-called metabolic interlock, which means co-regulation between enzymes of different amino acid biosynthetic pathways in fungi . This pha crassa \u201317 as crrevisiae . In N. cs et al. , 16 repos et al. found thno acids will cauno acids .glaA. Verdoes et al. [glaA into the genome of a laboratory A. niger strain to obtain glucoamylase-overproducing strains. Xin et al. [glaA and \u03b1-amylase encoding gene amyA substantially increases the expression and secretion of glucoamylase in A. niger. Furthermore, researchers were also focused on the improvement of protein secretion ability to increase glucoamylase production [glaA copies may overload host cells and fails to increase glucoamylase production. To efficiently improve the glucoamylase yield, cellular resources accumulated in primary metabolism require to be allocated coordinately to cell growth and product biosynthesis.Genetic engineering strategies were also applied in glucoamylase overproduction, among which one efficient strategy is the introduction of additional copies of the glucoamylase encoding gene s et al. introducn et al. also demoduction , 23. HowA. niger CBS 513.88. The framework of this study is summarized in Fig.\u00a0A. niger CBS 513.88, generating the mutants to the pentaketide 1,3,6,8-tetrahydroxynaphthalene (T4HN) [olvA leads to the color change of spores from black to olive [The model glucoamylase-producing strain ormation from A. A. niger . A. nigee (T4HN) . Interruto olive (AdditioE. coli cultivation. Potato dextrose agar (PDA) medium and culture conditions of 30\u00a0\u00b0C were used for A. niger plate cultivation. Potato dextrose agar (PDA) medium supplemented with 100\u00a0\u03bcg/ml of hygromycin and culture conditions of 30\u00a0\u00b0C were used for isolation and purification of the mutants.Luria Broth (LB) culture medium supplemented with 100\u00a0\u03bcg/ml of ampicillin and culture conditions of 37\u00a0\u00b0C and 220\u00a0rpm were used for Escherichia coli DH5\u03b1 cells. All the procedures were performed according to standard methods or the manufacturer\u2019s instructions. Donor DNA is required for CRSIPR-Cas9 mediated olvA deletion and target gene overexpression by homologous recombination. was obtained by PCR amplification from the fungal expression plasmid pAN 7-1 separately, generating the recombinant plasmids for overexpression of the candidate genes. After construction, the hygromycin gene in original pAN 7-1 was replaced by candidate gene so the gene was under the control of the constitutive strong promoter PgpdA. Afterwards, the expression cassette of the candidate gene (PgpdA+candidate gene+TtrpC) was obtained by PCR amplification from the recombinant plasmid and then was flanked by 1\u00a0kb homologous arm (HA) of olvA.All the primers used in this section were listed in Additional file olvA at the location of olvA. Regeneration medium supplemented with 150\u00a0\u03bcg/ml of hygromycin was used to generate selection pressure for the plasmid. The transformants, whose spores with olive color, were picked out for further single colony separation. PDA medium supplemented with 100\u00a0\u03bcg/ml of hygromycin was used for isolation and purification for the single colony. After genome extraction and PCR verification with three pairs of primers was inoculated with 102 0.076, MgSO4\u00b77H2O 1, EDTA-2Na 0.67, NaNO3 7.5, KH2PO4 3, NaH2PO4\u00b72H2O 1.69, MnSO4\u00b7H2O 0.04, ZnCl2 0.02, CuSO4\u00b75H2O 0.015, CoCl2\u00b76H2O 0.015, FeSO4\u00b77H2O 0.3, 0.1% antifoam reagent, initial pH\u2009=\u20095.5, 5 glass beads with diameter of 3\u00a0mm) was inoculated with 3\u00a0ml seed culture broth (pre-grown mycelia) and cultured under the conditions of 34\u00a0\u00b0C, 170\u00a0rpm.The fermentation medium was inoculated with 200\u00a0ml seed culture broth (pre-grown mycelia). The agitation and aeration were 375\u00a0rpm and 1\u00a0vvm, respectively. The pressure and temperature were 0.05\u00a0MPa and 34\u00a0\u00b0C, respectively. The fermentation pH was controlled at 4.5 by 5\u00a0M NaOH. When glucose concentration was decreased to around 10\u00a0g/l, supplemented medium was fed into the fermentor after batch culture. The feeding rate was initially calculated based on the glucose consumption rate in batch culture before feeding and was then adjusted based on the residual glucose in the broth to control the glucose concentration between 5 to 10\u00a0g/l.3.5\u00a0L fermentation medium as substrate. 230\u00a0\u03bcl of 2\u00a0g/l pNP-G (pre-warmed in 37\u00a0\u00b0C for 5\u00a0min) was mixed with 20\u00a0\u03bcl supernatant of the fermentation broth. 100\u00a0\u03bcl of 3\u00a0M Na2CO3 was added after an incubation in 37\u00a0\u00b0C water bath for 20\u00a0min. Enzyme activity was determined by the absorbance of the mixed solution at the wavelength of 405\u00a0nm. Before measuring samples, we established the standard curve of enzyme activity (R2\u2009>\u20090.999): enzyme activity\u2009=\u2009dilution ratio\u2009\u00d7\u2009(OD405\u2009+\u20090.01)/0.008.The enzyme activity of glucoamylase is expressed in AGI units (Amyloglucosidase). One AGI unit is defined as the amount of enzyme that produces 1\u00a0\u03bcmol of glucose per minute at pH 4.3 and at 60\u00a0\u00b0C from a soluble starch substrate. The glucoamylase activity was determined at 37\u00a0\u00b0C and pH 4.30 using the colorless chromophore p-nitrophenyl-\u03b1-\u2122 RT reagent kit with gDNA Eraser . PCR was performed by using TB Green Premix Ex taq\u2122 II (Takara) and Bio-rad CFX96 real-time PCR Detection system. The conditions were used: 95\u00a0\u00b0C for 30\u00a0s, followed by 39 cycles of 95\u00a0\u00b0C for 5\u00a0s and 60\u00a0\u00b0C for 30\u00a0s. The primers used in this section were listed in Additional file For RNA extraction, 1\u00a0ml fermentation samples were collected. For shake flask fermentation, samples were taken at 36\u00a0h and 72\u00a0h (oxygen-limited phase). For fed-batch fermentation in the 5\u00a0L fermentor, samples were taken at 36\u00a0h and 72\u00a0h (oxygen-limited phase in feeding stage). All procedures were performed according to the manufacturer\u2019s instructions. Fungal Total RNA Isolation Kit (Sangon Biotech) was used for RNA extraction after liquid nitrogen grinding. Total RNA was reverse-transcribed by PrimeScript13C internal standard solution for extraction in 95\u00a0\u00b0C water bath for 3\u00a0min [g for 2\u00a0min and the supernatant was collected for amino acids detection by GC\u2013MS .A rapid sampling device was used to take about 1\u00a0g (precisely determined) broth from the 5\u00a0L fermentor into 10\u00a0ml precooled \u2212\u200930\u00a0\u00b0C 40% methanol (v/v). After filtered and washed by 15\u00a0ml precooled \u2212\u200930\u00a0\u00b0C 40% methanol (v/v), the filter cake was thrown into 25\u00a0ml of 75\u00a0\u00b0C pre-warmed 75% ethanol (v/v), with the addition of 100\u00a0\u03bcl or 3\u00a0min . The extt test.Unless otherwise noted, all experiments were performed in three replicates and statistical significance was determined by two-tailed Student\u2019s olvA locus under the control of the constitutive strong promoter PgpdA. In addition, an olvA deletion strain CBS-\u0394olvA derived from A. niger CBS 513.88 was also generated as the control. The detailed information of the mutants was shown in Table\u00a0Five genes encoding the enzymes for the biosynthesis of four kinds of probable limiting amino acids, namely alanine transaminase, glutamate dehydrogenase, glycine/serine hydroxymethyltransferase, mitochondrial aspartate aminotransferase and cytosolic aspartate aminotransferase were overexpressed in the olvA showed no significant difference in glucoamylase production and biomass growth compared with CBS 513.88 during the fermentation in shake flask cultivation were measured to evaluate the yield of the mutants. First of all, mutant CBS-\u0394Fermentation in the shake flasks revealed that mutants OE-asp2, OE-gly and OE-glu exhibited a significant higher DCW at the time point of 36\u00a0h compared to CBS 513.88 is an important enzyme of glycine metabolism, which catalyses the reversible conversion of glycine and serine. In eukaryotes, the cytosolic and mitochondrial SHMT isoenzymes are encoded by distinct nuclear genes . In yeaso et al. reportedA. niger CBS 513.88 to improve the biosynthesis of these amino acids and the results are summarized in Fig.\u00a0A. niger.In this study, we constructed five mutants to overexpress the genes involved in the limited amino acid synthetic pathways in the strain A. niger under oxygen-limited conditions, we constructed five mutants to strengthen the biosynthesis of these limited amino acids and then tested the glucoamylase productivity in shake flask cultivation. Fed-batch fermentation in the 5\u00a0L fermentor revealed that the glucoamylase activity of mutant OE-asp2 increased 60.3% than that of the parental strain A. niger CBS 513.88. Moreover, profiles of intracellular amino acid pools demonstrated that the reinforcement of cytosolic aspartate biosynthesis in mutant OE-asp2 elevated most intracellular amino acid pools, especially the pools of alanine, glycine and leucine. Therefore, we speculated that the glucoamylase production was significantly increased in mutant OE-asp2 mainly due to the increased supply of intracellular aspartate and other limiting amino acid precursors derived from the improvement of cytosolic aspartate biosynthesis. Finally, our work demonstrated that aspartate plays an important role in the biosynthesis of amino acid precursors for glucoamylase production in A. niger under oxygen-limited conditions. Regulation of amino acid biosynthesis can efficiently relieve amino acid limitation in enzyme production. However, the amino acid ratio in biomass and protein products, together with the influence of amino acid biosynthetic pathways on intermediates and cofactors in global metabolism should be taken into consideration when selecting the target amino acids for engineering.Based on the previous integrative multi-omics prediction of limited amino acids for glucoamylase production in Additional file 1: Amino acid contents of biomass protein and glucoamylase.Additional file 2: Fig. S1. Color changes of spores of A. niger. a. The parental strain CBS 513.88 b. Gene olvA defective mutant. Fig. S2. Schematic diagram of constructions of donor DNA for olvA deletion and target gene overexpression. Fig. S3. Schematic diagram of the generation of the mutants for gene overexpression by CRISPR/Cas9 assisted homologous recombination. Fig. S4. Comparison of the mutant CBS-\u0394olvA and the parental strain CBS 513.88 in shake flask fermentation. Data represent the average values and standard deviations from three replicates. Fig. S5. Growth (a) and glucoamylase production (b) profiles of the two mutants OE-asp1 and OE-asp2 and the parental strain CBS 513.88 in the 5\u00a0L fermentor. The vertical line (at 40\u00a0h) shows the beginning of the oxygen-limited phase. Data represent the average values and standard deviations from three replicates. Fig. S6. a. Relative mRNA levels of gene An04g06380 in the mutant OE-asp1. b. Relative mRNA levels of gene An16g05570 in the mutant OE-asp2. Samples were taken at 36\u00a0h and 72\u00a0h (oxygen-limited phase) from the 5\u00a0L fermentor. Data represent the average values and standard deviations from three replicates.Additional file 3: Table S1. Primers for constructions of donor DNA for olvA deletion and target gene overexpression. Table S2. Primers for PCR diagnosis of transformants. Table S3. Primers for real-time quantitative PCR. Table S4. Plasmids used in the study"} +{"text": "Binge eating behavior is highly likely to progress to an eating disorder, with female students particularly at risk.This study aimed to verify the effect of a binge eating behavior management program, based on rational emotive behavior therapy (REBT), on binge eating behavior and related cognitive and emotional factors among female college students.2 tests, t tests, and analysis of covariance.The study, conducted from November 1 to December 2, 2016, involved a pretest-posttest design and nonequivalent control group. The sample included 24 and 22 first- to third-year students, from a college in South Korea, in the experimental and control groups, respectively. Data were collected using self-esteem, covert narcissism, perfectionism, body dissatisfaction, anxiety, depression, and binge eating scales and analyzed via frequency analysis, \u03c7The results indicated that the REBT-based binge eating behavior management program exerted positive effects on participants\u2019 self-esteem, reducing covert narcissism, body dissatisfaction, anxiety, depression, and binge eating. However, there was no significant difference in perfectionism, although the experimental group\u2019s mean score decreased from pretest to posttest.Based on the results, the program was considered to be effective, and is expected to be useful in preventing the development of eating disorders among female college students by treating binge eating behavior and related cognitive and emotional factors. This intervention could ultimately contribute to the improvement of female college students\u2019 health and quality of life. Binge eating behavior results from an individual\u2019s distorted body image, dissatisfaction with their physical appearance, and idealization of thin body types, and requires intervention before developing into an eating disorder. The purpose of this study was to address binge eating behavior and related factors among female college students in Korea, ultimately aiming to prevent the development of eating disorders. Our intervention integrated cognitive, emotional, and behavioral techniques used in rational emotive behavior therapy (REBT), aimed at treating binge eating behavior among female college students. The results show that the program was effective, with positive effects on participants\u2019 self-esteem, covert narcissism, perfectionism, body dissatisfaction, anxiety, depression, and binge eating.Over the past two decades, South Korean society has increasingly pursued values based on physical appearance. Consequently, women with normal body weight, particularly college students, have distorted body images, are dissatisfied with their physical appearance, and idealize the thin body type, resulting in inappropriate eating habits and high likelihood of developing eating disorders . AccordiAccording to the Health Insurance Review & Assessment (HIRA) Service, the incidence of eating disorders in Korea rose by 18.8% over 5 years from 2008 to 2012, with an annual increase of 4.5%. Of all those diagnosed with eating disorders, 49.2% are between their teens and 30s, highlighting the gravity of the problem among the younger generation. In another study, over 50% of female students attempted to manage their body weight through dieting; more female students are at risk for developing an eating disorder compared to their male counterparts [Binge eating behavior is characterized by both the amount of food consumed and loss of control . Binge eBinge eating behavior is particularly influenced by negative psychological factors . Women wIn particular, low self-esteem related to body image and outer appearance is reported to be a more potent predictor of binge eating behavior in women than in men . Gordon Women who exhibit binge eating behavior compare themselves to other women and idealize thin bodies, which is associated with their irrational belief that their value is determined by their weight or appearance. If these women fail to achieve that idealized image, they become dissatisfied with their bodies, which ultimately results in low self-esteem . AnxietyThe relationship between depression and eating problems has been consistently discussed , and parEllis developeREBT has been used in various areas such as clinical psychology, education, and counseling , and itsIn this study, a binge eating behavior management program based on the REBT theory was developed for female college students, and its effects were tested on self-esteem, covert narcissism, perfectionism, body dissatisfaction, anxiety, depression, and binge eating behavior.The study was approved by the research ethics committee at Sun Moon University prior to data collection (SM-201608-025-2). Prior to their participation, the study\u2019s aims and methods were explained to the participants, and they were informed about the voluntary nature of participation, and assured of confidentiality, after which written consent was obtained from each participant.This was a quasi-experimental study, following a nonequivalent control group pretest-posttest design. The sample size was based on an effect size of 0.5, which was derived from the results of a meta-analysis on the effects of a cognitive-behavioral counseling program ; a signiCurrent students of S university in C city were recruited through an on-campus recruitment poster. Those who provided informed consent to participate in the study were enrolled. The participant group allocation was determined by flipping a coin . Participants were included if they met the following inclusion criteria: tendency to engage in binge eating behavior, as demonstrated by eating attitude scores between 88 and 120 on the Bulimia Test Revised developed by Kang and Jeong , which w by Kang and .89 Perfectionism was measured using a multi-dimensional perfectionism tool, which was first developed by Hewitt and Flett and lateThe Cronbach\u2019s \u03b1 was 0.87 in a study conducted by Jeon and 0.91The Body Shape Questionnaire (BSQ), developed by Cooper and colleagues and lateThe State-Trait Anxiety Inventory developed by Spielberger, Gorsuch and Lushene , is a coDepression was measured using the Korean version of the Center for Epidemiologic Studies Depression Scale (CES-D) developed by Radloff to facilIn their development of the Bulimia Test, Smith and Thelen relied oTable\u00a0discussion and evaluation component was included at the end of each session, to allow participants to present their thoughts and feelings and develop confidence through mutual support and encouragement. This section of the program was designed to reflect participants\u2019 opinions regarding each session and improve program quality. Participants were asked to record the times and frequencies of their mealtimes; their thoughts, feelings, and behavior before/after the meal; and whether they were binging, in a dietary diary, to measure their eating habits and binging frequency. The participants were asked to write down a list of activities they liked and activities they did not like. Then, they were instructed to reward themselves with activities they liked when they did not binge eat, and engage in activities they did not like after binge eating.A Participants decided to enforce either compensation or punishment based on their own assessments of how well they maintained their dietary records. In the final session, participants presented their dietary diaries, and the researcher used a reinforcement technique involving awarding gifts to participants who had made regular notes and practiced compensation and punishment well.2 tests and t tests were performed in crosstabs analysis to assess homogeneity between the control and experimental groups. The Shapiro-Wilk test was used to assess the normality of the general characteristics and dependent variables. The normality of all variables at the baseline for the experimental group and control group was tested with skewness and kurtosis. The absolute skewness was smaller than 3, while the absolute kurtosis was smaller than 10, thereby satisfying the assumption of normality.SPSS 20.0 software was used to analyze the data . The measurement tools\u2019 internal consistency was calculated using the estimated Cronbach\u2019s \u03b1. A frequency analysis was performed to analyze participants\u2019 general characteristics, and \u03c7t-test was performed to determine whether the self-esteem, covert narcissism, perfectionism, body dissatisfaction, anxiety, depression, and binge eating variables were homogeneous between the control and experimental groups. An analysis of covariance (ANCOVA) was performed to control for the pre-score difference between groups for variables that differed between groups in the pre-homogeneity test. The ANCOVA was also performed to verify the effect of the REBT program.A p\u2009=\u2009.05); therefore, the two groups were homogeneous with regard to demographic variables as well as the control group (n\u2009=\u200922) were aged 19\u201321\u2009years, at 91.7 and 72.7%, respectively. Regarding school year, 33.3% in the experimental group and 40.9% in the control group were 1st- or 2nd-year students. The percentage of students majoring in food and medicine was 41.7% and that of students majoring in non-food and non-medicine subjects was 58.3% in the experimental group; the percentages were 27.3 and 72.7%, respectively, in the control group. Regarding living arrangements, 41.7% of participants lived in their homes, 37.5% in the dorm, and 20.8% in a boarding house or other in the experimental group; the percentages were 31.8, 45.5, and 22.7%, respectively, in the control group. Regarding religion, in the experimental group, 37.5% identified themselves as religious, while 62.5% did not; in the control group, 27.3% were religious, while 72.7% were not. In the experimental group, 58.3% of participants weighed under 60\u2009kgs, and 41.7% weighed 60\u2009kgs or more, with a mean body weight of 59.05\u2009kg. In the control group, 63.6% weighed under 60\u2009kgs, and 36.4% weighed 60\u2009kgs or higher, with a mean weight of 58.30\u2009kg.Most participants in the experimental group to posttest . However, the control group\u2019s scores increased from pretest to posttest was greater relative to that in the control group\u2019s scores (1.04), but this difference was nonsignificant. The experimental groups perfectionism scores decreased from pretest to posttest . Further, the control group\u2019s scores decreased from pretest to posttest to posttest . However, the control group\u2019s scores increased from pretest to posttest to posttest , while the control group\u2019s anxiety scores increased from the pretest to the posttest to posttest . However, the control group\u2019s scores increased from the pretest to the posttest were greater relative to those in the control group (6.09). Scores decreased from pretest to posttest in the experimental group. In addition, the control group\u2019s score on binge eating decreased from pretest to posttest for binge eating behavioral measures was low, other measures should be considered in future studies. Fourth, although we considered participants\u2019 weight, more restrictive selection criteria should be considered in future. Finally, because the study was conducted using the nonequivalent control group design, the results have limited generalizability. Despite these limitations, this study is significant in that it is the first to apply REBT in a binge eating behavior intervention for young female students. Furthermore, by examining the risk factors of binge eating, it informs the direction subsequent studies may take to develop binge eating behavior intervention programs.In summary, the REBT-based binge eating management program for female college students positively altered their self-esteem, and led to reduction in covert narcissism, body dissatisfaction, anxiety, depression, and binge eating. This program was applied to female students, but we suggest that subsequent studies expand the target population according to age, sex, and degree of symptoms based on the intervention components of the program. Since the ultimate aim of this program is to prevent eating disorders, we suggest future researchers to conduct long-term follow-ups to examine whether the effects of the program persist after its completion. In addition, while a group counseling technique was used in this study, subsequent studies may develop and assess interventions using social media or mobile phone applications to enable ease of participation for those who cannot participate in person."} +{"text": "From 2016, the Government of India introduced the oral rotavirus vaccine into the national immunization schedule. Currently, two indigenously developed vaccines are included in the Indian immunization program. We report the rotavirus disease burden and the diversity of rotavirus genotypes from 2005 to 2016 in a multi-centric surveillance study before the introduction of vaccines.A total of 29,561 stool samples collected from 2005 to 2016 were included in the analysis. Stools were tested for rotavirus antigen using enzyme immunoassay (EIA). Genotyping was performed on 65.8% of the EIA positive samples using reverse transcription- polymerase chain reaction (RT-PCR) to identify the G (VP7) and P (VP4) types. Multinomial logistic regression was used to quantify the odds of detecting genotypes across the surveillance period and in particular age groups.Of the 29,561 samples tested, 10,959 (37.1%) were positive for rotavirus. There was a peak in rotavirus positivity during December to February across all sites. Of the 7215 genotyped samples, G1P[8] (38.7%) was the most common, followed by G2P[4] (12.3%), G9P[4] (5.8%), G12P[6] (4.2%), G9P[8] (4%), and G12P[8] (2.4%). Globally, G9P[4] and G12P[6] are less common genotypes, although these genotypes have been reported from India and few other countries. There was a variation in the geographic and temporal distribution of genotypes, and the emergence or re-emergence of new genotypes such as G3P[8] was seen. Over the surveillance period, there was a decline in the proportion of G2P[4], and an increase in the proportion of G9P[4]. A higher proportion of mixed and partially typed/untyped samples was also seen more in the age group 0\u201311\u2009months.This 11\u2009years surveillance highlights the high burden of severe rotavirus gastroenteritis in Indian children <\u20095\u2009years of age before inclusion of rotavirus vaccines in the national programme. Regional variations in rotavirus epidemiology were seen, including the emergence of G3P[8] in the latter part of the surveillance. Having pre-introduction data is important to track changing epidemiology of rotaviruses, particularly following vaccine introduction. Rotavirus has been a major cause of mortality among children under 5\u2009years old, with approximately 128,500 deaths globally . AccordiSince 2006, two oral rotavirus vaccines, Rotarix and RotaTeq , have been commercially available in India only in the private market, and the coverage was less than 1% , 14. ROTPre- vaccine surveillance data on the epidemiology of rotavirus gastroenteritis is crucial to understand any shifting trends after vaccine introduction. We report the findings from different phases of a national multicentre hospital-based surveillance on rotavirus gastroenteritis in children <\u20095\u2009years from 2005 to 2016, focussing on the diversity, temporal and regional variation of circulating rotavirus genotypes.During November 2005 to June 2009, 10 hospitals from 7 Indian cities were included in the Indian Rotavirus Strain Surveillance Network, with testing for rotavirus being performed at 4 regional laboratories . The stuThese multi-centric surveillance data combine the results of the earlier two surveillance studies on rotavirus gastroenteritis in Indian children <\u20095\u2009years with that of 4\u2009years of surveillance from 2012 to 2016 in up to 28 sites across India to provide a description of the overall distribution and diversity of rotavirus genotypes before the introduction of the oral rotavirus vaccine into the national immunization schedule , 17. AllChildren aged \u226459\u2009months of age hospitalized for acute gastroenteritis (AGE) for at least 6\u2009h, and treated with oral and/or intravenous rehydration, were eligible for enrolment. An episode of AGE was defined as \u22653 episodes of watery stool within a 24\u2009h period. Eligible children were recruited after obtaining written informed consent from the parent/guardian. Stool samples were collected from the recruited children within 48\u2009h of admission to hospital to rule out nosocomial infection. Children \u226560\u2009months of age and those presenting with dysentery (blood in stool) were excluded from the study.The stool samples were either transported to the testing laboratory within 2\u2009h or stored at 4\u2009\u00b0C at the site. The samples which were stored at 4\u2009\u00b0C at the sites were transported to the testing laboratory every month in boxes with ice packs. On reaching the testing laboratory, samples were aliquoted, and tested for rotavirus antigen. The aliquots were then stored at -70\u2009\u00b0C for further testing.Stool samples were screened for rotavirus antigen at the testing laboratories using commercial enzyme immunoassay (EIA) kits recommended by the WHO . 65.8% of the EIA positive samples were genotyped to identify the VP7 (G type) and VP4 (P type) genes using reverse transcription polymerase chain reaction (RT-PCR) assays following published protocols , 18\u201320. All sites submitted completed case report forms on all participants recruited in the surveillance. All the case report forms were scrutinized for completeness. The clinical and laboratory data were entered in Excel 2003 (Microsoft), and were analyzed to evaluate the proportion of rotavirus associated diarrhoea, genotype diversity (G and P types), temporal and regional variation in rotavirus genotypes across the four geographical zones from 2005 to 2016. To evaluate the prevalence of rotavirus associated diarrhoea across the four regions, the proportion of diarrhoeal stool samples positive for rotavirus was calculated by region. To evaluate the regional variation in rotavirus positivity from 2005 to 2016, the monthly proportion of rotavirus positivity by EIA in the four zones were compared.We fitted mixed effect multinomial logistic regression models with genotype as the outcome variable and G1P[8] as the reference genotype. We included the year of surveillance, region , and age groups as independent variables in the model. As outcomes, we included the G1P[8], G2P[4] and G9P[4] genotypes, each of which had an overall proportion of >\u20095% among genotyped samples. In the outcome variable, the less common genotypes were grouped under \u201cothers\u201d. In addition, mixed and untyped/partially typed samples were also included as separate categories in the regression analysis. Age was categorized into three groups; 0\u201311\u2009months, 12\u201323\u2009months, and 24\u201359\u2009months. All statistical analysis was performed using IBM SPSS Statistics for Windows, version 21 . A p- value of <\u20090.05 was considered statistically significant.The study was approved by the institutional review boards (ethics committees) of Christian Medical College , National Institute of Virology , National Institute of Cholera and Enteric Diseases , All India Institute of Medical Sciences , and the site specific ethics committees associated with each hospital.From November 2005 through June 2016, stool samples collected from 29,561 enrolled children were tested for rotavirus. Overall, 10,959 (37.1%) samples were positive for rotavirus were genotyped. During 2005 to 2012, all the EIA positive samples were genotyped. For the surveillance from 2012 to 2016 involving 28 sites, the study protocol required that every third EIA positive sample be genotyped at the four reference laboratories. However, the reference laboratory at Vellore genotyped all the EIA positive samples as the site prepared and provided panels of genotyped samples for quality assurance. Hence, an overall of 65.8% samples were genotyped during the entire surveillance period in India from 2005 to 2016. Table\u00a0The distribution of G and P types showed interesting trends during the 11\u2009years surveillance. The proportion of G1 increased from 2005 to 2014 (78% of rotavirus genotypes during 2013\u20132014), but decreased subsequently to only 35.5% in 2016. G2 showed an increasing trend during 2005 to 2007 (45.5%), but decreased gradually during the subsequent years to 16.5% during 2015\u20132016. G3 emerged during the year 2013 (0.5%), which increased to 18.6% during 2015\u20132016. G9 did not show any specific trend, with the lowest being detected during 2007\u20132008 (9.1%), and highest during 2009\u20132010 (28.6%). During 2015\u20132016, G9 contributed to 25.8% of all rotavirus genotypes. However, G12 showed an increasing trend till 2008\u20132009 (3.6% during 2005\u20132006 to 23.7% during 2008\u20132009), but decreased substantially during the following years to only 3.6% during 2015\u20132016.Of the genotyped samples, P[4], P[6], and P[8] contributed to 99.5% of the P types. P[8] was the most common P type (63.6%). P[8] showed an increasing trend from 2005 to 2014 (81.3% during 2013\u20132014), after which it decreased to 49.4% in 2016. P[4] was the second most common P type, detected in 25.3% of the genotyped samples. There was no specific trend in the distribution of P[4] during 2005 to 2016. The proportion of P[4] increased from 2005 to 2007 (46% in 2007), after which it decreased to 27% in 2009. Subsequently, the proportion increased to 43.7% in 2010. From 2010 to 2014, P[4] decreased to 10.3% of all P types, after which the proportion increased to 38.1% in 2016. P[6] was detected in 10.6% of the genotyped samples. The proportion of P[6] increased from 8.4% during 2005\u20132006 to nearly 18% during 2007\u20132009. The proportion decreased during the subsequent years to 10.6% during 2016.p\u2009<\u20090.001). However, G9P[4] showed a significant increase in the later years of the surveillance .The distribution by year of rotavirus genotypes (G and P combinations) in the four geographical regions is provided in Figs. Proportional representation of partially typed and untyped samples consistently decreased over the surveillance period , probably explained by inclusion of additional approaches to reduce untyped samples in the later years of the surveillance .p\u2009=\u20090.043; partially typed/untyped: aMOR\u2009=\u20090.91, 95% CI 0.82\u20131, p\u2009=\u20090.053).Detection of less common genotypes was significantly more common in the age group 0\u201311\u2009months . Higher representation of mixed and partially typed/untyped samples was also seen more in the age group 0\u201311\u2009months as a cause of AGE. These findings are consistent with previous Indian findings of approximately 34% (inter study variation: 19\u201350%) positivity for rotavirus diarrhoea requiring hospitalization , 23. PriFrom 2005 to 2016, there was a notable shift in the rotavirus genotypes. The G12 genotypes, particularly G12P[6] in the north and G12P[8] in the south, showed an increase till 2013 but the proportion reduced during the subsequent years. The G12 genotype was first detected in children with diarrhoea in the Philippines during 1987\u20131988, and was subsequently reported from Thailand and USA (1998\u20131999), and from India in 2003 from the eastern region \u201333. In tIn this surveillance, G9 in association with P[4], P[6], and P[8] constituted 11% of the rotavirus positive samples, G9P[4] being the most common in the northern region (8%), compared to G9P[8] in the southern region (6%). G9 rotavirus genotype was first detected during 1983\u20131984 in Philadelphia, USA, causing diarrhoea in infants . SubsequG1P[8] and G2P4] were the two most common genotypes in this surveillance, and comprised more than 50% of the genotyped samples. These two genotypes have been commonly detected in other surveillance studies in India as well , 9\u201312. A were theIn our study, the proportion of G1P[8] decreased from 2014 onwards, which coincided with the emergence of G3P[8] genotype across all the geographical regions. Such changes in the distribution of rotavirus genotypes and the emergence of new genotypes before the introduction of rotavirus vaccine into the national immunization schedule will be important to consider while evaluating the change in rotavirus epidemiology after introduction of rotavirus vaccines. A recent review on viral gastroenteritis worldwide has found G3P[8] to be one of the six most common genotypes globally, causing 90% of the rotavirus associated diarrhoea requiring medical attention .The detection of uncommon genotypes such as G1P4], G1P[6], G2P[6], G2P[8], G3P[4], G3P[6], G10P[11], G12P[4], G12P[11], etc., and a high proportion of mixed infections (ranged from 5.2% in the south to 9.1% in the north) are an indication that children probably acquire rotavirus infections from various sources, and could serve as sources of new strains globally. Similar to our study, unusual rotavirus genotypes have been reported to cause approximately 4.9% of rotavirus diarrhoea worldwide . A revie, G1P[6],The strength of this surveillance is the use of a standardized protocol for recruitment of cases. One limitation of the study is the availability of different number of sites during the different periods of surveillance. While data from northern and southern regions is available for all the years of surveillance from 2005 to 2016 , no data is available from the eastern and western regions from August 2009 to September 2013. This could have led to potential delay in the detection of G3P[8] in the eastern region, where it was first reported during 2013\u20132014 (2.7%).The study highlights the substantial burden of rotavirus gastroenteritis in Indian children <\u20095\u2009years of age before the introduction of the oral rotavirus vaccine into the national immunization schedule. The study also demonstrates the diversity of circulating rotavirus genotypes causing diarrhoea in children across the different geographical regions of India, along with the emergence of new genotypes. With the introduction of the rotavirus vaccine into the national immunization program in 2016, continued surveillance will be important to evaluate the potential change in epidemiology of rotavirus gastroenteritis and the vaccine effectiveness against a broad range of genotypes.Additional file 1: Table S1: Year wise distribution of rotavirus genotypes in the northern region from 2005 to 2016. The table contains the year wise distribution of rotavirus genotypes causing diarrhoea in children <\u20095\u2009years of age in the northern region from 2005 to 2016.Additional file 2: Table S2: Year wise distribution of rotavirus genotypes in the southern region from 2005 to 2016. The table contains the year wise distribution of rotavirus genotypes causing diarrhoea in children <\u20095\u2009years of age in the southern region from 2005 to 2016.Additional file 3: Table S3: Year wise distribution of rotavirus genotypes in the eastern region from 2005 to 2016. The table contains the year wise distribution of rotavirus genotypes causing diarrhoea in children <\u20095\u2009years of age in the eastern region from 2005 to 2016.Additional file 4: Table S4: Year wise distribution of rotavirus genotypes in the western region from 2005 to 2016. The table contains the year wise distribution of rotavirus genotypes causing diarrhoea in children <\u20095\u2009years of age in the western region from 2005 to 2016."} +{"text": "Viral infections are among the main causes of morbidity and mortality of humans; sensitive and specific diagnostic methods for the rapid identification of viral pathogens are required. Surface-enhanced Raman spectroscopy (SERS) is one of the most promising techniques for routine analysis due to its excellent sensitivity, simple and low-cost instrumentation and minimal required sample preparation. The outstanding sensitivity of SERS is achieved due to tiny nanostructures which must be assembled before or during the analysis. As for specificity, it may be provided using recognition elements. Antibodies, complimentary nucleic acids and aptamers are the most usable recognition elements for virus identification. Here, SERS-based biosensors for virus identification with oligonucleotides as recognition elements are reviewed, and the potential of these biosensors is discussed. Viral infections are among the main causes of morbidity and mortality of humans, and incur significant financial costs on health care systems. In this regard, it is necessary to develop and apply sensitive and specific diagnostic methods for the rapid identification of viral pathogens.To date, a lot of methods of virus identification have been widely used; for example, detection via host cell lysis on Petri dishes, amplification-based techniques for specific regions of viral genomes, sequencing of viral genomes, immunoassays for the detection of specific viral proteins, etc. . HoweverWith PCR, it is possible to perform quantitative, multiplex analyses, etc. ,5. HowevNext generation sequencing is a highly sensitive and specific method that makes it possible to reveal novel genomic sequences and which has a great potential as a clinical method ,7. HowevImmunoassay methods are based on antigen-antibody interactions, e.g., widely used enzyme-linked immunoassays (ELISAs); they are sensitive and much faster than the methods discussed above, but require high affinity and specific antibodies, especially in the case of multiplex analysis. Despite the relatively low cost of the equipment, most recombinant antibodies are rather expensive, which is the sole weakness of immunoassays in point-of-care diagnostics . Low-cosNowadays, there is growing interest in the development of biosensors due to their portability, high speed analysis, flexible construction, sensitivity and many others advantages which make them suitable for point-of-care applications ,11,12,132\u2013106 enhancement, and surface-enhanced Raman spectroscopy (SERS), which results in up to 108 enhancement [Due to its excellent chemical specificity and ability to provide a fingerprint-like spectrum for complex aqueous solutions, Raman spectroscopy (RS) has become one of the most promising optical techniques. RS employs simple and cheap instrumentation, requiring minimal sample preparation. Raman scattering has not found wide application with biological matter due to its inherently weak signal. However, to date, two methods to enhance the sensitivity have been employed, namely, resonance Raman effects, which provides a 10ancement ,15. It iancement . Noble mIn this review, it is necessary to consider different types of biosensors: colloid nanoparticle solutions (NPs) of different particle morphologies, nanoparticles deposited on the surface, and combined multidimensional materials .12, while the SERS signals of the analyte decays to zero at 3 nm from the \u2018hot spots\u2019 [Colloids and nanostructured surfaces have fundamentally different signal amplification mechanisms. In the case of colloid biosensors, the short-range signals are amplified due to the fact that the colloid SERS are determined by localized surface plasmons and \u2018hot spots\u2019 arising from interparticle gaps in the nanocluster. The intensity of Raman scattering decreases with an increase of the distance from the \u2018hot spot\u2019 as follows, I ~ 1/dt spots\u2019 ,19.d\u03b5 and m\u03b5 are the real parts of the dielectric constant of the dielectric and metal, respectively, and \u03bb is the wavelength of the exciting electromagnetic field.The SERS effect at the nanostructured surfaces with the interface between the metal and the dielectric layers is not associated with \u2018hot spots\u2019 ; it is cWhile the aforementioned techniques for virus detection are sensitive, they are typically time-consuming and expensive. SERS is an outstanding technique in biological applications due to its excellent sensitivity and cheapness. With recent progress in the field, it is now possible to use portable equipment for highly sensitive diagnostics outside the scientific laboratory.Here, we overview SERS-based biosensors with oligonucleotides as recognition elements for virus identification; these include nucleic acid aptamers (onward-aptamers) and oligonucleotides that are complementary to viral genomes . Aptamers are considered low-cost analogues of antibodies, so aptamer-based biosensors (onward-aptasensors) are compared with antibody-based biosensors (onward-immunosensors).Oligonucleotides are the most promising agents in bimolecular recognition for SERS applications due to their small size and the availability of a wide range of chemical modifications.Antisense oligonucleotides (ASO) are conventional recognition elements; they are complementary sequences to some unique sites of viral genomes. Analyses require the destruction of viral particles to liberate the genome; and the signal from ASO must be different from the complex between the ASO and the viral genome. ASO production is simple, as it is sufficient to sequence the genomes of target viruses and choose a unique sequence for that particular strain ,23,24.Aptamers are oligonucleotides that are capable of recognizing a specific target, e.g., a protein. Aptamers have been widely used in many applications: separation, detection, imaging, diagnostics and therapeutics ,26,27,28The following advantages make oligonucleotides well suited for SERS applications. They can be chemically synthesized and easily purified, in contrast to most proteins. Aptamers and ASO can be easily modified with a tag, facilitating conjugation with metal- or carbon-based nanostructures that are used for SERS detection . SimilarSERS-based techniques can be divided into two types: direct and indirect. Techniques without reporter molecules (direct or label-free techniques) rely on the identification of the spectrum of an analyte itself. However, direct sensing in biofluids can result in spectra that are difficult to interpret due to the different and unpredictable enhancement of components , and due6 pfu/mL . The application of modern statistical analytical methods, such as principal component analysis (PCA), facilitates the classification of viruses based solely on their intrinsic spectra. Overall, the technique reached >98% sensitivity and 100% specificity for the measles virus. Different virus strains were readily identified [The development of statistics methods of spectra analyses has allowed us to distinguish among the SERS spectra of different strains of the same virus. For example, silver nanorod arrays were used as substrates for the SERS-based detection of several pathogenic viruses such as the adenovirus, rhinovirus, and human immunodeficiency virus . Additioentified .6 pfu/mL [Au/Ag multilayered nanorod arrays have been used for the detection of influenza A virus strains H1N1, H2N2 and H3N2. These strains were distinguished at concentration of 106 pfu/mL . Similar6 pfu/mL . The basNucleic acid aptamers were used as the primary recognition elements in label-free techniques. For example, substrates with silver nanorods with immobilized polyvalent aptamers that are capable of binding the envelope protein of influenza virus were used to detect several influenza virus strains, namely A/Uruguay/716/2007 NYMC X-175C, B/Brisbane/60/2008, A/Brisbane/59/2007 IVR-148 ,45. All These initial results demonstrated that SERS could be used in combination with multivariate statistical methods for the rapid identification and classification of viruses. The results suggested that SERS permits rapid and accurate virus identification, including differentiation of a single pathogen at the strain level. One of the biggest disadvantages of SERS-based techniques is the inability to determine virus titer, because of the nonlinear SERS enhancement at high analyte concentrations and nonuniform adsorption of molecules on the nanoparticle surface, as well as the formation of irregular \u2018hot spots\u2019 that decreases signal intensity.Indirect SERS-based techniques could be applied as an analytical tool for the quantitative identification of a target substance. Specificity is provided by molecular recognition elements such as antibodies, aptamers or other specific binding molecules immobilized on the surface of sensors which selectively capture analyte molecules, placing them close to the \u2018hot spots\u2019 on the surface.The sandwich-like construction of biosensors is used in indirect SERS biosensors. The SERS signal comes from a reporter molecule, not from the analyte itself. The Raman reporter molecules should be water soluble, easily conjugated or intercalated to oligonucleotides; in addition, narrow vibrational Raman bands, high photostability, and minimal autofluorescence are preferable. Detailed information on the synthesis of SERS reporter molecules, enhancing photostability and methods of conjugation may be found in several reviews ,47,48,49A particularly convenient feature of colloid-based biosensors is that analysis may be performed in a one-pot fashion. Au and Ag nanoparticles are preferable because of their stability and remarkable plasmonic properties. Notably, the aggregation of nanoparticles could be used to detect viruses ,51. In t\u221219 M (10\u221223 mole per probe), with the ability to distinguish a single base mismatch.In addition to color change, the aggregation of nanoparticles generates plasmonic coupling between nanoparticles and, as a result, the SERS effect. Most studies with colloid nanoparticles (NP) and oligonucleotides as recognition elements are connected with the detection of viral nucleic acids. In the study of Hu et al., two types of Au NP were used. The first NP was ASO-modified; it bound to the substrate in the presence of ASO, and the second NP bound to the first, producing dendrimer-like structures . These tGene mismatches in the H1N1 influenza virus were detected using colloid Au NP nanoparticles and ASO labeled with fluorescent dye . The sigA magnetic capture-based SERS assay for viral genome detection was developed using Au-coated, paramagnetic NPs that are useful both as SERS substrates and as an effective strategy to target viral genome purification from other components . One ASOA variety of substrates have been used for virus detection. Substrate-based detection has many advantages, including simplicity, low cost and miniaturization. Several examples with ASO as recognition elements are discussed further.ASO-modified, nanostructured surfaces were used to detect the genome of the hepatitis B virus . ASO werOne more biosensor with a similar setup was developed to detect the genome of the hepatitis B virus . Ag NPs The Mirkin group reported the detection of various virus genomes including hepatitis A, hepatitis B, human immunodeficiency, Ebola, and Variola . The assThe occurrence of false positives and false negatives is an important factor affecting further applications of biosensors. Therefore, a dual control system is useful with simultaneous measurement of fluorescence and SERS . The genSimilar techniques were developed to detect the genome of the avian influenza virus H5N1 ,60. The \u22121; therefore, the spectral lines of metal carbonyls did not overlap with lines of other objects [Raman dyes can be introduced through intercalating compounds that make a complex with double-stranded DNA during the assay setup, instead of chemical modification of ASO alone. The biosensor for the detection of the Epstein-Barr viral genome in blood plasma was made using this principle . The rhe objects ,63. ThusASO are intended to capture genome fragments. In contrast, nucleic acid aptamers are capable of capturing viral proteins. The aptamers were successfully used in indirect techniques as primary and secondary recognition elements due to the possibility of extensive modification of oligonucleotides. Very few studies have been done in this field.A sandwich-like assay was used for the whole virus capture and identification using aptamers to hemagglutinin of the influenza virus. The primary aptamers were immobilized on the SERS substrates with thiol-groups. After capturing the influenza viral particles, the secondary aptamers labeled with Raman dye were added . The usaA similar technique was applied using antibodies instead of aptamers. The immunosensor was developed to detect the hepatitis B virus . PolycloDiagnostic test strips based on lateral flow assays are widely used for self-diagnosis and in medical institutions, but in some cases, they have insufficient sensitivity. This problem can be solved by using test strips as biosensors for SERS .Fu et al. proposed test strips using gold nanoparticles with functionalized ASO for the genome of the human immunodeficiency virus . A test Similar tests could be performed without SERS detection, but additional amplification of the viral genome is necessary to increase the sensitivity of the technique. In the strip system, Au NPs were hybridized with the PCR amplified genome of the hepatitis C virus in the test zone; the accumulation of the hybrids results in a red line . PortablDespite the potential power of oligonucleotide-modified SERS substrates as a biosensing tool, there are three main critical issues that need to be solved before widespread application. The first problem is the need to produce inexpensive, chemically stable and reliable SERS substrates with uniformly high enhancement and reproducibility. We have mentioned that \u2018classic\u2019 nanomaterials are widely used, providing the sensitivity and stability of SERS-biosensors, for example, metal nanoparticles, metal nanoislands or nanorods on dielectric and other nanostructured materials. SERS enhancement and reproducibility also critically depends on the substrate morphology; therefore, the development of easy and low-cost fabrication methods for biocompatible nanostructures with specific sizes, shapes, alignments, and architectures is still a great challenge. To date, electron beam lithography (EBL) is the most common way to produce highly ordered nanostructured surfaces. However, it is a time-consuming and expensive process that is unable to produce batches of substrates, thereby hampering the mass production of SERS substrates. Photolithography solves these problems and makes possible the production of a large number of substrates in one cycle, thereby reducing the cost of production. It should be noted that photolithography cannot create structural elements with gaps below the diffraction limit (~300 nm). Nevertheless, the transition to longer laser excitation wavelengths (infrared radiation) makes it possible to use photolithographic structures with submicron characteristic sizes (~500 nm) in SERS measurements, with potential applications in biosensing .The controlled creation of metal-dielectric nanostructures plays a key role in the manufacture of reproducible SERS biosensors. The mass production of nanoscale substrates is possible due to modern nanoimprint techniques. A stamp template produced using EBL has been used to create SERS-substrates. One of techniques of direct superplastic nanoimprinting of crystalline metals used a temperature mode well below the melting temperatures of the metals . SERS-biThe second issue that may enhance the sensitivity and reproducibility of SERS-based biosensors is the application of statistics methods for automatic analyses of multiparameter spectral data. For example, the projection method on latent structures with linear discriminant analyses makes it possible to create a projection model that distinguishes similar spectra . Other mAs for the specificity of identification, oligonucleotide-based biosensors exhibit great promise. ASO are able to precisely detect the pathogen strain at the genome level, whereas aptamers are useful for the identification of intact virus particles. Chemical synthesis with a wide range of modifications is an advance of oligonucleotides rather than proteins. A comparison of ASO, aptamer and antibody-based biosensors for virus detection is provided in"} +{"text": "Protein quantification techniques such as immunoassays have been improved considerably, but they have several limitations, including time\u2010consuming procedures, low sensitivity, and extrinsic detection. Because direct surface\u2010enhanced Raman spectroscopy (SERS) can detect intrinsic signals of proteins, it can be used as an effective detection method. However, owing to the complexity and reliability of SERS signals, SERS is rarely adopted for quantification without a purified target protein. This study reports an efficient and effective direct SERS\u2010based immunoassay (SERSIA) technique for protein quantification and imaging. SERSIA relies on the uniform coating of gold nanoparticles (GNPs) on a target\u2010protein\u2010immobilized substrate by simple centrifugation. As centrifugation induces close contact between the GNPs and target proteins, the intrinsic signals of the target protein can be detected. For quantification, the protein levels in a cell lysate are estimated using similarity analysis between antibody\u2010only and protein\u2010conjugated samples. This method reliably estimates the protein level at a sub\u2010picomolar detection limit. Furthermore, this method enables quantitative imaging of immobilized protein at a micrometer range. Because this technique is fast, sensitive, and requires only one type of antibody, this approach can be a useful method to detect proteins in biological samples. An immunoassay technique based on direct surface\u2010enhanced Raman spectroscopy and similarity analysis is demonstrated in this study. The proposed method relies on the sedimentation of plasmonic nanoparticles on a protein conjugated substrate, providing sub\u2010picomolar quantification and reliable imaging of proteins. Because protein biomarkers can indicate the types and progression of diseases, the detection of proteins in body fluids is important for liquid biopsy of diseases. Accordingly, many protein assay techniques, such as the enzyme\u2010linked immunosorbent assay (ELISA), fluorescence, and localized surface plasmon resonance\u2010based detection techniques, have long led significant advances in protein detection.Protein detection is of considerable interest in biomedical engineering because proteins play vital roles as byproducts and mediators of various biochemical activities.1 Thus However, weak scattering intensity of Raman scattering is a major obstacle to make difficulty in detecting proteins sensitively. For the reason, surface\u2010enhanced Raman spectroscopy (SERS) has been introduced. SERS can enhance the subtle Raman scattering signal by an electromagnetic field in a nanogap between metallic nanostructures. Among the SERS approaches, direct SERS is the simplest because it detects the intrinsic Raman signal of analytes without indispensable tagging procedures. Moreover, the direct SERS is widely known to be an ultrasensitive method capable of femtomolar detection.Recently, spectroscopic methods such as Raman spectroscopy are in great interest as promising techniques for detecting proteins. Owing to the advantage of offering molecular vibrational and rotational information, Raman spectroscopy can provide insights associated with the structural properties of proteins as compared to other extrinsic analytical methods.4 Howe However, antibodies that adhere to the SERS substrate can be physical obstacles and may extend the distance from the SERS effective area to the target protein and become interfering substances, thereby diminishing the desired signal. For this reason, antibodies have been used restrictively in direct SERS detection. Therefore, locating the target protein close to the SERS probe is essential, to minimize the interference by antibodies. 2) The spectral features of proteins are usually complex and heterogeneous for each measurement. Different measurement setups and preprocessing methods among research groups can be linked to a disaccord of intensity and spectral shape. Moreover, because several factors such as interfacial properties of nanoparticles, structural flexibility of proteins, and different adsorption of proteins reduce reproducibility, they may make the analysis of complex spectral features more difficult.However, direct SERS has major limitations regarding application to biological samples: 1) Biological samples contain numerous substances that share Raman bands with the target protein. These molecules can produce a false\u2010positive signal and interfere with the detection of the target protein signal. The target protein can be purified and isolated from the sample, but the protein purification requires a complicated process and a large amount of the sample. Therefore, an immunological technique for capturing target proteins selectively with antibodies can be applied.8 HoweFigure\u00a0Herein, we present a direct SERS\u2010based immunoassay (SERSIA) that combines the SERS technique and similarity analysis. Our method can overcome the challenges of immunological application and heterogeneity in signal patterns. Our method is based on the close contact between gold nanoparticles (GNPs) and the target protein that is selectively immobilized on an antibody\u2010coated substrate Figure\u00a01A. We s22.1g, which can be easily performed using common centrifuge devices. First, 30, 50, and 100 nm GNP colloidal solutions were centrifuged to determine the size of nanoparticles that can be precipitated by the condition. After centrifugation for 3\u00a0min, sedimentation of 30 and 50 nm GNPs was not significant and most particles remained at the supernatant . By contrast, despite the short centrifugation time, the 100\u00a0nm GNPs were well\u2010precipitated, and the supernatant changed color from red to transparent . Thus, our substrate yielded comparable enhancement effect to aggregate nanoparticles without preconcentration or cumbersome chemical treatment. Moreover, because the plasmonic nanoparticles as SERS probes were coated on the entire substrate, SERS signals could be detected from anywhere. To evaluate the uniformity of SERS detection, the SERS spectra at 2 \u00b5m intervals and random locations were observed . As a result, the substrate exhibited a constant signal intensity at the characteristic Raman bands of 4\u2010ATP, suggesting the excellent signal uniformity of our SERS substrate.To characterize the SERS substrate, we attached 4\u2010aminothiophenol (4\u2010ATP) to the surface of the GNPs and then the enhancement effect and signal uniformity were evaluated. The enhancement factor (EF) of the substrate was calculated to be 2.0 \u00d7 10reatment.11 Mor2.2Our approach to detect the intrinsic SERS signal of target proteins was to locate GNPs close to the protein conjugated on the surface of the substrate. Therefore, the GNPs must be attached to proteins coated on the substrate, without additional linkers such as primary amine or thiol groups. As a proof of concept, we performed the same coating procedure on substrates in which bovine serum albumin (BSA) was bound on the surface. Before the SERS measurements, we examined the signal stability during our measurement, as excessive irradiation from a laser can lead to fluctuations in signals due to a burning event. During 10 s of exposure time, the fluctuations in the signals were within the natural noise range . Also, because Raman bands assigned to amorphous carbon did not appear, we confirmed that there was no burning event.12\u22121 assigned to citrate covering the nanoparticle surface . Notably, the APTES\u2010BSA\u2010GNP sample exhibited a different signal pattern from the APTES\u2010GNP sample was first chosen as the antigen. When the same volume of GNP colloidal solution was used, no difference was observed in the coverage of the GNP\u2010coated area between the antibody\u2010only and EGFR\u2010immobilized samples .The purpose of our method was to detect a target protein in a complex biological sample. The easiest means of capturing a specific protein in a biological sample is through conducting antibodies. As previously described, we confirmed that GNPs could be coated on a protein\u2010conjugated substrate using the centrifugation method. Therefore, we employed the same method with the antibody\u2010only and antigen\u2013antibody samples. To bind the antibody on an APTES\u2010functionalized substrate, we utilized the method of Vashist et\u00a0al. .16 EpiTo test the ability of our method to detect the target protein selectively, we compared the spectrum with those obtained from an off\u2010target sample . To observe the tendency of the signal difference, we employed principal component analysis (PCA), which converts high\u2010dimensional data such as a spectrum to dimension\u2010reduced data, and displays the tendency of data patterns. The results revealed that the off\u2010target sample exhibited no difference with the antibody\u2010only sample on the PCA score plot, whereas the target protein\u2010immobilized sample showed a different distribution. Because a separated distribution in the PCA results indicated that the samples exhibited different tendencies in their spectral patterns, these results support the claim that our method selectively identifies signal differences in the presence of target antigens. The resulting antibody\u2010coated substrates were immersed in diluted H1666 cell lysate, which contained EGFR. PCA results against the SERS spectra of the samples showed that in the control case, the signals that appeared before and after EGFR immobilization were not separated, and their 95%\u2010confidence ellipses were overlapped is a cytoskeleton\u2010related protein and reported to abundantly exist in tumor tissues. Also, because these proteins have no chromophores in their structure, identifying their Raman signal as compared with that proteins with chromophores is considerably more difficult. Because most proteins do not have chromophores, detecting such proteins for practical application to diverse proteins is critical. To capture the proteins, the glass substrates were coated with antibodies of the proteins. In SERS characterization, both samples exhibited discernable and various Raman peaks in the fingerprint regions of organic molecules. In particular, the characteristic peaks at \u22481243 cm\u22121 and \u22481673\u2009cm\u22121, which were derived from the predominant \u03b2\u2010sheet structure in immunoglobulin G, were observed in all antibody samples. The target\u2010protein\u2010immobilized samples shared similar Raman peaks with the corresponding antibody samples, but they had different signal intensities at several peaks.We then applied our method for multi\u2010protein detection. For the purpose, we attempted to test our method using proteins that have importance in practical biological studies. Accordingly, the SERS signals of four proteins\u2014CD63, CD9, EGFR, and TUBA1C\u2014were observed in an A549 cell lysate Figure\u00a0. Basical18 EGF2.5 In particular, the Euclidean distance is one of the practical methods to evaluate the similarity. The distance can indicate the numerical similarity between the measured and reference spectral data and reference data r = , the distance (d) between them can be expressed as follows n is the entire length of spectral data. An antigen\u2010conjugated sample may produce a different signal pattern from an antibody\u2010only sample, and the Euclidean distance in the signal pattern between both samples can indicate the protein level.We quantified the amount of target protein through signal similarity analysis between the antibody\u2010only and target\u2010protein\u2010immobilized samples. This similarity can be conceived in terms of distance.21 In R2 value of 96.7%. This result suggested that the similarity\u2010based quantification of the SERSIA has a reliability comparable to the gold standard method for protein quantification.We further applied this approach to multi\u2010protein quantification in a cell lysate. The four proteins mentioned earlier were observed. Simultaneously, the indirect ELISA test was performed using the same cell lysate. To identify the correlation between our SERSIA and ELISA results, we compared the absorbance in ELISA and the similarity in SERSIA Figure\u00a0. Interes\u22121 to 2.5\u00a0ng mL\u22121. The protein level estimated using the SERSIA decreased as the concentration of treated proteins decreased for analyzing multivariate data such as spectra are widely being introduced, we expect that the accuracy and reliability of our method can be improved by combining the techniques.3We demonstrated a SERS\u2010based immunoassay by centrifugation\u2010based GNP coating and similarity\u2010analysis\u2010based quantification. We coated GNPs onto a target protein\u2010coated antibody substrate through a simple centrifugation process and obtained the SERS signal of the target protein signal. Our method showed SERS signal amplification with repeated centrifugation steps. The proposed method exhibited advanced performance in the immunological approach of direct SERS. Thus, it allows substantially more effective detection of intrinsic signals from specific proteins in biological samples. Moreover, a Euclidean distance\u2010based similarity analysis was employed to identify the protein level. The detection sensitivity was comparable to that of the ELISA, and their correlation was well matched. In addition, this method proved the feasibility of protein imaging. We expect that our SERS approach can be utilized for protein quantification and imaging in various biological fields.44, sodium citrate dihydrate, APTES, BSA, 3\u2010mercaptopropionic acid (MPA), N\u2010hydroxysuccinimide, penicillin, and streptomycin were purchased from Sigma\u2010Aldrich . 1\u2010ethyl\u20103\u2010(3\u2010(dimethylamino)propyl)carbodiimide (EDC) was purchased from Daejung Chemicals (KR). Anti\u2010CD9 (sc\u201013118), anti\u2010CD63 (sc\u20105275), and anti\u2010EGFR (sc\u2010373746) antibodies were purchased from Santa Cruz Biotechnology . RPMI 1640 and fetal bovine serum (FBS) were purchased from GE Healthcare . A cell lysis buffer (10\u00d7) and phenylmethylsulfonyl fluoride (PMSF) were purchased from Abcam (UK). EGFR lyophilized powder (E2645) for the off\u2010target test and imaging was purchased from Sigma\u2010Aldrich . GNP colloidal solutions of one optical density were purchased from nanoComposix . Full\u2010length EGFR ELISA kit was purchased from Invitrogen HAuCl\u22121), and streptomycin (100 mg mL\u22121), and were incubated at 37\u00a0\u00b0C in 5% CO2. The FBS was obtained from supernatants through ultracentrifugation overnight at 4\u00a0\u00b0C. All cells were grown to 50% confluency and incubated for 48 h. Each supernatant was collected after 48 h. The cells in the flask were harvested using a cell scraper with cold (4\u00a0\u00b0C) PBS. The PBS, including the scraped cells, was centrifuged in 500\u00d7 g, and the pellet was stored at \u221280\u00a0\u00b0C until the downstream experiment was conducted. For lysis, PMSF (1\u00a0mm) and 50\u00a0\u00b5L of 1\u00d7 diluted cell lysis buffer were mixed and added to the pellet in a 1.5 mL tube. The resulting solution was stored on ice and gently shaken every 10\u00a0min. After 30\u00a0min, the tube was sonicated for 10\u201315 s, followed by centrifugation at 14\u00a0000\u00a0rpm for 10\u00a0min. The supernatant was used for SERS characterization.Cell lines were maintained in RPMI 1640 supplemented with 10% FBS, penicillin . To conjugate BSA on the APTES\u2010functionalized substrate, the cleaned substrate was immersed in APTES (1% v/v) for 2 h and then thoroughly rinsed with deionized water (DIW). The functionalized substrate was immersed in BSA (1% w/v) that was diluted in PBS for 8 h.All substrates were prepared by cutting cover glass to a size of 0.5 \u00d7 0.5\u00a0cm and cleaned using a piranha solution (H\u22121) in the PBS was mixed with APTES (1% v/v) at a ratio of 1:1. The cleaned substrate was then incubated in the mixed antibody solution for 30\u00a0min. Following washing with PBS, the antibody\u2010immobilized substrate was blocked with BSA (1% w/v) for 30\u00a0min at 37\u00a0\u00b0C, followed by washing with excessive PBS. Subsequently, the biological sample that contained antigens was treated with the antibody\u2010immobilized substrate for 1 h at 37\u00a0\u00b0C. Following the immobilization of the antigens, the substrate was thoroughly washed with excessive PBS.For the SERSIA, antibody solution (8\u00a0\u00b5g mLm) in ethanol for 12 h. After washing, EDC and NHS (both 0.2 m in PBS) were mixed at a ratio of 1:1 v/v. Next, the MPA\u2010coated GNP substrate was incubated in the mixed EDC and NHS solution (both 0.2 m in PBS) at a ratio of 1:1 for 1 h, followed by washing with PBS. Then, the antibody (100\u00a0\u00b5g mL\u22121) in PBS was treated with the substrate for 1 h and washed. The resulting substrate was blocked with BSA (1% w/v) for 20\u00a0min and washed. Then, the antigen solution was added, and the substrate was incubated for 1 h.For the control case in which the antigens were attached to the antibody\u2010immobilized GNPs, the cleaned substrate was immersed in APTES (1% v/v) for 1 h and then washed. In addition, the GNP colloidal solution was coated on the substrate using our centrifugation method. After the substrate was washed with DIW, the surface of the GNPs was modified through incubation in MPA purchased from Zeiss. The microscope was equipped with a spectrometer (Acton SP2300) from Princeton Instruments. The SERSIA substrate was irradiated with a 1.5\u2010mW 785\u2010nm laser, and then scattered light from the substrate passed through the 785\u00a0nm filter. A cooled spectrograph detector with a resolution of 1340 \u00d7 400 pixels was used to scan the Raman spectra. For protein quantification, the acquisition time was 10 s. For protein imaging, the acquisition region was 2 \u00d7 2\u00a0\u00b5m, and the acquisition time was 5 s. The spectral signals were adjusted and denoised using chromatogram baseline estimation and denoising using the sparsity (BEADS) method.22 AllThe authors declare no conflict of interest.Supporting InformationClick here for additional data file."} +{"text": "In the total explanatory model, for more than 13% of the variance in time spent sitting = 2.267; p < 0.01), the significant predictors were secondary education and the results of the UPDRS. The patients with secondary and vocational education, those starting treatment with DA and those with a less severe degree of Parkinson\u2019s symptoms (UPDRS), spent less time sitting in a day. It is possible to identify determinants of spontaneous PA. It may elucidate consequences in terms of influence on modifiable conditions of PA and the proper approach to patients with unmodifiable PA factors.Physical activity (PA) is a factor that may have an influence on the symptoms of Parkinson\u2019s disease (PD). The aim of this study was to identify the potential determinants of spontaneous PA in a PD patient group. A total of 134 PD patients aged 65.2 \u00b1 9.2 years with a Hoehn\u2013Yahr scale score \u22644 and a Mini Mental State Examination (MMSE) score \u226524 were examined. For the study\u2019s purposes, the authors analyzed age, sex, education, history of PD, dopaminergic treatment, the severity of PD symptoms using Unified Parkinson\u2019s Disease Rating Scale (UPDRS), and Hoehn\u2013Yahr scale. Additionally, all participants were evaluated through a set of scales for specific neuropsychiatric symptoms including depression, anxiety, apathy, fatigue, and sleep disorders. A linear regression analysis was used with backward elimination. In the total explanatory model, for 12% of the variability in activity (R Parkinson\u2019s disease (PD) is one of the most common neurodegenerative diseases and the most common form of parkinsonian syndrome. It affects 1% of people older than 60 years of age and 4% of people older than 80 years of age .The evidence collected thus far indicates that one of the important non-pharmacological interventions in the course of PD is the physical activity (PA) of the patients ,4,5. EveSo far, the literature has not elucidated the determinants of physical activity or inactivity in patients with PD. The present study analyzes the impact of sociodemographic factors, clinical features of the disease, and treatment on the time patients spend participating in spontaneous PA.A total of 134 PD patients aged 65.2 \u00b1 9.2 years , treated at the outpatient Neurology Clinic of the Silesian Medical University in Katowice, were examined. The study was approved by the Bioethics Committee of the Academy of Physical Education in Katowice. All participants signed the informed consent form. PD was diagnosed based on the principles of the United Kingdom Parkinson\u2019s Disease Society Brain Bank. All other diseases in stable patients were under medical supervision. In the examined group, the basic treatment was levodopa followed, in the frequency of use, by a DA (ropinirole or piribedil) The levodopa equivalent daily dose (LED) was calculated on the basis of the conversion ratios accepted based on the literature review . The parThe study used a diagnostic survey with an authorial questionnaire for the purpose of gathering information on demographics, the clinical picture of PD, concomitant diseases, and treatment of the PD patient. The questionnaire was verified in terms of accuracy and reliability in the pilot studies preceding the main study.The severity of PD symptoms was evaluated using the Unified Parkinson\u2019s Disease Rating Scale (UPDRS) scale and the Hoehn\u2013Yahr scale . For menFor the analysis of PA, the data coming from IPAQ and MLTPTo assess the results, the following operationalization of data concerning PA was conducted: The index of time of moderate weekly activity was calculated (ITMWA), which was established by the following formula: ITMWA = /2. On the basis of an empirical percentile division of the results obtained, the patients were divided into two groups: the 33% least active (ITMWA < 45 min a week) and the 33% most active (ITMWA > 2.5 h a week), which constituted the basis to characterize inactive and active patients.Moderate weekly activity (ITMWA), which is calculated using the result of MLTPAQ and IPAQ, is an author\u2019s concept developed for this work. In previous pilot studies, this parameter was verified by the method of competent judges used verified by Kendall test .p < 0.01) and less frequent treatment initiation with dopaminergic receptor agonist (DA) (1.9% (n = 1) vs. 21.8% (n = 12); p < 0.01). In addition, the PI-G had greater difficulties performing the activities of daily living (ADLs) assessed by UPDRS II , and the PI-G more often suffered from apathy vs. 49.1% (n = 27); p < 0.05). The other analyzed factors did not reveal statistically significant differences between the PA-G and PI-G. The results of the assessment of all the factors listed using methodology tools are presented in The physically \u201cinactive\u201d PD patient group (PI-G), compared to the physically \u201cactive\u201d PD patient group (PA-G), was characterized by a longer duration of the disease compared to ropinirole, separate analyses of the DA treatment in PI-G and PA-G group have been omitted.To determine the relationship between subitems in the total score of each part of the UPDRS scale, the correlation of coefficients in both groups was calculated. In UPDRS part I, the largest correlation with total score was found for point 4 (Motivation/Initiative) in both groups, respectively ; in part II, in PA-G with turning in bed r = 0.75, while in PI-G with walking r = 0.70. In part III of the UPDRS scale in PA-G, the total score showed the highest correlation with arising from a chair (r = 0.83), and in PI-G, with agility of the left leg (r = 0.78). In UPDRS part IV in PA-G\u2014with symptoms anorexia, nausea or vomiting\u2014r = 0.76, in PI-G, with the presence of \u201coff\u201d periods, r = 0.76, which was expected.As potential factors impacting the duration of PA expressed by means of ITMWA, the following factors were taken into account: age, sex, education, type of medication initiating the therapy and the current treatment, LED, stage of disease based on the Hoehn\u2013Yahr scale, severity of movement symptoms in part III of the UPDRS scale, degree of intensification of other Parkinson\u2019s symptoms based on the UPDRS scale parts I + II + III, anxiety, depression, apathy, fatigue, and sleep disorders.After analyzing the complete model, using an analysis of linear regression with the method of backward elimination for each explained variable, the optimum model of factors affecting PA was created to explain the largest variance of data. Only those models that explained more than 10% of variability are discussed below.2 = 0.125; F(16.133) = 2.185; p < 0.01). The only significant predictor was starting therapy with DA , which was associated with a longer duration of moderate PA. While performing the analysis to determine the best predictors in accordance with the principle of step-wise approach regression, it was found that six predictors, with the two most significant being beginning the treatment with DA and the severity of the disease based on the Hoehn\u2013Yahr scale = 5.585; p < 0.001), were responsible for 17% of the variability of ITMWA = 2.267; p < 0.01). Significant predictors in this model were secondary education, aggravation of movement disorders in part III of the UPDRS scale and intensification of Parkinson\u2019s symptoms in UPDRS scale, parts I + II + III. Assuming the best solution in accordance with the principle of step-wise approach regression, it was found that six predictors, the most significant of which were vocational and secondary education, commencement of treatment with DA and intensification of mobility symptoms in part III on the UPDRS scale = 5.429; p < 0.001), were responsible for over 19% of the variability of daily time spent sitting (in a sedentary way). Less time spent sitting during the day was observed in the patients with vocational and secondary education, whose first medication for the treatment of PD was DA and who had more intensified mobility symptoms were conducted.2 = 0.151; F(3.133) = 8.85; p < 0.001). However, the only significant predictor turned out to be the start of treatment with DA, which was connected with increased duration of PA.When using the ITMWA as a dependent variable in the model, the predictors were education, duration of the disease, starting treatment with DA, severity of the disease on the Hoehn\u2013Yahr scale, and the level of depression symptoms (BDI). The analysis using the method of backward elimination showed that the optimal model takes into account only three predictors: duration of the disease, starting treatment with DA, and advancement of the disease on the Hoehn\u2013Yahr scale. This model was significant and explained more than 15% of the variance = 5.509; p < 0.001). It was found that the patients with secondary and vocational education, who started treatment with DA and those who had less intense parkinsonian symptoms , spent less time in a sedentary way during the day . The analysis conducted with the method of reverse elimination excluded only the results from the PFS\u221216 scale, causing the obtained optimal model to be significant, which explained over 19% of the variance (R the day .p < 0.05). In previous research, the authors related a higher level of education with participating in health-oriented behaviors, including PA [The overall level of PA in the examined group of patients with PD was low, which is also indicated by the results of other authors . In termuding PA .p < 0.05). Other authors also pointed to the importance of education in participating in PA in adults [The results obtained in this study may indicate that patients with higher and secondary education have a greater awareness of pro-health behaviors and ways to create a healthy lifestyle. PA in this group may be a continuation of the previously practiced healthy behaviors, including various forms of PA. In addition, participating in PA in this group may also be a result of a better understanding of the nature of the disease and non-pharmacological measures to improve health conditions. However, patients with vocational education may treat PA as a continuation of work-related activity more often. Additionally, in the model explaining almost 20% of the variability in lack of time spent participating in PA during the day and secondary and vocational education were strong predictors (n adults and thosn adults .p = 0.030). It is possible that the group of patients starting treatment with DA have less initial motor dysfunction and experience motor problems that disrupt their activity to a lesser extent. This may not be associated only with the oligosymptomatic onset of the disease but also with its mild course. At the same time, it can be assumed that with the appropriate treatment, patients who report more severe parkinsonian symptoms with greater negative effects on daily functioning receive the levodopa drug first to initiate the treatment. LED did not affect the level of PA, which was also reported by other researchers [One of the observations of the present study is that increased levels of PA were connected with the commencement of dopaminergic therapy with DA. In the model explaining nearly 20% of the variability in time spent sitting during the day, the start of dopaminergic therapy with DA was one of the four significant explanations (earchers . It shouearchers . The autOther strong predictors of the number of days with moderate PA in the model described above explained over 15% of the variance in this variable, except for education and commencement of treatment with DA, and included the continuation of treatment with DA in this group. This phenomenon seems to be connected with the analogous mechanisms related to starting therapy with DA. In turn, in the model explaining almost 20% of the variability in time spent sitting during the day, except for education and commencement of treatment with DA, the intensification of Parkinson symptoms assessed by the sum of the points from part I, II and III of the UPDRS scale was the strongest predictor in this case. Therefore, with reference to patients with PD, the time spent participating in PA becomes shorter for the benefit of time spent sitting. The result of the degree of intensification of Parkinson symptoms, both in terms of mobility and non-mobility, and the limitations in everyday functioning are generally related to increased disability in the middle-aged period, which has also been noted by other authors .In the case of average weekly PA time, the model accounting for 17% of the variability included the following significant predictors: starting treatment with DA and lower stage of Hoehn\u2013Yahr disease, which were associated with a longer duration of physical activity. In turn, among all significant factors that explained over 19% of the variance in time spent sitting during the day, the following were notable: vocational and secondary education; lesser total intensification of symptoms from parts I, II and III of the UPDRS scale and, as previously discussed, commencement of treatment with DA. All of the factors mentioned above were predicators of less time spent sitting during the day.In this study, the importance of the Hoehn\u2013Yahr scale for scientists was confirmed. The subsequent degrees of the scale reflect the symptoms determining patient mobility. The result of the Hoehn\u2013Yahr scale also depends on the presence of posture disorders, which are usually connected with distinctive motor deterioration. The Hoehn\u2013Yahr scale turned out to be more useful in the analysis of the movement aspects of the disease than Part III of the UPDRS, the paradoxically lower result of which is associated with a longer time spent sitting. This result may be related to the fact that with lower intensity of tremors or by slowing down, patients need less time to perform daily activities; hence, they can spend more time sitting, and this effect disappears when assessing the results of parts I and II of the UPDRS.Despite the absence of differences between groups in the results for parts I, III and IV of UPDRS, it was found that in parts II, III and IV, other items for PA-G and PI-G correlated with the total score of these assessments. In addition, in part II, the total scores for PA-G and PI-G differed with statistical significance. The total result in PA-G had the highest correlation with night symptoms\u2014turning in bed\u2014while in PI-G, the highest correlation was found with gait disturbances, which can limit the activity of the subjects. However, in the case of PA-G in UPDRS part III, the highest correlation was shown by arising from a chair, a symptom that only applies to movement initiation, while in PI-G the highest correlation was found for disorders of the agility left leg, which is a symptom that persists constantly during activity. On the other hand, in the case of UPDRS part IV in PA-G, it was found that non-motor treatment complication\u2014anorexia, nausea or vomiting\u2014did not significantly affect motor activity, while in PI-G, a motor treatment complication\u2014off periods predictable\u2014was found to unequivocally impair the physical activity taken.The results obtained in this study indicate the association between the selected clinical, demographic and therapeutic factors and the PA undertaken by PD patients. It may allow for better identification of the patients threatened by lack of activity and may help increase their activity levels."} +{"text": "Helical polymer\u2013based molecular systems allow naked-eye determination of chirality and enantiomeric excess of chiral amines. Chirality plays a key role in the physiological system, because molecular functionalities may drastically alter due to a change in chirality. We report herein a unique color indicator with a static helicity memory, which exhibits visible color changes in response to the chirality of chiral amines. A difference of less than 2% in the enantiomeric excess (ee) values causes a change in the absorption that is visible to the naked eyes. This was further quantified by digital photography by converting to RGB values. This system relies on the change in the tunable helical pitch of the \u03c0-conjugated polymer backbone in specific solvents and allows rapid on-site monitoring of chirality of nonracemic amines, including drugs, and the simultaneous quantitative determination of their ee values. Chirality is an important aspect in living systems. For instance, a pair of enantiomers often exhibits totally different physiological activities depending on the homochirality of the biological molecule. Therefore, rapid and reliable methods for determining the chirality (configuration) and enantiomeric excess (ee) of chiral molecules, particularly chiral drugs, are highly demanded in the pharmaceutical industry Among the promising alternatives is the direct colorimetric discrimination between enantiomers and the simultaneous determination of ee values of the target chiral compounds, based on the differences in absorbance and fluorescence emission, thereby enabling the naked-eye detection of chirality. This is, however, challenging, and successful examples of these systems are limited due to the distinct conformational scaffold of low\u2013molecular weight chiral receptors, which exhibit a linear response (1-H) into a one-handed helix upon thermal annealing. The right (P)\u2013 or left (M)\u2013handed helical conformation induced in poly-1-H by nonracemic amines could be retained, namely, \u201cmemorized,\u201d after complete removal of the chiral amines, resulting in the formation of the one-handed helical P- or M-h-poly-1-H with static helicity memory, respectively - and (R)-1-phenylethylamine (S- and R-2a), namely, M-h-poly-1-S2a and M-h-poly-1-R2a, respectively, showed completely different colors in specific solvents.We recently reported that nonracemic amines in water induced the folding of \u03c0-conjugated fluorescent poly(diphenylacetylene) (PDPA) bearing carboxy pendants (poly-ectively (28). AsHere, we report an unprecedented helical polymer\u2013based versatile color indicator that allows not only the assignment of the absolute configuration of chiral amines but also the quantitative determination of their ee values in the full range of ee. This was achieved by digital photography by converting to the RGB values . The metM-h-poly-1-H and P-h-poly-1-H with static helicity memories were prepared from optically inactive poly-1-H based on the helicity induction and memory strategy that we developed previously -4-methylmorpholinium chloride (DMT-MM) as a condensing reagent, thereby quantitatively producing diastereomeric helices M-h-poly-1-S2a and M-h-poly-1-R2a, respectively were yellow in color and showed almost the same absorption and CD spectra (M-h-poly-1-H (fig. S8), suggesting that postmodification hardly affected the original helicity memory and helix-sense excess (hse) values even after introducing the enantiomeric amide pendant groups. When dissolved in tetrahydrofuran (THF)\u2013acetone , the absorption spectrum of M-h-poly-1-R2a notably red-shifted by ~100 nm and exhibited a remarkable hypochromic effect in the aromatic absorption region (260 to 300 nm). The CD spectrum also changed remarkably, and the solution color changed from yellow to deep red for M-h-poly-1-S2a and M-h-poly-1-R2a in THF-acetone were determined to be 30.9% and 8.0%, respectively, using quinine sulfate in aqueous sulfuric acid (0.1 M) as a standard material and DMF were completely independent of the polymer concentration , thus eliminating the possibility of the color change arising due to the formation of aggregates in THF-acetone .The solutions of spectra . These sdeep red . In contt 1.0 mM as well rescence . The absP-h-poly-1-S2a and P-h-poly-1-R2a prepared from the opposite right-handed helical P-h-poly-1-H were totally opposite to those of M-h-poly-1-R2a and M-h-poly-1-S2a, respectively, and mirror-image CD spectra were obtained in THF-acetone (fig. S10). In addition, the colorimetric and spectral differences between M-h-poly-1-S2a and M-h-poly-1-R2a were highly dependent on the hse of h-poly-1-H and decreased with decreasing hse values of h-poly-1-H before its modification (fig. S11). The reactions of h-poly-1-H with R- and S-2a using DMT-MM proceeded very quickly; the reaction reached completion within ~3 min, as confirmed by infrared (IR) spectroscopy (fig. S12). After diluting the reaction mixture with chloroform, we could visually discriminate the absolute configuration of 2a, without isolating the modified polymers (movie S1). Thus, a practically useful dual-mode, on-site chiral sensor could be developed.As expected, the spectral behaviors of 2b to 2j), amino alcohols (2k to 2s), and amino acid esters (2t1 to 2t10) upon their reaction with M-h-poly-1-H . The colorimetric response from the amino acid esters notably improved by introducing bulkier ester groups, particularly, the tert-butyl and benzyl ester groups, allowing the naked-eye detection of the enantiomers at 25\u00b0C (2t7 to 2t10) (2a to 2j), amino alcohols (2k to 2s), and amino acid esters (2t1 to 2t10) with the same configuration as R2a, S2k, and L2t1, respectively, exhibited more prominent color changes than their corresponding enantiomeric counterparts after functionalization with M-h-poly-1-H except for 2q and 2r, of which the (R)-enantiomers exhibited a more prominent color change because of difference in the priority sequences (see fig. S13B). This allowed the quick assignment of the chiral amine configurations by simple visible inspection. Enantiomers of representative drug-related compounds, such as amphetamine (2j)\u2014a stimulant drug and a metabolite of other stimulant drugs, and phenylpropylamine (norpseudoephedrine and norephedrine) (2s), could also be visually discriminated exhibited almost identical absorption and CD spectra (fig. S13A), suggesting that cooperative intramolecular H-bonding between the neighboring amide pendants along the helical backbone is crucial in the colorimetric chiral discrimination by this system and chiral secondary amines cannot be applied to this method.It was possible to develop a similar assay for the naked-eye detection of chirality for various chiral amines was obtained from amide hydrogen-deuterium (H/D) exchange experiments .Direct evidence for the difference between the intramolecular H-bonding among the adjacent amide pendants in red-colored cis-cisoidal M-h-poly-1-R2a can form regular intramolecular H-bonds between the neighboring amide pendants, whereas cis-cisoidal M-h-poly-1-S2a can only partially form these intramolecular H-bonds and stretched cis-transoidal conformation (yellow-colored), respectively reflections, suggesting a columnar pseudohexagonal packing . In contrast, the yellow-colored films showed no observable reflection in the same region. These results suggested that red-colored M-h-poly-1-R2a forms \u03c0-\u03c0 stacking of the pendant phenyl rings by adopting the contracted cis-cisoidal conformation through the formation of intramolecular H-bonds between the neighboring amide groups , naphthyl (2b and 2i), cyclohexyl (2c), and benzyl (2j and 2o)] over the other small substituents on the stereogenic center, the naked-eye detection of the enantiomers of the amines was possible at 25\u00b0C in polar THF and/or less polar chloroform solvents and methyl (2k) substituent, respectively, on the stereogenic center, the naked-eye detection of the enantiomers of 2d and 2k required low-temperature measurements in less polar chloroform as anticipated (2t1 to 2t4), the methyl ester residue is bulky but hydrophilic so that the amide residues are more easily solvated, although 2t2 and 2t4 have a bulky hydrophobic iso-butyl and benzyl substituent on the stereogenic center, respectively, requiring low-temperature measurements in less polar chloroform at \u221260\u00b0C (2t2) and \u221220\u00b0C (2t4) for the naked-eye detection of the enantiomers (2t5), isopropyl (2t6), tert-butyl (2t7), and benzyl (2t8) ester groups, strongly suppresses solvation of the amide residues, which enables to form the intramolecular H-bonding networks between the neighboring amide pendants along one of the diastereomeric helical polymer backbones, thus allowing the naked-eye detection of the enantiomers in chloroform, THF, or its mixture at 25\u00b0C (These results indicate that the degree of color change (difference) between the diastereomeric amide-bound one-handed helical polymers resulting from the formation or disruption (switching on/off) of the intramolecular H-bonding networks among the pendant amides is most likely determined by a delicate balance of the polarity and the intermolecular H-bonding ability (solvation) of the solvents with the chiral amide residues, temperature, and steric effect of the hydrophobic or hydrophilic substituents on the stereogenic center of the amide pendants derived from the enantiomers of primary amines. When one-handed helical icipated because at 25\u00b0C . In polaM-h-poly-1-H) for determining the ee of chiral amine 2a. The method was based on the simple and quick functionalization of the pendant carboxy groups of M-h-poly-1-H with various proportions of 2a, ranging from 100% S-2a (S100-2a) to 100% R-2a (R100-2a), in steps of 20% ee. The absorption spectra of M-h-poly-1-2a in THF-acetone red-shifted nonlinearly with increasing R-2a content, with a clear isosbestic point at 465 nm . As anticipated, upon further addition of acetone (table S1), the ee-dependent range for visible color change shifted to the R-rich side to the contracted cis-cisoidal structure (red). The conformational change is regulated by polar solvents (acetone in this case), which affect the intramolecular H-bonds as discussed above. M-h-poly-1-2a also exhibited a similar ee-dependent visible color change in the presence of an increasing amount of aprotic polar solvents, such as methyl isobutyl ketone (MIK), DMF, dimethylacetamide (DMA), and dimethyl sulfoxide (DMSO) in THF (fig. S28); polar solvents with higher dielectric constants showed a larger shift upon the addition of a small amount of the polar solvent is possible after simple and quick functionalization of the amines with M-h-poly-1, followed by their dissolution in appropriate solvent mixtures (figs. S29 and S30).We then used our helical polymer\u2013based color indicator = 0, 6, 12] could be clearly distinguished by naked eyes using a THF-acetone mixed solvent = 90 to 100, in steps of 2] prepared by the reaction of M-h-poly-1-H with the corresponding chiral amines RX-2a (table S3) showed substantial changes in the absorption intensity at 551 nm and apparent visible color changes with respect to the ee values of 2a in THF-DMF . This allowed quantification of the difference in the ee values with high accuracy = 90 to 100, in steps of 2], the P-h-poly-1-SX2a enantiomers (table S3) displayed absorption spectral changes and color changes (fig. S32) similar to those of M-h-poly-1-RX2a. It is also to be noted that optically active M- and P-h-poly-1-H compounds reacted with nonracemic 2a in a nonselective way (table S4). The ee difference between R90 and R100 of 2a could also be visually discriminated by their fluorescence (fig. S33).Taking advantage of the unique ee-dependent color changes of accuracy . Natural2a samples, ranging from R90 to R100, were estimated from their absorption spectra based on the calibration curve. The values were very close to the ee values determined by chiral HPLC, and the errors were relatively low (fig. S34 and table S5) (RX-2a (X \u2265 90) with high accuracy from the plots of the intensities of the G (green) component , M-h-poly-1-RX2a was also prepared (table S3). Apparent absorption spectral changes were observed for M-h-poly-1-RX2a (X \u2265 98) in THF-DMF (2a (P-h-poly-1-SX2a enantiomers with the opposite helicity memory (table S3) showed identical absorption spectral changes (fig. S35). These results demonstrated that using this unique colorimetric sensor, a difference in the ee values as small as 0.5% ee, even in a sample with a very high ee (\u226598), could be detected by acquiring the absorption spectra.To investigate whether this system can detect an extremely small difference in the ee in a sample with very high ee of 22, v/v) ; the plov/v) and (trimethylsilyl)diazomethane were purchased from Sigma-Aldrich . Tetraphenyltin (Ph4Sn) and L-(\u2212)- and D-(+)-mandelic acids were obtained from Tokyo Chemical Industry . Potassium hydroxide (KOH) and 2-methoxy-4-nitrobenzoic acid were purchased from FUJIFILM Wako Pure Chemical . Optically active amines . - and -norephedrine (1S2R- and 1R2S-2s) were purchased from FUJIFILM Wako Pure Chemical. (S)- and (R)-amphetamine (S- and R-2j) - and -norpseudoephedrine (1S2S- and 1R2R-2s) (Tungsten(VI) chloride or a Bruker Avance 400 spectrometer in CDCl3, DMSO-d6, DMF-d7, CD3OD, THF-d8, THF-d8\u2013acetone-d6 , and THF-d8\u2013CDCl3 using TMS or a solvent residual peak as the internal standard. IR spectra were recorded with a JASCO Fourier Transform IR-460 spectrophotometer . Absorption and CD spectra were measured in a 1.0-mm, 10-mm, or 0.1-mm quartz cell on a JASCO V-650 spectrophotometer and a JASCO J-725 spectropolarimeter, respectively. The temperature was controlled with a JASCO PTC-348WI apparatus. The concentration of polymers was calculated on the basis of the monomer units. Size exclusion chromatography (SEC) measurements were performed with a JASCO PU-2080 liquid chromatograph equipped with an ultraviolet (UV)\u2013vis (JASCO UV-970) detector at 40\u00b0C using a Shodex KF-805L SEC column. The temperature was controlled with a JASCO CO-1560 column oven. THF was used as the eluent at a flow rate of 1.0 ml/min. The molar mass calibration curves were obtained with polystyrene standards . Photoluminescence spectra were measured on a JASCO FP-8500 spectrofluorometer. The temperature was controlled with a JASCO ETC-815 apparatus. XRD measurements were performed on Nano Viewer (RA-MICRO7HFM) . Ee values of chiral amines were determined by HPLC equipped with a photodiode array detector (JASCO MD-4010) and a CD detector (JASCO CD-4095) at room temperature . A chiral column was connected, and n-hexane\u2013dichloromethane was used as the eluent at a flow rate of 0.5 ml/min. A chiral column was also used for ee determination of 2t7\u00b7HCl. pH 1.5 HClO4 aq./CH3CN was used as the eluent at a flow rate of 0.4 ml/min. Elemental analyses were performed by the Research Institute for Instrumental Analysis of Advanced Science Research Center, Kanazawa University, Kanazawa, Japan.Nuclear magnetic resonance (NMR) spectra were taken on a JNM-ECA 500 spectrometer and DMT-MM were added to a solution of M-h-poly-1-H in a DMSO-water mixed solvent (10 ml), and the resulting mixture was stirred at room temperature for 4 hours. The resulting polymer was precipitated into a large amount of methanol-water mixture , collected by centrifugation, washed with methanol-water mixture , and then dried in vacuo at room temperature overnight to yield M-h-poly-1-S2a . The side groups of M-h-poly-1-H were completely modified with S-2a as confirmed by its 1H NMR and elemental analysis.M-h-poly-1-H with R-2a and other various R and S amines (2b to 2u and 2aMe) were performed to afford the corresponding M-h-poly-1-R2s and M-h-poly-1-S2s. The complete modifications of the side groups were confirmed by 1H NMR, IR, and elemental analyses .In the same way, modifications of M-h-poly-1-RX2a (7.5 mM) in THF were prepared in 2-ml flasks equipped with a stopcock. A 200-\u03bcl aliquot of each M-h-poly-1-RX2a stock solution was transferred to three vials using a Hamilton microsyringe. THF was completely removed under a high vacuum to give three vials containing 1.5 \u03bcmol of M-h-poly-1-RX2a. A mixed solvent THF-DMF was prepared by mixing THF (140.30 g) and DMF (39.69 g) in a bottle with a Teflon screw cap. A 3-ml aliquot of the mixed solvent was added to the vials to keep the M-h-poly-1-RX2a concentrations at 0.5 mM. The absorption spectra were taken at 25\u00b0C using a 10-mm quartz cell for each vial, and the isosbestic point was observed at 456 nm. The concentration of the polymers was corrected using the \u03b5 (molar absorptivity) value (\u03b5456 = 2.0 \u00d7 103 M\u22121\u00b7cm\u22121). The average absorption intensity at 551 nm of each M-h-poly-1-RX2a sample was determined on the basis of the results of three vials. The average absorption intensities at 551 nm were plotted versus the % ee values of the samples determined by the chiral HPLC analysis (tables S2 and S3 and fig. S32C). This relationship was used as the calibration curve for ee determination of blind unknown M-h-poly-1-RX2a (X > 90) samples using absorption intensities at 551 nm (fig. S34B) (see below). The same procedure was used for the absorption spectral measurements of P-h-poly-1-SX2a .Stock solutions of M-h-poly-1-RX2a and P-h-poly-1-SX2a were carried out in a similar way by using a mixed solvent THF-DMF , which was prepared by mixing THF (138.53 g) and DMF (41.58 g) in a bottle with a Teflon screw cap. The absorption spectra were taken at 25\u00b0C using a 10-mm quartz cell for each vial, and the isosbestic point was observed at 456 nm. The concentration of the polymers was corrected using the \u03b5 (molar absorptivity) value (\u03b5456 = 2.0 \u00d7 103 M\u22121\u00b7cm\u22121). The average absorption intensity at 545 nm of each M-h-poly-1-RX2a sample was determined on the basis of the results of three vials. The average absorption intensities at 545 nm were plotted versus the % ee values of the samples determined by the chiral HPLC analysis (tables S2 and S3 and fig. S35D).The absorption spectral measurements of RX-2a samples [sample A (RX-2aA) and sample B (RX-2aB)] were prepared by mixing R90-2a and R100-2a at random with a Hamilton microsyringe. The ee values of RX-2aA and RX-2aB were determined to be 92.9% and 96.8% ee by chiral HPLC analysis (table S5) after derivatization with 2-methoxy-4-nitrobenzoic acid into the corresponding amide compound 3a in the same way as described in the Supplementary Materials (see section S3-1). The modification of M-h-poly-1-H with RX-2aA and RX-2aB was performed in the same way as described above [THF-water mixed solvent was used instead of DMSO-water mixed solvent] to afford M-h-poly-1-RX2aA and M-h-poly-1-RX2aB.Two blind unknown M-h-poly-1-RX2aA and M-h-poly-1-RX2aB (7.5 mM) in THF were prepared in 2-ml flasks equipped with a stopcock. A 200-\u03bcl aliquot of each M-h-poly-1-RX2aA and M-h-poly-1-RX2aB stock solution was transferred to three vials using a Hamilton microsyringe. THF was completely removed under a high vacuum to give three vials containing 1.5 \u03bcmol of M-h-poly-1-RX2aA and M-h-poly-1-RX2aB. A mixed solvent THF-DMF was prepared by mixing THF (140.30 g) and DMF (39.69 g) in a bottle with a Teflon screw cap. A 3-ml aliquot of the mixed solvent was added to the vials to keep the M-h-poly-1-RX2aA and M-h-poly-1-RX2aB concentrations at 0.5 mM. The absorption spectra were taken at 25\u00b0C using a 10-mm quartz cell for each vial, and the isosbestic point was observed at 456 nm (fig. S34A). The concentration of the polymers was corrected using the \u03b5 (molar absorptivity) value (\u03b5456 = 2.0 \u00d7 103 M\u22121\u00b7cm\u22121). The average absorption intensity at 551 nm of each M-h-poly-1-RX2aA and M-h-poly-1-RX2aB sample was determined on the basis of the results of three vials (table S5). The calibration curve was obtained from the relationship between the absorption intensities at 551 nm and the ee % values of the M-h-poly-1-RX2a samples (fig. S34B) (see above). By using this calibration curve, the % ee values of RX-2aA and RX-2aB were estimated to be 92.5% and 96.3% ee, respectively (fig. S34B and table S5).Stock solutions of M-h-poly-1-R2aA, M-h-poly-1-R2aB, and M-h-poly-1-RX2a samples were taken at 25\u00b0C under white light using a digital camera (CANON EOS Kiss M). Color values as RGB of the photo images of the solutions displayed on the screen were detected by using the Digital Color Meter (Apple Inc.), which is a built-in Mac utility. Ten spots of the photo images were randomly selected, and the color values as RGB of the solutions were determined by averaging them of the 10 spots. The calibration curve was obtained from the relationship between the G (green) values and the ee % values of the M-h-poly-1-RX2a samples (fig. S34C). By using this calibration curve, the ee % values of the blind samples (M-h-poly-1-R2aA and M-h-poly-1-R2aB) were determined from the G values of the blind samples (table S5). Solution samples for calibration curves were prepared on the same day to minimize errors during the measurements.Photographs of the solutions in 10-mm quartz cells of"} +{"text": "According to French Paradox, red wine was famous for the potential effects on coronary heart disease (CHD), but the specific compounds against CHD were unclear. Therefore, screening and characterization of bioactive compounds from red wine was extremely necessary. In this paper, the multi-activity integrated strategy was developed and validated to screen, identify and quantify active compounds from red wine by using ultra high performance liquid chromatography-fraction collector (UHPLC-FC), ultra fast liquid chromatography-quadrupole-time-of-flight/mass spectrometry (UFLC-Q-TOF/MS) and bioactive analysis. UHPLC-FC was employed to separate and collect the components from red wine, which was further identified by UFLC-Q-TOF/MS to acquire their structural information. Furthermore, the active fractions were tested for antioxidant activity, inhibitory activity against thrombin and lipase activities in vitro by the activity screening kit. As the results, there were 37 fractions had antioxidant activity, 22 fractions had thrombin inhibitory activity and 28 fractions had lipase inhibitory activity. Finally, 77 active components from red wine were screened and 12 ingredients out of them were selected for quantification based on the integration of multi-activity. Collectively, the multi-activity integrated strategy was helpful for the rapid and effective discovery of bioactive components, which provided reference for exploring the health care function of food. As a nutritious drink recommended by World Health Organization (WHO), red wine was widely recognized for its role in health care. According to Ben Cao Gang Mu (Compendium of Materia Medica) record, the famous classic book of traditional Chinese medicine, red wine could treat cold coagulation and blood stasis. Modern pharmacological studies showed that red wine had the function of antioxidant, anticoagulant and lipid-lowering ,2, whichIt was widely believed that the mechanisms of CHD were the result of the interaction of many complex factors, among them the \u201cresponse to injury hypothesis\u201d had been recognized by researchers ,6, whichThe famous French paradox proved that red wine can prevent CHD . AccumulIn this study, UHPLC-FC was used to separate, collect and enrich the fractions from red wine. Then antioxidant activity, inhibitory activity against thrombin and lipase activities of all fractions were performed for evaluating activities. Furthermore, UFLC-Q-TOF/MS was used to identify the components in fractions from red wine . As the In the study, an ultra high performance liquid chromatography-photo-diode array-fraction collector (UHPLC-PDA-FC) system was used to establish multi-activity integrated strategy for screening and evaluating the potentially bioactive components from red wine. The sample was divided into sixty fractions (60 s per fraction) by UHPLC-PDA-FC. Sixty fractions were used to screen the antioxidant activity, thrombin inhibitory activity and lipase inhibitory activity. The multi-activity integrated chromatogram consisted of chromatogram and multi-targets integrated column diagram of antioxidant activity, inhibitory activity of thrombin and lipase were established and the 5H9NO2) with MS/MS fragment of 70.0647 (C4H7N) [M+H-HCOOH]+, which was detected to be D-proline [4H9N) [M+H-HCOOH]+ and 14 (C5H3N) [M+H-HCOOH]+ were based on the same regular. Therefore, peaks 11 and 14 were identified as L-valine (C5H11NO2) [6H5NO2) [6H14N4O2) with MS/MS fragment of 70.0652 [M+H-HCOOH-2NH2-CN]+, which was detected to be L-arginine [6H12O6) with fragments of 99.0087 (C4H4O3) [M-H-3H2O-C2HO]\u2212, 71.0137 (C3H4O2) [M-H-3H2O-C2HO-CO]\u2212 and 59.0139 (C2H4O2) [M-H-3H2O-C2HO-CO-CH2]\u2212. Therefore, it was identified as D-glucose. Peak 4 had mass spectrum ion at 181.072 (C6H14O6) with MS/MS experiment gave fragmentation information for 83.0143 [M-H-C3H2O3]\u2212 and 71.0140 [M-H-C3H6O3-H2O]\u2212, which could be identified as D-mannitol [6H8O7) and the MS/MS fragments were 85.0296 (C4H6O2) [M-H-2CO2-H2O]\u2212 and 59.0134 (C3H8O) [M-H-3CO2]\u2212. Therefore, it was identified as citric acid [3H6O4) [M-H-CO2]\u2212 and 87.0085 (C3H4O3) [M-H-CO2-H2O]\u2212 were observed [9H8O3) had the MS/MS fragment of m/z 119.0502 (C8H8O) [M-H-CO2]\u2212. Therefore, Peak 40 was identified as 2-hydroxycinnamic acid [7H7NO2) and the fragment ions observed included m/z 120.0433 (C6H7N) [M+H-H2O]+, 78.0339 (C6H6) [M+H-HCOOH-NH2]+, which was identified to be 4-aminobenzoic acid [12H22O11) with MS/MS fragments of 161.0459 (C6H9O5) [M-H-C6H11O5-H2O]\u2212 and 71.0137 (C3H4O2) [M-H-C9H14O8-H2O]\u2212, which was detected to be trehalose \u2212 and 59.0133 (C2H2O2) [M-H-C3H8O2]\u2212. By comparing the characteristic fragmentation pattern with the reported in references, it can be identified as D-ribose. Peaks 10 (C4H6O5) was identified as L-malic acid \u2212 and 71.0137 (C3H4O2) [M-H-H2O-CO2]\u2212. Peaks 12 (C7H10O5) was identified as shikimic acid. MS/MS fragment was 137.0225 (C7H6O3) [M-H-2H2O]\u2212. The molecular weight of peak 13 was 150.0586 (C5H11NO2S) and ion m/z 74.0243 (C3H8O2) [M+H-NH3-C2S]+ was observed in MS/MS spectra, which could be identified as L-methionine \u2212 and 73.0291 (C3H4O) [M-H-CO2-H2O]\u2212. Hence, it can be identified as succinic acid [3H5O2]\u2212) with peak 15. The other fragment was 55.0191 [M-H-C3H5O2-H2O]\u2212, which can be identified as dimethyl succinate \u2212 and 91.0298 [M-H-CO2-H2O]\u2212), 23 (135.0560 [M-H-CO2]\u2212 and 89.0194 [M-H-CO2-CH3CO]\u2212), 24 (135.0454 [M-H-CO2]\u2212 and 117.0351 [M-H-CO2-H2O]\u2212), 25 (109.0298 [M-H-CO2]\u2212 and 91.0298 [M-H-CO2-H2O]\u2212), 29 (93.0340 [M-H-CO2]\u2212), 31 (163.0400 [M-H-2CO2]\u2212 and 119.0503 [M-H-3CO2]\u2212), 32 (119.0498 [M-H-CO2]\u2212 and 93.0347 [M-H-CO2-H2O]\u2212), 33 (163.0400 [M-H-2CO2]\u2212 and 119.0498 [M-H-3CO2]\u2212), 39 (108.0212 [M-H-CO2-CH3]\u2212), 42 (193.0490 [M-H-2CO2-C2H5O]\u2212 and 117.0341 [M-H-3CO2-C2H5O-CH3-H2O]\u2212), 43 (135.0449 [M-H-CO2]\u2212), 49 (119.0527 [M-H-CO2]\u2212 and 103.0565 [M-H-CO2-H2O]\u2212), 50 (93.0470 [M-H-CO2]\u2212 and 65.0473 [M-H-CO2-CO]\u2212), 52 (123.0436 [M-H-CO2]\u2212 and 108.0212 [M-H-CO2-H2O]\u2212), 55 (119.0503 [M-H-CO2]\u2212 and 93.0347 [M-H-CO2-H2O]\u2212), 56 (123.0450 [M-H-CO2]\u2212), 60 (257.0110 [M-H-CO2]\u2212 and 229.0126 [M-H-CO2-CO]\u2212) and 61 (109.0286 [M-H-CO2-C2H2]\u2212) were the same regular, which were identified as gallic acid [11H12O4). The MS/MS fragments were the same as peak 43 of 179.0433 [M-H-CH3]\u2212 and 135.0446 [M-H-CH3-CO2]\u2212. Therefore, it was identified as ethyl caffeate [9H8O3) and the product ion was 119.0479 [M+H-HCOOH]+, which could be identified as trans-4-hydroxycinnamic acid [8H11NO) with the MS/MS fragments at 121.0616 [M+H-NH3]+ and 95.0561 [M+H-NH3-C2H4]+, which was detected to be tyramine \u2212 ion at m/z 153.0558. In MS/MS mode, the produced ions were m/z 123.0085 [C8H10O3-CH2O]\u2212 and 108.0221 [C8H10O3-CH2OH-CH3]\u2212, which can be identified as hydroxytyrosol [\u2212 in the first-order mass spectrum. They had the same prominent ion at 125.0246 [C15H14O7-C9H8O4]\u2212. They were speculated to be (\u2212)-epigallocatechin [\u2212 ion at m/z 137.0255. The product ion was 108.0213 [M-H-CHO]\u2212. Therefore, peak 26 was identified as protocatechualdehyde. Peak 27 had the mass spectrum ion at 181.0506 (C9H10O4) with MS/MS fragment of 135.0453 [M-H-CH3-H2O]\u2212, which was identified as homovanillic acid [8H8O5). The product fragment ions were 124.0234 [M-H-CO2-CH3]\u2212 and 95.0136 [M-H-CO2-CH3-CHO]\u2212. Hence, it was identified as methyl gallate [\u2212 ion at m/z 353.0877 and MS/MS produced ions were at m/z 191.0616 [M-H-C6H5O5]\u2212 and 135.0439 [M-H-C6H5O5-CO]\u2212, it can be identified as chlorogenic acid [\u2212 and 108.0212 [M-H-CH]\u2212. Peaks 36, 38, 46 and 57 had same molecular weight of C30H26O12, which illustrated that they were isomers. Base on the MS/MS spectra, peak 36 , 38 , 46 and 57 were detected, which could be identified as procyanidin B1 [15H14O6) with fragments of 245.0843 [M-H-CO2]\u2212, 203.0713 [M-H-H2O-C3O2]\u2212 and 151.0401 [M-H-C8H10O2]\u2212, therefore it was identified as catechin [7H2O4]\u2212, 151.0386 [M-H-C7H4O4-C8H10O2]\u2212), 51 , 58 , 64 (285.0413 [M-H-C6H11O5]\u2212 and 151.0041 [M-H-C14H8O7]\u2212), 66 , 67 , 68 (151.0020 [M-H-C14H16O7]\u2212), 69 (285.0413 [M-H-C6H11O5]\u2212 and 151.0041 [M-H-C14H8O7]\u2212), 70 (301.0379 [M-H-C6H10O5]\u2212 and 151.0039 [M-H-C6H10O5-C7H6O2-CO]\u2212), 72 , 73 (245.0504 [M-H-H2O-2CO]\u2212 and 151.0035 [M-H-C8H6O4]\u2212), 75 , 83 (151.0034 [M-H-C8H6O3]\u2212), 85 (316.0231 [M-H-CH3]\u2212 and 151.0016 [M-H-C9H8O4]\u2212), 86 (151.0034 [M-H-C8H8O]\u2212), 88 (151.0034 [M-H-C8H6O2]\u2212) and 91 (300.0270 [M-H-CH3]\u2212 and 151.0016 [M-H-C9H8O3]\u2212) were identified as the type of ion of 151.0037 (C7H4O4), which were the characteristic fragment of flavonoid components. Indeed, they were epicatechin gallate [9H10O5) with MS/MS ions at 182.0256 [M-H-CH3]\u2212, 167.0014 [M-H-CH3-CO2]\u2212, 138.0317 [M-H-CH3-CO2-CH3]\u2212 and 123.0090 [M-H-CH3-2CO2-CH3]\u2212, which can be identified as syringic acid [\u2212 at m/z 325.0926 with the molecular formula C15H18O8. A loss of C6H10O5 in MS/MS was confirmed to be a hexose neutral loss. The other ions were 145.0294 [M-H-C6H10O5-H2O]\u2212 and 117.0343 [M-H-C6H10O5-CO]\u2212. Peak 47 was tentatively identified as p-coumaric acid glucoside [8H14O5) and the MS/MS fragment was 71.0503 [M-H-C2H5-CO2-C2H5O]\u2212. Therefore, it was identified as diethyl malate [9H10O5-C2H4]\u2212 and 125.0239 [C9H10O5-C2H4-CO2]\u2212 were observed [6H10O5]\u2212 and 316.0553 [M-H-C6H10O5-CH3]\u2212. Hence, peak 54 was tentatively identified as malvidin-3-O-glucoside [6H10O5]\u2212 and 316.0553 [M-H-C6H10O5-2H2O-CO]\u2212), 65 (303.0510 [M-H-2C6H10O5]\u2212), 74 (317.0648 [M-H-C6H10O5]\u2212 and 302.0429 [M-H-C6H10O5-CH3]\u2212), 78 (303.0767 [M-H-C6H10O5-CO2]\u2212) and 80 (477.1196 [M-H-C9H10O3]\u2212 and 331.0874 [M-H-C9H10O3-C6H10O5]\u2212) were displayed in MS/MS spectra, which could be identified as delphinidin-3-O-glucoside [\u2212 ion at the m/z 389.1242 with MS/MS spectra ions m/z 227.0994 [M-H-C6H10O5]\u2212 and 143.0505 [M-H-C6H10O5-C2H2O-C2H2O]\u2212. Peak 59 was tentatively identified as trans-piceid [6H10O5]\u2212 and 185.0606 [M-H-C6H10O5-C2H2O]\u2212), 76 (185.0587 [M-H-C6H10O5-C2H2O]\u2212 and 143.0501 [M-H-C6H10O5-C2H2O-C2H2O]\u2212) and 84 (143.0504 [M-H-C6H10O5-C2H2O-C2H2O]\u2212) were displayed in MS/MS spectra, which can be identified as cis-piceid [21H20O12). The product fragment ions were 301.0379 [M-H-C6H10O5]\u2212, 300.0277 [M-H-C6H11O5]\u2212, 271.0245 [M-H-C6H10O5-CHO]\u2212, 243.0291[M-H-C6H10O5-CHO2]\u2212 and 151.0031 [M-H-C6H10O5-C7H6O2-CO]\u2212. Therefore, it was identified as hyperoside [21H24O10) with MS/MS fragments of 273.0769 [M-H-C6H12O6]\u2212, 179.0349 [M-H-C15H14O5]\u2212 and 167.0352 [M-H-C6H12O6-C7H6O]\u2212, which was detected to be phloridzin [15H10O7) with fragments of 285.0398 [M+H-H2O]+, 257.0456 [M+H-H2O-CO]+, 229.0492 [M+H-H2O-2CO]+ and 153.0188 [M+H-C8H5O3]+, therefore it was identified as morin [2O]\u2212 and 153.0188 [M-H-C8H6O3]\u2212,which can be identified as delphinidin [2O-CO] and 137.0233 [M-H-C8H6O3]) and 90 (229.0498 [M-H-2H2O-CO-CH3] and 153.0188 [M-H-CH3-C8H6O3]) were showed in MS/MS spectra, which can be identified as cyanidin [3]\u2212 and 315.0153 [M-H-C2H6]\u2212 [13H20O) and the fragment ion was observed at m/z 56.9419 [M+H-C10H15]+, which was identified as ionone [10H20O2) with fragment of 76.0221 [M+H-HCOOH]+, therefore it was identified as ethyl caprylate (C10H20O2). The molecular weight of peak 94 was 195.1748. MS/MS ions of m/z 177.1634 [M+H-H2O]+ and 163.0394 [M+H-H2O-CH2]+ were observed, which revealed that it was theaspirane. Moreover, the MS spectrums of all compounds were shown in The components from red wine with antioxidant activity or inhibitory potential against thrombin and lipase activities were analyzed by UFLC-Q-TOF/MS to obtain structural information. The molecular compositions of ingredients were identified according to the literature and exact molecular weight. The peak number, retention time, MS/MS fragmentation ions information and identification of 94 components from red wine were listed in -proline ,15,16. P \u00d7 100%, where Asample was the fluorescence of the reactions with added fraction, Acontrol was the control, and Acontrol sample was the fluorescence of the solvent of the fraction [In this study, the inhibition of each fraction was assayed by measuring the fluorescence intensity of 4-methylumbelliferyl oleate (4-MU) (the hydrolytic product of 4-MUO). The assay was performed according to the method described with slight modifications. Initially, the concentrations of trypsin and time were investigated to establish linear range with the concentration of 4-MUO at 0.1 mM. Then, 25 \u03bcL fractions and 25 \u03bcL lipase solutions were mixed together and 50 \u03bcL 4-MUO was added to the mixture. Finally, the amount of 4-MU was measured by microplate reader at an excitation wavelength of 355 nm and an emission wavelength of 460 nm after incubating at 25 \u00b0C for 30 min. The inhibition of pancreatic lipase activity was calculated as follows: inhibition (%) = [(Afraction ,54.The results of precision and recovery have been measured in triplicates and the stability have been measured in sextuplicate, which were expressed as mean and RSD. The content of 12 compounds have been measured in triplicates and expressed as mean.In this study, the multi-activity integrated strategy was proposed to screen, identify and quantify components from red wine for prevention of CHD via UHPLC-FC and UFLC-Q-TOF/MS. The multi-activity integrated strategy of red wine was established and verified the availability of screening the preventing CHD compounds from the complex sample. Red wine exhibited potential antioxidant activity and inhibitory activity against thrombin and lipase, which could be used to prevent CHD. In addition, the system had advantages over traditional methods in screening potential active compounds. The multi-activity integrated strategy may help to discover bioactive components rapidly and efficiently, which provided reference for exploring the health care function in food."} +{"text": "Experimental and clinical therapies in the field of Alzheimer's disease (AD) have focused on elimination of extracellular amyloid beta aggregates or prevention of cytoplasmic neuronal fibrillary tangles formation, yet these approaches have been generally ineffective. Interruption of nuclear lamina integrity, or laminopathy, is a newly identified concept in AD pathophysiology. Unraveling the molecular players in the induction of nuclear lamina damage may lead to identification of new therapies. Here, using 3xTg and APP/PS1\u00a0mouse models of AD, and in vitro model of amyloid beta42 (A\u03b242) toxicity in primary neuronal cultures and SH\u2010SY5Y neuroblastoma cells, we have uncovered a key role for cathepsin L in the induction of nuclear lamina damage. The applicability of our findings to AD pathophysiology was validated in brain autopsy samples from patients. We report that upregulation of cathepsin L is an important process in the induction of nuclear lamina damage, shown by lamin B1\u00a0cleavage, and is associated with epigenetic modifications in AD pathophysiology. More importantly, pharmacological targeting and genetic knock out of cathepsin L mitigated A\u03b242 induced lamin B1\u00a0degradation and downstream structural and molecular changes. Affirming these findings, overexpression of cathepsin L alone was sufficient to induce lamin B1\u00a0cleavage. The proteolytic activity of cathepsin L on lamin B1 was confirmed using mass spectrometry. Our research identifies cathepsin L as a newly identified lamin B1 protease and mediator of laminopathy observed in AD. These results uncover a new aspect in the pathophysiology of AD that can be pharmacologically prevented, raising hope for potential therapeutic interventions. A\u03b242 works by binding to cell membrane, interacting with receptors, forming channel\u2010like structure or by directly entering into the cells to interact intracellular targets. Treatment of A\u03b242 did not induce apoptosis, rather induced lysosomal membrane permeabilization leading to release of lysosomal cathepsins. Cathepsin L cleaves nuclear LB1 and eventually evokes nuclear deformation/invagination. This results in exposing lamin\u2010associated histones susceptible for post\u2010translational modifications. The consequences of histone modifications deserve further studies. ADAlzheimer's diseaseA\u03b242amyloid beta42CASPcaspaseCTSBcathepsin BCTSDcathepsin DCTSLcathepsin LLB1Lamin B1MEFmouse embryonic fibroblastNLnuclear lamina1Alzheimer's disease (AD) accounts for ~64% of all dementias is a dense fibrillar protein layer that is located at the interface of nuclear envelope inner layer and chromatin. NL undergoes significant changes during cell division, proliferation, and differentiation as well as in different pathological conditions or superoxide dismutase resulted in hyperphosphorylation of Tau and changes in chromatin density. Tauopathy was associated with NL damage\u2010causing nuclear envelope invagination and aberrant gene expression that is activated after diminished thioredoxin (Trx1) reducing capacity Figure and in h) Figure C, D.n\u00a0=\u00a07\u20138, p\u00a0=\u00a00.059) and cathepsin L (CTSL) proteins in 3xTg mice and CTSB Figure ; however) Figure . Despite) Figure .2.2n\u00a0=\u00a05\u20136, p\u00a0<\u00a00.0001) as detected by LB1\u00a0labeled coffee\u2010bean shaped neuronal nuclei in comparison with the controls compared to control samples Figure . This was Figure . A ~50\u00a0k2.3To further investigate the underpinning mechanisms of LB1\u00a0cleavage observed in human AD and 3xTg mouse hippocampal samples, we used an A\u03b2 neuronal toxicity model by exposing SH\u2010SY5Y neuroblastoma cells to A\u03b242. Fibrillogenesis property of A\u03b242 was assessed by Thioflavin T assay Figure B. We firAlthough the appearance of 21\u00a0kDa cleaved product of LB1 was observed in A\u03b242\u2010treated cells, there was no apparent reduction in pro\u2010LB1 compared to control cells Figures and 4a. n\u00a0=\u00a03, p\u00a0<\u00a00.01), but was effectively prevented by z\u2010FY\u2010CHO were observed to have invaginated LB1 after treating with A\u03b242, which was prevented by CTSL inhibitor (p\u00a0<\u00a00.01) Figure . This fi) Figure .2.4n\u00a0=\u00a02 independent studies, p\u00a0<\u00a00.0001) compared to the vehicle\u2010treated cells . We also noted a preferential peri\u2010nuclear localization of lysosomes as identified by LAMP2 and CTSL staining in A\u03b242\u2010treated cells than the lysosomal acidic pH 5.0 and was not notably affected at pH 7.4 Figure . Distrib) Figure C. We als) Figure D (Noonan) Figure .n\u00a0=\u00a03 experiments, p\u00a0<\u00a00.01) Figure . No dete) Figure , cross\u2010r) Figure .2.5Despite robust decrease in pro\u2010casp6\u00a0level, administration of A\u03b242 in SH\u2010SY5Y and primary neuronal cultures in this study lack of the 46\u00a0kDa LB1 fragment negated the involvement of CASP6 in cleavage of LB1. To further confirm these results, we postulated that CASP6\u00a0might be degraded in this model, and therefore aimed to examine the interaction of CTSL and CTSB with CASP6 and LB1. Purified recombinant human LB1 (rhLB1) was incubated with rhCASP6, rhCTSL, and rhCTSB and their respective inhibitors at pH 7.4 to recapitulate the potential interaction of these enzymes after lysosomal membrane damage. Our data confirmed two distinct cleavage patterns of rhLB1 for CTSL and CASP6. CTSL\u2010cleaved rhLB1 with higher efficiency and produced a different cleaved product from CASP6 Figures D,E. ThisTo investigate the lack of CASP6\u2010mediated LB1 fragment (46\u00a0kDa) in A\u03b242 toxicity, rhCASP6 was incubated with purified rhCTSL and rhCTSB in the presence and absence of z\u2010FY\u2010CHO and CA074\u2010me, respectively. Our data showed that CTSL, but not CTSB can effectively digest both pro and active forms of CASP6 Figure . Loss ofn\u00a0=\u00a03, p\u00a0<\u00a00.01) Figure D,E.n\u00a0=\u00a03, p\u00a0<\u00a00.05) Figure H,I was s) Figure . Contrare Figure . These d2.7n\u00a0=\u00a02 experiments, p\u00a0=\u00a01.1744E\u2010138) and significant increase in DNA\u2010free space (p\u00a0=\u00a05.7328E\u201017) in SH\u2010SY5Y cells , although changes in DNA\u2010free space were not statistically significant (dark granulometry: p\u00a0=\u00a00.5810) Figure .n\u00a0=\u00a03 experiments, p\u00a0<\u00a00.05). The intensity of H3K9ac staining was specifically higher in areas where LB1 integrity was compromised , histone deacetylase 1 (HDAC1) was robustly decreased (p\u00a0<\u00a00.05) in these cells but returned to normal levels in the presence of CTSL inhibitor (p\u00a0<\u00a00.05) , which was effectively lowered by CTSL inhibition (p\u00a0<\u00a00.0001) upregulated at 6\u00a0months , HAT1 , and H3K9me2 than the wild\u2010type controls. These differences disappeared in 6\u2010month\u2010old mice that coincided with a significant increase in HAT1 in AD brain tissues which was ameliorated by CTSL inhibitor and induction of epigenetic changes in nuclear chromatin. We therefore asked whether changes in CTSL\u2010LB1 axis are involved in induction of such changes. 3D confocal microscopy in immunolabeled SH\u2010SY5Y cells was used to examine the effect of A\u03b242 treatment on the extent of histone modification. Using antibodies to H3K9ac and H3K9me2 as indicators of acetylation and methylation, representing epigenetic changes, we detected significant overall increase in histone acetylation Figure . A\u03b242\u2010in) Figure . We then) Figure . A signi) Figure . These r) Figure . To checs Figure A,B. Conve Figure A,B. The e Figure . We alsos Figure C,D. Simi) Figure E.3In the present study, we have identified CTSL as an important player in regulation of NL integrity in an in vitro model of A\u03b2 toxicity. This was associated with significant structural changes that altered chromatin organization and caused histone modifications. These changes were substantially alleviated after pharmacologic and genetic downregulation of CTSL. Further support for CTSL involvement in nuclear changes was obtained in the 3xTg mouse model of AD and in human postmortem hippocampal tissue. Neuronal NL damage is a newly identified frontier in AD pathology and our discovery unraveling the key role of CTSL in degradation of LB1 is an important finding that fills the gap in the field and may provide important information for developing new therapeutic strategies.3.1The importance of NL integrity in cell's functional and structural health is mediated by its anchoring role for nuclear chromatin, providing a functional compartmentalization for gene expression. The genome is divided into smaller sub\u2010domains A and B or overexpression (Tg+/+Ctsb), did not have any major effect on LB1\u00a0degradation. In fact, the \u2212/\u2212Ctsb cells displayed the worst degradation of LB1 when exposed to A\u03b242. There are conflicting reports on the role of CTSB in A\u03b2 toxicity in AD, identifying CTSB as a protective protease by digesting A\u03b2 and reducing its accumulation in the neuronal cells system for the first time with experimental evidence. The applicability of these findings in targeting CTSL as a therapeutic approach in AD animal models will be an exciting future research direction. This will require novel methods for in vivo modulation of the neuronal CTSL/LB1 axis.44.1Reagents and antibodies used for the study are listed in Tables 4.2APPswe, PS1M146V, and Tau P301L) and show evidence of amyloid plaque and NFT formation, with early signs of mild cognitive impairment at six months of age and cathepsin B (CTSB) enzymatic activity. The remaining supernatant was treated with protease and phosphatase inhibitors for Western blot sample preparation according to our routine procedures , were maintained on a C57BL/6 background for eight generations. Only homozygous mice for the transgene and their age\u2010matched wild\u2010type controls were used for the study. The 3xTg mice express three transgene ; Size\u00a0=\u00a00.1\u20131.5\u00a0\u00b5m4.4t test was used. One\u2010way ANOVA followed by Tukey's post hoc test was performed to compare more than two experimental groups. Sample size and significance level have been indicated in the figures and legends.We used GraphPad Prism version 6.00 for Windows. For comparing two experimental groups, Student's All the authors declare no competing of interests.MII conceptualized, designed and performed the experiments, analyzed the experimental data, and wrote the first draft of the manuscript. NP performed data acquisition and interpretation, and manuscript review and editing. TS took part in counting the 3D images for invagination. FC trained MII for 3D confocal microscopy and interpret the data. SM provided the facility for 3D confocal microscopy, review and edit the manuscript. BA provided the mouse hippocampal tissue, reviewed and edited the manuscript. MDB provided the human samples for IHC and WB, interpreted the data, reviewed and edited the manuscript. JFW provided the hippocampal samples for APP/PS1\u00a0mouse. MGS and RY provided samples and Western blot data for human hippocampal samples. ISP provided A\u03b242, reviewed and edited the manuscript. EE conceptualized, designed and supervised the experiments, data acquisition, and interpretation of results, edited the final version of manuscript, and acquired funding for the project. All authors read and approved the final manuscript.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Figure S5Click here for additional data file.Figure S6Click here for additional data file.Tables S1\u2013S3Click here for additional data file."} +{"text": "Renal medullary carcinoma (RMC) is a rare but highly aggressive malignancy that affects individuals of African descent with sickle cell trait (SCT). The driver of RMC pathogenesis is thought to be renal medullary ischemia from red blood cell sickling in the setting of SCT. Currently, no modifiable risk factors for RMC have been identified that explain why certain individuals with SCT develop RMC. Prior studies have demonstrated that high-intensity exercise increases adverse events related to red blood cell sickling. We hypothesized that high-intensity exercise may increase the risk of RMC. We used multiple sources of evidence including retrospective and prospective review of RMC patient exercise activity, objective measurements of skeletal muscle surface area, and measurement of hypoxia levels in the renal medulla of mice with SCT following high or moderate-intensity exercise. Our results suggest that high but not moderate-intensity exercise may be associated with the development of RMC among individuals with SCT.Renal medullary carcinoma (RMC) is a lethal malignancy affecting individuals with sickle hemoglobinopathies. Currently, no modifiable risk factors are known. We aimed to determine whether high-intensity exercise is a risk factor for RMC in individuals with sickle cell trait (SCT). We used multiple approaches to triangulate our conclusion. First, a case-control study was conducted at a single tertiary-care facility. Consecutive patients with RMC were compared to matched controls with similarly advanced genitourinary malignancies in a 1:2 ratio and compared on rates of physical activity and anthropometric measures, including skeletal muscle surface area. Next, we compared the rate of military service among our RMC patients to a similarly aged population of black individuals with SCT in the U.S. Further, we used genetically engineered mouse models of SCT to study the impact of exercise on renal medullary hypoxia. Compared with matched controls, patients with RMC reported higher physical activity and had higher skeletal muscle surface area. A higher proportion of patients with RMC reported military service than expected compared to the similarly-aged population of black individuals with SCT. When exposed to high-intensity exercise, mice with SCT demonstrated significantly higher renal medulla hypoxia compared to wild-type controls. These data suggest high-intensity exercise is the first modifiable risk factor for RMC in individuals with SCT. SMARCB1 expression, which is a component of the SWI/SNF chromatin remodeling complex . W. WS/\u03b2S sing IVIS b,c treading IVIS d,e. Whening IVIS d, there exercise e, howevel artery . As in hGiven the lethality of RMC, it is essential to identify modifiable risk factors that can inform future prevention strategies. Our clinical observations of frequently reported high-intensity exercise among our RMC patient population led us to hypothesize that high-intensity exercise may play a critical role in the pathogenesis of RMC. Our case-control analysis supports this hypothesis by demonstrating higher reported physical activity and objectively measured SM surface area among patients with RMC compared to matched controls. Additionally, we found significantly higher proportions of military service among patients with RMC compared to the general U.S. population of black SCT individuals. Our mouse models of SCT provide additional preclinical evidence of the consequence of high-intensity exercise on renal hypoxia in the setting of SCT, particularly in the right kidney, which is most frequently affected by RMC. Our experimental GEMM results suggest that moderate-intensity exercise is not harmful and may mitigate renal hypoxia, while increasing the intensity of exercise exacerbates renal hypoxia in SCT. These findings support human studies recommending low/moderate intensity exercise training to reduce complications related to sickle hemoglobinopathies ,27.SMARCB1 tumor suppressor in particular, remains to be determined. We have previously provided a testable conceptual model of how renal medullary hypoxia induced by RBC sickling can activate low-fidelity DNA repair mechanisms that can specifically induce the SMARCB1 deletions and translocations found in RMC tissues . It . It 2]. Our GEMMs provide the first evidence of the nature of this interaction by showing that only high-intensity exercise aggravates renal hypoxia in the setting of SCT. Because mouse models of RMC have not been developed to date, the exact steps of RMC pathogenesis remain to be elucidated. It is possible that the relationship between RMC and SCT is purely associative in nature and/or that hypoxic damage in the renal medulla induced by the sickling of RBCs is not the major driver of RMC pathogenesis. In that case, an alternative confounding causal mechanism would need to explain the distinct clinical features of RMC, such as the strong association with all sickle hemoglobinopathies and the distinct predilection towards the right kidney. These features are parsimoniously explained by renal medullary hypoxia due to RBC sickling being the key event of RMC pathogenesis, as represented in From a clinical practice standpoint, our findings support the current safe exercise recommendations by the Centers for Disease Control and Prevention (CDC), the National Athletic Trainers\u2019 Association (NATA), and the National Collegiate Athletic Association (NCAA) towards athletes with SCT, their coaches, and physicians ,43,44,45The major limitations of the clinical portion of this study are its retrospective observational nature and single center design. Additionally, given the rarity of RMC, our prospective cohort included only seven patients with exercise evaluation. Motivated by the present findings, further prospective multi-institutional evaluation of the association between exercise intensity and RMC is warranted. Another limitation is that four patients with RMC were unable to be matched. Matching patients with RMC to similarly aged patients with advanced genitourinary tumors seen in the same department and time period is challenging given the young age at which RMC affects individuals. Our inferences are strengthened by the multiple approaches we used to triangulate our findings and potentially refute our hypothesis. The retrospective evaluation of physical activity would be similarly biased in the RMC and matched controls as it was evaluated using the same approach over the same time period in a single department. These subjective reports were complemented by objective measures of SM surface area, which again showed significantly higher levels among patients with RMC compared to controls. This signal persisted after controlling for confounders including race, age, gender, and smoking. We additionally considered whether cachexia could have biased our findings. If that was the case, the results would have been biased against our hypothesis, because RMC is one of the deadliest genitourinary malignancies, with sarcopenia quickly manifesting following diagnosis ,7. This We supplemented these clinical observations with mouse models developed to test our hypothesis that high-intensity exercise will increase renal medullary hypoxia. We found that renal medullary hypoxia significantly increased predominantly in the right kidney following high- but not moderate-intensity exercise, consistent with the distinct predilection of RMC toward the right kidney in humans . The majIn conclusion, our data suggest that high-intensity exercise may be a risk factor for developing RMC in individuals with SCT. Prospective multi-institutional studies are warranted to validate these findings and elucidate the impact of specific exercise regimens on RMC risk. These findings should be considered when counseling and monitoring individuals with SCT, particularly those occupationally engaged in high-intensity exercise."} +{"text": "However, the ergogenic mechanisms supporting this ingestion strategy are strongly contested. It is therefore plausible that NaHCO3 may be ergogenic by causing beneficial shifts in the strong ion difference (SID), though the time course of this blood acid base balance variable is yet to be investigated. Twelve highly trained, adolescent swimmers consumed their typical pre-competition nutrition 1\u20133 hours before ingesting 0.3 g\u2219kg BM-1 NaHCO3 in gelatine capsules. Capillary blood samples were then taken during seated rest on nine occasions to identify the time course changes in HCO3\u2013, pH, and the SID. No significant differences were found in the time to peak of each blood measure ; however, a large effect size was calculated between time to peak HCO3\u2013 and the SID (g = 0.88). Considering that a difference between time to peak blood HCO3\u2013 and the SID was identified in adolescents, future research should compare the ergogenic effects of these two individualized NaHCO3 ingestion strategies compared to a traditional, standardized approach.The timing of sodium bicarbonate (NaHCO The specific roles of these authors are articulated in the \u2018author contributions\u2019 section.\u201dIf your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.\u00a0http://journals.plos.org/plosone/s/competing-interests).\u00a0 If this adherence statement is not accurate and\u00a0 there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: \"This does not alter our adherence to\u00a0 PLOS ONE policies on sharing data and materials.\u201d :[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1:\u00a0PartlyReviewer #2:\u00a0Partly**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified.The Reviewer #1:\u00a0NoReviewer #2:\u00a0Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0The authors have done a nice job in determining the difference in time to peak pH vs. HCO3 vs. SID following ingestion of sodium bicarbonate in a group of adolescent swimmers. The methods are generally sound although I have some concerns about the interpretation and the extrapolation of these results. I hope the authors will consider my suggestions.The rationale for using adolescents is rather weak. It feels like the authors are trying to justify using this population for a reason that may not have been the initial reason for using this population. Perhaps it was just a convenience sample? I may be wrong of course, but to me it currently appears this way. Likewise, there is no issue with that in my opinion, but it is best to be clear upfront if this was the case.The limitation of no performance measure should also be highlighted here as the authors focus somewhat on the suggestion that acidosis may not be limiting to performance, but do not investigate this in any way.I believe the authors must temper statements, extrapolations and conclusions to the sample population used here (adolescents) since this may at least in part explain some of the differences shown here compared to other literature. Specific comments are below.Line 42: In my opinion, the word \u201cadequate\u201d here undersells the strength of evidence regarding SB supplementation and performance. [See line 56 where the authors themselves state \u201cstrong ergogenic benefit\u201d]Line 65: Interestingly, 60 min post-supplementation in the data of Oliveira et al. showed only a 69% likelihood of having increased >5 mmol/L of HCO3, perhaps supporting these data?Line 76-77: \u201cA concomitant increase in muscular Cl\u2013 uptake is also observed \u201d \u2013 Following SB supplementation? Please clarify.https://doi.org/10.1123/ijsnem.2020-0031 as at least one example. Interestingly, this study also showed low bicarbonate increases at a standardised timepoint in adolescents . This study does somewhat try to report some individual variation which might help the authors here.Line 97-98: This statement is incorrect. Please see Line 106: \u201cadult-like NaHCO3 absorption\u201d sounds very odd. I suggest rewording.Lines 114-119: While this is interesting information and the sample is clearly top-class, is this necessary here since there are no performance measures? Does being an athlete change the variables measured here?178-181 (and 144-146): Did the authors also perform this analysis for the group as a whole? Why would the authors believe creatine and beta-alanine supplementation would influence the responses measured here? Certainly, in my experience working with both supplements, they do not affect any of these measures at rest.Line 181-182: \u201cAdditional effect sizes were calculated where appropriate using Hedges\u2019 g bias correction [41].\u201d \u2013 Could the authors please specify exactly where and why it was considered appropriate.Line 183-184: Coefficient of variation of what? Please specify here.Line 189-190: Despite no statistical significance, mean differences of as much as 35 min are fairly large in my opinion. It will be important to discuss this in my opinion.Line 245-247: This is very surprising considering my previous comment. Again, I would like to know the author\u2019s rationale for separating these two groups. Was this decision a priori?Line 269-271: How many individuals showed a rapid decrease following peak HCO3? And what are the authors considering a rapid decrease here?Line 271-273: Certainly, in the adolescent study here this might be the case. Please restrict these claims to the population employed.Line 287-288: Is the bicarbonate absorbed?Line 289: Although technically correct that creatine could increase buffering, its actual contribution to buffering capacity is extremely low. I suggest not calling them both buffering supplements.https://pubmed.ncbi.nlm.nih.gov/21407127/) has not shown beta-alanine to blunt bicarbonate increased with sodium bicarbonate. I cannot think of a mechanism via which beta-alanine supplementation would blunt these changes. Similarly, I am unaware of why creatine supplementation would either. I think it is important to provide some physiological and mechanistic reasoning and speculation as to why this difference occurred.Line 288-291: I would be intrigued to know the authors speculation as to why this occurred. Previous work with beta-alanine and sodium bicarbonate supplementation affect the repeatability of the two studies?Line 338: This should be changed to \u201cone hour following a standardised meal\u201d since they were not ingested together.Line 351-357: I believe some context needs to be provided. Could the absolute dose provided have contributed somewhat? Mean 20 g (0.3 * 65.3 kg) vs 23 g in reference 22? The authors themselves also speculate that co-ingestion of supplements contributed somewhat to the pharmacokinetics. Additionally, these data here are in adolescents and not in adults which might also have modified the results. Thus, some of these statements should be tempered and contextualised. In fact, there is very similar data here to that shown in reference 22 (of which I am a co-author). However, I believe conclusions here should be restricted to the adolescent athlete.It is also important to consider that the study of Oliveira performed robust statistical modelling of the data, providing statistical likelihoods and probabilities that bicarbonate increases were above +5 and +6 thresholds. This is entirely different to visually stating some people do not always stay above this threshold for prolonged periods. Again, I urge some tempering of statements because the analyses here were entirely different.Line 356: Since there are some differences in opinion between groups, I would suggest the authors make it clear that it is they specifically making this recommendation here, and not the entire scientific community . Certainly, this intriguing question cannot be conclusively answered either way right now until more work is performed.Line 369-371: \u201cGiven that the effects of acidosis on exercise performance are controversial, this finding suggests that using a time to peak SID approach could be a more appropriate NaHCO3 ingestion strategy in practice.\u201d \u2013 I don\u2019t believe that these data suggest this at all since you did not measure exercise performance and compare peak SID to peak HCO3. I suggest this statement be removed.Line 371-372: This is worded rather oddly (\u201chas an any further ergogenic benefits on exercise performance\u201d). Please consider rephrasing. Likewise, further ergogenic benefit compared to what? Please specify.Line 373-374: I suggest the authors make it clearer and isolate this only to the adolescent group studied here.Figure 1. The quality is quite poor and it is difficult to make out the individual timelines . Perhaps it is just the quality of the figure as it currently stands, but I am struggling to make out many individuals who reached peak HCO3 increases followed by quick decreases (Line 269-271). Could the authors identify individuals consuming other supplements and those not?Reviewer #2:\u00a0Newbury et al. rigorously investigated the pharmacokinetics of the increase in strong ions following ingestion of 0.3 g/kg BW sodium bicarbonate in highly trained swimmers. This research group has performed some innovative research on this topic during the last years and is considered as world-leading in the domain of sodium bicarbonate ingestion to improve exercise performance. The authors should be congratulated for using a highly ecological valid design making the study applicable for athletes. My primary concerns are related to the power of the study and to the validity of the measurements obtained.\u2022 L130: The authors try to perform the study as realistic as possible. However, all measurements are performed in a rested state. It would be interesting to investigate the impact of a warming-up on the pharmacokinetics of the strong ion increase.\u2022 L135-137: Did the authors perform some correlation analyses to assess whether the macronutrient composition of the pre-exercise meal is related to the pharmacokinetics of the increase in SID?\u2022 L171: A lot of results were almost significant and effect sizes were quite large. Did the authors perform an a-priori power analysis? I am afraid that a lot of statistical effects failed to reach significance as the power of the study was too low?\u2022 L178: Why did the authors perform a paired t-test and not an unpaired t-test when comparing participants that co-ingested other supplements and those who did not?\u2022 L198 \u2013 figure 1. In the legend of figure 1 is indicated that the individual response of 5 subjects is shown. However, more than 5 individual data lines are shown in figure 1.\u2022 L216 \u2013 figure 2. It seems that one figure is missing? Figure 2 doesn\u2019t show changes in blood bicarbonate, pH and SID but shows electrolytes?\u2022 Could the authors elaborate on the replicability of the SID measurements. E.g. if you would measure a subject twice, what is the variation in blood electrolytes?**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article digital diagnostic tool,\u00a0 19 May 2021Dear Dr. Sunderland, Thank you for allowing us to resubmit our manuscript titled \u2018The time to peak blood bicarbonate (HCO3\u2013), pH, and the strong ion difference (SID) following sodium bicarbonate (NaHCO3) ingestion in highly trained adolescent swimmers\u2019 with revisions. We have carefully considered the comments from both reviewers, subsequently making substantial revisions to the manuscript to ensure both reviewers\u2019 comments have been addressed. In summary, we feel the comments have enhanced the strength of the manuscript and we thank the reviewers for both their time and expertise in contributing to the review process. In addition, we can confirm that there are no ethical restrictions on the data set that was used to inform this study, therefore an anonymised Excel spreadsheet has been included with this resubmission.As requested, we have detailed below a point-by-point response to each of the comments with reference to both the revisions we have made, and the line numbers that the revisions can be found. In the attached revised manuscript, we have highlighted any changes made in red. We hope these responses and subsequent amendments to the manuscript are well received and now meet the required standard. However, we will of course be happy to address any further recommendations that may be required. Best regards, Author team Response to Reviewers\u2019 CommentsReviewer #1 General CommentsComment: The authors have done a nice job in determining the difference in time to peak pH vs. HCO3 vs. SID following ingestion of sodium bicarbonate in a group of adolescent swimmers. The methods are generally sound although I have some concerns about the interpretation and the extrapolation of these results. I hope the authors will consider my suggestions.Response: We thank the reviewer for providing constructive comments to assist our work. Below we have attempted to address the highlighted issues within the manuscript.Comment: The rationale for using adolescents is rather weak. It feels like the authors are trying to justify using this population for a reason that may not have been the initial reason for using this population. Perhaps it was just a convenience sample? I may be wrong of course, but to me it currently appears this way. Likewise, there is no issue with that in my opinion, but it is best to be clear upfront if this was the case. Response: The lead author is currently undertaking his PhD alongside a high-performance pathway swimming club, in which research questions are being raised in response to the competitive practices observed within an adolescent cohort. From a practical perspective, these athletes can be observed partaking in suboptimal supplement practices (including NaHCO3) during competition, which could be detrimental to both health and performance. Based on the latest developments in NaHCO3 research, it is therefore necessary to first identify the pharmacokinetics of HCO3\u2013 following ingestion to best inform NaHCO3 supplement practice. Lines 90 \u2013 107 of the introduction has been amended to better justify the identification of time course changes in adolescent blood analytes following NaHCO3 ingestion. We are happy to consider further revisions on this if required.Comment: The limitation of no performance measure should also be highlighted here as the authors focus somewhat on the suggestion that acidosis may not be limiting to performance, but do not investigate this in any way. Response: Upon review of our manuscript, we understand that discussing the possible ergogenic effects may be misleading based upon the contents off the study. We have therefore made attempts to temper our arguments regarding the applicability of a time to peak SID approach in practice.Comment: I believe the authors must temper statements, extrapolations and conclusions to the sample population used here (adolescents) since this may at least in part explain some of the differences shown here compared to other literature.Response: The authors agree with this comment and have attempted to make this clearer throughout the manuscript.Reviewer #1 Specific Comments:Line 42: In my opinion, the word \u201cadequate\u201d here undersells the strength of evidence regarding SB supplementation and performance. [See line 56 where the authors themselves state \u201cstrong ergogenic benefit\u201d]Response: We have updated these statements on lines 39 and 53 to better reflect the strength of NaHCO3 supplementation.Line 65: Interestingly, 60 min post-supplementation in the data of Oliveira et al. showed only a 69% likelihood of having increased >5 mmol/L of HCO3, perhaps supporting these data?Response: Whilst this interesting data certainly supports the lack of blood HCO3\u2013difference found 60 min post-ingestion in the Boegman et al. study, we felt that adding the Oliveira et al. data here would have required further discussion point that would have significantly enhanced the word count of the present introduction. Line 76-77: \u201cA concomitant increase in muscular Cl\u2013 uptake is also observed \u201d \u2013 Following SB supplementation? Please clarify.Response: Line 76 has been updated to clarify this change.https://doi.org/10.1123/ijsnem.2020-0031 as at least one example. Interestingly, this study also showed low bicarbonate increases at a standardised timepoint in adolescents . This study does somewhat try to report some individual variation which might help the authors here.Line 97-98: This statement is incorrect. Please see Response: Thank you for bringing this study to our attention, we were unaware of this publication at the initial time of writing. Lines 90 \u2013 107 and the discussion section (completely reworked) have been amended based upon these findings.Line 106: \u201cadult-like NaHCO3 absorption\u201d sounds very odd. I suggest rewording.Response: This sentence has now been removed from the manuscript.Lines 114-119: While this is interesting information and the sample is clearly top-class, is this necessary here since there are no performance measures? Does being an athlete change the variables measured here?Response: Though this did not change the variables being measured, we included this information to better inform future studies of our participant sample. Furthermore, we hoped that that this information would add justification for the lack of placebo/control comparison since it was not possible to remove all participants from an additional training session in the short time between testing and British Championships . The reworked discussion section also references the potential early maturation of our participant sample, in which this data may be useful for comparison with adults and other adolescent cohorts.178-181 (and 144-146): Did the authors also perform this analysis for the group as a whole? Why would the authors believe creatine and beta-alanine supplementation would influence the responses measured here? Certainly, in my experience working with both supplements, they do not affect any of these measures at rest.Line 245-247: This is very surprising considering my previous comment. Again, I would like to know the author\u2019s rationale for separating these two groups. Was this decision a priori?https://pubmed.ncbi.nlm.nih.gov/21407127/) has not shown beta-alanine to blunt bicarbonate increased with sodium bicarbonate. I cannot think of a mechanism via which beta-alanine supplementation would blunt these changes. Similarly, I am unaware of why creatine supplementation would either. I think it is important to provide some physiological and mechanistic reasoning and speculation as to why this difference occurred.Line 288-291: I would be intrigued to know the authors speculation as to why this occurred. Previous work with beta-alanine and sodium bicarbonate supplementation of our study. We have amended lines 181 \u2013 182 to make this clear to the reader.Line 183-184: Coefficient of variation of what? Please specify here.Response: Lines 183 \u2013 184 have been amended to explain that this analysis was carried out for each blood analyte.Line 189-190: Despite no statistical significance, mean differences of as much as 33 min are fairly large in my opinion. It will be important to discuss this in my opinion.Response: This was the key finding from the results, however, the authors agree that this may have been overlooked somewhat in the discussion. Large parts of the discussion have been rewritten to reflect the importance of this outcome.Line 269-271: How many individuals showed a rapid decrease following peak HCO3? And what are the authors considering a rapid decrease here?Response: Based on the vagueness of this sentence, we have amended lines 291 \u2013 294 to include the number of participants that spent a particular amount of time each participant spent above the 5 \u2013 6 mmol\u2219L-1.Line 271-273: Certainly, in the adolescent study here this might be the case. Please restrict these claims to the population employed.Response: The interpretation of the study results has been tempered to an adolescent cohort in lines 294 \u2013 296.Line 287-288: Is the bicarbonate absorbed?Response: This discussion point has been moved later in the manuscript, but has been reworded to suggest \u2018NaHCO3 uptake\u2019 rather than \u2018absorption\u2019 on line 358. Line 289: Although technically correct that creatine could increase buffering, its actual contribution to buffering capacity is extremely low. I suggest not calling them both buffering supplements.Response: The authors agree with this statement; however, this section has been removed based upon the co-ingestion of supplements no longer being a feature of this manuscript.Line 302-304: I believe there may be, as mentioned previously [Comment Line 97-98].Response: We have attempted to provide an explanation for the differences between adults vs. adolescent blood analyte responses in lines 318 \u2013 343 and 381 \u2013 392.Line 315-316: The authors cannot state this since they made no comparison to not ingesting a meal. Please modify this statement, perhaps indicating simply that incidence and intensity of side-effects was low.Response: Thank you for this recommendation, we have amended lines 374 \u2013 375 to include this more appropriate statement.Line 317: What is the definition of \u201cminor\u201d here?Response: We based this definition on the participants recording less than 5/10 on the visual analogue scales. However, upon reflection, we understand that some may interpret a 4/10 rating as a \u2018moderate\u2019 severity. This statement has therefore been amended to state that the participants rated the severity at \u201cless than 5 out of 10\u201d on line 376. Line 317-319: Was the food intake prior to supplement ingestion recorded? This could provide useful and important information. Particularly when comparing the two groups separated according to creatine and beta-alanine co-supplementation.Response: Food intake was recorded, and this information has now been presented in the methods section . A correlation analysis has also been conducted upon recommendation from reviewer 2 and this has been included in the results section (lines 251 \u2013 272) and referred to during the discussion.Line 319: What are the authors referring to here with \u201cThis\u201d? The meal in general or the fact it was a high CHO meal?Response: This was a reference to the co-ingestion of a meal. A comparison could have been made to the similarity in CHO content between the present study and Carr et al. (1.2 vs. 1.5 g/kg BM CHO); however, each participant had a different fat and protein intakes. We decided not to make the comparison between CHO content within the manuscript. Line 377 \u2013 379 have now been amended to make \u201cthis\u201d clear to the reader.Line 338-339: \u201cStark difference\u201d to what? Between sessions? To data from reference 21?Response: This statement was meant in reference to the difference in reliability outcomes (excellent vs poor) found within Boegman et al. and Oliveira et al. Lines 404 \u2013 406 have been reworded to make the differences between studies clearer.Line 338-340: Could the authors please elaborate why they believe venous vs. capillary blood samples might lead to different time course responses. Likewise, why would the different time frames (180 vs 240 min) affect the repeatability of the two studies?Response: Based upon the discussion points in Oliveira et al., it appears as though the sensitivity/accuracy of venous blood samples were capable of detecting fluctuations across the sampling time frame. Whilst this is not thought to affect the time course response, the long-lasting plateaus mentioned in this study could have meant that a \u201ctrue peak\u201d could have been identified anywhere between 20 \u2013 240 min. A sampling time frame of shorter duration would therefore have shortened the window available for this fluctuation to occur, and therefore reduced the technical error between trials. We acknowledge that these are strengths of the Oliveira et al. study as opposed to criticisms, however, a venous sampling approach with a 4-hour window may not always be an appropriate strategy for athletes. Although this is an interesting avenue of discussion, we decided not to elaborate on this topic in lines 404 \u2013 411 since this is an area of research that requires much further investigationLine 338: This should be changed to \u201cone hour following a standardised meal\u201d since they were not ingested together.Response: This statement in lines 402 - 402 has now been amended to accurately represent the NaHCO3 ingestion conditions of Oliveira et al. study.Line 351-357: I believe some context needs to be provided. Could the absolute dose provided have contributed somewhat? Mean 20 g (0.3 * 65.3 kg) vs 23 g in reference 22? The authors themselves also speculate that co-ingestion of supplements contributed somewhat to the pharmacokinetics. Additionally, these data here are in adolescents and not in adults which might also have modified the results. Thus, some of these statements should be tempered and contextualised. In fact, there is very similar data here to that shown in reference 22 (of which I am a co-author). However, I believe conclusions here should be restricted to the adolescent athlete.It is also important to consider that the study of Oliveira performed robust statistical modelling of the data, providing statistical likelihoods and probabilities that bicarbonate increases were above +5 and +6 thresholds. This is entirely different to visually stating some people do not always stay above this threshold for prolonged periods. Again, I urge some tempering of statements because the analyses here were entirely different.Line 356: Since there are some differences in opinion between groups, I would suggest the authors make it clear that it is they specifically making this recommendation here, and not the entire scientific community . Certainly, this intriguing question cannot be conclusively answered either way right now until more work is performed.Line 356: Since there are some differences in opinion between groups, I would suggest the authors make it clear that it is they specifically making this recommendation here, and not the entire scientific community . Certainly, this intriguing question cannot be conclusively answered either way right now until more work is performed.Response: The majority of the discussion section has been restructured and reworded in an attempt to temper the statements made in the original manuscript. We have provided more discussion surrounding the potential differences in adult vs adolescent physiology that may have affected our results, therefore limiting our claims to the population that was studied. We accept reviewer 1\u2019s concerns and hope that the amended manuscript has satisfactorily addressed the above issues. However, we are happy to further amend based upon recommendation.Line 369-371: \u201cGiven that the effects of acidosis on exercise performance are controversial, this finding suggests that using a time to peak SID approach could be a more appropriate NaHCO3 ingestion strategy in practice.\u201d \u2013 I don\u2019t believe that these data suggest this at all since you did not measure exercise performance and compare peak SID to peak HCO3. I suggest this statement be removed.Response: This statement has been tempered to suggest that a time to peak SID approach should be tested versus a time to peak HCO3\u2013 approach to determine whether a difference exists in performance (lines 417 \u2013 420).Line 371-372: This is worded rather oddly (\u201chas an any further ergogenic benefits on exercise performance\u201d). Please consider rephrasing. Likewise, further ergogenic benefit compared to what? Please specify.Response: We have reworded this sentence in lines 418 \u2013 420 to state: \u201cfuture studies should seek to identify NaHCO3 dosing strategies based upon time to peak SID could therefore provide a more optimal approach to supplementation compared to strategies based upon an increase in blood HCO3\u2013\u201c.Line 373-374: I suggest the authors make it clearer and isolate this only to the adolescent group studied here.Response: This has been attempted throughout the manuscript.Figure 1. The quality is quite poor and it is difficult to make out the individual timelines . Perhaps it is just the quality of the figure as it currently stands, but I am struggling to make out many individuals who reached peak HCO3 increases followed by quick decreases (Line 269-271). Could the authors identify individuals consuming other supplements and those not?Response: Based on this feedback, we have changed the figures to represent the vastly different time course changes in HCO3\u2013 and the SID of four different participants. Reviewer #2: Newbury et al. rigorously investigated the pharmacokinetics of the increase in strong ions following ingestion of 0.3 g/kg BW sodium bicarbonate in highly trained swimmers. This research group has performed some innovative research on this topic during the last years and is considered as world-leading in the domain of sodium bicarbonate ingestion to improve exercise performance. The authors should be congratulated for using a highly ecological valid design making the study applicable for athletes. My primary concerns are related to the power of the study and to the validity of the measurements obtained.Response: The authors would like to thank reviewer 2 for their kind words and feedback. We would also like to extend our gratitude for noticing a key error in statical analysis (using a paired samples t-test on independent groups) that has completely reshaped the discussion following the study results. Each of the below comments has been addressed to a level that we deemed satisfactory; however, we are open to further recommendations in order to strengthen the robustness of this investigation.L130: The authors try to perform the study as realistic as possible. However, all measurements are performed in a rested state. It would be interesting to investigate the impact of a warming-up on the pharmacokinetics of the strong ion increase.Response: The inclusion of a warm-up has recently been shown to have a significant effect on the time and magnitude of HCO3\u2013 following NaHCO3 ingestion , therefore it certainly would be interesting to see the possible changes that occur in the SID. The next step following this research is to investigate the time to peak SID approach on exercise performance (which is currently in progress \u2013 COVID dependant), where the effects of a warm-up would become apparent. However, a pre-determined time to peak is first required at rest prior to this approach being used in a performance situation. This highlights a limitation of personalised dosing strategies since further testing may be necessary in practice, which in turn increases the logistical and financial burden on the swimmers/swimming club. Though we could not find a suitable place to include this discussion point, we have made a reference that warm-up may have an effect in line 411.L135-137: Did the authors perform some correlation analyses to assess whether the macronutrient composition of the pre-exercise meal is related to the pharmacokinetics of the increase in SID?Response: We have included a correlation analysis based upon your request. This has been included in the results section on lines 251 \u2013 271, and used for discussion points later in the manuscript.L171: A lot of results were almost significant and effect sizes were quite large. Did the authors perform an a-priori power analysis? I am afraid that a lot of statistical effects failed to reach significance as the power of the study was too low?Response: Our sample size was determined by the availability of highly trained adolescent swimmers that volunteered and provided parental consent to participate in this research (n = 12). This sample size was deemed appropriate based upon previous NaHCO3 dose response research , especially when considering the novelty of the participant cohort. Though we did not perform an a-priori power calculation, Jones et al. (2016) stated \u201cbased on an a priori power calculation ; a minimum of 12 participants were required to achieve 95% power at p < .01\u201d. Nonetheless, the authors agree that the statistical power of the study may have been too low, which prompted the inclusion of bias corrected (Hedges g) effect sizes to discuss changes on most occasions. We also attempted to include as much individual data as possible to be transparent with our results, since we understand that there is a possibility of type I and type II errors occurring with a sample size of n = 12. L178: Why did the authors perform a paired t-test and not an unpaired t-test when comparing participants that co-ingested other supplements and those who did not?Response: This was a genuine error that has not been detected on peer review on multiple occasions. We would like to thank the reviewer for bringing this to our attention since this has significantly altered the outcome of the supplement vs. non-supplement data. Based on these results, we have significantly altered the content included within the results and discussion sections.L198 \u2013 figure 1: In the legend of figure 1 is indicated that the individual response of 5 subjects is shown. However, more than 5 individual data lines are shown in figure 1.L216 \u2013 figure 2: It seems that one figure is missing? Figure 2 doesn\u2019t show changes in blood bicarbonate, pH and SID but shows electrolytes?Response: The authors made a late change to the figures in order to reduce the number of figures presented in the manuscript. However, this change was mistakenly not reflected in the figure titles. These have now been amended in the results section.Comment: Could the authors elaborate on the replicability of the SID measurements. E.g. if you would measure a subject twice, what is the variation in blood electrolytes?Response: Based upon the novelty of this measurement, and the time limitations of the participant cohort, this was unable to be provided for the present study. Previous work by Gough et al. (2017) has shown that the replicability of blood sodium changes following NaHCO3 ingestion is poor, however, which may suggest that blood electrolyte changes may not be reliable. Considering that a time difference may exist between time to peak HCO3\u2013 and the SID, this study could act as a catalyst for future research to determine whether an approach based upon the peak SID is actually viable in applied practice. Though we have not discussed this directly, a recommendation for further research regarding repeatability of the data is included in lines 393 \u2013 411. 17 Jun 2021The time to peak blood bicarbonate (HCO3\u2013), pH, and strong ion difference (SID) following sodium bicarbonate (NaHCO3) ingestion in highly trained adolescent swimmersPONE-D-21-06382R1Dear Dr. Sparks,We\u2019re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you\u2019ll receive an e-mail detailing the required amendments. When these have been addressed, you\u2019ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they\u2019ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Caroline SunderlandAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the \u201cComments to the Author\u201d section, enter your conflict of interest statement in the \u201cConfidential to Editor\u201d section, and submit your \"Accept\" recommendation.Reviewer #1:\u00a0All comments have been addressedReviewer #2:\u00a0All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified.The Reviewer #1:\u00a0NoReviewer #2:\u00a0Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0I congratulate the authors on an excellent response to my concerns and suggestions and thank them for treating them with consideration. I believe the updated version of the manuscript reads excellently and has benefitted from the updated analysis and removal of the supplementation subgroups. Congratulations on the work!I have a couple of minor edits below that the authors may wish to consider, but they are just related to wording or sentence structure.Line 252: I believe this should read, \u201cThe total energy content of the pre-ingestion MEAL also had a moderate\u2026\u201dLine 327: I believe this should read, \u201c\u2026capacity TO increase blood\u2026\u201dLines 411-414: This sentence reads rather awkwardly. Perhaps restructure the sentence to something more along the lines of: \u201cGiven that the effects of acidosis on exercise performance are controversial, future studies should seek to identify WHETHER NaHCO3 dosing strategies based upon a peak SID concentration could be more ergogenic than standardised ingestion strategies, or COMPARED TO individualised approaches based upon a time to peak blood HCO3\u2013.\u201dReviewer #2:\u00a0All my previous concerns have been successfully addressed by the authors. I thank the authors for these adjustments.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article , pH, and the strong ion difference (SID) following sodium bicarbonate (NaHCO3) ingestion in highly trained adolescent swimmers The time to peak blood bicarbonate (HCODear Dr. Sparks:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Caroline Sunderland Academic EditorPLOS ONE"} +{"text": "Evidence from in-vivo and in-vitro studies demonstrated stimulatory or inhibitory roles in mitral valve prolapse development, aortic leaflet fusion, and calcification pathways, specifically osteoblastic differentiation and transcription factors modulation. Tissue expression assessment and comparison between physiological and pathological phenotypes of different disease entities, including mitral valve prolapse and mitral chordae tendineae rupture, emerged as the best strategies to address miRNAs over or under-representation and thus, their impact on pathogeneses. In this review, we discuss the fundamental intra- and intercellular signals regulated by miRNAs leading to defects in mitral and aortic valves, congenital heart diseases, and the possible therapeutic strategies targeting them. These miRNAs inhibitors are comprised of antisense oligonucleotides and sponge vectors. The miRNA mimics, miRNA expression vectors, and small molecules are instead possible practical strategies to increase specific miRNA activity. Advantages and technical limitations of these new drugs, including instability and complex pharmacokinetics, are also presented. Novel delivery strategies, such as nanoparticles and liposomes, are described to improve knowledge on future personalized treatment directions. They usIn aortic stenosis, miRNAs were also found to be altered in stenotic aortic valve leaflets compared to insufficient ones by Nigam et al. . The redSeveral studies have then recorded an important association of miRNAs with post-transcriptional regulation of gene expression in aortic valve stenosis. miR-141 was found to be a regulator of the levels of bone morphogenetic protein 2 (BMP-2), whereby unrestrained activity led to calcification of the aortic valve mediated by a stimulation of osteogenesis. miR-141 was markedly attenuated in patients with aortic stenosis associated with the bicuspid aortic valve . YanagawZhang et al. demonstrated the role of miR-30b in reducing osteoblast differentiation activity induced by bone morphogenetic protein 2 . The latSong et al. analysed the progression of calcified aortic valve disease and suggested the crucial role of myofibroblastic and osteoblast-like phenotypes . The autPlasma levels of miRNAs have been used to monitor degenerative disease of the mitral valve (MV) but have not been widely adopted by the cardiology community ,9,10,11.Mitral valve prolapse is a debilitating disease with a worldwide prevalence of 2\u20133% ,21. In yMitral valve prolapse severity can be assessed by transthoracic echocardiography. Although the information provided by 2D and 3D echography yields a comprehensive assessment ,27,28, tThe landmark study by Mayeux et al. has highlighted the emergence of biomarkers as an important tool but there is still no consensus for using specific circulating biomarkers for mitral valve prolapse diagnosis in clinical practice .The degenerative process involving the mitral valve has been addressed by several investigators looking for circumstantial associations between the presence of biomarkers and the histopathological alteration. Songia et al. suggested the possible association between osteoprotegerin and mitral valve degeneration in Barlow syndrome without recording a specific correlation ,15. Tan Calcium phosphate crystals are responsible for abnormal accumulation in either native or prosthetic valves, leading to valvular calcification (VC), loss of elasticity, and ischemic conditions . IntimalSeveral coding genes have been studied and linked to the specific development of calcific valves. Osteopontin (OPN), osteocalcin (OC), bone morphogenetic proteins (BMPs), alkaline phosphatase (Alp), and transcription factor Runx2 were demonstrated to be upregulated in calcific processes . Other dPro-inflammatory activity has also been related to VC. Tumor necrosis factor-alpha (TNFa), interleukins including IL-1B and IL-6, tumor growth factor-beta 1 (TGFB1), and other cytokines mediate vascular smooth muscle cells transition into osteoblast-like cells . The pheMicroRNAs, with the exception of miR-29, have been categorized into either activating or promoting valvular calcification. Some authors claim miR-29a/b repression is a pivotal factor in calcification generation. A preclinical study by Du et al. revealed how increased expression of ADAMTS-7 , caused marked upregulation of calcifying rat vascular smooth muscle cells, and was linked to miR-29 inhibition . CalcifiRecently, Lin et al. demonstrated miR-34c/5p to be downregulated in calcific tissues (in vitro high glucose-induced human aorta VSMCs) and inhibiting BMF as the primary target . In factRecently, investigators have provided proof of upregulated levels of miRNAs in calcific vessels. An in vitro demonstration of human and murine aortic tissue and, specifically, of smooth muscle cells expressing miR-29b at increased levels comes from a Japanese investigation and a SpCalcium deposition in human VSMCs favored by phosphorus (Ph) was evaluated by Sudo et al. to determine the impact of miR-29. Real-time quantitative PCR (RT-qPCR) analysis on Pi-induced calcific VSMCs was performed and showed decreased levels of elastin with consequent osteoblast-related genes expression. Of note, miR-29 was found to elicit elastin suppression, thereby closing the circle .Panizo et al. induced vessel calcification in rats by using the common experimental method of feeding them a high phosphate diet. They found low levels of miR-133b and miR-211 alongside high levels of miR-29b . The forTo further evaluate the role of miRNAs in valvular calcification, exosomes from VSMCs have been evaluated by several authors through RT-qPCR. Interestingly, preclinical studies show differences in the expression of hundreds of miRNAs when comparing mice calcific models versus the normal population ,52.p-values of comparison between the two populations\u2019 expression [Pan et al. established a cellular calcification model using the mouse line MOVAS-1 . To searpression .Over the recent years, miRNAs have represented an emerging class of widely labored circulating biomarkers in various pathological states including degenerative mitral valve disease ,15,16.Concerning the prolapse of the mitral valve, only a small number of circulating miRNAs have been studied focusing on the degenerative disorders of the MV. These findings were limited to the experimental animal models. Hulanicka et al. evaluated plasma miRNA levels as potential biomarkers of chronic myxomatous mitral disease (MMVD) in dachshunds. Investigators evaluated the expression of 9 miRNAs that had already been discovered and were involved in cardiovascular diseases . Using aIn greater detail, according to the American College of Veterinary Internal Medicine (ACVIM), the plasma levels of miRNAs were compared in three groups of dogs. The authors recorded that miR-30b expression differed between dogs of the ACVIM group in stage B (asymptomatic n = 8) and those that were included in the unaffected stage A group (control N = 8) . The expA second study reported the miRNA expression profile in dogs suffering from MMVD . Li et The major concern with the use of animal models to evaluate the expression of miRNA is the increased risk of discrepancy with human pathoanatomic conditions. Bulent et al. worked around this problem by investigating the expression profile of circulating miRNAs in the development of mitral chordae tendineae rupture in humans . 22 miRNSongia et al. performed the first study using human plasma from patients with degenerative mitral valve prolapse and noted a strong correlation between several circulating miRNAs and mitral valves with myxomatous prolapse . Some ofThe authors working on circulating biomarkers have provided valuable information on the etiology of degeneration and prolapse of MV alongside the possibility of stratifying patients affected by the disease . DeroyerIn another study, Tan et al. performed an analysis using proteomic evaluation on two pooled plasma samples from 24 individuals affected by mitral valve prolapse and MR compared to 24 individuals with no MV prolapse and failure . All enrUnlike the studies cited above, the results reported in the analysis by Songia et al. were supported by solid statistical evidence, underlined by a marked change in plasma level of the miRNAs studied using AUROC curves . The keyAmong other things, the study of Songia et al. confirmed the existence of well-characterized signaling pathways involved in cell migration and proliferation of endothelial cells ,16,55,56Although the report of Songia et al. claimed that specific circulating biomarkers could be interpreted as molecular signatures, the study has some critical points . The stuHowever, given the reported evidence, the future direction postulates that miRNAs identified in plasma could be used soon and is an inexpensive screening tool for patients with progressive degenerative mitral valve disease and severe mitral regurgitation.Congenital heart diseases (CHD) comprise a large group of functional and structural disorders, namely atrial septal defects (ASD), ventricular septal defects (VSD), pulmonary valve atresia (PVA), coarctation of the aorta (CoA), tricuspid atresia (TA), tetralogy of Fallot (TOF) and several others ,64,65. mSeveral other preclinical studies, conducted on zebrafish ventricles, denoted the role of miR-133 to diminish cardiac regenerating processes . FollowiCardiogenesis is also suppressed by the miR-15 family, which, specifically inhibited, has shown to promote myocyte proliferation after myocardial infarction . On the Several miRNAs also regulate the signals of insulin-growth factor 1 (IGF-1) in skeletal muscle, contributing to muscle development or atrophy . SeveralSpecific miRNAs are also differentially expressed in bicuspid aortic valve (BAV), the most common congenital heart disease. Aortic valve endothelial cells on the ventricular side are frequently exposed to high shear forces while on the aortic side turbulent blood flow and high levels of antioxidant enzymes are present . ConversMiR-133 can be used for guiding the therapeutic management of aortic stenosis, due to its potential role in predicting left ventricular hypertrophy ,76,77. SThe key factor involved in calcium metabolism and inflammatory pathways was found to be pidermal growth factor receptor (EGFR) . SeveralmiR-181 is another important miRNA. Aortic valve endothelial cells were associated with its increased expression but decreased levels of targets, including SIRT1 and GATA6 that negatively affect vascular SMCs elastin production . SeveralRegulating miRNAs expression is an attractive therapeutic challenge. Valvular calcification is currently not a direct target for pharmacological action. Endothelin receptor antagonists have emerged as possible molecules of interest in preclinical studies ,82. ChroConcerning statin treatment, several studies reported increased rates of calcification . Possibl\u2212/\u2212 mice with aortic valve calcification demonstrated that the intragastric administration of melatonin for 24 weeks improved aortic valvular parameters. It reduced thickness and calcium deposition in the leaflets and ameliorated echocardiographic markers, namely transvalvular peak jet velocity and aortic valve area [Another possible strategy recently proposed, involves melatonin administration. In vitro studies confirmed melatonin reduces the level of CircRIC3, a circular RNA with procalcific effects. It acts as a miR-204-5p sponge to stimulate and increase expression levels of the procalcification gene dipeptidyl peptidase-4 (DPP4). A preclinical in vivo study involvinlve area . At the The authors also demonstrated melatonin caused in vitro suppression of calcification in human valvular interstitial cells (hIVICs) .The primary concerns in utilizing miRNAs as therapeutic targets, either to positively or to negatively affect them, arise from the need of achieving stability and resistance to degradation enzymes. We named expression vectors, antisense nucleotides (ASOs), small molecules and miR-mimics as novel approaches under current experimentation. Expression vectors are also defined as miRNAs sponges and constitute artificial binding sites for miRNAs to reduce their effect on miRNAs ,88. OligA possible strategy to improve stability is the modification with 2-O-methyl (2-OMe) . This caTo decrease nuclease degradation, modifications including 2-O-methoxyethyl (2-MOE), 2-fluoro (2-F), and locked nucleic acid (LNA) have also been tested . In partIn vitro studies conducted on oligonucleotides had limited pharmacological effects due to unfavorable kinetic characteristics, notably poor tissue distribution and fast excretion. Thus, appropriate delivery systems have been developed, functioning as carriers for in vivo molecular directing .A good delivery system should achieve the following features: evading the immune system response, avoiding nucleases degradation, directed to target cells, and releasing the content for incorporation into RNA processing machinery ,93,94,952O, glycerol, and propylene glycol, in the presence of perfluorobutane gas [Other emerging technologies to improve kinetic parameters are nanoscale drug delivery systems using liposomes . Lower stane gas ,106,107.Local miRNA delivery results have been published and discussed in an Israeli study for metastatic breast cancer prevention by miR-96 and miR-182 treatment . In vivoAn Italian group discussed the potential use of miRNAs for mitral valve diseases. In fact, miRNAs appear as predictive diagnostic and prognostic biomarkers but, most promisingly, as potential targets for personalized treatments .With regards to the structural and molecular changes in extracellular matrix composition, the miRNA investigations may be relevant in knowing the progression of altered ECM during aging. This process leads to calcification or myxomatous degeneration of cardiac valves and a preventive diagnosis based on miRNA might establish a pharmacological therapeutic target or a basis to improve existing prosthetic devices and treatment options ,114,115.At present, no clinical data on therapeutic approaches targeting miRNAs are available and hypotheses formulation for future miRNAs practical use remains difficult.A preclinical study investigAdditionally, the reduction in valvular inflammation was detected as decreased tissue levels of Il-6 and Il-17 and fibrosis, both in valves and rat serum.Unfortunately, even though preclinical studies are very promising, further evidence is needed to understand how signaling pathways directly modulate the pathophysiology of degenerative mitral and aortic valve diseases. Furthermore, with regard to degenerative disease of the mitral valve, more in-depth investigations are needed to clarify the possible role of circulating miR-150-5p and define its causal relationship with the different pathophysiological expressions of degenerative mitral valve prolapse. The main objective is to direct both the early diagnosis and the most suitable therapies through the design and application of clinical experimental and observational studies.New insights into circulating miRNAs may open up new scenarios for treatment and diagnostic screening. About the therapy, the possibility of identifying different possible pharmacological targets able to slow down or even stop the progression of aortic and mitral degenerative diseases is outlined. Instead, regarding the new diagnostic procedures, miRNAs can be useful as specific circulating molecular signatures to be used as a rapid first line and less expensive screening tool for the identification of severe aortic and mitral disease.We hope that in the coming years, further emphasis, interest, and funding will be allocated for these fascinating molecular approaches."} +{"text": "Mathieu Jaboulay (1860\u20101913) was a professor of clinical surgery in Lyon, France who is best known for his development of vascular anastomosis and for conducting the first reported renal xenotransplantation experiments on humans, using pig and goat kidneys to treat end\u2010stage renal failure in 1906. His insights and pioneering techniques contributed significantly to allotransplantation and contemporary attempts at xenotransplantation. He is also credited with inventing several surgical instruments and novel surgical techniques that continue to influence vascular, general, and urological surgery to this day. However, this article will focus specifically on his notable contributions to xenotransplantation research and surgery. In 2020, 129 681 organs were transplanted worldwide,One promising alternative to allotransplantation\u2014and a means of addressing the organ shortage problem\u2014is xenotransplantation. Xenotransplantation is the cross\u2010species transplantation of organs, tissues, or cells between two different species.2.Jaboulay Figure\u00a0 was bornJaboulay was a popular lecturer and surgeon who was revered by his students and internationally among his peers, primarily for his originality and pioneering surgical techniques.3In 1902, Emerich Ullmann (1861\u20101937) performed the first \u201csuccessful\u201d kidney transplant, when he removed and autotransplanted a dog's kidney to the vessels of its neck where it produced urine for a short period.In 1906, Jaboulay conducted the first reported clinical kidney xenotransplant that resulted in the production of urine. He believed that the use of animal organs could establish urine production as a means to help treat individuals with renal failure. The first transplant took place on the 24th of January in a 48\u2010year\u2010old woman whose clinical features included\u2014oliguria, hypertension, headache, and hearing and vision loss.Following dissection of the vessels in the fold of the patient's elbow, the pig kidney was grafted into the left antecubital space and left uncovered. The renal artery was connected to the central end of the humeral artery,On the third day, Jaboulay observed that the kidney was no longer functioning; it was removed on the same day. Following examination, Jaboulay identified the cause of the failure to be vascular thrombosis and erroneously lamented that the cause was his suturing technique.The second of Jaboulays\u2019 kidney xenotransplants took place nearly 3 months later on the 9th of April 1906. Although Jaboulay gives no definitive reason for changing his animal of choice from a pig to a goat, he does note that the goat kidney was smaller and had better quality vessels.4Jaboulay\u2014like Ullman and Decastello\u2014would later abandon his work on xenotransplantation to continue his work on cancer, which was his primary focus in the latter years of his life.5The kidneys have historically been one of the primary organs of interest for transplantation. Because of their great need\u2014the kidney is consistently the most transplanted organ in many countries. The immediate production of urine may indicate a successful transplant operation. They also have the surgical benefit of being vascularized by a single main vessel, the renal artery. It is perhaps easy and intuitive to deem Jaboulays\u2019 two kidney xenotransplants to have been prima facie unsuccessful since neither resulted in long\u2010term function and patient survival. Nevertheless, arguably, they were in some meaningful sense \u201csuccessful,\u201d since in both cases the xenografts were vascularized and produced urine. Some of the human kidney transplants performed over the next few decades never produced any urine.After many failed xenotransplantation experiments using primarily non\u2010human primates as the source of organs. Jaboulay's early use of a pig for xenotransplantation would be re\u2010explored. Non\u2010human primates were a rational choice due to their genetic similarities to humans. However, the heightened risk of zoonotic disease transmission in primates, difficulty of large\u2010scale breeding, and greater hesitancy among the public for the use of primates in experimentation, in part led researchers away from non\u2010human primates. Surely a primary reason why Jaboulay used a pig in his initial experiment was because of its ready availability and few people would object to his killing a pig for this purpose. Nonetheless, his choice of pigs as the source of kidneys for clinical transplantation was taken up by others many years later.Despite the complications and failures that plagued attempts at xenotransplantation for decades, Jaboulay had initiated the exploration of the pig\u2014or goat\u2014as a source of organs for humans. It has required solutions to numerous technological, immunological, and ethical hurdles, which over 100 years later, are now beginning to show the potential that many early proponents foresaw. Jaboulay, like many other pioneers before him, was willing to try something unconventional and risky in the hope of success and the restoration of health."} +{"text": "Perianal abscesses are a common surgical emergency. Due to their perceived ease, drainage is often delegated to junior trainees with varying levels of experience. The purpose of this study is to evaluate the current trend in perianal abscesses management at our institution, and identify factors that predict subsequent fistula formation or abscess recurrence.All acute patients admitted to a major teaching hospital who required surgical drainage of a perianal abscess were analysed over a two\u2010year period from January 2019 to December 2020. Patient demographics, clinical and laboratory findings were retrospectively reviewed. Proceduralist experience, operative management strategy and recurrence rates (fistula or abscess) were analysed.P\u00a0=\u00a00.01). Searching for a fistula tract was performed in 41% of cases but did not reduce recurrence (P\u00a0=\u00a00.9). Seton insertion occurred in 10%, and fistulotomy in 2%.The mean age of patients was 43\u2009years old, and 73% were male. Trainees performed 96% of the procedures. Re\u2010presentation with a fistula or abscess recurrence requiring further surgery was 31%. Comorbidities of IBD, diabetes, or malignancy were present in one\u2010third of patients and significantly increased the risk of recurrence (Perianal abscess drainage at our institution is almost exclusively performed by trainees, the majority of which occurs after\u2010hours. Patients who present with a fever, inflammatory bowel disease, diabetes mellitus or malignancy are at an increased risk of recurrent abscess or a subsequent fistula after drainage, and input from an experienced surgeon may be of value when considering seton insertion or fistulotomy. Perianal abscess management is primarily done by trainees of varying experience. This retrospective review evaluates current trends at our institution and identify factors that predict abscess recurrence. Perianal abscesses are a daily presentation to emergency departments. They are most commonly assessed and drained by surgical trainees of varying experience. The underlying pathophysiology is well taught in surgical texts; they arise due to obstruction of the anal glands which traverse the internal sphincter and open at the dentate line.Definitive management is perhaps mistakenly perceived as a \u2018low acuity\u2019 or \u2018simple\u2019 incision and drainage of sepsis, often delegated to the junior trainee or performed after\u2010hours. While this approach may apply to abscesses elsewhere on the body, the intricate sphincter anatomy can be deceiving as evidenced by the fact that one\u2010third will re\u2010present with fistula in ano.The purpose of this study therefore is to evaluate the current trend of perianal abscess management at our institution, and identify clinical or operative factors which can assist trainees reduce subsequent fistula formation or abscess recurrence.P\u00a0=\u00a00.05.All acute patients admitted to a major teaching hospital who required surgical drainage of a perianal abscess were analysed over a 2\u2010year period from January 2019 to December 2020. Patient demographics, clinical and laboratory findings were retrospectively reviewed. Proceduralist experience and operative strategy were documented. Searching for a fistula tract was defined as using an anal retractor and gently passing a Lockhart\u2010Mummery probe or instillation of hydrogen peroxide and identifying an internal opening. A recurrence was defined as a previous or subsequent presentation with a perianal abscess requiring surgery, or a subsequent examination under anaesthesia which identified a fistula tract. Statistical analysis was performed using Chi\u2010squared tests with a significance value HREC approval was obtained for this project \u2013 low/negligible risk pathway (Ref 2022/ETH00214). The data that support the findings of this study are available from the corresponding author upon reasonable request.A total of 78 patients required perianal abscess drainage during the 2\u2010year period. The mean age was 42.9\u2009years and 73% were male. Trainees performed 96% of the procedures. Wound swab for culture was performed in 77%. Abscess recurrence rate was 31%, and these surgical drainage procedures were also all performed by trainees.P\u00a0=\u00a00.01). Interestingly, a documented fever (T\u2009>\u200938\u00b0) in the emergency department was present in 17%, and these patients had a significantly higher risk of recurrence (P\u00a0=\u00a00.04). Gender, symptom duration or elevated inflammatory markers did not significantly affect recurrence rate.Comorbidities of inflammatory bowel disease (IBD), diabetes, or malignancy were present in one\u2010third of patients and significantly increased the risk of subsequent fistula formation or abscess recurrence (P\u00a0=\u00a00.9). Seton insertion occurred in 10%, and fistulotomy in 2%. Management using a seton or fistulotomy was performed at similar rates both in\u2010hours and after\u2010hours, 15% compared with 11% respectively.After\u2010hours surgery was performed in 58% of cases. Searching for a fistula tract was performed in 41% of cases but did not reduce recurrence (Trainees perform most perianal abscess drainages which may be complex procedures due to the anatomy involved, but this practice appears safe as overall recurrence rates are in keeping with current literature.From a practical viewpoint, skin incisions close to the anal verge ensure that any potential fistula will have a short length. Any search for a fistula tract, using anal retractors and Lockhart\u2010Mummery probes must be performed carefully to avoid creating false tracts. Wound packing is often up to the discretion of the proceduralist and the cavity size; however, randomized trials have demonstrated no significant benefit other than increasing post\u2010operative pain.Performing a fistulotomy or seton insertion at the time of drainage remains a controversial strategy. A number of trials have reported conflicting outcomes, and the traditional teaching is that delaying fistula management is preferred, as the acute inflammation and oedematous tissue have resolved. The advertised benefits of faster wound healing, decreased abscess recurrence and fistula formation must be balanced against the three\u2010fold increased risk of incontinenceWound cultures are not considered routine when draining perianal abscesses, although they can differentiate patients into those with bacteria arising from the colon versus the skin. The former suggests an abscess of cryptoglandular origin which traditionally was thought to increase the risk of recurrence or fistula formation; however, this has not been consistently demonstrated.Surgical examination under anaesthesia is traditionally considered the gold standard for defining an abscess or fistula tract. The use of imaging in select patients with suspected complex pathology however can provide an anatomical road map to avoid sphincter damage or creation of false tracts. Readily available and rapid computed tomography differentiates an abscess from severe cellulitis, or identifies extension into the supralevator space; the limitation being its ability to identify fistula tracts as they share the same appearance as inflammatory stranding.A limitation to this present study is the undetermined effect on anal sphincter function and incontinence post\u2010surgery. Short\u2010 and long\u2010term function following perianal abscess drainage presents itself as a topic of future research to ensure trainees are producing acceptable results.Perianal abscess drainage at our institution is almost exclusively performed by trainees, the majority of which occurs after\u2010hours. This practice appears to successfully manage sepsis and produces comparable recurrence rates to the known literature. Trainees would do well to recognize patients who present with a fever, IBD, diabetes mellitus or malignancy as they significantly predict a risk of recurrent abscess or a subsequent fistula after drainage. Input from an experienced surgeon or pre\u2010operative imaging is a good strategy for the stable patient identified at high risk of hiding complex pathology. In addition to simple drainage, searching for a fistula tract, seton insertion or fistulotomy did not significantly reduce recurrence in our series, and since our study has not assessed continence after treatment this should not be routinely done by surgical trainees.None declared.Mina Sarofim: Conceptualization; data curation; formal analysis; investigation; writing \u2013 original draft; writing \u2013 review and editing. Kevin Ooi: Conceptualization; project administration; supervision; writing \u2013 review and editing."} +{"text": "The original version of this article unfortunately contained a mistake. In figure\u00a03, the electrode localizations of all patients do not match the one from the table.The corrected Fig."} +{"text": "The complex physical structure and abundant repeat sequences make it difficult to assemble the mitogenomes of seed plants, especially gymnosperms. Only approximately 33 mitogenomes of gymnosperms have been reported. However, as the most widely distributed and the second largest family among gymnosperms, Cupressaceae has only six assembled mitogenomes, including five draft mitogenomes and one complete mitogenome, which has greatly hindered the understanding of mitogenome evolution within this large family, even gymnosperms.Thuja sutchuenensis, with a size of 2.4\u00a0Mb. Multiple sequence units constituted its complex structure, which can be reduced to three linear contigs and one small circular contig. The analysis of repeat sequences indicated that the numbers of simple sequence repeats increased during the evolutionary history of gymnosperms, and the mitogenome of Thuja sutchuenensis harboured abundant extra-long repeats (more than 5\u00a0kb). Additionally, the longest repeat sequence identified in these seven gymnosperms also came from the mitogenome of Thuja sutchuenensis, with a length of up to 47\u00a0kb. The analysis of colinear blocks and gene clusters both revealed that the orders of mitochondrial genes within gymnosperms was not conserved. The comparative analysis showed that only four tRNAs were shared by seven gymnosperms, namely, trnD-GUC, trnE-UUC, trnI-CAU and trnY-GUA. Furthermore, four genes have undergone potential positive selection in most gymnosperm species, namely, atp8, ccmB, mttB and sdh4.In this study, we assembled and validated the complete mitogenome of Thuja sutchuenensis. The investigation of Thuja sutchuenensis\u2019s mitogenome in our study provides new insight into further understanding the complex mitogenome architecture within gymnosperms.We successfully assembled the second complete mitogenome within Cupressaceae and verified that it consisted of multiple sequence units. Our study also indicated that abundant long repeats may contribute to the generation of the complex conformation of the mitogenome of The online version contains supplementary material available at 10.1186/s12870-023-04054-9. Larix sibirica Ledeb. was 33 times larger than that of Ginkgo biloba L. (G. biloba) [Pinus taeda L. (Pi. taeda) and Welwitschia mirabilis Hook. f. (W. mirabilis), differ by 12 protein-coding genes [Due to the complicated physical structure, sequence composition, and abundance of repeat sequences, there are still certain challenges associated with the assembly and annotation of vascular plant mitochondrial genomes \u20135. An in biloba) , 12. The vs. 29) .Gymnosperms can be classified into five main lineages, including Cycads, Ginkgo, Pinaceae, Gnetophytes, and Conifer II (non-Pinaceae conifers or Cupressophyta), according to recent studies . CupressThuja sutchuenensis Franch. (Th. sutchuenensis), belonging to an astern Asian-North American disjunct genus, was first discovered by French missionary P. G. Farges in 1892 and officially published by Franche in 1899 [Th. sutchuenensis was not rediscovered until 1999, during a comprehensive investigation of rare and endangered plants in Chengkou County [Th. sutchuenensis were rare, and reproductive barriers were the main reason for its population decline [Th. sutchuenensis is now assessed as an endangered species and the I-class national key protected wild plant in China. Previous research has demonstrated that there is a close relationship between the mitogenome and cytoplasmic male sterility (CMS), and abortion occurred during the growth of ovulate strobilus, microstrobilus and seeds of Th. sutchuenensis [Th. sutchuenensis is helpful to understand the molecular mechanism of the reproductive barriers, and is of great significance for the protection of this endangered plant. in 1899 , 16. Sin in 1899 \u201319. Th. decline \u201322. Theruenensis . Hence, Th. sutchuenensis was sequenced, assembled and validated. Then, we compared it with multiple reported mitogenomes of gymnosperms. Our research provides more evidence that the plant mitochondrial genome contains several sequence components with complex structures. The investigation of the Th. sutchuenensis' mitogenome also provides supporting data for the study of the reproductive obstacles of this endangered species and the genetic evolution of the gymnosperm mitogenome.In this study, the complete mitogenome of Th. sutchuenensis is 2.46\u00a0Mb, including 4 contigs. Bandage [Assembly results indicated that the complete mitochondrial genome of Bandage was usedTh. sutchuenensis, PCR and Sanger sequencing were carried out. We designed four pairs of specific primers for PCR amplification. The two connected regions of contig 1 and contig 3 should successfully generate PCR products by primer pair AF\u2009+\u2009AR and BF\u2009+\u2009BR, respectively, while the connected regions of contig 1 and contig 2, contig 2 and contig 3 should successfully generate PCR products by primer pairs CF\u2009+\u2009CR and DF\u2009+\u2009DR, respectively. The PCR amplification results showed that the lengths of bands were consistent with that expected , two large subunits of ribosome genes (rpl-) and one succinate dehydrogenase gene . In addition, three rRNA genes, five tRNA genes of mitochondrial origin , and one tRNA gene of chloroplast origin (trnW-CCA) were also annotated in the mitogenome. The relative order and direction of these genes are shown in the mitogenome map were found in contigs1-4 of Th. sutchuenensis. The results indicated that all species had the highest proportion of tetranucleotide polymers except for W. mirabilis and Taxus cuspidata Sieb. et Zucc. (Ta. cuspidata), which were dominated by pentanucleotide polymers and dinucleotide polymers, respectively and G. biloba had the fewest SSRs (both less than 200), Pi. taeda and W. mirabilis had a moderate number of SSRs (500\u2013700), while Ta. cuspidata, Platycladus orientalis (L.) Franco and Th. sutchuenensis harboured the most SSRs . Comparative analysis of dispersed repeats indicated that forward repeats and palindromic repeats accounted for the largest proportion in gymnosperms , far more than the other five gymnosperms were found, which appeared in the mitochondrial and plastid genomes simultaneously (rrn18/rrn16 and rrn26/rrn23). Therefore, this could not represent the existence of sequence migration between the two genomes, as these rRNA genes may be of a common origin. In conclusion, no significant sequence migration was detected between the chloroplast and mitochondrial genomes of Th. sutchuenensis.The blast analysis for the mitogenome and plastid genome indicated that 17 homologous fragments were identified, with a total length of 3,481\u00a0bp, accounting for 0.14% of the total mitogenome , which had three identical gene clusters.Synteny analysis indicated that there were a large number 10,496) of homologous collinear blocks among the seven gymnosperms, especially between 96 of homTh. sutchuenensis with 6 other representative gymnosperm species to show the different gene compositions among gymnosperm mitogenomes. Among the 7 species representing 5 lineages of gymnosperms, C. taitungensis, G. biloba and Pi. taeda had the highest number of PCGs (41), while the fewest PCGs were found in W. mirabilis . A comprehensive reannotation of tRNA for the mitogenomes of seven gymnosperms showed that compared with the basal group of gymnosperms (C. taitungensis and G. biloba), numerous loss events of mitochondrial-derived tRNA occurred in the evolutionary history of gymnosperms. Only four tRNAs were found to be conserved in seven gymnosperms, namely, trnD-GUC, trnE-UUC, trnI-CAU and trnY-GUA was conserved in all species except for W. mirabilis, whereas trnfM-CAU (cp origin) was found only in this species.We compared the mitogenomes of of PCGs 4, while t29, Fig.\u00a0.1. In addN/dS values were less than 1.0, with the exception of W. mirabilis vs. Pi. Taeda and Pl. orientalis vs. Th. sutchuenensis, indicating that almost all gymnosperms were negatively selected . The nucleotide substitution rate at the species level was calculated using a supermatrix concatenating 28 genes. The results showed that most Th. sutchuenensis was assembled and validated in this study, which consists of one circular contig and three linear contigs with overlapping regions among each other Carri\u00e8re, which were composed of 2, 9 and 13 segments, respectively [The National Center for Biotechnology Information (NCBI) released 7,943 chloroplast genomes and 470 mitochondrial genomes as of July 28, 2022. As an increasing number of mitogenomes are published, studies have found that the size of plant mitogenomes varies greatly, ranging from 66\u00a0kb to 11.7\u00a0Mb , 27. Addectively , 13.C. taitungensis and G. biloba (both less than 200), Pi. taeda and W. mirabilis (500\u2013700), and Ta. cuspidata, Pl. orientalis and Th. Sutchuenensis , which may indicate that the size of the SSR sequence of gymnosperms has gradually increased during evolution , which may cause high-frequency recombination, leading to isomerization of the genome into multiple major forms , 29, 30.nosperms .G. biloba having the most, with 8 gene clusters, and W. mirabilis having the fewest, with just 2 company and included sample quality detection, library construction, library quality detection, library sequencing and other processes. In total, 15\u00a0Gb of sequence reads were obtained, and 13.71\u00a0Gb remained after filtering and qualification. The average read length of filtered reads was 8.16\u00a0kb (N50\u2009=\u200917.69\u00a0kb), and the longest read was 733.08\u00a0kb.Ta. cuspidata (MN593023) and Pl. orientalis (OL703044 and OL703045) were used as the query sequence, and the BLASTn program [Th. sutchuenensis to assem program was used program software2O. After an initial denaturation at 94\u00a0\u00b0C for 2\u00a0min, PCRs were performed for 35 cycles. Each cycle consisted of denaturation at 94\u00a0\u00b0C for 30\u00a0s, annealing at 55\u00a0\u00b0C for 30\u00a0s, and elongation at 72\u00a0\u00b0C for 1\u00a0min. The PCR products were sequenced by Sanger sequencing.PCR experiments with specific primer pairs were used to amplify the connected regions of three contigs, which could verify the presence of this complex conformation. Four pairs of specific primers were designed for 4 connecting regions using Primer blast of NCBI, which are listed in Table STh. sutchuenensis was submitted to NCBI under accession numbers ON603305-ON603308.Geseq was usedTh. sutchuenensis. The online website MISA (https://webblast.ipk-gatersleben.de/misa/) was used to identify SSRs, with minimum repetition numbers of mono-, di-, tri-, tetra-, penta-, and hexanucleotides was 10, 5, 4, 3, 3, and 3, respectively [https://bibiserv.cebitec.uni-bielefeld.de/reputer), with the hamming distance\u2009=\u20093, maximum computed repeats\u2009=\u20095,000, and minimal repeat size\u2009=\u200930 [https://tandem.bu.edu/trf/trf.html) to identify tandem repeats with default settings [C. taitungensis (AP009381), G. biloba (KM672373), W. mirabilis (KT313400), Pi. taeda (MF991879), Ta. cuspidata (MN593023) and Pl. orientalis . These six mitogenomes, together with Th. sutchuenensis, were analysed for repeat sequences.The repeat sequences of each contig were annotated separately. Then, the long repeats (>\u2009500\u00a0bp) were annotated for the whole mitogenome of ectively . Dispersize\u2009=\u200930 . We alsosettings . AdditioTh. sutchuenensis, we used BLASTn [Th. sutchuenensis and six other gymnosperms based on sequence similarity. Moreover, we searched for gene clusters common to gymnosperms by simple visual inspection to evaluate the conservation of gene orders for the above seven gymnosperms.To identify the potential homologous sequences that may be transferred between the plastid (MH784400.1) and mitogenome of d BLASTn to compad BLASTn was usedd BLASTn was usedTh. sutchuenensis. The corresponding nucleotide sequences were aligned and concatenated by using Mafft (v7.450) [dN), synonymous substitution rate (dS), and ratio of dN to dS. Single-gene matrix and concatenated matrix were used as input files to calculate the nucleotide substitution rates at the gene level and species level, respectively. TBtools and R-package (ggplot) were used to draw boxplots and heatmaps for pairwise dN/dS values [We used Phylosuite (V1.2.1) to identv7.450) and Phyl(v7.450) was used and PhyS values .Additional file 1:\u00a0Figure S1. Gel electrophoresis imagefor the PCR products. M, marker; 1-6, the ID of the duplicated biologicalsamples. The expected lengths of each fragment are shown at the bottom of thegel.\u00a0Figure S2. Sanger sequencing results. Panels\u00a0A, B, C and D are the alignment of corresponding genomic regions (the first row)\u00a0with the PCR\u00a0products\u00a0. A: the connection region 1 between contig 1 and contig 3; B: the\u00a0connection region 2 between contig 1 and contig 3, C: the connection region between\u00a0contig 1 and contig 2; D the connection region between contig 2 and contig 3.\u00a0Figure S3. The histogram of simple\u00a0sequence repeats (SSRs) identified in the 4 contigs of Thuja sutchuenensis.\u00a0Figure S4. The histogram of dispersed repeats identified in the\u00a04 contigs of Thuja sutchuenensis.\u00a0Figure S5.\u00a0The distribution of long repeats (>500 bp) in the\u00a0whole mitogenome of\u00a0Thuja sutchuenensis.\u00a0Figure S6. The histogram of simple sequence repeats (SSRs) and\u00a0dispersed repeats identified in the 7 gymnosperm mitogenomes. A and B shows the\u00a0comparison of SSRs and dispersed repeats among the 7 gymnosperm mitogenomes,\u00a0respectively.\u00a0Figure S7. The histogram of long repeats identified in the 7\u00a0gymnosperm mitogenomes.\u00a0Figure S8. Homologous fragments\u00a0between the chloroplast and mitochondrial genome of Thuja sutchuenensis.\u00a0The red, blue and green fragments represent homologous fragments with the\u00a0percent of identity more than 90, more than 80 but less than 90, less than 80,\u00a0respectively.\u00a0Figure S9. The distribution of mitochondrial gene clusters\u00a0in 7 gymnosperms.\u00a0Figure S10. Heatmap of pairwise dN/dSratios between each pair of sequences in the multigene nucleotide alignment.\u00a0Figure S11.\u00a0Distribution diagram of 4 pairs of specific\u00a0primers.Additional file 2:\u00a0Table S2. 1 The simple sequence repeats (SSRs) identified in contig 1 of Thuja sutchuenensis.\u00a02\u00a0The simple sequence repeats (SSRs) identified in contig 2 of Thuja\u00a0sutchuenensis.\u00a03 The simple sequence repeats (SSRs) identified in contig 3 of Thuja sutchuenensis.\u00a04 The simple sequence repeats (SSRs) identified in contig 4 of Thuja sutchuenensis.\u00a05 The dispersed repeats identified in contig 1 of Thuja sutchuenensis.\u00a06 The dispersed repeats identified in contig 2 of Thuja sutchuenensis.\u00a07 The dispersed repeats identified in contig 3 of Thuja sutchuenensis.\u00a08 The dispersed repeats identified in contig 4 of Thuja sutchuenensis.\u00a09 The tandem repeats identified in contig 1 of Thuja sutchuenensis.\u00a010 The tandem repeats identified in contig 2 of Thuja sutchuenensis.\u00a011 The tandem repeats identified in contig 3 of Thuja sutchuenensis.\u00a012 The tandem repeats identified in contig 4 of Thuja sutchuenensis.\u00a013 The long repeats\u00a0 (>500 bp) identified in the whole mitogenome of Thuja sutchuenensis.\u00a014 The simple sequence repeats (SSRs) identified in the mitogenome of Cycas taitungensis.\u00a015 The simple sequence repeats (SSRs) identified in the mitogenome of Ginkgo biloba.\u00a016 The simple sequence repeats (SSRs) identified in the mitogenome of Pinus taeda.\u00a017 The simple sequence repeats (SSRs) identified in the mitogenome of Welwitschia mirabilis.\u00a018 The simple sequence repeats (SSRs) identified in the mitogenome of Taxus cuspidata.\u00a019 The simple sequence repeats (SSRs) identified in contig 1 of Platycladus orientalis.\u00a020 The simple sequence repeats (SSRs) identified in contig 2 of Platycladus orientalis.\u00a021 The dispersed repeats identified in the mitogenome of Cycas taitungensis.\u00a022 The dispersed repeats identified in the mitogenome of Ginkgo biloba.\u00a023 The dispersed repeats identified in the mitogenome of Pinus taeda.\u00a024 The dispersed repeats identified in the mitogenome of Welwitschia mirabilis.\u00a025 The dispersed repeats identified in the mitogenome of Taxus cuspidata.\u00a026 The dispersed repeats identified in contig 1 of Platycladus orientalis.\u00a027 The dispersed repeats identified in contig 2 of Platycladus orientalis.\u00a028 The long repeats (>500 bp) identified in the whole mitogenome of Platycladus orientalis.\u00a029 Pairwise dN/dS ratios between each pair of sequences in the multigene nucleotide alignment of 7 gymnosperms plants.\u00a030 Pairwise dN/dS ratios in different mitochondrial genes of 7 gymnosperms plants.Additional file 3:\u00a0Table S3. 1 General features of seven gymnosperm mitogenomes.\u00a0Table S3. 2 Primers used in this study."} +{"text": "Actually, the quality of water is one of the most important indicators of the human environmental impact, the control of which is crucial to avoiding irreversible damage in the future. Nowadays, in parallel to the growth of the chemical industry, new chemical compounds have been developed, such as dyes and medicines. The increasing use of these products has led to the appearance of recalcitrant pollutants in industrial wastewater, and even in the drinking water circuit of our populations. The current work presents a photoreactor prototype that allows the performance of experiments for the decomposition of coloured pollutants using photocatalysis at the laboratory scale. The design of this device included the study of the photometric technique for light emission and the development of a software that allows monitoring the dye degradation process. Open-source hardware platforms, such as Arduino, were used for the monitoring system, which have the advantages of being low-cost platforms. A software application that manages the communication of the reactor with the computer and graphically displays the data read by the sensor was also developed. The results obtained demonstrated that this device can accelerate the photodegradation reaction in addition to monitoring the changes throughout the process. Photocatalysis represents a powerful tool to achieve more sustainable processes and products followin2-coated quartz tube for the degradation of three different dyes. Similarly, Abhang et al. [Several systems for water pollution monitoring have beeg et al. presenteg et al. , UV LEDsg et al. proposedg et al. showed ag et al. measuredEven if several designs have been proposed in literature for conducting lab scale photoreactions, the interesting idea of monitoring the photocatalysis process for the decomposition of a dye in a solution on a computer screen has been never considered. Based on this innovating milestone, in this work a compact low-cost device to monitor wastewater decontamination has been developed taking as reference a handmade simple prototype . This inIn the following sections, the architecture of the system, lab-scale considerations and design feasibility are presented, since the work is a set that integrates the improved design of a photoreactor and the software that controls it.Initially, the photocatalysis reactions were carried out by holding a test tube in the reactor displayed in 2 catalyst. All these chemicals were purchased from Sigma-Aldrich and used as received. Methyl red is a commercially available azoic dye widely used in the textile industry that was chosen as a model compound for pollutant removal [2O2), it was selected as an oxidant with green credentials. Its decomposition leads to the formation of water (H2O) and oxygen (O2), which, in the last term, is responsible for the colourant degradation by oxidation.All the experiments carried out in the photoreactors consisted of studying the photocatalytic degradation of coloured liquid samples formulated by mixing 1 mL of stock solution (5 mg of methyl red dissolved in 100 mL of ethanol), 20 mL of ethanol, 1.2 mL of hydrogen peroxide, and 30 mg of P25 TiO removal . The relTo achieve all the proposed challenges, the reactor was equipped with ultraviolet LEDs, a light emitter and sensor, a motor to which magnets were attached, a microcontroller mounted on a prototyping board, an internal support housing the electronics and test tubes, an external container that protects the internal parts and a lid. Transmittance LED. The Lumex SSL-LX5093UEGC [5093UEGC LED was Ultraviolet LED. As a source of ultraviolet light, two 380\u2013390 nm Epiled UV LEDs of 1W power and a wide viewing angle were chosen to cover the maximum surface area of the test tube. Two aluminum heatsinks were incorporated into the LEDs to increase the surface area and reduce their working temperature.Operational Amplifier. An operational amplifier was necessary to condition the signal coming from the LDR sensor. A Burr-Brown OPA241 [n OPA241 with raiMicrocontroller. Arduino is a platform that develops open hardware prototyping boards and offers multiple options depending on the field of application. In our case, it was the main component that controlled all the subsystems that made the reactor work. An Arduino Nano Every was used because all these operations do not require considerable processing power. Moreover, it was not necessary to have a large number of inputs and outputs, and a compact and minimalist design was also one of the objectives. The Arduino Nano Every board is based on the ATMega4809 microcontroller (MCU). This MCU provides the following features: 46 KB of Flash, 6 KB of SRAM, a 256 bytes EEPROM, 20 MHz clock frequency, SPI connectivity, I2C, micro USB, 8 digital inputs with a 10-bit DAC and 14 digital outputs.Stirrer. It was necessary to have a compact and quiet motor for the stirrer function. For this purpose, a computer axial fan of 50 \u00d7 50 mm at 5 V was used [was used , to whicPhotosensor. At the beginning of this work, the aim was to determine what kind of sensor would be suitable. To this end, the responses of various sensors to little light transmittance variations were studied. The sensors tested were a cadmium sulfide photoresistor (LDR) [or (LDR) ; a BPW21or (LDR) , which rThe main items that were used to assemble the electronic reactor are briefly described as follows:An electronic circuit was designed specifically for this device. Its schematic was made with Fritzing software and is shown in The part of the circuit dedicated to transmittance measurement had to be able to illuminate the solution sample with the necessary intensity to allow the sensor to appreciate the colour changes, but without saturating the sensor at any point of the reaction. The transmittance LED was connected to one of the PWM outputs of the microcontroller, as the maximum current (8.1 mA) was well below the limits of its outputs. Unlike other technologies tested, such as photodiodes, an LDR provides a large enough signal that does not require conditioning in most applications in which it is used. However, in this case it was desirable to measure subtle changes in light, so signal amplification was required, especially at the beginning of the reaction.The control of the speed of the stirrer and the intensity of the ultraviolet LEDs was performed by pulse-width modulation through the PWM outputs D3 and D5 of the Arduino board that acted on the gates of two IRLZ44 MOSFET transistors.The choice to connect the transistor in charge of the stirrer control to output D3 was motivated by the ability of the ATMega4809 to change its working frequency individually. Otherwise, it would interfere with the time counting functions of the firmware. This output generated a modulated pulse of 62.5 kHz, instead of the 976 Hz normally used on this microcontroller, which is outside the human-audible range and prevents the motor from emitting a coil whining noise.There are three 3D printed parts of which the reactor is composed: a housing, internal support, and a lid see . The reaThe outer part was designed with two holes, one for connecting the USB cable that connects the reactor to the computer where the control software is installed, and the other for connecting the cable to a 5 V power supply, such as from a mobile phone charger. The USB connector icon and 5 V were engraved above these holes; see The reactor enclosure was designed with repairability in mind. The inner support d can be The design of the internal support also considers the dimensions of the tube and its shape. For this reason, the upper inner support has a hole in which the test tube fits, avoiding the need for laboratory clamps. The LEDs and sensors are housed in the inner wall of the holder.The electronic circuitry and the fan were attached to cylindrical brackets at the right height so that the micro USB connectors could match the holes for the cables in the outer body.The firmware of the MCU was programmed to be able to perform several actions commanded from the PC and give the sensor readings and the status of the device. The commands would make the reactor do the pooling cycle of the reaction sample at a certain period, stop it, adjust the UV light intensity and the stirrer speed or perform the zero adjustments. During sampling, the stirrer and the UV LEDs are switched on for most of the cycle. In the remaining time, the light and the stirrer are turned off, preventing the sensor from being affected by the intense UV light and the electrical noise generated by the stirrer motor. Then, it waits 3 s for the possible particles to settle, turns on the transmittance LED and takes the readings. Finally, the average value obtained is returned to the PC.During the zero adjustment process, the transmittance of the solution without the contaminant is measured. Internally, the intensity of the transmittance LED is adjusted to avoid saturation of the sensor during the final stage of photocatalysis.The software that controls the reactor from the PC is responsible for managing the communication with the reactor and for displaying the readings. It was written in Python and employs various libraries, such as Tkinter for the GUI and Matplotlib for the plotting. After addressing all electronic concerns and casing, a prototype reactor was finally assembled. The estimated cost of the materials was \u20ac96.10 ($96.72) excluding manual labour. Several experiments were conducted in order to evaluate the behaviour and effectiveness of the designed photoreactor against a dye-decomposition-catalysed process. Indeed, the proposed goals of the designed device were: (1) to allow accelerating a photodegradation process through the controlled incidence of ultraviolet light during a catalysed experiment and (2) to monitor the evolution of the process through the change in colour due to dye degradation.The purpose of the first experiments carried out with the photoreactor was to make sure that the photocatalysis process occurs. This fact was tested out by simply visually checking up, as shown Photocatalysis was performed both in the herein designed reactor and the previous device shown in Once it was verified that the photocatalytic process was working correctly, the next step was to check that the sensors used were able to measure the transmittance level of the solution as the pollutant was decomposing. This is made possible by the colour change that occurs in the solution during the decomposition of the pollutant.The first experiments showed a very high level of noise in the transmittance signals obtained by the three different sensors used see . With thThe graph of the The aim of this comparison was to select the one that offered the best cost\u2013quality trade off. These measurements were taken during a photocatalysis experiment in which the three selected sensors were used simultaneously. As can be seen in the graph, the TSL257 sensor provided a noisier signal at the end of the process than the other two sensors. On the other hand, the similarity of the response between the LDR and the BPW21 photodiode is striking, despite them being logarithmic and linear, respectively. However, this may be because the illuminance variation in the light passing through the solution is limited enough for the response to approximate a linear function. Finally, LDR sensor was chosen, due to its reduced fluctuations and its reduced cost.The reactor presented in this work decomposed the pollutant after about 23 min, whereas the previous handmade device achieved similar results after 10 min. Even if the new design was slower, it used eight times less power, but the UV LED strip gets hot enough to burn and to cause premature failure of the LEDs. However, the heat produced and also propagated along the reaction tube, could be the reason for the acceleration of the catalytic reaction.The main objectives in the present work were the design, prototyping and fabrication of a lab-scale reactor for monitoring the photocatalytic decomposition of a dye in solution. The design presented satisfied the requirements for a safe and compact reactor that could improve the experience and logistics of laboratory experiments. In addition, the developed system was demonstrated to be able to monitor the decomposition of the dye with a simple to use program and display it on a computer screen, allowing the potential data recording for later post-processing. This work also involved the assessment of the behaviour of the different sensors. It has been demonstrated that the use of an LDR better meets the proposed expectations. This work resulted in a contribution to the photoreactor research field in the sense that it effectively provides a low-cost photoreactor fabricated by 3D printing, allowing the user to monitor the evolution of a coloured pollutant decomposition in real time through customised software. The designed photoreactor is able to synchronise the on/off timing of the LED which accelerates the reaction, the sensor that measures the transmittance and the integrated magnetic stirrer, to avoid interference due to catalyst particles in suspension. In short, the presented design would be easily modular and scalable both on a laboratory and industrial scale. It would also be possible to synchronise the data with the cloud to be able to monitor the evolution of the photocatalysis online."} +{"text": "We aimed to evaluate the effectiveness and safety between high-power short-duration (HPSD) radiofrequency ablation (RFA) and conventional RFA in patients with atrial fibrillation (AF). Clinicaltrials.gov. Studies comparing HPSD and traditional applications in patients undergoing initial catheter ablation for atrial fibrillation from inception through December 2021 were searched on Pubmed, Medline, Cochrane, and P < 0.001), RF duration , fluoroscopy duration , and acute pulmonary vein reconnection (Odds ratio (OR) 0.40, P < 0.001), while improving the freedom from atrial arrhythmia at one year 1.12\u20131.94, P=0.005) and rates of first-pass isolation . Compared with the conventional group, freedom from atrial arrhythmia at one-year follow-up was higher in the HPSD group without the guidance of AI/LSI and studies with a power setting of 40\u201350\u2009W . Nevertheless, the two groups had similar effectiveness with a power setting of 50\u2009W in the HPSD RFA . There was no difference in complications between the two groups (P=0.71). The meta-analysis included seventeen studies with a total of 4934 patients. HPSD group decreased procedure duration (mean difference (MD) \u221238.28\u2009min, HPSD RFA was associated with shorter procedure duration, higher freedom from atrial arrhythmia, and comparable safety compared to conventional RFA. Catheter ablation is recommended as an effective therapy for atrial fibrillation (AF) to reduce the risk of stroke, heart failure, and mortality and improve the quality of life . As the Clinicaltrials.gov from inception through December 2021 by two reviewers (Shuyu Jin and Yumei Xue) independently. The search involved the following keywords: AND (\u201cHigh power\u201d OR \u201cHigh-power shorter-duration\u201d OR HPSD)\uff0eThis meta-analysis was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines PRISMA . An all-The studies included fulfilled the following criteria: (1) cohort study, case-control study, cross-sectional study, or randomized controlled trial (RCT) conducted in patients with age\u226518, with paroxysmal and/or persistent AF undergoing initial catheter ablation; (2) comparison between HPSD RFA and conventional RFA; (3) studies must meet the following for each ablation strategy: HPSD settings: Power\u226540\u2009W, with ablation duration of 5 to 15\u2009s per site including the posterior wall; conventional settings: Power \u226435\u2009W, duration >10\u2009s for any ablation; (4) reported outcome data including but not lcomparesdure time, freedom from atrial arrhythmia, total complications, redo-ablation procedure; (5) the follow-up duration was at least 6 months.The exclusion criteria were as follows: (1) conference abstracts, case reports, review articles, meta-analyses, editorials, or nonEnglish articles; (2) an equivocal study design or group allocation.A standardized data collection form was extracted by two investigators independently to obtain the following data from each study including name of the first author, year of publication, country of origin, study population, inclusion and exclusion criteria, demographic data of participants, and ablation procedure details. Disagreements were arbitrated by a third person in rereview. The original author was contacted by mail for access if the full text could be obtained. For literature in which the same study populations were reported many times or repeatedly published, only one with the most complete data was included. The quality of these studies was evaluated by two investigators (Shuyu Jin and Yumei Xue) using the Newcastle Ottawa scale (NOS) for observational studies and the \u2009 HPSD RFA\u2009:\u2009 ablation power \u226540\u2009W, including the posterior wall, with ablation duration of 5 to 15\u2009s per site.\u2009 Conventional RFA\u2009:\u2009 ablation power limited to 20\u201335\u2009W, with a longer ablation duration of 10\u201330\u2009s per site.\u2009 Procedure time: time from the application of local anesthesia to the withdrawal of all catheters.\u2009 RF time: total time from the first to the last ablation site.\u2009 Fluoroscopy time: total time for fluoroscopy from the start to the end of the procedure.\u2009 First-pass PVI\u2009:\u2009 rate of complete PVI after first-pass circumferential RF delivery.\u2009 Atrial arrhythmia recurrence: any symptomatic or asymptomatic atrial arrhythmia lasting >30\u2009s after completing the blanking period post ablation.\u2009 Acute PVR\u2009:\u2009 acute reconnection was assessed at 20\u201330 minutes post ablation, and adenosine was administered intravenously (dosed to achieve transient heart block) or waiting for 30 minutes following the last RF application to assess PV reconnection, including spontaneous reconnection and dormant conduction.\u2009 Major complication: complications that required any intervention or prolonged hospital stay including pericarditis, complete atrioventricular block, sinus node dysfunction, phrenic nerve palsy, stroke, ptocardial effusion, vascular access issues, steam pop, esophageal lesions, and death.P-value \u22640.05 was considered statistically significant. The fixed-effects model and the random-effects model were considered based on the level of heterogeneity. The heterogeneity of studies was evaluated by Cochran's Q and the I2 statistic. I2 lies between 0% and 100% with larger values showing increasing heterogeneity. I2 value\u2009>\u200950% was considered high degrees of heterogeneity and the random model was used in the subgroup analysis or sensitivity analysis excluding the trials that potentially biased the results to avoid publication bias, otherwise, a fixed-effects model was used and lower PVR , conversely, no difference was found in this endpoint between the two groups with the power setting of 50\u2009W (P=0.52) , total RF duration (P < 0.001), and fluoroscopy duration (P < 0.001) with no major complications. The QDOT-FAST trial [However, the appropriate power for the RF ablation is not clear. One study used higST trial used 90\u2009ST trial . Winkle ST trial reportedPrevious studies indicate force-time integral (FTI) as a target value to achieve permanent PVI, while not considering power settings. Consequently, only 72% of PVs remained isolated in 3 months . AI is aSafety during elective PVI procedures is of worthwhile concern. Radiofrequency catheter ablation is a technique where conductive and resistive heating are delivered through electrode catheters to myocardial tissue creating a thermal lesion. Irreversible myocardial tissue injury with cellular death occurs once the temperature of approximately 50\u00b0C has been reached, whereas conductive heating transfers thermal energy directly to deeper tissue . Unlike In terms of procedure duration, RF duration, and fluoroscopy time, the HPSD RFA strategy represents distinct advantages compared with the conventional RFA strategy whether in the subgroup analysis or not. Additionally, the reduction in procedure times can decrease the intravenous fluid volumes administered to patients which may benefit patients with cardiac insufficiency. Finally, less radiation exposure will also benefit both patients and physicians .To conclude, our results of the pooled analysis favor the use of HPSD settings over conventional settings. However, more RCT studies are needed to further assess the above results.We acknowledge several limitations in our study. First, we have only one RCT included in our meta-analysis while the rest were nonrandomized comparative studies. Although, all included studies were of good quality based on the Newcastle Ottawa scale, reflecting a real-world experience, more randomized controlled trials would provide better evidence for the difference in outcomes between two groups. Second, there were variations in each study in terms of power, types of catheters, contact force, target temperature, antiarrhythmic drug use, and the definition of freedom from atrial arrhythmia, resulting in significant heterogeneity between groups. And seldom included studies analyzed total energy during ablation procedure which we could not compare between two groups. Third, the included studies not only performed PVI but also additional linear ablation and different surgical methods might affect the maintenance of sinus rhythm. At last, we have a limited number of studies that reported PVR during redo procedures and with the guidance of AI/LSI. Finally, the exact anatomical locations of PVR were not clearly described in each study, so we could not analyze the specific locations of PVR.High-power short-duration RFA was related to better procedural effectiveness and higher freedom from atrial arrhythmia with comparable safety when compared with conventional RFA. Additionally, HPSD RFA decreases procedural, RF, and fluoroscopy durations. Meanwhile, in the subgroup analysis, HPSD RFA demonstrates a feasible, effective and safe approach for AF ablation."} +{"text": "The application of polymeric and composite materials in two-phase passive heat transfer devices is reviewed critically, with a focus on advantages and disadvantages of these materials in thermal management systems. Recent technology developments led to an increase of the power density in several applications including portable electronics, space and deployable systems, etc., which require high-performance and compact thermal management systems. In this context, passive two-phase systems are the most promising heat transfer devices to dissipate large heat fluxes without external power supply. Usually, heat transfer systems are built with metals due to their excellent thermal properties. However, there is an increasing interest in replacing metallic materials with polymers and composites that can offer cost-effectiveness, light weight and high mechanical flexibility. The present work reviews state-of the-art applications of polymers and composites in two-phase passive thermal management systems, with an analysis of their limitations and technical challenges. Thermal management systems play a vital but often overlooked role in several high-tech systems and devices, ranging from small portable electronic devices to large data centeres ,3,4,5,6,Two-phase passive thermal management systems are heat transfer devices where a process of liquid\u2013vapour circulation is established between an evaporator and a condenser by exploiting both sensible heat and latent heat in the heat transfer process. They are highly thermal conductors of which their thermal conductivities are many times greater than thermal conductivities of solid materials . Therefore, they have been widely used as thermal control systems for several advanced applications .Similar to other thermal management systems, passive heat transfer devices are usually built with metallic materials, often with high thermal conductivity such as copper. However, recent advances in technology such as foldable and flexible electronic components and devices, soft robotics, as well as spacecraft components, often have additional requirements of mechanical flexibility, low-cost, and/or low weight, which sometimes are difficult to achieve using metallic materials. Thus, there is a growing interest in replacing metals in full or in part with polymer and/or composite materials, which offer cost-effectiveness, light weight, high mechanical flexibility, resistance to corrosion and ease of manufacturing, at the price of a generally much lower thermal conductivity.This paper presents a review of the existing applications of polymer and composite materials in two-phase passive thermal management systems. Firstly, an overview of the material properties relevant to heat transfer devices is presented. Secondly, a survey of state-of-the art two-phase passive heat transfer devices fabricated using polymer or composite materials, with focus on natural circulation, gravity-driven heat transfer devices (thermosyphons) and on capillary-driven systems . Finally, the main limitations and technical challenges of polymer and composite materials relative to their use in thermal management systems are discussed, with an outlook towards potential new research trends.n times to form a long linear chain, as shown in Polymer materials are constituted by high molecular weight molecules, which are composed of a large number of units called monomers, connected by covalent bonds, resulting in high molecular weights that can exceed several millions . A commoThe most distinctive characteristic of polymeric materials as compared with metallic materials, which generally exhibit an elastic response to an applied stress or deformation, is their visco-elastic behaviour . In partMost polymeric materials can be sorted into one of the following three categories: thermoplastics, thermosetting materials, elastomers. The names of the most commonly used polymers belonging to each of these categories are listed in To improve certain properties of polymeric materials , they are often composited with different polymers and/or other materials . This results in a class of composite or engineered materials which often exhibit exceptional properties in terms of tensile, compressive, flexural and impact strength, Young\u2019s modulus, thermal expansion coefficient, corrosion resistance, and fatigue resistance .In many cases, composite materials consist of two or more materials, one of which acts as a continuous matrix and the others are dispersed fillers, selected according to criteria depending on the application. In thermal management applications, high thermal conductivity fillers are often chosen to enhance the conduction heat transfer rate of the material. To date, several types of high thermally conductive fillers are being used, such as metallic, carbon, and ceramic fillers ,22. MetaPolymeric composites can be processed with either thermoplastic or thermosetting polymers using a range of advanced manufacturing techniques, such as high-pressure injection, stamping, hot compression molding, resin injection, low-temperature and pressure compression molding, centrifugal molding, pultrusion, continuous impregnation, filament and tape winding, hand layup, spray layup, autoclaving, extrusion, additive manufacturing and many more ,41,42,43Two phase passive thermal management systems are heat transfer devices which can transfer heat from one point to another thanks to the thermally-induced circulation and/or oscillation of a heat transfer fluid without using any external forces, due to the interplay effects of three physical phenomena: phase change, capillarity and gravity. There are several types of two-phase passive heat transfer devices which can be distinguished based on the peculiar geometry and working principle: the conventional heat pipe, the loop heat pipe and the capillary-pumped loop heat pipe, the thermosyphon, and the pulsating heat pipe ,44,45. WA thermosyphon (TS) is a passive heat transfer device which exploits the buoyancy induced by a temperature gradient to circulate a heat transfer fluid, either in an open or in a closed loop. This concept, which has been known for centuries, equally applies to single-phase and two-phase flows; however, the latter can usually achieve better performances by exploiting the phase transition (latent) heat. Since the fluid circulation is due to buoyancy, these devices require gravity assistance, and their performance strongly depends on their spatial orientation. Modern thermosyphons can be dated back to the 1800s , althougIn the past few decades, several conceptual and experimental studies have been conducted on thermosyphons ,56,57,58Conventional heat pipes (HPs) are a common type of two phase passive heat transfer device which can achieve large heat transfer rates over relatively long distances and under relatively small temperature differences between the heat source and the heat sink. The concept of HP was first introduced by Gaugler in 1944s , and wasSince they were introduced, heat pipes have been manufactured according to different designs, using mainly metallic materials such as copper and aluminium ,74,75,76Oshman et al. developeHsieh and Yang fabricatYang et al. producedChao et al. developeYang et al. developeLoop heat pipes (LHP) and capillary pumped loops (CPL), schematically displayed in LHP and CPL are efficient systems and are suitable for several applications, including aerospace, cryogenics, solar systems and electronics ,97,98,99Pulsating heat pipes (PHPs), also known as oscillating heat pipes (OHPs), initially proposed by Akachi in the 1990s , are theThe PHP has many advantages with respect to the other two phase passive devices, such as: a completely passive operation without the need of a wick structure, structure simplicity and compactness, ease of manufacturing, and also the ability to operate against gravity . BecauseA limited number of attempts have been carried out to fabricate PHPs using polymer materials. Lin et al. developeAlthough polymer materials represent an interesting and often desirable alternative to metals, they also have some peculiar properties that can make their use difficult in thermal management systems. In the followng sections, the main issues that jeopardize the use of polymer materials in thermal systems are discussed, along with solutions to mitigate their impact on the performance and/or the lifetime of two-phase passive heat transfer devices.Thermal conductivity measures the ability of a solid, liquid or gaseous medium to transfer heat by means of molecular diffusion, i.e., by random collisions at molecular level. Due to their high molecular weight and the large number of internal degrees of freedom of their molecules, polymer materials have low thermal conductivities, typically less than 1 W/m\u00b7K ,141. TheOne common method to improve the thermal conductivity of polymers is to realign polymer chains in order to increase the crystalline to amorphous ratio using either mechanical stretching , nanoscale templating, or electrospinning . HoweverCarbon-based fillers, such as graphite, are characterized by high thermal conductivity, low weight, low cost and fair dispersability into a polymer matrix ,149. ExpA sensible target is to develop conductive polymer composites with thermal conductivity \u226510 W/m\u00b7K and containing relatively low amount of fillers (\u226420% volume fraction). This could be achieved using graphene nanoplatelet fillers, which have a thermal conductivity of 800 W/m\u00b7K contact line, which is usually expressed in terms of interfacial tensions between the solid and the vapor, +\u03b3LVcos\u03b8 , where \u03b8e system ,164. Acct angles . The twoWhile metals and most ceramics typically have large surface energies, therefore they are wetted by most liquids, the surface energy of polymer materials is significantly smaller, which makes their surface poorly wettable . In compSince wettability is directly responsible for capillary forces , it is aTo improve polymers surface wettability, it is necessary to modify the molecular structure of the surface using a range of surface treatment processes such as laser beam and plasma surface treatments ,168, or Although it is desirable in a number of applications , the mechanical flexibility of polymer materials can have unwanted effects on the operation of heat transfer devices. In particular, the deformation of polymeric walls due, e.g., to variations of the ambient pressure affects the pressure of the heat transfer fluid, both because of the volume change, and because the pressure difference between the external and the internal pressure must balance the stress buildup in the deformed channel walls, which acts to recover the initial shape. More importantly, the channel wall deformation affects the hydraulic diameter; this is of particular relevance in the case of pulsating heat pipes, where a variation of the hydraulic diameter may cause the Bond number to fall outside of the optimal range, reducing the thermal performance of the device . An addiThe use of polymer and composite materials to fabricate two-phase passive thermal management systems has been reviewed critically, with an assessment of their limitations and technical challenges. Although two-phase passive systems are often built with metallic materials because of their good mechanical and thermal properties, recent advances in technology such as the develoment of portable or deployable systems, including foldable and flexible electronics, are making the use of polymer and composite materials more and more attractive in order to meet technical requirements of mechanical flexibility and low weight, as well as cost-effectiveness. Consequently, several attempts to use these materials to replace metals in passive thermal management systems are documented in the literature.Polymeric two-phase passive heat transfer devices and thermal management systems are generally efficient and exhibit relatively high thermal performances. However, due to the low thermal conductivity, the poor surface wettability, the high gas-vapour permeability, and the viscoelastic behaviour of most polymer materials, the performances are usually not comparable to those achieved using metals. In addition, the decay of the thermal performances in time significantly affects the lifetime of devices, despite these materials are not subject to corrosion. At present, the most promising route to overcome these limitations is the development of polymer-based composites."} +{"text": "Additionally, the MgO/PDMS composites exhibited adequate electrical insulating properties, with a room-temperature resistivity of 7.92 \u00d7 1015 \u03a9\u2219cm.Owing to the increasing demand for the miniaturization and integration of electronic devices, thermal interface materials (TIMs) are crucial components for removing heat and improving the lifetime and safety of electronic devices. Among these, thermal pads are reusable alternatives to thermal paste-type TIMs; however, conventional thermal pads comprise a homogeneous polymer with low thermal conductivity. Composite materials of thermally conducting fillers and polymer matrices are considered suitable alternatives to high-performance pad materials owing to their controllable thermal properties. However, they degrade the thermal performance of the filler materials at high loading ratios via aggregation. In this study, we propose novel nanocomposites using densely aligned MgO nanowire fillers and polydimethylsiloxane (PDMS) matrices. The developed nanocomposites ensured the enhanced thermal conducting properties, while maintaining mechanical flexibility. The three-step preparation process involves the (i) fabrication of the MgO structure using a freeze dryer; (ii) compression of the MgO structure; and (iii) the infiltration of PDMS in the structure. The resulting aligned composites exhibited a superior thermal conductivity (approximately 1.18 W m With the continuous increase in the performance and miniaturization of advanced electronic devices, generated heat has become a major problem that degrades the performance and reduces the lifetime of devices . Thermal\u22121K\u22121. Composite materials composed of fillers and flexible polymeric matrices with high thermal conductivity are suitable alternatives in realizing high-performance thermal pads ,30(1)k=\u03c168 vol%) c. Theref68 vol%) d. This s68 vol%) e [31,32,The applicability of the developed composite to actual devices was evaluated by analyzing the heat dissipation, insulation, and mechanical properties of the pure PDMS, random composites, and aligned composites. The heat dissipation performance of the composites was examined by measuring their surface temperature using an infrared thermal imaging camera under constant heating at 90 \u00b0C. The pure PDMS, aligned composites, and random composites were placed on a hot plate (90 \u00b0C), and the changes in the temperature distribution of the surface were recorded for 30 s using a fixed thermal imaging camera a. The teAnother requirement for thermal pads applied to electronic devices is electrical insulation to prevent the electrical hazards caused by the leakage current of electronic devices. The resistivity of the two composites and pure PDMS was measured by applying a direct current. The fabricated 3D composites demonstrated an enhanced compressive strength compared to the random composites and pure PDMS . The aliIn this study, we developed a thermal pad composite with superior thermal, mechanical, and electrical properties using densely aligned MgO filler structures without aggregation problems. The densely aligned thermal pads enhanced the thermal conductivity by 136% compared with the randomly dispersed thermal pads at the highest filler content (11.68 vol%). The aligned thermal pad composites also demonstrated improved heat dissipation characteristics when applied to practical electronic devices. Therefore, the proposed thermal pad composite can serve as an efficient and excellent thermal management material for next-generation devices, such as curved, flexible, and stretchable devices, owing to its exceptional electrical insulation, flexibility, and adhesion. Furthermore, unique and outstanding composites produced through the 3D structuring of different filler materials can be applied to various applications, such as electromagnetic shielding materials (copper), transparent conductive flexible substrates (indium tin oxide), and high-strength structural materials (carbon fiber)."} +{"text": "Hemangiomas are benign blood vessel and capillary tumor growths which are widespread in many organs but extremely rare in the bladder, making up just 0.6% of all bladder tumors. To the best of our knowledge, few cases of bladder hemangioma are associated with pregnancy in the literature, and no bladder hemangiomas have been discovered incidentally after abortion. The use of angioembolization is well established; however, postoperative follow-up is crucial to identify tumor recurrence or residual disease. Case presentation: In 2013, a 38-year-old female was referred to a urology clinic with an incidental finding after an abortion of a large bladder mass identified incidentally using ultrasound (US). The patient was recommended for CT, which reported a polypoidal hypervascular lesion, as previously described arising from the urinary bladder wall. Diagnostic cystoscopy showed a large, bluish-red, pulsatile, vascularized submucosal mass with large dilated submucosal vessels, a wide-based stalk, and no active bleeding in the posterior wall of the urinary bladder, measuring about 2 \u00d7 3 cm, with negative urine cytology. Due to the vascular nature of the lesion and no active bleeding, the decision was made not to biopsy. The patient underwent angioembolization and scheduled for US every six months with regular diagnostic cystoscopy. In 2018, at 5 years of follow-up, the patient developed recurrence after a successful pregnancy. The angiography revealed recanalization of the previously embolized left superior vesical arteries from the anterior division of the left internal iliac artery, resulting in arteriovenous malformation (AVM). The second angioembolization was performed, with the total exclusion of AVM without residual. By the end of 2022, the patient had remained asymptomatic and without recurrence. Conclusion: Angioembolization is a safe treatment technique, minimally invasive, and has less effect on the quality of life, especially in young patients. Long-term follow-up is essential for detecting tumor recurrence or residual disease. Hemangiomas are benign blood vessel and capillary tumor growths widespread in many organs but extremely rare in the urinary bladder, where they make up just 0.6% of all bladder tumors ,2. FewerA 38-year-old, medically free female, was referred to a urology clinic in 2013 with an incidental finding of a large bladder mass by ultrasound after an abortion A. The paComputed tomography (CT) with intravenous contrast medium in 2013: A 17.1 \u00d7 24.2 mm, well-defined, homogenous hypo- to isodense polypoidal soft tissue lesion was revealed, arising from the posterior wall of the urinary bladder to the left of the midline and bulging inside the urinary bladder. After IV contrast injection, the lesion showed significant contrast enhancement with mild heterogeneity at its base. The rest of the urinary bladder wall was unremarkable.Impression: A polypoidal hypervascular lesion, as previously described, arising from the urinary bladder wall showed a distended UB with a polypoidal soft tissue mass measuring 16 \u00d7 16 mm with internal vascularity, nidus in the afferent and efferent vessels, and turbulence in the flow. CT showed a polypoidal hypervascular lesion in the same site of the previously embolized AVM in the posterior urinary bladder wall A,B.Second angioembolization in 2018: After informed consent was obtained from the patient, under aseptic conditions and local anesthesia, the right common femoral artery was punctured and a 6F standard sheath was inserted. Selective catheterization of the left internal iliac artery using a C2 angiocatheter followed by selective angiography, revealed recanalization of the previously embolized left superior vesical arteries approaching from the anterior division of the left internal iliac artery and supplying the known intra-resulting AVM. Selective catheterization of the left superior vesical artery using a 3F microcatheter, followed by embolization of the artery, was executed using ONYX. Final control showed total exclusion of the AVM without residual disease and with no complications A,B.The patient completed almost five years of follow-up\u2014with regular ultrasonography every six months\u2014without recurrence, hematuria, or lower urinary tract symptoms. During this period, the patient went through a successful pregnancy for the second time. Mellow et al. state thTo the best of our knowledge, there have only been a few cases of pregnancy-related vertebral hemangiomas in the literature ,7,8,9. HCheng et al. mentioned that size ranged from 0.2\u20133 cm, and about 10% (two cases) were of arteriovenous hemangioma (AVM) . A thirdThe traditional methods for diagnosing bladder hemangiomas include excretory urography, cystography, and cystoscopy. Given that these lesions were vascular, a biopsy could be dangerous due to the possibility of bleeding. In addition, because they extend into the bladder submucosa, their size is sometimes underestimated. Establishing the proper diagnosis may be facilitated by using CT and the sonographic features of significant bladder wall thickening, intramural anechoic gaps, and calcification . HemangiRegarding our case, incidentally, the ultrasound reported a large bladder mass, and the CT reported a polypoidal hypervascular lesion, as previously described, arising from the urinary bladder wall.The diagnosis was strongly supported by cystoscopic observations of a lobulated, bluish-red, vascularized, submucosal mass in a patient with recurrent hematuria. Nevertheless, endometriosis, melanoma, and sarcoma can have comparable symptoms .In our case, the diagnostic cystoscopy showed a large bluish-red, pulsatile, vascularized; submucosal mass with large, dilated, submucosal vessels; a wide-based stalk, and no active bleeding in the posterior wall of the urinary bladder, measuring about 2 \u00d7 3 cm, with negative urine cytology. Moreover, because of the probability of a difficult-to-control hemorrhage or the recurrence of bleeding after the biopsy, and considering the patient\u2019s situation following fulguration and expected blood loss after abortion, a biopsy was not taken. Instead, the decision was taken to perform angiography and angioembolization for diagnosis and treatment.The size, location, and depth of penetration of a urinary bladder hemangioma are essential considerations for treating patients . SurveilTreatment varies widely; observation , transurAlthough urinary bladder hemangioma has a benign course, postoperative follow-up is essential for identifying tumor recurrence or residual disease; this can be achieved with flexible cystoscopy, CT scans, or even ultrasonography ,25. ChenWhereas a partial cystectomy may reduce storage function, a partial cystectomy plus bladder augmentation can preserve storage function; however, this treatment may worsen voiding function . ConversOur reported case is a rare case of incidental bladder hemangioma after abortion. Angioembolization is a safe treatment technique, minimally invasive, and has less effect on quality of life, especially in young patients. Long-term postoperative follow-up is essential for detecting tumor recurrence or residual disease."} +{"text": "Visual simultaneous localization and mapping is a widely used technology for mobile robots to carry out precise positioning in the environment of GNSS technology failure. However, as the robot moves around indoors, its position accuracy will gradually decrease over time due to common and unavoidable environmental factors. In this paper, we propose an improved method called RTABMAP-VIWO, which is based on RTABMAP. The basic idea is to use an Extended Kalman Filter (EKF) framework for fusion attitude estimates from the wheel odometry and IMU, and provide new prediction values. This helps to reduce the local cumulative error of RTABMAP and make it more accurate. We compare and evaluate three kinds of SLAM methods using both public datasets and real indoor scenes. In the dataset experiments, our proposed method reduces the Root-Mean-Square Error (RMSE) coefficient by 48.1% compared to the RTABMAP, and the coefficient is also reduced by at least 29.4% in the real environment experiments. The results demonstrate that the improved method is feasible. By incorporating the IMU into the RTABMAP method, the trajectory and posture errors of the mobile robot are significantly improved. In recent years, the demand for high-precision mapping and positioning has significantly increased due to the advancement and evolution of autopilot, AR and VR, UAV, and robotics. Mapping and positioning are crucial components in various innovative technologies, and their accuracy and robustness are of great importance. The field of simultaneous localization and mapping (SLAM), also known as Concurrent Mapping and Localization (CML), is a popular area of study for scholars both domestically and internationally. SLAM is used to process and construct maps in unknown environments and to determine the location of robots within these maps ,2,3,4. CIn fact, it is not easy to construct an efficient and accurate map and obtain its own precise location in it . It requMourikis et al. proposedLabb\u00e9 et al. proposedIn indoor SLAM, cameras have an advantage over 3D lidar in terms of cost and semantic information. Meanwhile, wheel odometry is also one of the common sensors in mobile robots. An improved algorithm for RTABMAP is presented in this paper. The main idea is to use an EKF to fuse the IMU with the wheel odometry and provide a new state estimation model to achieve more accurate location estimation.We have validated the improved method through experiments conducted in public datasets (indoors) as well as real environments, which include enclosed rooms and open corridors.However, we found that RTABMAP\u2019s own localization accuracy in the constructed maps is not very high in practical tests, especially in the environment with multiple interference factors. So, in this paper, we hope to improve the robustness and real-time performance of the RTABMAP algorithm by improving the algorithm to adapt to diverse scenes. At the same time, it provides a new reference for research and application in the field of SLAM. This paper aims to optimize the RTABMAP method and integrate data from various sensors. By combining the strengths of different sensors, their weaknesses can be compensated for, resulting in more comprehensive, accurate, and reliable positioning results. This article makes the following main contributions:The inertial measurement unit (IMU) mainly consists of sensors such as accelerometers, gyroscopes, magnetometers, and others. It is generally categorized as 9-DOF and 6-DOF (excluding the magnetometer). In addition to violent vibrations and shocks, IMUs are hardly disturbed by other forms of external signals and extreme weather and strong light. Due to the redundancy between the measured values of angular velocity and linear acceleration, the \u201crelative position change in a short period of time\u201d of the IMU output has a very high degree of confidence. The IMU is an independent system that can estimate the relative position without relying on any external device to assist the derivation. In contrast, lidar, camera, etc., are dependent on external sensing and have some instability.X, Y, Z) of the n coordinate system are commonly achieved by sequentially rotating the angles of roll (\u03c8), pitch (\u03b3), and yaw (\u03b8). The conversion process is shown in In the IMU system, usually in order to explain the robot\u2019s position, movement speed, direction, etc., the corresponding coordinate system must be selected. In this paper, we use the IMU\u2019s installation plane as the carrier coordinate system (b coordinate system). Additionally, we select the geographical coordinate system as the navigation coordinate system (n coordinate system) to simplify the attitude calculation and improve comprehension . The thrThe transformation matrix between the n coordinate system and the b coordinate system is often referred to as the direction cosine matrix. The coordinate transformation of the direction cosine matrix can be expressed as follows:In this formula, the subscript b represents the carrier coordinate system, the subscript n represents the navigation coordinate system, and This representation method has a flaw known as gimbal lock, which can result in the loss of one posture\u2019s degree of freedom. To avoid this issue and provide a more concise description of the robot\u2019s motion, the four-element method is commoThe quaternionic representation of the rotation matrix is shown in Equation (4).\u03b8, \u03b3, and \u03c8 can be obtained, as shown in Equations (5)\u2013(7).By jointly solving Equations (2) and (4), the quaternion representations of The IMU has both advantages and disadvantages. In an IMU, both accelerometers and gyroscopes use an integral method to measure data. For example, integrating linear acceleration can obtain linear velocity, and integrating again can obtain position information. The rotation angle can also be obtained by integrating the angular speed measured using the gyroscope. However, when attempting to solve the state quantity through integration, particularly in discrete form, it is inevitable that various errors will be introduced. The IMU errors are mainly divided into systematic errors and random errors. The former mainly include the scale factor error, axis deviation error, bias error, and temperature error, while the latter mainly include random walk and bias instability. Systematic errors usually follow certain patterns, which can be eliminated through real-time compensation. Random errors, on the other hand, typically refer to noise, and it is often challenging to identify a suitable function to describe the noise, making the processing quite complex. Usually, the manufacturer corrects the systematic errors in the IMU chip when leaving the factory . As for Random walk and bias instability are key parameters in the fused positioning system. There are related calculations in many fused SLAM algorithms involving the IMU (such as VINS and ORB-SLAM3). Once the above parameters are obtained, a covariance is set on the pre-integrated error term of the IMU , which iThe steps of the Allan variance calculation are as follows.Acquire and group the data.Set a fixed sampling frequency 2.Calculate the average value of the data.Calculate the average value of each set of raw output data, i.e., the average value of the dataset 3.Obtain the Allan variance.Defining the sampling time of each group as In this paper, data were collected by keeping the IMU stationary for about 2.5 h, using a frequency of 200 Hz. The output data were processed according to the Allan variance formula described above. The Allan variance curve is shown in Because the Kalman Filter (KF) is more suitable for linear systems, and our system has many sensors, there are many uncertainties, so we cannot continue to use this algorithm. The EKF applies linear analysis to the KF model, allowing for the use of a nonlinear model in the prediction and update process of the linear model. Suppose there is a robot that measures information such as linear velocity through a wheel odometry and uses an IMU to measure information such as acceleration and angular velocity. This paper presents a new odometry that utilizes the EKF for data fusion from two sensors, enabling accurate estimation of the robot\u2019s attitude and position. The EKF divides the system into prediction and update steps, which are implemented based on the system\u2019s motion model and observation model, respectively ,31.\u03b8, \u03b3, \u03c8 are the Euler angles of the robot. The state of the robot is represented by the state vector, and the state equation is shown in Equation (11).The motion of the robot is described by a nonlinear system model, as shown in Equation (12).The observation equation of the robot is shown in Equation (13).Define the prior estimate, as shown in Equation (14).Define the prior estimation error covariance matrix, as shown in Equation (15).Define posterior estimation as shown in Equation (16).I is the identity matrix.Define the posterior estimation error covariance matrix, as shown in Equation (18).Finally, the prediction state and the covariance matrix are divided into the following four steps:From the calculation process of the above formula, it is clear that the EKF can effectively incorporate the conducive information in the observation value e vector . In thise vector is used The IMU is not affected by the environment when solving poses. Therefore, it can estimate the speed, position, and attitude of all parameters solely based on the inertial information generated by the carrier\u2019s motion. The improvement method in this paper introduces the IMU for fusion in the framework of the RTABMAP algorithm based on visual-wheel-odometry. The IMU can estimate motion parameters that can be utilized to rectify the absence of scale information in the camera. It can also offer high-frequency attitude estimation data (acceleration and angular velocity) and real-time rapid response positioning . On the The proposed system has two main parts: multi-sensor fusion localization and 3D dense point cloud mapping. The multi-sensor fusion localization section integrates position estimation from wheel odometry, the IMU, and cameras. The robot obtains displacement and velocity through trajectory extrapolation with the help of wheel odometry, and at the same time obtains angular velocity, angular displacement, and acceleration with the integration algorithms of the IMU\u2019s gyroscopes and accelerometers. Finally, the EKF is utilized to fuse the above data into a motion model of the robot, and the robot\u2019s state is updated in real-time by the prediction step and update step of the EKF. Due to the different locations of the sensors in space, there is also a translation of the coordinate system between the sensors, so the EKF will eventually provide a TF coordinate translation as well as an odometry called Odom_combined, which is then time-synchronized with the camera\u2019s raw data and deposited into the node created by the STM. The node also holds visual words for loop closure and similarity detection and a local occupation grid for building a global map. Finally, the corresponding solution is performed based on the data in the node, and the solution result will be used as the constraints for the graph optimization (next node). When there is a new loop closure or proximity link added to the graph, graph optimization propagates computational errors throughout the map to reduce odometry drift and performs ranging corrections via TF transformation to correct the robot\u2019s localization in the map. For the 3D dense point cloud mapping, the original RTABMAP method is used to generate a real-time dense three-dimensional point cloud map of the robot\u2019s environment in rtabmapviz. The specific system framework is shown in To validate the improved method\u2019s effectiveness in real-world scenarios, this paper assesses localization accuracy through two dataset experiments and three real-world experiments. The dataset is the V1 series in EuRoC, the real experimental scenarios are all indoors, and each experimental site is of different sizes and contains complex environmental conditions to verify the feasibility under different settings. In this paper, the original method is still called RTABMAP. The improved method is called RTABMAP-VIWO, abbreviated as VIWO. We use the EVO to compaIn the calculation of APE, the translation error to evaluate the accuracy, which is calculated as shown in Equation (23).In real-world scenarios, we use RGB-D cameras as the primary sensor, but for dataset testing purposes, we rely on stereo cameras as the primary sensor due to the types of sensor limitations of the EuRoC dataset. Despite the difference in sensors, we are still able to validate the feasibility of our proposed method.The dataset utilized in this article is the EuRoC MAV visual inertial dataset , which wFor this experiment, we use the V1_01_easy sequence (V101) to assess and compare the trajectories of VIWO and RTABMAP. To ensure a fair and convenient comparison, we compare VIWO with the original method using the actual trajectories from the dataset, and compare one or more APEs using the evo_res tool in EVO. In the V101 sequence, the camera direction remains constant regardless of the drone\u2019s attitude, resulting in minimal stimulation of the three-axis gyroscope in the IMU. The absolute pose error map of the V101 sequence is shown in In the V101 sequence, this paper demonstrates the feasibility of making improvements to RTABMAP, and the performance enhancement effect of VIWO is further demonstrated subsequently. In this test, we conduct comparison experiments using the V1_03_difficult sequence (V103) and include ORB-SLAM2 in the comparison. Both the RTABMAP and ORB-SLAM2 methods utilize feature extraction to extract and match feature points in the image. Both methods are based on nonlinear optimization to optimize the camera position and map, and both include complete loop closure detection and support multiple cameras. Therefore, they share many similarities in the algorithm\u2019s details. Because ORB-SLAM2 produces sparse point cloud maps, its performance is superior. During the experiment, the trajectory of ORB-SLAM2 appears smoother due to its recording of more pose keyframes compared to RTABMAP. The V103 sequence was recorded in a challenging environment, ranging from slow flight with good visibility to dynamic flight with motion blur and poor lighting. This posed a significant increase in difficulty compared to the V101 sequence. Upon comparing the blue mark ring in In order to better validate the robustness and accuracy of the positional trajectories of RTABMAP-VIWO in the real world, we built a wheeled mobile robot driven by four DC reduction motors with encoders. The motor operating voltage is 24 V, and the steering method is differential steering. The controller chip of the lower system is STM32F407VET6, and the controller used by the upper system is the same laptop that was used to run the dataset, CPU: i5-7300HQ, 2.50 GHz \u00d7 4; RAM: 16 GB; GPU: NVIDIA GTX1050Ti; camera: realsenseD435; RGB FOV (H \u00d7 V): 69\u00b0 \u00d7 42\u00b0; fps: 30; IMU: HiPNUC Hi226 (6-DOF); refresh rate: 200 Hz; operating system: Ubuntu 18.04; ROS: Melodic. The mounting position of each sensor is shown in During test 1, the mobile robot navigated an S-shaped path indoors. The experiment involved two methods of turning: turning on the spot and turning at a differential speed. Scenario 1 is a large, connected laboratory (approximately 12 m \u00d7 11 m in size) consisting of Room 1 and Room 2, where Room 1 has no air conditioning vibrations and Room 2 has air conditioning vibrations, and the rest of the conditions are the same in both rooms. The dividing walls and connecting passages of the two rooms are shown in Mobile robots are typically assigned closed-loop routes to travel, regardless of the scenario they are used in. Hence, in test 2, this paper assesses the VIWO via a closed-loop pathway. The entirety of test 2 in Scenario 2 takes place in Room 2, so the environment in test 2 encompasses light reflection and air-conditioner vibration. Refer to As shown in In indoor settings, it is inevitable to encounter long, narrow corridors or tunnel-like structures with a high degree of repetitive features. In test 3, we test the mobile robot in this scene. As shown in As shown in The results of the dataset experiments are shown in In this paper, we conduct experiments on the EuRoC dataset with the main purpose of verifying the feasibility of our method. However, the dataset experiments are different from the real environment, and the purpose of our improvement is to enhance the performance of RTABMAP and to be able to be applied in real life, so we conducted the experiments in a real environment with multiple common interferences. Five experiments ranging from simple to difficult finally proved that the RTABMAP-VIWO system utilizing the EKF fusion IMU is superior to RTABMAP and exhibits higher positioning accuracy and anti-jamming.There are limitations that exist in our method. The individual sensors in the wheeled robot we built are mainly fixed by aluminum as well as 3D-printed non-standard parts, which creates a considerable mounting error for the TF conversion in the whole system. In addition, when operating in the environment shown in Aiming at the complex indoor environment, this paper proposes an improved RTABMAP method. To achieve precise and reliable robot pose estimation in indoor environments, this paper integrates the IMU into the original system using the EKF to minimize positioning errors caused by the primary sensor. By combining the 3D dense point cloud map, constructed using RTABMAP, with the proposed visual inertial wheel odometry estimation, mobile robots can achieve more accurate indoor repositioning. This provides more precise sensory information for further path planning and autonomous navigation.This paper conducts tests and evaluations in both common and challenging indoor environments. The performance of three SLAM methods is compared in both public datasets and real-world scenarios. In RTABMAP, motion estimation during localization or loop closure detection is mainly performed visually. The experimental results demonstrate that both RTABMAP-VIWO systems, using stereo and RGB-D cameras as the primary sensors, respectively, exhibit low drift error and high robustness. In addition, the main work of this paper is the improvement in the localization accuracy of the mobile robot, while the map construction and navigation have not been studied in depth. In the future, we will incorporate low-cost 2D lidar, which will improve the robot\u2019s localization accuracy and build a higher-quality map, creating the basis for subsequent robot navigation. In addition, we will combine SLAM with deep learning to further reduce the impact of reflective ground lighting and passing pedestrians on localization."} +{"text": "Cities are critical to a sustainable future for our planet; still, the construction and operation of cities rely on intensive resource and energy use and transformation, leading to the generation of waste, effluents, and pollution, representing negative externalities outside and inside the city. Within every process, transformation implies the use of energy and the increase of entropy. In an urban system, the transformation of energy and materials will trigger the creation of entropic landscapes, mainly in the informal city and in unguarded natural landscapes, even hundreds of kilometers away, which generates substantial economic, social, and environmental impacts. In this sense, cities are significant contributors to the environmental crisis. Upstream, degradation of landscapes and ecosystems is frequent. Cities\u2019 externalities and exogenous consumptions are directly linked with entropy and entropic landscapes, which are recognized as pollution or waste and in the degradation of natural ecosystems and communities. Through a systematic review of existing literature, this paper first outlines briefly how entropy has been applied in different disciplines and then focuses on presenting recent developments of how entropy has been defined, used, and characterized in urban studies concerning sustainability in cities and architecture, and presents a definition of the concept in relation to urban systems and key aspects to consider. Cities depend on unsustainable linear flows ,2,3 thatHowever, the way entropy is framed in literature makes the concept alien and not easy to approach for architects, urban designers, and cities\u2019 policy makers. The concept is perceived as negative or intanBy using a systematic literature review ,14,15 anAccordingly, this paper is structured in five sections. First, Entropy is a broad concept that has undergone progressive development. It was used primarily in the nonsocial sciences, and then its use extended to other disciplines, including the urban and regional system studies . The worIn thermodynamics, entropy measures a system\u2019s disorder or randomness. For example, organized, usable energy has low entropy, whereas disorganized entropy, such as heat, has high . TherefoEntropy from a thermodynamic perspective was later redeveloped as uncertainty, with the principle of entropy maximization applied as a statistical inference method for supporting spatial location and spatial interaction models . In infoIn economics, scholars highlighted that economic development depends on the use (and degradation) of limited natural resources. Nicholas Georgescu-Roegen mentioned that \u201ca living organism does not need just energy but low entropy which it sucks from the environment and degrades into high entropy (waste)\u201d , in otheRegarding arts, Arnheim Rudolf refers to entropy with the idea of a clash of orders, where disorder is not the absence of all order but rather the clash of uncoordinated orders. He adds that any progress requires a change of order; for example, a revolution must aim at the destruction of the given order and will succeed only by asserting an order of its own . MoreoveFinally, regarding ecology and biology, entropy is used from a different perspective. In this perspective, life processes degrade input resources to create new information, build an organization, add structure, or upgrade energy. The resources, once degraded, will become available again for new life cycles, driven by the entropic degradation of solar energy. In addition, the higher the ecological diversity, the lower the production of entropy per unit of biomass, because resources are better utilized and support the growth of the whole spectrum of ecosystem hierarch . More reFrom all these areas of knowledge, the use of entropy within a broad biological perspective is central for this literature review since the approach to the city will be a systemic approach; even more, the city will be understood as a living organism. What is central to underline is that biological organisms are open thermodynamical systems exchanging energy and matter with the ecosystem and that after entropy, order, even life could occur. The evolution and growth of biological systems cannot be explained by the Second Law of thermodynamics of an isolated system but with the open system perspective, where an organism requires an exchange of matter and energy with their environment . The cheCentral concepts that could be adapted to the urban perspective of entropy were developed and postulated by Prigogine, including complex system, self-organization, equilibrium, the arrow of time, and especially his theory of far-from-equilibrium systems, irreversibility, and dissipative structures. This is especially true for urban systems, which are located inside a natural ecosystem taking and dissipating energy . For Prigogine, entropy is much more than merely a disorder and its irreversibility, it is a mechanism for producing order and could be directed to a new organism and system , creatinEntropy research is a developing subject in different disciplines, and in recent years, the concept was adapted to urban studies with different perspectives. To identify and describe current practices and incorporate front-line knowledge, a systematic literature review was chosen as a method of research, integrating findings and perspectives in relation to entropy from many studies . The sysAfter the general study of the entropy concept in other disciplines , it can The definition of the search strategy for the systematic review includes specifying the sources for consultation, the keywords, the filter criteria, the number of search rounds, and the exclusion standards. The search was performed using three prestigious academic search engines . For the key words\u2019 definition, three steps were applied. The first step was to define two main keywords used for this research. The first word was entropy and to find a second word, five searches in SCOPUS were conducted . AccordiThe first bibliometric analysis was performed three times, changing the number of occurrences . The code for the software was: (Map created based on text data (RIS file) > Title and abstract > Ignoring structured abstract labels and copyright > Full counting > 20998 terms in total > (25/50/100) occurrences: (215/76/31) words > 60% > (129/46/19) words included) . We can Since there is a high occurrence of words \u201cmeasure\u201d and \u201cmodel\u201d, they will be included in the final search. In contrast, some missing keywords essential for the present literature review are \u201cflows\u201d, \u201csystem\u201d, \u201csustainability\u201d , \u201ccircularity\u201d, and \u201cmetabolism\u201d. These words will also be included using the Boolean AND to reduce the search scope and be more precise. Finally, the second main keyword should be \u201curban\u201d, but since \u201ccities\u201d also presented a high number of results, it will be used [\u201curban\u201d OR \u201ccities\u201d]. With all these insights from the first search, the next step was to execute several preliminary searches in Scopus using a combination of identified keywords , aiming From Search #8 included the results from the preliminary search . Originally, 1071 papers were obtained, which was considered high for a systematic and detailed literature review. Then, filters were applied, including the exclusion by area and language, bringing a still high number of results (659). Later, titles were scanned, and it could be inferred that most of the results were not related to the research topic. Therefore, two actions were carried out: first, the exclusion of the words (\u201cmeasure\u201d or \u201cmodel\u201d) since they were leading to broad results and to results out of the scope of the research question studies. Second, the search strategy needs to be narrowed down. Subsequently, the next step was to use two levels of keywords for search. In the first level, the main words \u201centropy\u201d and (\u201curban\u201d or \u201ccity\u201d) should be present in the title or keywords. The second level, keywords \u201cflow\u201d or \u201csystem\u201d AND \u201csustainable\u201d or \u201cmetabolism\u201d or \u201ccircular\u201d or \u201cdissipative\u201d should be present in the abstract or the title or the keywords. This coding was used in Scopus (239 documents) and adapted to Science Direct (92 documents) and Taylor and Francis (14 documents).To compare the results from the original search , #1A witThe next step was to apply distinct layers of filters . First fIn Zotero, the repeated papers were excluded, obtaining 110 papers. Then, a second bibliometric network analysis using VOSviewer version 1.6.18 was performed to evaluate the relevance of the results . For thiEven using a narrow search code , #17, thIn the last step, a final bibliometric network analysis using VOS viewer was made at 9, 6, and 4 occurrences to identify the main themes for review. From the four occurrences analysis, some conclusions could be presented. First, \u201ccity\u201d and \u201centropy\u201d appeared in the center of the diagram, closely connected with the system and process. Second, the present literature review research aim is between city and entropy. Using this area as the center, we can draw layers of closely related words and observe three clusters. The red cluster is linked to the urban from a sustainable perspective and includes urban sustainability, urban system, open system, or thermodynamics concepts. The second cluster (green) relates to the city\u2019s systemic vision from a territorial approach and spatial analysis. It includes central words such as \u201cstructure\u201d, \u201carea\u201d, \u201corder\u201d, or \u201cdevelopment\u201d, and \u201curban growth\u201d or \u201ccomplex system\u201d that is eccentrically located. Finally, the third cluster (blue), is linked to processes and planning. A relevant word in this cluster is urban entropy, close to the systemic approach .For the content analysis of this research, three themes linked to the previously identified clusters were defined. First, the definition of entropy in relation to sustainability. Second, the connection between entropy and the vision of the city as a system, including identification of analyzed flows . Third, the process or measure method (and instruments). An additional theme is related to the solution/ideas for circularity from each paper.The literature review results are presented in three sections. The first is a descriptive analysis of the results in terms of year of publication, area of knowledge, and country of origin. The next section is a content analysis focusing on three main topics: entropy definition, entropy in urban context, and entropy characterization. The final section is a table with a summary of the 38 reviewed papers.The 38 resulting papers were processed to analyze the year of publication, the subject of knowledge, and the country of origin . First, The next section is a content analysis focusing on three main topics: entropy definition, entropy in urban context, and entropy characterization.In the revised papers, entropy is related to change with two main connotations, the first is related to change in energy and the second to change in order . Regardiit is produced in relation to the urban system. A common categorization throughout the studies is the internal and the external entropy , the production of entropy should be directly related to energy flows and cannot be expressed in terms of material waste or pollution; in this sense, the entropy that returns to the natural environment could only be waste heat, since \u201cthermodynamics does not include the notion of usefulness or utility, and because of this, failure to capture the human degradation of resources\u201d . Hence, About the relation between irreversibility, utility, and entropy, some authors argue that only irreversible processes produce entropy while otFinally, in relation to change in order, in the revised studies, there are two main approaches. The first is related mainly with spatial arrangement, measuring the level of dispersion of an urban area, that is translated then into an indicator of risk in urban systems . Within As other authors noted, entropy within urban studies is linked to two different approaches: the first approach is connected with information theory and regularly employs the formula developed by Shannon. Here, entropy is usually a measure related to information and aesthetics; the second approach is related to thermodynamic laws with a tangible and scientific nature; it could be called physical entropy and is related to energy, waste, and heat or pollution that the cities generate. One of the biggest challenges within urban studies is to relate these two different approaches . For theConnected with the thermodynamic approach, a critical aspect concerning entropy and urban systems is the vision of the city as an open system. This vision includes the definition of the boundary, the identification of the flows and the effects on the urban and natural systems. In this vision, it is central to recognize three main points. First, the city-ordered structure is possible due to the generation of more significant disorder in the environment out of the open system. Second, the city absorbs external input (negentropy) and emits internal output . Third, the non-equilibrium steady state of the city should not be confused with thermodynamic equilibrium . Most ofHowever, entropy flow is not always presented as a negative aspect; it could also represent the harmonious capacity of systems, and the entropy production could reflect the ability for system reduction and reproduction in the metabolic process and can indicate the vitality of the system . In compSimilarly significant, the concept of dissipative structures was consFinally, some other aspects of the urban system and entropy addressed by revised papers are that entropy addresses three main features in the urban system: the position or location, the mechanic flow network, and the system scaling or size . These tNo single practice was found or a method that is repeated in two or more papers. Despite general equations from open system theory being referred to in documents related to the non-equilibrium system, it was more to establish a broad context (city as an open system). There are some coincidences in general perspectives regarding characterization, such as the approach or some other similitudes, such as recognizing that there is an external and an internal entropy. Still, no common base was founded for the proposed elements, indicators, proxies or indexes for entropy. In parallel, it was noted that indicators were more related to urban systems, and measuring is more related to energy and efficiency .The entropy measurement method for open systems continuously refers to the Prigogine equations for open systems, where the entropy is the sum of the negative entropy produced inside the system with the positive entropy created in the environment. Here, entropy change depends on the interactions among constituents of the system and exchanges with the external environment. The equations presented in the analyzed papers are summarized in Another aspect that is frequently measured and related to entropy is energy. For instance, an entropy indicator was presented as the ratio between the entropy variation due to energy losses of buildings and the entropy variation due to the solar energy gain of a mesh area . In anotThis is exemplified by some figurative strategies that have also been developed in the literature, aiming to measure entropy from another perspective (not in terms of joules per kelvin). For instance, some general strategies were presented by ,43,51,52Other diverse methods have been proposed to measure entropy, even if these methods do not measure entropy in thermodynamics units. For instance, referredIn contrast, a list of general entropy indicators based on the \u201clow-entropy city principle\u201d was developed. Some strategies are using renewable and local energies, local food chains, investment in public transport and smart mobility, and intelligent management systems for blocks of buildings, among others . In the This section reviews the 38 papers in relation to entropy and its definition, the connection to urban studies, and the characterization and measure methods. Entropy is a broad concept that is still under development in many disciplines. It has been used in numerous fields, including thermodynamics, economics, statistics, communication, arts, and urban studies. For the present literature review, the focus was on the entropy that entails a physical manifestation in the natural or urban system. Hence, those papers related to statistics and information theory and not directly linked to a physical indicator of entropy are not part of this review. However, some exceptions were made to papers that passed through the search filter strategy. Those papers explore the connection between the statistical method and urban sustainability indicators.The results from the descriptive analysis regarding the field of knowledge confirm that the three main areas are related to urban studies , which infers a growing interest in this study of entropy within the field. In addition, many of the studies are based in Italy (40%), where entropy is more related to the perception of negative output and from a figurative approach to thermodynamics. It is also valuable to point out the minor presence of countries from the global south. Excluding China, 3 out of 38 are from that region , representing a gap in the literature and an opportunity to develop studies in those regions.Three main aspects arise from the results concerning the definition of entropy. First is the idea of considering the city as an open system. Second, there are two main zones where entropy could be produced (inside and outside); note that the boundary is usually not considered. Third, any transformation process of any kind will require energy, and the use of energy entails entropy production. These three points are closely related since the exchange of energy (and matter) between systems (inside/outside) is associated with the conversion of energy (heat) and matter related. It is also important to point out that some of these aspects have been studied for decades (not necessarily with the thermodynamic-entropy approach). In an open system, the order of the internal system increases (due to exergy and matter consumption) and the entropy decreases (in a specific location and for a short time). Still, the overall net effect increases the disorder . Cities One key aspect to fully incorporating entropy as a valuable tool towards sustainable cities is to clarify the relation between energy and matter concerning entropy. As previously reviewed, cities could be defined as a dissipative structure since they are open systems, far from the equilibrium state, with fluctuations and a nonlinear interaction mechanism . In thisHowever, this idea introduces some failures because entropy\u2019s original conception was related to thermodynamics and linked to heat and energy. In the case of material waste or pollution, the link with energy is not clearly established yet, and the association with the classical concept of entropy is vague. Still, it could be possible that every system or material affected by urban development could be measured not as the quantity of energy used (exergy) but as the quantity of energy needed to restore the system\u2019s original state. For instance, restoring a forest to its original condition before deforestation will require an enormous amount of energy (and time) to replenish the energy taken and transported to the city, not only for the wood but also for biodiversity lost. What is important to understand is the link between entropy and pollution, system degradation, or waste, considering that in systems , a decrease in energy is correlated with the increase in disorder, hence an increase in entropy. The biological system develops in the opposite direction of entropy, towards a lower entropy and away from equilibrium . HoweverConcerning irreversibility, some initial concepts described as anti-entropic processes that \u201ctakes diffuse material and contrate them\u201d were explained in contrast to the entropic process that takes concentrated materials and diffuses them through the ocean, the earth\u2019s surface, or the atmosphere . The disAnother concept related with irreversibility is the idea that matter is embodied energy , and is In the same line, one of the main problems of the use of entropy as an evaluation measure in urban development towards sustainability is that the original application of the concept needs to be related to energy. Even the initial approach of disorder or randomness resulted from the loss of energy. The energy here is required to maintain a system\u2019s \u201cordered\u201d state. Thus, if you inject more energy into a system, there is more chance that this system will be more \u201cordered\u201d. Moreover, a system with more energy tends to function better, but this is not a straightforward law. For instance, the energy contained in a rainforest is potentially higher than in a desert, so the level of entropy of such a system is lower than in the desert.On the other hand, when we remove a tree from the jungle, we move the energy contained in that tree from the forest to the city. Hence, we inject more \u201cenergy\u201d or order in the city but create disorder or entropy in the forest. Moreover, in the case of the desert example, underground, we might find petroleum. However, this material is not part of the \u201cdessert\u201d ecosystem since it is inside the Earth\u2019s crust, and from a sustainability perspective, it should remain there . In any One opportunity identified is the development of tools for measuring the entropy of the urban flows for each subsystem . It is evident in the case of energy since entropy was initially formulated in that context. However, the entropy generated is still a blurry concept for water, air, or materials. Ideally, one single measure that includes time, area, and energy use should be used. For instance, the unit of measurement could be related to footprint; in this case, the entropy could be the piece of land, the energy, and the time needed to absorb the output flows or produce what is required to restore a system to the initial state. In this scenario, entropy measured could be the area of the territory affected by an urban activity in the function of the time it could take to regain equilibrium or initial state, or the amount of energy or work required to return to the initial state. In this case, entropy is the disturbance in ecosystem services and functions used , activitThe present research had the objective of finding and presenting how the concept of entropy has been utilized in urban studies concerning the sustainability of cities. Therefore, to reach this goal, first, a broad definition of entropy was presented, and second, a systematic literature review was conducted, analyzing 38 articles related to the research objective.Entropy is a broad concept, and there is no established definition of the concept for urban studies. However, two main types of studies were identified, those that deal with the entropy from a thermodynamic perspective and those that understand entropy from a statistical/information perspective. The thermodynamic entropy approach is less developed and usually employs a figurative form. Even more, entropy related to energy is rarely present in urban studies. It is generally unrelated to matter (and components) or too narrow when presented. In addition, a lack of a tool for visualizing urban entropy was observed. This is probably due to the absence of a unique definition.One aspect that is always present is that the concept of entropy within urban studies should be contextualized in relation to non-equilibrium systems. The non-equilibrium thermodynamics is a developing science that is still evolving. However, this perspective allows a positive approach to the concept, where entropy could be related to creating life or new systems out of the chaos, not only to disorder or uncertainty. Within this perspective, irreversibility is a crucial concept to develop. The introduction of the arrow of time in the urban process is key to understanding the entropy in the urban systems and establishing that the irreversibility of a process entails positive and negative aspects. One of the main remaining challenges is identifying tools and methods to quantitatively measure the entropy of this open non-equilibrium urban system and link this to energy and matter. One of the key observations of this paper is precisely to highlight the dependence of cities on natural systems. Energy from natural systems is injected into urban systems in order to prolong the life of the city and prevent degradation. Understanding or being able to monitor or control the growing entropy can allow for more sustainable development to be tracked within the dynamics of urban systems.Concerning the characterization process and the measuring method (and instruments), the most evident conclusion is that there is no established method or theory to measure and characterize the entropy within the urban system and specifically for the thermodynamic approach and does not have an established corpus.Finally, a definition of entropy, considering the reviewed literature, the main current uses of the term and the origin of the word (en + \u201ctropos\u201d (the Greek word for transformation)), is proposed to develop further research about entropy in the urban context. Hence, the proposed definition is: \u201centropy is the number of irreversible changes introduced in nature by urban systems, each time an energy or material transformation is required by cities to continue operating\u201d.Entropy as part of urban system studies is a little explored topic, especially in the Global South. In this region, cities are projected to grow exponentially and will require enormous energy and matter transformation processes. Hence, vast quantities of entropy will be generated and physically manifested in the natural and urban systems. Accordingly, the study and understanding of entropy are vital. Part of the future work is precisely defining what part of this physical manifestation in the natural and urban systems is and is not entropy. A key aspect is to explore the city as a dissipative structure. In this context, the energy needed to restore a system\u2019s original state before the transformation could be measured. Alternatively, the disturbance of the affected natural ecosystem could be measured. A central aspect to consider is that entropy effects are related to time, space, and matter. This should be considered when developing tools for measuring the entropy of urban flow subsystems, such as energy materials or water.The remaining problem is identifying and measuring the irreversible changes upstream and downstream, in the past and the future, improving understanding of entropy within the urban system, and developing specific instruments, indicators, and methodologies to measure entropy inside and outside the cities. Then, entropy will help to identify and illustrate the parallel and correlated landscapes between a city and nature."} +{"text": "China is one of the countries hardest hit by disasters. Disaster shocks not only cause a large number of casualties and property damage but also have an impact on the risk preference of those who experience it. Current research has not reached a consensus conclusion on the impact of risk preferences. This paper empirically analyzes the effects of natural and man-made disasters on residents\u2019 risk preference based on the data of the China Household Financial Survey (CHFS) in 2019. The results indicate that: (1) Both natural and man-made disasters can significantly lead to an increase in the risk aversion of residents, and man-made disasters have a greater impact. (2) Education background plays a negative moderating role in the impact of man-made disasters on residents\u2019 risk preference. (3) Natural disaster experiences have a greater impact on the risk preference of rural residents, while man-made disaster experiences have a greater impact on the risk preference of urban residents. Natural disaster experiences make rural residents more risk-averse, while man-made disaster experiences make urban residents more risk-averse. The results provide new evidence and perspective on the negative impact of disaster shocks on the social life of residents. Disasters are usually divided into two categories according to their causes: natural disasters and man-made disasters. Both of them have brought a large number of casualties and property losses around the world. Furthermore, disaster experiences may also affect an individual\u2019s perspective, mood, and risk preference , 2.Risk preference is defined as the psychological attitude of decision-makers toward risks. It is important for saving, consumption, investment, and other behaviors. People\u2019s risk preference may change under the influence of many factors, such as income , 4, age As to natural disasters, some studies suggest that more residents tend to be risk-averse after experiencing the impact of natural disasters , 9, evenAs to man-made disasters, it refers to disasters caused by human factors, such as wars, terrorist violence, car accidents, shipwrecks, nuclear accidents, and so on. . There iAfter reviewing the above literature, it is found that scholars mainly focus on natural disasters in terms of the impact of exogenous shocks on risk preference, and almost no comparative studies have been conducted on the impact of natural and man-made disasters on residents\u2019 risk preference under a unified framework. Meanwhile, in addition to single disasters, overall annual disasters can also cause serious losses, so does it also have an impact on residents\u2019 risk preference? In addition, it remains to be explored whether there are differences in the direction, degree, and path of impact of the two types of disasters on residents\u2019 risk preference.So we analyze the effects of natural and man-made disasters on residents\u2019 risk preference based on the data from the 2019 China Household Financial Survey (CHFS) questionnaire, reveal the differences in the impacts of the two types of disasters on residents and the reasons for them, and further discuss the main channels through which disaster experiences affect residents\u2019 risk preference by grouping residents. We expand the research on the impacts of disaster experiences on risk preferences in the existing literature by analyzing natural and man-made disasters in the same framework. With the frequency of disasters, understanding and grasping the impacts and mechanisms of the negative shocks of different disasters on residents\u2019 risk preference is crucial for effective economic recovery after disasters.The remainder of this paper is organized as follows. In the next section, we formulate the research hypotheses. Section 3 follows with the research design. The empirical analysis and robustness tests are then performed to identify the disaster shocks that affect residents\u2019 risk preference. Section 5 shows the extension and further discussion of the model. Section 6 contains the conclusions and recommendations.Risk perception is a concept used to describe people\u2019s attitudes and intuitive judgments about risk . GeneralHypothesis 1: Both natural and man-made disaster experiences make residents more risk-averse.Due to the different occurrence characteristics and impact degrees of natural disasters and man-made disasters, the risk perception of residents may be different, which in turn has different effects on risk preference. Firstly, residents will get help from the government and the community after natural disasters, which will increase their trust in the outside world and incrSecondly, from the perspective of post-disaster psychology, when natural disasters break out, residents\u2019 behaviors will evolve into group behaviors based on common disaster impact and consistent post-disaster psychology, and the influence degree of their risk preference may be weakened. As for man-made disasters, most of them are individual disasters with strong destructiveness but a narrow scope of influence. This is different from group disasters represented by natural disasters. Individual disasters may be more risk-averse due to the \u201cpsychological gap\u201d after the disaster. In summary, we proposes that:Hypothesis 2: The impact of natural disasters on residents\u2019 risk preference is weaker than that of man-made disasters.The data is the micro household data in the China Household Financial Survey database (Hereinafter referred to as CHFS.), collected in 2019 by the Southwestern University of Finance and Economics in China. The CHFS conducts a random national sample every two years, covering the entire country. To reduce the interference with the results, the samples are processed as follows: (1) Eliminate samples with missing key indicators; (2) Eliminate the samples that cannot directly judge the risk preference of residents; (3) Considering the characteristics of venture capital participation groups and referring to the way Liu and Tian handle tWe set residents\u2019 risk preferences as the dependent variable. In previous literature, the measurement of risk preference mainly adopts residents\u2019 subjective answers to a question that can reflect residents\u2019 risk preference and is easy to answer, such as investment choice preference. Similarly, in the CHFS survey, one of the questions the respondents were asked is \u201cIf you have a sum of money for investment, which investment project would you most like to choose?\u201d. 5 options can be chosen, which are A: high risk, high return project, B: Slightly high risk, slightly high return project, C: average risk, average return project, D: slightly lower risk, slightly lower return project, and E: not willing to take any risk. Referring to the index design method in Xu et al. , subjectThe main independent variables are natural and man-made disaster experiences. We used a question from the CHFS questionnaire to describe natural and man-made disaster experiences. It\u2019s a multiple-choice question. The question is: \u201cSince 2014, has there been at least one event in your household that had a significant impact on you?\u201d There are three options available for answering, among which, A: My family has experienced some natural disasters , B: My family has experienced some man-made disasters , and C: None. To discriminate the effects of natural and man-made disasters. Control variables generally contain individual basic characteristics and household factors. Based on previous studies, we use six indicators to represent the basic characteristics of individuals: age, gender, educational background, marital status, degree of economic attention, and happiness. Considering that residents\u2019 age may have a non-linear influence on their behavior, the squared term variable of age is introduced with reference to the study of Yu et al. . Four faWith reference to the study of Ntanos , we madeResidents\u2019 subjective risk preference is taken as the dependent variable, and residents\u2019 experiences of natural and man-made disasters are taken as the core independent variables. The following model is established to identify the parameters to be estimated:i * is the latent variable of residents\u2019 subjective risk preference. r0\u3001r1\u3001r2\u3001r3\u3001r4 are the intercepts that satisfy r0