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+{"text": "PLoS Computational Biology [Science [Platynereis dumerilii introns are at the same positions as those in humans seems to support the results in [P. dumerilii was similar to that in humans, where two-thirds of the introns were already present in the last common ancestor and the remaining one-third was gained late after divergence from this ancestor.We thank Cs\u0171r\u00f6s for his thoughtful remarks about our recent paper in  Biology ,2. Altho Biology   intron patterns. Denote in (i = 0..V) to be the number of intron sites for pattern i. Note that n0, which is the number of unobserved intron sites, is unknown. The log-likelihood function in our method  and ixL(1) for states 0 (intron absence) and 1 (intron presence), respectively, using the post-order tree traversal. After that we compute:\u03bb is the probability of introns being present at the root node and irL(0) and irL(1) are the two conditional likelihoods of the root node for pattern i.Suppose that there are ion 7 in ) can be ion 5 in ) appearssenstein . For eaci along each branch k, ik,g and ik,l as well as the expected intron counts at each node ih,h, o can also be computed in linear time with N using the pruning technique, but this time with the pre-order tree traversal. Finally, Equations 9 and 10 in [k given the data; h given the data; and i. In this way, our algorithm also has a linear time with N and V. In fact, the code that was released together with our paper [Since all the conditional likelihoods for every node are now already known, the expected counts of intron gain and loss for each intron pattern nd 10 in  can be rur paper  has alreCs\u0171r\u00f6s  also comN\u22122 optimal solutions (see our Proposition 2 in [ep(0 \u2192 1) + ep(1 \u2192 0) < 1 in [k\u03b1 + k\u03b2 < 1 in our terms, where k\u03b1 and k\u03b2 are the probabilities of intron gain and loss along branch k, respectively [C and D are external nodes  and Co = 420 and Do = 160, where Xo shows the number of introns at node X. We suppose further that P = 1,000 and that one optimal solution for node B has the following parameters: Bo = 400, yg = 60, yl = 40, zg = 120, and zl = 360. In this case, y\u03b1 = 0.1, y\u03b2 = 0.1, z\u03b1 = 0.2, z\u03b2 = 0.9, and z\u03b1 + z\u03b2 > 1. According to our Protocol S2, the other optimal solution for node B has the following parameters: Bo = 600, yg = 360, yl = 540, zg = 40, and zl = 480. In this case, y\u03b1 = 0.9, y\u03b2 = 0.9, z\u03b1 = 0.1, z\u03b2 = 0.8, and y\u03b1 + y\u03b2 > 1. That is, both optimal solutions for node B violate the condition k\u03b1 + k\u03b2 < 1, and the method of Cs\u0171r\u00f6s may not find an optimal solution in this case. Therefore, to always find the most biologically meaningful solution in linear time using our method, we propose to use a new definition: the most biologically meaningful solution is the one that has the least total number of intron gains and losses. Now we can compute the most biologically meaningful solution from any arbitrary optimal solutions by using the post-order tree traversal, and for each internal node B choose the optimal solution for which the sum yg + yl + zg + zl is smaller . The algorithm clearly has a linear time with N.Although the use of Equation 1 in [n0  by means of likelihood. Therefore, a trial-and-error procedure, which basically tries all possible values for n0, was used. Our method, however, suggests that n0 can be optimized by means of likelihood. The problem may be that the method in [n0 is invariant, these two terms are constant and can be omitted. However, they cannot be omitted when inferring intron evolution (where n0 is unknown) if we want to optimize n0 by means of likelihood. One advantage of using our log-likelihood function is that we can use conventional methods  for optimizing n0, which are more efficient than the trial-and-error method employed in [Cs\u0171r\u00f6s  stated tethod in  attemptsloyed in . Anotherloyed in . The metloyed in  does notOur intent when comparing the number of possible patterns with the number of intron sites in the dataset in  was to s"}
+{"text": "H-cyclo\u00adpenta\u00ad[\u03b1]phenanthren-3-one], C31H50O, is a triterpenoid which was isolated from Skimmia laureola. The three six-membered rings adopt chair, slightly distorted half-chair and distorted boat conformations, and the five-membered ring is in an envelope conformation. All the rings are trans fused. In the crystal structure, there is a weak C\u2014H\u22efO hydrogen bond.The title compound [systematic name: 17--4,4,10,13,14-penta\u00admethyl-1,5,6,10,11,12,13,15,16,17-deca\u00adhydro-2 \u00c5                           b = 19.4804 (5) \u00c5                           c = 20.5035 (5) \u00c5                           V = 2684.36 (10) \u00c53                                                   Z = 4                           K\u03b1 radiationMo \u22121                        \u03bc = 0.06 mmT = 173 K                           0.30 \u00d7 0.05 \u00d7 0.04 mm                                 Nonius  diffractometer with Bruker APEXII CCDSORTAV; Blessing, 1997T                           min = 0.981, T                           max = 0.997Absorption correction: multi-scan (6101 measured reflections3485 independent reflectionsI > 2\u03c3(I)2918 reflections with R                           int = 0.036                                                            R[F                           2 > 2\u03c3(F                           2)] = 0.055                           wR(F                           2) = 0.122                           S = 1.15                           3485 reflections297 parametersH-atom parameters constrainedmax = 0.21 e \u00c5\u22123                        \u0394\u03c1min = \u22120.20 e \u00c5\u22123                        \u0394\u03c1                                 COLLECT (Nonius, 1998HKL                DENZO (Otwinowski & Minor, 1997SCALEPACK (Otwinowski & Minor, 1997SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008ORTEP-3 for Windows (Farrugia, 1997SHELXL97.Data collection: 10.1107/S1600536810005118/lh2983sup1.cif            Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536810005118/lh2983Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:"}
+{"text": "Using transgenic Drosophila expressing human tau, we found that RNAi\u2013mediated knockdown of milton or Miro, an adaptor protein essential for axonal transport of mitochondria, enhanced human tau-induced neurodegeneration. Tau phosphorylation at an AD\u2013related site Ser262 increased with knockdown of milton or Miro; and partitioning defective-1 (PAR-1), the Drosophila homolog of mammalian microtubule affinity-regulating kinase, mediated this increase of tau phosphorylation. Tau phosphorylation at Ser262 has been reported to promote tau detachment from microtubules, and we found that the levels of microtubule-unbound free tau increased by milton knockdown. Blocking tau phosphorylation at Ser262 site by PAR-1 knockdown or by mutating the Ser262 site to unphosphorylatable alanine suppressed the enhancement of tau-induced neurodegeneration caused by milton knockdown. Furthermore, knockdown of milton or Miro increased the levels of active PAR-1. These results suggest that an increase in tau phosphorylation at Ser262 through PAR-1 contributes to tau-mediated neurodegeneration under a pathological condition in which axonal mitochondria is depleted. Intriguingly, we found that knockdown of milton or Miro alone caused late-onset neurodegeneration in the fly brain, and this neurodegeneration could be suppressed by knockdown of Drosophila tau or PAR-1. Our results suggest that loss of axonal mitochondria may play an important role in tau phosphorylation and toxicity in the pathogenesis of AD.Abnormal phosphorylation and toxicity of a microtubule-associated protein tau are involved in the pathogenesis of Alzheimer's disease (AD); however, what pathological conditions trigger tau abnormality in AD is not fully understood. A reduction in the number of mitochondria in the axon has been implicated in AD. In this study, we investigated whether and how loss of axonal mitochondria promotes tau phosphorylation and toxicity  Abnormal phosphorylation and toxicity of a microtubule-associated protein tau are involved in the pathogenesis of Alzheimer's disease (AD). Tau is phosphorylated at multiple sites, and phosphorylation of tau regulates its microtubule binding and physiological functions such as regulation of microtubule stability. Abnormal phosphorylation of tau occurs in the AD brains and is thought to cause tau toxicity; however, what pathological conditions trigger abnormal phosphorylation and toxicity of tau in AD is not fully understood. Since a reduction in the number of mitochondria in the axon has been observed in the AD brains, we investigated whether and how loss of axonal mitochondria promotes tau phosphorylation and toxicity. Using transgenic flies expressing human tau, we found that knockdown of milton or Miro, an adaptor protein essential for axonal transport of mitochondria, enhanced human tau-induced neurodegeneration. This study demonstrates that loss of axonal mitochondria caused by milton knockdown increases tau phosphorylation at an AD\u2013related site through partitioning defective-1 (PAR-1), promotes detachment of tau from microtubules, and enhances tau-mediated neurodegeneration. Our results suggest that loss of axonal mitochondria may play an important role in tau phosphorylation and toxicity in the pathogenesis of AD. In neuronal axons, these requirements need to be addressed locally, and the proper distribution of mitochondria is essential for neuronal functions and survival Mitochondria are principal mediators of local ATP supply and CaTau is a microtubule-associated protein that is expressed in neurons and localizes predominantly in the axons, where it regulates microtubule dynamics. Tau is phosphorylated at a number of sites, and a fine-tuned balance between phosphorylation and dephosphorylation of tau is critical for its physiological functions, such as microtubule stabilization, in the axons Hyperphosphorylated tau is found in neurofibrillary tangles, the intracellular protein inclusions that are associated with a range of neurodegenerative diseases including AD Drosophila, mitochondrial transport is facilitated by milton and Miro, which regulate mitochondrial attachment to microtubules via kinesin heavy chain Drosophila, in the absence of milton or Miro, synaptic terminals and axons lack mitochondria, although mitochondria are numerous in the neuronal cell body Mitochondrial transport is regulated by a series of molecular adaptors that mediate the attachment of mitochondria to molecular motors Drosophila as a model system, we investigated the effects of knockdown of milton or Miro, an adaptor protein essential for axonal transport of mitochondria, on tau phosphorylation and toxicity. We demonstrate that loss of axonal mitochondria caused by milton knockdown increases tau phosphorylation at Ser262 through PAR-1, promotes detachment of tau from microtubules, and enhances tau-mediated neurodegeneration.In this study, using in vivo, we used transgenic Drosophila expressing human tau To test whether loss of axonal mitochondria enhances human tau toxicity It has been reported that overexpression of tau alone can reduce anterograde transport of a variety of kinesin cargos, including mitochondria Milton is a component of an adaptor complex that links mitochondria to kinesin heavy chain and is essential for axonal transport of mitochondria  [19]. PrGD) dramatically enhanced neurodegeneration in the lamina at 3-day-old compared to fly eyes expressing tau alone (TRiP) was used. Expression of this RNAi in neurons reduced milton mRNA levels in the fly brain  iai) that targets a different region of Miro. Expression of Miro RNAiiai reduced Miro mRNA levels  and the mammalian homolog of PAR-1, microtubule affinity-regulating kinase (MARK), are reported to phosphorylate tau at Ser262 in vivoWe investigated the role of tau phosphorylation at Ser262 in the enhancement of tau-induced axon degeneration caused by milton knockdown. We first examined whether PAR-1 knockdown enhances or suppresses tau-induced axon degeneration in the milton knockdown background. RNAi-mediated knockdown of PAR-1 significantly suppressed neurodegeneration in the lamina of flies expressing human tau and milton RNAi  expressed at the levels similar to the expression of wild-type tau  were useOur results demonstrate that knockdown of milton or Miro enhances tau-induced neurodegeneration and increases tau phosphorylation at Ser262. PAR-1 mediates the increase in tau phosphorylation at Ser262, and tau phosphorylation site Ser262 and PAR-1 are critical for the enhancement of tau-induced neurodegeneration caused by milton knockdown. To further investigate the relationship between loss of axonal mitochondria and PAR-1, the effect of knockdown of milton or Miro on PAR-1 activity was examined.TRiP)  . FurtherTRiP) . These eTRiP) .DrosophilaA previous report showed that total PAR-1 level increased when it was phosphorylated at Thr408 in Milton knockdown did not cause non-specific activation of kinases, since it did not increase the level of phosphorylated, active p44mapk in fly eyes . MoreoveWe also observed that the phenotype induced by PAR-1 overexpression was enhanced by milton knockdown. Overexpression of PAR-1 in fly eyes has been reported to cause eye degeneration Although knockdown of milton or Miro without human tau overexpression did not cause neurodegeneration in the young flies , we founWe quantified age-dependent progression of neurodegeneration caused by milton knockdown in the lamina, where neurodegeneration was the most prominent. Degeneration in the lamina is undetectable at 3-day-old, which is in line with the previous observation in milton mutant flies TRiP) was used. Expression of milton RNAiTRiP also caused age-dependent neurodegeneration in the lamina . Accumulation of amyloid-\u03b2 peptides is thought to be causative for AD and has been suggested to cause tau abnormality It has been reported that, in non-neuronal cultured cells or primary-cultured hippocampal neurons with virus-mediated overexpression of human tau, an excess of microtubule-bound tau blocks microtubule-dependent transport of vesicles and organelle including mitochondria and causes synaptic degeneration in vivo, we used a Drosophila model of human tau toxicity In contrast, this study examined whether and how specific loss of axonal mitochondria promotes tau phosphorylation and toxicity. To address this question Using this model system, we found that milton knockdown significantly enhanced tau-mediated neurodegeneration. Milton knockdown increased the levels of active PAR-1 and tau phosphorylation at Ser262, and promoted detachment of tau from microtubules. If the enhancement of tau toxicity caused by milton knockdown in our model is due to an additive reduction in the number of axonal mitochondria, blocking tau phosphorylation at Ser262, which increases tau binding to microtubules and blocks microtubule-dependent transport, would enhance neurodegeneration. However, our results have shown that blocking tau phosphorylation at Ser262 by PAR-1 knockdown rescues the enhancement of tau-mediated neurodegeneration in the milton knockdown background. These results suggest that the enhancement of tau toxicity in the milton knockdown background is not likely to be due to an additive reduction of axonal transport of mitochondria caused by an excess of microtubule-bound tau. Rather, this study suggests that, when axonal mitochondria are chronically depleted, increased free, microtubule-unbound, Ser262-phosphorylated tau promotes neurodegeneration.A fine-tuned balance of microtubule-binding of tau is critical for its physiological functions. It has been suggested that both an excess of microtubule-bound tau and an excess of free, microtubule-unbound tau can cause toxicity DrosophilaDrosophila homolog of adducin, a cytoskeletal protein involved in regulating actin filament dynamics We found that the levels of active PAR-1 are increased by milton knockdown . PAR-1 iDetachment of tau from microtubules has been suggested to initiate its abnormal metabolism and toxicity of tau in AD pathogenesis In summary, this study highlights a potential role of loss of axonal mitochondria in tau phosphorylation and toxicity in AD pathogenesis. Reductions in the function and number of mitochondria in the axon have also been implicated in several neurodegenerative diseases such as Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis 5\u2032-CCGGAATTCGATATGGGCTGAATACAAATCACAGAATCG-3\u2032, rev, 5\u2032-CTAGTCTAGATTCATTAAAACCGGGAGGTAGATGAGATGT-3\u2032 ), and the resulting constructs were subcloned into the pUAST Drosophila transformation vector and microinjected into fly embryos of the 1118w genotype. Transgenic fly lines carrying UAS-Miro RNAi were established by microinjecting the Miro RNAi construct ) into fly embryos of the 1118w genotype. The transgenic fly lines carrying S262A mutant tau was described previously Transgenic fly lines carrying UAS-luciferase RNAi were established following the method described previously en bloc with 0.5% aqueous uranyl acetate for 1 hr, dehydrated with ethanol and embedded in Epon. Thin-sections (70 nm) of laminas, in which photoreceptor axons were cut longitudinally, were collected on copper grids. The sections were stained with 2% uranyl acetate in 70% ethanol and Reynolds' lead citrate solution. Electron micrographs were obtained with a VELETA CCD Camera (Olympus Soft Imaging Solutions GMBH) mounted on a JEM-1010 electron microscope (Jeol Ltd.).Probosces were removed from decapitated heads, which were then immersion-fixed overnight in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer at 4\u00b0C. Samples were post-fixed 1 hr in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer on ice. After washing, samples were stained Preparation of paraffin sections, hematoxylin and eosin staining, and analysis of neurodegeneration were described previously Twenty fly heads for each genotype were homogenized in SDS-Tris-Glycine sample buffer, and the same amount of the lysate was loaded to each lane of multiple 10% Tris-Glycine gels and transferred to nitrocellulose membrane. The membranes were blocked with 5% milk (Nestle), blotted with the antibodies described below, incubated with appropriate secondary antibody and developed using ECL plus Western Blotting Detection Reagents  or imaging with an Odyssey system. One of the membranes was probed with anti-tubulin, and used as the loading control for other blots in each experiment. Anti-tau monoclonal antibody , and anti-tau pSer262 , phospho-Thr231 , anti-HA (Santa Cruz), anti-myc (Millipore), anti-active p44mapk (Promega), anti-tubulin (Sigma), anti-GFP (Clontech) were purchased. Anti-tau pS202 (CP13) was a kind gift from Dr. Peter Davis (Albert Einstein College of Medicine). Anti-PAR-1 pT408 was described previously Quantitative real-time PCR analysis was performed as described previously 5\u2032-GGCTTCAGGGCCAGGTATCT-3\u2032milton for 5\u2032-GCCGAACTTGGCTGACTTTG-3\u2032milton rev 5\u2032-AAAAGCACCTCATTCTGCGT-3\u2032Miro for 5\u2032-CCTCAGGTGAGGAAACGCT-3\u2032Miro rev 5\u2032-AAGCCCGGTGGCGGTGAGAA-3\u2032dTau for 5\u2032-GCGCCAGAAGCCGTCATGGA-3\u2032dTau rev 5\u2032-TGCACCGCAAGTGCTTCTAA-3\u2032Actin for 5\u2032-TGCTGCACTCCAAACTTCCA-3\u2032Actin rev 5\u2032-GCTAAGCTGTCGCACAAATG-3\u2032rp49 for 5\u2032- GTTCGATCCGTAACCGATGT-3\u2032rp49 rev 5\u2032- GCGGCTGTGATTATGCGAAT-3\u2032TBP for 5\u2032-AGGGAAACCGAGCTTTTGGA-3\u2032TBP rev Microtubule binding assay was performed using a previously reported procedure with a modification Statistics was done with the JMP software (SAS) with Student's t or Tukey-Kramer HSD. Values are given as mean \u00b1 standard deviation or standard error.Figure S1Expression of human 0N4R wild-type tau causes late-onset, progressive neurodegeneration in the lamina. The lamina expressing tau at 3-day-old (A) or 10-day-old (B), or the lamina of control flies (gmr-GAL4 driver only) at 10-day-old (C). Compare vacuoles indicated by arrows in A (3-day-old) and B (10-day-old). (D) Quantification of the area of vacuoles in the lamina (arrows in A and B), mean \u00b1 SEM, n\u200a=\u200a14\u201320. *, p<0.05, Student's t-test. Genotypes are as follows: (tau) +/+;gmr-GAL4/+;UAS-tau/+ and (control) +/+;gmr-GAL4/+;+/+.(TIF)Click here for additional data file.Figure S2Mitochondria are observed in the presynaptic terminals of photoreceptor neurons expressing tau. Transmission electron micrographs of presynaptic terminals in the lamina of flies expressing human tau. Presynaptic terminals are colored green to accentuate the structures. Arrows indicate mitochondria in synaptic terminals. Flies were 3 days-after-eclosion (day-old). Genotype is: +/+;gmr-GAL4/+;UAS-tau/+.(TIF)Click here for additional data file.Figure S3GD). Flies were 3 days-after-eclosion (day-old). Genotypes are as follows: (control) +/+;gmr-GAL4/+;+/+ and (milton RNAiGD) UAS-milton RNAiGD/+;gmr-GAL4/+;+/+.Presynaptic terminals in milton knockdown flies have larger vesicles. Transmission electron micrographs of presynaptic terminals in the lamina of control flies bearing the gmr-GAL4 driver only (control) and flies with milton knockdown (milton RNAi(TIF)Click here for additional data file.Figure S4GD/+;gmr-GAL4/+;UAS-tau/+.Milton knockdown causes loss of axonal mitochondria in the photoreceptor neurons expressing human tau in the fly brain. A representative transmission electron micrograph of a presynaptic terminal of a photoreceptor neuron in the lamina of the fly co-expressing human tau and milton RNAi. The presynaptic terminal is colored green to accentuate the structures. Note that mitochondria are not observed (compare to (TIF)Click here for additional data file.Figure S5TRiP reduces milton mRNA levels and causes mislocalization of mitochondria in the fly brain. (A) Reduction in milton mRNA levels by the expression of milton RNAiTRiP in eyes and neurons. Expression of UAS-luciferase (control) or UAS-milton RNAiTRiP (milton RNAiTRiP) was driven by a combination of two drivers, the pan-retinal gmr-GAL4 driver and pan-neuronal elav-GAL4 driver. More than thirty flies for each genotype were collected and frozen. Heads were mechanically isolated, and total RNA was extracted. Milton RNA levels were quantified by qRT-PCR . Note that milton RNAi is only expressed in eyes and neurons, while endogenous milton is ubiquitously expressed. Genotypes are as follows: (control) elav-GAL4/Y;gmr-GAL4/+;UAS-luciferase/+ and (milton RNAiTRiP) elav-GAL4/Y;gmr-GAL4/+;UAS-milton RNAiTRiP/+. (B) Milton RNAiTRiP reduces axonal mitochondrial in the fly brain. (Top) A schematic view of the mushroom body structure, where axons (orange) can be easily identified in the fly brain. (Bottom) Mito-GFP signal in the lobe tips. Ratios relative to control are shown . The mito-GFP signal in the axons was significantly decreased in the milton RNAiTRiP fly brains. Representative images are shown. The method for mito-GFP analysis in the brain is described in TRiP) elav-GAL4/Y;mito-GFP/+; milton RNAiTRiP/+.Milton RNAi(TIF)Click here for additional data file.Figure S6KK reduces axonal mitochondria in the fly brain. Mito-GFP signal in the lobe tips (axons) of the mushroom body structure in the brains of control and Miro RNAiKK flies. Representative images are shown at the top, and the ratio of mito-GFP signal in the lobe tips relative to control are shown at the bottom . Genotypes are as follows: (control) elav-GAL4/Y;mito-GFP/+;+/+ and (Miro RNAiKK) elav-GAL4/Y;mito-GFP/UAS- Miro RNAiKK;+/+.Miro RNAi(TIF)Click here for additional data file.Figure S7iai causes a reduction in Miro mRNA levels in the fly brain. Expression of UAS-Miro RNAiiai (Miro RNAiiai) was driven by the pan-neuronal elav-GAL4 driver. More than thirty flies for control flies (the elav-GAL4 driver only) or Miro RNAi flies were collected and frozen. Heads were mechanically isolated, and total RNA was extracted. Miro mRNA levels were quantified by qRT-PCR . Note that Miro RNAiiai is only expressed in neurons, while endogenous Miro is ubiquitously expressed. Genotypes are as follows: (control) elav-GAL4/Y;+/+;+/+ and (Miro RNAi) elav-GAL4/Y;+/+;UAS-Miro RNAiiai/+.Miro RNAi(TIF)Click here for additional data file.Figure S8Tau-mediated neurodegeneration in the lamina is not enhanced by RNAi targeting CG30106, CG4395, CG6064, or CG30340. Quantification of neurodegeneration measured by the area of vacuoles in the lamina, presented as mean \u00b1 SEM, n\u200a=\u200a10\u201312. The asterisk indicates significant difference between tau and tau+milton RNAi . Flies were 3 days-after-eclosion (day-old).(TIF)Click here for additional data file.Figure S9GD) (C), co-expressing human tau and Miro RNAiiai (tau+Miro RNAi) (D), or expressing milton RNAi alone (milton RNAiGD) (E). Genotypes are as follows: (control) +/+;gmr-GAL4/+;+/+, (tau) +/+;gmr-GAL4/+;UAS-tau/+, (tau+milton RNAiGD) UAS-milton RNAiGD/+;gmr-GAL4/+;UAS-tau/+, (tau+Miro RNAi) +/+;gmr-GAL4/+;UAS-tau/UAS-Miro RNAiiai and (milton RNAiGD) UAS-Milton RNAiGD/+;gmr-GAL4/+;+/+.Knockdown of milton or Miro does not enhance tau-mediated reduction in the external eye size. Eyes from flies carrying the pan-retinal gmr-GAL4 driver only (control) (A), expressing human tau alone (tau) (B), co-expressing human tau and milton RNAi (tau+milton RNAi(TIF)Click here for additional data file.Figure S10Expression of S262A tau causes late-onset, progressive neurodegeneration in the lamina. The lamina expressing S262A tau at 3-day-old (A) or 10-day-old (B). Vacuoles in A are indicated by arrows. (C) Quantification of the area of vacuoles in the lamina, mean \u00b1 SEM, n\u200a=\u200a10\u201312. *, p<0.05, Student's t-test. Genotype is: +/+;gmr-GAL4/+;UAS-S262Atau/+.(TIF)Click here for additional data file.Figure S11GD. Blots were probed with anti-phospho-mapk (P-p44mapk) or anti-tubulin. No significant differences were found . The asterisk indicates non-specific bands. (B) Western blots of eyes from flies expressing HA-tagged p44mapk alone or flies co-expressing p44mapk and milton RNAiGD. Blots were probed with anti-HA (p44mapk) or anti-tubulin. No significant differences were found . (C) Western blots of eyes from flies expressing GFP alone or flies co-expressing GFP and milton RNAiGD. Blots were probed with anti-GFP or anti-tubulin. No significant differences were found . (D) Western blots of eyes from flies expressing myc-tagged APP alone or flies co-expressing APP and milton RNAiGD. Blots were probed with anti-myc (APP) or anti-tubulin. No significant differences were found . All flies were 3 days-after-eclosion (day-old). Genotypes are as follows: (p44mapk) +/+;gmr-GAL4/+;UAS-p44mapk-HA/+, (p44mapk+milton RNAi) UAS-Milton RNAiGD/+;gmr-GAL4/+;UAS-p44mapk-HA/+, (GFP) +/+;gmr-GAL4/UAS-GFP;+/+, (GFP+milton RNAi) UAS-Milton RNAiGD/+;gmr-GAL4/UAS-GFP;+/+, (APP) +/+;gmr-GAL4/UAS-APP-myc;+/+ and (APP+milton RNAi) UAS-Milton RNAiGD/+;gmr-GAL4/UAS-APP-myc;+/+.Milton knockdown does not cause non-specific activation of kinases, non-specific accumulation of overexpressed proteins, or an increase in the expression of proteins under the control of GAL4/UAS system. (A) Western blots of eyes from flies expressing HA-tagged p44mapk alone or flies co-expressing p44mapk and milton RNAi(TIF)Click here for additional data file.Figure S12GD (F), and expressing milton RNAiGD alone (G) are shown. (H) Quantification of neurodegeneration (arrows in F), mean \u00b1 SEM, n\u200a=\u200a10\u201312. *, significant difference between PAR-1 and PAR-1+milton RNAiGD . Genotypes are as follows: (control) +/+;gmr-GAL4/+;+/+, (PAR-1) +/+;gmr-GAL4/+;UAS-PAR-1-myc/+, (PAR-1+milton RNAiGD) UAS-Milton RNAiGD/+;gmr-GAL4/+;UAS-PAR-1-myc/+ and (milton RNAiGD) UAS-Milton RNAiGD/+;gmr-GAL4/+;+/+.Milton knockdown enhances neurodegeneration caused by PAR-1 overexpression. (A\u2013D) PAR-1 overexpression causes late-onset, progressive neurodegeneration in the lamina. The lamina expressing PAR-1 at 3 days-after-eclosion (day-old) (A) or 10-day-old (B), or the lamina of control flies at 10-day-old (C). (D) Quantification of the area of vacuoles in the lamina (arrows in B), mean \u00b1 SEM, n\u200a=\u200a10\u201312. *, p<0.05, Student's t-test. (E\u2013H) Milton knockdown enhances PAR-1-induced lamina degeneration. The lamina of flies expressing PAR-1 alone (E), co-expressing PAR-1 and milton RNAi(TIF)Click here for additional data file.Figure S13Tau RNAi causes a reduction in tau mRNA levels in the fly brain. Expression of UAS-luciferase (control) or UAS-tau RNAi (tau RNAi) was driven by a combination of two drivers, the pan-retinal gmr-GAL4 driver and pan-neuronal elav-GAL4 driver. More than thirty flies for each genotype were collected and frozen. Heads were mechanically isolated, and total RNA was extracted. Tau mRNA levels were quantified by qRT-PCR . Genotypes are as follows: (control) elav-GAL4/Y;gmr-GAL4/+;UAS-luciferase/+, and (tau RNAi) elav-GAL4/Y;gmr-GAL4/+;UAS-tau RNAi/+.(TIF)Click here for additional data file.Table S1Fly stocks.(DOC)Click here for additional data file.Table S2Genotypes of the flies that were used in each experiment.(DOC)Click here for additional data file.Text S1(DOC)Click here for additional data file."}
+{"text": "Lotus corniculatus grasslands. Regeneration scenario, fertilizer treatment, and their interaction influenced soil microbial richness, diversity and evenness indices. Labile carbon pool 2 was a significant factor affected plant and microbe communities in July, suggesting that plants and microbes may be competing for nutrients. The higher ratios of positive to negative association were found in soil bacteria and total microbe than in archaea and fungi. Stronger clustering of microbial communities from the same regeneration scenario indicated that the vegetative composition of regeneration site may have a greater influence on soil microbial communities than fertilizer treatment.The soil microbial community in reclaimed mining areas is fundamental to vegetative establishment. However, how this community responds to different regeneration scenarios and fertilizer treatments is poorly understood. This research evaluated plant and soil microbial communities from different regeneration scenarios and different fertilizer treatments. Regeneration scenarios significantly influenced soil bacterial, archaeal, and fungal rDNA abundance. The ratios of fungi to bacteria or archaea were increased with fertilizer application. The diversity of both plants and microbes was lowest in  The considerable growth of the mining industry in China over the last few decades has generated a vast amount of solid waste, which occupies a huge area of land The majority on studies of the effects of mine reclamation on microorganisms have focused on specific fungal groups (particularly mycorrhizae) Recently, genetic profiling methods have produced more information on soil microbial ecology compared to the cultivation of isolated microbes Bacteria, archaea, and eukaryota form the three main domains of the phylogenic tree of life Research was conducted at Antaibao Mine in Northwest Plateau Loess . The climate is terrestrial temperate, and the area experiences monsoons. Annual average precipitation is 480 to 510 mm, with rainfall occurring mainly in the summer. The annual average air temperature is about 10.1\u00b0C, with the lowest average minimum temperature in January (\u22125.6\u00b0C) and the highest average maximum temperature in July (23.7\u00b0C). The frost-free season ranges in length from 180 to 200 days. Undisturbed soil in the area is classified as Cumulic Anthrosol (WBR).Lotus corniculatus (CO), Medicago sativa (SA) grasslands, Pinus tabulaeformis plantation (TA), and Salix matsudana -Sabina chinensis mixed forest (MF). The grass seed was sow in rows , and the seeding rates of L. corniculatus and M. sativa were 5 and 10 kg\u00b7hm\u22122, respectively. 3-year-old P. tabulaeformis and S. chinensis, and 5-year-old S.matsudana seedling were planted 2 m apart. There were four fertilizer treatments in total: CK\u200a=\u200ano fertilizer added, IN\u200a=\u200ainorganic fertilizer  added, OR\u200a=\u200aorganic fertilizer  added, and IO\u200a=\u200acombination of inorganic and organic fertilizer (375 kg/hm2 inorganic fertilizer +22.5 m3/hm2 organic fertilizer) to soils.Ecological reconstruction was initiated on the abandoned land in 2009, and there were four regeneration scenarios including We only investigated plant species in the field; we did not sample plant species. There were no endangered or protected species involed in this study. The location is Antaibao Mine Company that is state-owned enterprise. No specific permits were required for the described field studies.To study the characteristics of the various plant communities under different restoration types, quadrats were set up in the study areas. Quadrats of 20 m\u00d720 m, and 1 m\u00d71 m were established in forest and grassland communities, respectively. There were 3 replications for each of the 16 treatments, resulting in a total of 48 quadrates. Sampling occurred on two occasions, April and July 2011. The cover, height, diameter at breast height (DBH), individual number for each tree species, and the cover and height for herbs were recorded in each quadrat [1).From each site, 6 soil cores (7.5 cm diameter\u00d710 cm depth) were randomly collected in the middle of rows and mixed from the profile of each plot and bulked \u22123) for soil bulk density (BD) were obtained using the gravimetric method. Soil pH in dH2O was measured in subsets of field-moist soils at a soil:solution ratio of 1\u22365 (g:ml) after 0.5\u20131 h. Soil organic carbon (SOC) was determined using the dichromate oxidation method. Total nitrogen (TN) was analyzed by the Kjeldahl method 2O4Values  and stored at \u221220\u00b0C. The DNA extracts were 10-fold diluted and used as template with a final content of 1\u201310 ng in each reaction mixture to amplify soil bacterial, archaeal, and fungal rRNA genes.DNA was extracted from 0.5 g fresh soil samples using Ultra-clean TM soil DNA Isolation Kits  following the manufacturer\u2019s protocol. The extracted DNA was eluted with Premix Ex Taq\u2122  were applied into bacterial 16S rRNA gene quantitative assay. The reaction mixture for quantifying archaeal and fungal rRNA gene included SYBR\u00ae Premix Ex Taq\u2122 with Green I . Primers were labeled at the 5\u2032 end with the reporter dye FAM (6-carboxy-fluorescein) for T-RFLP analysis. The detailed information on primer, probe, and PCR condition are listed in Real-time PCR was performed on an iCycler iQ 5 thermocycler (Bio-Rad). The probe TM189F and HhaI, HaeIII and MspI  and incubated at 37\u00b0C for 3 h in the manufacturer\u2019s recommended reaction buffer. Digestions were in a total volume of 25 \u00b5l, including 4 U of enzyme and about 500 ng of DNA. The digestion products were further purified, and a portion was mixed with deionized formamide and the internal standard GeneScan-ROX1000 (bacteria)/LIZ 500 (archaea and fungi) (Applied Biosystems). The mixtures were denatured for 3 min at 95\u00b0C, and the DNA fragments were size separated using a 3130xl Genetic Analyzer (Applied Biosystems).All PCR products were verified using 1% agarose gel electrophoresis and purified with Wizard\u00ae SV Gel and PCR Clean-Up System . Purified products were digested in separate reactions with restriction endonucleases The effects of regeneration scenario and fertilizer treatment on plant and soil microbe characteristics were examined with separately one-way analyses of variance (ANOVA). A three-way ANOVA was applied to analyses the effects of season, regeneration scenario and fertilizer treatment on soil microbial communities. Plant species-pairs and microbial RF (Restricted fragment)-pairs association were analyzed by Spearman rank correlation. These statistical analyses were performed using SPSS 13.0 for Windows.Plant species importance values (IV) for each quadrat were calculated using the following formulas To reveal their variation among different restoration types, plant species, and RF richness, diversity and evenness in each quadrat were determined. Four species diversity indices were employed:S)Species number as the richness index  on soil pH in all vegetation types except SA grassland . Soil pHPlant community composition and the coverage of vegetation were investigated . The vegThe seasonal effects on the abundance of soil microbial rRNA genes were significant . FertiliHhaI, HaeIII and MspI. The effects of sampling date, regeneration scenarios, and fertilizer treatments on microbial diversity indices produced by HhaI are listed in T-RFLP profiles of soil bacterial, archaeal, and fungal rRNA genes from study sites were produced using the endonuclease enzymes HhaI, the 77 bp RF had the highest relative abundance and there were RFs unique to each regeneration scenario. In T-RFLP profiles of soil archaeal 16S rRNA restricted using HhaI, the 320 bp RF had the highest relative abundance at all sites. Fragments of 136 and 141 bp were found in regeneration areas where trees grew, but not in grasslands; and the fragments lower than 88 bp were only observed in fertilizer treatments. Both regeneration scenario and fertilizer treatment demonstrated an influence on T-RFLP profiles of soil fungi 18S rRNA restricted using HhaI enzyme  and richness index (S) were the most and least susceptible for these factors, respectively.Soil bacterial and total microbial diversity indices inferred from fragments restricted using ng HaeIII. Soil arPlant species-pair and microbial inter-RF ratios of positive and negative association of the Spearman rank correlation test are listed in The results of CCA showed that the eigenvalues of axes 1 and 2 was respectively 62.3% and 44.0%, and the first two axes explained 64.7% of species\u2013environment relations . Liable carbon pool and bulk density were significantly linked to the plant community variability (P<0.05), and positively correlated with the first axes .HhaI, were shown by the CCA method (HaeIII and MspI enzymes (results not shown).The relationships between environmental factors and soil microbial communities, according to T-RFLP profiles restricted using A method . In ApriA method . Soil pHA method . TreatmeL. corniculatus demands high fertility soil, and we observed that fertilizer significantly improved plant coverage in CO grasslands. M. sativa has been widely planted in reclamation sites in the Loess Plateau due to its strong resistance to drought and barren soil M. sativa grew well, the coverage being over 90% with the available space almost used; other species, therefore, could not easily invade and plant richness, diversity and evenness was significantly lower in these grasslands  increases in soil organic and recalcitrant carbon levels and nitrogen content . FertiliSoil pH was significantly (P<0.05) decreased as a result of fertilizer treatments, especially in the mixed forest . HoweverUsing a Monte Carlo permutation test in CCA, we showed that LC2 was a significant environmental factor affecting both plant and microbial communities in July. However, labile carbon was not the only significant environmental factor that affected soil microbial communities in April . The teset al. In our study, soil microbial diversity was more affected by the vegetative composition of the regeneration sites than by fertilizer treatments . We alsoet al. Higher ratios of positive to negative association from the Spearman rank correlation test indicate that biological community is more steady The results partly confirmed our hypothesis that soil microbial communities were significantly influenced by regeneration scenarios, however, fertilizer treatments produced less significant influence on soil microbial communities. Regeneration scenarios produced a significant effect on soil microbial rDNA copy numbers and microbial communities. Season showed pronounced effects on soil microbial growth and composition. There may be nutrient competition between vegetation and microbes for their growth during initial rehabilitation period, but mutual benefits would be demonstrated between vegetation and soil microbe with succession progressed in reclaimed mining areas of Shanxi, China.Figure S1Ratios of soil bacterial, archaeal and fungal log numbers of rRNA gene copy number in the reclaimed mining area. Points show the means of three replicates, and vertical bars show standard deviations. Lower case letters indicate that the means are not significantly different among fertilizer treatments for the same regeneration scenario (P<0.05). Capital letters indicate that the means are not significantly different among regeneration scenarios for the same fertilizer treatment (P<0.05). CO, SA, TA and MF respectively represent Lotus corniculatus, Medicago sativa, Pinus tabulaeformis and Salix matsudana\u2013Sabina chinensis mixed forest. CK, IN, IO and OR respectively represent no, inorganic, organic and a combination of inorganic and organic fertilizer added to soils.(TIF)Click here for additional data file.Figure S2Relative fluorescence of soil bacteria, archaea and fungi populations measured by T-RFLP electropherogram target on rRNA gene sequences digested using HhaI restriction enzymes in the reclaimed mining area. CO, SA, TA and MF respectively represent Lotus corniculatus, Medicago sativa, Pinus tabulaeformis and Salix matsudana\u2013Sabina chinensis mixed forest. CK, IN, IO and OR respectively represent no, inorganic, organic and a combination of inorganic and organic fertilizer added to soils.(TIF)Click here for additional data file.Figure S3CCA ordination biplot of 16 quadrats and environmental factors for soil bacterial, archaeal, fungal, and total microbial communities in the reclaimed mining area in April. Arrows indicate the direction and magnitude of measurable variables associated with soil microbial communities structures. Circle, Star, Diamond and Up-triangle symbol types respectively represent Lotus corniculatus, Medicago sativa, Pinus tabulaeformis and Salix matsudana\u2013Sabina chinensis mixed forest. White, green, blue and black respectively represent no, inorganic, organic and a combination of inorganic and organic fertilizer added to soils. BD, SOC/TN, and LC2 are the soil bulk density, ratio of soil organic carbon to total nitrogen, and labile carbon pool 2, respectively.(TIF)Click here for additional data file.Table S1Plant community composition and coverage in reclaimed mining area. CO, SA, TA and MF are respectively Lotus corniculatus, Medicago sativa, Pinus tabulaeformis and Salix matsudana -Sabina chinensis mixed forest. CK, IN, IO and OR are respectively no, inorganic, organic and combination of inorganic and organic fertilizer added to soils(DOC)Click here for additional data file.Table S2Results of three-way ANOVA showing the effects of season, regeneration scenarios and fertilizer treatments for Soil bacteria, archaea, fungi and total microbe RFs richness index (S), Shanon-Wiener diversity index (H\u2019), Simpson diversity index (D) and Pielou evenness index (E). ** Effect is significant at the 0.01 level; * Effect is significant at the 0.05 level. ns Effect is not significant.(DOC)Click here for additional data file.Table S3Ratios of positive and negative association from Spearman rank correlation test of the inter-species correlation of plant and inter-RFs correlation of soil bacteria, archaea, fungi and total microbes in the reclaimed mining area. CO, SA, TA and MF represent Lotus corniculatus, Medicago sativa, Pinus tabulaeformis and Salix matsudana\u2013Sabina chinensis mixed forest. CK, IN, IO and OR represent no, inorganic, organic and a combination of inorganic and organic fertilizer added to soils.(DOC)Click here for additional data file."}
+{"text": "Actinobacteria, Fusobacterium, Bacteroidetes and Proteobacteria were the most predominant in all soil samples. The order of Shannon-Wiener index (H) of all soil samples was in the order of EBF>CF>SDF>AM, whereas bacterial species richness as estimated by four restriction enzymes indicated no significant difference. Principal component analysis (PCA) revealed that the soil bacterial communities\u2019 structures of EBF, CF, SDF and AM were clearly separated along the first and second principal components, which explained 62.17% and 31.58% of the total variance, respectively. The soil physical-chemical properties such as total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP) and total potassium (TK) were positively correlated with the diversity of bacterial communities.Soil microbes are active players in energy flow and material exchange of the forest ecosystems, but the research on the relationship between the microbial diversity and the vegetation types is less conducted, especially in the subtropical area of China. In this present study, the rhizosphere soils of evergreen broad-leaf forest (EBF), coniferous forest (CF), subalpine dwarf forest (SDF) and alpine meadow (AM) were chosen as test sites. Terminal-restriction fragment length polymorphisms (T-RFLP) analysis was used to detect the composition and diversity of soil bacterial communities under different vegetation types in the National Natural Reserve of Wuyi Mountains. Our results revealed distinct differences in soil microbial composition under different vegetation types. Total 73 microbes were identified in soil samples of the four vegetation types, and 56, 49, 46 and 36 clones were obtained from the soils of EBF, CF, SDF and AM, respectively, and subsequently sequenced. The  Soilmunities . Vegetatmunities . Weand eon types .A deeper understanding about the effects of the vegetation types on the composition of the soil microbial communities in a forest ecosystem can help devise better strategies and management practices to improve soil health and plant growth . HoweverThe diversity of microorganisms in soil is very abundant; however, traditional microbial culture method has its limitations to study the composition of microbial communities because only 1% of the soil microorganisms present can be cultured . TerminaThis study has been approved by the Wuyi Mountain National Nature Reserve Management Committee, which takes care of the planning and protecting of Wuyi Mountain. The study did not involve any endangered or protected species. All the data in this study can be published and shared.2 forested area in the subtropical monsoon region of China [The Wuyi Mountain National Natural Reserve has the largest subtropical primordial forest ecosystem at the same altitude of the world, and is one of the key regions of biodiversity protection worldwide. It is located in Fujian Province , a 565 kmof China . The annof China . The annet al. [Three replicate soil sampling plots (20 m\u00d720 m) were selected in each site along an altitude gradient in August 2013. Soil samples were randomly collected from 0\u201320 cm soil depths in each plot using a soil core sampler (diameter of 2.0 cm). Twenty cores were composited into one soil sample, which were then sieved (2 mm) to remove soil impurities, hand-mixed and stored in plastic bags. Half of each soil sample was stored at 4\u00b0C until analysis, and another half was air-dried and sieved to determine soil pH, moisture content, total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP) and total potassium (TK) as described by Wu et al. .MspI, HaeIII, RsaI and AluI were used to digest the purified 16S rRNA fragments, followed the method as described by Nithya & Pandian [We used the high salt/SDS method to extract the DNA from soil samples as reported previously . The amo Pandian . Samples Pandian .S), Shannon-Weiner index (H), Pielou\u2019s evenness index (E) and Simpson index (D) were calculated as described previously in order to measure species diversity of bacterial communities [et al.[P < 0.05) through SPSS 17.0.The GeneMarker Version 1.2) was used to analyze the T-FRs. Fragments smaller than 50 or larger than 1200 bp were deleted from the analysis. The relative abundance of each T-RF was calculated as the peak area of the respective T-RF divided by the total peak area of all T-RFs . Affiliamunities . The cor.2 was usProteobacteria, Bacteroidetes, Fusobacterium, Actinobacteria, Cellulophaga, Arthrobacter, Lactobacillus, Clostridium, Mycoplasma, Nitrospira, Streptococcus, Desulfobacter, Staphylococcus and Chloroflexi. The Actinobacteria, Fusobacterium, Bacteroidetes and Proteobacteria were four dominant phylum in all soil samples (Acidobacteria (21.37%) and Fusobacterium (16.81%), whereas Acidobacteria (26.87%) and Fusobacterium (26.37%) were also the most dominant in CF. Compared with the clone sequences retrieved from EBF and CF samples, the predominant T-RFs were affiliated to Acidobacteria (21.33%) and Proteobacteria (20.56%) in SDF samples, whereas Acidobacteria (19.22%) and Bacteroidetes (18.75%) were found in AM samples.All the experimental data of T-RFLP were listed in Supporting Information . Accordi samples . For EBFAlu I was 37.25% lower than those (51) with Hae III. A similar tendency of variation was also found in the CF, SDF and AM samples. The Species Richness index (S), Shannon-Weiner index (H), Pielou\u2019s evenness index (E) and Simpson index (D) were then used to show the species diversity of bacterial communities. Correlation coefficients among species indices in the bacterial communities showed that the Pielou\u2019s evenness index had no correlation with other diversity indexes. The Shannon-Weiner index was significantly correlated with Species Richness index and Simpson index, and it could be regarded as the key indicator of species diversity of the bacterial communities  of T-RFLP data in different vegetation types along an altitude gradient was showed in P<0.05). The soil pH values ranged from 4.56 to 4.97, indicating that all study sites were acidic. The soil moisture contents followed the sequence order of EBF>CF>SDF>AM, suggesting that soil moisture content decreased with increasing altitude. The average concentrations of the TOC, TN, TP and TK in the EBF soil were 141.19, 0.75, 0.32 and 24.76 g\u00b7kg-1, respectively, which were 105.63%, 56.25%, 166.67% and 92.39% higher than those in the AM soil, respectively.Soil nutrients are important carbon and nitrogen sources for soil microorganisms, especially the soil organic matter. To explore the relationship between soil nutrient and the diversity of soil microbial communities, a correlation analysis between soil physical-chemical properties and diversity of microbial community were conducted . The resProteobacteria, Actinobacteria, Acidobacteria, Chloroflexi, Bacteroidetes, Firmicutes, Planctomycetes, Verrucomicrobia and Gemmatimonadetes [Proteobacteria, Bacteroidetes, Fusobacterium, Actinobacteria, Cellulophaga, Arthrobacter, Lactobacillus, Clostridium, Mycoplasma, Nitrospira, Streptococcus, Desulfobacter, Staphylococcus and Chloroflexi. Similar results were also reported in other mountains [Actinobacteria, Fusobacterium, Bacteroidetes and Proteobacteria were the most predominant phyla in all the soil samples.Previous studies indicated that the soil bacterial communities were normally comprised of nine major bacterial phyla: onadetes . We obseountains . In the p <0.05). Therefore, the most important reason for the difference in soil microbial diversity along an altitude gradient is the decline of soil nutrient content. Furthermore, the soil tiny animals, plant root and seasonal variation might also cause or contribute to the differences [The diversity of soil bacterial communities decreased with increasing elevation, and showed a general trend of EFB>CF>SDF>AM. A correlation analysis of microbial diversity and soil physicochemical properties also indicates that the soil nutrient indicators such as TOC, TN, TP and TK tend to decrease with an increment in altitude. Our results demonstrated close links between soil nutrient contents and bacterial diversity, and the correlation coefficients between the four diversity indexes and TOC or TN were all greater than 0.86  and phospholipid fatty acid (PLFA). This study also reveals the interactions between bacterial communities\u2019 composition and certain soil characteristics, and the roles of microorganisms involved in the biogeochemical cycling of nutrient elements in this forest ecosystem.S1 FileTable A). All T-RFs detected using T-RFLP (Table B). Bacterial species detected by Hae III (Table C). Bacterial community detected in all soil samples (Table D). Principal components analysis (Table E). T-RFLP profiles digested by four restriction enzymes in rhizosphere soil .The data includes five tables and one figure as follows. T-RFLP database for four enzyme digestions ((ZIP)Click here for additional data file."}
+{"text": "P<0.05), indicating that inorganic fertilization had different impacts on these C transformation enzymes. Compared with CK, the M, MN1 and MN2 treatments exhibited higher enzyme activities, soil TOC, total nitrogen, dissolved organic C, and microbial biomass C and N. The fertilization regime of the MN1 treatment was identified as optimal because it produced the highest yields and increased soil quality, ensuring sustainability. The results suggest that inorganic fertilizer alone, especially in high amounts, in greenhouse fields is detrimental to soil quality.In order to discover the advantages and disadvantages of different fertilization regimes and identify the best management practice of fertilization in greenhouse fields, soil enzyme activities involved in carbon (C) transformations, soil chemical characteristics, and crop yields were monitored after long-term (20-year) fertilization regimes, including no fertilizer (CK), 300 kg N ha-1 and 600 kg N ha-1 as urea (N1 and N2), 75 Mg ha-1 horse manure compost (M), and M with either 300 or 600 kg N ha-1 urea (MN1 and MN2). Compared with CK, fertilization increased crop yields by 31% (N2) to 69% (MN1). However, compared with CK, inorganic fertilization  also caused soil acidification and salinization. In the N2 treatment, soil total organic carbon (TOC) decreased from 14.1\u00b10.27 g kg-1 at the beginning of the long-term experiment in 1988 to 12.6\u00b10.11 g kg-1 (P<0.05). Compared to CK, N1 and N2 exhibited higher soil \u03b1-galactosidase and \u03b2-galactosidase activities, but lower soil \u03b1-glucosidase and \u03b2-glucosidase activities ( Food crop yield as well as soil sustainability therefore remain among the most important issues considered by Chinese people. Field greenhouse cultivation is a major means of vegetable production in China particularly important to ensure a vegetable supply in winter. A survey in 2008 showed that over 90% of the world field greenhouse cultivation area is in China (ca. 26700km2). Greenho2). In rece2),5. ThereSoil carbon is considered an important indicator of soil quality tied to major soil functions, such as aggregate stability, nutrient retention and availability, and nutrient cycling. Although much research on the effects of N applications on soil C content has been conducted \u201310, greeEnzymes catalyze all biochemical reactions and are an integral part of nutrient cycling in soil; they respond to soil management changes long before other soil quality indicator changes are detectable. Soil glycosidase activity is due to a group of enzymes involved in the hydrolysis of soil glycosides. It facilitates the breakdown of low-molecular-weight carbohydrates and produces the end product-glucose, important in terrestrial C cycling for providing necessary energy for proliferation of microorganisms. Among tLong-term fertilization experiments are valuable assets for studying changes in soil nutrient dynamics and balance, predicting soil carrying capacity, and assessing soil quality and system sustainability. Liaoning Province, located in the northeastern China, has a long winter time (165\u2013175 days per year) and therefore, greenhouse fields are the preferred means of cultivating vegetables in this region. In greenhouse fields, vegetable productivity is sustained by fertilization with either organic sources, such as composted manures, or inorganic materials, such as synthetic fertilizers. To date, few studies have assessed the effects of long-term synthetic fertilizer use and alternative fertilization regimes, such as manure, and combined manure and chemical fertilizer use, on soil enzyme activities and soil chemical properties. After imposing fertilization treatments for 20 years at an experimental vegetable production site in a greenhouse, we addressed the following questions: (1) How do chemical properties, especially soil C, change under different fertilization regimes; (2) how do C transformation-related enzyme activities change under different fertilization regimes; and (3) what is the optimal fertilization regime for this greenhouse field cultivation system?2) established in 1988 at Shenyang Agricultural University of China. The authority of the field is Shenyang Agricultural University of China who issued the permission for the research work in this field. The region has a continental monsoon climate, with a mean annual precipitation of 705 mm and a mean annual temperature of 7.0\u20137.9\u00b0C. The soil is classified as Hapli-Udic Cambisol (FAO Classification), with the basic properties of 14.1 g kg-1 total organic C (TOC), 1.16g kg-1 total N (TN), pH(H2O) 6.75, and sandy loam texture in the 0\u201320 cm layer. The mean annual air temperature inside the greenhouse is 17.6\u00b0C.The experimental site  is located in a greenhouse vegetable field (400 m2 each), with three replicates of each of the following six treatments: unfertilized control (CK), organic manure alone (M), 300 kg ha-1 N (N1), (4) 600kg ha-1N (N2), and combined applications of organic manure with chemical fertilizer N (MN1 and MN2). The manure was a horse manure compost, with 48.3% water content and containing 150 g C kg-1, 7 g N kg-1, 1.75 g P kg-1, and 2.49 g K kg-1 on a dry weight basis. The manure compost was incorporated in the 0\u201315 cm soil layer at a rate of 75 Mg ha-1 before planting (April 22nd). Thirty and 50 days after transplanting, chemical fertilizer (urea) was applied as sidedress to a depth of 15 cm.In 1988\u20131997, the field was planted with radish, onion, cucumber, potato, mustard, pimiento, cabbage and bean, in rotation twice a year. After 1997, the field was changed to a single crop rotation of cucumber and tomato under conventional tillage (0\u201320 cm). All the plots were planted the same crop types and with the same crop rotations. After every crop harvest, the produce was weighed and all the weights of the vegetables planted from the first to the last day of the year were considered the annual crop yield. The groundwater extracted from well was the water source and furrow irrigation was applied. The experiment followed a randomized block design consisting of eighteen plots , and using KEC and KEN values of 0.45 and 0.54 to calculate the Cmic and Nmic, respectively[+. Soil cation exchange capacity (CEC) was measured by ammonium acetate (pH 7) method[2 and pH 12.0 modified universal buffer were added to extract p-nitrophenol. The amount of p-nitrophenol released by glycosidases was determined colorimetrically at 410 nm. The substrates for \u03b1- glucosidase, \u03b2-glucosidase, \u03b1-galactosidase and \u03b2-galactosidase were \u03b1-D-glucopyranoside, \u03b2-D-glucopyranoside, \u03b1-D-galactopyranoside and \u03b2-D-galactopyranoside, respectively.Soil samples from 0\u201320 cm were taken from each plot in January 2008. Each soil sample was a composite comprising five soil cores (2.5-cm diameter). The samples were stored in individual plastic bags, and transferred to a 4\u00b0C cold room. Soil total organic carbon (TOC) was determined by using an elemental analyzer . Soil total nitrogen (TN) was measured by Kjeldahl digestion-distillation. Soil microbial biomass C (Cmic) and N (Nmic) were determined by fumigation-extraction,14, the pectively. Soil dipectively. Soil pH7) method. Soil \u03b1-P<0.05 level were considered to be statistically significant. The relationships between soil enzyme activities and soil chemical characteristics were analyzed by bivariate correlation analysis. Principal component analysis was performed by using the data reduction analysis in SPSS statistical software (SPSS 16.0). Multivariate analysis of variance was conducted to determine the effects of manure, fertilizer N and the interactions between manure and fertilizer on soil properties and enzyme activities. Manure and fertilizer N were independent variables and soil properties and enzyme activities were dependent variables.All statistical analyses were performed by SPSS statistical software (SPSS 16.0). Differences at The mean annual crop yields over the 20 years of experiment followed the order MN1 > MN2 = M = N1 > N2 > CK . CompareAfter 20 years of fertilization treatments, the soil properties changed greatly . The soi-1 TOC and the TOC/TN ratio of 12.2 in 1988, the TOC in CK and N2 was significantly lower , and the TOC/TN decreased by 44% and 74% in N1 and N2, respectively , thus decreasing the soil\u2019s nutrient supplying capacity. In contrast, all the manure treatments  increased the soil CEC. Regular additions of organic matter, such as manure, have been shown to increase soil stability, cohesion, and water retention, as well as CEC.The principle component analysis indicated that manured soils were distinct from the soils without manure application, showing the importance of manure application in enhancing soil enzyme activities. In this study, the activities of soil \u03b1- and \u03b2-galactosidase, \u03b1- and \u03b2-glucosidase all increased in the manure treatments, which was consistent with results by Bandick and Dick(1999), Ekenler and Tabatabai(2003) and Mandal(2007)\u201340. The Positive correlations between soil TOC and Cmic and soil enzyme activities were significant, indicating the important role of organic matter in maintaining enzyme activity. Furthermore, organic matter can play an important role in the immobilization of soil extracellular enzymes in the three-dimensional network of clay-humus complexes . The incIn this study, N application increased soil \u03b1- and \u03b2-galactosidase activities. Under high soil N availability, soil microbes allocate more N toward the production of enzymes used for acquiring energy, such as melibiose and lactose, among other nutrients ,43. A heLong-term N fertilization caused decreased soil \u03b1- and \u03b2- glucosidase activities which is consistent with results conducted by Eivazi and Tabatabai (1990) who found that adding inorganic N during the assay can partially inhibit \u03b2 -glucosidase activity . PreviouThe contrasting results of the effect of N fertilization on the activities of enzymes involved in C transformation showed that inorganic fertilization can have different impacts on the turnover rate of organic matter. The higher \u03b1- and \u03b2-galactosidase activities in our experiment indicated that more melibiose and lactose, while less maltose and cellobiose were decomposed after long-term inorganic fertilization. Long-term fertilization can therefore potentially alter the composition of different fractions of organic C in greenhouse field systems.Multivariate analysis showed a significant interaction between manure and inorganic fertilizer on enzyme activity, as compared with inorganic N alone. The long-term combined application of manure and inorganic fertilizers could be a viable option to couple soil C and N cycles for sustained crop production and the maintenance of environmental quality . In our Twenty years of fertilization in a greenhouse field increased crop yields significantly, with the highest yields observed when manure and inorganic fertilizer were applied together. Overall, this combination improved soil chemical and biological properties, increasing soil C and generally favoring greater enzyme activity. Long-term application of inorganic fertilizer alone caused a decrease in soil organic C and soil acidification, as indicated by our soil pH measurements. The treatments that included manure exhibited the highest TOC levels. We propose crop yield and soil sustainability as criteria to evaluate fertilization regimes. Our study showed that the most appropriate strategy for long-term successful fertility management of greenhouse fields is a combination of organic manure and chemical fertilizer N because such a regime not only increased crop yield but also maintained soil quality, ensuring soil sustainability."}
+{"text": "Yet little is known about the exact antibiotic concentration range that can effectively select for resistant organisms in animal gastrointestinal (GI) tract. In this study, the effect of different dosages of enrofloxacin on resistance and mutation development in rat GI tract E. coli was investigated by determining the number of resistant E. coli recoverable from rat fecal samples. Our data showed that high dose antibiotic treatment could effectively eliminate E. coli with single gyrA mutation in the early course of treatment, yet the eradication effects diminished upon prolonged treatment. Therapeutic and sub-therapeutic dose (1/10 and 1/100 of therapeutic doses) of enrofloxacin could effectively select for mutation in GI tract E. coli at the later course of enrofloxacin treatment and during the cessation periods. Surprisingly, very low dose of enrofloxacin (1/1000 therapeutic dose) could also select for mutation in GI tract E. coli at the later course of enrofloxacin treatment, only with slightly lower efficiency. No enrofloxacin-resistant E. coli could be selected at all test levels of enrofloxacin during long term treatment and the strength of antibiotic treatment does not alter the overall level of E. coli in rat GI tract. This study demonstrated that long term antibiotic treatment seems to be the major trigger for the development of target mutations in GI tract E. coli, which provided insight into the rational use of antibiotics in animal husbandry.It has been suggested that bacterial resistance is selected within a mutation selection window of antibiotics. More recent studies showed that even extremely low concentration of antibiotic could select resistant bacteria  The use of antibiotics in treatment of bacterial infections represents one of the most important inventions in human history. Since its discovery in the 1940s, antibiotics have saved millions of human lives and have also been widely used in the fields of veterinary medicine and agriculture in the past 70 years. However, due to the extensive use and misuse of antibiotics in various settings, most agents have lost their efficacy to bacteria as a result of emergence and spread of multiple-drug-resistant bacterial pathogens . In the in vitro. The concentration can be even lower than the concentration of antibiotics used for growth promotion purpose in animals, and the concentration of environmental antibiotic residues due to natural production by microorganisms and human contamination . The antibiotic was dissolved in 0.1 M NaOH at 10 mg/mL stock solution on the day of use. 11\u201315 weeks old, specified pathogen-free (SPF) male SD rats with body weight 250\u2013350 g were used in all experiments. Animals were purchased from Guangdong Medical Laboratory Animal Center and were housed individually and allowed free access to food and water. They were examined twice every day for any clinical signs such as behavior, gastrointestinal (GI) function, respiratory distress, food, and water intake etc. The experimental protocol was approved by the Research Animal Care and Use Committee of the Hong Kong Polytechnic University.E. coli in animal GI tracts. Thirty male rats were equally divided into six groups: one group was treated with therapeutic dose of enrofloxacin (10 mg/kg body weight), one was treated with saline as control and other groups were treated with doses of 10-fold, 1/10, 1/100, and 1/1000 of the therapeutic dose.Since fluoroquinolones are concentration-dependent antibiotic , we checThe oral antibiotic treatment regimen lasted about 1 month including three antibiotic treatments and three cessation gaps between treatments. All the rats were subjected to different doses of enrofloxacin treatment for 5 days, then cessation of antibiotic treatment for 4 days, another enrofloxacin retreatment for 5 days, followed by another cessation of antibiotic treatment for 8 days, then enrofloxacin retreatment for another 5 days, and tracing for 3 more days without antibiotic treatment.E. coli on MacConkey plate  were considered as E. coli and some of which were confirmed by 16SrRNA sequencing using primers .At the indicated time intervals, fresh feces (250\u2013500 mg) were collected and re-suspended in 1 ml of saline. The suspension was mixed and diluted by 10-fold in saline. 100 \u03bcl of suspension was plated on MacConkey agar containing 0 mg/L, 0.125 mg/L, 0.5 mg/L, and 2 mg/L of enrofloxacin. The plates were incubated at 37\u00b0C for 12 h and the total colony counts were recorded. The colony forming units (CFUs) per gram of feces were determined. Colonies that showed typical morphology of E. coli strain ATCC 25922 as quality control. The MICs of enrofloxacin were determined following CLSI guidelines  of the escribed .E. coli of these rats were checked by plating 200~300 mg of fecal samples of the rats on MacConkey plates with 0, 0.125, 0.5, and 2 mg/L of enrofloxacin. All rats were found to contain a similar amount of E. coli in their GI tract (data not shown). Five rats exhibited a background of less susceptible E. coli which can grow on MacConkey plate containing 0.5 mg/L of enrofloxacin. No fecal E. coli was able to grow on MacConkey plate with 2 mg/L concentration of enrofloxacin (data not shown). Twenty colonies with typical morphology of E. coli from MacConkey plates were randomly selected and confirmed to be E. coli by 16SrRNA sequencing. Similar E. coli confirmation was performed for the following each group of experiment by randomly selecting 20~40 E. coli for 16SrRNA sequencing. All the checked colonies were confirmed to be E. coli (data not shown).Thirty SPF male SD rats were selected for this experiment. Resistance background of GI tract E. coli with reduced susceptibility to enrofloxacin were grouped into one category and treated with a high dose of enrofloxacin (10-fold therapeutic dose). The rest of the 25 rats were randomly separated into 5 groups with one control group and 4 different treatment groups including therapeutic dose, 1/10, 1/100, and 1/1000 of the therapeutic dose treatments. The background of the high dose group animals is different from the others. The purpose of the high dose group study is different from other groups and intended to check the efficiency of higher dose of enrofloxacin on the clearance of less susceptible E. coli in animal GI tract.Upon background check, these rats were separated into six experimental groups. All five rats with a low background of E. coli. The numbers of GI tract E. coli decreased gradually during the first three days of treatment; the reduction became dramatic at the fourth day of treatment and the number of E coli recoverable remained at a low level for the first treatment period. After withdrawing the antibiotics, E. coli appeared again at the second day after the cessation of antibiotic treatment and increased on the third and fourth day. At the beginning of the second course of treatment period, the numbers of GI tract E. coli almost returned back to the normal level. During the whole 5-days antibiotic treatment, the numbers of GI tract E. coli were not affected, and remained at the normal level, which lasted throughout the second antibiotic cessation period. The GI tract E. coli gradually decreased again upon the start of the third antibiotic treatment. The number of E. coli became dramatically reduced again at the third day of the third course of the treatment and then gradually recovered at the fourth and fifth days of the antibiotic treatment. The number of E. coli kept recovering upon cessation of antibiotic treatment .For the 10-fold therapeutic dose treatment group, the application of enrofloxacin caused gradual eradication of rat GI tract E. coli was recorded. The number of less susceptible E. coli, which can grow at MacConkey plate with 0.125 mg/L was high before antibiotic treatment and slightly decreased on the second and third days of antibiotic treatment. The number of less susceptible E. coli became dramatically decreased on the fourth and fifth days of treatment. After withdrawing the antibiotic, the number of less susceptible E. coli reverted back to the normal level and remained so until the beginning the third course of the antibiotic treatment. The less susceptible E. coli slightly decreased at the first day of the third course of the antibiotic treatment and became undetectable at the third day of the treatment, then recovered to normal level throughout the rest of the treatment and non-treatment period .The effect of the antibiotic treatment to the development of fluoroquinolone-resistant E. coli that can grow on MacConkey with 0.5 mg/L enrofloxacin exhibited different response to high dose enrofloxacin treatment. The numbers of E. coli that can grow on MacConkey with 0.5 mg/L enrofloxacin reduced on the seconf day of treatment and became almost undetectable throughout the rest of the first, second and third treatments and non-antibiotic treatment period except for a period of brief appearance during the later course of the third treatment and antibiotic cessation period . Throughout the whole course of treatment, no E. coli isolates could be recovered on MacConkey plate with 2 mg/L of enrofloxacin, suggesting that high dose antibiotic could not select for fluoroquinolone-resistant GI tract E. coli.The E. coli isolates that grew on MacConkey with 0.125 mg/L and 0.5 mg/L enrofloxacin, respectively, were selected to check their MIC and target mutation profiles. E. coli that grew on MacConkey with 0.125 mg/L enrofloxacin exhibited a MIC range of enrofloxacin of 0.006~0.03 with no mutation in any of the four target genes gyrA, gyrB, parC, and parE; yet all E. coli strains that grew on MacConkey with 0.5 mg/L enrofloxacin exhibited MIC of enrofloxacin of 0.25~1 mg/L with single mutation in gyrA . Therefore, the MacConkey plate with 0.5 mg/L enrofloxacin could be used to check for the mutation rate of GI tract E. coli upon antibiotic treatment.Each of 20 E. coli exhibited slightly less susceptible E. coli background. Hence the therapeutic treatment dose of enrofloxacin did not have any effect on the numbers of E. coli in rat GI tract . However, after antibiotic treatment, the number of less susceptible E. coli decreased slightly and then increased to high level throughout the first course of antibiotic treatment and antibiotic cessation periods. During the second course of treatment, the numbers of less susceptible E. coli reduced gradually during treatment and remained at a lower level during the second antibiotic withdrawal period. During the third course of antibiotic treatment, the number of less susceptible E. coli increased to a high level and remained so until the end of the experiment .For the therapeutic treatment group, the GI tract E. coli that grew on MacConkey agar with 0.5 mg/L enrofloxacin (mutation rate) increased to around half of the total E. coli at the second day upon the treatment of enrofloxacin and remained at this level during the first course of the treatment. E. coli strains with mutation disappeared during the antibiotic withdrawal period and reappeared during the second course of treatment albeit at a lower level. E. coli strains with mutation increased to a high level at the second day of the third course of treatment, then decreased and reappeared again during the antibiotic cessation period . The data showed that mutation in E. coli might mainly triggered by antibiotic treatment and release of the antibiotic pressure can reduce the numbers of E. coli with mutation, which suggested that without antibiotic pressure, the E. coli with mutation may be less competitive than other normal E. coli in animal GI tract. The long term antibiotic treatment may make E. coli with mutation to adapt to animal GI tract, which can be seen from the increased number of E. coli with mutation at the end of third course of antibiotic treatment and the following cessation period.The numbers of E. coli in rat GI tract . The less susceptible E. coli strains emerged upon antibiotic treatment and remained at a stable level throughout the course of the experiment . The numbers of GI tract E. coli with target mutation slightly increased upon antibiotic treatment and disappeared during the antibiotic cessation period for the first two courses of treatments. The mutation rate was significantly increased in E. coli upon the third course of antibiotic treatment .Similar to the effect of the therapeutic dose of antibiotic treatment, sub-therapeutic dose of enrofloxacin did not have any impact on the overall numbers of E. coli could be observed throughout the experiment . The less susceptible E. coli could be selected during the second and third courses of antibiotic treatment and the E. coli with mutation could also be selected during second and third courses of the antibiotic treatments, with a higher rate at the end of the third course of the treatment, suggesting long term antibiotic treatment is the major trigger for the development of resistance in GI tract E. coli . For the control group, neither less susceptible E. coli nor E. coli with target mutation could be selected . Throughout the whole experiment, no E. coli that can grow on 2 mg/L of enrofloxacin could be obtained (data not shown).For the lower-level antibiotic treatment groups, namely 1/100 and 1/1000 of the therapeutic doses, no change in the number of GI tract E. coli isolates that grew on MacConkey with 0.125 mg/L and 0.5 mg/L enrofloxacin from different treatment experiments were selected to check their MIC and target mutation profiles. Different from the results obtained from the high dose treatment group, E. coli that grew on MacConkey with 0.125 mg/L enrofloxacin showed MIC range of enrofloxacin of 0.015~0.5, a little bit higher MIC than E. coli from high dose treatment group, with no mutation on any of the four target genes gyrA, gyrB, parC, and parE for most of the strains, but not all strains; on the other hand, E. coli that grew on MacConkey with 0.5 mg/L enrofloxacin exhibited MIC of enrofloxacin of 0.25~1 mg/L with a single mutation on GyrA (S83L) and no mutation at other target genes for all test strains, similar to the results from high dose treatment group (Table 1). The results further confirmed the use of MacConkey plate with 0.5 mg/L enrofloxacin as a tool to check for the mutation rate of GI tract E. coli upon antibiotic treatment.Each of 20 E. coli, develop resistance upon encountering different levels of antibiotic pressure.Improper uses of antibiotics from clinical applications and promotion of animal growth are the main causes for high prevalence of antimicrobial resistance in bacterial pathogens . EvidencE. coli and their background level of susceptibility to enrofloxacin. Five rats with a predominant background of low level resistance to enrofloxacin  were arranged into one group and treated with high dose of enrofloxacin to check whether high dose of antibiotic could eradicate organisms with intermediate enrofloxacin resistance, while other groups of mice with lower or no background of less resistant E. coli were treated with different doses of antibiotic. This study has come up with several conclusions that may be useful for the understanding of bacterial resistance development in animal GI tract. Firstly, high dose of antibiotic treatment can eradicate less susceptible E. coli (with target mutation) and prevent mutation development in E.coli in the early course of enrofloxacin treatment, while became less effective for long term treatment; therapeutic dose of enrofloxacin could select for the less susceptible E. coli and those containing target mutations. It is commonly accepted that intake of therapeutic dose of antibiotics for the whole course of treatment could prevent the development of resistance in bacterial pathogens. Our data showed that only high dose of antibiotic could effectively eliminate E. coli with target mutation, which are present in rat GI tract before treatment. However, therapeutic dose, 1/10 and 1/100 of therapeutic doses of enrofloxacin could select for less susceptible E. coli without target mutation and E. coli with de novo target mutation. Extremely low dose of enrofloxacin treatment, such as 1/1000 of therapeutic dose, can also select for less susceptible E. coli without target mutation and E. coli with de novo target mutation, but with lower efficiency. From the data obtained, we can also see that most of the target mutations in E. coli were selected during the later course of antibiotic treatment and antibiotic cessation periods. Long term antibiotic treatment seems to be the major trigger for the development of target mutations in GI tract E. coli. Another interesting finding for this study is that antibiotic treatment, except for those with high dosage, did not affect the total number of GI tract E. coli. Even under high dosage antibiotic treatment, the number of GI tract E. coli decreased upon treatment and then became normal even during the second treatment.To obtain meaningful interpretation of the data, all rats were checked for the initial load of GI tract in vitro  E. coli could be selected. In addition, no double target mutations in gyrA or parC could be detected in E. coli that could grow on MacConkey with 0.5 mg/L enrofloxacin. Our data is consistent with an early study in chicken in which treatment with enrofloxacin at doses routinely prescribed (50 ppm) rapidly reduced the fecal counts of colonized E. coli below the detection limit and did not induce resistance, whereas high frequencies of fluoroquinolone-resistant colonized C. jejuni were selected due to the de novo mutation at the target genes (E. coli with S83, D87, or both mutations have commonly been reported in in vitro selection experiments and contributed to fluoroquinolone resistance in E. coli (E. coli with resistant to enrofloxacin could be selected and only E. coli with reduced susceptibility to enrofloxacin and S83F target mutation could be selected, which is probably due to the unique GI environment. These data suggested that the generation of double target mutations in GyrA and/or ParC, which is the major mechanism of fluoroquinolone resistance in E. coli, may be associated with a fitness cost that hampers its survival in animal GI tract (E. coli in mice GI tract. The fact that the high prevalence of fluoroquinolone-resistant E. coli in animal GI tract may be due to the long term selection under antibiotic selective pressure in animal gut or colonization of fluoroquinolone-resistant E. coli that has been selected outside the animal gut. Lastly, the development of enrofloxacin-resistant E. coli in animal GI tract may possibly require the acquisition of plasmid mediated resistance determinant, which may further facilitate the development of target mutation and therefore development of enrofloxacin resistance in E. coli. Recent study has shown the high carriage of different PMQR genes in animal E. coli isolates, which contribute to the development of enrofloxacin resistance in E. coli (E. coli development resistance to fluoroquinolone require further investigations.Recent studies have reported the selection of resistant bacteria under very low concentration of antibiotics in vitro . However mutants . The selerations . Similaret genes . E. coli E. coli . HoweverGI tract . In our  E. coli . DiffereE. coli, which provided insights into the rational use of antibiotics in animal husbandry.In conclusion, this study demonstrated that long term antibiotic treatment seems to be the major trigger for the development of target mutations in GI tract 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": "Aedes aegypti and Ae. albopictus mosquitoes, the emergence of ZIKV suggests an ongoing intensification of environmental and social factors that have given rise to a new regime of arbovirus transmission. Here, we review hypotheses and preliminary evidence for the environmental and social changes that have fueled the ZIKV epidemic. Potential drivers include climate variation, land use change, poverty, and human movement. Beyond the direct impact of microcephaly and GBS, the ZIKV epidemic will likely have social ramifications for women\u2019s health and economic consequences for tourism and beyond.Since Zika virus (ZIKV) was detected in Brazil in 2015, it has spread explosively across the Americas and has been linked to increased incidence of microcephaly and Guillain-Barr\u00e9 syndrome (GBS). In one year, it has infected over 500,000 people (suspected and confirmed cases) in 40 countries and territories in the Americas. Along with recent epidemics of dengue (DENV) and chikungunya virus (CHIKV), which are also transmitted by  Flaviviridae, genus flavivirus), which began reappearing globally in the late 20th century, more than quadrupled in reported incidence from the 1980s\u20132000s and now causes an estimated 96 million symptomatic cases yearly . 2002 Sep 14;360(9336).Hotez PJ. Neglected Tropical Diseases in the Anthropocene: The Cases of Zika, Ebola, and Other Infections. PLoS Negl Trop Dis. 2016 Apr 8;10(4):e0004648.Stewart Ibarra AM, Ryan SJ, Beltr\u00e1n E, Mej\u00eda R, Silva M, Mu\u00f1oz \u00c1. Dengue Vector Dynamics (Aedes aegypti) Influenced by Climate and Social Factors in Ecuador: Implications for Targeted Control. 2013 Nov;8(11).Musso D, Cao-Lormeau VM, Gubler DJ. Zika virus: following the path of dengue and chikungunya? The Lancet. 2015 Jul;386(9990):243\u20134."}
+{"text": "DRNL function affects gynoecium development. Some of the mutant phenotypes present similarities to those observed in plants treated with exogenous cytokinins, and AHP6 has been previously proposed to be a target of DRNL. Therefore, we explored the response of drnl-2 developing gynoecia to cytokinins, and found that the loss of DRNL function affects the response of the gynoecium to exogenously applied cytokinins in a developmental-stage-dependent manner. In summary, this gene participates during gynoecium development, possibly through the dynamic modulation of cytokinin homeostasis and response.The gynoecium is the female reproductive system in flowering plants. It is a complex structure formed by different tissues, some that are essential for reproduction and others that facilitate the fertilization process and nurture and protect the developing seeds. The coordinated development of these different tissues during the formation of the gynoecium is important for reproductive success. Both hormones and genetic regulators guide the development of the different tissues. Auxin and cytokinin in particular have been found to play important roles in this process. On the other hand, the AP2/ERF2 transcription factor BOL/DRNL/ESR2/SOB is expressed at very early stages of aerial organ formation and has been proposed to be a marker for organ founder cells. In this work, we found that this gene is also expressed at later stages during gynoecium development, particularly at the lateral regions . The loss of  In contrast to many animals, plants can make new organs postembryonically. Stem cells produce signals to maintain a certain group of cells in an undifferentiated state with active cell division, which we call a meristem  is an AP2/ERF transcription factor that functions at early stages of organogenesis (35S::ESR2-ER) and gain or loss of function of this gene (bol-D and drnl-2)  function in Arabidopsis causes cotyledon fusions, though this phenotype does not present full penetrance  . It is e drnl-2) . This in drnl-2) . The losnetrance . Moreovel whorls . In the l whorls . The valance 6%, , while gFigure 1A), at the top it has a stigma with the style below, then the ovary with valves that protect the ovules, and finally the gynophore at the bottom. In Arabidopsis, the ovary is formed by two fused carpels. The floral meristem gives rise to the carpel primordia, and two congenitally fused carpels will arise and form a kind of hollow tube that during development will close at the top, followed by differentiation at the apical end, where the style and stigma will be formed. Inside the hollow tube, two meristematic regions will be formed along the side where the carpels are fused; these regions are also called the carpel margin meristem (CMM). The CMM gives rise to all the internal tissues, septum, placenta, ovules, transmitting tract, tissues that are crucial for the reproductive competence of the plant  and Columbia (Col); mutants bol-D  ecotypes Landsberg ts bol-D , drnl-2   under long-day conditions  in a growth chamber at 22\u00b0C. Two weeks after germination, the plants were transferred to a greenhouse with a temperature range from 22 to 28\u00b0C, and natural light conditions. Day length varied in different seasons.drnl-2 and Ler plants, which were germinated and grown under the same conditions as in the rest of the experiments. The numbers of fruits (siliques) and pistils that did not develop into fruits per plant were registered (n = 14 plants). Fruits were collected and classified according to their phenotype (n = 205 fruits). For the phenotypic analysis of pistils that did not develop into fruits, 199 pistils present along inflorescence stems were analyzed. Images were captured using a Stemi 2000-C microscope .Fruits were evaluated in BOL expression was observed under a DM6000B microscope coupled with a DFC420 C camera (both from Leica).\u03b2-Glucuronidase staining was performed for 24\u2013168 h at 37\u00b0C in a 2 mM X-Gluc solution (Gold Biotechnology), using established protocols . BOL expAHP6 expression in response to BOL activity, one drop of \u03b2-estradiol or mock solution was applied per inflorescence in DRNL-ER AHP6::GFP plants. The \u03b2-estradiol solution contained 10 \u03bcM \u03b2-estradiol (Sigma\u2013Aldrich) with 0.01% Silwet L-77 (Lehle Seeds). A solution containing DMSO and Silwet L-77 in the same concentration as in the \u03b2-estradiol solution was used for the mock treatment.To analyze the regulation of Transverse sections of the gynoecia were made 48 h after the treatments, according to \u00ae 7100 according to the manufacturer\u2019s instructions . Using a rotary microtome , 10 \u03bcm thick transverse sections of Ler and drnl-2 inflorescences were made and stained with alcian blue  for 25 min and neutral red (0.5%) for 5 min. Micrographs were obtained using a DM6000B microscope coupled with a DFC420 C camera (both from Leica).Tissues were fixed in FAE  with vacuum  and incubated for 120 min at room temperature. The material was rinsed with 70% ethanol and incubated overnight at 4\u00b0C, followed by dehydration in a series of ethanol solutions  for 60 min. each and embedded in TechnovitTM MiniPrep Kit (Zymo Research). The samples were treated with DNase I, included in the kit. Reverse transcription and amplification were performed using a KAPA SYBR FAST One-Step qRT-PCR Kit (Sigma\u2013Aldrich) in a StepOneTM thermocycler (Applied Biosystems). Three biological replicates and three technical replicates were included in the analysis. Target gene expression levels were normalized to ACTIN 2. Data was analyzed using the 2T-\u0394\u0394C method  were grown simultaneously under the same conditions. When the first fruits were observed in the inflorescence stem, those fruits were removed, leaving only closed buds in the inflorescences. Once this was done, BAP solution drops  and 0.01% Silwet L-77 (in distilled water) were applied on the inflorescences for five consecutive days. Sixteen days after treatment the gynoecia were collected and analyzed in chronological order of development. The mock solution contained Silwet L-77 and the same concentration of NaOH (0.2 mN) used to prepare the hormone solution.Cytokinin treatments were performed in a similar way as described by DRNL during gynoecium development, drnl-2 . This mutant presents reduced fertility . Only about 12% of drnl-2 gynoecia developed into a fruit in our growth conditions , in comparison to WT plants, where most gynoecia are fertilized and become fruits. Out of this 12% of gynoecia that converted into fruits, different altered phenotypes along the apical\u2013basal axis of the fruits were observed, and were most evident in the ovary region. These phenotypes were classified in 4 types: WT-like, reduced valves (RV), very reduced valves (VRV), and one valve (OV) . The 81.5% of the total number of fruits presented a WT-like phenotype. These fruits had symmetrical valves like those of WT plants. The rest of the fruits presented defects in the symmetry of the valves: 5.9% had reduced valves, 2% had very reduced valves, and interestingly, the percentage of fruits with only one valve was greater than that of fruits with asymmetric valves (10.7%) .To obtain information about the role of , drnl-2  mutant gertility , and a lFigure 1C, middle). These structures also presented equivalent phenotypes as those observed in developed fruits, but the frequency of the one-valve phenotype was higher . The 61.98% of these pistils presented a WT phenotype, 5.21% had reduced valves, 2.08% very reduced valves, and 30.73% presented the one-valve phenotype.Besides fruits, we also analyzed the phenotype of the gynoecia that stayed in the stem but did not develop as fruits . On the other hand, interestingly, in some of the gynoecia or fruits presenting \u201cone valve,\u201d this \u201cvalve\u201d had almost the width of two valves. While we could clearly distinguish the replum and margins of the valve on one side of the fruit or gynoecium, these structures were not visible on the opposite side. In some fruits and gynoecia it was possible to partially distinguish the presence of these structures in some regions, and appeared to be absent in others . As observed in those gynoecia and fruits, the defects observed in drnl-2 developing gynoecia, do not appear to be 100% penetrant, and also vary in severity from pistil to pistil. Examples of altered growth were present from early gynoecium developmental stages. In Figure 2D, a doughnut-shaped, possibly stage 8 young drnl-2 gynoecium can be observed, presenting a roundish central opening. In contrast, stage 8 . Examples of valve asymmetrical growth in stage 9 and stage 10 drnl-2 are presented in Figures 2E,F. The image in Figure 2F possibly corresponds to the \u201cone valve\u201d phenotype presented in Figures 1G,H), because the same structure was observed along the apical\u2013basal axis of the gynoecium in different sections. Developmentally retarded and deformed drnl-2 stamens, were also observed, as previously described . On the other hand, as observed in fruits and old gynoecia, many developing gynoecia presented normal valve symmetry at their middle region . Some gynoecia also presented uneven development of their inner tissues, and examples are shown in Figures 2L,N,P,R, compared to their equivalent WT counterparts in Figures 2K,M,O,Q. Figure 2P  shows an example of a drnl-2 ovary where the septum presents cell death and the transmitting tract presents the characteristic staining with alcian blue of a gynoecium at late developmental stages. However, ovule development seems to be delayed since the integuments of the ovules have not yet grown to enclose the female gametophyte, as occurs in the WT at this point . Figure 2R  shows the transmitting tract region of an ovary where one side presents the characteristic blue staining and cell death (observed as an empty space in the septum), but the other not, which is uncommon in WT ovaries . In summary, valve defects can be detected in drnl-2 gynoecia throughout development, and some gynoecia also present asymmetric or asynchronic development of other tissues.After observing the external phenotypes of the escribed . Alteratdrnl-2 mutants presented clear alterations in gynoecium morphology at different stages during gynoecium development, we analyzed DRNL expression throughout this process to know whether it was also expressed at intermediate stages of development.Since DRNL in the reproductive tissues of developing flowers, we performed GUS staining of a BOL::GUS line . The activity of the DRNL promoter was maintained at the valves of developing gynoecia until stage 11 , although as development proceeded, it became weaker and more restricted to the adaxial cell layers. At these stages, GUS activity was also found in stamens, mostly at the apical zone and, afterward, throughout the anther  as reported by DRNL promoter activity was no longer found in the gynoecium . However, expression was still detectable in the anthers in microspores and the tapetum , as previously reported . This resemblance suggested that there could be a possible relation of DRNL with cytokinins during gynoecium development. Therefore, we sought reported genes that participate in the cytokinin pathway and that have been found to be connected to DRNL. One of these genes is AHP6, which encodes a histidine phosphotransfer protein that negatively modulates cytokinin signaling , its expression began to decrease in this region, and then at stage 9, a more confined expression of AHP6 in the valves was observed. AHP6 expression disappeared in the valves at stage 12, but it remained in the medial vasculature .We used an ter line  and obseAHP6 and DRNL have very similar expression patterns. Both are expressed in the prospective valves during early stages of development. Their expression is lost in the lateral domain of the gynoecium at stage 12, when the tissue is mature. In addition, DRNL and AHP6 not only shared expression locations at the gynoecium, but also in other structures such as stamen primordia . We then sought to determine whether this upregulation of AHP6 expression through DRNL was occurring at the gynoecium. For this, a cross between a DRNL activity inducible line (AHP6 reporter line (DRNL-ER AHP6::GFP) was performed. We observed that induction of DRNL activity caused a change in the AHP6 expression pattern in the gynoecium, detectable 48 h after the \u03b2-estradiol treatment . In stages 8\u20139 gynoecia, AHP6 expression was not detected in the provasculature of the medial region, but when DRNL activity was induced we could observe that the expression of AHP6 appeared in this tissue, and was slightly increased in its normal domain of expression .After this similarity in the expression patterns in the gynoecium was observed, we sought to determine whether the gain or loss of ble line  to the AAHP6 expression in the drnl-2 loss of function mutant. qRT-PCR analysis was performed in mutant and WT inflorescences, and no evident decrease in AHP6 expression in the inflorescences of drnl-2 could be detected .For this, inflorescences of Figure 5). Compared to untreated gynoecia and fruits , according to the developmental stage at which the gynoecia were when they were treated, and the resulting WT phenotype upon cytokinin treatment: Class I, gynoecia that were at late stages of development at the beginning of the treatment (around stages 12\u201313) , which became short and wide after the treatment; Class II, gynoecia that were at intermediate stages of development (around stages 9\u201311) , which presented tissue proliferation in their external medial region at the end of the treatment; and Class III, which were at early stages of development (around stages 6\u20138) at the beginning of the treatment, and presented apical\u2013basal defects at the end of the treatment . Both Ler and Col WT gynoecia presented these classes of phenotypes, though they were more severe in the Col ecotype.From this experiment, it became evident again that the response to cytokinins depends on the developmental stage in which each gynoecium was at the time of treatment. It should be noted that the treated gynoecia did not continue with their normal development to fruit. These gynoecia were small and showed a gradient of phenotypes as we previously reported  . This lack of overproliferation indicates that these drnl-2 gynoecia are less responsive to cytokinins. However, younger drnl-2 gynoecia that were equivalent to class III WT gynoecia, presented the opposite. We detected more severe apical\u2013basal defects than those observed in treated WT gynoecia . These defects were so severe that some drnl-2 gynoecia did not even develop valves, whereas in WT plants this defect was not observed in the conditions used for this experiment . Therefore, while older drnl-2 gynoecia presented a reduced response, younger drnl-2 gynoecia presented an increased response to the cytokinin treatment, compared to WT gynoecia. These results suggest that drnl-2 gynoecia may be more sensitive or responsive to cytokinins at early stages of development (stages 6\u20138), whereas at later stages (stages 9\u201311) mutant gynoecia are less sensitive or responsive to the external application of this phytohormone.When ahp6 gynoecia appear to be more sensitive to cytokinins than WT gynoecia at the stages analyzed, particularly young gynoecia. Both class II and class III gynoecia show a more severe response than those of Col WT plants . Class II gynoecia develop the tissue proliferations, and this ectopic tissue was even more evident in the ahp6 mutant than in WT gynoecia . For class III, while WT gynoecia presented the apical\u2013basal defects described in previous reports , most treated ahp6 gynoecia presented the lack of both valves , indicating an increase in the severity of the response.On the other hand, drnl-2 and their respectively WT gynoecia. It became clear that the ahp6 and drnl-2 mutants both increased sensitivity or response of early gynoecia to cytokinins. The response of both mutants was severe and produced many gynoecia without valves. However, the effects of these mutants in the response of treated gynoecia at intermediate stages of development (Class II) was opposite, with ahp6 increasing and drnl-2 decreasing sensitivity or response to the treatment. We also noticed another conspicuous difference between drnl-2 and the other genotypes : the three phenotype classes developed in chronological order and were easily identified in Ler WT, Col WT, and ahp6 gynoecia. The response per developmental stage was clear and generally uniform in these genotypes  and lateral organs (leaves) emerging from it  genes  in cotyledons and stamens, possibly through N) genes . TherefoJAGGED, NUBBIN, and members of the YABBY, KANADI, and HD-ZIP III families. They have been reported to be related to polarity or organ growth and most of their mutants present phenotypes that are different to the ones observed in drnl-2  or plants treated with auxin transport inhibitors do not develop lateral organs or develop them with severe defects . However, in inflorescences of the loss of function mutant drnl-2, we could not detect a clear decrease in AHP6 expression by qRT-PCR, contrary to what we had expected. This may be because the regulation of AHP6 by DRNL is tissue-specific, restricted to very narrow domains, and the RNA used for this analysis was isolated from inflorescences and not from isolated gynoecium tissues. Another possible explanation is that drnl-2 is not a null allele , an enzyme that inactivates cytokinins, also proposed to be a direct target of DRNL , or regulating other genes. For example, it has been reported that increased expression of DRNL/ESR2 caused decreased expression of some type A ARRs . Moreover, in lfs mutant plants, the development of leaf primordia is not recovered through auxin micro application nor by the expression of LFS under the DR5 promoter. The authors suggest LFS might be also regulating cytokinin homeostasis, because genes that participate in the cytokinin pathway (such as type A ARRs and CKXs) were found to be altered in the mutant in global expression analyses (The lower sensitivity to cytokinins in more developed gynoecia suggests that e A ARRs , which ne A ARRs . Also, ve A ARRs . The autanalyses .drnl-2 gynoecia, in comparison to the rest of evaluated genotypes. This response may be reflecting that the loss of DRNL function is not only affecting the morphology of floral organs but also affects the organ spatio-temporal order of initiation or development in the floral meristem. The exacerbated asynchrony observed in treated drnl-2, and not detected in ahp6 mutants, further suggests that DRNL regulates more elements in the cytokinin pathway. Therefore, it might be that Arabidopsis DRNL, and possibly the tomato LFS, have functions in modulating the cytokinin pathway, or the response to it, through differential gene regulation at different stages of development. Figure 6 shows a model of the participation of DRNL as a modulator of cytokinin homeostasis and response during gynoecium development, playing at least two different roles as gynoecium development progresses.Finally, another interesting observation was the asynchronous response of young DRNL participates at further stages during gynoecium development. It differentially modulates the response of the gynoecium to cytokinins at distinct stages, having possibly a dual role during development. It would be interesting to test whether this is the case for other plant species and organs, and to further clarify the mechanisms through which this modulation is achieved. Moreover, considering that DRNL has been previously associated to other hormones and is also modulating cytokinin homeostasis or response, DRNL may orchestrate different hormonal pathways during the development of the gynoecium and, possible new organs in general.Besides being expressed at the gynoecium initiation stage, NM-M conceptualized the research, designed experiments, participated in writing, editing, and revising the manuscript. YD-M conceptualized the research, designed and performed experiments, wrote the original drafts, and prepared figures. JS performed experiments, prepared figures, wrote parts, and revised the manuscript. JIR-O performed experiments. SdF conceptualized the research, designed experiments, participated in writing, editing, and revising the 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": "Introduction: With increasing survival of vertically HIV\u2010infected children and ongoing new horizontal HIV infections, the population of adolescents (age 10\u201319 years) living with HIV is increasing. This review aims to describe the epidemiology of the adolescent HIV epidemic and the ability of national monitoring systems to measure outcomes in HIV\u2010infected adolescents through the adolescent transition to adulthood.Methods: Differences in global trends between younger (age 10\u201314 years) and older (age 15\u201319 years) adolescents in key epidemic indicators are interrogated using 2016 UNAIDS estimates. National population\u2010based survey data in the 15 highest adolescent HIV burden countries are evaluated and examples of national case\u2010based surveillance systems described. Finally, we consider the potential impact of adolescent\u2010specific recommendations in the 2016 WHO Consolidated Guidelines on the Use of Antiretroviral Drugs for Treating and Preventing HIV Infection.Discussion: UNAIDS estimates indicate the population of adolescents living with HIV is increasing, new HIV infections in older adolescents are declining, and while AIDS\u2010related deaths are beginning to decline in younger adolescents, they are still increasing in older adolescents. National population\u2010based surveys provide valuable estimates of HIV prevalence in older adolescents and recent surveys include data on younger adolescents. Only a few countries have nationwide electronic case\u2010based HIV surveillance, with the ability to provide population\u2010level data on key HIV outcomes in the diagnosed population living with HIV. However, in the 15 highest adolescent HIV burden countries, there are no systems tracking adolescent transition to adulthood or healthcare transition. The strength of the 2016 WHO adolescent\u2010specific recommendations on antiretroviral therapy and provision of HIV services to adolescents was hampered by the lack of evidence specific to this age group.Conclusions: Progress is being made in national surveillance and global monitoring systems to specifically identify trends in adolescents living with HIV. However, HIV programmes responsive to the evolving HIV prevention and treatment needs of adolescents can be facilitated further by: data disaggregation to younger and older adolescents and mode of HIV infection where feasible; implementation of tools to achieve expanded national case\u2010based surveillance; streamlining consent/assent procedures in younger adolescents and consensus on indicators of adolescent healthcare transition and transition to adulthood. Globally in 2015, there were 1.2 billion adolescents aged 10\u201319 years , accounting for 16% of the world's population . AdolescCompared to HIV\u2010infected children and adults, HIV\u2010infected adolescents have poorer retention in care, lower rates of virologic suppression and higher rates of mortality \u20139. DiffeThe adolescent developmental transition to adulthood coincides in some settings with a healthcare transition from paediatric to adult HIV services. There is concern that outcomes following this healthcare transition are worse than in paediatric HIV services prior to the transition \u201315. HoweThe most recent estimates and trends in the size of the global adolescent HIV epidemic are reviewed. We interrogate global and country\u2010specific trend differences (for the five highest adolescent HIV burden countries) between younger and older adolescents in the total number living with HIV, the number of AIDS\u2010related deaths and the number of new infections using the 2016 UNAIDS estimates for 1990\u20132015. Estimates from 1990 through 2015 are used and are based on the most recent 2016 UNAIDS Spectrum model  for the Annually UNAIDS publishes global, regional and national estimates of key HIV epidemic indicators including age\u2010 and sex\u2010specific estimates of the number of people living with HIV, the number of people newly infected with HIV and the number of people dying from AIDS\u2010related causes . As it iIt is estimated that 50% of HIV\u2010infected adolescents live in just six countries and that 15 countries account for 75% of all HIV\u2010infected adolescents globally, 13 countries in sub\u2010Saharan Africa, one in Asia and one in South America  Figure . In 2015During the last decade, with expansion of paediatric antiretroviral therapy (ART) and its survival benefits, in combination with approximately 250,000  new infections in older adolescents annually, the number of adolescents living with HIV has risen 28% between 2005 and 2015 . Sixty\u2010fAIDS\u2010related deaths in all adolescents 10\u201319 years of age peaked in 2012 at approximately 43,900  deaths globally and have since seen a small decline each year to 40,800  deaths in 2015 . This paThe increasing AIDS related deaths among adolescents between 2005 and 2013 are potentially a reflection of the \u201cbulge\u201d of vertically HIV\u2010infected children aging into the adolescent age group. Following expansion of ART services, particularly in sub\u2010Saharan Africa, between 2005 and 2015 over 100,000 additional children living with HIV aged into adolescence each year, as the \u201cbulge\u201d of children vertically infected 10 years earlier (between 1995 and 2005) survived into adolescence. The number of children living with HIV that aged into adolescence increased from 53,000 in 2000 to 100,000 in 2005. Thus the proportion of adolescents living with HIV and at risk of AIDS deaths increased. The number of adolescents living with HIV entering adolescence is starting to decline, reflecting the increase in vertical HIV transmission prevention services from 2005 until 2015 and the dramatic decline in children newly infected with HIV 10 years earlier.It is assumed that deaths in older adolescents are predominantly among vertically and not newly horizontally infected adolescents since horizontally infected adolescents will only recently have been infected; however, we are not aware of direct data to confirm this. As more children are reached with ART in future years, the adolescents living with HIV who were vertically infected will live longer. However, the contribution of this increased longevity to the total number of adolescents living with HIV will be offset by the rapid decline in the entrants to the adolescent HIV population as the number of children infected vertically between 2005 and 2015 has declined ,26. ThusThe Collaborative Initiative for Paediatric HIV Education and Research (CIPHER) pooled individual retrospective data from 12 cohort networks, representing 50 countries and five regions of the world . MortaliEstimates indicate that HIV\u2010associated mortality is declining for younger vertically infected adolescents. Although this may seem positive, it is possible that the mortality burden is being displaced from younger to older adolescence as cohorts age up, with older adolescent mortality continuing to increase in the majority of high\u2010burden countries. Older adolescence coincides with the potential healthcare transition from paediatric to adult HIV services, as well as the most challenging developmental transitional stages on the road to adulthood. Disentangling the contribution of the healthcare transition and the transition to adulthood to rising mortality in older adolescents is going to require identification within surveillance systems of the younger and older adolescent age groups as well as indicators of adolescent transition to adulthood and, if relevant, the time of transfer between paediatric and adult HIV care services.National surveys conducted periodically in countries with high HIV prevalence can be a valuable source for understanding the status of the HIV epidemic in individual countries. These are most often household surveys conducted in a nationally representative sample of the population and can include interviews as well as minimally invasive investigations such as blood pressure measurements and blood sampling for HIV. Due to challenges with obtaining consent for HIV testing in children and younger adolescents, as well as the large sample sizes required for robust estimates in these relatively low HIV\u2010prevalence age groups, until recently national surveys have seldom included people younger than 15 years of age. With the recent release of preliminary results for Malawi, Zambia and Zimbabwe from the Population\u2010Based HIV Impact Assessments (PHIA) being conducted in the President's Emergency Plan for AIDS Relief (PEPFAR)\u2010supported countries, progress is being made through age disaggregation of the data to better understand HIV prevalence in adolescents and particularly the differences between younger and older adolescents \u201330. PrioWhere available, HIV\u2010prevalence estimates in younger adolescents range from 0.6%  0.2\u20130.9) in Kenya to 2.9% (95% CI 1.7\u20133.9) in Zimbabwe, with little difference in this age group by gender \u201332. In oComplementary to surveys that provide detailed demographic, behaviour and other health\u2010related information on a sample of the population, national case\u2010based surveillance systems are essential to obtain reliable individual\u2010level HIV\u2010specific outcome data on the entire population living with HIV. These case\u2010based surveillance systems, when implemented nationally, can provide the absolute numbers of people diagnosed with HIV, the proportion that ever received ART, that remain on ART and that are virologically suppressed. Such a surveillance system can also provide data for specific geographic areas and more routinely than a household survey. In the absence of national case\u2010based data, estimation of outcomes using mathematical models is required. For the adolescent population living with HIV, case\u2010based national monitoring has the potential to be very useful and avoids relying on modelled estimates that due to the low prevalence of HIV in this age group are hampered by imprecision. Capacity is emerging for countries to perform case\u2010based surveillance of HIV programmes. Examples from the United Kingdom/Ireland, South Africa and Brazil are outlined below.One of the first examples of national paediatric HIV surveillance was the National Study of HIV in Pregnancy and Childhood (NSHPC) in the United Kingdom and Ireland ,43. SincAnother example of case\u2010based surveillance information captured at the point of care is the South African 3\u2010Tier ART Monitoring and Evaluation System ,48. The South Africa is also establishing a national cohort through the National Health Laboratory Service (NHLS) electronic database. Since 2004, the NHLS has been the primary provider of public sector laboratory testing in South Africa, including CD4 and HIV viral load tests. Through sophisticated record linkage techniques patients are either directly or probabilistically linked to test records that can be used to identify points in the cascade of HIV care. For example, the earliest recorded CD4 count for a patient would indicate entry into HIV care and the first recorded HIV viral load would indicate initiation of ART, as viral load testing is only conducted at or following initiation of ART and not prior to initiation, according to South African National HIV treatment guidelines . This apIn Brazil, epidemiologic surveillance for HIV is conducted through the integration of national electronic information systems that are not necessarily specific to HIV. These include the notifiable disease information system, mortality information system, laboratory tests control system for CD4 and virologic data and the medication logistics control system for ART data . Annual While these case\u2010based surveillance systems offer tremendous potential they require consistent and complete reporting including from key populations often hesitant to be in contact with official health systems. Using case\u2010based surveillance for monitoring the outcomes of adolescents must be done with a comprehensive understanding of the limitations of the system.The 2016 revision of the WHO consolidated guidelines on The Use of Antiretroviral Drugs for Treating and Preventing HIV Infection, includes three adolescent specific recommendations addressing when to start ART, what antiretroviral regimens to use and guidance on the delivery of adolescent friendly health services see  22]. Th. Th22]. The additional effort required to fulfil ethical requirements related to research on people younger than 18 years of age can be a deterrent to their inclusion in pharmaceutical and other studies . Even whIncreasing numbers of adolescents living with HIV will be starting ART and requiring adolescent\u2010appropriate ART services . There iUntil recently, global and national programmatic reporting has traditionally disaggregated HIV data above and below 15 years of age. The recent WHO Consolidated Strategic Information Guidelines strongly recommend age disaggregation in 5\u2010year age bands, and with expanding electronic monitoring systems this is becoming feasible . Where pThe population of adolescents aged 10\u201319 years living with HIV continues to grow as survival of vertically HIV\u2010infected children and adolescents improves and new HIV infections, although declining, are still substantial . AlongsiTo facilitate HIV programming that is responsive to the vulnerabilities of this transitioning age group and ensure safe transitioning to adulthood for all adolescents living with HIV we recommend :Disaggregation of routine monitoring, surveillance and research data by age  and where feasible by likely mode of HIV transmission, especially in older adolescents and young adults. This will aid identification and understanding of differences in trends in key outcomes including new infections, proportion on ART, virologic suppression, retention in care, mortality and differing service delivery needs in younger and older as well as vertically and horizontally HIV\u2010infected adolescents;Continued expansion of case\u2010based national HIV surveillance particularly in high HIV burden countries, through the development and implementation of appropriate systems and tools. This will generate reliable population level data on the HIV epidemic as a whole and particularly for lower prevalence age groups including children and adolescents where modelled estimates lack precision;Streamlining of processes to receive consent and assent for inclusion of children and adolescents in all forms of research including national population\u2010based surveys and pharmaceutical studies;For indicators of adolescent transition to be included in and reported on by national HIV programs, consensus is needed across settings on how to define and measure successful transition through adolescence to adulthood and successful healthcare transition where applicable.With advances in the efficacy of and access to ART regimens, this growing generation of adolescents living with HIV, both vertically and horizontally acquired, have the opportunity unlike generations before, to meaningfully contribute to society and thrive into adulthood. To fulfil this potential, a new paradigm of health system programming and monitoring is required, with the central involvement of adolescents in determining this trajectory.ALS and MD conceived of the review and conducted background research. MM provided additional UNAIDS data and contributed to analysis and interpretation of the data. AA contributed intellectual content. ALS drafted the manuscript and all authors critically reviewed and approved of the manuscript.We would like to thank the following people for their guidance related to national surveillance and monitoring programs: Ali Judd (UK), Mhairi Maskew (South Africa), Meg Osler (South Africa), Jorge Pinto (Brazil), Gayle Sherman (South Africa), Lyson Tenthani , Claire Thorne (UK), Hannock Tweya , Rachel Vreeman (Kenya)."}
+{"text": "In order to draw reliable conclusions about the temperatures at which transformations occur, we address how to accurately measure the bulk temperature of the samples under microwave irradiation. A new temperature calibration method merging data from four independent techniques is developed to obtain the bulk temperature as a function of the surface temperature in thermal processes under microwave conditions. Additionally, other analysis techniques such as Differential Thermal Analysis (DTA) or Raman spectroscopy are correlated to dielectric permittivity measurements and the temperatures of thermal transitions observed using each technique are compared. Our findings reveal that the combination of all these procedures could help prove the existence of specific non-thermal microwave effects in a scientifically meaningful way.In this study, real-time and  For many years, microwave energy has been increasingly and successfully used to process materials with unique properties at high temperatures (>1000\u2009\u00b0C) in a broad range of traditional and emerging manufacturing fields of materials such as ceramics, metals or in nanotechnology.The benefits of using microwaves for heating materials are well-known and have been widely recognized over the last decades. In microwave processing, energy is rapidly transformed into heat inside the materials through electromagnetic (EM) radiation, whereas in conventional heating, thermal energy is merely propagated throughout the volume of the specimen by an external heating source.7. Moreover, an important decrease has been reported in the reaction temperature of hundreds of degrees centigrade in comparison to conventional heating means1 which was associated to a substantial decrease in the apparent activation energy.Numerous studies on microwave processing have revealed that microwave technology significantly reduces reaction time and influences material properties12. While there is no doubt that thermal microwave effects do exist, the specific or non-thermal microwave effects are more difficult to justify and are subject to constant speculation and discussion, as many previously reported non-thermal microwave effects have turned out to be purely thermal in nature8. Solving this significant yet basic problem is thus a major challenge for researchers in the field of microwave processing.There is a constant debate, however, on whether the observed rate enhancement attained with this ultra-fast microwave dielectric heating is caused by purely thermal effects, or if this is connected to specific or non-thermal microwave effects arising from the interactions between the radiation fields and the materialin-situ monitoring of microwave reactions in combination with other characterization techniques for solid materials as X-Ray Diffraction, Neutron Scattering, FTIR or Raman Spectroscopy16. Schmink and Leadbeater15 and Vaucher et al.16 employed in-situ Raman spectrometers to examine heating selectivity during microwave exposure. However, although they are ground-breaking studies, additional experimental evidence is still required.Exploiting the many facets of microwave processing requires a thorough and comprehensive understanding of the underlying processes at atomic level for which new and complex instruments and measurement capabilities are needed. In this regard, several approaches have been developed for the 19.The interaction between electromagnetic waves and matter is quantified by complex physical parameters such as electrical conductivity and dielectric properties (or permittivity). These interactions depend on the structure of the materials and chemical bonds between their atoms as electromagnetic fields couple directly to the electrons in atoms, molecules and crystals or cluster structures of materials. Hence, it has also been suggested that the measurement of dielectric properties and the variations of this measurement with other process-related parameters  could shed light on the thermal processes of materials under microwave fields18). For most of the permittivity experiments in the GHz range, especially around the Industrial Scientific and Medical (ISM) frequencies 0.915\u2009GHz and 2.45\u2009GHz, the thermal process was carried out by increasing the temperature of the materials using conventional means  to moderate temperatures (200\u2009\u00b0C to 500\u2009\u00b0C), which does not allow us to observe microwave effects with the monitoring of permittivity changes with temperature.There is a wide range of reported experimental techniques to determine the dielectric properties of materials as a function of temperature from very low frequencies to several hundred gigahertzes  the ultrafast nature of microwave reactions; (ii) the compatibility of experimental methods with microwave technology; and (iii) the complexity of the temperature measurement procedures.8.In order to demonstrate the existence of the above mentioned specific microwave effects, the bulk temperature reached during the microwave reaction process must be determined accurately. The vast majority of experiments conducted under microwave conditions have demonstrated that what controls the outcome of a chemical reaction is solely the bulk temperature in the material24. In the past, the reaction temperatures under microwave heating conditions were very often measured inappropriately which led to many misguided claims of certain non-thermal microwave effects. When the experiments were repeated using adequate temperature monitoring and controls, it was proven that some of the originally proposed specific non-thermal effects were simply thermal in nature21.The precise measurement of reaction temperature is a non-trivial and complex matter which entails basic knowledge of the effects caused by microwave dielectric heating24. There are also reasonable doubts about the accuracy of temperature measurements, given that microwave heating can lead to important temperature gradients between the center and the surface of the sample, which increase particularly at high temperatures.Apart from the singularities of the microwave heating process, what makes measuring reaction temperatures under microwave conditions such a complicated task is the inconvenience of using classical temperature measurement instruments such as thermocouples, since these devices are metallic and will couple with the EM fields. Also, as fiber-optic sensors are extremely fragile, the sample temperature is usually measured with an IR pyrometer pointing at the sample surface from outside the microwave heating chamberThus, determining the real bulk temperature during the microwave irradiation could allow us to verify whether specific non-thermal microwave effects actually exist in a scientifically robust way.in-situ permittivity measurements under intense microwave electromagnetic fields together with the simultaneous determination of dielectric properties and synchronous recording of some thermo-physical and mechanical properties as a powerful technique for the study of thermal processes in materials.In this study, a new temperature calibration method merging data from four independent techniques is developed to obtain the bulk temperature as a function of the surface temperature. We propose real-time and In order to draw reliable conclusions from the temperatures at which the transformations occur, we first address the accurate measurement of the bulk temperature under microwave conditions. Also carried out are comparative permittivity measurements with other analysis techniques such as Differential Thermal Analysis (DTA) or Raman spectroscopy to contrast thermal transitions with some selected materials.Our findings allow for deeper theoretical insight into the high-temperature ultrafast interaction of materials with microwaves and its kinetics, and most importantly, provide a solid experimental basis which, for the first time, could assess the existence of thermal or non-thermal effects in upcoming microwave heating experiments leading to new developments in microwave technology.in-situ permittivity measurements of dielectric samples consisted of a dual-mode microwave cylindrical cavity capable to heat and measure with two separate microwave systems without interferences. The first of these systems, used for the heating process and coupled to the cavity by the horizontal probe located on the back sidewall of the cavity, delivered a microwave power signal (~150\u2009W) around the ISM frequency of 2.45\u2009GHz (from 2.4 to 2.5\u2009GHz), while the second system, coupled to the vertical probe located at the bottom wall, generated low power microwaves between 1.8 and 2.2\u2009GHz to characterize the dielectric properties of the materials by the Cavity Perturbation Method (see Methods section).The experimental setup Fig.\u00a0 used in Samples of dielectric materials were prepared in a cylindrical shape  to fit inside the quartz vials  which were inserted into the center of the cavity through a hole located on the top wall. The reactor was designed to ensure a strong and uniform electric field distribution in the cavity where the sample was positioned during both operation modes (heating and measuring). Quartz holders were employed as they are transparent to microwave radiation and can withstand temperatures above 1000\u2009\u00b0C.in-situ structural transformations. In addition, a video camera was installed in a third hole  to highly conductive like aluminium were employed to encase the heater during the experimental work.To incorporate the effect of temperature gradients from the core of the sample to the surface of the sample and container which dielectric samples of different composition might show, the cartridge heater was also surrounded by a hollow cylinder of a second material with different thermal conductivity which filled the space between it and the quartz holder see Fig.\u00a0. MateriaAs the microwaves were switched off during the calibration procedure, we were able to use a metallic thermocouple capable of reading temperatures over 1000\u2009\u00b0C to measure the temperature of the cartridge heater core. Figure\u00a0The uncertainty range in the determination of the bulk temperature from the measurement of the holder surface temperature is also represented in Fig.\u00a021.Fiber optic (FO) sensors represent an accurate alternative to determine the temperature of bodies under microwave heating by placing them in direct contact with the material of interest. Unlike thermocouples, FO measurement devices are immune to electromagnetic interferences and do not require shielding. However, FO probes are extremely fragile and their main limitation, as compared to IR thermometers is that their narrow temperature measurement range normally only reaches up to 300\u2009\u00b0CThe FO calibration procedure consisted in heating a sample within the microwave cavity described in Fig.\u00a0The materials selected for this technique were distilled water and silicon carbide (SiC), which have good microwave absorbing properties and dissimilar thermal conductivities. Samples of these materials were poured into the quartz container to a height of 15\u2009mm and subjected to microwave heating. The microwave power was adjusted at an approximate rate of 5\u2009\u00b0C/min.The ratio between the measured surface and FO bulk temperature is also plotted in Fig.\u00a0The temperature calibration procedure described in this section consisted in using the information given by the continuous measurement of dielectric properties as a function of the surface temperature during microwave heating to identify thermal phenomena in some reference materials with well-known transition bulk temperatures Fig.\u00a0. For thi2SO4), which undergoes a change in its crystalline structure from an orthorhombic configuration to a hexagonal one at 425\u2009\u00b1\u20094.8\u2009\u00b0C, according to bibliography30. A sample of this salt was poured into the quartz tube. It was then heated by microwave irradiation in the microwave cavity shown in Fig.\u00a0For our first experiment we chose silver sulfate . This material undergoes a sharp orthorhombic to cubic phase transition at a bulk temperature of 298.9\u2009\u00b1\u20090.9\u2009\u00b0C30. The phase transition was also identified by a strong change in the dielectric properties when the pyrometer indicated a surface temperature of 245\u2009\u00b0C  which belongs to the Aurivillius family and shows ferroelectric and piezoelectric properties up to high temperatures. Its Raman response reflects an orthorhombic to tetragonal phase transition at a bulk temperature of 677.5\u2009\u00b0C\u2009\u00b1\u20092.5\u2009\u00b0C33.Following a similar procedure as before to identify thermal effects this time we used reference materials with well-known Raman shifts. We first selected bismuth titanate . This salt undergoes a change in its crystal structure from an orthorhombic to a hexagonal system at a bulk temperature of 585\u2009\u00b1\u20092\u2009\u00b0C34. The Raman response of the mode around 980\u2009cm\u22121 experienced a sharp shift at a holder surface temperature of 463\u2009\u00b0C, which was correlated with the dielectric measurements , a borosilicate glass known for its interesting physical and chemical features and commonly used to calibrate equipment for testing viscosity and other glass properties in accordance with ASTM procedure C 965\u20138138.Annealing and strain temperatures are both thermal parameters directly related to the stress in the molecular structure of glass and have a direct influence on the variation of viscosity of this material at these temperatures. Some studies that have measured the electric properties of glass have revealed that the viscosity of this material can be directly correlated with its internal structure. As the glass temperature rises, there is an increase in the polarization of atomic bonds and in the bonding distance between atoms, which means that there is a continuous increase in the dielectric constant, while the viscosity of the sample diminishes35, are 470\u2009\u00b1\u20099\u2009\u00b0C and 513\u2009\u00b1\u20096\u2009\u00b0C, respectively.The strain and annealing temperatures of 717\u2009A glass, certified by NIST and several other laboratoriesTo measure the variation of the dielectric properties of borosilicate glass under microwave irradiation, we produced a cylindrical rod 10\u2009mm wide and 15\u2009mm high which was inserted into the quartz vial at the cavity center. As expected, the changes in glass viscosity due to the increasing temperature led to perceptible changes in the dielectric constant.Figure\u00a0A second derivative of the dielectric constant was employed to allow a rapid identification of the temperatures at which the growth tendency changes. The peak temperatures (479\u2009\u00b0C and 511\u2009\u00b0C), visible in Fig.\u00a041.Based on these results, the measurement of dielectric properties as a function of the temperature at microwave frequencies can be also considered as a suitable methodology to determine thermal parameters of interest in glass, such as strain and annealing temperatures, especially for those types of glass that present limitations when more common measurement procedures at lower frequency ranges (in the KHz-MHz) are employed. These measurement problems are due to the reduced sensitivity since viscosity can be masked by conductivity at low frequenciesFor a second microwave experiment we used a ceramic frit composed of a mixture of raw materials , which undergoes phase transformations at high temperatures.A sample was placed in the quartz container, inside the microwave reactor, and heated to more than 1000\u2009\u00b0C. In addition to measuring this material\u2019s dielectric properties, a DTA measurement was carried out separately for the comparison of the phase transformations and transition temperatures.Figure\u00a042. Moreover, the increase of dielectric properties of the frit sample could be caused by the movement of charges in the sample until the accommodation of the final structure during the dolomite decarbonation process.Although different mechanisms have been attributed to this process, practically all of them show the existence of alkaline-earth cation diffusion in the sample up to achieve the complete de-carbonation of the sampleIn addition, a second change is observed in the dielectric properties response around 1100\u2009\u00b0C, especially noticeable in the loss factor, which may be explained by the change in the viscosity of the sample due to melting. It should be noted that in changes of state at high temperature there is an initial increase of the atomic bond distances and, in some cases, an atomic breakdown. Furthermore, in the case of ceramic frit, the mixture of different raw materials triggers chemical reactions and mass transport in the sample. Hence, in our case, the variations in the dielectric properties cannot be attributed to a single cause but to all the above mentioned ones.The DTA measurement revealed a first transition at approximately 700\u2009\u00b0C, which is related to the dolomite decarbonation together with an endothermic reaction, and a second one around 1100\u2009\u00b0C associated to the frit melting.As in the first experiment, after temperature corrections, the transition temperatures identified by the dielectric measurements under microwave heating in both thermal phenomena of the ceramic frit were in good agreement with the results obtained by conventional techniques as DTA.Again, the material studied in the second microwave heating experiment underwent similar transformations to those which occur under conventional heating and no special mechanisms were evidenced due to the presence of the microwave fields.Several studies have recently reported that microwave heating could trigger transformations in materials at temperatures several hundred degrees centigrade lower than under conventional heating, leading them to claim that non-thermal or specific microwave effects do exist. Nevertheless, our findings revealed that differences between the bulk temperature and the surface temperature of the sample can be of the order of several hundred degrees. Consequently, there are reasonable doubts about the accuracy of the temperature measurement procedures employed in these studies, which needs to be corrected.The temperature calibration during microwave heating in our system has been achieved, among others, by measuring materials with phase changes. In the phase transition, structural changes occur in the solids at fixed temperatures that entail changes in their polarizability. Despite of the fact that the calibration procedure made use of solid materials, the calibration line defines the real bulk temperature of a material during the heating process using our recently developed microwave reactor described here and it can be extended to liquids, solids and their mixtures. The results presented open a field of work that allows discriminating accurately the different phenomena during the heating with microwave energy, eliminating the uncertainty about temperature. Considering the above findings, we believe that the new approach to calibrate temperature measurements in our microwave reactor serves not only to assess the bulk temperature of materials when heated by microwaves, but can also be used as a basis for research about thermal and non-thermal effects under microwave irradiation in upcoming experiments.The samples employed in the measurements are summarized in Table\u00a0To prepare the Bi4Ti3O12 sample, commercial Rutile (TiO2)  and \u03bc-Bi2O3  were used as starting materials. The appropriate stoichiometric amounts of the starting materials were mixed in an attrition-mill (in a PTFE Teflon container) with 100\u2009g of 1.2\u2009mm zirconia (ZrO2) balls in water for 3\u2009hours. T5003 Rohm&Haas dispersant (0.6\u2009wt%) was added to improve homogenization. The powders were overnight dried at 75\u2009\u00b0C and sieved through a 0.1\u2009mm mesh and sintered at 1200\u2009\u00b0C for 2\u2009hours in an Agni GmbH electrical furnace with a 3\u2009\u00b0C/min heating rate.Borosilicate glass (Standard Reference Glass 717\u2009A) employed in the experiments was provided by NIST  as a piece with dimensions 20\u2009cm\u2009\u00d7\u200910\u2009cm\u2009\u00d7\u200910\u2009cm. The sample was machined with a crown drill bit with adequate dimensions to be inserted in the quartz holder .43. For the dielectric properties measurement under microwave heating, the samples were prepared in a cylindrical shape  and placed at the center of the microwave cavity inserted in a quartz tube.Dielectric properties of materials and their variation with the temperature were calculated by the Cavity Perturbation Method (CPM)20.For the dielectric properties measurement, the low power microwave source of a vectorial network analyzer (VNA) coupled at the bottom wall of the microwave cavity see Fig.\u00a0 generateThe heating rate was 5\u201310\u2009\u00b0C/min depending on the sample. The microwave heating system is based on another frequency sweeping source (from 2.2 to 2.6\u2009GHz) and a microwave amplifier that, together with an adjustable coupling device (electric probe with variable penetration) and a double directional coupler (MECA\u2019s 722\u201340\u20131.950), allows for generating, adjusting and measuring the microwave forward and reflected signals in the microwave cavity as a function of the frequency and temperature. By selecting the appropriate frequencies of the source around the frequency peak of the cavity, we provide an excitation with a pulsed shape to supply the required microwave power depending on a desirable level of heating rate in the sample (maximum delivered power was 150\u2009W). More detailed information has been included in supplementary information.The infrared (IR) pyrometer (Optris CT-Laser LT) was used to measure the surface temperature of the quartz holder. Since the temperature calibration is performed through the described procedures, the emissivity was fixed to 0.97 during the experiments.\u22121, and maximum power of 100\u2009mW. It was calibrated following the procedure that involves the measurement of two NIST traceable standards: silicon and acrylonitrile.The portable Raman spectrometer (ProRaman-L-532-B1S) has a 532\u2009nm excitation, typical linewidth of 2\u2009cm2O3 was used as reference material. The temperature range for the measurement was 30\u20131250\u2009\u00b0C, under air flux of 0.04\u2009l/min, and the heating speed was 10\u2009\u00b0C/min.For the DTA measurements, an equipment Netzcsh model STA-409 was used. The furnace of the equipment has a temperature controller TASC 414/2 Netzsch. The samples were placed on platinum crucibles. Calcined \u03b1-AlSupplementary Information"}
+{"text": "Chromatin looping allows enhancer-bound regulatory factors to influence transcription. Large domains, referred to as topologically associated domains, participate in genome organization. However, the mechanisms underlining interactions within these\u00a0domains, which control gene expression, are not fully understood. Here we report that activation of embryonic myogenesis is associated with establishment of long-range chromatin interactions centered on Pax3-bound loci. Using mass spectrometry and genomic studies, we identify the ubiquitously expressed LIM-domain binding protein 1 (Ldb1) as the mediator of looping interactions at a subset of Pax3 binding sites. Ldb1 is recruited to Pax3-bound elements independently of CTCF-Cohesin, and is necessary for efficient deposition of H3K4me1 at these sites and chromatin looping. When Ldb1 is deleted in Pax3-expressing cells in vivo, specification of migratory myogenic progenitors is severely impaired. These results highlight Ldb1 requirement for Pax3 myogenic activity and demonstrate how transcription factors can promote formation of sub-topologically associated domain interactions involved in lineage specification. Chromatin looping allows for enhancer\u2013promoter interactions that regulate transcription. Here the authors show that activation of embryonic myogenesis is associated with establishment of long-range chromatin interactions centred on Pax3-bound loci and find that Ldb1 functions as the mediator of looping interactions at a subset of Pax3 binding sites. In the last decade, unbiased genome-wide analysis of chromosome conformation using Hi-C has identified that different parts of the genome with similar histone marks form two compartments characterized either by active transcription, being gene rich and having higher chromatin accessibility (type A), or being gene poor, displaying lower gene expression and repressive histone marks (type B)2. Importantly, these studies demonstrated the existence of topologically associated domains , which are chromosomal subunits displaying a higher contact frequency and defined by the binding of the Ctcf-Cohesin complex at the contact domain border5.Chromatin topology is an essential element of gene regulation because it establishes the framework for nuclear chromosome organization and ultimately, interactions between distal regulatory elements (enhancers) and gene promoters , the extent to which transcription factors (TF) shape the three-dimensional organization of the genome during differentiation is not clearly defined. In fact, while the ubiquitously expressed Yin Yang 1 (YY1) has been shown to mediate distinct enhancer\u2013promoter interactions independently of CTCF in multiple cell types15, only a few studies have investigated the mechanisms underlying the establishment of tissue-specific looping using a model of lineage specification. In situ Hi-C during macrophage activation identified a correlation between AP1 occupancy and establishment of new looping interactions16. Similarly, B cell activation requires Myc for the shift from long- to short-range interactions, which in turn facilitate enhancer\u2013promoter contacts regulating gene expression17. More recently, Monahan and colleagues reported that increased expression of the olfactory receptor genes observed during olfactory neuron differentiation involves strengthening of intra- and inter-chromosomal interactions between the selected gene promoter and several enhancers bound by the Lhx2-Ebf-Ldb1 complex18.Despite the existence of loci where looping interactions control gene expression  cells and next-generation sequencing-based technologies, we find that Pax3-mediated activation of the myogenic program occurs through a time-dependent establishment of long-range interactions involving PAX3 binding sites. PAX3 genomic occupancy is associated with an increased deposition of histone marks (H3K4me1 and H3K27Ac) normally found at active enhancer regions, and overlaps to elements capable of driving gene expression in developing embryos. Using mass spectrometry, we then identify PAX3 interaction with members of the chromatin looping complex, including the LIM-domain binding protein 1 (LDB1). We demonstrate that LDB1 is recruited to a subset of PAX3-bound elements characterized by increased levels of H3K4me1 deposition. Reduced Ldb1 expression impairs Pax3-dependent myogenic specification both in vitro and in vivo, and decreases deposition of H3K4me1 and chromatin looping of PAX3-bound enhancers. Importantly, our study show that forced recruitment of LDB1 to PAX3 enhancers is sufficient to induce gene expression, chromatin looping and H3K4me1 deposition, thus supporting that changes in genomic architecture are capable of driving transcription of Pax3 target genes during myogenesis.19  showed common and cell-type specific PAX3-bound loci20. Upon annotation of both datasets, we observed that the vast majority (~90%) of PAX3 ChIP-seq peaks are more than 5 kilobases (>5\u2009kb) away from the transcription start site (TSS) of the nearest annotated gene  and myogenic progenitors (6-days), along with control non-induced cells. After subtracting the HiChIP contacts called in non-induced Pax3 cells, we identified 3227 and 7987 loops in Pax3-induced mesodermal cells and myogenic progenitors, respectively , poised (H3K27me3 and H3K4me3), and active (H3K27Ac and H3K4me3) promoters and enhancers (H3K27Ac and H3K4me1)me3 Fig.\u00a0. To deteme3 Fig.\u00a0, which dme3 Fig.\u00a0, which bme3 Fig.\u00a0. PAX3 bime3 Fig.\u00a0, a phenoion Fig.\u00a0. Clusterion Fig.\u00a0. These rion Fig.\u00a0. In agreion Fig.\u00a0.Fig. 2Ep27. The VISTA catalog contains a list of annotated enhancers examined based on their ability to drive regional-specific LacZ expression in transgenic E11.5 embryos. Out of 52 enhancers annotated as active in the somites, 9 overlapped with PAX3 ChIP-seq peaks  and the \u03b2-globin genes38. LDB1 is expressed broadly and its function goes beyond globin gene expression, as it also interacts with ISL1 in cardiac progenitors and pancreatic \u03b2 cells40 and LHX2 during olfactory receptor choice18. Co-immunoprecipitation followed by western blot confirmed interactions between PAX3 and LDB1, ASH2L and the Cohesin subunit SMC1 , which displayed equal myogenic activity as the untagged protein , whereas non-induced displayed 6317 LDB1 and 19082 SMC1 peaks  resulted in robust myogenic differentiation, as measured by MYOG immunostaining on terminally differentiated cultures from 6-day dox-induced cells . Based on these results, we conclude that forced recruitment of Ldb1 to Pax3 sites mediates the formation of long-range interactions responsible for gene activation.Based on our proteomic, epigenetic, and transcriptomic data, Pax3 specifies the skeletal myogenic program by cooperating with Ldb1 and the HMT complex Fig.\u00a0. Nonethe46 Fig.\u00a0. Importa46 Fig.\u00a0, while s46 Fig.\u00a0. Therefo46 Fig.\u00a0. ASH2L ills Fig.\u00a0. Intereslls Fig.\u00a0. This walls Fig.\u00a0. To inveMyf5 \u2212111\u2009kb enhancer) following induction of full length PAX3, whereas the \u0394TAD mutant lacked this activity  displayed extensive migration at this time point, but Ldb1-deleted littermates  had severely reduced numbers of PAX3+ migrating progenitors, and most of those that were observed did not express MYF5  into the NotI site of the p2lox-Pax3-noStopCodon (described in ref. 46). The HaFlag G-block sequence is provided in Supplementary Table\u00a061). To purify cells transduced with the knockdown constructs, all pLKO plasmids were modified by replacing the selection gene (puromycin or blasticidin) with the sequence encoding the EGFP (referred hereafter as pLKO-shRNA-PGK-GFP).The p2lox-Pax3 and p2lox-Pax3-\u0394TAD-3xFLAG vectors were previously described62. The recombination cassette, located next to the Hprt gene, contains the tet-responsive-element (TRE) driving the expression of one single copy of cDNA, thus ensuring quasi-physiological expression levels. mES cells were maintained on mitotically impaired mouse embryonic fibroblasts (MEFs) in knock-out DMEM (Invitrogen) supplemented with 15% FBS , 1% penicillin/streptomycin (Invitrogen), 2\u2009mM Glutamax (Invitrogen), 0.1\u2009mM non-essential aminoacids (Invitrogen), 0.1\u2009mM \u03b2-mercaptoethanol (Invitrogen), and 1000\u2009U/ml LIF (Millipore). A detailed version of the myogenic differentiation protocol has been described63. Briefly, the ES/MEF cell suspension was preplated in a gelatin-coated dish for 30\u2009min in order to remove fibroblasts and the resulting supernatant (enriched for mES cells) was then diluted to 40,000\u2009cells/ml in EB differentiation medium and incubated in an orbital shaker at 80\u2009RPM. EB differentiation medium: IMDM (Invitrogen) supplemented with 15% FBS , 1% penicillin/streptomycin (Invitrogen), 2\u2009mM Glutamax (Invitrogen), 50\u2009\u03bcg/ml ascorbic acid (Sigma-Aldrich), 4.5\u2009mM monothioglycerol . Trangene induction was achieved by adding doxycycline (Sigma-Aldrich) to day-3 EBs cultures , and then maintained throughout the differentiation protocol by replacing the media (including dox) every 2 days. At day 5, EBs were disgregated and single cells were incubated for 20\u2009min with PDGFR\u03b1-PE and FLK1-APC conjugated antibodies (e-Bioscience). PDGFR\u03b1\u2009+\u2009FLK1\u2212 cells were sorted using FACSAriaII (BD biosciences) and replated on gelatin-coated dishes using EB differentiation media supplemented with 1\u2009\u00b5g/ml doxycycline and 10\u2009ng/ml mouse basic-FGF (Preprotech). Skeletal myogenic differentiation was assessed in cells cultured 4 days  as monolayer by withdrawing bFGF and doxycycline from the cultures (in order to shutdown transgene expression) followed by additional 2 days culture in the same serum- or serum-free media. HEK293T cells were maintained in DMEM (Invitrogen) supplemented with 10% FBS (Millipore), 1% penicillin/streptomycin (Invitrogen) and 2\u2009mM Glutamax (Invitrogen).Inducible mES cell lines were generated by Cre-loxP mediated recombination of the p2lox targeting plasmids into A2lox-cre mouse ES cellsIntracellular staining using MYOD and MYOG antibodies was performed on non-induced (day-4 EBs), 1-day and 6-day Pax3-induced cells. Cells were collected and fixed with 1% paraformaldehyde/PBS for 15\u2009min. Fixed cells were then incubated on ice for 1\u2009h with antibodies diluted in 0.3% Triton X-100/0.5% BSA/PBS solution. The list of antibodies used in this study and relative dilutions are provided in Supplementary Table\u00a0g, 30\u2009\u00b0C. Ldb1 knockdown was achieved by transduction of day-5 PDGFR\u03b1\u2009+\u2009FLK1\u2212 sorted cells with shSCR and shLdb1 lentiviral particles. Transduction efficiency  was assessed 3 days post-transduction using FACS. Replated day-5 PDGFR\u03b1\u2009+\u2009FLK1\u2212 cells from Ldb1 knockdown experiments were cultured 4 days as monolayer  and collected for ChIP, HiChIP and gene expression analysis or withdrawn of bFGF and dox for 2 days and collected for immunostaining and western blot.Lentiviruses were produced in HEK293T cells by co-transfection of pVSV-G, \u03948.9 and lentiviral constructs using Lipofectamine LTX-Plus reagent (Invitrogen). Supernatants containing lentiviral particles were filtered using 0.45\u2009\u00b5m filters and applied to cells cultured in 6-well plates. To facilitate transduction, 6-well plates were centrifuged for 90\u2009min at 1100\u2009\u00d7\u2009All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal work was approved by the University of Minnesota Institutional Animal Care and Use Committee (protocol number 1702-34580A).Ldb1 deletion in the Pax3 lineage was achieved by timely mating Pax3cre/+; Ldb1fl/+ males with Ldb1fl/fl females. Importantly, for these experiments the Cre was inherited through the paternal lineage. For immunostaining, embryos were collected at 9.5, 10.5, or 11.5\u2009d.p.c., embedded in Tissue-Tek\u00ae O.C.T. compound (Sakura\u00ae Finetek\u2014VWR) and frozen. For all experiments, genotyping was performed using genomic DNA isolated from Yolk Sacs.64. Briefly, d4 EBs  were trypsin-treated at 37\u2009\u00b0C for 1\u2009min with gentle shaking and reaction was inhibited by adding 10%FBS/PBS. Single cells were washed once with PBS, resuspended in 10%FBS/PBS and supplemented with formaldehyde  for crosslinking of protein-DNA complexes (10\u2009min at RT) followed by quenching with glycine and staining with PDGFR\u03b1-PE antibody. PDGFR\u03b1+ cells were sorted using FACSAriaII, snap-frozen in liquid nitrogen and stored at \u221280\u2009\u00b0C if not processed immediately. For 6-day experiments, PDGFR\u03b1+FLK1\u2212 cells were FACS sorted from d5 EBs  and then cultured for additional 4 days (as described in the Cell culture section) before collecting them for formaldehyde crosslinking. Cell pellets were incubated in lysis buffer LB1 supplemented with protease inhibitors  for 10\u2009min at +4\u2009\u00b0C followed by incubation in buffer LB2 supplemented with protease inhibitors  for 10\u2009min at +4\u2009\u00b0C. Cell pellet was then resuspended in LB3 supplemented with protease inhibitors  and then sonicated. For Histone marks, cells were sonicated using Bioruptor Pico  to achieve an average size of 200\u2009bp. For transcription factors and cofactors, cells were sonicated with a Branson sonicator at 18% power for 1\u2009min with intervals of 10\u2009s ON\u201310\u2009s OFF to achieve an average chromatin size of 300\u2009bp. After shearing, samples were centrifuged for 10\u2009min at 16,000\u2009\u00d7\u2009g and snap-frozen in liquid nitrogen if not processed immediately. For Histone ChIP, 12\u2009\u00b5g of chromatin (diluted to 250\u2009\u00b5l) were precleared for 4\u2009h at 4\u2009\u00b0C with 10\u2009\u00b5l of BSA-blocked Protein A-conjugated sepharose beads . For TF-cofactor ChIP, 25\u201340\u2009\u00b5g of chromatin (diluted to 500\u2009\u00b5l) were precleared for 4\u2009h at 4\u2009\u00b0C with 20\u2009\u00b5l of BSA-blocked Protein A (or Protein G)-conjugated sepharose beads . Samples were supplemented with 1/10 volume of 10% Triton X-100 and incubated overnight with antibodies for immunoprecipitation. The list of antibodies used in this study and relative dilutions are provided in Supplementary Table\u00a0g, pellet were washed with 75% ethanol, air dried and dissolved in 45\u2009\u00b5l H2O. qPCR was performed in a final volume of 10\u2009\u00b5l using SYBR Premix Ex Taq II (Clontech), 0.5\u2009\u00b5l of 1.4\u2009\u00b5M primer stock, and 0.3\u2009\u00b5l sample/reaction and run on a 384-well plate on a ABI7900HT instrument (Applied Biosystems). The primer sequences used for qPCR are provided in Supplementary Table\u00a0ChIP was performed following the protocol described by Young and colleagues with minor modificationsLibraries were generated following a gel-free protocol using AMPure XP beads (Beckman Coulter) for all the purification and size selection steps. Ten nanograms or less of DNA were end repaired using End-it DNA end repair (Epicentre), then A-tailed using Klenow Fragment (3\u2032\u2009\u2192\u20095\u2032 exo-NEB) followed by adapter-barcode ligation using T4 DNA ligase (Enzymatics). Illumina compatible adapter-barcodes were purchased from BIOO scientific. After ligation, DNAs were negatively size selected using 0.5\u00d7 Ampure XP beads and unbound DNAs were positively size selected by adding 0.4\u00d7 Ampure XP beads . Libraries were amplified using Phusion High Fidelity PCR master mix 2x (NEB) with a 16 cycles program. Ldb1 and H3K4me1 (from \u0394TAD and \u0394TAD-Ldb1 lines) ChIP-seq libraries were generated using the NEBNext UltraII DNA library prep kit (NEB) following manufacturer\u2019s instructions. Purified libraries were then submitted to the University of Minnesota Genomic Center (UMGC) for quantification, quality control, and sequencing. Libraries were pooled and sequenced on Single-End runs of the HiSeq2500 operated at High Output mode (Illumina). Libraries for TF/cofactors were sequenced to an average depth of 25 million reads/sample. Libraries for histone marks were sequenced to an average depth of 45 million reads/sample  and 35 million reads/sample . The sequencer outputs were processed using the computer cluster managed by the Minnesota Supercomputing Institute (MSI).65 followed by removal of PCR duplicates using Samtools66. Peak calling was performed using MACS67 with the following parameters: \u2013bw 300 -p 1e-3. Similar results were obtained by performing peak calling using QESEQ68 with the following parameters: transcription factors/cofactors -s 100 -c 15 -p 0.01; histones marks -s 100 -c 20 -p 0.001 (replicate 1) -s 100 -c 15 -p 0.001 (replicate 2). To identify the list of high confidence PAX3 and LDB1 peaks we performed three independent ChIP-seq experiments and, using the MACS output and the bedtools intersect function, only common regions between two experiments were further considered69. The Myf5 \u2212111\u2009kb region was detected in only one of three biological replicates generated for 1-day PAX3 and 1-day LDB1 ChIP. However, upon validation by qPCR 71. Functional annotation of ChIP-seq clusters was performed using GREAT (http://bejerano.stanford.edu/great/public/html/)41.Each sample\u2019s reads were aligned to the mouse genome (mm10) using Bowtie2https://enhancer.lbl.gov/)27. Identification of the Pax3 peaks overlapping to Vista enhancers was performed using bedtools intersect. Resulting bed files were loaded on IGV for visualization.Genomic coordinates and embryo pictures for the enhancers annotated to Somite, Limb, and Neural tube were downloaded from the Vista browser , 6-day\u2009+\u2009shSCR and 6-day\u2009+\u2009shLdb1 and non-induced 1-day \u0394TAD-Ldb1-induced cells were harvested using trypsin for 1\u20132\u2009min, followed by inactivation with PBS/10% FBS, centrifuged for 5\u2009min at 400\u2009\u00d7\u2009g, washed with PBS twice, and then counted. Thirteen million cells, diluted to 1\u2009million/ml in PBS, were crosslinked for 15\u2009min using methanol-free formaldehyde (Pierce\u2014Thermoscientific). In situ contact generation was performed by digesting chromatin with MboI (NEB) followed by blunting with dNTP mix containing biotin-dATP  and ligation using T4 DNA ligase (NEB). After centrifugation, nuclei were resuspended in 880\u2009\u03bcL in Nuclear Lysis Buffer , transferred in a Millitube and sonicated using a Covaris S220 . Chromatin was cleared by centrifugation, diluted 1:2 in ChIP Dilution Buffer , precleared for 4\u2009h at +4\u2009\u00b0C with 75\u2009\u00b5l of Protein G-conjugated magnetic beads and then incubated with 30\u2009\u00b5g of anti-Pax3 antibody overnight at +4\u2009\u00b0C. For \u0394TAD-Ldb1 HiChIP, chromatin was precleared with Protein A beads and then incubated with 15\u2009\u00b5g of anti-Ldb1 antibody (Abcam) overnight at +4\u2009\u00b0C. Immune complexes were recovered by incubation with 60\u2009\u00b5l of BSA-blocked Protein G (or Protein A for Ldb1 HiChIP)-conjugated sepharose beads for 5\u2009h at +4\u2009\u00b0C, then washed five times with RIPA wash buffer  and one time with TEN buffer . Due to the large sample volume during Pax3/Ldb1 HiChIP, Protein-A/G:Antibody-chromatin bound beads were splitted in two 1.5\u2009ml tubes during the previous procedure. Immunoprecipitated chromatin was recovered by incubating beads with 200\u2009\u00b5l/tube of Elution buffer  for 30\u2009min at 65\u2009\u00b0C. ChIP samples and Input  were reverse crosslinked overnight at 65\u2009\u00b0C, then diluted 1:1 with TE  supplemented with 4\u2009\u00b5l of RNaseA 20\u2009mg/ml and incubated for 2\u2009h at 37\u2009\u00b0C followed by Proteinase K treatment (4\u2009\u00b5l of 20\u2009mg/ml stock for each sample) for 30\u2009min at 55\u2009\u00b0C. DNA was purified by phenol\u2013chloroform\u2013isoamyl alcol extraction (twice) followed by chloroform extraction, then supplemented with 1/10 of volume of 3\u2009M Sodium Acetate pH 5 and 1.5\u2009\u00b5l of glycogen and precipitated with two volumes of 100% ethanol at \u221280\u2009\u00b0C for >1\u2009h. Following 30\u2009min centrifuge at 16,000\u2009\u00d7\u2009g, pellets were washed with 75% ethanol, air dried and the combined DNA pellet were dissolved in 40\u2009\u00b5l H2O. Before proceeding with the biotin capture and transposase mediated library generation, ChIP samples were analyzed by qPCR to ensure enrichment of Pax3 sites  vs. control region (Myf5 \u2212257\u2009kb). Samples were quantified using Pico-green (Invitrogen), resuspended in binding buffer and then incubated with 20\u2009\u00b5l of Streptavidin T1-conjugated magnetic beads (Invitrogen). After washing, DNA-bound streptavidin beads were incubated with 1.5\u2009\u00b5l of Tn5 transposase in a final volume of 50\u2009\u00b5l for 10\u2009min at 55\u2009\u00b0C. This amount of transposase was selected based on the picogreen results of the ChIP, which yielded 25\u2009ng of DNA. Following quenching and washing, transposed DNA was incubated 5\u2009min at 65\u2009\u00b0C with activated NEBNext UltraII QS Taq (+Illumina adaptor/barcodes) to allow primer extension and then amplified for five cycles (15\u2009s 98\u2009\u00b0C followed by 1.5\u2009min at 65\u2009\u00b0C). Additional cycles for library amplification were determined by qPCR using T5-T7 primers, then libraries were purified with 1.5\u00d7 AMPure beads and eluted in 50\u2009\u00b5l of H2O. After Quality Control, libraries were pooled and sequenced on a Paired-End run of the HiSeq2500 operated at High Output mode (Illumina) which yielded ~80 million reads for each of the two biological replicates.Long-range interactions involving Pax3 binding sites were analyzed following the detailed protocol described by the Chang group72. Contact matrices were generated for 10-kb bin pairs using default parameters in HiC-Pro to map the reads to the mm10 genome, remove duplicate reads, assign reads to Mbol restriction fragments, and acquire valid interactions. Statistically significant bin pairs and number of contacts were determined using the Fit-Hi-C contact caller using default settings and interaction distance <2\u2009Mb73.HiChIP paired-end samples were processed using the HiC-Pro pipeline74.Raw contact matrices from the HiC-Pro pipeline were normalized by read depth. The resulting normalized matrices were used to generate heat maps. The matrix acquired for the condition with no addition of doxycycline (non-induced) was subtracted from the matrices for 1-day and 6-day in doxycycline conditions. This enhanced the visualization of the heat maps for contacts specific to the manipulated condition. Similarly, the matrix for the Ldb1 knockdown sample was subtracted from the shSCR matrix to emphasize loss of contacts with the knockdown of Ldb1. R Bioconductor package \u201cHiTC\u201d was used to assist the generation of the contact heat maps75. Initially, peaks were called from the aligned reads generated by the HiC-Pro pipeline. Using these called peaks along with the default parameters of the FitHiChIP algorithm, significant interactions were collected with a stringency q-value cutoff of 0.01. The interactions from the control conditions were subtracted (bedtools intersect \u2013v \u2013f 0.9 \u2013F 0.9) to filter for interactions specific only to the condition of interest. The resulting interaction coordinates for both anchors of each loop were overlapped with ChIP-seq peaks to quantitatively compare the HiChIP results with ChIP-seq analysis. Visualizations of these significant loops were viewed on WashU Epigenome Browser76.FitHiChIP was used for the determination of statistically significant interactionsg), cell pellets were washed with ice cold PBS and incubated 30\u2009min in 10 packed cell volumes (PCV) of ice cold PBS supplemented with 5\u2009\u00b5g/ml Digitonin (Sigma-Aldrich). Cells were centrifuged 5\u2009min at 500\u2009\u00d7\u2009g, gently resuspended in five PCV of ice cold Hypotonic buffer , and phosphatase (PhoStop\u2014Roche) inhibitors), incubated 10\u2009min on ice and vortexed for 10\u2009s. After centrifuge (10\u2009min at 500\u2009\u00d7\u2009g), the previous step was repeated once and the nuclei preparation was checked on a microscope using Trypan blue. Nuclei were then gently resuspended in one PCV of ice cold low salt buffer  and then supplemented drop-by-drop with one PCV of ice cold High Salt Buffer  and 4\u2009\u00b5l of Benzonase (Sigma-Aldrich). After 30\u2009min incubation on ice, sample were centrifuged 30\u2009min at +4\u2009\u00b0C 16,000\u2009\u00d7\u2009g and the supernatant was transferred into 3\u2009ml Slide-A-Lyzer\u00ae G2 Dialysis Cassette\u2014cutoff 7000 MWCO (Thermoscientific) and dialyzed overnight at +4\u2009\u00b0C with gentle stirring using Dialysis buffer . Nuclear extracts were recovered, centrifuged 30\u2009min at +4\u2009\u00b0C 16,000\u2009\u00d7\u2009g to remove precipitated proteins and precleared 2\u2009h with 300\u2009\u00b5l of Agarose IgG beads. This method yielded ~10\u2009mg of nuclear extract, which was then incubated with 200\u2009\u00b5l of anti-FLAG M2 magnetic beads (Sigma-Aldrich) for 4\u20135\u2009h at +4\u2009\u00b0C with gentle rotation. Aliquots were collected before (Input), after (FT) FLAG IP for quality control. Beads were washed five times with 10 volumes of Wash buffer_1  and then incubated for 1\u2009h with 150\u2009\u00b5l of 0.2\u2009mg/ml 3xFLAG peptide diluted in Wash buffer_1. This step was repeated four more times and, ultimately, all elutions were pulled and incubated with 100\u2009\u00b5l of BSA-blocked anti-HA magnetic beads (Pierce\u2014Thermoscientific) for 5\u20136\u2009h at +4\u2009\u00b0C with gentle rotation. Aliquots were collected before (termed IP \u03b1FLAG) and after (FT 2nd IP) HA immunoprecipitation for quality control. Beads were washed five times with 10 volumes of Wash buffer_2  and then incubated for 1\u2009h with 100\u2009\u00b5l of 0.2\u2009mg/ml HA peptide diluted in Wash buffer_1. This step was repeated three more times with 50\u2009\u00b5l of 0.2\u2009mg/ml HA peptide diluted in Wash buffer_1 and, ultimately, all elutions were pulled. An aliquot of the final elution (IP \u03b1HA) was collected for quality control. Aliquots from the immunoprecipitation were analyzed by western blot using a monoclonal Pax3 antibody (DHSB), and silver staining using a 4\u201315% gradient polyacrylamide gel (Biorad). Mass spectrometry analysis was performed at the Taplin Mass Spectrometry facility . Two biological replicates were submitted as protein pellets following trichloroacetic acid (TCA) precipitation. However, due to the lower yield from the control sample (IP from untagged Pax3 line), these two replicates were combined before mass spectrometry analysis to ensure the detection of non-specific interactors.Proteins were extracted from cultured cells using RIPA buffer  supplemented of Protease inhibitors (Complete\u2014Roche) and quantified with Bradford reagent (Sigma-Aldrich). Protein samples were prepared in Laemmli buffer and loaded on gels for SDS-PAGE. Proteins were transferred on PVDF membranes (Millipore) for the detection with the indicated antibodies. The list of antibodies used in this study and relative dilutions are provided in Supplementary Table\u00a0Pellets were resuspended with ammonium bicarbonates and reduced by adding DTT at a 1\u2009mM concentration (in 50\u2009mM ammonium bicarbonate) for 30\u2009min at 60\u2009\u00b0C, then cooled to room temperature and supplemented with iodoacetamide (stock in 50\u2009mM ammonium bicarbonate) to a concentration of 5\u2009mM followed by incubation for 15\u2009min in the dark at room temperature. DTT was then added to a 5\u2009mM concentration to quench the reaction. Digestion was performed by adding sequence grade trypsin at a concentration of 5\u2009ng/\u00b5l and overnight incubation at 37\u2009\u00b0C. Samples were then desalted by an in-house made desalting column.77. After equilibrating the column each sample was loaded via a Famos auto sampler  onto the column. A gradient was formed and peptides were eluted with increasing concentrations of solvent B . As peptides eluted, they were subjected to electrospray ionization and then entered into an LTQ Orbitrap Velos Pro ion-trap mass spectrometer . Peptides were detected, isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions for each peptide.On the day of analysis, samples were reconstituted in 5\u201310\u2009\u00b5l of HPLC solvent A . A nano-scale reverse-phase HPLC capillary column was created by packing 2.6\u2009\u00b5m C18 spherical silica beads into a fused silica capillary (100\u2009\u00b5m inner diameter\u2009\u00d7\u2009~30\u2009cm length) with a flame-drawn tip78. All databases include a reversed version of all the sequences and the data was filtered to between 1\u20132% peptide false discovery rate (FDR). Search settings: peptide_mass_tolerance\u2009=\u20092.0; digest_mass_range\u2009=\u2009600.0000 35000.0000; max_num_internal_cleavage_sites\u2009=\u20092; diff_search_options\u2009=\u200915.9949146221\u2009M 14.0157\u2009C; add_C_Cysteine\u2009=\u200957.0214637236. The list of proteins detected by mass spectrometry (available in Supplementary Data\u00a0www.expasy.org), which enabled the identification of protein complexes associated to Pax3. List of proteins used for STRING analysis and the results are available in Supplementary Data\u00a0Peptide sequences (and hence protein identity) were determined by matching protein databases (UniProt) with the acquired fragmentation pattern by the software program, Sequest Samples were resuspended in Trizol (Invitrogen) prior to RNA isolation using the PureLinkTM RNA Mini kit (Invitrogen) following the manufacturer\u2019s instructions for \u201cTrizol samples\u201d (including in-column DNase treatment). RNAs were retro-transcribed using Superscript Vilo (Invitrogen). Gene expression analyses were performed using an amount of cDNA corresponding to 12.5\u2009ng of starting RNA for each reaction. qRT-PCR were performed using Premix Ex Taq (Probe qPCR) Master Mix (Takara) and TaqMan probes (Applied Biosystems).20. For heatmap in Supplementary Fig.\u00a0t test analysis  and read count >10 and fold induction/repression >2. Heatmap was generated using the R package pheatmap.The 1-day/6-day Pax3 induction transcriptomic analysis was previously describedImmunofluorescence staining was performed by fixing cells with 4% Paraformaldehyde/PBS for 10\u2009min at 4\u2009\u00b0C, then permeabilized with 0.1% Triton/PBS and blocked with 5% BSA/PBS before incubating with the primary antibodies. The list of antibodies used in this study and relative dilutions are provided in Supplementary Table\u00a079. Analysis of myogenic progenitor migration and differentiation in E10.5 and E11.5 forelimbs was performed as follow: color channels were separated and threshold level for the green and blue channels were adjusted in order to select the area positive respectively to PAX3 or MYHC (green) and DAPI (blue). The area positive for each channel was analyzed using Analyze Particle using 0-Infinity as Size parameter. Finally, the value representing PAX3+ and MYHC+ area for each image was normalized based on nuclear staining (DAPI+). After calculating mean, all values were reported as % relative to mean for graphical display. Data represent mean\u2009\u00b1\u2009s.e.m. of two representative pictures from three independent experiments. Statistical analyses between control and treated group in Fig.\u00a0t-test. P values\u2009<\u20090.05 were considered to be statistically significant.Analyses were performed using the ImageJ distribution FijiFurther information on research design is available in the\u00a0Supplementary informationDescription of Additional Supplementary FilesPeer Review FileSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Reporting SummarySource Data"}
+{"text": "Alphaproteobacteria-infected hepatopancreatic tissues of the crab Eurypanopeus depressus. The circular virus genome  encodes 14 hypothetical proteins, some similar to other bacteriophages (Microviridae). Based on its relatedness to other Microviridae, this virus represents a member of a novel genus.A single-stranded DNA (ssDNA) virus is presented from a metagenomic data set derived from  Alphaproteobacteria-infected hepatopancreatic tissues of the crab Eurypanopeus depressus. The circular virus genome  encodes 14 hypothetical proteins, some similar to other bacteriophages (Microviridae). Based on its relatedness to other Microviridae, this virus represents a member of a novel genus.A single-stranded DNA (ssDNA) virus is presented from a metagenomic data set derived from  Microviridae is a viral family with two subfamilies and 6 genera , a panopeid crab from meso- and euryhaline locations across the Gulf of Mexico and Atlantic North America. The specimen was collected from a euryhaline site in North Carolina  in December 2018. A total of 1\u2009\u03bcg of DNA was used to prepare a NEBNext Ultra DNA library for Illumina HiSeq (10\u00d7) sequencing  with a PE150 cartridge. This resulted in 11 million reads (50 to 150 bp) that were assembled using SPAdes v.3.13.0  . The genome  of a Microviridae sp. was identified based on high coverage , with a GC content of 33% and 14 hypothetical open reading frames (ORFs)  on homogenized hepatopancreatic tissues of and 127) . This res (ORFs) . The gen0 on homona HiSeq \u00d7 sequenc insects .Microviridae (Ciona robusta (Tunicata) (marine) .Of the 14 hypothetical ORFs, 5 showed similarity to other proteins in GenBank . The prooviridae . One viroviridae , and two(marine) . Two gen(marine) . Seven Ooviridae . Based oAnaplasmataceae which parasitizes the host hepatopancreas, identified via histology, electron microscopy, and genomics (our unpublished data). It may constitute a useful model system for understanding the effect of phage therapy relative to an intracellular bacterium causing disease in crustaceans.To conclude, we present the genome of a bacteriophage likely to infect an undescribed member of the MN335165, BioProject number PRJNA574411, and BioSample number SAMN12567204.The complete genome, annotation, and associated forward and reverse reads for this novel virus can be found under accession number"}
+{"text": "These meso-trifluoromethyl derivatives of \u03b2-octaalkylporphyrins underwent smooth metalation, similar to other common porphyrins, however, the corresponding zinc complexes underwent a type of solvolysis, whereby the trifluoromethyl groups were converted into methoxycarbonyl groups by the methanol used as solvent. UV-visible absorption spectra and X-ray crystal structure analyses revealed that the presence of a methoxycarbonyl substituent did not influence the deformation of the molecular framework and its absorption properties; this is because the methoxycarbonyl has a planar and perpendicular geometry, as opposed to the relatively bulky trifluoromethyl substituent.5,10-Bistrifluoromethyl substituted \u03b2-octamethylporphyrins were synthesized via a scrambling side reaction of a dipyrromethane precursor in the presence of a large excess of trifluoroacetic acid. Compared with the  A solution of 7 and large excess of zinc acetate in CHCl3 and MeOH (3:1 v/v) zinc acetated was stirred at room temperature until 7 was completely consumed. The resulting solution was washed with water twice and dried over anhydrous sodium sulfate. After removal of the solvent, 7Zn was obtained almost quantitatively. Recrystalization from CHCl3\u2013MeOH gave an analytical sample of 7Zn as purple solids 1H-NMR (CDCl3): \u03b4 = 1.70 , 1.80 , 1.82\u20131.87 , 3.89\u20133.96 , 4.00 , 4.05\u20134.10 , 9.83  and 9.92  ppm; 19F-NMR (CDCl3): \u03b4 T = \u221231.77 (CF3) ppm; UV-vis (CH2Cl2) : \u03bbmax (\u03b5 [M\u22121cm\u22121]): 416 , 550 , 567 , and 611  nm; HR-ESI-TOF-Mass (positive-mode) (%intensity): C37H43F2N4Zn ([M \u2212 F]+), calcd: 645.2742, found: 645.2733 (100%); Elemental analysis calcd for C37H43F3N4Zn: C 66.70, H 6.77, N 8.69; found: C 66.71, H 6.51, N 8.41.5,10-Bis(trifluoromethyl)-2,3,7,8,12,13,17,18-octamethylporphyrin zinc complex (9Zn). A solution of 9 and large excess of zinc acetate in CHCl3 and MeOH (3:1 v/v) zinc acetated was stirred at room temperature until 9 was completely consumed. The resulting solution was washed with water twice and dried over anhydrous sodium sulfate. After removal of the solvent, 9Zn was obtained almost quantitatively. Recrystalization from CHCl3-MeOH gave an analytical sample of 9Zn as purple solids 1H-NMR (CDCl3): \u03b4 = 3.31 , 3.33 , 3.40 , 3.46 , and 9.67  ppm; 19F-NMR (CDCl3): \u03b4 T = \u221235.12 (CF3) ppm; UV-vis (CH2Cl2): \u03bbmax (\u03b5 [M\u22121cm\u22121]): 360 , 429 , 578 (9300), and 623  nm; HR-ESI-TOF-Mass (positive-mode) (%intensity): C30H26F6N4Zn ([M]+), calcd: 620.1348, found: 620.1350 (100%); Elemental analysis calcd for C30H26F6N4Zn: C 57.94, H 4.21, N 9.01; found: C 57.73, H 4.32, N 8.89.5-Methoxycarbonyl-2,3,7,8,12,13,17,18-octaethylporphyrin zinc complex (11Zn). A solution of 7Zn (10.0 mg) in CHCl3 (5 mL) and MeOH (5 mL) was refulxed for 1 day. After evaporated to dryness, recrystalization from CHCl3/MeOH afforded 11Zn as purple solids . 1H-NMR (CDCl3): \u03b4 = 1.71 , 1.91\u20131.96 , 3.80\u20133.84 , 4.08\u20134.15 , 4.53  10.13 , and 10.21  ppm; UV-vis (CH2Cl2): \u03bbmax (\u03b5 [M\u22121cm\u22121]): 338 , 406 , 537 , and 574  nm; HR-ESI-TOF-Mass (positive-mode) (%intensity): C38H46N4O4Zn ([M]+), calcd: 654.2907, found: 654.2911 (100%); Elemental analysis calcd for C38H46N4O2Zn: C 69.56, H 7.07, N 8.54; found: C 69.72, H 6.73, N 8.60.5,10-Bis(methoxycarbonyl)-2,3,7,8,12,13,17,18-octamethylporphyrin zinc complex (12Zn). A solution of 9Zn (10.9 mg) in CHCl3 (2 mL), MeOH (10 mL), and H2O (0.5 mL) was refulxed for 1 day. After evaporated to dryness, recrystalization from CHCl3/MeOH afforded 12Zn as purple solids . 1H-NMR (CDCl3): \u03b4 = 3.33 , 3.35 , 3.41 , 3.54 , 4.51  and 9.74 ppm; UV-vis (CH2Cl2): \u03bbmax (\u03b5 [M\u22121cm\u22121]): 345 , 409 , 541 , and 577  nm. Elemental analysis calcd for C32H32N4O4Zn: C 63.84, H 5.36, N 9.31; found: C 63.21, H 5.17, N 9.14.3 substituted \u03b2-octamethylporphyrins in a cis-arrangement and the metal-dependent solvolysis of meso-CF3 functionalities. The synthesis of such a cis-configured porphyrin is usually challenging because more complicated oligo-pyrrolic precursors containing CF3 substituent may be required. Compared with its trans-analogue 5, the cis-configured 9 showed more red-shifted absorption bands, suggesting that a cis-arrangement worked more effectively than a trans-arrangement for extension of the absorption range meso-positions, and even fewer examples with \u03b2-octaalkylporphyrins [3 into CO2Me including the role of the metal is still unclear (We have demonstrated the formation of bis-CFrphyrins ,20,21. A unclear , our syn"}
+{"text": "An electroencephalography (EEG) coherence network is a representation of functional brain connectivity, and is constructed by calculating the coherence between pairs of electrode signals as a function of frequency. Typical visualizations of coherence networks use a matrix representation with rows and columns representing electrodes and cells representing coherences between electrode signals, or a 2D node-link diagram with vertices representing electrodes and edges representing coherences. However, such representations do not allow an easy embedding of spatial information or they suffer from visual clutter, especially for multichannel EEG coherence networks. In this paper, a new method for data-driven visualization of multichannel EEG coherence networks is proposed to avoid the drawbacks of conventional methods. This method partitions electrodes into dense groups of spatially connected regions. It not only preserves spatial relationships between regions, but also allows an analysis of the functional connectivity within and between brain regions, which could be used to explore the relationship between functional connectivity and underlying brain structures. As an example application, the method is applied to the analysis of multichannel EEG coherence networks obtained from older and younger adults who perform a cognitive task. The proposed method can serve as a preprocessing step before a more detailed analysis of EEG coherence networks. EEG records the electrical activity of the brain by attaching electrodes to the scalp of a subject at multiple locations. Synchronous electrical activity in brain regions is generally assumed to imply functional integration. Such synchronization occurs over a large range of scales. For example, local synchronization occurs during visual processing, synchronization between neighboring temporal and parietal cortical regions is observed during multimodal semantic processing, and long-range fronto-parietal interactions occur in working memory retention and mental imagery . A largeVisualization provides a visual representation of the data to help people carry out analysis tasks effectively. It happens at an early stage in the process, usually before a statistical or computational analysis, and it allows people to explore their data before knowing exactly what kind of questions to ask . After tAn EEG coherence network represents functional brain connectivity, more precisely, the coherences between pairs of signals recorded by the electrodes. However, visualization of high-density or multichannel EEG (at least 64 electrodes) coherence networks is not always managed well . TypicalTo study connectivity patterns in the coherence graph, researchers often employ a hypothesis-driven or semi-data-driven definition of certain regions of interest (ROIs), in which all electrodes are assumed to record similar signals because of volume conduction effects . Howeverfunctional units (FUs). An FU is represented in the coherence graph by a spatially connected maximal clique (a clique is a vertex set in which every two-element subset is connected by an edge). Because larger ROIs are assumed to correspond to stronger source signals, larger FUs are considered to be more interesting. Therefore, the maximal clique based (MCB) method of maximal cliques, for which vertex sets are as large as possible. Since the MCB method is very time-consuming (time complexity O(3n/3), with n the number of vertices), as an alternative a watershed based (WB) method was proposed that detects spatially connected cliques in a greedy way (O(n2 logn)) was proposed that merges FUs if they are spatially connected and if their union is a clique  with identical gray value, with different gray values for adjacent FUs  map, in exactly the same way as for the FUMCB and FUIWB maps.A drawback of the MCB and IWB methods is that the analysis of local synchronization is difficult, since these methods detect maximal cliques, that is, groups of spatially-connected electrodes that are as large as possible. Specifically, the MCB method views coherences above or equal to the pre-defined threshold equally while the IWB method clusters electrodes based on their neighbours which have the largest coherence value with them, without considering how strong the electrodes are connected with the other members of the group. Therefore, in this paper we propose an alternative method, based on detecting densely interconnected groups of brain regions known as network networks . The comA preliminary version of this paper appeared in Overall, the new community-based method for detecting functional units is not only expected to reduce the drawbacks of the conventional hypothesis-based approaches, but also to allow a more detailed analysis of the relationship between functional connectivity and underlying brain structure than the data-driven MCB and IWB methods.The principal concept in our approach is to visualize brain connectivity, and to extract meaningful information from this representation for further analysis. The challenge in visualization often lies in the analysis of a huge amount of data, in our case the large number of EEG channels.A straightforward method would be to visualize functional brain connectivity data as 3D node-link diagrams: ROIs are shown as nodes and the relationships between these nodes are encoded in the edges. But this approach suffers from visual clutter, and side effects of 3D rendering such as occlusion are hard to remedy .An alternative approach is to depict the connectivity data by a 2D representation, which could reduce the work of 3D rendering. A wide variety of methods has been developed to map data on 2D space to visualize neuronal interactions or relations between brain regions. To preserve the spatial information of the data to some extent, a node-link diagram based on a biologically meaningful layout has been used . In thisSome methods were proposed to remedy the visual clutter by eliminating overlaps and reducing the number of long-distance edges employing graph drawing. For example, for 2D node-link diagrams the layout can be calculated by multidimensional scaling or force-directed algorithms . HoweverSome studies explored the capabilities of node-link diagrams and matrix representations, and these suggested that the matrix representations outperform node-link diagrams for most tasks when the number of nodes becomes very large .Based on the basic node-link and matrix representations, several other useful tools have been developed for visualizing brain networks. To take advantage of both node-link diagrams and matrix representations, some visualization methods provide a dual representation to explore networks . ChristoAlthough there are many unsupervised graph clustering methods, they either aim to find a predetermined number of clusters or do not consider the spatial information of the data . ten CaaIn this section, we first provide some background on EEG coherence and the data representation of the EEG coherence network. Then we describe the community clique-based method of detecting dense spatially-connected groups of electrodes. Then, we briefly describe the MCB and IWB methods for later comparison in the \u201cDuring an EEG experiment, brain activity is recorded by electrodes attached to the scalp of a subject at different locations. The electrodes are placed based on the 10/20 system and the position of an electrode is indicated by its label which is a combination of letters indicating brain regions , and digits indicating lateralization  and distance from the midline (higher numbers are farther away) see Fig.\u00a0. A conduActivity from one source can result in a strong signal recorded by multiple electrodes, and nearby electrodes usually record similar signals due to volume conduction and reference electrode effects . Often, c\u03bb as a function of frequency \u03bb for two continuous time signals x and y is defined as the absolute square of the cross-spectrum fxy normalized by the autospectra fxx and fyy , defined by a set of vertices V and a set of edges E\u2286V\u00d7V where vertices represent electrodes. Since weak coherences may represent spurious connections and these connections tend to obscure the topology of strong and significant connections  represents the weight  of the edge between nodes v and v\u2032, v, L(v) is the community label of vertex v, the \u03b4\u2212function \u03b4 is 1 if i=j and 0 otherwise, and Q is equal to the fraction of the sum of weights of edges that connect nodes in the same community minus what that fraction would be on average if the communities remained fixed but the edge weights were randomly distributed. The higher the value of Q, the more confident one can be that a significant community structure has been found  and placing it in one of the other communities. The node v is then placed in the community for which this gain is maximum and positive. If no positive gain is possible, nothing is done. This process is applied repeatedly and sequentially for all nodes until no further improvement can be achieved and the first phase is then complete.The optimal community structure for a given network is typically estimated with optimization algorithms rather than computed exactly ). A simp\u0394Q when removing one node v from its community CL(v) to an arbitrary community Ci (Equation unity Ci : 2\\docuCL(v), Ci, and Kv is the sum of the weights of the links incident to node v.where The second phase of the algorithm consists of building a new network whose nodes are the communities found in the first phase. To do so, the weights of the links between the new nodes are given by the sum of the weights of the links between nodes in the corresponding two communities. Links between nodes of the same community lead to self-loops for this community in the new network. Once this second phase is completed, the first phase of the algorithm is reapplied to the new network. The combination of both phases is called a \u201cpass\u201d. The passes are iterated until there are no more changes.Here, we extend the method proposed by Assign a unique community to each node of the network.\u0394Q for node v caused by removing node v from its community and placing it in another community such that the node v is connected to each node of that community and has at least one Voronoi neighbour in that community.Use v in the community for which the gain is the highest and positive. If no positive gain is available, nothing is done.Place the node Continue repeating steps (2) and (3) until every node is processed.Q is achieved.Repeat steps (2) -(4) until no further improvement of the modularity index The outline of our method can be summarized as follows. The difference with Blondel\u2019s method is in step 2, the calculation of the modularity gain, where an extra condition is applied which ensures that the resulting communities are spatially connected cliques (see the introduction for the motivation): Note that the algorithm\u2019s output depends on the order in which the nodes are processed in step (2). The ordering does not have a significant influence on the modularity that is obtained, but can influence the computation time . In our Ci contains a sorted list of the vertices in community i;L(v) is the community label of vertex v;Hi contains a list of vertices (sorted by vertex number) that are connected to each of the vertices in Ci;Ri contains a list of vertices which have at least one Voronoi neighbor in Ci.Algorithm 1 shows the pseudocode of the community clique detection procedure. It maintains the following dynamic vertex sets in the coherence graph of significant electrode connections: remove) removes the node v from the community CL(v) and returns a set consisting of the remaining nodes. Similarly, add inserts node v into the community Ci and returns the updated community. The operation isVor(Ci) returns \u201ctrue\u201d if the community Ci is empty, or if it only has one vertex, or if each pair of vertices in Ci is Voronoi-connected when |Ci|>1; and it returns \u201cfalse\u201d if not. The size of a vertex set is denoted by |\u00b7|.The operation Initialization (lines 1-7).-Initially, every node of the network is a singleton community (line 3).-Ci is identical to the set of vertices which are connected to the node v=V(i) (line 5)The set of vertices which are connected to vertices of community -Ci is the set of vertices that are connected Voronoi-neighbours of the node v=V(i) (line 6).The set of vertices which are connected Voronoi-neighbours of vertices of community -flag as true (line 8). flag is used to indicate that the modularity can be improved. If flag is false, there is no improvement of modularity.Set Main Procedure (line 8-line 37). This consists of the following steps. -ith node v=V(i) from V. If after removing v from its original community CL(v) any pair of remaining vertices is not Voronoi-connected, nothing is done, and the procedure continues with a new node (line 13). Otherwise, set the maximal gain of modularity max\u0394Q to zero, and take the jth community Cj.Take the -In case Cj is empty or v is not connected to any nodes of Cj or v has no Voronoi neighbors in Cj, nothing is done, and the procedure continues with a new community (line 16). Otherwise, compute the modularity gain \u0394Q (line 17).-\u0394Q is higher than max\u0394Q, which means the modularity can be improved, set the label of the current community j to the destination community label d to which community the node v will move (line 21). Otherwise, nothing is done, and the procedure continues with a new community.If the current gain -\u0394Q, and update community CL(v) by removing node v from its original community CL(v) (line 26); replace HL(v) by vertices that are connected to each node of the updated community CL(v) (line 27); replace RL(v) by the vertices that are connected Voronoi neighbours of nodes of the updated community CL(v) (line 28); move node v into the destination community Cd (line 29); replace HL(v) by its intersection with the set of vertices that are connected with v in the coherence graph (line 30); replace RL(v) by its union with the vertices that are connected Voronoi neighbours of v (line 31); v receives label d (line 32).After all communities are traversed, select the first community which has the highest -flag=false after all nodes have been processed.This procedure is repeated until no improvement is obtained, which means We now turn to a more precise analysis of the algorithm: Figure\u00a0T1, the initial stage, each of these twelve vertices correspond to a unique community represented by a specific colored symbol: L(a)=1, L(b)=2, L(c)=3, L(d)=4, L(e)=5, L(f)=6, L(g)=7, L(h)=8, L(i)=9, L(j)=10, L(k)=11, L(l)=12. Then, we calculate the modularity gain \u0394Q caused by removing k  from its community to the other communities; all the values of \u0394Q are listed on the right in Fig.\u00a0At T2-T12. At every next step, the next vertex v will be chosen and the gain of removing v from its community CL(v) to the remaining communities will be computed. If the positive highest gain max\u0394Q results from the movement of node v to the community in which v has at least one Voronoi neighbour and is connected with each vertex in that community, then the vertex v will be removed from its original community to the destination community CDes, CL(v) is updated by deleting v from CL(v) and CDes is replaced by the union of itself with v. At T2, v=h, CL(v)=C8=\u2205, CDes=C12={h,l}. At T3, v=a, CL(v)=C1=\u2205, CDes=C5={a,e}. At T4, v=f, CL(v)=C6=\u2205, CDes=C5={a,e,f}. At T5, v=e, nothing is done since max\u0394Q is negative when merging e and i into one community. At T6, v=b, CL(v)=C2=\u2205, CDes=C5={a,b,e,f}. At T7, v=l, nothing is done since all the communities except C12, the original community, have no connected Voronoi neighbours of l. At T8, v=d, CL(v)=C4=\u2205, CDes=C12={d,h,l}. At T9, v=g, CL(v)=C7=\u2205, CDes=C12={g,d,h,l}. At T10, v=i, CL(v)=C9=\u2205, CDes=C5={a,b,e,f,i}. At T11, v=j, CL(v)=C10=\u2205, CDes=C5={a,b,e,f,i,j}. At T12, v=c, and at T13, v=k, nothing is done since the vertex v has no connected Voronoi-neighbours.T14 on, all vertices will be traversed again. The gain \u0394Q can be easily computed and it can be observed that there is no more positive gain, which means the modularity can not be improved anymore. So the detection procedure stops. Finally, we obtain two community cliques {a,b,e,f,i,j} and {g,d,h,l} at T25.From compsub contains an increasing or decreasing clique.The set currentcand contains the candidates that are a Voronoi neighbor of at least one element in compsub, and only these can be added to compsub at the current step.The set complcand is the complement of currentcand in candidates containing vertices that are connected to all vertices in compsub.The set not contains vertices that are connected to all vertices in compsub and that were added to compsub previously.The set The maximal clique based (MCB) method  is an excurrentcand that has the largest number of connections with the other candidates (currentcand\u222acomplcand) is added to compsub. Let this element be v (in the coherence graph). The set newcurrentcand is the intersection of currentcand and the neighborhood of v (in the coherence graph), united with the Voronoi neighbors of v in complcand. The set newcomplcand is the intersection of complcand and the neighborhood of v (in the coherence graph), minus the Voronoi neighbors of v in complcand. The set (new)not is the intersection of not and the neighborhood of v. This is repeated until newcurrentcand is empty. If newnot is also empty, then compsub is a Voronoi-connected maximal clique.At each call, the element from Figure\u00a0A. starts with all twelve vertices in the set candidates (not illustrated), and with not=\u2205. A1. Then, the vertex g with the highest degree ; otherwise, they are in complcand . A2-A6. At every step, the element, say, v, from currentcand that has the largest number of connections in the coherence graph with the other candidates (currentcand\u222acomplcand) is added to compsub. In case of ties, one vertex is selected randomly. The spatial neighbours of v in complcand, denoted by \u039b(v), are moved from complcand to currentcand. Furthermore, vertices not adjacent to v in the coherence graph, denoted by \u0393c(v), are removed from both currentcand and complcand. This continues until currentcand is empty. At A2, v=f, \u039b(v)={b,e,j}, and \u0393c(v)={d,h,l}. At A3, v=e, \u039b(v)={a,i}, and \u0393c(v)=\u2205. At A4, v=a, \u039b(v)=\u2205, and \u0393c(v)=\u2205. At A5, v=b, \u039b(v)=\u2205, and \u0393c(v)=\u2205. At A6, v=i, \u039b(v)=\u2205, and \u0393c(v)=\u2205. At A7, v=j, \u039b(v)=\u2205, \u0393c(v)=\u2205, and compsub={a,b,e,f,i,j,g} is a Voronoi-connected maximal clique, because currentcand=\u2205 (and not=\u2205).The following detailed description contains (row and column) references to Fig.\u00a0B. A later iteration for the MCB method returns to the situation preceding A2, with vertex g in the compsub, and vertex f put into the set of not. Then, at B1. only the vertex h is retained in currentcand, and complcand is the same as in A1. . At B2, v=h, \u039b(v)={d,l}, \u0393c(v)={a,b,e,i,j}, and not=\u2205. At B3, v=d, \u039b(v)=\u2205, \u0393c(v)=\u2205, and not=\u2205. At B4, v=l, \u039b(v)=\u2205, \u0393c(v)=\u2205, not=\u2205, and compsub={g,d,h,l} is a Voronoi-connected maximal clique, because currentcand=\u2205 (and not=\u2205).g belongs to Voronoi-connected maximal cliques {a,b,e,f,i,j,g} and {g,d,h,l}). To assign a unique label to every vertex, a quantity total strength is defined for a (sub)graph G= as the sum of all edge values. For a vertex detected in more than one clique, it will be assigned to the clique which has the largest total strength. Then, the final cliques in above example are {a, b, e, f, i, j, g}and {d, h, l } (see C1).As it can be seen from the above example, every vertex can be part of multiple (Voronoi-connected) maximal cliques.  between markers, which are defined as nodes having locally maximal average coherence, and their Voronoi neighbors. These edges are sorted in a descending order based on their values. Each marker corresponds to a basin and is assigned a unique label.Main Procedure. Remove the first edge, e=, from the queue, and determine the label of node v\u2032. In case the node v\u2032 is unlabelled, v\u2032 receives the label of v and the queue is extended with the edges between v\u2032 and its unlabelled connected Voronoi-neighbours, if v\u2032 is connected to every node of the basin where v is in. In case v\u2032 was also labelled, check if the two basins that contain v and v\u2032 can merge into a single basin. If so, then merge them, otherwise nothing is done.This IWB method contains two main steps: queue is empty. Each basin then corresponds to an FUIWB.The main procedure is repeated until Figure\u00a0S1, three markers, a,f,h, are detected, and they are represented by a green diamond, red star, and magenta up-triangle, respectively. Each is then assigned a unique number: L(a)=1, L(f)=2, L(h)=3; edges (corresponding to significant coherences) between markers and their unlabelled Voronoi neighbours represented by blue squares are added in the queue; the edge with the highest value in the queue is c and is shown at the top on the right of S1.At S2-S11. At every next step, the edge, say, c, with the highest value in the queue is removed. Then, the vertex v\u2032 is labelled. The edges (denoted by \u03a6(v\u2032)) between v\u2032 and its unlabelled Voronoi-neighbours are inserted in the queue and will be highlighted in bold. At S2, v=h, v\u2032=l, L(v\u2032)=L(h)=3, and \u03a6(v\u2032)=\u2205. At S3, v=h, v\u2032=d, L(v\u2032)=L(h)=3, and \u03a6(v\u2032)=\u2205. At S4, v=f, v\u2032=g, L(v\u2032)=L(f)=2, and \u03a6(v\u2032)=\u2205. At S5, v=h, v\u2032=g, h which was labelled already in the previous stage. But, the basin of g and the basin of h can not merge since their union is not a clique in the coherence graph =L(a)=1, and \u03a6(v\u2032)=\u2205. At S7, v=a, v\u2032=e, L(v\u2032)=L(a)=1, and \u03a6(v\u2032)={}. At S8, v=f, v\u2032=b, b was labelled already in the previous stage. But the basins of f and b can merge since their union is a clique in the coherence graph. Hence, vertices in the basin of b will be moved to the basin of f. L(v\u2032)=L(f)=3, L(a)=L(f)=3, L(e)=L(f)=3, and \u03a6(v\u2032)=\u2205. At S9, v=f, v\u2032=e, L(v\u2032)=L(e)=3, and \u03a6(v\u2032)=\u2205. At S10, v=e, v\u2032=i, L(v\u2032)=L(e)=3, and \u03a6(v\u2032)=\u2205. At S11, v=i, v\u2032=j, L(v\u2032)=L(j)=3, and \u03a6(v\u2032)=\u2205. At S12, v=f, v\u2032=j, L(v\u2032)=L(j)=3, and \u03a6(v\u2032)=\u2205. Now, two basins have been detected, { a, b, e, f, i, j, g} and { d, h, l}, because the queue is empty.inter-FU coherence\u03bb between two FUs C1 and C2 is defined as the sum of coherence values between one vertex in C1 and the other vertex in C2, divided by the maximal number of edges between C1 and C2: Given FUs, the any pair of vertices are taken into account to normalize for the size of the FUs even if their coherence is below the predefined threshold.Note that coherences between C1 reflects its average coherenceAn FU map visualizes each FU as a set of Voronoi cells with identical gray values and with different gray values for adjacent FUs. See Fig.\u00a0In our case, only FUs larger than five cells are considered. White Voronoi cells are part of smaller FUs.In this subsection, we will first compare the proposed method with two other methods using a synthetic EEG coherence network see Fig.\u00a0. The comtotal strength when it is part of multiple cliques, no matter how strong the vertex is connected to these cliques. Hence, the vertex will be placed in the clique with more vertices when two cliques have an equal average coherence. For example, at the stage of maximal clique detection, we found two overlapping maximal cliques, {a,b,e,f,i,j,g} and {g,d,h,l}, in Fig.\u00a0g is removed from {g,d,h,l} at the assignment stage, and the final cliques are shown in C1.For the MCB method, at the stage of (Voronoi-connected) maximal clique detection, coherences are considered equally when their values are above or equal to the threshold. This method intends to find all possible maximal cliques in a coherence network. Then, at the stage of vertex assignment, the vertex is assigned to the clique which has the highest S3 in Fig.\u00a0g is assigned to f since their connection value is 0.71 while the connection value with h is 0.69. The final cliques are shown in S12.For the IWB method, the edges connecting a labelled vertex and an unlabelled vertex will be placed in the queue first if these vertices are significantly connected Voronoi neighbours. Then, the unlabelled vertex will be placed in the clique in which one of its Voronoi neighbours has the highest coherence with this vertex compared to others in the queue. For example, at T8 in Fig.\u00a0g has a stronger connection with clique {d,h,l} than with clique {a,b,e,f}. At T21, vertex g still has a stronger connection with clique {d,h,l} than with clique {a,b,e,f,i,j}. The final cliques are shown in T26.The community clique-based (CCB) method detects the dense spatially connected cliques in a coherence network. It first calculates the degree of connections between nodes and community cliques, which is quantified by modularity. Then, the node will be placed in the clique which has the strongest node-community connection. For example, at In this section, we will first describe the experimental setup, before applying the CCB method to twelve participants for an example application. To compare the three methods when applied to real EEG coherence networks, we selected four of these sixteen participants (two young and two old) to demonstrate any differences.L target tones were analyzed in L segments of 1 second, sampled at 256Hz. A procedure from Neurospec was adopted to compute the coherence (www.neurospec.org). A detailed description of the procedure is given in Brain responses were recorded during an auditory oddball detection experiment, in which older and younger participants were instructed to count target tones and ignore standard tones. After the experiment, each participant had to report the number of perceived target tones. In our data, brain responses to L repetitive stimuli, the EEG data can be separated into L segments. A significance threshold for the estimated coherence is then given by In the present study we do not consider ongoing EEG but the event-related potential (ERP) which is an EEG recording of the brain response to a sensory stimulus. To calculate the coherence for an ERP with p is a probability value associated with a confidence level \u03b1, such that p=1\u2212\u03b1.where p=0.01, and L=13 segments. In addition, we set the inter-FU coherence threshold to the same value as the significance threshold \u03b8.Throughout this section, we use For the young participants, it can be observed that there is no big difference between FU maps obtained from these three methods. In the Hz frequency band, FU maps are very similar for both young participants in terms of, for example, the number of FUs and their location. Similarly, for the Hz frequency band, there are no big differences between the methods either.3 both MCB and IWB methods detect two large FUs located anteriorly and posteriorly: FUsMCB1, 5 and FUsIWB1, 3. In the CCB method, however, these FUs are split into small FUs due to a weak inter-community connection. For example, FUMCB5 splits into FUsCCB6 and 7, while FUMCB1 splits into FUsCCB1 and 2. From these splits, we can see that FUsCCB6 and 7 have higher average coherence than FUMCB5, and the inter-FU coherence between FUsCCB6 and 7 is also lower than their average coherence. This is also true for FUMCB1 and FUsCCB1, 2. From a global point of view, FUCCB7 has the highest average coherence, followed by 1 and 2, and there are higher inter-FU coherences among these FUs. For participant 4, the MCB method detects two large FUs located anteriorly and posteriorly, with significant inter-FU coherence between them. The IWB method has a similar result, except for the frontocentral connection. The CCB method, in contrast, finds a total of seven FUs with size above five. Compared to the CCB method, FU 1 obtained by the MCB and IWB methods is split into four FUs 1, 2, 3, 4 in the CCB method due to the weak inter-community connections with each other. FUCCB1 in the CCB method has the highest average coherence among these four FUs. From a global point of view, the two FUsCCB having the strongest connection are 1 and 7, which are located at the frontal and parietal-occipital areas of the brain, respectively. In the Hz frequency band, the MCB and IWB methods produce similar results, except in the frontocentral area of the brain. The main difference between methods is that FUMCB1 splits into FUsCCB1 and 2 in the CCB method due to weak inter-community connections. In addition, the average coherence of FUCCB1 and 2 is higher than FUMCB1. FUMCB6 is an extension of FUCCB7, but it can be seen that their average coherence differs.For the older participants, however, there are large differences between FU maps for the different methods. In the Hz frequency band, the three methods result in a similar number of FUs. In the Hz frequency band for participant Besides comparing the three methods when applied to the synthetic EEG network in the \u201cIn this example, the FU maps obtained from older participants generally have larger FUs and the coherence within-and between FUs is also high compared to the young participants. This could be interpreted as the older participants having higher local and global synchronization. All the methods are trying to find cliques and for the young participants brain areas are apparently less synchronized, which results in lower coherence. In this case we end up with small(er) FUs. This also explains why the FU maps obtained from the three methods are similar for the younger participants. For the older participants, there is apparently high synchronization between brain areas. In this case, more electrodes make up larger cliques. The MCB and IWB methods then detect larger FUs. Consequently, the detected FUs are less suited for analyzing local synchronization. In this case, however, the CCB method still considers the community properties: if an electrode can be put into several FUs, it will be added to the FU with which it has a strong connection rather than to the largest FU as in the other methods.In this subsection, we apply (only) the CCB method to the data of six younger and six older participants in the experiment described in the \u201cCCB maps of the younger and older adults, respectively. In general, the color of circles and edges is lighter for older participants than for younger participants over the three frequency bands. This probably corresponds to earlier findings using a hypothesis-driven method  concept that attempts to preserve spatial relationships between functional brain regions and allows analysis of functional connectivity within and between regions.A new community clique based (CCB) method was proposed that first partitions an EEG coherence network into dense groups of spatially connected electrodes recording pairwise significantly coherent signals. The resulting communities (groups of electrodes) were visualized in an FU map, which makes it possible to investigate the relationship between functional brain connectivity and underlying brain structure. The novelty of this method is that it is helpful to analyze the local and global connectivity without any a priori hypotheses. Community cliques found by the CCB method can be used for further analysis, e.g., the analysis of ERP components across FUs and synchronization between FUs.groups, e.g., old and young participants. Our method is a visually aided pre-processing method that can be used before analysis questions about data are well defined. Although our method is specific to EEG coherence networks, we believe that it can be easily adapted to other network visualizations which need to capture the whole structure of networks and that do not only depend on the analysis of single nodes or specified connections between pairs of nodes.As topics for future work we first mention the influence of the order in which nodes are traversed in the CCB procedure, which needs to be further analyzed. Second, differences between FU maps were assessed only visually in our study. However, there is still the need to develop methods for comparing FU maps quantitatively, and to discriminate not only between single subjects, but also between different"}
+{"text": "Buchnera aphidicola and diverse facultative symbionts. The symbiotic associations for one aphid species, especially for polyphagous species, often differ across populations. In the present study, by using high-throughput 16S rRNA sequencing, we surveyed in detail the microbiota in natural populations of the cotton aphid Aphis gossypii in China and assessed differences in bacterial diversity with respect to host plant and geography. The microbial community of A. gossypii was dominated by a few heritable symbionts. Arsenophonus was the most dominant secondary symbiont, and Spiroplasma was detected for the first time. Statistical tests and ordination analyses showed that host plants rather than geography seemed to have shaped the associated symbiont composition. Special symbiont communities inhabited the Cucurbitaceae-feeding populations, which supported the ecological specialization of A. gossypii on cucurbits from the viewpoint of symbiotic bacteria. Correlation analysis suggested antagonistic interactions between Buchnera and coexisting secondary symbionts and more complicated interactions between different secondary symbionts. Our findings lend further support to an important role of the host plant in structuring symbiont communities of polyphagous aphids and will improve our understanding of the interactions among phytophagous insects, symbionts, and environments.Aphids live in symbiosis with a variety of bacteria, including the obligate symbiont The online version of this article (10.1007/s00248-019-01435-2) contains supplementary material, which is available to authorized users. Buchnera aphidicola, which inhabits specialized bacteriocytes and provides aphids with important nutrients for their growth and reproduction [Buchnera is strictly maternally inherited [Aphids are well known for their symbiotic associations with bacteria. Almost all aphid species harbor the primary endosymbiont oduction \u20134. Buchnnherited , 6 and hnherited \u201314.Arsenophonus, Fukatsuia symbiotica, Hamiltonella defensa, Regiella insecticola, Rickettsiella viridis, and Serratia symbiotica from the Gammaproteobacteria; Rickettsia and Wolbachia from the Alphaproteobacteria; and Spiroplasma from the Mollicutes [Buchnera in certain aphid species, such as Serratia symbiotica, Erwinia haradaeae, Fukatsuia symbiotica, Hamiltonella defensa, and Sodalis in some Lachninae species [Wolbachia in Pentalonia nigronervosa [Aphids also host multiple secondary (or facultative) bacterial symbionts that are generally not essential for their survival or reproduction. Some are commonly studied, such as llicutes \u201321. Thesllicutes  and are llicutes , 23, 24.llicutes \u201329, protllicutes \u201334 and fllicutes \u201337, inflllicutes \u201340, modillicutes , and affllicutes , 43. Morllicutes , Zytynskllicutes , and Guollicutes . In addi species \u201351 and Wonervosa , 53.Acyrthosiphon pisum, which consists of at least eleven biotypes adapted to specific host plants [Hamiltonella defensa and alfalfa and Regiella insecticola and clover. A nonrandom distribution of bacterial symbionts across host plants has also been reported in other polyphagous aphid species such as Aphis craccivora [Phylloxera notabilis [Aphis citricidus [Acyrthosiphon pisum in Japan, particularly for Regiella. Jones et\u00a0al. [Aphis gossypii and Pentalonia caladii varied across aphid populations from different Hawaiian islands. Some studies also indicated correlations between aphid symbionts and other factors, including developmental stage of aphids [The associations between microbial symbionts and aphids are quite different in different aphid species. Symbionts carried by one aphid species also often vary across populations. There seems to be a widespread pattern in polyphagous aphids that the populations feeding on different host plants differ in their symbiont communities \u201356. Mostt plants . Facultat plants , 58\u201361. accivora , 62 and otabilis  and Aphitricidus . Neverthtricidus  revealeds et\u00a0al.  found thf aphids , 68, reaf aphids , plant sf aphids , and seaf aphids .Aphis gossypii Glover, is a cosmopolitan insect pest causing serious economic losses in agriculture. It feeds on many important crops, including cotton, cucurbits, citrus, eggplant, peppers, potato, and flowering ornamental plants such as Hibiscus [Bt cotton in northern China. A. gossypii is currently controlled primarily by insecticides, which have been reported to influence the bacterial communities associated with aphids [A. gossypii.The cotton aphid, Hibiscus . SeveralHibiscus  and JoneHibiscus  investigHibiscus  also fouh aphids , 76. In h aphids  uncovereA. gossypii, samples used in previous studies were restricted to a few plants. A detailed and deep exploration of the microbiota in natural populations of A. gossypii is still lacking. In this study, using Illumina sequencing of 16S rRNA gene, we characterized the microbial communities of A. gossypii collected from diverse plants and different regions in China, assessed differences in bacterial community according to host plant and geography, and discussed the interactions between symbionts.Although geography has been proposed to have a role in structuring the bacterial communities of Aphis gossypii feeding on plants belonging to 25 families were collected from 23 regions of China  following the manufacturer\u2019s protocol. A blank sample of sterile ultrapure water was also processed through the same extraction protocol to serve as a negative control during the DNA extraction. The standard cytochrome oxidase subunit I (COI) barcodes were amplified by universal primers   to test DNA was amplified using the universal primers of the V3\u2013V4 region of 16S rRNA gene . The first polymerase chain reaction (PCR) was carried out in a 50-\u03bcL volume containing 1.5\u00a0\u03bcL (10\u00a0\u03bcM) of each primer, 0.4\u00a0U Q5 High-Fidelity DNA Polymerase , 10\u00a0\u03bcL 5\u00d7 Q5 Reaction Buffer (New England Biolabs), 10\u00a0\u03bcL 5\u00d7 Q5 High GC Enhancer (New England Biolabs), 1\u00a0\u03bcL dNTPs (New England Biolabs), and 40\u201360 ng DNA extract. The reaction conditions were as follows: initial denaturation at 95\u00a0\u00b0C for 5\u00a0min, followed by 15\u00a0cycles of 95\u00a0\u00b0C for 1\u00a0min, 50\u00a0\u00b0C for 1\u00a0min, 72\u00a0\u00b0C for 1\u00a0min, and final elongation at 72\u00a0\u00b0C for 7\u00a0min. The PCR products were purified using VAHTS\u2122 DNA Clean Beads . In the next step, 10\u00a0\u03bcL of the purified product was ligated to adapter and sample barcode in a 40-\u03bcL volume containing 1\u00a0\u03bcL (10\u00a0\u03bcM) of each fusion primer and 20\u00a0\u03bcL of 2\u00d7 Phusion High-Fidelity PCR Master Mix (New England Biolabs). The second PCR conditions were as follows: 98\u00a0\u00b0C for 30\u00a0s, 10\u00a0cycles of 98\u00a0\u00b0C for 10\u00a0s, 65\u00a0\u00b0C for 30\u00a0s, and 72\u00a0\u00b0C for 30\u00a0s, followed by a final extension at 72\u00a0\u00b0C for 5\u00a0min. Negative amplification controls (sterile ultrapure water) were also included in all PCR reactions. The final PCR products were recovered using 1.8% agarose gel electrophoresis, purified with VAHTS\u2122 DNA Clean Beads (Vazyme Biotech) and then quantified by NanoDrop 2000 . All positive PCR products were mixed at a mass ratio of 1:1. Finally, the library pool was submitted to an Illumina HiSeq 2500 platform  for paired-end sequencing. The raw reads have been deposited in the NCBI Sequence Read Archive (SRA) database under BioProject accession number PRJNA543947.Paired-end reads were assembled using FLASH v1.2.11 . The merdiversity function in the R package vegan [decostand function of vegan. To better investigate the symbiont and secondary symbiont communities, all OTUs assigned to the reported symbionts of aphids were screened out from the OTU table, and the relative abundance of each symbiont was calculated.Alpha diversity indices  for each sample were calculated using the ge vegan . The relA. gossypii were grouped according to geographic region and host plant ; therefore, we performed the non-parametric Kruskal\u2013Wallis test to check for significant differences across all groups and conducted the non-parametric Wilcoxon tests for pairwise group comparisons.All samples of nt Table . First, vegdist function in vegan. In the analyses of symbiont community, to reduce the influence of the most abundant Buchnera, the relative abundance data were logarithmically transformed with the decostand function of vegan. For the grouping scheme of geographic region, we assessed variation in community composition across all 23 groups, across ten groups with a sample size \u2265\u20093 , and across three groups colonizing Rhamnaceae (sample size \u2265\u20093) . For the grouping scheme of host plant, we assessed variation in community composition among all 25 groups, among eleven groups with a sample size \u2265\u20093, among six groups with a sample size \u2265\u20095, and among eight groups from Beijing (sample size \u2265\u20093) .Next, we investigated the patterns of beta diversity to address the relative importance of geography and host plant on symbiont and secondary symbiont communities. Beta diversity, i.e., the variation of symbiont community composition among differently grouped samples, was quantified using Bray\u2013Curtis dissimilarity which considered the presence/absence and relative abundance of the individual symbiont. The Bray\u2013Curtis distance was calculated between each pair of samples using the prcomp function in the R package stats to visualize variation among different groups in symbiont and secondary symbiont community compositions. PCA reduces the dimension of multivariate data and interprets such data diagrammatically. The resulting ordination was plotted with the R package ggbiplot [metaMDS function in vegan and presented two-dimensional plots by the R package ggplot2 [capscale and anova.cca functions in vegan and the resulting ordination was visualized by ggplot2. These ordination techniques are useful in representing community variation in response to environmental factors, such as geography and host plant in this study. In an ordination, samples that are close are more similar to one another than those that are far apart.Principal component analysis (PCA) was firstly performed on the relative abundance matrix using the ggbiplot . Then, w ggplot2 . NMDS is ggplot2 . For theanosim function and adonis function in vegan, respectively, and P values were obtained using 999 permutations. To further identify which symbionts were driving the differences in secondary symbiont community, we carried out the analysis of variance (ANOVA) tests in STAMP v2.1.3 [P values were used to control the false discovery rate.Based on the Bray\u2013Curtis distance matrices, differences in symbiont and secondary symbiont community structures were also statistically analyzed with analysis of similarities (ANOSIM) and permutational multivariate analysis of variance (PERMANOVA). These two analyses are both resemblance-based permutation methods widely used in ecology and PERMANOVA is generally more powerful than ANOSIM to detect changes in community composition . ANOSIM P v2.1.3  based onvegan. The geographic distance matrix was generated using the Geographic Distance Matrix Generator v1.2.3 [Furthermore, to test the effect of geographic distances among sampling sites in structuring the symbiont and secondary symbiont communities, the Pearson correlation coefficient between geographic distance matrix and Bray\u2013Curtis distance matrix was calculated using Mantel test in r v1.2.3 . Mantel A. gossypii, the Spearman correlation coefficients (\u03c1) between symbionts were calculated based on their relative abundances using the cor function in stats and were visualized in a heatmap with the R package corrplot [Finally, to explore potential interactions among different symbionts associated with corrplot .A. gossypii was dominated by the primary endosymbiont Buchnera aphidicola , followed by the secondary symbiont Arsenophonus (1.11%) and the bacteria Acinetobacter (0.99%) . A total of 1524 OTUs were identified at 97% similarity and were assigned into 39 phyla , 104 classes , 180 orders , 310 families , and 630 genera Table . The bac9%) Fig.\u00a0.Fig. 1BaBuchnera aphidicola. Along with Arsenophonus, they were also infected with Rickettsia , Serratia symbiotica (0.07%), Wolbachia (0.04%), Hamiltonella defensa (<\u20090.005%), Regiella insecticola (<\u20090.005%), and Spiroplasma (<\u20090.005%) , followed by Wolbachia (68/110), and Serratia symbiotica (32/110). Hamiltonella defensa, Regiella insecticola, and Spiroplasma were found to be low in both infection rate and abundance (Table\u00a0Arsenophonus and Wolbachia was the most common type (30/110), followed by multiple infections with Arsenophonus, Serratia symbiotica, and Wolbachia (13/110).The alpha diversity of the symbiont community was very low  Table . A total5%) Fig.\u00a0, Table\u00a01P\u2009=\u20090.710 for Shannon index, P\u2009=\u20090.770 for Simpson index, Kruskal\u2013Wallis test; P\u2009=\u20090.216\u20130.978 for Shannon index, P\u2009=\u20090.295\u20131.000 for Simpson index, Wilcoxon test). However, the Kruskal\u2013Wallis test revealed statistical differences in the populations occupying different host plants (P\u2009=\u20090.006\u2009<\u20090.01 for Shannon and Simpson indices). The symbionts within cotton aphid samples feeding on Buxaceae and Cucurbitaceae showed significantly higher and lower alpha diversities than samples on other plants, respectively .No significant difference was detected among the alpha diversity indices of aphid symbionts from different geographic regions  and across three groups feeding on Rhamnaceae (\u2265\u20093 samples) . In the Mantel test, no significant correlation between Bray\u2013Curtis dissimilarity and geographic distance could be observed . For the symbiont communities associated with aphid populations occupying different host plants, although the PCA and NMDS ordinations did not show significant structuring patterns (figures not shown), both ANOSIM and PERMANOVA uncovered a strong effect of host plant on symbiont composition , except for the cPCoA ordination of three groups feeding on Rhamnaceae (sample size \u2265\u20093), which showed that the samples from the same geographic region tended to cluster together and separate from others Fig.\u00a0. ANOSIM P\u00a0=\u20090.026\u2009<\u20090.05, Fig.\u00a0r\u00a0=\u20090.011, P\u00a0=\u20090.413).The barplot of secondary symbiont compositions of different geographic populations is shown in Fig.\u00a0P\u2009=\u20090.001\u20130.002\u2009<\u20090.01, Figs.\u00a0P\u2009<\u20090.01, Table\u00a0Arsenophonus and Wolbachia were found to significantly differ across different host plant groups  in the communities from Acanthaceae, Asteraceae, Buxaceae, Melastomataceae, Rosaceae, and Rutaceae and was absent from the Caprifoliaceae and Polygonaceae groups . However, in the NMDS and cPCoA analyses of six groups with a sample size \u2265\u20095 and cPCoA of eight groups from Beijing (sample size \u2265\u20093), the communities within aphids feeding on Cucurbitaceae were clearly separated from other samples  and Wolbachia   Table . Both poA. gossypii microbiota was dominated by a few bacterial taxa. Out of the top ten abundant genera, three were symbiotic bacteria, namely, Buchnera, Arsenophonus, and Rickettsia . The presence of multiple phylotypes may therefore be correlated with mutation accumulation in the reduced Buchnera genomes, which seems to be caused by loss of DNA repair genes and fixation of slightly deleterious mutations through genetic drift [As expected, partners , the ubiic drift , 97. HowSpiroplasma, which was not reported in A. gossypii previously, was identified in our sequencing data. Spiroplasma had extremely low relative abundance (<\u20090.005%) in two aphid samples, which may explain why it was not found before. The defensive symbiont Hamiltonella defensa was reported to infect all A. gossypii samples examined by Zhao et\u00a0al. [Arsenophonus was the predominant facultative symbiont, with the highest infection prevalence and abundance. It has been reported in previous studies of A. gossypii [Arsenophonus in aphids and revealed its high prevalence in the genus Aphis. Our results confirm their conclusion that Arsenophonus is a major bacterial partner of aphids. Most Arsenophonus in insects, including A. gossypii, was found to be associated with the lysogenic bacteriophage APSE [Hamiltonella to confer protection against parasitoid wasps [Arsenophonus has presented a positive correlation with parasitism, indicating a potential defensive role of Arsenophonus [Arsenophonus in A. gossypii may play a similar role in providing resistance against parasitoids, especially in the case of rare Hamiltonella infection. Further experiments are required to determine the function of Arsenophonus in aphids.Seven secondary symbionts were detected in this study, although their relative abundances were very low. o et\u00a0al. , Ayoubi o et\u00a0al. , and Zhao et\u00a0al. . Howevergossypii , 73\u201377. gossypii  surveyedage APSE , which iid wasps , 101. Innophonus . TherefoA. gossypii feeding on a limited number of plant species [Geography has been reported to influence the microbial profiles of the Japanese, Australian, Hawaiian, and Chinese populations of  species , 73, 74.A. gossypii. The alpha diversity of symbionts was found to be significantly different across aphid populations exploiting different plants. ANOSIM and PERMANOVA tests also revealed a strong effect of the host plant on both symbiont and secondary symbiont communities. These findings are consistent with previous studies that showed that the populations of polyphagous aphids colonizing different plants tended to harbor different symbiont communities  [Compared with the limited impact of geography, the host plant appeared to have played a greater role in shaping the symbiotic bacterial community associated with horbiae) \u201356.Arsenophonus but high-abundance Wolbachia within the Cucurbitaceae-feeding populations. Correlations between certain endosymbionts and host plants have been repeatedly reported in polyphagous aphids, especially in the extensively studied pea aphid Acyrthosiphon pisum [Regiella insecticola around the world [Arsenophonus-bearing locust populations were reported in Aphis craccivora [A. gossypii is a typical polyphagous species with a very wide range of host plants. Genetic differentiation has been found to occur among its host-associated populations [A. gossypii. The special symbiont communities within cucurbits-feeding populations support the ecological specialization of A. gossypii on Cucurbitaceae from the perspective of symbiotic bacteria. However, it is not clear whether the associated symbionts have played a substantive role in host plant specialization of A. gossypii. Some studies suggested that facultative symbionts had an important influence on the host plant use of aphids [It is worth noting that the Cucurbitaceae-feeding cotton aphids hosted unique symbiont communities. They showed lower alpha diversity and were clustered together and separated from other samples in some ordination analyses. The post hoc Scheff\u00e9 tests revealed significantly low-abundance on pisum , 60, 62.he world , 58, 65,accivora , 62. A. ulations \u2013105. Botulations , 107 andulations  have conf aphids \u201340; somef aphids , 110. FuBuchnera aphidicola and secondary symbionts. In the pea aphid, Serratia symbiotica and Rickettsia have been reported to suppress the population density of Buchnera [Hamiltonella on the abundance of Buchnera in A. gossypii. These findings indicate competition between the primary and secondary symbionts for resources and survival niches within the same host aphid.We conducted correlation analysis to assess the interactions between different symbionts. The correlation coefficients suggested antagonistic interactions between Buchnera , 112. ZhBuchnera  also fouA. gossypii is heteroecious holocyclic in China, alternating between primary host plants such as Punica, Hibiscus, and Rhamnus and various herbaceous secondary host plants [A. gossypii. Co-infections may bring ecological benefits for the host aphids. Acyrthosiphon pisum co-infected with Hamiltonella\u2013Serratia or Hamiltonella\u2013Fukatsuia exhibited greater resistance to parasitoids [Acyrthosiphon pisum strain co-infected with Rickettsiella viridis and Hamiltonella defensa was more exposed to ladybird predation than the singly Rickettsiella-infected strain. Ayoubi et\u00a0al. [Hamiltonella\u2013Arsenophonus combination in A. gossypii conferred no resistance against parasitism by Aphidius matricariae. Therefore, the interactions between facultative symbionts seem very complicated, either synergistically or antagonistically, which was also indicated by the positive and negative Spearman correlation coefficients in our study.Multiple infections with secondary symbionts occurred commonly in our examined cotton aphids. Co-infections are often unstable , 113. Wet plants . The sext plants , 116 creasitoids , 32. Howasitoids  reportedi et\u00a0al.  found thAphis gossypii using Illumina sequencing of 16S rRNA gene. The microbiota of A. gossypii was dominated by heritable symbionts, among which Buchnera aphidicola and Arsenophonus were the predominant symbiont and facultative symbiont, respectively. The symbiont diversity was found to vary with the host plant rather than geography, suggesting an important role of the host plant in shaping the bacterial community structure. The cucurbits-adapted aphid populations harbored unique symbiont communities, which provide a good model to explore the direct or indirect impacts of facultative symbionts on host specialization. Moreover, the interactions between coexisting symbionts within A. gossypii were revealed to be very complicated.Based on an extensive sampling from different plants and regions in China, we analyzed the diversity of symbiotic bacteria within ESM 1(DOCX 1.07 mb)"}
+{"text": "Myzus persicae (Sulzer). Ninety-two aphid samples collected from different host plants in various regions of China were examined using high-throughput amplicon sequencing. We comprehensively characterized the symbiont diversity of M. persicae and assessed the variations in aphid-associated symbiont communities. We detected a higher diversity of symbionts than has been previously observed. M. persicae hosted the primary endosymbiont Buchnera aphidicola and seven secondary symbionts, among which Wolbachia was the most prevalent and Rickettsia, Arsenophonus, and Spiroplasma were reported for the first time. Ordination analyses and statistical tests revealed that the symbiont flora associated with M. persicae did not change with respect to host plant or geography, which may be due to frequent migrations between different aphid populations. These findings will advance our knowledge of the microbiota of polyphagous insects and will enrich our understanding of assembly of host-microbiome systems.Aphids are known to be associated with a variety of symbiotic bacteria. To improve our knowledge of the bacterial diversity of polyphagous aphids, in the present study, we investigated the microbiota of the cosmopolitan agricultural pest The online version of this article (10.1007/s00248-020-01622-6) contains supplementary material, which is available to authorized users. Buchnera aphidicola. Buchnera is the primary endosymbiont of aphids, resides in bacteriocytes, and can provide essential amino acids and vitamins for its hosts  or geography .In general, the host plant and geography appeared to have no significant impacts on the symbiont flora of P = 0.077\u20130.680 for OTU, P = 0.180\u20130.860 for ASV; Fig. P = 0.035; Fig. P = 0.006 for OTU, Fig. P = 0.025 for ASV, Fig. P = 0.012; Fig. P > 0.05, Table P < 0.05 for ASV; PERMANOVA: P < 0.05 for OTU and ASV; Table P < 0.05 for OTU and ASV; Table Buchnera (P = 0.049), Rickettsia (P = 0.037), and Wolbachia (P = 0.027) were present in significantly different relative abundances among these groups. The Asteraceae-feeding aphid populations in Beijing harbored a significantly low abundance of Buchnera  or secondary symbiont communities  was observed in the Mantel tests.Furthermore, at the OTU level, no correlation between geographic distances and Bray\u2013Curtis dissimilarities of symbiont  [Rickettsia, Arsenophonus, and Spiroplasma were also detected for the first time in the present study. Gallo-Franco et al. [M. persicae samples, which may have been due to improper processing of the data. In their study, many OTUs were only annotated to bacterial families, and OTUs with relative abundances lower than 1% were ignored. However, secondary symbionts may occur at very low titers in their host aphids  , 28, Rico et al.  did not M. persicae samples on C. sonchifolium (Asteraceae) harbored a high abundance of Rickettsia. In most cases, the relative abundance of Rickettsia in C. sonchifolium-feeding aphid populations was greater than that of Buchnera  in a study of Henry et al. [. The relative abundances of S. symbiotica and R. insecticola were extremely low (< 0.02% across infected samples). H. defensa was only detected in four of 92 samples; however, it showed an average relative abundance of 3.45% across infected samples  cannot be discarded. Among all seven secondary symbionts associated with M. persicae, Spiroplasma showed the lowest infection frequency and relative abundance across all samples. Its relative abundance across infected samples was also extremely low (0.006%). To date, Spiroplasma has rarely been detected in aphids [A. pisum, Spiroplasma was shown to be abundant [Spiroplasma detection . The widespread sharing of haplotype H1 and the absence of an IBD pattern suggests that M. persicae in China have undergone repeated migrations between populations. Because the host plants of M. persicae are primarily crops and ornamental plants, human activities may have greatly facilitated its dispersal. It appears that the absence of distinct patterns of symbiont community is normal within such a large panmictic population of M. persicae. In addition, secondary symbionts can be horizontally transmitted between aphids during sexual reproduction [M. persicae, suggesting that horizontal transmission may have occurred. Frequent migrations between different populations, coupled with intraspecific horizontal transmission of secondary symbionts, may have caused the absence of distinct symbiont community patterns in M. persicae.climates . This spoduction  as well oduction  or parasoduction . Same phM. persicae, a representative polyphagous aphid species. Heritable bacterial symbionts comprised the major components of the M. persicae microbiome. The symbiont compositions were not significantly altered across natural aphid populations. Specific biological characteristics of M. persicae and human activities may together have contributed to the absence of distinct patterns in symbiont community. Thus, the results of our study highlight the importance of the host and environment in microbiome assemblage.Characterizing symbiont communities is fundamental to understanding host-symbiont systems. In the present study, based on a broad sampling, we provide the first comprehensive survey of symbiont diversity within ESM 1(DOCX 6620 kb)"}
+{"text": "We aimed to investigate the hazard of hospitalization for heart failure (hHF) according to the transitions in metabolic health and obesity status.The Korean National Health Insurance Service datasets from 2002 to 2017 were used for this nationwide, longitudinal, population-based study. The hazard of hHF was analyzed according to the eight groups stratified by stability in metabolic health and transition in obesity status among initially metabolically healthy adults who underwent two cycles of health examinations in 2009\u20132010 and 2013\u20132014 .During two examinations, 48.43% of the initially metabolically healthy obese (MHO) individuals and 20.94% of the initially metabolically healthy non-obese (MHNO) individuals showed changes in their metabolic health and obesity status. During a mean follow-up of 3.70\u00a0years, 3151 individuals were hospitalized for HF. When stable MHNO individuals were set as the reference, transition to metabolically unhealthy phenotype was associated with an increased hazard of hHF; the hazard ratio (HR) and 95% confidence interval (CI) in the individuals who transformed from MHO to metabolically unhealthy non-obese was 2.033 (1.579\u20132.616). The constant MHO group had a 17.3% increased hazard of hHF compared with the stable MHNO group [HR (95% CI) 1.173 (1.039\u20131.325)]. Individuals who shifted from MHO to MHNO showed a 34.3% lower hazard of hHF compared with those who maintained the MHO category [HR (95% CI) 0.657 (0.508\u20130.849)].Dynamic changes in metabolic health and obesity status were observed during a relatively short interval of 3\u20135\u00a0years. Loss of metabolic health was significantly associated with an increased hazard of hHF. Even if metabolic health was maintained, persistent obesity remained as a risk factor for hHF, and transition from MHO to MHNO had a protective effect against hHF. Therefore, the prevention and control of obesity while maintaining metabolic health would be crucial in preventing hHF. Heart failure (HF) is a complex clinical syndrome that results from any structural or functional impairment in ventricular filling and ejection of blood . HF is aObesity is a well-known risk factor for incident HF \u20137. AmongDue to the limitations of BMI, which cannot distinguish fat mass and lean mass , 9, receTo establish an appropriate preventive strategy for HF with regard to obesity and metabolic health, we compared the hazards of incident hospitalization for heart failure (hHF) according to the transitions in obesity sub-phenotypes during the two cycles of health examinations using the Korean National Health Insurance Service (KNHIS) database.We used the KNHIS datasets of claims and preventive health examinations from January 2002 to December 2017 for this study. The KNHIS covers all residents in Korea as a single-insurer organization operated by the Korean government. The KNHIS operates two major programs to offer universal coverage to all residents of Korea: National Health Insurance (NHI) encompassing approximately 97% of the population and Medical Aid (MA) covering the remaining 3% of the population . The Ins2) at the time of the first health examination between 2009 and 2010 and those who had, at or before baseline, claims for diabetes mellitus (ICD-10 codes E10\u201314), atrial fibrillation (AF) (ICD-10 codes I48.0\u20134 or I48.9), myocardial infarction (MI) (ICD-10 codes I21\u201322), HF (ICD-10 code I50), and/or rheumatic mitral valve disease (ICD-10 codes I05), and those who had cardiac/vascular implants or grafts (ICD-10 codes Z95) at or before baseline. Furthermore, individuals with any malignancy, hypothyroidism or hyperthyroidism at or before baseline, and missing data in at least one variable were excluded  underwent at least one health examination between 2009 and 2010 and were aged\u2009\u2265\u200920\u00a0years at the time of this initial health examination, (2) underwent another health examination between 2013 and 2014, and (3) were metabolically healthy (did not have metabolic syndrome [MetS]) at the time of the initial health examination between 2009 and 2010. The time point of the second health examination between 2013 and 2014 was considered as the baseline. Among these, we excluded individuals who were underweight . Blood tests, including plasma glucose levels and lipid profiles were performed using venous samples obtained after an overnight fast. These health examinations were carried out only in hospitals certified by the KNHIS.Data on smoking history, alcohol consumption, and regular exercise were obtained using questionnaires. Heavy alcohol consumption was considered as an average daily alcohol ingestion of\u2009\u2265\u200930\u00a0g. Regular exercise was defined as performance of a high-intensity physical activity  for\u2009>\u200920\u00a0min per session,\u2009\u2265\u20093\u00a0days/week, and/or moderate-intensity physical activity  for\u2009>\u200930\u00a0min per session,\u2009\u2265\u20095\u00a0days/week. Low-income status was regarded as being registered in the MA program for the lowest income population or being part of the lowest one-fifth of the population registered in the NHI, based on the monthly household income. BMI was calculated as body weight in kilograms divided by the height in meters squared , MHO, MUNO, and metabolically unhealthy obesity (MUO). Since only individuals who were metabolically healthy at the initial health examinations conducted between 2009 and 2010 were included in the current study, all participants were MHNO or MHO at the time of initial examination. At the second examination conducted between 2013 and 2014 (baseline), they transitioned to one of the four obesity sub-phenotypes . All individuals were categorized into eight groups according to the transition in obesity sub-phenotypes from the first to the second health examinations .According to the obesity guidelines for the Korean population , BMI\u2009\u2265\u20092criteria , using tcriteria  . Categorical variables are shown as frequencies and percentages.The SAS software  was used for all statistical analyses. Two-sided -phenotypes were compared using the Kaplan\u2013Meier curves; the differences among the groups were assessed using a log-rank test. A multivariate Cox regression analysis was performed to calculate the hazard ratios (HRs) and 95% CIs for the hHF incidence rates according to the eight groups of transition in obesity sub-phenotypes: model 1 was unadjusted, while model 2 was adjusted for age, sex, smoking history, alcohol consumption, regular exercise, and eGFR. Furthermore, additional model (model 3) was constructed which was adjusted for systolic BP, low-density lipoprotein cholesterol (LDL-C), household income in addition to the potential confounders reflected in model 2. These regression models were constructed to include various risk factors for HF as potential confounders, referring to previous reports [-phenotypes were calculated in subgroups divided by age  and sex. In these subgroup analyses, the Cox regression models were adjusted for the same potential confounders reflected in model 2 of the main analysis. The potential effect modification by age group and sex was assessed through this stratified analysis and the p for interaction was calculated.The incidence rate of hHF was derived from the number of incident cases divided by the total follow-up duration (person-years). The cumulative incidence rates of hHF according to the eight groups of transition in obesity sub reports , 29\u201331. Next, to compare the effect of transition in each MetS component, we selected individuals who satisfied no MetS component at the initial health examination, and then, calculated the HRs (95% CI) of incident hHF according to the transition in each MetS component.We also conducted a sensitivity analysis after excluding individuals who developed hHF within 1\u00a0year of follow-up. Furthermore, we performed a sensitivity analysis after excluding individuals with hypertension or dyslipidemia at the first examination. The presence of hypertension and dyslipidemia was defined according to a previous study . In addiA total of 7,148,763 individuals were included in the study , 3151 participants were hospitalized for HF. The cumulative incidence of hHF is presented according to the eight groups of transition in obesity sub-phenotypes using the Kaplan\u2013Meier curves  and sex . Compared to the stable MHNO group (reference), individuals who had shifted to metabolically unhealthy category at the second examination showed a trend of consistently higher hazard of hHF in all subgroups. However, the increase in the hazard of hHF associated with the transition to metabolically unhealthy category was more prominent in the youngest age group (20-44\u00a0years). The higher hazard of incident hHF in the stable MHO category compared with the stable MHNO category was prominent only in individuals aged\u2009<\u200965\u00a0years and women. The MHO to MHNO group demonstrated significantly lower hazard of hHF compared to the reference group only in women.The hazard of hHF according to the eight groups of transition in obesity sub-phenotypes was assessed in subgroups stratified by age  for hHF incidence were calculated according to the development of each MetS component among individuals who satisfied no MetS component at the initial health examination  [Several limitations of this study should be acknowledged. First, because of the observational nature of this study, it is inevitably limited to clarify the causal relationships. However, to minimize the possible reverse causality effect, individuals with claims for HF at or before baseline were excluded, and the transition in obesity sub-phenotype was assessed prior to the baseline examination. Furthermore, a sensitivity analysis after excluding people who developed hHF within 1\u00a0year of follow-up demonstrated consistent results. Second, considering that the highest mean BMI of the participants according to the eight groups of transition in obesity sub-phenotypes was 28.06\u00a0kg/ms HFrEF) . DespiteIn this large population-based study, loss of metabolic health was associated with an increased hazard of hHF. In addition, even when metabolic health was maintained, persistent obesity was associated with a higher hazard of hHF than was stable MHNO category. Transition from MHO to MHNO may have a protective effect against future hHF. Therefore, obesity as a predictor of hHF should be considered, accounting for its dynamic changes as well as the transition in metabolic health status. As a clinical implication, the development of obesity should be prevented, and the prevalent obesity should be managed actively while strictly maintaining metabolic health for the prevention of hHF.Additional file 1. Additional figure legend and tables.Additional file 2. Additional figure S1."}
+{"text": "Huntiella species are wood-infecting, filamentous ascomycetes that occur in fresh wounds on a wide variety of tree species. These fungi are mainly known as saprobes although some have been associated with disease symptoms. Six fungal isolates with typical culture characteristics of Huntiella spp. were collected from wounds on native forest trees in Greece and South Africa. The aim of this study was to identify these isolates, using morphological characters and multigene phylogenies of the rRNA internal transcribed spacer (ITS) region, portions of the \u03b2-tubulin (BT1) and translation elongation factor 1\u03b1 (TEF-1\u03b1) genes. The mating strategies of these fungi were also determined through PCR amplification of mating type genes. The study revealed two new species; one from Platanusorientalis in Greece and one from Colophospermummopane and Senegalianigrescens in South Africa. These novel taxa have been provided with the names, H.hellenicasp. nov. and H.krugerisp. nov., respectively. The former species was found to have a homothallic and the latter a heterothallic mating system. Huntiella species are members of the family Ceratocystidaceae  as defined by Ambrosiella, Berkeleyomyces, Bretziella, Catunica, Ceratocystis, Chalaropsis, Davidsoniella, Endoconidiophora, Huntiella, Meredithiella, Phialophoropsis, Solaloca, Tielaviopsis, Toshiolenlla and Wolfgangiella  in Texas, USA . Moreover, one species (H.bhutanensis) is found in association with the bark beetle Ips schmutzenhoferi  J. L\u00e9onard and Senegalianigrescens (Oliv.) P. Hurter in South Africa. These fungi displayed typical culture characteristics of Huntiella spp., including rapid growth on agar medium, white fluffy mycelia when young, as well as the production of fruity aroma. Identification was accomplished, based on morphology and multigene phylogenies for the ITS, BT1 and TEF-1\u03b1 gene regions. Furthermore, we considered the mating biology of these isolates in order to complement our taxonomic studies.The objective of this study was to identify two fungal isolates collected from Huntiella isolates were collected from fresh wounds of Colophospermummopane in Kruger National Park in April 2009 and another one of the South African isolates was obtained from a broken branch of a Senegalianigrescens tree damaged by elephants in Kruger National Park in June 2010. The isolates from Greece were obtained from the stump of a Platanusorientalis tree that was cut about two months before sampling, in a natural forest along the banks of the Spercheios River in Phthiotis Regional Unit during November 2018. Isolation from wood samples was performed using a trapping technique originally described by Ceratocystisplatani  Engelbrecht & Harrington using freshly-cut twigs of P.orientalis as bait , using a sterile needle under a dissection microscope . The South African isolate was obtained by transferring mycelial strands from infected wood on to MEA. Primary isolations were incubated for 3\u20137 d at 25 \u00b0C. From these isolations, purified cultures from single hyphal tips were prepared for morphological characterisation, phylogenetic analyses and mating-type studies. All purified isolates were deposited in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa and the living culture collection (PPRI) of the South African National Collection of Fungi (NCF), Roodeplaat, Pretoria, South Africa. The dried-down type specimens were deposited in the National Collection of Fungi (PREM), Roodeplaat, Pretoria, South Africa.Isolates from Greece were made by transferring ascospore masses from the tips of the ascomata on the surface of ITS) regions 1 and 2, including the 5.8S rRNA, a partial \u03b2-tubulin 1 gene (BT1) and a partial Translation Elongation factor-1\u03b1 gene (TEF-1\u03b1), amplified using the set of primers as described by All the isolates obtained in this study were used for DNA sequence-based characterisation. Total genomic DNA was extracted from the mycelium of isolates grown on 2% MEA for 3\u20134 d at 25 \u00b0C, using Prepman Ultra Sample Preparation Reagent  following the manufacturer\u2019s protocols. Three gene regions were amplified for sequencing and phylogenetic analyses. These included the Internal Transcribed Spacer  to remove excess primers and dNTPs. Amplicons were sequenced in both directions using an ABI PRISM\u2122 3100 DNA sequencer  at the Sequencing Facility of the Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria, South Africa.A total volume of 25 \u03bcl PCR reaction mixture contained 1 \u03bcl of DNA template, 0.5 \u03bcl (10 pM) of each primer (Forward and Reverse), 5 \u03bcl MyTaq PCR buffer  and 0.3 \u03bcl of MyTaq DNA Polymerase . The PCR reactions were conducted using an Applied Biosystems ProFlex PCR System . The PCR programme for amplification of the Huntiella spp. (except H.decorticans) were downloaded from GenBank (http://www.ncbi.nlm.nih.gov). The sequences were aligned using MAFFT v. 7 with an online FFT-NS-i strategy , Maximum Parsimony (MP) and Bayesian Inference (BI). The appropriate substitution model for each dataset was obtained using the software package jModeltest v. 2.1.5  chains were run from a random starting tree for five million generations and trees were sampled every 100 generations. Twenty-five percent of the trees sampled were discarded as burn-in and the remaining trees were used to construct 50% majority rule consensus trees. Ceratocystiscercfabiensis (isolate CMW 43029) was used as the outgroup taxon for all the phylogenetic analyses. The resulting trees were visualised using MEGA v. 7.Single gene sequence datasets of the v. 2.1.5 . The ML L v. 3.0 . Confide. 4.0b10 . Gaps we. 4.0b10  on the Ci, SMZ 18, Nikon, Tokyo, Japan) mounted with a camera (Nikon DS Ri-2) were used for all observations. Fifty measurements of each relevant microscopic structure were made when available and these are presented as minimum\u2013maximum and average \u00b1 standard deviation.Morphological features were studied on the isolates grown on 2% MEA. The fruiting structures were initially mounted in water and this was later replaced with 85% lactic acid and in which measurements were made and images captured. Nikon microscopes  taken from an actively-growing colony was placed at the centres of Petri dishes. Four replicates per isolate were used to study growth rate and the experiment was repeated once. Colony diameters were assessed by taking two measurements perpendicular to each other for all isolates daily and growth rates were calculated. Colony characteristics were described on the same medium used for the growth studies and colours were assessed using the colour charts of MAT) of the studied Huntiella spp. was determined, based on the results of the mating type PCR reactions .All six isolates, included in this study, were successfully sequenced at all three selected gene regions for phylogenetic analyses, resulting in DNA sequence data of approximately 614, 574 and 830 bp for the ML, MP and BI were concordant and showed similar phylogenetic relationships amongst taxa , TEF-1\u03b1 , 157\u2013493 \u00b5m wide (avg. 218.2 \u00b5m), ornamented with spine-like structures, dark brown, conical, 12\u201329 \u00b5m long, 4\u20139 \u00b5m wide at base becoming attenuated; ostiolar necks upright, straight, occasionally situated at off-centre of base, darker than base when young, 344\u2013616 \u00b5m long (avg. 515.5 \u00b5m), 34\u201360 \u00b5m wide (avg. 46.6 \u00b5m) at base, gradually tapering towards apex; ostiolar hyphae hyaline, straight to divergent, 15\u201339 \u00b5m long, 1\u20133 \u00b5m wide, tapering towards apex. Asci evanescent. Ascospores hyaline, subglobose, aseptate, covered with sheath giving a hat-like feature in side view, 4\u20135.5 \u00d7 3\u20134.5 \u00b5m (5 \u00b1 0.23 \u00d7 4 \u00b1 0.28 \u00b5m) excluding sheath.Asexual state.Thielaviopsis-like Conidiophores macronematous, simple or branched; when branched radiating from basal cell once, often reduced to conidiogenous cells. Conidiogenuscells endoblastic, hyaline, varying from lageniform to cylindrical depending spore shape; in case of thick barrel-shaped conidia, apex often becoming wider than base. Conidia hyaline, 1-celled, in two recognisable shapes; majority ellipsoidal to barrel-shaped , typical fat barrel-shaped 5\u20138 \u00d7 4.5\u20137.5 \u00b5m (5.9 \u00b1 0.61 \u00d7 5.3 \u00b1 0.55 \u00b5m), width of some barrel-shaped ranging 2.5\u20134 \u00b5m wide; rectangular-shaped , not commonly found, 5\u20139 \u00d7 1\u20133 \u00b5m (6.9 \u00b1 1.18 \u00d7 2.3 \u00b1 0.38 \u00b5m). Aleurioconidia not observed.Cultures on 2% MEA in dark in 8 d showing circular growth with even edge, mycelium flat, superficial, medium dense and texture becoming pelt-like with age, colour above not uniform, salmon (11f\u2019) to ochreous (15b\u2019) with inner half irregularly umber (13m), below ochreous (15b\u2019) with inner half irregularly umber (13i\u2019) at centre. Optimum growth temperatures at 30 \u00b0C at 9.6 mm/d, followed by at 25 \u00b0C (7.6 mm/d), 35 \u00b0C (7.2 mm/d), 20 \u00b0C (4.7 mm/d), 15 \u00b0C (3.2 mm/d), 10 \u00b0C (1.1 mm/d) and 5 \u00b0C (0.2 mm/d).Platanusorientalis in a natural forest along the banks of the Spercheios River, Nov. 2018, P. Tsopelas & N. Soulioti, PREM 62889, holotype (dried culture of CMW 54800), culture ex-holotype CMW 54800 = PPRI 27982, other cultures CMW 54801 = PPRI 27983.Greece, Phthiotis, near the village Kastri, occurring on freshly-cut stumps of H.hellenica developed at temperatures over 25 \u00b0C. Cultures incubated at 20 \u00b0C and below produced only the asexual state. Huntiellahellenica is closely related to H.savannae  ascomatal necks than H.savannae (average 579 \u03bcm long) and H.pycnanthi (average 673 \u03bcm long). Huntiellahellenica had larger (average 6.9 \u00d7 2.3 \u03bcm) barrel-shaped conidia than H.savannae (average 4.8 \u00d7 3 \u03bcm) and H.pycnanthi (average 6 \u00d7 3 \u03bcm). Optimal temperature for growth of H.hellenica was 30 \u00b0C, similar to H.savannae and H.pycnanthi, but H.hellenica differed from H.pycnanthi in growing minimally at 10 \u00b0C and below.The sexual state of savannae , H.pycnycnanthi  and H.kTaxon classificationFungiMicroascalesCeratocystidaceaeF.F. Liu. Marinc. & M.J. Wingf.sp. nov.60B4330B-1F8B-575A-AAA0-A8F5AE420CACMycoBank No: 835638The name refers to the Kruger National Park in South Africa, where this fungus was collected.Heterothallic with isolates having either a MAT1-1-1 gene or a MAT1-2-1 gene.Sexual state. Not observed.Asexual state. Produced on 2% MEA in 3 weeks. Thielaviopsis-like. Conidiophores macronematous, upright, simple or branched in one tier, 29\u201337 \u00b5m in length, often reduced to conidiogenous cells; cellsConidiogenous  enteroblastic, lageniform, 10\u201320 \u00b5m long, 1.5\u20133 \u00b5m wide, tapering towards apex. Conidia hyaline, rectangular-shaped, usually straight, with top-end conidium often club-shaped, 4\u201311 \u00d7 1\u20132 \u00b5m (avg. 6.2 \u00d7 1.7 \u00b5m). Aleurioconidia hyaline, holoblastic, mostly terminal, ellipsoidal to subglobose with an extended tube-like base, club-shaped, 4\u20137 \u00d7 2\u20133 \u00b5m (5.6 \u00b1 0.76 \u00d7 2.5 \u00b1 0.24 \u00b5m).Cultures on 2% MEA in dark in 8 d showing circular growth with even edge, mycelium superficial, flat, dense, colour above uniformly white, below luteous (19). Optimum growth temperatures were at 30 \u00b0C at 9 mm/d, followed by at 25 \u00b0C (8.2 mm/d), 35 \u00b0C (6.2 mm/d), 20 \u00b0C (6 mm/d), 15 \u00b0C (3.4 mm/d), 10 \u00b0C (0.9 mm/d) and 5 \u00b0C (0.3 mm/d).Senegalianigrescens, June 2010, M. Mbenoun, PREM 62883, holotype (dried culture of CMW 36849), culture ex-holotype CMW 36849 = CBS 131676 = PPRI 27952.South Africa, Mpumalanga, Kruger National Park, Satara rest camp, Colophospermummopane, April 2009, M. Mbenoun, CMW 55933, CMW 55934, CMW 55935.South Africa, Mpumalanga, Kruger National Park, Punda Maria, Huntiellakrugeri is closely related to H.hellenica described in the present study, H.cryptoformis  were longer than those of H.hellenica (average 5.9 \u00d7 5.3 \u03bcm) and H.cryptoformis (average 5.5 \u00d7 2.5 \u03bcm). In addition, H.krugeri produced hyaline aleurioconidia, which are absent in other closely-related species in the genus.toformis  and H.ssavannae . Due to Huntiella species isolated from Platanusorientalis in Greece, Colophospermummopane and Senegalianigrescens in the Kruger National Park of South Africa. These two species, provided with the name H.hellenica and H.krugeri, respectively, were shown to reside in the African Clade of Huntiella , as well as their distinct morphological characteristics. Mating studies showed that Huntiellahellenica and H.krugeri were homothallic and heterothallic, respectively. All indications were that these two species are saprobes that grow on the freshly-exposed surfaces of trees.This study led to the discovery of two novel untiella . The ideP.orientalis, from which H.hellenica emerged, was sampled approximately two months after tree felling and it was also infected by the pathogen Ceratocystisplatani, which causes a devastating disease in natural stands of P.orientalis in Greece. Colonszation of the stump with H.hellenica could have occurred on the freshly-cut surface with a contaminated tool as occurs for C.platani (The stump of platani  or was tHuntiella spp. They grew rapidly in culture; their mycelium was white when young and turned dark with age. The one species that displayed a sexual state - H.hellenica, produced hat-shaped ascospores and had short conical spines on the ascomatal bases. Temperature is known to influence the ability of Huntiella spp. to produce a sexual state (H.hellenica, which did not produce acomata below 25 \u00b0C.The novel species described in this study showed typical characteristics of al state  and thisHuntiella (BT1 and TEF-1\u03b1, provided the best resolution for species identification of Huntiella, while ITS sequences provided little information or no support.Comparison of DNA sequence data for multiple gene regions is essential when seeking to identify species in untiella . The thruntiella . Howeveruntiella . Thus prHuntiella spp. (H.hellenica has both mating-type idiomorphs and this explains the presence of sexual structures in all isolates derived from single hyphal tips. In contrast, the isolate of H.krugeri contained one mating gene and is clearly a heterothallic species of Huntiella, also consistent with the fact that the isolate produced only an asexual state. This result is also consistent with those of Huntiella spp. can have different mating strategies. Collectively, Huntiella spp. have a remarkable range of mating strategies, including homothallic and heterothallic species, as well as those exhibiting unisexuality (Huntiella.Primers, developed to identify the mating type idiomorphs in lla spp. , were efexuality . The newHuntiella, discovered in this study, bring the total number of species in the genus to 31. These are found in many different regions of the world and on a wide variety of woody substrates (Huntiella species in the future.The two new species of bstrates . The renbstrates  and reprbstrates . The desbstrates , should"}
+{"text": "In this paper, we propose a new unsupervised attention-based cycle generative adversarial network to solve the problem of single-image dehazing. The proposed method adds an attention mechanism that can dehaze different areas on the basis of the previous generative adversarial network (GAN) dehazing method. This mechanism not only avoids the need to change the haze-free area due to the overall style migration of traditional GANs, but also pays attention to the different degrees of haze concentrations that need to be changed, while retaining the details of the original image. To more accurately and quickly label the concentrations and areas of haze, we innovatively use training-enhanced dark channels as attention maps, combining the advantages of prior algorithms and deep learning. The proposed method does not require paired datasets, and it can adequately generate high-resolution images. Experiments demonstrate that our algorithm is superior to previous algorithms in various scenarios. The proposed algorithm can effectively process very hazy images, misty images, and haze-free images, which is of great significance for dehazing in complex scenes. In hazy weather, there are usually a large number of impurities (such as particulates and water droplets) in outdoor scenes. Due to the absorption and scattering of the atmosphere, haze, fog, and smoke are generated. The influence of these factors causes a certain degree of degradation in the image quality acquired by a camera, reducing the sharpness and contrast of the captured image. In severe cases, these factors can also distort the color information in the image, making computer vision technologies such as image recognition and image segmentation more difficult to apply. In recent years, with the popularization of computer vision systems and their application in various industries, these systems have played an important role in roads, aviation, and other fields. To enable these systems to function normally in various severe weather conditions, image clarity processing is essential. After the image acquisition device collects the hazy image, in order to facilitate the further operation of a program, image enhancement preprocessing operations are usually performed on the original image to improve its contrast, while rendering the target object clearer and easier to identify. For hazy images, this image enhancement operation is called image dehazing.The dehazing operation for a single image has always been a key concern. Traditional methods are usually based on the parameter estimation of an atmospheric scattering model ,2, repreAs algorithm synthesis makes it easier to create large-scale datasets that meet the needs of the network, most existing methods use artificially synthesized hazy images as the dataset. A synthetic hazy image is created according to an algorithm, and the distribution of the hazy areas has a certain regularity, which is very different from natural haze characteristics. The models and algorithms designed on the basis of this training have certain limitations in practical applications, and they cannot adequately remove natural haze. Therefore, this experiment uses the O-HAZE  dataset.In actual hazy images, the concentrations of haze in different regions and depths are inconsistent; using a simple method for overall image conversion results in uneven haze removal, with the resulting image having serious chromatic aberration. The existing overall image conversion methods do not pay attention to specific parts of an image, failing to focus on the area to be converted; thus, they are unable to retain the original features in mist- and haze-free areas. These algorithms may change areas that do not need to be processed, thereby leading to \u201cfalse dehazing\u201d.In view of the above problems, this paper combines the classic dark-channel prior dehazing algorithm , noting Aiming at the characteristics of uneven concentrations of haze in different areas of an actual image, the proposed method enhances the network structure of CycleGAN , introduThe proposed method innovatively uses the enhanced dark channel as the attention map and introduces the dark-channel enhancement coefficient. Through the training of the network, the enhanced dark-channel map can more effectively and accurately mark the hazy areas and their concentrations in an image, as well as increase the difference between different haze concentrations.Benefiting from the characteristics of the dark channel and attention mechanism, the proposed network can better retain the original features and details of an image, and it is more robust to mist- and haze-free image conversion.Our innovations can be summarized as follows:The subsequent chapters introduce work related to image dehazing. In Image dehazing has always been a popular research topic, especially as it relates to single-image dehazing. Traditional single-image dehazing methods are mostly based on image-dehazing theory, studied using prior information-based methods. Through many observations, He et al.  discoverWith the development of deep learning, convolutional neural networks (CNNs) have been successful in many computer vision tasks, and many single-image dehazing methods incorporating deep learning have emerged ,14,15. TWith the rise of GANs , great pThe goal of image dehazing is to improve the clarity of the whole image; thus, past methods applied a global process to the image. For haze images synthesized by algorithms, the haze distribution and size are often regular; hence, the overall conversion method has a better dehazing effect. However, actual haze has characteristics of different image depths, different hazy areas, and uneven haze concentrations. A uniform conversion of the overall image is, thus, not particularly ideal, as it is impossible to distinguish between very hazy areas, misty areas, and haze-free areas. The proposed method implements the previously described CycleDehaze  network,Attention mechanisms are often used to focus on specific objects or areas within an image. In our work, the transformed area is taken as the hazy area of an image, whereby the degree of transformation depends on the haze concentration. This is a local concern; therefore, it is necessary to introduce the attention mechanism into this dehazing approach.v to enhance the attention map. To improve the adaptability of the attention map to network changes, the proposed method trains the coefficient v along with the network.In view of the characteristics of actual hazy images, attention maps need to solve two main problems: (1) the labeling of hazy areas, and (2) the quantification of haze concentration. In response to the first problem, the proposed method uses dark channels to effectively reflect the hazy area. A larger value in a dark-channel map denotes greater haze concentration in the original image and a whiter area reflected in the attention map. However, the dark channel shown in v. Similar to CycleDehaze [mask_x being obtained. Finally, GA(x), x, and mask_x are calculated using an elementwise product to obtain the final result. x and y\u2032 into haze-free images x\u2032 and y\u2033, whereas the GB generator is responsible for converting haze-free images x\u2032 and y into hazy images x\u2033 and y\u2032. DA and DB are the two discriminators, while \u0298 represents the elementwise product operation. This figure also shows the cyclic generation process of the network, including the conversion between the target domain and the source domain; the upper-left shaded region describes the focus of our study through which we obtained results.The proposed method implements CycleDehaze  for geneleDehaze , this neleDehaze , and norThe attention mechanism is an important part of this paper. It has been widely used in image conversion. Mejjati et al.  proposedHowever, in image dehazing, the hazy area covers almost the whole image, while the level of haze is different; thus, the attention map obtained via training usually focuses on the sharp and prominent parts of the image. It is impossible to notice the actual hazy areas in the whole image, thereby making it impossible to accurately identify the haze concentration. Therefore, the dehazing effect is not ideal, as situations usually arise where the local effect is better, but the overall effect is biased, whereas some areas are not noticed.The attention map in this paper was inspired by the dark-channel method of He et al. . The darEquation (1) describes the calculation method of the original dark-channel method , where \u03a9Equation (2) allows enhancing the original dark channel to obtain a new dark channel. The proposed method applies a dark channel enhancement coefficient This network introduces cyclic consistency loss and perception loss. Similar to CycleGAN , images The proposed method divides the final desired image into two parts; the first is the part of the original image that does not need to be converted, while the second is the part generated by the generator. The next step is to calculate and combine both parts.For the final synthesis of the haze-free image, the proposed model uses the method of Mejjati et al. . First, CycleGAN , the conTo retain details in the image, the proposed method introduces the VGG16  model tox, y, x\u2033, and y\u2033 in Equation (3), using the VGG16 [The proposed method characterizes the values of he VGG16  model toion loss , as showOur final loss function ultimately includes three parts: the adversarial loss of the GANs , the cycy.Equation (5) describes the adversarial loss of the GANs . We set Equation (6) describes the cyclic consistency loss on the basis of CycleGAN . In the xA and yA represent two attention mechanisms.Equation (7) describes the calculation of all losses, where Finally, we can obtain the optimal parameters of In this section, the proposed model is compared with various generative adversarial network models, including CycleDehaze , pix2pixMost current datasets use synthetic hazy images . HoweverTaking the O-HAZE  dataset Due to the small number of images in the dataset, the proposed method enhanced the dataset by sequentially cropping the 45 original hazy images. To ensure the robustness of the dataset to different scales and textures, the position of the crop origin and the size of the crop were randomly generated. The cropped area was always a square, with length ranging from 256 pixels to the value of the smaller side of the image. If the distance between the crop origin and the image boundary was smaller than the length of the crop area, the image was discarded. To ensure that each image had the same weight in the dataset, the proposed method cropped each image to obtain 200 subimages, which were then scaled to a size of 256 \u00d7 256 pixels. The same method was used to randomly crop the haze-free images to form an unpaired dataset.The results of the experiment were compared with six other methods using the O-HAZE dataset . AlthougThe peak signal-to-noise ratio (PSNR)  is the rThe structural similarity (SSIM)  consider of SSIM  denotes The CIEDE2000  color diAccording to \u2212112, the value of In order to prove the advantages of the proposed method, we performed a statistical test on the experimental results in Then, we performed post-hoc test to further prove that the proposed method is significantly different from the method of Mejjati et al.  and Engii et al.  is 1.282i et al.  is 1.563We also compared the performance of the proposed method and CycleDehaze  using thIn order to test the effect of our method across datasets, we conducted crossover experiments using models trained on the I-HAZE  and O-HAIn addition, v, an enhanced dark-channel attention map was obtained, as shown in the figure after training. It can be seen that the improved dark-channel attention map better shows the distribution and concentration of hazy areas than the attention map trained using the method of Mejjati et al. [We also compared the results using different attention maps applied to hazy areas, mainly focusing on two types of attention maps: one obtained via self-training of the network and the other obtained via calculation using the dark channel. Mejjati et al.\u2019s  model wai et al. .To verify that the model could achieve the expected dehazing effect in any situation, we verified that the image was completely haze-free in extreme cases, as shown in In summary, as a result of the introduction of the attention mechanism, the proposed method can better approach this issue such that changes in the area are hardly noticeable; thus, there is no need to distinguish the haziness of the input image, thereby improving the robustness of the model.We compared the results obtained using the proposed method and others in the literature on the same dataset, where it was found that the proposed method\u2019s sharpness, dehazing effect, and visual effect were better than those of other methods. When dehazing the misty and haze-free image areas, due to the introduction of the attention mechanism, the dark channels in the areas that were haze-free or that had little haze were closer to gray and black; thus, the value of each pixel in the attention map was biased toward 0. On the other hand, for the reserved area of the original image with no need for conversion, its weight was biased toward 1; thus, the characteristics of the original image could be better preserved. As shown in The proposed method innovatively uses an enhanced dark-channel graph as the attention map added to CycleGAN . This al"}
+{"text": "In addition, we extended these findings to show that patients with the highest frequency of 127-hi cells at diagnosis were significantly more likely to maintain \u03b2 cell function. Moreover, in patients treated with alefacept in the T1DAL clinical trial, the probability of responding favorably to the antiinflammatory drug was significantly higher in those with a higher frequency of 127-hi cells at diagnosis than those with a lower 127-hi cell frequency. These data are consistent with the hypothesis that 127-hi cells maintain an antiinflammatory environment that is permissive for partial remission, \u03b2 cell survival, and response to antiinflammatory immunotherapy.Transient partial remission, a period of low insulin requirement experienced by most patients soon after diagnosis, has been associated with mechanisms of immune regulation. A better understanding of such natural mechanisms of immune regulation might identify new targets for immunotherapies that reverse type 1 diabetes (T1D). In this study, using Cox model multivariate analysis, we validated our previous findings that patients with the highest frequency of CD4 Type 1 diabetes (T1D) is a progressive heterogeneous autoimmune disease resulting in the destruction of insulin-secreting \u03b2 cells by T cells \u20136. In an+ T cell population that expresses CD25 and a high density of CD127 that is present at a higher frequency in children with a long remission compared with children with short or no remission (+CD25+CD127hi (127-hi) cells are predominantly memory cells with a mix of antiinflammatory  Th2 cells and proinflammatory (IFN-\u03b3\u2013 and IL-2\u2013secreting) Th1-type cells, with a bias toward Th2 cells ; Medical College of Wisconsin (MCW); San Raffaele Diabetes Research Institute (SRDRI); and the University of Queensland (UQ), we tested the validity of our original finding that the relative frequency of 127-hi cells correlates with length of partial remission (LoR) .+ and CD8+ T cells fall into 3 main functional groups. Proinflammatory cells can be either IFN-\u03b3\u2013secreting Th1 (CD4+ T cells) and Tc1 (CD8+ T cells) cells or IL-17\u2013 and IL-22\u2013secreting Th17/Tc17 cells; antiinflammatory Th2/Tc2 cells secrete IL-4, IL-5, and IL-13 and regulatory cells secrete IL-10 and TGF-\u03b2 . Within inflamed tissue precommitted cells are thought to commit to a specific lineage on encounter with specific antigen . To reciRetrospective analyses of clinical trial data show that patients with T1D are more likely to respond well to immunotherapy if it is given soon after diagnosis \u20136. HowevThe source, number, sex, and age of the participants in all figures and tables are summarized in n = 12 for ITN, n = 39 for SRDRI, n = 22 for MCW, and n = 11 for UQ). Our group sent each collaborative group a schematic  so that we could quantify the same cell subset using the same flow cytometry data. All investigators also quantified the relative frequency of established T cell populations, CD4+ T cells, and Tregs. The data were then exchanged and unblinded, and the frequency of 127-hi cells, CD4+ T cells, and Tregs in each sample was compared among investigators at different sites for consistency using linear regression (https://doi.org/10.1172/jci.insight.136114DS1).Investigators at all 4 research sites, ITN, SRDRI, MCW, and UQ, were asked to identify flow cytometry data from their own group generated using PBMCs from people within 3 months of diagnosis with T1D stained with a panel of antibodies that were specific for CD3, CD4, CD25, and CD127 , whereas age, BMI, baseline IDAA1c, and sex did not . Multivariate analysis also identified baseline 127-hi cell frequency as the only significant correlate with LoR , and sex with LoR. When using Cox model single covariate analysis adjusted for study variability, 127-hi cell frequency at diagnosis correlated with LoR . To examine the effect of age, patients were stratified by being older than 17 years, between 9 and 17 years, and younger than 9 years at diagnosis. Patients diagnosed with T1D when they were older than 17 years had a significantly longer end of remission compared with children younger than 9 years (P = 0.01). There was no significant difference in end of remission between the group aged between 9 and 17 years at diagnosis and either of the other groups. Using the log-rank test for trend, there was also a significant association between earlier end of remission in younger patients and longer end of remission in patients 17 years and older (P = 0.003). The end of remission was not different in patients stratified by having an IDAA1c level at baseline of less than 7.5, between 7.5 and 9, or greater than 9 . However, when the analysis included all patients tested (n = 42), including those with poor glucose control (n = 14), the relationship was no longer significant.We next tested the relationship between 127-hi cell frequency at baseline and future \u03b2 cell function by measuring the effect of 127-hi cell frequency on probability of preserving \u03b2 cell function, where the limit of survival was fasting C-peptide level at 12 months after diagnosis . The fasr = 0.71, P < 0.0001).Fasting C-peptide is less commonly used to assess \u03b2 cell function than stimulated C-peptide. In our study, fasting C-peptide was the measurement of choice because stimulated C-peptide was measured in only 12 of the 84 participants, whereas fasting C-peptide was measured in 42 patients. To determine the relationship between levels of fasting and stimulated C-peptide, we compared these 2 measurements in the same blood samples collected at baseline. Only patients with good glucose control were included. The data showed a strong correlation between levels of fasting C-peptide and stimulated C-peptide AUC in this population  and pre-Th1 (CXCR5\u2013CXCR3+CCR4\u2013) cells and comparing these frequencies with CD25\u2013 and total CD4+ memory T cells. As seen in healthy people, 127-hi memory cells had a significantly higher pre-Th2 frequency than either the CD25\u2013 or the total CD4+ memory cell populations, whereas the frequency of pre-Th1 cells was not different between the 127-hi and CD25\u2013 memory cell compartments . Whole blood was collected in heparin and processed within 2 hours. PBMCs were isolated using standard methods and frozen in liquid nitrogen.A standard formula, HbA1c (%) + 4\u00d7 insulin dose (U/kg per 24 hours), was used to take into account both insulin requirement and HbA1c levels in a single value, the IDAA1c. An IDAA1c equal to or less than 9 indicates the partial remission period . In thisFor some experiments, patients were stratified for analysis into those with either good glycemic control, defined as having a LoR longer than 1 month, or poor glycemic control, defined as having a LoR shorter than 1 month.Fluorochromes, vendors, catalog numbers, and registry identifiers for all antibodies used in flow cytometry experiments, including isotype control antibodies, are as shown in +, 127-hi, CD4+CD25\u2013 (CD25\u2013) T cells, and CD25+CD127lo (Tregs), we used either the BD Biosciences Treg cocktail or individual antibodies for CD3, CD4, CD25, and CD127 , CM , and EM  cells in total CD4+, 127-hi, and CD25\u2013 cells was determined by cell surface phenotype using anti-CCR7, anti-CD45RA, and anti-CD45RO. Tregs were identified as CD3+CD4+CD25hiCD127lo as previously described  and precommitted Th2 (CD45RO+CXCR5\u2013CXCR3\u2013CCR4+) (6 to 3 \u00d7 106 cell per ml with 50 ng/ml PMA (MilliporeSigma) and 1 \u03bcM Ionomycin (MilliporeSigma). 1 \u03bcl Brefeldin A (BD Bioscience) per ml medium was added at the beginning of the culture. After 4 hours, cultured cells were washed twice. Intracellular expression of GATA-3, T-bet, and ROR\u03b3t was measured. Data were acquired on an LSR II , FACSCanto II , LSR Fortessa and LSR II , LSRFortessa X20 , and LSRFortessa (BD) or CytoFlex S (Beckman Coulter) (used at SDBRI) and analyzed using FlowJo version 10. Isotype controls were used in every experiment and for every antigen-specific antibody.To identify CD4D127 see . The MFIed PBMCs . The relescribed . To iden3\u2013CCR4+) , 77 memo\u2013 memory and CD25+CD127hi memory cells were identified using the antibodies described in the section above. The 4 cell populations were sorted on a BD FACSAria high-speed cell sorter. Gates used to sort cell subsets are shown in Pre-Th1 and pre-Th2 CD25Sorted T cell subsets were incubated at 5000 cells per well in 96-well plates for 48 hours with 2 \u03bcl per well anti-CD3/CD28 coated beads (BD Biosciences), 10% human serum (Gemini), HEPES (Gibco BRL), glutamine, penicillin, streptomycin (Irvine Scientific), and 2-mercaptoethanol (MilliporeSigma) in RPMI (Invitrogen). Different plates were set up for different time points. After 48 hours of culture, 100 \u03bcl culture supernatant was collected and concentrations of IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IFN-\u03b3, and TNF-\u03b1 were determined by Flow Cytometry and the Biolegend LegendPlex Human Th2 panel.t test. One-way ANOVA followed by Sidak\u2019s multiple comparison test was used to compare Th2/Th1 ratios between more than 2 cell populations. Differences in cytokine secretion between cells subsets was determined using Wilcoxon\u2019s matched-pairs signed-rank test. Differences in transcription factor expression were calculated using Welch\u2019s ANOVA (P = 0.003) and Wilcoxon\u2019s matched-pairs signed-rank test. The effect of alefacept on the frequency of circulating 127-hi cells over 24 months was assessed using repeated-measures ANOVA. All analyses were performed with Graphpad Prism and SPSS V21.0 . A P value of less than 0.05 was considered statistically significant.The relationship between the relative frequency of cell subsets determined at different research sites was determined using both Spearman\u2019s correlation and linear regression. Associations between T cell subsets and probability of remaining in remission and the probability of preserving \u03b2 cell function were assessed using the log-rank (Mantel-Cox) test and the log-rank test for trend. Univariate and multivariate Cox proportional hazards regression analyses were also used to assess the relationship between T cell subsets and clinical variables with LoR as the outcome variable. Covariates included the 127-hi cell subset frequency at baseline (time 0), age at diagnosis, sex, baseline IDAA1c, and BMI. These results are reported as hazard ratios, with 95% confidence intervals. Multivariate analysis was performed for up to 5 variables and adjusted for study variability. The relationship between covariates was determined with linear regression. The relationship between levels of fasting C-peptide and stimulated C-peptide AUC was determined using Spearman\u2019s correlation. Changes in 127-hi cell frequency and IDAA1c and C-peptide levels at 6, 12, and 24 months compared with baseline were determined using the paired 2-tailed Student\u2019s All data reported in this study were collected by each collaborative organization as part of studies conducted prior to the study described, generated at SDBRI by reanalysis of previously generated data, or generated at SDBRI from staining vials of frozen PBMC samples obtained from ITN and collected by ITN as part of their prior studies. The sample and data analysis completed specifically for this study was approved by the SDBRI IRB, under exemption 4. Human subject protocol and consent forms were reviewed and approved by the TSRI IRB and the SDBRI IRB.AN generated the data for"}
+{"text": "The CDMSS could measure kLa, an index for evaluating the performance of aerobic culture incubators, and kG, an indicator of the degree of CO2 ventilation in the flask gas phase. We observed that cylindrical flasks provided a different culture environment, yielded a much higher kG than the Erlenmeyer and Sakaguchi flasks, and yielded kLa equivalent to that by Erlenmeyer flask by setting the ring-type baffle appropriately. Baffled cylindrical flask used for Escherichia coli K12 IFO3301 shake culture maintained lower CO2 concentrations in the headspace than conventional flasks; therefore, CO2 accumulation in the culture broth could be suppressed. Cell growth in baffled cylindrical flask  was about 1.3 and 1.4 times that in the Erlenmeyer and Sakaguchi flasks, respectively. This study focused on the batch culture at the flask scale and designed the headspace environment with low CO2 accumulation. Therefore, we conclude that redesign of flasks based on kLa and kG may contribute to a wide range of fields employing microorganism culture.The circulation direct monitoring and sampling system (CDMSS) is used as a monitoring device for CO In 1932, shake-flask culture was developed for submerged culture of fungi to overcome the biomass yield limitations of surface culture . CDMSS was also used to calculate the total oxygen transfer capacity coefficient (kLa), which is an indicator of the capacity to supply oxygen from the headspace to the liquid via the gas\u2013liquid interface, and kG, which is an indicator of CO2 ventilation from the headspace through the breathable stopper into the atmosphere, in Erlenmeyer and Sakaguchi flasks. Based on these findings, we utilised and evaluated the flasks of cylindrical shape with CO2 ventilation capacity in the headspace, which cannot be obtained in Erlenmeyer and Sakaguchi flasks.Our study examined the behaviour of Escherichia coli K12 IFO3301 was selected as the experimental organism. The LB medium (pH 7.0) used to culture E. coli K12 IFO3301 consisted of: (in g/L) tryptone, 10; yeast extract, 5; and NaCl, 5. A loop-full of E. coli K12 IFO3301 slant culture was inoculated into a 500-mL Erlenmeyer flask containing 100 mL of LB medium. The sample was then cultured at 30 \u00b0C on a rotary shaker with 70 mm shaking diameter at 200 rpm for 7.5 h. Glycerol stocks were prepared by adding the culture medium to glycerol  and stored at \u2212 80 \u00b0C.E. coli.Erlenmeyer, Sakaguchi, cylindrical, and baffled cylindrical flasks were selected for the study. All flasks were 500-mL in size. A detachable, O-ring shaped baffle was selected for constructing the baffled cylindrical flask (6 cm from the bottom). One mL each of glycerol stock was inoculated into a 500-mL Erlenmeyer flask and a 500-mL cylindrical flask containing 100 mL of LB medium, respectively, and cultured at 30 \u00b0C on a rotary shaker with 70 mm shaking diameter at 200 rpm. In the case of the 500-mL Sakaguchi flask containing 100 mL of LB medium, 1 mL of glycerol stock was inoculated and cultured at 30 \u00b0C on a reciprocating shaker with 70 mm shaking diameter at 120 strokes/min. Lastly, 50 mL of LB medium was added to the 500-mL baffled cylindrical flask and then 0.5 mL of glycerol stock was inoculated, cultured in the same way as Erlenmeyer flask and cylindrical flask. The shaking conditions of the Erlenmeyer flask and the Sakaguchi flask were those of the most frequently used rotary type and reciprocating type, respectively, which are standard culture conditions of 2 transfer capacity coefficient (kLa) was calculated as follows:t is time (s), C is the measurement value of dissolved oxygen concentration, C0 is the dissolved oxygen concentration at t\u2009=\u20090 (mg/L), and Cmax is the maximum dissolved oxygen concentration under the above-mentioned conditions. The maximum dissolved oxygen concentration was determined just before the measurements were taken for all conditions. When the decision coefficient value (termed R2) of the approximate expression of Eq. kLa.Dissolved oxygen concentration was measured with the CDMSS using the sulfite oxidation method, and the total O2 concentration in the headspace of all the flasks decreased during measurement. The headspace was continuously suctioned (200 mL/min) by using the CDMSS and the suctioned gas was exhausted without circulation to the headspace to prevent a decrease in the O2 concentration. In all the experiments for kLa determination, dissolved oxygen was measured while confirming that the O2 concentration in the headspace was equivalent to that in the atmosphere. The shaking conditions were constant at 200 rpm for the Erlenmeyer and cylindrical flasks and 120 strokes/min for the Sakaguchi flask. The working volume was set to 100 or 50 mL, and the kLa for each condition was calculated.Preliminary experiments confirmed that the O2 concentration in the headspace of shake culture in the Erlenmeyer and Sakaguchi flasks was very high compared with that in the atmosphere. Consequently, we measured CO2 concentration in the headspace of flasks with shaking in a non-steady state by using the CDMSS. kG, an indicator of CO2 ventilation capacity, was calculated as follows:t is time (s), 2 concentration at t\u2009=\u20090 (mg/L), and 2 concentration in fresh air. The minimum CO2 concentration, a very important factor for Eq. In this study, it was observed that the CO2 concentration decreased from 3.5 to 0.5%. When the R2 value of the approximate expression of Eq. kG. CO2 was added to the headspace of each flask via breathable culture stoppers until its level reached at least 5%. Shaking was initiated and after the CO2 concentration in the headspace had decreased to 3.5%, headspace CO2 concentration was measured every 20 s.Under all conditions, we obtained data until CO660  and pH of the culture broth were measured, which was sampled without interrupted shaking by CDMSS, using V-570 spectrophotometer  and pH meter , respectively. To ensure minimal decrease in the volume of the culture broth owing to sampling from the same flask, the total sampling volume was maintained at\u2009<\u200910% of the total amount of the initial culture medium. All measurements were performed in duplicates.The UOD2 and O2 in the gas\u2013liquid phases were monitored using CDMSS in shake culture  and the maximum growth in the Sakaguchi flask was 1.1 times that in the Erlenmeyer flask ([UOD660 of Sakaguchi flask: 3.67/UOD660 of Erlenmeyer flask: 3.36]). The maximum cell growth rate, kG, and kLa in baffled cylindrical flask were 2 times higher , identical, and 5.9 times higher  than those in the cylindrical flask, respectively.Oxygen supply capacity and ventilation capacity were quantified for comparison in different flasks and shaking conditions. The E. coli K12 IFO3301 cultivation. These shaking conditions are standard about batch culture on flask scale. It was suggested that the log-phase growth of E. coli K12 IFO3301 can be predicted by monitoring CO2 in the headspace of conventional shake flasks, because the CO2 concentration and the growth of E. coli K12 IFO3301 showed a very good correlation. Similar data have been reported by using a fluorescent sensor  and Sakaguchi (reciprocating shaker with 70 mm shaking diameter at 120 strokes/min) flasks for shaking conditions of 2 accumulation in aerobic culture. We reported that E. coli growth improves in shake culture in Erlenmeyer and Sakaguchi flasks when the CO2 of flask gas phase, which accumulates due to respiratory activity, is maintained at low concentration by CDMSS with gaseous CO2 adsorbent  was 6.4 (8.9/1.4) and the maximum growth ratio  was 1.4 (4.7/3.4). The CO2 concentration in baffled cylindrical flask and cylindrical flask was identical, despite the former supporting higher cell growth. This may be due to not only lower amount of medium and larger headspace but also sufficient ventilation in the baffled cylindrical flask. It is true that E. coli K12 IFO3301 growth requires a high kLa, but we conclude that not only high kLa but also high kG is required by comparison of various flasks. It was noted that the detailed monitoring of flask gas phase in shaking culture became difficult in the case of the flask with high ventilation capacity.We have an approach about shake-flask culture that goes beyond monitoring. It is possible that conventional shake culture using Erlenmeyer and Sakaguchi flasks has not previously taken into consideration the effect of CO2 has been reported to have a significant impact on culture growth (Blombach and Takors 2 concentration in the headspace can be maintained at low levels by adding adsorbent to the bypass part of CDMSS (Takahashi et al. 2 accumulation without using CO2 adsorbent. However, the handling of flask set on the shaking table was the same as that of the conventional flask, but when the medium volume was 100 mL without baffled, the oxygen supply capacity was significantly reduced due to the lack of formation of thin water film on the flask wall. Hence, we ensured that kLa increased by inserting a baffle and lower working volume, and allowing the culture broth that climbed up the wall of the flask to hit the baffle during rotary. At that time, the working volume was also changed, but the oxygen supply capacity was quantified so that comparison was possible even under different flask conditions. Therefore, we expect that redesign of flasks based on not only kLa but also kG (as shown in Fig. CO"}
+{"text": "We describe the kinetics of up-regulation of several memory-, anergy- and suppression-related markers such as CD44, CD73, FR4, CD25, CD28, PD-1, Egr-2, Foxp3 and CTLA-4 in this process. The conversion into suppressive Tr1 cells correlates with the transient intracellular CTLA-4 expression and required the restimulation of anergic cells in a short-term time window. Restimulation after longer time periods, when CTLA-4 is down-regulated again retains the anergic state but does not lead to the induction of suppressor function. Our data require further functional investigations but at this stage may suggest a role for anergic T cells as a circulating pool of passive cells that may be re-activated into Tr1 cells upon short-term restimulation with high and systemic doses of antigen. It is tentative to speculate that such a scenario may represent cases of allergen responses in non-allergic individuals.T cell anergy is a common mechanism of T cell tolerance. However, although anergic T cells are retained for longer time periods in their hosts, they remain functionally passive. Here, we describe the induction of anergic CD4 Although the molecular details, how anergy is induced and maintained, are increasingly understood , 2, aner+ Th1 T cell clones that were stimulated with only via CD3 antibodies  without co-stimulation and defined as functional unresponsiveness to further stimulation despite intact antigen presentation by MHC/peptide  and full costimulation via CD80/CD86   producing IL-10 may induce regulatory function in anergic T cells. In vitro exposure or injection of immature or semi-mature DC maturation stages of different DC subsets have been shown to induce either T cell anergy or the development into different subsets of Tregs, such as induced Foxp3+ Tregs (iTregs) or Foxp3- IL-10+ type 1 regulatory (Tr1) cells  cells , 13.+ T cells also appear in normal healthy mice. They can be identified by the surface marker profile CD44high CD73high folate receptor 4 (FR4)high and could convert into Foxp3+ Tregs after adoptive transfer where they prevented autoimmunity .C57BL/6, B6.OT-II.Rag1ain ITIB  was kind6 cells were resuspended in R10 medium (RPMI 1640 (Sigma) with 10% heat-inactivated FCS (Gibco), 100 \u00b5g/ml Penicillin-Streptomycin, 2 mM L-glutamine and 50 \u00b5M 2-mercaptoethanol ) containing 1.3 \u00b5M CCF solution (Invitrogen) and 3.6 \u00b5M Probenecid (Sigma) and incubated for 90\u00a0min at RT in the dark. After incubation, the cells were washed with FACS buffer and subsequently stained in FACS buffer containing 10% 2.4G2 hybridoma cell line supernatant . Surface staining was performed with antibodies to CD25 (PC61), CD28 (E18), CD4 (GK1.5), CD44 (IM7), CD73 (TY/11.8), FR4 (12A5), PD-1 (RMP1-30), V\u03b12 (B20.1), DO11.10 TCR clonotype (KJ1-26)  and V\u03b25.1, 5.2 (MR9-4) (BD Biosciences). In the case of staining with biotinylated antibodies, surface staining was followed by a second incubation with fluorophore-conjugated streptavidin. Subsequently, the cells were fixed with 1% formaldehyde and analysed on a BD LSR II flow cytometer.For CCF4-substrate loading, 1x10Cells were stimulated in R10 medium containing 10 ng/ml PMA, 1 \u00b5g/ml Ionomycin and 5 \u00b5g/ml Brefeldin A  for 4 hours at 37\u00b0C. After CCF4-substrate loading and surface staining, the cells were fixed and permeabilized using the Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent (eBioscience) according to manufacturer\u2019s instructions. Intracellular and intranuclear staining was performed in 1x Permeabilization Buffer (eBioscience) using the following antibodies: CTLA-4 (UC10-4B9), Foxp3 (150D), IFN-\u03b3 (XMG1.2), Ki-67 (16A8)  and Egr-2 .7 bulk lymph node and spleen cells from DO11.10 or (ITIB.)OT-II mice or 4.5-7x106 (ITIB.)OT-II.Rag1-/- cells into BALB/c or C57BL/6 recipient mice, respectively. One day later, anergy was induced by injection of 275 \u00b5g OVA327-399 peptide (China Peptides) or 400-1000 \u00b5g OVA protein . Control mice received PBS injections instead of OVA. For Tr1 conversion experiments, the mice received a second OVA injections 3 days  or 11 days  after the first one.Adoptive T cell transfer was performed by i.v. injection of 1-2x10in vivo, 5 days after the last OVA injection, erythrocyte-lysed spleen cells were directly FACS analysed for their frequency of CD4+ KJ1-26+ and further for the intracellular CTLA-4 or intranuclear Foxp3 among them. For determination of suppression in vitro, 5 days after the last OVA injection, erythrocyte-lysed spleen cells were enriched for CD4+ cells by magnetic cell separation (MACS) according to the manufacturer\u2019s instructions. To test their suppressor capacity these CD4+ cells were added to CFSE-labelled MACS-enriched CD4+ T cells from BALB/c lymph nodes stimulated with anti-CD3 antibodies and irradiated spleen APCs as we performed before  or 11 days  or PBS. For anergy determination, 3 days after the last OVA injection erythrocyte-lysed spleen cells were labelled with CFSE and restimulated with 100 U/ml mouse IL-2 (Peprotech), anti-CD3 alone or anti-CD3 and anti-CD28 antibodies  or 10 \u00b5M OVA peptide. FACS analysis for CD4, KJ1-26 and CFSE of spleen cells was performed after 5 days to analyse the proliferation history of the DO11.10 T cells. For determination of suppression d before . After 5Data are presented as mean \u00b1 SEM or SD as indicated in the figure legend. Unpaired t-tests were used for comparison of cytokine production after first and second OVA injection. The statistical differences between more than two groups were calculated using one-way ANOVA with Tukey\u2019s test for multiple comparisons. All statistical analyses were performed using PRISM 7 (GraphPad).in vivo by intravenous injection of high dose antigen  on day 3 after OVA injection. However, after accumulation, the cells collapsed rapidly again resulting in a frequency of only 2% of OT-II cells within all CD4+ T cells on day 5   . We did  setting -acetoxym setting . After e setting . Therefo\u2212 IL-10+ phenotype supporting our hypothesis of conversion into Tr1 cells after a second antigen encounter within a short time interval.Taken together, anergic T cells acquire a Foxp3in vitro that anergic cells up-regulated CTLA-4 and its high expression was maintained and required for subsequent Tr1 generation. When the second antigen stimulation occurred during the phase when CTLA-4 was already down-regulated again, the cells remained anergic but failed to become IL-10+ Tr1 cells . The mild stimulation by OVA injection in the absence of adjuvants did not allow their survival. Anergic cells undergo deletion during the contraction phase, an accompanying mechanism that has been reported to be associated with T cell anergy induction in vivo   as compared with earlier ones  and 3 (Egr-3) for expression of the anergy-associated transcriptional program, as identified in clonal anergy and in y models , 28, 29. and simultaneously suppressed effector cell proliferation. This may indicate a role for persisting anergic T cells as a memory cell pool for Foxp3\u2212 Tr1 cells in vivo. The long-term protocol for the OVA injections led to persistence of the anergic state but not the acquisition of regulatory functions. A deeper understanding of the mechanisms that allow for reactivation of this memory compartment will be useful for the development of optimized vaccination protocols that may allow for prevention or suppression of autoimmune or allergic diseases.Here we characterized a population of anergic CD4The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Regierung von Unterfranken, AZ 52/14.AT, TS, LC, and IE performed the experiments and analysed the data. AT, AK, and ML compiled the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the DFG grant LU851/14-1 for MBL and the Interdisciplinary Center for Clinical Research (IZKF) project A-408 for AK and MBL and AdvCSP-2 for AK. We thank Marion Heuer for her expert technical assistance on the project. This publication was supported by the Open Access Publication Fund of the University of W\u00fcrzburg.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": "Background: For children with acute appendicitis (AA), a clear diagnosis is a challenge. The purpose of this study is to explore whether inflammatory markers in the blood combined with symptom duration are helpful in the diagnosis of acute appendicitis and in predicting the severity of acute appendicitis.Methods: All the selected patients underwent appendectomy between November 10, 2011 and November 15, 2019, in whom preoperative WBCC, CRP, and NE% had been measured in a short time. All patients were divided into two groups: uncomplicated AA and complicated AA, postoperatively.Results: For our standards, 813 patients were selected, 442 of them had complicated AA. The mean [standard deviation (SD)] age for the uncomplicated AA group was 9.78 \u00b1 2.02 years and for the complicated AA group was 9.69 \u00b1 2.16 years (P = 0.55). Elevated WBCC, CRP, and NE% had a higher relatively sensitivity in complicated AA than uncomplicated AA especially when WBCC, CRP, and NE% were at normal levels, which had a sensitivity of 100% in uncomplicated AA, but this only applied to nine patients. CRP values were significantly different in three time groups, whether uncomplicated or complicated AA.Conclusion: The combination of WBCC, CRP, and NE% values is very sensitive for the diagnosis of acute appendicitis, and when we predict complicated AA using the CRP value, we also need to consider the time of symptom onset. Acute appendicitis (AA) is one of the most common acute abdominal diseases in children . HoweverAA is a rapidly progressing disease that can adversely affect patients if it is not accurately diagnosed and properly treated. And mostly luminal occlusion due to various etiologies and bacterial infections of the appendix are held responsible in the pathogenesis of this disease which is mostly seen in adolescents . As wellThe inflammatory response to AA is progressive , white bPatients undergoing appendectomy in the General Surgery Department at the single institution of Chongqing Children's Hospital were identified. We reviewed medical records of the surgical patients who underwent appendectomy between November 10, 2011 and November 15, 2019. The study was approved by the ethics committee of children's Hospital Affiliated to Chongqing Medical University. The patients were not required to provide written informed consent for this study. And the information we obtain does not relate to any patient's personal data. All patients were selected for the diagnosis of AA, which was obtained through surgical records and pathological diagnosis. If we find that the diagnosis of intraoperative findings or postoperative histological findings are inconsistent, then we choose a more advanced pathological diagnosis as the final diagnosis. Patients were considered eligible for entry into the study upon meeting the following inclusion criteria: age <18 and >7 years, the first diagnosis was AA with concomitant surgical treatment rather than elective surgical treatment for AA, no antibiotics were given before admission, no severe sepsis, no steroids, or immunosuppressive medication administration. Exclusion criteria included patients who have diseases of the blood system and other infectious diseases, the patient's postoperative pathological specimen showing a normal appendix, and admission of more than 12 h without operating. Standard perioperative variables were collected including age, gender, time interval between symptom onset and admission, time interval from admission to operation, preoperative white blood cell count, C-reactive protein, neutrophil percentage, intraoperative, and pathological diagnosis. The more serious diagnoses were chosen. The time when fever, nausea, vomiting, or abdominal pain was reported by the patients was defined as the time of symptom onset. All blood specimens were obtained after admission, and all patients were tested for blood inflammatory markers. From admission to surgery, we used antibiotics once in our institution . Reference intervals for WBCC were dependent on the age of patients as previously reported  Table 1Table 1, do score , the uppIn General, operative and pathologic findings were graded G1 for simple or suppurative AA, G2 for gangrenous AA, G3 for perforated AA or a phlegmon, and G4 for a periappendicular abscess . In ordet-test or one-way ANOVA test, respectively. Results were considered statistically significant if p < 0.05.The SPSS 24.0  software was used to perform all the statistical analyses. Means [standard deviations (SDs)] were used to describe the numerical variables. Categorical variables were described as frequencies with percentages and were analyzed with Chi-square test or Fisher exact test, as appropriate. Continuous data were expressed as the means \u00b1 (standard deviations), maximum and minimum for normally distributed data, which were tested with the Student's n = 441, 54.2%). In both groups, the vast majority of patients were boys, and there was no difference in male-female age between the uncomplicated and uncomplicated AA groups.A total of 813 children aged 7\u201318 years were finally studied after inclusion and exclusion criteria. The number of patients, gender, and mean age for each pathology grade group are shown in The estimated sensitivity of elevation of WBCC, elevation of CRP, elevation NE%, as well as the normal levels of the three inflammatory markers  in pathologically confirmed AA is shown in Analysis of type of AA and elapsed time is shown in According to pre-hospital time, we divided the patients into three groups. Values of WBCC, CRP, and NE% in children undergoing appendectomy for AA according to surgical and pathologic findings are shown in The relationship between WBCC, CRP, and NE% values in different periods of time in the same type of AA is shown in AA is one of the most common acute abdominal issues in pediatric surgery , 23. TimMohammed Elniel et al. pointed out 72 h is the time critical point to operate in AA . SometimIn our study, the sensitivity of elevation of WBCC, elevation of CRP, and elevation of NE% in complicated AA was more than in uncomplicated AA. However, we found that elevated CRP had a relatively low sensitivity in uncomplicated AA and upper sensitivity in complicated AA. It seems that CRP is not a useful inflammatory marker for uncomplicated AA, though it does seem useful for detecting complicated AA. Monsalve et al.  in a stuOn the other hand, an elevation of at least one inflammatory marker  appeared to be the most sensitive indicator of AA, with 97.6% in uncomplicated AA, 100% in complicated AA, and 98.9% in total. However, the sensitivity of the three combined tests is extremely high, this does not completely rule out AA. This seems to confirm Jasper J. Atema's viewpoint that no level of inflammatory markers could sufficiently exclude the presence of AA in his article .WBCC value, CRP value, and NE% value are higher in complicated AA than in uncomplicated AA, in accordance with most previous reports , 19, 34.The present study has certain limitations that need to be considered when interpreting the data. First, the data were collected locally from a single institution, and as a retrospective study, there are some records that are not so accurate, such as whether antibiotics were clearly used before admission, duration of symptoms, and so on. Second, there were only 11 patients in the group of uncomplicated AA with symptoms lasting from 48 to 72 h, which brought some limitations to our analysis. In addition, we chose children between 7 and 18 years old, which brings some limitations to our analysis of children's AA and age factors.The WBCC, CRP, and NE% levels together are very useful laboratory tests for diagnosing AA. A combination of normal WBCC, CRP, and NE% values seems to be very useful in predicting uncomplicated AA, and the severity of AA is related to the duration of symptoms, but a small time interval between admission and operation seems to not have a significant impact on the severity of AA. CRP and NE% values are superior to WBCC value in predicting complicated AA. But when we use CRP value to predict complicated AA, we have to consider the duration of symptoms. In this respect, it seems that the NE% value is better than the CRP value.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the ethics committee of Chongqing Medical University. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.JL and QL designed and analyzed the data and evaluated the manuscript. JL, HZ, and CG performed the statistical measurements and analyzed the data. CG and JL analyzed the data and wrote the paper. All authors contributed to the article and approved the submitted version.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": "Concern around declining bee populations globally has become an environmental issue of mainstream importance. Policymakers, scientists, environmental non-government organizations, media outlets and the public have displayed great interest in conservation actions to support pollinators. As with many environmental causes, green washing, or in this case \u2018bee washing\u2019, has become rampant. Bee washing can lead to multiple negative consequences, including misinformation, misallocation of resources, increasing threats and steering public understanding and environmental policy away from evidence-based decision-making. Here I will discuss the multiple potential consequences of bee washing on efforts to conserve declining wild bees and promote wild bee health.  \u2022Concern around declining bee populations globally has become an environmental issue of mainstream importance.\u2022Policymakers, scientists, environmental non-government organizations, media outlets and the public have displayed interest in conservation action to support pollinators.\u2022\u2018Bee washing\u2019, has become rampant.\u2022Narratives and actions tend to focus on low-hanging fruit, actions which are easy to address and/or the selling of commercial items where industry benefits but the species of concern do not.\u2022Negative consequences include misinformation, misallocation of resources, increasing threats and steering environmental policy away from evidence-based decision-making. Over recent decades, the plight of wild bees and other pollinators has gone from a niche area to one of the most mainstream environmental topics . Scienti\u2018Bee washing\u2019, a term coined by The crux of the matter is that when conservation actions are misplaced, as in the case with bee washing, they can actively harm populations meant to be conserved, waste limited resources , misinform the public and/or de-legitimize scientific evidence . MisplacBee washing initiatives which promote managed species contribute to one of the main threats to wild bee health. The spillover of pathogens from managed species to closely related species has been documented in a variety of taxa, often with devastating consequences for wild populations . ApproxiOsmia, Megachile), often under the bee washing guise of solitary bee support, and their disease dynamics are completely unstudied , the Buff-tailed Bumblebee (Bombus terrestris) as well as other managed bee species outside their native ranges has resulted in multiple negative impacts to local bee fauna associated with competition and invasion. Hives of bumblebees and honeybees are moved around within and between countries and the escape of bees and interactions with local fauna are not monitored (While there are several other documented and suspected drivers of wild bee decline globally, the proliferation and widespread use managed bees has become an issue of increasing conservation concern and one that bee washing activities may exacerbate. With the increase in mainstream concern about bees, the popularity of managed honeybees has also increased in urban areas as part of \u201csustainability\u201d initiatives . A recenonitored . Numerouonitored  and devaonitored . This isonitored , especiaonitored  and qualIn order to better protect wild bee health and biodiversity, conservation actions and policy will have to shift away from bee washing to more evidence-based, nuanced and precautionary approaches. Efforts should focus on reducing the reliance of systems on managed bees and reducing the impacts of managed bee use on wild bees. This will require policy which acknowledges and values the importance of wild bee health biodiversity for pollination services for crop plants and for resilience under climate change. Critical actions to protect wild bee health includes screening and monitoring of pathogens among commercial stock and in adjacent wild populations. Legal frameworks should be developed for the restrictions of bee movement outside of native ranges  and densIt is increasingly understood that human dimensions of conservation issues can have a significant impact on conservation outcomes . Law, poNone."}
+{"text": "This review provides a summary of recent progress in the development of different nano-platforms for the efficient synergistic effect between photodynamic therapy and chemotherapy. In particular, this review focuses on various methods in which photosensitizers and chemotherapeutic agents are co-delivered to the targeted tumor site. In many cases, the photosensitizers act as drug carriers, but this review, also covers different types of appropriate nanocarriers that aid in the delivery of photosensitizers to the tumor site. These nanocarriers include transition metal, silica and graphene-based materials, liposomes, dendrimers, polymers, metal\u2013organic frameworks, nano emulsions, and biologically derived nanocarriers. Many studies have demonstrated various benefits from using these nanocarriers including enhanced water solubility, stability, longer circulation times, and higher accumulation of therapeutic agents/photosensitizers at tumor sites. This review also describes novel approaches from different research groups that utilize various targeting strategies to increase treatment efficacy through simultaneous photodynamic therapy and chemotherapy. Table of ContentsIntroduction ........................................................................................................................................................................31. \u20031.1. Principles of Photodynamic Therapy .........................................................................................................................3\u20031.2. Mechanism of Photodynamic Therapy ......................................................................................................................4\u20031.3. Photosensitizers ............................................................................................................................................................5Combination of Photodynamic Therapy and Chemotherapy .....................................................................................52. \u20032.1. Combination of Photosensitizers and Chemo-Drugs without External Carriers..................................................5\u2003\u2003\u20032.1.1. Photosensitizers as Carriers .............................................................................................................................5\u2003\u2003\u2003\u2003\u2003MXenes..................................................................................................................................................................6\u2003\u2003\u20032.1.2. Photosensitizer-Drug Materials .......................................................................................................................9\u20032.2. Combination of Photosensitizers and Chemo-Drugs with External Carriers .......................................................9\u2003\u2003\u20032.2.1. Transition Metal Based Nano-Platforms .......................................................................................................12\u2003\u2003\u2003\u2003\u2003Synthesis Routes of Transition Metals Nano-Platforms.................................................................................12\u2003\u2003\u2003\u2003\u2003Application of Transition Metals in PDT.........................................................................................................13\u2003\u2003\u20032.2.2. Silica ..................................................................................................................................................................14\u2003\u2003\u2003\u2003\u2003Synthesis Routes of Silica..................................................................................................................................15\u2003\u2003\u2003\u2003\u2003Application of Silica in PDT..............................................................................................................................15\u2003\u2003\u20032.2.3. Graphene ..........................................................................................................................................................16\u2003\u2003\u2003\u2003\u2003Application of Graphene in PDT......................................................................................................................17\u2003\u2003\u20032.2.4. Liposomes ........................................................................................................................................................18\u2003\u2003\u2003\u2003\u2003Synthesis Routes of Liposomes........................................................................................................................19\u2003\u2003\u2003\u2003\u2003Application of Liposome in PDT......................................................................................................................19\u2003\u2003\u20032.2.5. Dendrimers ......................................................................................................................................................20\u2003\u2003\u20032.2.6. Polymers ..........................................................................................................................................................20\u2003\u2003\u2003\u2003\u2003Application of Dendrimers in PDT..................................................................................................................20\u2003\u2003\u20032.2.7. Metal-Organic Frameworks ...........................................................................................................................20\u2003\u2003\u2003\u2003\u2003Main Synthesizing Methods.............................................................................................................................20\u2003\u2003\u2003\u2003\u2003Application of Polymers in PDT......................................................................................................................20\u2003\u2003\u20032.2.8. Biological Nanocarriers .................................................................................................................................24\u2003\u2003\u2003\u2003\u2003Preparation Method of Metal\u2013Organic Frameworks....................................................................................24\u2003\u2003\u2003\u2003\u2003Application of Metal\u2013Organic Frameworks in PDT......................................................................................24\u2003\u2003\u20032.2.9. Nanoemulsions ...............................................................................................................................................25\u2003\u2003\u2003\u2003\u2003Preparation of Red Blood Cells Membranes-Derived Vesicles....................................................................25\u2003\u2003\u2003\u2003\u2003Application of Biological Nanocarriers in PDT.............................................................................................25\u2003\u2003\u20032.2.10. Nano Emulsions............................................................................................................................................26\u2003\u2003\u2003\u2003\u2003Synthesis Routes of Nano Emulsions..............................................................................................................26\u2003\u2003\u2003\u2003\u2003Application of Nano Emulsion in PDT...........................................................................................................26\u20032.3. Targeting Strategy......................................................................................................................................................26\u2003\u2003\u20032.3.1. pH Triggered...................................................................................................................................................27\u2003\u2003\u20032.3.2. Enzyme Triggered...........................................................................................................................................28\u2003\u2003\u20032.3.3. Redox Triggered Agents.................................................................................................................................28\u2003\u2003\u20032.3.4. Chemical and Biological Targeting Agents...................................................................................................29Conclusions and Outlook ..............................................................................................................................................303. In 2019, 1,762,450 new cancer cases and 606,880 cancer deaths were projected to occur in the United States alone ,4,5. DueSome studies have recently investigated the efficacy of combining chemotherapy and PDT . For exaIn 1903, von Tappeiner and Jesionek proposed the first published report on the use of PDT as a treatment for skin tumors by using tropical eosin and exposing it to light ,16. TheyIn PDT, a photosensitizer should be excited by a specific wavelength of light that causes two different types of reactions to occur: type I and type II photochemical reactions . As showType I reactions occur where the triplet state forms radicals with biomolecules such as lipid radicals that can further react with other biomolecules and then oxygen to form reactive oxygen species (ROS) such as hydroxyl radicals and hydrogen peroxides. Additionally, these excited electrons can react directly with molecular oxygen to produce the superoxide anion radical, which can form other ROS species . Type II1O2 yield (singlet oxygen), the distribution of PSs, depth penetration of the light, and molecule stability. The generation of singlet oxygen is a very important factor for PDT because of its extreme cytotoxicity in PDT [An\u2212x/n]\u00b7zH2O as a cationic nanocarrier to deliver the anti-cancer prodrug c,c,t-[diamine-dichlorodisuccinato-platinum(IV)] and photosensitizer chlorin-e6 to improve the activity of cisplatin in cisplatin-resistant human cancer cells 4-zinc \u03b2-tetra-(4-carboxyl benzyloxyl)phthalocyanine (PDCZP) that showed better in vitro and in vivo anticancer effects under lighting on MCF-7, SW480, and HepG2 cells and the murine hepatocellular carcinoma H22 mode.Albumin is a versatile protein with a unique structure that can be conjugated to hydrophobic and hydrophilic components ,232. In 6 clusters coordinated with terephthalic acid to form UiO-66, which has microporous cages and excellent stability, so this UiO-66 can be considered as a suitable candidate for drug loading [Nanoscale metal organic frameworks are different types of hybrid porous nanomaterials that can be prepared by the coordinated interaction of metal ions and bridging ligands. Metal\u2013organic frameworks have great potential in drug delivery systems due to their large surface area, high porosity, and modifiable surface chemistry . Metal\u2013o loading ,237.In the solvo-thermal method, metal and organic precursors are added to an organic solvent and are stirred at room temperature until a clear solution is formed. Then, the homogenous mixture is transferred to a Teflon-lined autoclave and heated for 12 or 24 h. Finally, the desired product is separated via centrifugation or filtration. These MOFs can be modified by different organic ligands in the next levels ,239.3 NMOFs), and bioreductive banoxantrone (AQ4N), which was anchored to the nanocarriers by a phosphate ion-sensitive bond. Photosensitizers such as photochlor (HPPH) and azide were anchored to UiO-66 by the solvo-thermal method [2-depleting (consuming) PDT process does indeed aggravate intracellular/tumor hypoxia that activates the cytotoxicity of AQ4N through a cascade process, consequently achieving PDT-induced and hypoxia-activated synergistic therapy. Benefiting from the localized therapeutic effect of PDT and hypoxia-activated cytotoxicity of AQ4N, this hybrid nanomedicine exhibits enhanced therapeutic efficacy with negligible systemic toxicity, making it a promising candidate for cancer therapy. In 2020, Zhang and coworkers [In 2019, He and co-workers  designedl method . Moreoveoworkers   and shape make them different from conventional emulsions. They consist of two immiscible liquids that are mixed by different types of surfactants.There are several methods for the preparation of nano emulsions. However, all methods can be classified into two main methods: high-energy emulsification such as stirring, ultrasonic emulsification, high-pressure homogenization, micro-fluidization, and low-energy emulsification such as phase inversion temperature, emulsion inversion point, and spontaneous emulsification . In the In 2018, Maria Candido and co-workers  preparedVarious strategies such as pH triggered, enzyme triggered, chemical targeting agents, biological targeting agents, and redox triggered agents have been used as targeting strategies to deliver drugs to a specific part of the body . PhotoseThe important point in this strategy is the physiological differences in pH. Internal cellular structures such as lysosomes have reduced pH (~pH = 4) and certain cancerous cells are more acidic than normal healthy cells. Materials can exploit this feature and are considered pH responsive and upon reaching a lower pH environment, this trigger should be able to collapse, swell, or change the nanocarrier according to the change in the pH of their environment ,258,259.l-aspartate) (PBLA) to encapsulate zinc(II) phthalocyanine and doxorubicin via an acid-labile hydrazone linker for HepG2 human hepatocellular carcinoma cells. In 2014, Shi and co-workers [cis-aconitic anhydride bond between Zn and PEG was cleaved and caused DOX to be released.For example, Gao and co-workers  introduc-workers  prepared-workers . Then, t-workers . In all -workers  developep-hydroxyphenyl) porphine (acryloyl-THPP) reacted with 4,4\u2032-trimethylene dipiperidine (TMPD) to form pH-responsive crosslinked biodegradable \u03b2-amino esters (BAEs) that can release DOX into the tumor microenvironment. Ruan and co-workers  synthesi3O4 nano-platform was prepared and DOX was loaded to be released by the enzymatic degradation of gelatin by the presence of gelatinase. It was observed that this nano-platform not only increased the accumulation of drugs in human breast cancer cells (MCF7), but also increased the level of ROS production. In 2019, Cui and co-workers [Different cancerous cells upregulate the expression of certain enzymes on their surface such as metalloproteinase and cathepsins. This phenomenon requires future studies to help researchers develop novel targeting agents according to those upregulated enzymes . Along t-workers  designed-workers . HypoxiaThere are various reduction/oxidation (redox) reactions in cells, but cells continually regulate ROS levels to maintain natural physiological baseline levels. However, when cells turn cancerous, they alter their microenvironment, which can be exploited to develop drug delivery systems. There are special microenvironments in cancerous cells. In these cells, the level of glutathione and reactive oxygen species are higher than in healthy cells. Therefore, according to the level of glutathione in tumor cells, redox responsive drug delivery systems have been introduced to enhance the targeting ability of DDSs and decrease the side effects of therapeutic agents . For exaPlatin prodrugs can also act as redox responsive materials. Their bond can be reduced to facilitate the anti-cancer drug platin to be released. For example, Lim and co-workers and Wang and co-workers used oxaliplatin prodrug cis-platinum to develop a redox responsive nano-platform. Both groups observed that platin could be released only by changing the levels of glutathione in tumor cells, which is very significant, in order to decrease the side effects of highly toxic platin-based drugs . In otheTargeting ligands can be conjugated on the surface of nanocarriers to enhance accumulation and selectivity for certain cells or tissue types. Different materials such as hyaluronic acid, proteins/peptides, antibodies, carbohydrates/polysaccharides, aptamers, and folic acid have been tested for their ability to improve the selectivity of drug delivery systems . For exaCD44 usually binds with hyaluronic acid, which is one of the main materials of the extracellular matrix. Moreover, hyaluronic acid based materials can be degraded by hyaluronidase, which is abundant in tumor cells ,267. In Tumor homing peptides can be considered as other promising chemical targeting agents, for example, Lyp-1, which has the p32 protein (HABP1 or C1QR protein) as its receptor, can be conjugated to various drug delivery systems to increase their targeting abilities. Lyp-1 has nine amino acids and its receptors are overexpressed on the surface of cancer cell lines such as the 4T1, MDA-MB-435, and MCF-7 cell lines. Another peptide that can be considered as the targeting agent is the KE108 peptide, a synthetic nanopeptide that can efficiently target all five subtypes of somatostatin receptors overexpressed by neuroendocrine tumor cells. For example, Li and co-workers  conjugatchlorin-e6 targeted with an OV-TL16 mAb in OVCAR-3 carcinoma xenografts. The OV-TL 16 antibody can identify the OA-3 antigen, which is expressed on OVCAR-3 cells and on most human ovarian carcinomas, and dramatically increased the accumulation of nanocarriers in tumors. In 2008, Hongrapipat and co-workers [chlorin-e6 over nontargeted conjugates. In 2018, Wang and co-workers [Antibody-targeted nanocarriers for cancer therapy have an exceptional role because of their specificity and vital advantages. Most monoclonal antibodies (mAbs) are used to target nanocarriers to cancer-specific antigens, deliver chemodrugs and photosensitizers in the form of antibody\u2013drug conjugates, and recruit cytotoxic T cells to combat cancer cells . For ins-workers  confirme-workers  investig-workers  with the-workers  on the s+ T cells and CD4+ T cells in distant tumors.There are also other biological targeting agents. In 2016, He and co-workers  fabricatIn the present review, various types of photosensitizers, their synthetic strategies, and the synergistic effect of PDT and chemotherapy were explored. This review split photosensitizers into two main categories that can be defined as with or without external carriers. In the first category, photosensitizers can act as either the drug or carrier, so there is no need for external carriers or drugs to achieve the synergistic effect of PDT and chemotherapy. In the second category, various external carriers such as transition metals, silica, graphene, liposomes, dendrimers, polymers, metal\u2013organic frameworks, and different types of biological carriers are used to deliver photosensitizers and chemo\u2013drugs to specific tumor sites. Furthermore, there are other strategies to increase the efficiency of treatment and decrease the side effects. For example, pH, redox, and enzyme triggered methods and/or surface modification by different chemicals have been used to increase the selectivity of photosensitizers and their carriers and potentially reduce or eliminate unwanted side effects. Although lots of photosensitizers have been proven to be efficient, the assessment of their cytotoxicity, biocompatibility, bio-distribution, and excretion are very important and are currently under investigation. Finding more convenient ways for treatment such as the oral administration of photosensitizers can further expand PDT to gain even more attraction. Finally, the mechanism of cellular uptake and the fate of different types of photosensitizers in the human body is key to their utilization. Appropriate strategies and model systems are required to obtain comprehensive knowledge about such complicated processes before using these materials in clinical trials. Eventually, we believe that with all of these therapeutic strategies and methods, the combination of PDT and chemotherapy has great potential and may soon move beyond model organisms."}
+{"text": "Previous research and treatment of coronary heart disease mostly focused on the large epicardial vessels, with limited research on the small endocardial coronary arteries or arterioles that could not be detected by coronary angiography, especially microvascular angina caused by microvascular stenosis or microcirculation dysfunction. Conventional Western medicine therapies have no specific efficacy, but traditional Chinese medicine has significant advantages in this regard. In particular, traditional Chinese medicine of supplementing Qi and activating blood circulation protects the vascular endothelium, relaxes coronary microvessels, reduces myocardial no-reflow after ischemia-reperfusion, increases myocardial hypoxia tolerance, constrains the aggregation of platelet, and increases the rate of blood flow. Moreover, these treatments can significantly improve patients' symptoms through multitarget comprehensive intervention. Here, we analyzed the pathogenesis of microvascular angina pectoris, the treatment status of modern medicine, and the research on the multitarget intervention of traditional Chinese medicine to provide new research ideas for correctly identifying the role of coronary microcirculation in coronary artery disease to solve clinical problems and prevent cardiovascular events. Therefore, interventional therapy can only solve the stenosis of large vessels and not the stenosis and dysfunction of small arterioles and microarteries. Furthermore, arterioles, microarteries, and venules together constitute the microcirculation of the coronary artery, which is the primary resistance vessel bed and myocardial metabolism site of the coronary artery and the main (95%) contributor to coronary resistance [Previous studies  on coronsistance . Therefosistance .Even if there is no obvious stenosis of large blood vessels in clinical, an increase in microcirculation resistance can also lead to insufficient myocardial perfusion and ischemic heart disease events. A clinical study on the evaluation of symptoms of myocardial ischemia in the United States  demonstrThe anterior arteriole is the main functional site for regulating myocardial blood perfusion. When the regulatory mechanism of anterior arterioles is unbalanced due to local nerve and body fluid factors or abnormal contraction of blood vessels distributed in sheets, the release of adenosine from distal local myocardial tissue will increase due to ischemia, which acts on afferent nerves and causes chest pain. Simultaneously, the arterioles regulated by metabolism expand and the intraluminal pressure decreases, resulting in further narrowing of anterior arterioles and arteriolar branches. The continuous increase in adenosine concentration in myocardial tissue leads to chest pain, which cannot be relieved . Patient\u03bcm\u20135\u2009mm epicardial coronary artery) and the resistance vessel (with a diameter of <500\u2009\u03bcm). These tiny coronary arteries, which cannot be directly observed by angiography, constitute coronary microcirculation; the aforementioned provides important vascular resistance under normal conditions and determines the blood flow, redox, inflammation, agglutination, and other reactions of the coronary artery through metabolic regulation to meet the oxygen supply to the myocardium and myocardial perfusion [Coronary microcirculatory dysfunction (MCD) is currently considered  to be imerfusion . BecauseAt present, the main methods to evaluate myocardial microcirculation , 16 inclUnder physiological conditions, the coronary microcirculation forms a mutually restrictive balance regulation mechanism by myogenic, blood flow-dependent, and metabolic contraction and expansion of coronary arterioles to maintain normal blood perfusion. Coronary microcirculation is an important factor in the development of coronary heart disease. Smoking, the elderly, hyperlipidemia, diabetes, and mental stimulation can all affect the function of microcirculation in small coronary artery endothelial cells , 18.Current studies  believe Coronary microvascular endothelial dysfunction (CED) is an important link in the occurrence and development of MVA. Normal coronary endothelial cells can not only act as a mechanical barrier but can also receive and transmit information. In addition, they also have endocrine and metabolic functions and control and release a series of vasoactive substances, such as nitric oxide, prostacyclin, and endothelin. Angiotensin II is used to maintain the steady state of vasomotor and contraction and achieve the balance of myocardial perfusion and oxygen supply . MaintaiBecause the diameter of the small coronary artery exceeds the resolution of angiography, there are no effective research technical means to confirm whether microvascular lesions occur based on coronary arteriosclerosis or are caused by microvascular stenosis. Supporting evidence  related Patients with microvascular angina pectoris often have increased levels of various inflammatory factors , especiaThe imbalance of cardiac autonomic nerve function changes coronary artery microcirculation, which is considered to be related to MCD . In partAmong the above mechanisms, CED is considered the primary pathogenesis of MVA caused by MCD. Endothelial cells usually regulate blood flow by releasing diastolic and systolic factors, which act on the coronary smooth muscle and change the lumen diameter and blood flow, resulting in chest pain symptoms and ST segment changes. Coronary endothelial cell disorder causes excessive secretion of systolic factors and insufficient secretion of diastolic factors , which l\u03bcm. The thinner the vessels, the smaller the dilation effect. Although calcium channel blockers [MVA is a recent research hotspot, and its pathogenesis includes many of the aspects mentioned above. Therefore, drug intervention should target multiple pathways, including the microvascular endothelium, microcirculation, inflammatory factors, platelet function, and myocardial and vascular function and regulation. At present, Western medicine has no specific therapeutic drugs for MVA. The existing drugs for treating coronary heart disease have a single mechanism, which can only intervene during disease unilaterally and not be adjusted from the whole mechanism. For example, traditional nitrates , 37 can blockers  can prevblockers \u201340 can sblockers  have shoblockers , 42. Nicblockers  can alleblockers \u201346 can pblockers .MCD is an important pathogenesis of MVA and an independent predictor of adverse cardiovascular events. The dysfunction of coronary microvascular endothelial cells is considered  to be thAstragalus flavone, Astragalus saponin, and Astragalus polysaccharide, and the extracts of blood-activating drugs [Uncaria total alkaloids, and curcumin, have protective effects on vascular endothelial cells.Traditional Chinese medicine focuses on syndrome differentiation and treatment under the guidance of the overall concept. It aims to regulate the balance of Yin and Yang and to prevent diseases. For example, for MVA, traditional Chinese medicine can prevent cardiovascular events by multitarget comprehensive interventions, such as protecting the vascular endothelium, alleviating vasospasm, and improving microcirculation, with both anti-inflammatory and antiplatelet effects. Compared to Western medicine, the targeted effects of traditional Chinese medicine monomer or compound on diseases are relatively mild; this is reflected in the \u201cmicroeffect,\u201d which reduces the possibility of toxic side effects and drug residues while pursuing a long-term curative effect. Recently, traditional Chinese medicine has made remarkable achievements in the prevention and treatment of microvascular angina pectoris  and has ng drugs , such asng drugs , such as\u03b1 and IL-6 to increase the level of serum NO and improve the vasomotor function of vascular endothelial cells. Moreover, Danshen decoction [The components of traditional Chinese medicine are complex, with various pharmacological activities and different combinations. Therefore, its treatment mechanism can be reflected in many aspects, which are embodied in \u201cmultiple causes.\u201d Recently, many studies have shown that traditional Chinese medicine decoctions, Chinese patent medicines, and traditional Chinese medicine injections commonly used in clinics have beneficial effects in the prevention and treatment of MVA. For example, clinical observation results show that Baoyuan decoction , compounecoction , Ginsengecoction , and Zhiecoction  can signecoction  can signSalvia miltiorrhiza polyphenolate [Salvia miltiorrhiza dropping pills [Salvia miltiorrhiza dropping pills [Chinese patent medicine has the advantages of a definite curative effect, less toxic side effects, and convenient use for treating microvascular angina pectoris. Clinical data show that Tongmai Yangxin pill , Yixinshhenolate , compounng pills \u201373, Danhng pills , 80, comng pills \u201373, and ng pills  prevent ng pills \u201363, Shexng pills , 65, Tonng pills , Shenqi ng pills , and Sheng pills  can inhiIn conclusion, traditional Chinese medicine can restore the balance of the body through \u201cmulticause and microeffect,\u201d integration and coordination, and comprehensive intervention to restore the balance of the body, representing significant advantages in the treatment of diseases and providing new ideas for the prevention and treatment of coronary heart disease. Additionally, by summarizing the relevant literature, we found some problems in the current research. Firstly, there are few clinical studies of traditional Chinese medicine in this area, particularly clinical studies with high-level evidence-based medicine. Most clinical studies have problems such as small sample size, short research cycle, insufficient attention to the prognosis of the disease, and limited follow-up cycle. Secondly, the current clinical diagnostic technology of microvascular angina pectoris mostly depends on the evaluation of microvascular function. Most of these tests are radioactive, invasive, and expensive. The clinical use rate is low, resulting in an insufficient diagnosis of patients with MVA and inconsistent diagnostic standards of research objects. Thirdly, many experimental drugs are self-made prescriptions, with diverse treatment methods, but there is a lack of unified consensus, and the efficacy evaluation standards of each family are not unified, which is not conducive to their widespread clinical promotion. We should comply with the development tendency of integrated traditional Chinese and Western medicine and pay close attention to the top-level design of clinical research while continuously improving clinical diagnostic technology.Future research should consider large sample survey and syndrome differentiation as the premise and conduct a large sample, multicenter, and randomized controlled trials by combining prescriptions and syndromes and selecting a compound of traditional Chinese medicine as the research goal so as to better serve the advantages of \u201cmicroeffect and multicause\u201d and comprehensive intervention of traditional Chinese medicine. Additionally, paying attention to the follow-up of adverse drug reactions and outcome events and standardizing the clinical treatment methods and efficacy evaluation standards of traditional Chinese medicine will provide new research ideas to correctly identify the role of coronary microcirculation in coronary artery disease, thereby solving clinical problems and preventing and treating cardiovascular events."}
+{"text": "Plasmodium falciparum. However, there is limited information on the C-terminal 19-kDa region of Plasmodium ovale MSP1 (PoMSP119). This study aims to analyze the genetic diversity and immunogenicity of PoMSP119.Merozoite surface protein 1 (MSP1) plays an essential role in erythrocyte invasion by malaria parasites. The C-terminal 19-kDa region of MSP1 has long been considered one of the major candidate antigens for a malaria blood-stage vaccine against Plasmodium ovale isolates including Plasmodium ovale curtisi and Plasmodium ovale wallikeri imported from Africa into China and collected during the period 2012\u20132016 were used. Genomic DNA was used to amplify P. ovale curtisi (poc) msp119 (pocmsp119) and P. ovale wallikeri (pow) msp119 (powmsp119) genes by polymerase chain reaction. The genetic diversity of pomsp119 was analyzed using the GeneDoc version 6 programs. Recombinant PoMSP119 (rPoMSP119)-glutathione S-transferase (GST) proteins were expressed in an Escherichia coli expression system and analyzed by western blot. Immune responses in BALB/c mice immunized with rPoMSP119-GST were determined using enzyme-linked immunosorbent assay. In addition, antigen-specific T cell responses were assessed by lymphocyte proliferation assays. A total of 49 serum samples from healthy individuals and individuals infected with P. ovale were used for the evaluation of natural immune responses by using protein microarrays.A total of 37 clinical pomsp119 were found to be thoroughly conserved in all the clinical isolates. rPoMSP119 proteins were efficiently expressed and purified as\u2009~\u200937-kDa proteins. High antibody responses in mice immunized with rPoMSP119-GST were observed. rPoMSP119-GST induced high avidity indexes, with an average of 92.57% and 85.32% for rPocMSP119 and rPowMSP119, respectively. Cross-reactivity between rPocMSP119 and rPowMSP119 was observed. Cellular immune responses to rPocMSP119 (69.51%) and rPowMSP119 (52.17%) induced in rPocMSP119- and rPowMSP119-immunized mice were found in the splenocyte proliferation assays. The sensitivity and specificity of rPoMSP119-GST proteins for the detection of natural immune responses in patients infected with P. ovale were 89.96% and 75%, respectively.Sequences of pomsp119. In addition, naturally acquired humoral\u00a0immune\u00a0responses against rPoMSP1 were observed in P. ovale infections, and high immunogenicity of rPoMSP119 in mice was also identified. These instructive findings should encourage further testing of PoMSP119 for rational vaccine design.This study revealed highly conserved gene sequences of The online version contains supplementary material available at 10.1186/s13071-021-05086-6. Plasmodium ovale is one of the five species of Plasmodium that regularly infect humans, and accounts for 0.5\u201310.5% of all malaria cases [Plasmodium ovale curtisi and Plasmodium ovale wallikeri [Plasmodium ovale has a similar morphology and life cycle to Plasmodium vivax [P. ovale malaria is underestimated because of the low density of these parasites in infected subjects, mild clinical symptoms (e.g. fever), and mixed infections with other species of Plasmodium [P. ovale infection can evolve into severe malaria with severe anemia and may become fatal, especially in areas where malaria is endemic [P. falciparum malaria vaccines [P. ovale malaria. China has eliminated malaria within its borders [P. ovale infection, increased in recent years [Malaria is one of the most severe infectious diseases that threaten human health. According to the World Health Organization, there were 229 million clinical cases of malaria and 490,000 deaths from this disease in 2019 [allikeri . Plasmod endemic , 7. Althvaccines \u201310, no w borders , and wasnt years \u201314.33) and 19-kDa (MSP119) fragments; the latter remains attached to the merozoite surface and enters the erythrocytes [P. falciparum MSP1 (PfMSP119) [P. vivax MSP1 (PvMSP119) [19 can prevent the invasion of merozoites into erythrocytes [19 have been associated with protection from malaria in pregnant women, infants and older children naturally infected with P. falciparum [19 is a promising candidate antigen for a blood-stage vaccine.During the invasion of human erythrocytes by malaria parasites, the merozoite surface proteins (MSPs) (including MSP1) are exposed to the host immune system . Moreovehrocytes \u201322. SevefMSP119) \u201325 and PvMSP119) , 27. GenvMSP119) . Moreovehrocytes . Antibodlciparum \u201332. ThesP. ovale isolates from Thailand were analyzed and showed low sequence diversity [19. In the present study, sequences of pomsp119 from clinical P. ovale curtisi and P. ovale wallikeri isolates from patients with P. ovale malaria imported into China from Africa were investigated. In addition, the immunogenicity of PoMSP119 was assessed in mice, and the levels of immune responses against PoMSP119 assessed in serum samples of patients infected with P. ovale.Genetic polymorphisms of MSP1 in clinical iversity . HoweverP. ovale curtisi or P. ovale wallikeri infection by polymerase chain reaction (PCR) tests [pomsp119 genes. In addition, serum samples of the P. ovale-infected patients (n\u2009=\u200929) who had returned from Africa and those of healthy individuals (n\u2009=\u200920) from China were also obtained from the local hospitals of Jiangsu Province.Blood samples of febrile patients who had returned from work in malaria-endemic areas of sub-Saharan Africa during the period 2012\u20132016, and who were confirmed for R) tests , were obR) tests , 35. GenP. ovale isolates  were collected for PCR amplification  msp1 (pocmsp1) (KC137343) and P. ovale wallikeri (pow) msp1 (powmsp1) (KC137341) genes from the GenBank database of the National Centre for Biotechnology Information were used as reference sequences [pomsp119 were identified via matching with similar sequences, as previously reported [pomsp119 forward (5\u2032-AGT AAG GAA AAA GAT TTG ACA A-3\u2032) and pomsp119 reverse (5\u2032-AAG TAA GTT AAA TAG GAT GAT-3\u2032). The primers for the nested PCR were as follows: pomsp119 forward (5'-ATG GGA TCT AAA CAT AAA TGT-3') and pomsp119 reverse (5'-GAA AAC ACC TTC GAA GAA TGG-3'). All the amplification reactions had the same reaction conditions, as follows: 98\u00a0\u00b0C for 3\u00a0min, followed by 35 cycles of 98\u00a0\u00b0C for 10\u00a0s, 45\u00a0\u00b0C for 1\u00a0min, and 72\u00a0\u00b0C for 1\u00a0min, and final extension at 72\u00a0\u00b0C for 5\u00a0min. PCR products were electrophoresed on 1.2% agarose gels for visualization under an ultraviolet transilluminator , then purified and sequenced by Genewiz . In addition, PCR-amplified fragments were also cloned into the pUC57 vector and sequenced by Genewiz using the following primers: M13F, 5\u2032-TGT AAA ACG ACG GCC AGT-3\u2032; M13R, 5\u2032-CAG GAA ACA GCT ATG AC-3\u2032. To evaluate genetic diversity within sequences of different isolates, sequences of pocmsp119 and powmsp119 were aligned using GeneDoc version 2.7.0.A total of 37 clinical equences , 34. Thereported , 37, andpocmsp119 and powmsp119 were subcloned into the pGEX-6p-1 expression vector which contained a glutathione S-transferase (GST)-tag fusion protein. Recombinant pGEX-6p-1pomsp119 were transferred into Escherichia coli strain BL21 pLysS cells to express PoMSP119 proteins. Colonies of recombinants were cultured in Luria Bertani broth supplemented with ampicillin 50\u00a0\u00b5g/ml by shaking at 250\u00a0r.p.m. at 37\u00a0\u00b0C until the optical density (OD) at 600\u00a0nm reached 0.6\u20130.8. To induce the expression of recombinant PoMSP119 (rPoMSP119) proteins, isopropyl \u03b2-D-1-thiogalactopyranoside (0.1\u00a0mM)  was added and the culture was allowed to grow for another eight hours. Proteins were purified by Tanlen-bio Scientific . The rPoMSP119-GST proteins were separated using 10% sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis (SDS\u2013PAGE) and detected via western blot and Coomassie brilliant blue staining . For western blot analysis, the proteins were electrophoresed on a polyvinylidene difluoride (PVDF) membrane . Nonspecific bindings were blocked by incubation with 5% skimmed milk in Tris-buffered saline supplemented with 0.1% Tween-20 (TBST) at room temperature for 2\u00a0h. The membranes were incubated with anti-GST rabbit monoclonal antibody (CWBio Biotech) as the primary antibody at 1:2000 dilution overnight at 4\u00a0\u00b0C, and then washed three times with 0.1% TBST. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) (CWBio Biotech) was used as the secondary antibody for detection (at 1:5000 dilution for 1 h). Finally, the membranes were visualized using the ChemiDoc MP imaging system (Bio-Rad).Genes including 19-GST, rPowMSP119-GST, GST, or phosphate-buffered saline (PBS) mixed with Freund\u2019s complete adjuvant  as the primary immunization. The same amount of recombinant protein was mixed with incomplete Freund\u2019s adjuvant then injected at days 21 and 42 after the initial injection to boost immunization. Mouse serum samples were collected and stored at \u2212\u00a080 \u2103 on days 0, 7, 14, 28, 35, and 49 after the initial injection.Female BALB/c mice  aged 6\u20138\u00a0weeks were intraperitoneally injected with 50\u00a0\u03bcg of rPocMSP119-GST from the sera of immunized mice. Concretely, rPoMSP119\u2013GST and GST proteins were transferred from SDS-PAGE onto PVDF membranes. The membranes were incubated with the sera of mice immunized with rPoMSP119-GST as the primary antibody, with sera of the GST immunized group, or with the sera of those injected with PBS, the negative control group, and then incubated with HRP-conjugated goat anti-mouse IgG (Cowin Biotech) at 1:5000 dilution for detection.Western blot analysis was performed to detect antibodies directed against rPoMSP119-GST and GST in the sera of immunized mice were evaluated through enzyme-linked immunosorbent assay (ELISA) as previously described [19-GST or GST was immobilized on 96-well plates overnight at 4\u00a0\u00b0C in coating buffer (15\u00a0mM sodium carbonate and 35\u00a0mM sodium bicarbonate in distilled water) and blocked with 5% (weight/volume) non-fat milk in TBST at room temperature for 2 h. A 100-\u00b5l volume of a twofold serial dilution  of anti-rPoMSP119-GST or anti-GST mouse sera was added to each well and incubated for 1 h at room temperature. The plates were washed three times with PBS containing 0.1% of Tween-20 (PBST) then incubated with HRP-conjugated goat anti-mouse IgG antibodies (Southern Biotech) at 1:5000 dilution for 1 h at room temperature. Finally, the plates were washed again and incubated with 100\u00a0\u00b5l of 3,3\u2019,5,5\u2019-tetramethylbenzidine (Invitrogen) substrate for 8 min in the dark, and the reaction was stopped with 50\u00a0\u00b5l of 2\u00a0M H2SO4 in each well. The absorbance was read at 450\u00a0nm. All samples were tested in duplicate and the mean absorbance was calculated.Levels of IgG antibodies against rPoMSP1escribed , 38. For19-GST IgG antibody was performed in accordance with the above-mentioned ELISA test with the exception that the test here was repeated in a 96-well plate coated with the same recombinant proteins. More specifically, sera were incubated for 90\u00a0min at room temperature, following by washing of one of the plates with PBST while the other plate was first incubated with 100\u00a0\u00b5l of TBST containing 6\u00a0M urea for 10\u00a0min at room temperature before final washing with PBST. Finally, all the plates were incubated with HRP-conjugated goat anti-mouse IgG antibodies at 1:5000 dilution for 1 h at room temperature. The reaction was stopped with 50\u00a0\u00b5l of 2\u00a0M H2SO4 in each well and absorbance was measured at 450\u00a0nm. The avidity index (AI) was calculated as follows [The affinity test of the anti-rPoMSP1 follows :\\documen19-GST, GST and PBS (5\u2009\u00d7\u2009105 cells/well) were treated with 10 \u00b5L of rPocMSP119-GST (5\u00a0\u00b5g/mL), 10 \u00b5L of rPowMSP119-GST (5\u00a0\u00b5g/mL), 10 \u00b5L of GST (5\u00a0\u00b5g/mL) or 10 \u00b5L of concanavalin A  as the positive control in 96-well flat-bottomed microtiter plates then incubated for 72\u00a0h at 37\u00a0\u00b0C with 5% CO2. A 10-\u00b5l volume of CCK-8 was added to each well and the plates were incubated at 37\u00a0\u00b0C for 2 h. Finally, cell proliferation was measured at 450\u00a0nm by a microplate reader.Assays for lymphocyte proliferation were performed using Cell Counting Kit-8 , as previously described , 40. BriP. ovale-infected and 20 healthy individuals were screened by well-type amine arrays. The screening was performed as previously described [19-GST, rPowMSP119-GST and GST solution in PBS (100\u00a0ng/\u00b5l) was spotted into each well of the arrays and incubated for 2\u00a0h at 37\u00a0\u00b0C. Array slides were washed three times with PBST for 10\u00a0min and were blocked with 5% of bovine serum albumin in PBST at 37\u00a0\u00b0C for 2\u00a0h. The arrays were washed again and probed with 1\u00a0\u00b5l of serum samples at 1:200 dilution. Finally, 1\u00a0\u00b5l of Alexa Fluor 546 goat anti-human IgG  in PBST was added to the arrays for antibody detection. The intensity of the serological responses was measured by a fluorescent microarray scanner . The positive cut-off value was calculated as the mean fluorescence intensity (MFI) of negative controls plus 2\u00a0SD. A Mann\u2013Whitney U-test was performed to compare differences in MFI between groups.Serum samples from 29 cases of escribed , 42. Bri t-test was used for the comparison of mean values between samples; differences were considered statistically significant when P-values\u2009<\u20090.05.GraphPad Prism software  was used for the statistical analyses and graphing. An unpaired two-tailed Student19 and PowMSP119 showed only one amino acid difference for each   that were used as sources of genomic DNA for PCR amplification are listed in Additional file pomsp119 genes were successfully amplified and showed a single 258-bp band on electropherograms  and PowMSP1 (KC137341) were predicted to be 1727 and 1672 amino acids, respectively [3 and Pow19-GST was estimated as approximately 35\u00a0kDa . Recombinant proteins were efficiently expressed and purified, as shown in Fig.\u00a019-GST proteins  in mice, ELISA was performed using rPoMSP119-GST and GST proteins as the coating antigens. The results showed that both rPocMSP119-GST and rPowMSP119-GST were immunogenic  than immunization of mice with GST  .To measure levels of immune responses against rPoMSP1nic Fig.\u00a0. The ave000 Fig.\u00a0a. After GST Fig.\u00a0b, c. The19-GST were assessed through spleen lymphocyte proliferation assays. The proliferation of splenocytes in vitro under rPocMSP119-GST, rPowMSP119-GST, GST, and ConA (positive control) stimulations was determined. Cell proliferation induced by rPocMSP119-GST was 69.51% and that induced by rPowMSP119-GST was 52.17%. Collectively, rPoMSP119-GST proteins induced a stronger proliferation response of spleen cells  than ConA. GST failed to induce cell proliferation . However, anti-GST IgG antibodies showed a significantly lower level of cross-reactivity and avidity indices of cross-reaction with rPocMSP119-GST than in reaction of rPocMSP119-GST with anti-rPocMSP119-GST IgG    and 75% , respectively. Anti-rPowMSP119-GST antibodies showed sensitivity and specificity of 89.96%  and 75% , respectively. However, no statistically significant difference was observed in the MFI of total IgG against GST protein between P. ovale-infected and healthy individuals  Fig.\u00a0a, b. Ant19 is carried into the invaded erythrocytes. In a later study, antibodies against MSP119 were shown to prevent merozoites from invading erythrocytes in vitro [pomsp119 were completely conserved in 20 P. ovale curtisi and 17 P. ovale wallikeri clinical isolates. These results are consistent with those from previous studies which reported genetic conservation of msp119 across malaria parasites [19 is under relatively lower selective pressure than the N-terminal of PoMSP1, which has also been shown to have low genetic diversity [Molecules of blood-stage malaria parasites are components of interest for the development of effective malaria vaccines . Severalin vitro . Antigenin vitro . In the arasites , 36, 37.arasites . In the iversity .19 might protect children from high levels of blood-stage parasitemia and clinical malaria [19-GST and rPowMSP119-GST was detected. This cross-reaction indicates that rPocMSP119 and rPowMSP119 share similar antigenic determinants and, therefore, PoMSP119 might possess species-specific efficacy as a vaccine candidate. In addition, as rPoMSP119-GST protein contains the GST tag, rPocMSP119-GST and rPowMSP119-GST could be detected by using the sera of mice immunized with GST protein as a non-specific control. Of the five classes of immunoglobulins, IgG is well-known as one that plays a critical role in malaria immunity [19-GST proteins than in the sera of the control group of mice immunized with GST protein  and specificity (75%). The differences in immune responses were not related to the presence of GST protein as no significant difference was observed in the MFI of total IgG against GST protein between P. ovale-infected and healthy individuals (Fig.\u00a019-GST, which may indirectly reflect or contribute to protection against P. ovale malaria infection as humoral immune responses are partly involved in preventing clinical malaria [19 and PvMSP119 as serological markers to detect the prevalence of malaria parasites [19 as a biomarker of exposure is worthy of further study.A number of studies have reported serologic analyses that investigated IgG antibodies against specific antigens of y levels , 51, 52;vity 89.9% and spe malaria . Seroepi malaria . Previouarasites , 56. Duepomsp119 sequences were completely conserved in clinical P. ovale curtisi and P. ovale wallikeri isolates. Furthermore, the immunogenicity of rPoMSP119 in mice was comprehensively analyzed. The informative results presented here contribute to the knowledge base for the development of a PoMSP119-based vaccine. In addition, the naturally acquired antibody responses against rPoMSP119 shown here indicate that further study of PoMSP119 could provide new insights for serological research. The cross-reactivity of PoMSP119 with sera from patients infected with P. falciparum or P. vivax needs to be explored in future studies, as does the clinical protection against malaria afforded by anti-PoMSP119 antibodies.This study demonstrated that Additional file 1: Table S1. Information on imported Plasmodium ovale curtisi and Plasmodium ovale wallikeri isolates used in this study.Additional file 2: Figure S1. Alignments of amino acid sequences of Plasmodium ovale curtisi and Plasmodium ovale wallikeri MSP119 in all amplified clinical isolates.Additional file 3: Table S2. Information on serum samples from patients infected with Plasmodium ovale used for the microarrays."}
+{"text": "In an effort to provide a suitable inoculum, microbial consortia were harvested from saline estuaries and enriched. However, it was found that bioaugmentation was not necessary because native communities from tannery wastewater were selected over exogenous communities from the enriched consortia. Overall, Dethiosulfovibrio sp. and Petrimonas sp. were strongly selected , while Desulfobacterium autotrophicum (57%), and Desulfobacter halotolerans (27%) dominated the sulfate reducing bacteria. The presence of elemental sulfur reducing genera such as Dethiosulfovibrio and Petrimonas is not desirable in HLFCRs, and strategies to counter their selection need to be considered to ensure efficiency of these systems for pre-treatment of tannery effluent.Recent research has demonstrated that hybrid linear flow channel reactors (HLFCRs) can desulfurize tannery effluent via sulfate reduction and concurrent oxidation of sulfide to elemental sulfur. The reactors can be used to pre-treat tannery effluent to improve the efficiency of downstream anaerobic digestion and recover sulfur. This study was conducted to gain insight into the bacterial communities in HLFCRs operated in series and identify structure-function relationships. This was accomplished by interpreting the results obtained from amplicon sequencing of the 16S rRNA gene and quantification of the dissimilatory sulfite reducing ( HoweverNaCl) [\u2212 . During vourable . Therefo2S gas), precipitation or oxidation are effective at removing HS\u2212. However, drawbacks include the high energy, capital and chemical costs, as well as long reaction times and the generation of large volumes of sludge that require disposal [42\u2212, which can be converted to HS\u2212 under anaerobic conditions. During conventional treatment of tannery effluent, sulfide is commonly dealt with during primary treatment, where suspended solids and metals are also removed . Active disposal ,6. These\u2212 to elemental sulphur (S0) have been investigated for removal of S from a range on industrial effluents. Aerobic/chemotrophic and anaerobic/phototrophic bioreactors have been shown to be highly cost effective and capable of high removal efficiencies while producing minimal sludge for disposal at laboratory scale [Biological methods for partial sulfide oxidation (SO) of HSry scale . However42\u2212 from tannery wastewater (TWW) has received limited attention. In an up-flow anaerobic sludge bed reactor, completely stirred tank reactor and trench reactor, a group of researchers [42\u2212 removal from a TWW feed with a SO42\u2212 concentration of 1800 mg/L. Others inoculated an upflow anaerobic sequencing batch reactor with Citrobacter freundii CZ1001 and reached a SO42\u2212 removal efficiency of 90% from tannery effluent with a SO42\u2212 loading of 1069 mg/L [Similarly, the removal of SOearchers  were all42\u2212 rich effluents are the incomplete organic oxidising genera Desulfovibrio, Desulfobulbus, Desulfomicrobium and the complete organic oxidizer, Desulfobacter [32\u2212) and thiosulphate (S2O32\u2212) is also common, as well as nitrate (NO3\u2212) and nitrite (NO2\u2212) for some [The most common genera found in bioreactors treating SOfobacter . Apart ffor some .\u2212 to S0 to occur concurrently [2 mass transfer and creates a microaerobic environment within the biofilm where the pH and redox conditions are suitable for partial SO [0 that can then be harvested as a value-added product from the FSB [The novel hybrid linear flow channel reactor (HLFCR) is a semi-passive system that allows sulfate reduction (SR) and partial SO of HSurrently . The prirtial SO . At labo the FSB . It was hypothesized that HLFCRs may be suitable for pre-treatment of TWW to reduce the concentration of S species to non-inhibitory concentrations while preserving sufficient organics for downstream AD using anaerobic sequencing batch reactors . To thisdsrB gene as a phylogenetic marker. To gain insight into the contribution of other bacterial taxa, including SO species, amplicon sequencing of a fragment of the 16S rRNA gene was also performed on selected samples. The relationship between microbial community structure and process performance is critical, particularly in complex matrices such as tannery effluent. This is the first time that microbial communities in HLFCRs treating TWW have been investigated. The main focus of this study was to evaluate the SRB community compositions and succession in the HLCFRs using molecular tools based on amplification of a ~350 base-pair (bp) fragment of the \u03b2-subunit of the 42\u2212 for the experimental reactor feed. Details about the biological TWW treatment system used to obtain the partially treated effluent cannot be provided for reasons of confidentiality. Five batches of both raw and partially treated TWW  were obt2SO4), 0.67 g/L potassium chloride (KCl), 0.2 g/L sodium hydrogen carbonate (NaHCO3), 0.03 g/L boric acid (H3BO4) and cultured in increasing volumes over 39 days. Thereafter, the consortia were assessed for their SO and SR ability (data not shown), and the two most promising candidates (designated A and B) were chosen. The characteristics of the estuarine water at sites A and B were: pH 7.3 and 6.6; conductivity 43 and 49 mS/cm; HS\u2212 0.59 and 0.30 mg/L, and SO42\u2212 2295 and 2300 mg/L, respectively. After selection, consortia A and B were adapted to TWW (batch 2) for 39 days to form the final inoculum (inoculum AB).Five consortia (consortia A\u2013E) were cultured from samples sourced from various saline estuaries in South Africa (sites A\u2013E). In order to select for saline-adapted SR and SO microbial species, the samples were inoculated into lactate-supplemented artificial seawater . During start-up, HLFCR1 was operated in batch and continuous modes intermittently as previously described [Initially, HLFCR1 was filled with 100% TWW, while HLCFR2 was filled with a mixture of inoculum AB culture and TWW (50% escribed  until st\u2212), pH and redox measurements were performed immediately. Thereafter, the samples were centrifuged and the sulfate (SO42\u2212) and soluble COD concentrations were determined on filtered (0.45 \u03bcm) supernatant fluid. The HS\u2212 and SO42\u2212 concentrations were determined using N,N-dimethyl-p-phenylenediamine (DMPD) and barium chloride (BaCl) techniques, respectively [\u00ae  test reagents and kits according to the manufacturers\u2019 instructions as previously described [Liquid samples were drawn from the bulk liquid using a syringe and hypodermic needle through the sampling ports. The hydrogen sulfide , bulk liquid of the HLFCRs (16 mL) and TWW (4 mL) as well as the biofilm (0.25 g FSB) samples using Qiagen  PowerLyzer Ultraclean DNA extraction kits and Powerlyzer PowerSoil DNA isolation kits for the liquid and solid samples, respectively, according to the manufacturers\u2019 instructions. Each of the extractions were performed in duplicate and the DNA concentrations were measured using a Jenway Genova  NanoDrop spectrophotometer. Equimolar amounts of each duplicate were combined for molecular studies. dsrB) using the primer pairs dsr2061F [Amplicon sequencing was performed at Molecular Research laboratories (MR DNA)  according to their established in-house protocols. Briefly, metagenomic DNA was used to amplify: (i) the V4 region of the small subunit (SSU) of the 16S rRNA gene using the primer pairs 515F-Y  and (ii)dsr2061F  and dsr4dsr2061F , with thInitial denaturation steps at 95 \u00b0C for 5 min were followed by 30 cycles of denaturation (95 \u00b0C for 30 s), annealing (53 \u00b0C for 40 s) and extension (72 \u00b0C for 1 min) and final elongation (72 \u00b0C for 10 min) using the Qiagen HotStarTaq Plus Master Mix kit. The PCR products were quality checked by visualisation in 2% agarose gel. Aliquots of samples were multiplexed using unique dual indices. Based on their DNA concentrations and molecular weights, samples were pooled in equal ratios, purified using calibrated Ampure XP beads  and used to prepare the DNA library according to the Illumina TruSeq protocol. http://rdp.cme.mus.edu and http://www.ncbi.nlm.nih.gov accessed on 15 August 2022). Rarefaction is a widely applied method of normalizing the sequencing output data by randomly discarding reads so that all the samples contain the same number of reads as the sample with the lowest number of reads [Sequencing was performed using an Illumina MiSeq instrument according to the manufacturer\u2019s instructions. The data was analysed using the MR DNA analysis pipeline. The sequences were joined together, the barcodes were removed, and those with <150 bp and/or ambiguous base calls were removed. Quality filtration was performed by applying a maximum expected error threshold of 1.0, after which the sequences were dereplicated and denoised by removing unique sequences identified by PCR error points and chimeras in order to generate zero radius operational taxonomic units (zOTUs). The zOTUs were assigned taxonomic classification using BLASTn against a curated database derived from the Ribosomal Database Project II (RDP II) and the National Centre for Biotechnology Information (NCBI) databases . The relative abundances (RA) of the zOTUs obtained from analysis of dsrB and 16S rRNA amplicon sequencing were used to calculate univariate diversity indices. Various multivariate statistical analyses were also performed. Data was square root transformed and used to construct Bray\u2013Curtis similarity plots. The similarity plots were used for: (i) one-way unordered analysis of similarity (ANOSIM), (ii) cluster analyses (group average linkages), and (iii) non-metric multidimensional scaling (nMDS). Results were either tabulated (ANOSIM) or used to construct nMDS plots overlayed with the results of the cluster analyses.All statistical analyses were conducted using Primer 7Fourth root transformed and normalised physicochemical (abiotic) data was used to construct similarity matrices based on Euclidian distances. The data was then analysed using principal component analyses (PCA) and one-way unordered ANOSIM. To determine which abiotic parameters were the most significant drivers of microbial community selection, BEST analyses of Spearman rank correlations were performed on the afore-mentioned Bray\u2013Curtis (biotic) and Euclidian distance (abiotic) similarity matrices. If significant, the \u2018best\u2019 correlated parameters were used to construct LINKTREE plots. Significance levels for all data is defined as: <0.05 * \u2265 0.01 > ** 0.005 \u2265 *** throughout the manuscript unless otherwise stated. \u2212 species. Briefly, HLFCR1 reached a steady-state during continuous operation on day 40 of the 140-day experimental period, with an average SO42\u2212 reduction rate (SRR) of 444 mg/L.day\u22121. Pseudo-steady state, specifically with respect to sulfide in the effluent, was maintained for the remaining 100 days, with only minor disruption due to effluent port blockages. Higher SRR were achieved in HLFCR1, the first reactor in series (up to 1049 mg/L.day\u22121) than in HLFCR2 (up to 513 mg/L.day\u22121). The higher rate in HLFCR1 was attributed to the presence of higher concentrations of SO42\u2212 and more readily biodegradable carbon substrates (soluble COD). Near complete removal of HS\u2212 was achieved, with final effluent values of <5 mg/L when the flow out of the reactor was unrestricted (n = 4), demonstrating the potential of the reactors for S recovery [2 ingress is restricted to the point where sulfide oxidation within the biofilm becomes limiting, resulting in steady increase in effluent sulfide concentration.Pre-treatment of TWW for removal and recovery of S species using HLFCRs has been conceptually proven and detailed performance results have been published elsewhere . It was stricted . Occasiorecovery ,13. ThisThis is the first time that microbial communities in HLFCRs treating TWW have been investigated. The main focus of this study was to characterize the environmental enrichments and endogenous TWW populations and evaluate the SRB and SO community compositions and succession in the HLCFRs using molecular tools as outlined in the graphical abstract. It was hypothesized that the enriched consortia obtained from saline estuaries would contain SRB and SO species able to proliferate in the saline TWW .viz. all the other samples, Overall, the SRB communities in the enriched consortia were significantly different from the SRB communities in the HLFCRs and the batches of TWW  grouped well away from those of the TWW, bulk liquid and FSB of both HLFCRs A. v/v) batch 2 TWW, and the results indicate that the SRB community in HLFCR2 was dominated by endogenous zOTUs from the, TWW, not from the enriched consortia A or B, because (i) data points denoting the SRB community compositions in the initial samples from HLFCR2, batch 2 TWW and Inoculum AB grouped closely together, and (ii) data points denoting the SRB community compositions in Inoculum AB grouped away from those denoting enriched consortia A and B. This suggests that there is no need for bioaugmentation of HLFCRs treating TWW from the study facility.The SRB community compositions in Inoculum AB also differed from those in the enriched consortia B,C. In cThe data points representing the SRB in different batches of TWW grouped away from one another in the nMDS plots, but the data point representing batch 1 (day 1\u201333) grouped closely with the data points representing all the samples from HLFCR1, as well as HLFCR2 towards the end of the experiment (day 80\u2013122), sharing 50% similarity B. These Gluconobacter, 32.8% Acetobacter and 26.3% Lactobacillus, an average COD removal efficiency of 95.2% was achieved [The most noteworthy finding was that the SRB communities in both HLFCRs stabilized from day 80 to the end of the microbial study (day 122) and were highly similar to the original communities in HLFCR1 C, indicaachieved . ResultsChanges in the overall bacterial community composition were determined by evaluating the results of 16S rRNA amplicon sequencing. The results mimicked those found with the SRBs in that the original communities in HLFCR2 were highly similar to those in Inoculum AB and TWW batch 2, and the communities in both HLFCRs evolved initially and then stabilized so that the communities in both HLFCRs were >70% similar between day 80 and 122 of the study D. dsrB amplicon sequencing  for the range of parameters analyzed, although the highest R value was obtained for pH (0.595) and combined pH and HS\u2212 concentration (0.518). In contrast, a significant overall correlation was found between the SRB community structures and the abiotic data . This was attributed to the fact that the parameters that were measured were likely to have a significant influence on the SRB communities, but not necessarily the non-SRB communities. No significant overall correlation was found between the bacterial community composition and the abiotic data  substantiated the results obtained with the nMDS and PCA plots\u2014that the primary drivers of SRB community structures were pH and COD concentration. In this instance, selection of the final (day 80 and day 122) communities in both HLFCRs was firstly by pH (<7.6) and then COD (>3.36 g/L) .Proteobacteria were the most abundant phyla in the TWW and inoculum AB (84% RA in each). Firmicutes (7\u20138%) and Bacteriodetes (7\u20139%) were also relatively well represented phyla in these samples (Synergistetes (from <1% in TWW and Inoculum AB to 15\u201330% in the HLFCRs by days 80 and 122), and Bacteroidetes (21\u201329% RA by days 80 and 122). samples . As allu samples , that thDethiosulfovibrio (14\u219229% and <1\u219212%), Petrimonas (21\u219224% and 17\u219226%) and Desulfomicrobium (8\u21929% and <1\u219225%) were found during the course of the experiment. Petrimonas sulfuriphila (type strain BN3T) and Petrimonas mucosa (type strain ING2-E5AT = DSM 28695T = CECT 8611T) were first described in 2005 [Dysgonomonadaceae [Petrimonas spp., like all members of the Dysgonomonadaceae family, are capable of fermenting a range of complex proteins and carbohydrates to CH3COO\u2212 and CO2 [Petrimonas are strictly anaerobic chemoorganotrophs commonly found in mesophilic anaerobic digesters [D. mucosa has been correlated with unstable reactor performance. It appears that the metabolic versatility of the genus provides it with superior stress resistance, allowing it to out-compete other genera [P. sulfuriphila can use S0 as a terminal electron acceptor, reducing it to H2S, and growth is stimulated by the presence of S0 [0 from HS\u2212 oxidation would have provided an ideal competitive environment for proliferation of P. sulfuriphila in the HLFCRs. Indeed, strains of Petrimonas and Desulfovibrio have recently been found co-dominating in a SO42\u2212 and aromatic hydrocarbon contaminated environment and hypothesized synergistic metabolic interactions between the two [In terms of bacterial genera in HLFCR1 and HFLCR2, notable respective increases in the RA of  in 2005 , respect and CO2 ,27. BothDethiosulfovibrio, currently includes five known species that all share metabolic similarities with Petrimonas. Dethiosulfovibrio peptidovorans was described in 1997 [Dethiosulfovibrio russensis, Dethiosulfovibrio marinus, Dethiosulfovibrio acidaminovorans [Dethiosulfovibrio salsuginus [D. marinus, D. acidaminovorans and D. russensis were isolated from sulfur mats, similar in nature to the FSB, where they were initially present in filamentous forms [Dethiosulfovibrio are strict anaerobes capable of utilizing proteins, peptides and amino acids as energy sources, but unlike Petrimonas cannot utilize sugars, although some can ferment organic acids [0 (like Petrimonas) but can also reduce thiosulfate (S2O32\u2212) to H2S [Petrimonas spp., the metabolic capabilities of Dethiosulfovibrio spp. clearly explains their competitive selection in the HLFCRs. In HLFCRs, the selection and dominance of these S0 reducing genera is not desirable as it counters the formation of S0 for recovery and removal of HS\u2212 as a pre-treatment measure for AD. Their proliferation is most likely a consequence of slower or incomplete biofilm formation which resulted in increased O2 penetration. This led to partial SO in the upper layer of the bulk liquid, rather than exclusively within the biofilm. The existence of colloidal S in the anaerobic liquid bulk volume would have provided a source of S for the functional bacteria. Another strongly selected genus,  in 1997  followednovorans  and Dethlsuginus . D. marius forms . All speic acids ,32,33. A) to H2S ,33,34. LArcobacter, detected in high RA in this study, has been shown to dominate in biofilms in highly sulfidic saline environments [Another noteworthy finding was that ronments , and hasClostridium (2\u2192<1% and 17\u2192<1%) and Arcobacter (13\u21922% and 13\u21924%) decreased in HLFCR1 and HLFCR2, respectively, while Desulfobotulus decreased in HLFCR1 (10\u2192<1%), and other genera such as Desulfuromonas remained relatively stable in both HLFCRs (Desulfovibrio was found in high RA in the inoculum (61%) and during start-up of HLFR2 (39%) but decreased to <1% by the end of the study. In general, although all the most abundant genera were present in both batch 2 TWW and Inoculum AB, the RA profiles bore little resemblance to those in the HLFCRs at the end of the study  and two other genera known to contain SRB species, namely, Clostridium [Tissierella [Thiomicrospira [Arcobater [Thermoanaerobacter , the batches of TWW (38\u201379%) and the inoculum (76%) but decreased notably in HLFCR1 (23\u21926%) and HLFCR2 (31\u21929%) during the course of the experiment. This decrease, together with the increase in Desulfomicrobium and similar trends being found with Desulfobotulus substantiated the results obtained using universal bacterial primers. However, failure of the latter to detect Dethiosulfovibrio and Desulfuromonas was notable and highlights the need to use more than one primer set when evaluating microbial community compositions because primer pairs shown to have good coverage in one environment may not amplify critical OTUs from other environments [Examination of the results obtained with the SRB specific primer set B showed ronments . Desulfobacterium autotrophicum and Desulfobacter halotolerans were strongly selected, and Desulfomicrobium orale remained resilient within the HLFCRs, reaching maxima of 57%, 27%, and 65% RA, respectively in the HLFCRs. At species level, the temporal changes in the RA of the most abundant bacterial species in the TWW, inoculum AB and HLFCRs were assessed to ascertain which species were out-competed, preferentially selected or were resilient within the HLFCRs . DesulfoD. autotrophicum is a complete organic substrate oxidizer, capable of heterotrophically degrading a variety of carbon sources, including acids, alcohols and long chain fatty acids [2O32\u2212 and SO42\u2212 as electron acceptors for heterotrophic growth [42\u2212 at both high and low concentrations [2 and CO2 [2 and completely oxidize acetyl-CoA to CO2 [D. autotrophicum is incapable of utilising S0 as an electron acceptor [ty acids ,43. It uc growth  and is ctrations . It is aacceptor , it cannD. halotolerans is capable of growth in hypersaline environments containing up to 13% NaCl [D. autotrophicum, it is a complete CH3COO\u2212 oxidizer [D. orale has been found to oxidize lactate and pyruvate incompletely to CH3COO\u2212 [D. orale has previously been shown to be inhibited by HS\u2212 [\u2212 were found. 13% NaCl , a factooxidizer ,46. D. o CH3COO\u2212 , and it d by HS\u2212  an asserdsrB gene were determined in the eluted DNA from centrifuged pellets of inoculum AB, batches of TWW, and the bulk liquid of the HLFCRs  of the dsrB copy numbers and SRR after the start-up of HLCFR1 (day 12\u201338), which stabilised to some degree thereafter. In contrast, there was a decreasing trend in dsrB copy numbers as well as SRR in HLFCR2, corresponding to the decrease in SO42\u2212 concentration in the bulk liquid, suggesting that the substrate concentration impacts on the proliferation of the SRB. The competitive selection of S0 utilising Dethiosulfovibrio sp. and Petrimonas sp. is consistent with the reduced FSB formation from day 75 [dsrB copy numbers in HLFCR2. The proliferation of Petrimonas sp. also explains how the bulk concentration of HS\u2212 in HLFCR2 (430 mg/L) remained similar to that in HLFCR1 (520 mg/L) despite receiving an influent with less SO42\u2212 (1000\u20131700 mg/L) than HLFCR1 (2400 mg/L). In the HLFCRs, there was a notable increase in DNA concentration within each reactor over the course of the experiment, strongly suggesting an increase in microbial biomass. There was also a general increase in m day 75  as well dsrB copy numbers and SRR in both bioreactors (r = 0.627), and in HLCFR1 on its own (r = 0.802), but sample numbers used in the statistical analysis were low , and the results were not statistically significant (p > 0.05). There appeared to be a moderate correlation (Pearson\u2019s) between The results of the study showed that the endogenous functional bacteria in TWW were more resilient in the HLFCRs than those harvested from saline estuaries. This indicates that bioaugmentation of HLFCRs during start-up is not necessary. The non-augmented HLFCR achieved a SRR of 600 mg/L.day and near complete SO, similar to results obtained using active methods . To ensuDethiosulfovibrio sp. and Petrimonas sp. are consistent with the slow or incomplete biofilm formation and offer a plausible explanation for declining S0 recovery. This would not have been evident if only chemical results were available viz. if the systems were treated as \u2018black boxes.\u2019 The findings provide a key to improving the performance of the HLFCRs, either by promoting more rapid and complete biofilm formation or by determining means of inhibiting the growth of Dethiosulfovibrio sp. and Petrimonas sp. The proliferation of The HLFCRs were able to pre-treat TWW sufficiently to reduce S-related inhibition of downstream AD. In terms of the bacterial communities, temporal community stabilization coincided with more stable system performance.0 (Dethiosulfovibrio sp. and Petrimonas sp.) and reducing SO42\u2212 . The TWW and AB inoculum contained little S0, but this accumulated in the FSB and as colloidal sulfur in the HLFCRs. Biofilm formation was slower than in previous studies using simple, synthetic feed solutions and reduction of biofilm S0 could reduce S0 recovery and increase the risk of undesirable HS\u2212 concentrations in the effluent. Future studies should focus on varying operational conditions to promote more rapid biofilm formation and discourage the selection of So reducing bacteria. The microbial succession in the HLFCRs showed a notable increase in the RA of species capable of utilizing proteinaceous substrates and reducing S"}
+{"text": "Severe hypothyroidism (SH) is a rare but life-threatening endocrine emergency. Only a few data are available on its management and outcomes of the most severe forms requiring ICU admission. We aimed to describe the clinical manifestations, management, and in-ICU and 6-month survival rates of these patients.We conducted a retrospective, multicenter study over 18\u00a0years in 32 French ICUs. The local medical records of patients from each participating ICU were screened using the International Classification of Disease 10th revision. Inclusion criteria were the presence of biological hypothyroidism associated with at least one cardinal sign among alteration of consciousness, hypothermia and circulatory failure, and at least one SH-related organ failure.Eighty-two patients were included in the study. Thyroiditis and thyroidectomy represented the main SH etiologies , while hypothyroidism was unknown in 44 patients (54%) before ICU admission. The most frequent SH triggers were levothyroxine discontinuation (28%), sepsis (15%), and amiodarone-related hypothyroidism (11%). Clinical presentations included hypothermia (66%), hemodynamic failure (57%), and coma (52%). In-ICU and 6-month mortality rates were 26% and 39%, respectively. Multivariable analyses retained age >\u200970\u00a0years [odds ratio OR 6.01 (1.75\u201324.1)] Sequential Organ-Failure Assessment score cardiovascular component \u2265\u20092 [OR 11.1 (2.47\u201384.2)] and ventilation component \u2265\u20092 [OR 4.52 (1.27\u201318.6)] as being independently associated with in-ICU mortality.SH is a rare life-threatening emergency with various clinical presentations. Hemodynamic and respiratory failures are strongly associated with worse outcomes. The very high mortality prompts early diagnosis and rapid levothyroxine administration with close cardiac and hemodynamic monitoring.The online version contains supplementary material available at 10.1186/s13613-023-01112-1. Hypothyroidism is a pathological condition related to a deficiency in circulating thyroid-hormone concentrations . EtiologThe term myxedema was first used by Ord in 1877 to describe a severe disorder associated with dry skin, swelling, profound hypothermia, and neurocognitive impairment among other clinical features in previously healthy women. A link was made with cretinism description and symptoms observed after thyroidectomy , 5. SincPrognosis has been markedly improved by thyroid hormone replacement therapy, with in-hospital mortality reaching 30\u201340% in the most recent series \u201313. IndeThis study retrospectively included SH patients hospitalized in 32 ICUs between 2000 and 2017  and complied with French research Reference Methodology MR003 regarding health-data privacy, and the French National Commission on Informatics and Liberty (CNIL)t test or the Wilcoxon test, as appropriate. Categorical variables [expressed as number (%)] were compared with \u03c72 tests. Patients\u2019 demographic, clinical, management characteristics and laboratory results were tested in bivariate analyses for association with in-ICU mortality and the presence of circulatory failure. Thereafter, factors achieving p\u2009\u2264\u20090.10 in bivariate analyses were entered into logistic regression models to investigate variables associated with ICU and 6-month mortality. Logistic regression analyses using backward-stepwise variable elimination were run (with the variable exit threshold set at p\u2009>\u20090.05). Multiple backward-stepwise logistic-regression analyses were used to select the final regression model using the Akaike information criterion. Multicollinearity was assessed by calculating a variance inflation factor of each variable and was ruled out if the variance inflation was lower than 4 and >\u20090.2. Variables associated with one another were not included in the model. No assumptions were made for missing data ] were compared with Student\u2019s During the 18-year study period, 447 adult patients with at least one organ failure and TSH and/or FT4 concentration outside the reference range were admitted to 32 ICUs. Among this population, SH diagnosis was confirmed for 82 of them  versus 50 (45\u201364) beats/min, p\u2009=\u20090.05], higher arterial lactate  and were more likely to have cardiac arrest and aspiration pneumonia before ICU admission than those without hemodynamic impairment. On the other hand, patients without cardiovascular failure had more frequent hypercapnia   vs 68 (58\u201376) years, p\u2009=\u20090.015] and had higher SAPS II and SOFA scores. Moreover, patients with aspiration pneumonia and hemodynamic failure [OR\u2009=\u200910.9 (2.3\u2013104.8) p\u2009<\u20090.001] had a lower ICU survival. Similarly, ICU nonsurvivors had significantly lower hemoglobin levels and higher arterial lactate , p\u2009=\u20090.007], hemodynamic failure  and ventilation impairment  were independently associated with in-ICU mortality Table . SimilarHerein, we report the largest multicenter cohort of SH patients admitted to ICU. SH is an extremely rare thyroid emergency, associated with a significant in-ICU and 6-month mortality of 26% and 39%, respectively. Though clinical presentation may encompass multiple known signs of hypothyroidism, SH-related hemodynamic and respiratory failures at ICU admission were strongly associated with a higher likelihood of mortality. Thyroid hormone replacement (levothyroxine) was consistently provided, although the route of administration and loading dose varied. Moreover, this treatment was frequently combined with steroids.Thyroiditis and thyroidectomy were the two main identified causes of hypothyroidism leading to SH. Autoimmune thyroiditis is considered the first etiology of hypothyroidism in iodine-sufficient worldwide areas which affects preferentially middle-life women , 19. IndSH should be recognized promptly to initiate thyroid replacement, monitoring, and treatment of potentially related life-threatening organ failures. Developing reliable diagnosis scores may help to identify earlier patients with SH. Indeed, our results externally validate the performance of the diagnosis scoring system of myxedema coma proposed by Popoveniuc et al. , as the Among clinical manifestations reported in our cohort of SH patients, neurological impairment was ubiquitous with, however, various severity forms, such as coma, somnolence, and seizures. Importantly, coma affected only one-half of our patients which is consistent with findings from a national database Japanese cohort reporting that only one-third of SH patients presented a coma at hospital admission . Myxedem3) was associated with more side effects and hemodynamic instability in this setting. However, the initial dose and the route of administration are still matters of debate [For decades, levothyroxine remains the first line treatment for thyroid replacement in SH, whereas triiodothyronine (Tf debate  as aggref debate . As illuf debate . Moreovef debate .Our study\u2019s strengths include the large cohort investigated and characterized in detail, and its multicenter design, with 6-month post-ICU-admission follow-up. However, it has several limitations that should be highlighted. The first is inherent to its retrospective design. Missing follow-up thyroid-hormone dosages precluded analysis of any potential relationship between clinical evolution and thyroid-hormone-level kinetics. In addition, some residual confounding factors might have biased our results. Second, data collection spanned 18\u00a0years. Therefore, we cannot rule out that the standard of care for critically ill patients has not changed over the study. However, European and American guidelines regarding the first-line treatment of hypothyroidism did not change during the study period , 33. ThiSH is a rare life-threatening endocrine emergency with various clinical presentations leading to ICU admission. Half of these severe patients have a coma but neurological clinical features could be limited to mild consciousness alteration or seizures. Based on 82 patients with SH admitted to ICUs, overall ICU, and 6-month post-admission mortality rates were 26% and 39%. Older age, hemodynamic and respiratory failure, but not neurological failure were strongly associated with fatal outcomes. This very high mortality for a reversible disease prompts early diagnosis and rapid levothyroxine administration with close cardiac and hemodynamic monitoring. Data are still warranted to better define the appropriate dose and route of administration of this necessary treatment.Additional file 1: Figure S1. Flowchart of patient selection from participating ICUs. Table S1. Amount of Missing Data for Each Variable Included in the Analysis. Table S2. Characteristics of Severe Hypothyroidism Patients According to the Presence of a Circulatory Failure at ICU Admission. Table S3. Clinical and Biological Features at ICU Admission according to ICU survival. Table S4. Predictive Patient Factors Associated with 6-month Mortality in Critically ill Adults with Severe Hypothyroidism."}
+{"text": "Caryocar brasiliense) endocarp. The biochar was characterized, before and after adsorption, by infrared spectroscopy (FTIR), scanning electron microscopy (SEM), X-ray diffractometry (XRD), and thermogravimetric analysis (TG). The surface load of the materials was performed by the point of zero charge (pHPZC) method. The study also included analyses of contact time parameters and adsorbed concentration in the adsorption process. Morphological analysis showed that a more significant and profound number of fissures and pores appeared in the activated biochar compared to the biochar. Residual mass analysis evidenced that biochar lost about 15% more mass than the activated biochar, indicating that activation occurred satisfactorily. The adsorption process was well adjusted by pseudo-second-order kinetics and Langmuir\u2019s isothermal model. The activated biochar achieved an excellent adsorption capacity of 476.19 mg.g\u22121, thus demonstrating to be a sound system for removing dyes from an aqueous medium.Given the increase in environmental pollution, especially of water, the emergence of studies that seek to develop strategies to mitigate/treat such effects have gained prominence in the world scientific community. Among the numerous adsorption processes, those made from biochar production stand out. This study analyzed the adsorption properties of the blue methylene model dye in the aqueous solution of biochar and activated biochar developed from pequi ( The current context, marked by the advance of economic activity and worldwide population growth, has led to increased environmental pollution, especially water pollution. It has generated concern and received attention from society and the scientific community . In partA significant contaminant of effluents is wastewater from the dye and paint industries, which should be classified as possible environmental pollutants. The dyes widely used in the textile industry are primarily synthetic and challenging to study or recover. They can generate a series of mutagenic and carcinogenic actions in humans and living organisms in aquatic environments. Pollution caused by dyes, when they react with complexes of chemicals present in the environment, becomes even more resistant to degradation. In particular, studies have shown that Methylene Blue, the dye used in this study, is widely used in the local textile industry in dyeing fabrics . Thus, rThe indiscriminate environmental pollution of dyes by the textile, pharmaceutical, cosmetic, and food industries has become a concern for the scientific community in recent years, which has led to research on techniques for removing these pollutants, with an emphasis on adsorption. The adsorption process is a versatile and affordable alternative for treating various effluents, including those of the textile industry ,8,9.One of the most used materials in dye adsorption processes is activated carbon or biochar. It can effectively remove pollutants, mainly organic, in adsorption processes and by the economic viability of production mechanisms and less polluting and generating fewer intermediaries ,11,12.Caryocar brasiliense), whose carbonization of biomass via pyrolysis is a necessary thermal modification process [Biochars can be produced from materials of plant origin (biomass), which are lignocellulosic, renewable, abundant, and have some degree of porosity . The bio process ,15.Caryocar brasiliense) stands out because it has significant economic value through its fruits both in cooking and in the manufacture of liqueurs and medicinal syrups [The Cerrado biome of Brazil, one of the biomes richest in plant resources, is still minimally used in the daily life of Brazilians. This biome has diverse fruit-producing plant species that humans and animals can consume. Among the fruits of Cerrado, the pequi , characterizing them by technical trust and applying them in removing a model organic dye through adsorption tests, varying the time, pH, and concentration parameters.16H18SN3Cl, Din\u00e2mica, Brasilpa\u00eds), Sodium Hydroxide , Hydrochloric Acid , Sodium Chloride , Distilled Water (Brazil). All reagents were analytical in degree, and no previous purification was required. The pequi endocarp residues were obtained at the free fair of the municipality of Floriano (PI), Avenida Bucar Neto, Centro, Floriano (PI), CEP 64800-000.For this work, the following reagents were used: methylene blue . The biochar was produced in five steps from the base of the pequi endocarp. Initially, pequi was collected, and any other residue was physically removed. In the second step, each part of the pequi  was separated, and the endocarp was dried for 2 h by sunlight (30 \u00b1 2 \u00b0C). Then, dried endocarp was crushed in a mill. Third, drying was performed in an oven for 6 h (80 \u00b1 2 \u00b0C). In the fourth step, the dry material was placed in porcelain crucibles and charred in a muffle with a heating ratio of 10 \u00b0C min\u22121 until the temperature of 800 \u00b0C. Soon after, the material was submitted to a cooling process with a rate of 5 \u00b0C min\u22121. Finally, the material that underwent the activation heat treatment was placed in an agitator with distilled water for 1 h and then filtered the material on qualitative paper, drying it in an oven for 12 h at 105 \u00b0C. The activated material was called activated biochar (ABE). NaOH was chosen as a precursor because of its ease of acquisition, economic advantage, and use in works involving activated biochar [In the fifth step, the BE underwent a chemical activation with sodium hydroxide (NaOH), in which the ratio between BE and NaOH was 1:3 (m:m). The activation process involved mixing the biochar with the precursor (NaOH). The mixture was submitted to an activation heat treatment, with a heating rate of 5 \u00b0C min biochar ,24,25,26\u22121. The TG curves were obtained using a thermal analyzer   using a heating rate of 10 \u00b0C min\u22121 between 25 and 800 \u00b0C in an inert nitrogen atmosphere. X-ray diffraction was performed using a Shimadzu instrument, model Labx-XDR 6000  [The biochars were characterized by Scanning Electron Microscopy (SEM), Infrared Spectroscopy (FTIR), and Thermogravimetry (TG). The infrared spectra were obtained using an Vertex 70 FTIR spectrophotometer , by the insert method, with 60 scans in the range of 600 to 4000 cm, Japan) ,28,29,30PZC method determines the pH by balancing loads between the adsorbent surface and a solution. In the test, 20.0 mg samples of activated carbons were added to 20.0 mL of NaCl (0.1 mol.L\u22121). The pH was adjusted to 2 to 12, adding HCl solution at 1.0 mol.L\u22121 and/or NaOH at 1.0 mol.L\u22121. The initial pH was read using one pH meter. For 24 h, the mixtures were left under constant agitation at 140 rpm at 25 \u00b0C. After the described period, the solutions were centrifuged for 10 min at 5000 rpm, and the final pH of the solution was measured. The difference between the initial and final pH was calculated (\u0394pH) (Equation (1)) and plotted from the obtained data, and then the intersection where \u0394pH = 0 was determined, corresponding to the point of zero charge [The pHo charge .(1)\u0394pH=\u22121. The system was agitated for 24 h. After contact time, the solutions were centrifuged for 1 min at 14,000 rpm and diluted. The new concentrations were determined by reading the aliquots in a UV-vis spectrophotometer  using a pre-established calibration curve (665 nm). Finally, the adsorbed amount qe and (mg.g\u22121) and adsorption efficiency R (%) were calculated according to Equations (2) and (3), respectively [The effect of pH on dye adsorption was studied at pHs 4, 7, and 10 in triplicate, in which pH was adjusted with NaOH and HCl solutions. Biochar samples were in contact with 40.0 mL of a solution containing the methylene blue model dye (MB), with a concentration of 300.0 mg.Lectively .(2)qe=C\u22121) of the dye, respectively; V is equivalent to the volume in liters of the dye solution used.-1 for biochar (BE) and 800 mg.L\u22121 for activated biochar (ABE). The solutions were constantly shaken at 140 rpm in 10, 20, 40, 60, 120, 220, 260, and 300 min intervals. Next, the samples were centrifuged, and the resulting solutions were measured in a UV-vis spectrophotometer to calculate the concentrations, according to Equation (2). The experimental data were adjusted to two kinetic models (pseudo-first-order and pseudo-second-order) to verify the best fit. The linearized form of the pseudo-first and pseudo-second-order kinetic models are expressed in Equations (4) and (5), respectively [eq and tq are the amount of dye adsorbed (mg.g\u22121) at equilibrium and time t (min), respectively; k1 represents the adsorption constant of the pseudo-first-order model (min\u22121), and k2 is the constant of the pseudo-second-order kinetic model (mg.g\u22121min\u22121).The kinetic adsorption assay was performed in triplicate at 25 \u00b0C, with pH adjusted according to the best adsorption capacity. Different masses of biochar were added in different vials containing 40.0 mL of the dye solution, with a concentration of 100 mg.Lectively ,33.(4)l\u22121, which agitated the best equilibrium time found in the kinetics experiment. Then, the samples were centrifuged, and measurements of UV-vis spectrophotometer solutions were performed to calculate the equilibrium concentrations resulting from adsorption, according to Equation (2). Finally, we investigated which adsorption isotherm model best fits the experimental data (Langmuir or Freundlich). Equation 6 expresses the linearized form of the Langmuir equation, and Equation (7) presents the linearized form of the Freundlich equation [q is the amount of species adsorbed by mass of the bioadsorbent (mg.g\u22121), q0 is the maximum amount of species adsorbed by mass of the bioadsorbent (mg.g\u22121), C represents the equilibrium concentration of the adsorbent (mg.L\u22121), LK is the Langmuir adsorption constant related to the chemical balance between adsorbent and adsorbent (mg.L\u22121), FK is the Freundlich adsorption constant related to adsorption capacity, n is the parameter related to the intensity of the adsorption process.The test was performed in triplicate at a temperature of 25 \u00b0C, with pH adjusted according to the best adsorption. First, 70.0 mg of biochar was added to 40.0 mL of the dye in solution, varying the concentrations between 50 and 1000 mg.Lequation ,35.(6)CPequi endocarp was used to generate the biochars, which have yellowish placement, according to the methodology presented, characterizing them as a fine black powder with low density, characteristic of this type of material. The color change after the thermal process indicates the production of biochar. Visually, it was impossible to verify differences between biochars after activation, so several characterizations were made to verify the properties of the materials obtained before and after adsorption.PZC for the studied biochar samples of the endocarp (BE) and activated biochar (ABE).\u22121 is attributed to C=O vibrations in aromatic structures for biochar samples produced from the BE. After activation, the ABE presented a band around 1000 cm\u22121, usually found in oxidized biochars, and attributed to the elongation of C-O vibrations in acids, ethanols, phenols, ethers, and esters. Another band around 690 cm\u22121 is related to Na-O vibration from changes in the activation process, which indicates structural changes in the biochar [The FTIR spectra of BE and ABE are presented in  biochar ,37,38.The thermogravimetric analysis (TG) of the biochars is presented in PZC) determines an index at which a surface tends to be positively or negatively charged as a function of pH. PZC curves of the biochar obtained. The pHPZC value of the BE was 5.7, while the pHPZC value of the ABE was 6.4. For pH values below pHPZC of biochars BE (5.7) and ABE (6.4), the surface charge is positive, indicating that adsorption is favored for anionic species, while values above pHPZC favor adsorption of cationic species, which is the case of the Methylene Blue dye, used in the research. The results confirm that the medium\u2019s pH influences the biochar\u2019s surface. Ions (H+ or OH\u2212) present in the solution can interact with the active sites of the biochars, thus altering their charge balance [The pH of the medium affects the surface charge of the adsorbent and its degree of ionization and affects the adsorbed species. The pH at the point of zero charges , whose adjustment parameters are presented in The kinetic adsorption studies of BE and ABE are presented in 2), 0.98920 for BE and 0.99990 for ABE. The results of eq calculated by the PSO model were close to the experimental values of eq, thus indicating that the adsorption of the MB dye involved chemical interactions. The analysis showed that the ABE adsorption had a value of eq equal to 476.19 mg.g\u22121, much higher than that of the BE, with q equal to 7.78 mg.g\u22121.\u22121 for BE and 432.3 mg.g\u22121 for ABE, indicating a significant increase in adsorption capacity after activation. The isotherms were adjusted against the Langmuir and Freundlich models. The linear form of Langmuir and Freundlich models is presented in 2), whose statistical parameters are presented in The adsorption isotherms of biochars are presented in 2), 0.8784 for the BE and 0.9962 for the ABE. Thus, the analysis suggests that the adsorption process occurs homogeneously on the adsorbent surface in the monolayer and by chemical interactions, corroborating the kinetic data.2 adjustments were similar for both Langmuir and Freundlich in the case of BE; this suggests the presence of residues that promote a mixture between chemical and physical adsorption processes. As for the ABE biochar, which was better adjusted to the Langmuir model, the result suggests chemical adsorption in a monolayer [The experimental values for each biochar B follow onolayer ,45.After the dye adsorption, the materials were characterized to verify possible changes in characterizations and to prove the fixation of the dye in the biochars. The same acronyms were used to facilitate identification, adding A after the acronyms of the material, BE and ABE for biochar after adsorption, and activated biochar after adsorption, respectively.\u22121 attributed to C=O vibrations in aromatic structures was only found for the BE. For ABE, the bands remained around 1000 cm\u22121, which indicates the elongation of C-O vibrations in acids, ethanols, phenols, ethers, and esters, and around 690 cm\u22121 related to Na-O vibration [The FTIR spectra of the materials after adsorption A presentibration .The thermal analysis of the BE biochar before and after adsorption is presented in \u22121, which revealed the feasibility of using it as a sustainable source to produce high performance activated carbon. It is noticed that the use of biochar produced from the endocarp of pequi presented itself as an excellent option in the adsorption process. Therefore, future work is suggested to analyze the production of biochar from the other compounds of pequi, almond, and mesocarp.Biochar of the pequi endocarp was successfully prepared by pyrolysis of biomass at 500 \u00b0C. The biochar underwent an efficient activation process, with an activating agent NaOH at 800 \u00b0C, proven by the characterizations with alteration in structure, change of thermal profile by removing some impurities present before activation, and alteration of some FTIR bands, due to the removal of some groups with activation. Both were applied in the adsorption of a model dye, with a better fit to the kinetic model of pseudo-second-order, Langmuir isotherm, and adsorption capacity at pH 10 for BE and 7 and 10 for ABE. The activated material, derived from biomass, renewable, and often discarded in nature without any proven use, is a promising adsorbent for the removal of MB from an aqueous environment with a preferential adsorption capacity of 476.19 mg.g"}
+{"text": "The present study investigated the prevalence of osteoporosis (OP) among patients with essential hypertension (EH) in the Changchun community and analysed the correlation between EH and OP.The study included 425 subjects with EH and 425 age- and sex-matched healthy controls. Bone mineral density (BMD) and serum creatinine (CR) levels were measured, and the subjects' current EH and OP statuses were surveyed to analyse the correlation between EH and OP.p\u2009<\u20090.05). A significant sex difference in the BMD T-score was observed among the subjects . In both the EH group and the control group, the rate of having OP in females was greater than that in males. However, the OP prevalence among subjects with EH varied significantly by age, body weight, fracture history, nocturnal urination frequency, depression and anxiety status, duration of hypertension, and antihypertensive medication use (p\u2009<\u20090.05). Two-way analysis of variance suggested an effect of the interaction between different EH statuses and bone mass conditions on the serum CR values .The EH group exhibited lower BMD and a higher rate of having OP than the control group, and this difference was statistically significant (The prevalence of OP and low BMD were significantly higher among subjects with EH than among healthy controls. Additionally, the findings indicate that age, weight, fracture history, nocturnal urination frequency, depression and anxiety, duration of hypertension and antihypertensive drug use may be correlated to having OP in EH subjects, requiring further studies. Moreover, serum CR levels in subjects with different bone mass profiles were strongly influenced by the presence or absence of EH, and the serum CR levels differed significantly with the interaction of these two factors. Osteoporosis (OP) and essential hypertension (EH) are common in both the middle-aged and elderly populations and often occur as comorbid diseases . With inThe present study included 425 subjects between 50 and 80\u00a0years old who were diagnosed with EH and had at least a 2-year duration of chronic disease. The control group comprised an equal number of healthy individuals in the same age group. We used questionnaires to recruit hypertensive subjects, as they have a better awareness of whether they already have hypertension. In addition, we screened patients with a history of hypertension for at least 2\u00a0years and ascertained their hypertension and medication status through an interview, which provided basic data support for subsequent analysis of the correlation between EH and OP. The control group comprised an equal number of non-EH subjects with the same sex and age distribution. Finally, 1786 subjects were screened, of whom only 425 could be included as controls. In addition, we ensured that the sex and age distribution was exactly the same between the EH and control groups.Subjects with the following conditions were excluded: pituitary, thyroid, parathyroid, adrenal, or gonadal diseases or tumours; severe heart, liver, kidney, central nervous system or psychiatric diseases; type I or type II diabetes or chronic metabolic disorders other than OP; dependence on drugs other than antihypertensive medication during the study period; spinal idiopathic disease; and other major medical conditions, such as infection or cancer. Patients who had received anti-osteoporosis treatment and pregnant or lactating women were also excluded from the study.The studied population comprised residents of the local community in the Changchun urban area who were recruited from October 2017 to December 2017. Cluster sampling was performed to include a diverse sample of subjects in the community who met all the inclusion criteria. The general information, sociodemographic data, and data on the medical status of all the participants were collected through a questionnaire survey. The detailed medical history of the participants was recorded, and physical examination and laboratory tests were conducted to exclude subjects with abnormal results or certain medical conditions. The study procedures were explained in detail to the participants, and signed informed consent forms were obtained from all the participants. All procedures were conducted in accordance with the relevant guidelines and regulations. The Institutional Review Board of Changchun University of Traditional Chinese Medicine Hospital approved the study.The subjects of this study are all from Luyuan District and Chaoyang District of Changchun City, both of which are old urban areas that cover a wide range of risk exposures. Therefore, the representativeness of the sample was improved, and the systematic error that occurs in the selection process of research subjects is reduced.BMI was calculated based on weight and height, and related studies \u201312\u00a0have http://slideplayer.com/slide/4493823/) that provides the relationship between the BMD measurement value  and OP, the subjects were classified into the following groups: normal bone mass , osteopenia , and osteoporosis  [BMD levels in the lumbar spine and left hip were determined through dual energy X-ray absorptiometry (DXA) by using a Discovery Wi , produced by American Hologic. According to the World Health Organization website (\u2009\u2009\u2212\u20092.5) . The antIn this study, specific professionals who were designated to perform BMD testing conducted strict quality control every day before testing subjects. After completing the diagnostic tests for some functional indicators and scanning the lumbar vertebral phantom provided by the manufacturer, the diagnostic report was evaluated. If a diagnostic report is not generated, the test should be performed a second time. In the case of two consecutive failures or if the difference between the two consecutive scans and the baseline value is greater than 1.5%, the researcher must contact the manufacturer to identify the cause. In addition, if a deviation from the baseline trend is observed in the quality control chart, the inspector should check whether it is a calibration drift. The height and weight of the subjects must be measured before the bone density measurement; in addition, the date of birth (year/month/day) must be collected to obtain more accurate measurement results. Furthermore, any accessories worn by patients, such as watches, bracelets, and rings, should also be removed while measurements are taken.(1) Independent sample t tests were used to analyse group differences in age and body weight between the EH group and the control group.U test was used to analyse differences in the BMD T-score and CR levels between the EH group and the control group.(2) The Mann\u2013Whitney (3) For the count data, Pearson\u2019s correlation coefficient and the chi-square test were used if the expected frequency of all cells was\u2009>\u20095, whereas Fisher's exact test was used if the expected frequency was\u2009<\u20095. Analysis of covariance was used to control for the effects of confounding variables, and Bonferroni correction was applied to adjust for multiple tests.(4) The chi-square test was used to analyse differences between the EH group and the control group in terms of sex and bone mass. The clinical correlations of sex, age, fracture history, nocturnal urination frequency, depression and anxiety status, duration of EH, and antihypertensive drug use with bone mass status were also analysed within the EH group. Additionally, the column proportions were compared, and the p values were adjusted (through the Bonferroni method) in the Z-test to compare between-group differences in the demographic and clinical variables according to different bone mass profiles and intragroup differences (within the EH group) in nocturnal urination frequency, depression and anxiety status, duration of EH, and antihypertensive medication use in a two-by-two comparison.R.(5) The Mantel\u2013Haenszel chi-square test was used to determine whether there was a linear correlation between age and the rate of OP within the EH group, and the strength of the association was determined according to Pearson\u2019s correlation coefficient (6) Two-factor analysis of variance (ANOVA) was used to evaluate the effect of different EH statuses and bone mass conditions on CR. Box plots were used to test for outliers, the Shapiro\u2013Wilk test was used to determine the normality of the data distributions, and Levene\u2019s chi-square test was used to determine isotropy.EpiData 2.1b was used to establish the database, and SPSS (version 26.0) 10 was used for statistical processing with a two-sided p value of 0.05. The observations in the present study were independent of each other. No significant outlier was present in the observed variables, which were nearly normally distributed within each group and exhibited equal variance. The following tests were selected according to the type of variable and the need for clinical analysis:The effects of confounding factors such as sex and age were excluded from the present study. Table 2\u2009=\u200912.968; p\u2009=\u20090.002) (Table p\u2009<\u20090.001), the CR levels between the two groups did not vary significantly (p\u2009=\u20090.934).The prevalence of OP in the EH group was significantly higher than that in the control group , and the occurrence of OP increased with age (R\u2009=\u20090.146 and p\u2009=\u20090.003). Additionally, the body weight of the non-OP subgroup was approximately similar to that of the OP subgroup . However, the OP prevalence were higher in subjects with previous fractures . Other factors identified to be related to OP were nocturnal urination frequency, depression and anxiety status, duration of hypertension, and antihypertensive medication use. The percentage of EH subjects who never woke up at night to urinate, who were not depressed and anxious was lower in the OP subgroup than in the non-OP subgroup. Based on the duration of EH, the proportion of EH subjects with 6\u201310\u00a0years of illness or more was higher in the OP subgroup . Furthermore, Fig.\u00a02\u2009=\u200936.722, p\u2009<\u20090.001).As shown in Table U\u2009=\u200915,489.000, p\u2009=\u20090.002). An effect of the interaction between EH status and bone mass condition on the CR level was observed . The separate effect analysis suggested that the effect on CR values differed according to the different bone mass conditions and the presence or absence of EH. Among the EH study subjects, significant differences in CR expression were observed according to the different bone mass conditions . Additionally, serum CR levels in the study subjects with different bone mass conditions were more strongly affected by the presence or absence of EH. The CR levels varied significantly under the interaction of EH and OP, and use of the CR concentration may seriously reduce the diagnostic efficacy of OP in subjects with EH.No significant difference was observed in the CR levels between the EH and control groups Table ; howeverEH and OP are two major age-related diseases  that conSIRT1) expression. In experimental models [SIRT1 expression was found to be positively associated with bone mass. In patients with EH, the use of RAAS inhibitors, including angiotensin-converting enzyme (ACE) inhibitors, (for 2\u00a0years or less) was associated with considerable rates of fracture development; however, their long-term use was associated with a reduced fracture risk [2+ by this class of drug. Because antihypertensive medications are widely used with elderly patients and EH and OP are often comorbid, the effects of antihypertensive therapy on bone and fracture rates should be considered prior to its clinical application.The mechanism underlying the occurrence of OP in patients with HTN is not fully understood. However, the renin-angiotensin\u2013aldosterone system (RAAS) is known to play a crucial role in blood pressure regulation and fluid homeostasis . The roll models , SIRT1 eure risk , 15, 17 The present study focused on body weight and CR levels. On the one hand, body weight and CR levels show a complex trend under the influence of both diseases. The impact of obesity on the odds of EH development may exceed its protective impact on skeletal health in middle-aged and elderly individuals . AlthougThe present study has certain limitations. First, although there are many factors that can jointly influence OP and EH, due to limited data, this study could adjust for only a limited number of confounders and did not use propensity score matching to better eliminate them. Second, our cohort was relatively young for the OP study. We should allow more time to recruit a larger number of subjects, especially in for the control group, as it is very difficult to find older individuals free of either EH or OP. Third, the study was an analytical cross-sectional study, so it is difficult to establish causality. The existing data are also insufficient to determine the association of some hypertension (HTN) drugs with OP. Although we tried our best to obtain objective data, some bias still existed in the individual lifestyle data. Therefore, based on the present study, prospective cohort studies should be considered for further research in this domain.Both EH and OP have become common in the elderly population, and EH may be a strong causative factor for OP. The extent to which the shared factors for these diseases affect bone mass status warrants further definitive quantitative analysis in the future. Therefore, we recommend that along with effective screening for hypertension, bone density testing should be promoted in the elderly population, especially for patients diagnosed with hypertension, for early screening and early intervention. Considering the closely related manifestations of these two diseases, proper blood pressure control and rational use of medications in the middle-aged and elderly populations may improve bone health and prevent the occurrence of fractures in patients with hypertension."}
+{"text": "The hexagonal crystal structure of HAp supports the adsorption mechanisms including ionic exchange reaction, surface complexation, the co-precipitation of new partially soluble phases and physical adsorption such as electrostatic interaction and hydrogen bonding. However, nano HAp has some drawbacks such as agglomeration and a significant pressure drop during filtration when used in powder form. Therefore, instead of using nano HAp alone, researchers have worked on modificationsand composites of nano HAp to overcome these issues and enhance the adsorption capacity. The modification of cationic doping and organic molecule grafting for nano HAp can promote the immobilization of ions and then increase adsorption capacity. Developing nano HAp composite with biopolymers such as gelatin, chitosan and chitin has proven to obtain a synergetic effect for improving the adsorption capacity of composites, in which nano HAp fixed and dispersed in polymers can playmuch more of a role for adsorption. This review summarizes the adsorption properties and adsorbent applications of nano HAp as well as the methods to enhance the adsorption capacity of nano HAp.Nano hydroxyapatite (Ca The dipole\u2013dipole hydrogen bonding interaction occurs between the azoic group of the dye molecule and the hydroxyl groups of HAp, as well as the electrostatic interactions which may occur between the cationic groups of a dye such as Na+ with anionic groups of HAp such as OH\u2212 and PO43\u2212 [The adsorption of reactive red 198 dye on HAp can be explained as follows. The dye molecules are adsorbed through the electrostatic attractions between the ionized sulphonyl groups of the dye molecule and the positively charged Cand PO43\u2212 .The mechanism of the HAp adsorption of methylene blue can be explained as follows: under alkaline conditions, the negative charge on the surface of hydroxyapatite and the key group of methylene blue play a dominant role. Under acidic conditions, the electrostatic effect does not exist because the surface of hydroxyapatite is positively charged, and the adsorption is achieved by the (P-OH) group of HAp-particles and the (N) of the methylene blue molecule. Therefore, the adsorption mechanism is related to the external condition .The pH of the adsorption medium is considered the most critical parameter in the phenol adsorption process by nano HAp, because the charge of both the adsorbate and the adsorbent depends on the pH of the solution. Lin et al. studied the effect of the initial pH of the solution on the phenol adsorption onto nano HAp and found that the adsorption capacity is decreased with increasing pH up to 8.2, and then the adsorption capacity is increased by further increasing the pH to the alkaline value . The maxIn the nitrobenzene adsorption process, the pH of the adsorption medium is the most vital parameter. Wei et al. found that the adsorption capacity of nanocrystalline HAp decreases sharply when the pH is higher than 6.0, and the maximum adsorption capacity of nanocrystalline HAp on nitrobenzene was at pH 2.0 .Vasugi et al. studied the reactive red 198 dye adsorption capacity of nano HAp under different pH values and learned that nano HAp showed a better dye removal capacity in acidic pH and that the maximum dye removal capacity was observed at pH 6 .Ciobanu et al. examined the removal rate of reactive blue 19 dye by nano HAp under different pH values . CiobanuAllam et al. (2016) proposed that when the pH value in the solution is higher or lower than the HAp isoelectric point, the charge on the surface of the nanoparticles will change because the HAp isoelectric point is between 6 and 7.2, which will affect the mechanism of organic matter adsorption .Lin et al. studied the effect of contact time on the phenol adsorption by HAp nanopowders . They foWei et al., examined the effect of contact time on the adsorption of the nitrobenzene on nanocrystalline HAp for initial nitrobenzene concentrations of 5, 10 and 50 mg/L . For theVasugi et al. investigated the effect of contact time on the reactive red 198 dye removal percentage by HA . They obLin et al. investigated the effect of the nano HAp adsorbent dose on the percentage of phenol removal . They idWei et al. examined the effect of the adsorbent dose on the nitrobenzene removal by nano HAp over the 2.0\u201310 g/L range of the adsorbent, keeping all other parameters constant . They obVasugi et al. evaluated the effect of nano HAp adsorbent dosage on the percentage of reactive red 198 dye removal . At a pHCiobanu et al. studied the effect of nano HAp adsorbent dose on the reactive blue 19 dye removal . The dyeEven though the adsorption by nano HAp is recognized as a promising method for nitrobenzene removal from wastewater, there are some drawbacks of nano HAp, such as poor strength, low stability and a significant pressure drop during filtration when used in powder form . To prevVasugi et al. investigated the reactive red 198 dye adsorption by nano HAp and a trivalent cation (yttrium) substituted HAp (Y-HAp) . The stuHou et al. prepared the composite of HAp and chitosan, which was used to adsorb Congo red dye . Compare6H11Na3O12P2\u00b78H2O, DFP) as a phosphorus source [2/g, so it shows good adsorption performance for organic excitements such as methyl orange and Congo red [Guan et al. prepared polyalcohol modified HAp nanoparticles with a core-shell structure using D-Fructose-1, 6-phosphate trisodium salt octahydrate ):The adsorption of FThe fluoride removal of the n-HAp by ion exchange could be expressed by the following reactions (Equations (10)and(11)) :Ca10):Several factors such as the solution pH, contact time, adsorbent dose and other anions in the medium affect the adsorption of fluoride ions on nano HAp.\u2212 kg\u22121 at pH 3. At pH 11, the defluoridation was only 570 mgF\u2212 kg\u22121. Accordingly, the defluoridation capacity increases with the decreasing pH [Among the factors mentioned above, the pH value plays an important role in the adsorption of fluoride ions at the water adsorbent interface. Sundaram et al. studied the fluoride adsorption on nano HAp under different pH values from 3 to 11 at room temperature with an initial fluoride concentration of 10 mg/L . The remasing pH . This phThe adsorption of aqueous fluoride ions is also strongly influenced by contact time. Sundaram et al. examined the fluoride adsorption on nano HAp, varying the contact time in the range of 10\u201360 min with an initial fluoride concentration of 10 mg/L at room temperature . \u00d7(ln X)\u22122.2(\u00b10.2); R2 = 0.996, where qe is the adsorption capacity of Hap [Jim\u00e9nez-Reyes and Solache-R\u00edos conducted experiments using hydroxyapatite to adsorb 5 mg/L fluoride ions . It has y of Hap . \u2212, NO3\u2212 and SO42\u2212 HCO3\u2212 on the defluoridation capacity of nano HAp by keeping the initial concentrations of these ions ranging from 100\u2013500 mg/L and 10 mg/L as initial fluoride concentration at 303 K [\u2212, NO3\u2212 and SO42\u2212 ions [3\u2212 ions, the defluoridation capacity considerably decreased. Sundaram et al. concluded that the decrease in the defluoridation capacity is due to the competition of HCO3\u2212 ions with the fluoride ions [\u2212 ions from the NaHCO3 hydrolysis and their competition with fluoride ions for active sites on nano HAp [Co-existing anions in the aqueous solution may compete with fluoride ions for adsorption sites during defluoridation and cause a negative effect on the defluoridation capacity. Sundaram et al. investigated the effect of the co-anions including Clat 303 K . Sundara42\u2212 ions . In preside ions . In anotnano HAp . Although nanohydroxyapatite has been identified as a promising defluoridating material, its use is limited. The reasons are its brittleness and the difficulty for it to be used directly in fixed bed columns because of the significant pressure drop it causes in field applications. To overcome such technological barriers and to enhance the fluoride adsorption capacity of nanohydroxyapatite, different techniques have been used.\u2212 kg\u22121 than nano HAp alone, which has a DC of 1296 mgF\u2212kg\u22121 [\u2212/kg in comparison to nano HAp, which possessesa DC of 1296 mg F\u2212/kg [\u2212/kg compared to that of nano Hap [2+ in the gelatin polymatrix via electrostatic attraction [\u2212 ions of the n-HAp lattice are replaced by F\u2212 ions by means of ion exchange [2+ does not occur and, consecutively, permits the entrapping of fluoride ions from the solution as a result of the electrostatic adsorption as well as the strong Lewis acid\u2013base interaction [The collective effect of biopolymer and inorganic material has the ability to increase the mechanical properties of the composite . Sundaramg F\u2212/kg . Pandi amg F\u2212/kg . They obnano Hap . Pandi atraction . In addiexchange . Howevereraction . Pandi aeraction .3O4 /HAp /Chitosan as adsorbents to remove fluoride ions from the solution [\u2212 and OH\u2212 and the electrostatic adsorption of Ca2+ and F\u2212, the adsorption mechanism also involved the complexation of Fe3+ and F\u2212 [The excepted compound with organic compounds, adding inorganic substances to improve the adsorption capacity, is also a potential method. Pandi and Viswanathan prepared Fesolution . Compare+ and F\u2212 . Moreove+ and F\u2212 . 3+, Mg2+ and La3+ to adsorb fluoride ions [3+ and La3+ are greater than the two valence cations of Mg2+, the surface part has a more positive charge, which makes its adsorption ability stronger [- in the hydroxyapatite crystal [Cationic doping can change the microstructure and properties of the crystal. This method is considered to improve the defluorination ability of hydroxyapatite ,150. Cheide ions . The reside ions . Becausestronger . The dopstronger . At the  crystal . +, DTA+ and HDP+ added more positive adsorption sites to it. When a positively charged surface such as HAp is modified with a cationic surfactant, the hydrophilic groups orient away from the similarly charged substrate and towards the solution. By these means, the hydrophilic character of the adsorbent is increased, and, thus, more fluoride ions are attracted. The fluoride adsorption capacities of the three adsorbents followed the order: CTAB-HAp > DTAB-HAp > HDPC-HAp [Surface modification technology has been verified to be efficient and effective in enhancing the adsorption capacities of adsorbents. Muthu Prabhu and Meenakshi investigated the fluoride adsorption capacity of nano HAp surface-modified by cationic surfactants, namely, cetyltrimethyl ammonium bromide (CTAB), hexadecylpyridinium chloride (HDPC) and dodecyltrimethyl ammonium bromide (DTAB) . The amiHDPC-HAp . The lowHDPC-HAp  The acceHDPC-HAp . Muthu PHDPC-HAp . They suHDPC-HAp . The review presents the adsorption applications of nano HAp and its composites in the removal of heavy metal ions, radionuclides, organic pollutants and fluoride ions from wastewater. Nano HAp adsorbent shows a high and constant removing ability in an alkaline medium(pH 7\u201310)for metal ions and radionuclides. A higher alkaline region is not beneficial for the adsorbent of nano HAp; however, the acidic condition facilitates the removing of organic pollutants and fluoride ions. Usually, the sorbent capacity of nano HAp increases with a rising adsorbent dosage, but too of a high dosage cannot lead to an improvement in capacity due to the agglomeration of nano HAp. Nano HAp has the advantage of removing low concentrations, especially trace pollutants. Nano HAp shows a quick adsorption, and the adsorption rate gradually declines with the extension of contact time and reaches the platform in 30\u201360 min for ions; it takes more time (2\u20136 h) to reach the adsorption equilibrium for the majority of organic pollutants. In the future, nano HAp and its composites may be advantageous as adsorbents due to their high adsorption capacities compared to other adsorbents. Furthermore, many researchers are branching out with the modification of nano HAp, using techniques such as surface modification. However, the brittleness of HAp limits its applications. Moreover, nano HAp powder causes excessive pressure drops in field applications and therefore cannot be directly used in fixed bed columns. To overcome these technological problems, the use of composites or modification for HAp has been extensively studied. Nano-sized hydroxyapatite has its disadvantage in that the small particle size  makes it very difficult to separate from an aqueous solution. However magnetic adsorbents can avoid this problem, as they can be separated easily from the solution by using an external magnetic field. Hence, combining both nanotechnology and the magnetic separation technique in developing nano HAp based adsorbents would overcome the above drawback. On the other hand, the separation of nanoparticles from aqueous solutions limits the application of HAp with magnetic materials such that it only can be used in pollution removing at small volumes, such as a bottle. For its application in rivers and lakes, HAp manufactured in a layer or block is necessary. Sintering is a common forming method which can efficiently process large-scale production. However, it is difficult to preserve the excellent properties of nanoparticles. So, composites of HAp and polymers are considered as a promising method for forming. The high dispersion of nano HAp in composites should be focused on. It can be concluded that HAp is a promising material as an adsorbent for heavy metal ions, radionuclides, organic pollutants and fluoride ions in wastewater treatment. More research efforts on the practical applications development of these fascinating materials are highly recommended for the future."}
+{"text": "The processing of faba beans generates great quantities of hulls, which are high in bioactive compounds with demonstrated radical-inhibiting properties. There is no research on the impact of using faba bean hull nanoparticles (FBH-NPs) to improve the quality and extend the shelf-life of beef products. Hence, the target of this investigation was to assess the inhibiting influence of adding FBH-NPs at two different concentrations (1 and 1.5%) on the physical attributes, lipid and protein oxidation, colour degradation, and microbiological safety of burgers during refrigerated storage (4 \u00b1 1 \u00b0C/12 days). The FBH-NPs presented great phenolic content (103.14 \u00b1 0.98 mg GAE/g dw) and antioxidant potential. The water holding capacity and cooking properties in burgers including FBH-NPs were improved during storage. The FBH-NPs significantly (p < 0.05) decreased the reduction rate of redness and lightness during the burger refrigerated storage and the FBH-NPs were more beneficial in preventing cold burger discolouration. In the FBH-NPs-treated burgers, peroxide values, TBARS, and protein carbonyl content were lower than in the control (up to 12 days). The microbiological load of burgers including FBH-NPs was lower than the load of the control during refrigerated storage. The findings revealed that FBH-NPs were more efficient in enhancing the cooking characteristics, retarding lipid or protein oxidation, preventing colour detrition and improving the microbial safety of burgers. Many nations throughout the world have seen a surge in the production and consumption of processed meat products such as patties, sausages, and burgers in recent years. Lipid oxidation is a primary source of quality degradation and shelf-life loss in foods containing fats such as meat products . Food liVicia faba, is a flowering plant that belongs to the Fabaceae family and Vicia genus [The faba bean, also known as broad bean, fava bean, or ia genus . The fabia genus  and Egypia genus . G\u00fczel aia genus  stated tia genus  and theyia genus . Nanotecia genus , includiia genus . There iFBH were bought from the feed market in the governorate of Kafr El-Sheikh, Egypt. FBH was converted to nanoparticle size using the method of Khataee et al.  with a sv/v, HCl: MeOH) were combined with five grams of FBH-NPs. The homogenate was maintained at 2 \u00b0C in the dark for 12 h before being centrifuged at 660\u00d7 g for 10 min and filtrated with Whatman paper No. 1. The filtrate solutions were kept at 20 \u00b0C until they were analysed.The FBH-NPs methanolic extract was made using a modified version of the process described by Barakat et al. . Fifty mThe total phenolic (TP) concentration was determined using the method described by Ghasemzadeh et al. , and theDifferent in vitro techniques such as DPPH, ABTS, FRAP, and \u03b2-carotene bleaching were used to assess the antioxidant capacity of the FBH and FBH-NPs. The DPPH scavenging activity was established by the procedure of Turkmen et al. . The ABTFresh beef  and back fat  were provided by the regional retail store . Ten minutes after purchasing it, the meat was taken to the laboratory and placed inside an ice box. The beef flesh and back fat were ground twice in a meat grinder , first through an 8 mm plate and then through a 4 mm plate. The burger was made using the following ingredients: 85% minced beef, 13% minced fat, 1.3% salt, 0.65% seasoning mixture  and 0.05% sodium tripolyphosphate. All of the ingredients were combined and stirred together for five minutes to produce a homogenous dough, which was then split into four groups. The first and second groups contained 1 and 1.5% of FBH-NPs, respectively. The third group had a BHT/BHA mixture in a 1:1 ratio at 0.2% as a control, while the fourth group had no antioxidants (control). Beef burgers weighing 72 \u00b1 4 g were formed into 10 cm in diameter and 1 cm thick shapes using a press burger maker. The prepared burgers were placed into Styrofoam platters, wrapped with plastic film, and kept refrigerated at (4 \u00b1 1 \u00b0C) for 12 days. The samples were tested every three days.The pH for the burgers was determined using a pH-metre  and the technique described by Abdelhakam et al. . The WHCTo analyse the burger cooking attributes reported earlier by Essa and Elsebaie , the burThe yellowness (b*), redness (a*), and lightness (L*) were measured objectively on the raw burger surface by the Hunter Lab Colorimeter apparatus  as previously stated by Essa and Elsebaie .The oxymyoglobin and metmyoglobin percentages in uncooked burgers were determined using the technique of Carlez et al. . The bel\u22121 samples.The IDF technique was used to assess the peroxide value (PV) of the burger samples . The datTBARS levels were measured in milligrams of malonaldehyde (MDA) per kilogram of specimen using spectrophotometric technique of Yoon et al. .\u22121.cm\u22121).Using 2,4-dinitrophenylhydrazine (DNPH), the protein oxidation was evaluated using the technique described by Mohamed et al. . The carA burger sample of 10 g was taken aseptically and transmitted to 90 mL of sterile peptone water . For 2 min, the mixture was vortexed. After that, sequential decimal dilutions were made from the initial dilution and peptone water. To determine the total plate count (TPCs), psychrophilic bacteria, and lipolytic bacteria, 100 \u00b5L of each dilution was aseptically transmitted and spread on the surface of plate count agar medium . All agar plates were then incubated for 48 h at 37 \u00b0C in the case of TPCs and for 5 days at 7 \u00b0C for psychrophilic bacteria. In terms of lipolytic bacteria, the plates were incubated for 5 days at 37 \u00b0C. Subsequently, they were flooded with a 20% copper sulphate solution and the bacterial colonies with blue colour zones were enumerated .p < 0.05) amongst the treatments.All treatments and analyses were performed in triplicate. All of the data were presented as mean\u00b1standard deviation (SD). Using SPSS software , the data was analysed for variance (ANOVA) and the Duncan test to determine the significance (\u22121 (on a dry basis), respectively. Hashemi and Ebrahimzadeh [\u22121. The difference in total phenolic content was related to the extraction technique used. The seed coats of other legumes likewise have higher phenolic content and antioxidant activity [\u22121, respectively. These findings are consistent with the information provided by Dawi et al. [As indicated in himzadeh  found thactivity . Accordii et al. .p < 0.05) less than that of the FBH-NPs. Methanolic extract of FBH-NPs presented lower antioxidant activity values than the artificial antioxidants when they were examined with the DPPH\u2022 technique (IC50 = 112.51 \u00b1 0.48 \u00b5g/mL) or with ABTS\u2022+ (226.66 \u00b1 1.31 \u00b5mol g\u22121 of Trolox). In the DPPH\u2022 and ABTS\u2022+ assays, alpha-tocopherol had the strongest antioxidant activity. The FBH-NPs had very low IC50 values (112.51 g/mL), indicating that they had potent antioxidant activity  for FBH methanolic extract, which is lower than the values found in this investigation  and alpha-tocopherol) using the Tukey test, it was discovered that the FBH had the poorest antioxidant capacity, followed by the FBH-NPs (+2 g\u22121) in the FRAP technique, whereas the industrial antioxidants (BHA/BHT mixture) had greater values. The purity of the commercial additives, which differs from the complexity of the examined FBH-NPs, might explain this disparity. Furthermore, due to worries about the negative health consequences of synthetic antioxidants, people increasingly prefer items with natural ingredients [During the \u03b2-carotene bleaching test, the \u03b2-carotene was oxidised, and as a result, smaller molecules were broken, the solution colour disappeared, and the yellowing discoloration of the \u03b2-carotene could be spectrophotometrically quantified . The FBH FBH-NPs . The FBHredients . The antredients .p < 0.05) in the pH of the various burger samples  compared to the control. Furthermore, during storage at 4 \u00b1 1\u00b0C, there were no significant variations in pH values (p < 0.05) between the burgers containing BHA/BHT and others containing 1.5% FBH-NPs. After nine cold-storage days, the pH of the burger control group climbed fast, reaching 6.80. Samples containing BHA/BHT and FBH-NPs at various concentrations, on the other hand, showed a small rise in pH throughout cold storage. After twelve storage days, the pH of the burger samples containing BHA/BHT was 6.30, while samples with 1% FBH-NPs and 1.5% FBH-NPs exhibited pH values of 6.63 and 6.43, respectively. The findings show an elevation in pH which may have been caused by the breakdown of nitrogenous substances via endogenous or microbiological enzymes [ enzymes .p < 0.05) greater in samples containing FBH-NPs than in the control sample. There was an increase in WHC values as a consequence of the addition of more FBH-NPs. The high dietary fibre content of the FBH-NPs might be the most likely cause of the samples\u2019 increased WHC. According to Kaya et al. [One of the most essential indices for both manufacturers and customers is the meat\u2019s WHC, which is defined as its capacity to retain all or a portion of its own and additional water . Figure a et al. , FBH inca et al. , 1 g of a et al.  proved ta et al. , the ratThe favourable effect of FBH-NPs on the burger samples might be related to the water-binding characteristics of the FBH powder. The current findings are consistent with those reported by Embaby et al. .p < 0.05) than in the samples containing FBH-NPs. Cooking yield was considerably greater (p < 0.05) in the beef burgers containing FBH-NPs. When compared to other treatments, the beef burger that contained 1.5% FBH-NPs had the maximum cooking yield. This is most likely due to the FBH-NPs hydrocolloidal fibre\u2019s ability to produce a three-dimensional matrix that holds not only water, but also fat added to the formula, preventing fat and water losses throughout the cooking process [As shown in  process . Fat was process . The samp < 0.05) in the beef burgers with FBH-NPs. Beef burgers containing 1.5% FBH-NPs gave the lowest cooking yield percentage in all tested specimens. In fact, the high cooking loss percentages were from the control group and burgers containing BHA/BHT. This might be attributable to the considerable loss of fat and moisture during the cooking process. In addition, there was an increase in the cooking loss for all tested samples throughout the whole storage period. Additionally, as shown in the same figure, cooking loss increased marginally in the burger samples made with FBH-NPs compared to the control sample over the cold storage time. Nevertheless, the burgers containing FBH-NPs appeared to have a lower cooking loss percentage than the other treatments. The samples with 1.5% FBH-NPs had the lowest cooking yield (21.07%) after 12 days of cold storage.However, as shown in Generally, the cooking yield and cooking loss of beef burgers formed with FBH-NPs followed the same pattern as the WHC findings. The addition of FBH-NPs had a good influence on the cooking characteristics of the produced burger specimens. These findings might be attributed to the functional capabilities of FBH powder as a water-binding substance in general, rather than polyphenols specifically .p < 0.05).p < 0.05). The control sample showed the greatest hunter L* value (p < 0.05) when compared to the other treatments. As FBH-NPs were added to the burgers, they darkened, resulting in lower hunter L* values when compared with the control sample. Furthermore, increasing the concentration of FBH-NPs to 1.5% resulted in a steady decrease in hunter L* values (41.85). The dark hue of FBH caused a drop in hunter L* values for burgers reinforced with FBH-NPs. After storage, the burgers with the BHT/BHA mixture displayed a small decline in hunter L* value (40.87) when compared with the control specimen (41.10). In terms of changes in the hunter L* values over storage (zero to twelve days), all analysed samples showed a progressive reduction trend, suggesting increased darkening . The oxymyoglobin content of the burger-FBH-NPs was greater than that of the control. These findings revealed that the FBH-NPs significantly raised the oxymyoglobin content of burgers as compared to the control (p < 0.05).p < 0.05) greater than in the other treatments. Similarly, the proportion of metmyoglobin in all treatments was raised significantly with storage prolongation (p < 0.05). There was a significant change (p < 0.05) between the FBH-NPs-treated and control burgers. After 12 days of storage, the conversion of oxymyoglobin to metmyoglobin was reduced in the burgers containing 1% FBH-NPs and 1.5% FBH-NPs as compared with the control. There was a strong correlation  among burger redness (hunter a*) reduction, metmyoglobin production, and oxymyoglobin% decrease (data not presented).The hunter a* value  and oxym2+ in oxymyoglobin is known to be oxidised into Fe3+ in metmyoglobin via primary and secondary lipid oxidation products [The presence of oxymyoglobin is what gives minced beef its attractive red colour . Metmyogproducts ,53. Accop < 0.05) influence on PV. The burger included polyphenol components from the spice mixture; the control group (no antioxidants were added) was tested.\u22121 fat for all raw burger samples. On the sixth day, the PV of the control group surpassed the critical limit value, followed by a quick decline; however, the PV in burgers containing BHA/BHT, 1% FBH-NPs, or 1.5% FBH-NPs remained below the limit value until the 12th day of chilled storage. The results indicate that the control specimen displayed evident lipid-oxidation until the 6th day of storage and the highest PV as an indicator for the primary auto-oxidation termination. The PV reduced after 6 days of cold storage, probably owing to hydroperoxide breakdown to create secondary lipid oxidation products [The beginning PV ranged from 0.44 to 0.46 meq peroxide Kgproducts . A smallproducts .p < 0.05). During chilled storage, the lowest amount of TBARS was found in the burgers containing artificial antioxidants (BHA/BHT) and 1.5% FBH-NPs, while the highest value was found in the control specimen. The beginning TBARS value of all produced burger samples was \u223c0.20 mg MDA/kg, which elevated fast in the control sample to 0.98 mg MDA/kg on the 9th day of storage, followed by 1% FBH-NPs (0.60 mg MDA/kg) on the 12th day, 1.5% FBH-NPs (0.492 mg MDA/kg) on the 12th day, and BHA/BHT (0.488 mg MDA/kg) on the 12th day.p < 0.05) difference in the PV and TBARS values between the burgers containing BHA/BHT and others containing 1.5% FBH-NPs under cold storage at 4 \u00b1 1\u00b0C. FBH-NPs\u2019 inhibitory impact on lipid oxidation is ascribed to their phenolic components, which exhibit antioxidant activity by preventing radical chain reactions during the oxidation reaction [The use of artificial and/or natural antioxidants considerably reduced the TBARS\u2032 values even on the first day of storage. Furthermore, lipid oxidation-decrement was greatest in the 1.5% FBH-NPs burger when compared to the control or 1% FBH-NPs samples. reaction .p < 0.05) in all stored burger specimens, while the highest content was achieved for the control group without antioxidants. After nine days of storage, the protein carbonyl concentration in the control specimen increased steadily from 1.87 to 5.94 nanomoles of carbonyl/milligram of protein. During chilled storage, the burgers containing 1.5% FBH-NPs had the smallest carbonyl concentration when compared to 1% LPP-NPs-treated. During storage, there were no significant variations in carbonyl content (p < 0.05) between burgers that were 1.5% FBH-NPs-treated and those with BHA/BHT. These findings show that FBH-NPs have a favourable influence on microbial growth suppression, particularly in proteolytic bacteria that promote protein degradation. The rise in the control sample carbonyl content observed after cold storage might be attributable to amino acid oxidation [As shown in xidation .The TPCs, psychrotrophic count, and lipolytic bacteria count in the treated and control burgers raised significantly as the storage duration increased . The ratThe inhibitory effect of bioactive and phenolic components found in FBH-NPs resulted in significantly lower TPCs, psychrotrophic, and thermophilic counts in all treated burgers when compared to the control at the end of the chilled storage duration. During the storage duration, there was no significant difference in the TPCs, psychrotrophic count, or lipolytic bacteria count among the burgers containing BHA/BHT and those containing 1.5% FBH-NPs. This could be related to the phenolics found in FBH-NPs, which have antioxidant and antimicrobial properties .The phenolic content and antioxidant capacity of FBH-NPs were investigated in this work. FBH-NPs methanolic extract has a significant phenolic content and excellent antioxidant effects. During the chilled storage duration, the addition of FBH-NPs (1% or 1.5%) to the burgers, effectively enhanced their physical properties such as WHC and cooking yield as well as minimising cooking loss. Additionally, the FBH-NPs (1% or 1.5%) addition enhanced the colour stability and reduced the metmyoglobin content of the burgers compared to the other samples. Furthermore, the same levels of FBH-NPs were sufficient to inhibit protein and lipid oxidation throughout the storage days. Adding FBH-NPs (1% or 1.5%) to the burgers enhanced their microbiological stability under refrigerated storage by reducing the TPCs, psychrotrophic count, and lipolytic bacteria count compared with the other samples. Moreover, according to the obtained results, the addition of FBH-NPs to the burgers at 1.5% was the best concentration. Using FBH-NPs as natural antioxidants, as revealed in this study, might be a useful technique for improving burger quality."}
+{"text": "Purpose: In order to overcome the problem that conventional pharmacological treatments of periodontitis cannot effectively synergizing antimicrobial and immunomodulation, inspired by the critical role of toll-like receptor 4 (TLR4) in bacterial recognition and immune activation, we demonstrated a combined antibacterial-immunoregulatory strategy based on biomimetic nanoparticles.Methods: Functioned cell membranes and silk fibroin nanoparticles (SNs) loaded with minocycline hydrochloride (Mino) were used to prepare a biomimetic nanoparticle (MSNCs). SNs and MSNCs were characterized by Scanning Electron Microscope, size, zeta potential, dispersion index. At the same time, SNs were characterized by cell counting kit-8 and real-time Polymerase Chain Reaction (RT-PCR). TLR4-expressing cell membranes were characterized by RT-PCR and western blot (WB). Cell membrane coating was characterized by Transmission Electron Microscope (TEM), the Bradford staining and WB. Then, Laser confocal, flow cytometry and agar plate coating were evaluated in vitro with antibacterial effects, RT-PCR was simultaneously evaluated with immunoregulatory effects. Finally, Anti-inflammatory treatment of MSNCs was evaluated in a ligature-induced periodontitis (LIP) mouse model.Results: Successfully prepared cell membranes overexpressing TLR4 and constructed MSNCs. In vitro studies had shown that MSNCs effectively targeted bacteria via TLR4 and acted as molecular decoys to competitively neutralize lipopolysaccharide (LPS) in the microenvironment as well as inhibit inflammatory activation of macrophages. In vivo, MSNCs effectively attenuated periodontal tissue inflammation and alveolar bone loss in a LIP mouse model.Conclusion: MSNCs have good targeted antibacterial and immunoregulatory effects, and provide a new and effective strategy for the treatment of periodontitis and have good potential for application in various types of pathogenic bacterial infections. The high prevalence of periodontitis is not only a major cause of tooth loss, but also a potential risk factor for diseases such as rheumatoid arthritis, cardiovascular disease and gastric cancer . The patIt is well known that innate immunity, as the first line of defense against bacterial invasion, relies heavily on pattern recognition receptors (PRRs) . In partetc. Surface membrane biomimetic modification of nanoparticles is a novel and efficient way. The cell membrane is coated around the nanoparticle surface by physical extrusion, ultrasonic or electroporation methods, which gives the nanoparticles bionic properties and preserves the protein function of the source cell membrane  and can release antibacterial drugs to kill bacteria efficiently. Moreover, they effectively inhibited the inflammatory activation of macrophages by neutralizing LPS in the microenvironment. In vivo studies showed that MSNCs effectively suppressed periodontal inflammation and reduced alveolar bone resorption in a ligature-induced periodontitis (LIP) mouse model  cocoons were donated from Southwest University ; TLR4 lentivirus was purchased from Genechem Co. ; FITC and LPS were purchased from Sigma-Aldrich ; Cell counting kit-8 was purchased from MCE ; Sodium carbonate, lithium bromide, paraformaldehyde and hematoxylin, and eosin staining kit were purchased from Solarbio Science and Technology Co. ; acetone was purchased from CHUANDONG CHEMICAL ; Membrane and Cytosol Protein Extraction Kit, Dil, BCA Protein Assay Kit and DAPI were purchased from Beyotime Biotechnology .2 atmosphere. Gram-negative bacteria E. coli (ATCC 43888) was grown in LB medium at 37\u00b0C in an aerobic environment, and bacteria used in all experiments were in the mid-exponential growth period. The bacterial suspension was diluted to 1*106 colony forming units (CFU per mL) for experimental use after measuring the optical density at 600\u00a0nm using a microplate reader .RAW264.7 cells is a macrophage cell line  that was established from a tumor in a male mouse induced with the Abelson murine leukemia virus. And the cell line was used for macrophage membranes collection in the current study. RAW264.7 cells cultured in Dulbecco\u2019s modified Eagle\u2019s medium  supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin  at 37\u00b0C in a 5% COSilk fibroin solution was prepared as previously described . BrieflyAdd the corresponding mass of Mino in SNs suspension at different mass ratios  and stir away from light overnight at room temperature. The supernatant was centrifuged at 32,000\u00a0g for 30 min, and the double-distilled water was resuspended and repeatedly washed twice before being stored at 4\u00b0C, protected from light. The supernatant was collected and used to calculate the encapsulation efficiency and drug loading efficiency. The equations of drug loading and encapsulation efficiency were described as follows:Lentiviral transfection of RAW264.7 cells was performed according to the product instructions. Briefly, RAW264.7 cells (50 000/well) were incubated for 12 h, then appropriate H-DMEM, TLR4 lentivirus (MOI = 50), and empty lentivirus were added separately to the culture medium for 10\u00a0h. After 48\u00a0h of incubation, the cells were harvested for the TLR4 mRNA levels by RT-PCR. Among them, the medium-treated group served as the control group for the experiment, the blank vector lentivirus group served as the negative control (NC) group, and the TLR4 lentivirus-treated group served as the TLR4 group. The sequences of the primers  were shown in E. coli in one group. The dialysis bags were completely submerged in PBS (PH = 7.4) and drug release was monitored at a constant temperature of 37\u00b0C for 0, 1, 4, 8, 12, 24, and 48\u00a0h.CMs extraction was performed according to Membrane and Cytosol Protein Extraction Kit. Briefly, functionalized macrophages were repeatedly rinsed with PBS after collection and cell counting. The cells were resuspended with Membrane Protein Extraction Reagent A containing PMSF. After 10\u00a0min of an ice bath, the cell suspension was homogenized using an ultrasonic disruptor and centrifugated at 700\u00a0g for 10 min, then the supernatant was collected and centrifugated at 14,000\u00a0g for 30 min, and resuspended the precipitate with PBS. Eventually, the concentration of CMs suspension was determined by the BCA method, and the suspension was stored at \u221280\u00b0C for use. The extracted CMs and SNs were mixed in a 1:1 mass ratio and repeatedly extruded through a polycarbonate membrane with the aid of a liposome extruder to obtain MSNCs. The morphology and structure of MSNCs were separately observed by scanning electron microscopy and transmission electron microscopy . Mean particle size and potential were detected by DLS. Finally, the Bradford method and WB were used to verify the successful coating of the CMs. After preparation of MSNCs, two equal groups of MSNCs were added into dialysis bags with appropriate amount of E. coli suspension was fixed with paraformaldehyde and washed twice with PBS. Dil-SNCs were added to the experimental group, incubated at 37\u00b0C for 1 h, stained with DAPI for 3 min, and resuspended after centrifugation to obtain the suspension. The suspension was dropped on slides for sample preparation and then images were acquired by laser confocal microscopy . Meanwhile, after paraformaldehyde fixation of E. coli, the experimental groups were incubated with FITC-SNs and FITC-SNC individually for 1\u00a0h at 37\u00b0C and washed for flow cytometry detection .The red fluorescent dye Dil and the green fluorescent dye FITC were used to label CMs and SNs, respectively. Dil -SNCs were prepared by a liposome extruder after mixing Dil with CMs for 2\u00a0h at room temperature; FITC-SNs were prepared by mixing SNs with FITC overnight away from light and purified by dialysis. E. coli were inoculated on 24-well plates, and SNCs and MSNCs were added to the experimental groups, respectively, and incubated for 24\u00a0h in a 37\u00b0C bacterial incubator. The incubated mixture was serially diluted and coated on nutrient agar plates, incubated for 24\u00a0h in a 37\u00b0C bacterial incubator, and photographed to observe the growth of colonies in the plates.RAW264.7 cells were used to detect SNCs biocompatibility and LPS neutralization. Cells were inoculated with a complete Medium in the control group and 40, 80, 120, 160, and 200\u00a0\u03bcg/mL of SNCs H-DMEM medium suspension in the experimental group, and cell viability was detected using the CCK-8 kit after 24\u00a0h of incubation. In addition, the experimental groups were incubated with LPS, LPS + SNs, and LPS + SNCs for 12 h, after being incubated in the cell incubator for 24\u00a0h. RT-PCR was performed as before.6-week-old male C57 mice were provided by the laboratory Animal Center of Chongqing Medical University. Periodontitis molds were completed by ligating the cervical portions of the bilateral maxillary first and second\u00a0M of C57 mice for 1 week using 5\u20130 silk wire. To study the diffusion of MSNCs at the site of inflammation, we prepared FITC-labeled MSNCs and injected FITC-MSNCs into the palatal gingiva of mice with ligature-induced periodontitis, collected at 0, 4, and 24 h, and observed their diffusion in the periodontium by fluorescence distribution under a stereomicroscope. Then, all mice were divided into four groups, no treatment in the control group, no treatment in the periodontitis group after completion of periodontal ligation, and the rest of the experimental groups were administered with the effective drug concentration of 0.5\u00a0mg/ml in MSNs and MSNCs suspension respectively at the ligated tooth position by drops of 5\u00a0\u03bcl on each side for 1 week. Mice were anesthetized and executed after 1 week, and periodontal tissues were taken from bilateral ligated tooth sites for RT-PCR, WB, and Hematoxylin Eosin (HE) staining to detect the inflammatory changes. An antibody against IL-1\u03b2  was used to detect markers in cell homogenization by WB. Animal handling and surgical procedures were performed following the protocol approved by the Ethics Committee of the Affiliated Stomatological Hospital of Chongqing Medical University (2022 (No. 062)).post hoc test. A value of p < 0.05 was considered statistically significant .All experimental data are presented as the mean \u00b1 standard error of the mean (SEM), and the results were analyzed using a one-way analysis of variance (ANOVA) among groups followed by Tukey\u2019s p > 0.05) to the increased concentration of SNs (p > 0.05) to the increased concentration of SNs , demonstrating that macrophage membrane camouflaging could improve the adherence to E. coli and achieve precise delivery of Mino  to the increased concentration of SNCs (p < 0.001), indicating that SNCs competitively neutralized LPS in the microenvironment and effectively inhibited the activation of pro-inflammatory macrophages , and WB assay suggested that IL-1\u03b2 protein expression had significantly decreased in MSNCs  overexpressing TLR4 by genetic engineering and proposed a new antibacterial and immunoregulatory synergistic therapy for periodontitis. The TLR4 was used to achieve precise targeted drug delivery and killing of pathogens, while nanoparticles with TLR4 acted as ideal decoys for LPS to reduce the activation of immune cells. The synergistic therapy based on MSNCs made up for the defects of the current treatment and realized the effective control of periodontal inflammation.Nanoparticles are an effective platform to achieve precise drug delivery and cellular regulation . NanoparE. coli combined with MSNCs, the initial release rate of the drug slowed down significantly, but this effect diminished significantly after 8\u00a0h. And similar to Gou\u2019s study (To achieve functions including precise drug delivery and immune escape, nanoparticles often require complex functionalization modifications . As an e\u2019s study , the druE. coli precisely (We still verified that SNCs have good biocompatibility. We next verified that MSNCs have good antibacterial effects based on their targeted drug-release properties. On the one hand, the functionalized cell membrane on the surface of the nanoparticles could bind specifically to the LPS on bacterial due to TLR4, which made them have significant bacterial binding ability; on the other hand, MSNCs could release antibacterial drugs in a short period of time to kill recisely . LPS actrecisely . The RT-To verify the inhibitory effect of MSNCs on inflammation in periodontal tissues, a LIP mouse model was constructed in this study, and its therapeutic effect was evaluated after the topical administration of treatment. MSNCs have good local storage capacity and tissue penetration and can selectively diffuse to the periodontal inflammation site with good target diffusion of inflammation. The results showed that the level of inflammation in periodontal tissues was significantly lower and tissue destruction was significantly reduced in the MSNCs group compared with the no-treatment and MSNs groups, suggesting that MSNCs effectively reduced inflammation and inhibited bone loss in periodontitis.In summary, we constructed an engineered cell membrane-encapsulated nanosystem MSNCs for precise targeting and inhibition of bacteria, and to neutralize LPS in the microenvironment to inhibit inflammatory cell activation. MSNCs effectively inhibited periodontal inflammation and bone loss through dual regulation of antibacterial and immunity. Based on this nanoplatform, genetically engineered modifications with different ligand-receptor patterns can be constructed to respond to immune diseases infected by different pathogens. In addition, this system still has some potential for improvement, such as the drug loading rate can be further optimized and the therapeutic effect can be evaluated over a longer period of time. In conclusion, MSNCs provide a new therapeutic platform for periodontitis, and the dual functional design of antibacterial and immunoregulation provides additional consideration for the treatment of inflammatory diseases with bacterial infections."}
+{"text": "Physarum polycephalum can choose diets based on protein\u2010to\u2010carbohydrate (P:C) content to support optimal growth rate. Yet, the role of genetic background  in mediating growth rate response to dietary P:C ratios in the slime mould is unknown. Here, we studied the effects of interactions between mitochondrial and nuclear DNA haplotypes and diet (i.e. G\u2009\u00d7\u2009G\u2009\u00d7\u2009E interactions) on the growth rate of P.\u2009polycephalum. A genetic panel of six distinct strains of P.\u2009polycephalum that differ in their mitochondrial and nuclear DNA haplotypes was used to measure growth rate across five diets that varied in their P:C ratio and total calories. We first determined the strains' growth rate  when grown on a set menu with access to a particular diet. We then assessed whether the growth rate of strains increased on a buffet menu with access to all diets. Our findings show that the growth rate of P.\u2009polycephalum is generally higher on diets containing more carbohydrates than protein and that total calories negatively affect the growth rate. Three\u2010way interactions between mitochondrial, nuclear haplotypes and dietary P:C ratios affected the strains' surface area of growth but not biomass. Intriguingly, strains did not increase their surface area and biomass when they had access to all diets on the buffet menu. Our findings have broad implications for our understanding of the effect of mitonuclear interactions on trait expression across diverse eukaryotic lineages.Trait expression in metazoans is strongly influenced by the balance of macronutrients  in the diet. At the same time, an individual's genetic background seems to regulate the magnitude of phenotypic response to a particular diet. It needs to be better understood whether interactions between diet, genetic background and trait expression are found in unicellular eukaryotes. A protist\u2014the slime mould,  Three\u2010way interactions between mitochondrial, nuclear haplotypes and dietary P:C ratios affected the slime mould's surface area of growth but not biomass. Our findings have broad implications for our understanding of the effect of mitonuclear interactions on trait expression across diverse eukaryotic lineages. When given a choice of 11 different isocaloric diets that differed in P:C ratio  and three nDNA haplotypes . We used the hierarchy in the mating\u2010type locus (matA) of the haplotypes to induce uniparental mitochondrial inheritance in the crossing scheme \u2009>\u2009TU9 (matA11)\u2009>\u2009Tu111 (matA12)\u2009>\u2009DP89 (matA15)\u2009=\u2009DP246 (matA16). Thus, the haplotypes AI35 and TU9 served as the mitochondrial donors because they expressed dominant matA alleles than the nDNA haplotypes  ratios. The ratios were 3:1, 1:1, 1:3, 1:5 and 1:8, and the corresponding total calories in each diet were 194, 110, 65, 108 and 102\u2009kcal respectively Table\u00a0. In thes2.3set menu design. On each experimental day, an individual plasmodial fragment (2\u2009\u00d7\u20092\u2009cm) from the oatmeal agar media was transferred onto the centre of the petri dish  containing the particular experimental diet. We assayed up to four individual plasmodial fragments per strain per diet on each experimental day. The petri dishes were stored in a dark room maintained at 24\u00b0C and ~40% relative humidity to allow the growth of macroplasmodia for 48\u2009h. We retrieved the Petri dishes from the darkroom at the end of the 48\u2010h growth period. Then, we photographed each petri dish (with a black background and standard light settings) using a digital camera . A standard ruler  was included in the background. Immediately after the photographs, we measured the wet biomass (to the nearest 0.001\u2009g) of the macroplasmodia in each petri dish (Mettler Toledo PL403). We assayed 30 plasmodial fragments per strain per diet combination across 10 experimental days.In the first assay, we studied the growth rate of each of the six slime mould strains when grown on each of the five experimental diets\u2014the 2.4buffet menu option for the slime mould strains to determine if the slime moulds could achieve a higher growth rate when choosing from all diets. Each petri dish (14.5\u2009\u00d7\u20092\u2009cm) contained equal\u2010sized wedges of the five experimental diets, with a 2\u2009\u00d7\u20092\u2009cm piece of macroplasmodium placed in the middle of the petri dish , to calculate the surface area of the macroplasmodia present on the experimental diets. First, we estimated the total surface area (a) of each photo (in cm2) using the ruler as a reference. Second, we calculated the total number of pixels in the photo (b), which included the total surface area of the petri dish. Third, the macroplasmodia were distinguished from the diet background using the quick selection and magic wand tools in the Photopea software. We then estimated the number of pixels in the selected area that shows the portion of the photo that the macroplasmodium occupied (c). Lastly, we calculated the surface area (cm2) of the macroplasmodia using the equation described below.For our second measure, growth rate, we extracted the surface area of individual macroplasmodia grown on the experimental diets in both menu options. We estimated surface area of the macroplasmodia separately from each diet wedge in the buffet menu design. We analysed all photos using an online photo editing software, Photopea , indicating that the variables should be analysed separately. Moreover, the response variables did not follow a normal distribution and so were square\u2010root transformed before the analyses. We then built separate lmer models for each response variable that included mtDNA haplotypes (two levels), nDNA haplotypes (three levels), experimental diets (five levels) and the interaction between these factors as fixed effects. The full model also included factors explaining the hierarchical structuring of the data\u2014plasmodial replicate (30 levels) denoted as \u2018plasmodia\u2019 in the model, experimental replicate , and interactions between random and fixed effects as random intercepts. We derived a final model by sequentially eliminating interaction terms (random and then fixed effects) that explained non\u2010significant (p\u2009>\u2009.05) variation in the response variables. We used a log\u2010likelihood ratio test to compare models. We selected the best model using the Akaike information criterion and p\u2010values from the model comparison test  models in the lme4 package  attributed to each fixed effect term using the F\u2010to\u2010f function in effectsize R package. The F\u2010to\u2010f function uses the F\u2010value, numerator and denominator degrees of freedom from the Type\u2010III F\u2010test results of the final models to calculate the Cohen's f effect size  of growth rate parameters adjusted for differences in total calories across all combinations of nDNA and mtDNA haplotypes. We further tested for significant pairwise contrasts across nDNA and mtDNA haplotypes using the emmeans package.Second, we tested the effect of calories on slime mould growth rate using a similar lmer approach as explained above. Here, the models included all fixed and random effect terms described above, with the exception of the categorical variable \u2018diet\u2019, which was replaced with the fixed covariate\u2014\u2018calories\u2019. The parameter estimates for fixed and random effects were estimated using the Type\u2010III ggplot2 and ggpmisc packages in R. We generated a third\u2010order polynomial equation (with the associated coefficient of determination (R2) and p\u2010value significance) for each pairwise comparison using stat_poly_line and stat_poly_eq functions in ggpmisc package . We found significant mitonuclear interactions (mtDNA\u2009\u00d7\u2009nDNA) for both surface area  and that the strain can distribute its biomass over different diets on offer to achieve optimal growth on their preferred P:C ratio ; data curation ; formal analysis ; project administration (lead); validation (lead); visualization (lead); writing \u2013 original draft (lead); writing \u2013 review and editing (lead). Natalie Cordina: Data curation ; investigation ; methodology ; software (lead); writing \u2013 review and editing (supporting). Madeleine Beekman: Conceptualization (supporting); formal analysis (supporting); funding acquisition (lead); methodology (supporting); project administration (supporting); resources (lead); writing \u2013 review and editing .The authors declare that we have no known competing interests that could have appeared to influence the work reported in this paper.https://doi.org/10.6084/m9.figshare.21286647.v2.This article has earned an Open Data badge for making publicly available the digitally\u2010shareable data necessary to reproduce the reported results. The data is available at Data S1:Click here for additional data file."}
+{"text": "We describe transition of HIV-positive children from efavirenz- or nevirapine-based antiretroviral therapy (ART) to optimal dolutegravir (DTG) or lopinavir/ritonavir (LPV/r) (solid formulation)-based ART in Lesotho.We followed a cohort of children less than 15 years of age who were initiated on ART on or after January 1, 2018 from 21 selected health facilities in Lesotho. From March 2020 to May 2022, we collected data retrospectively through chart abstraction and prospectively through caregiver interviews to cover a period of 24 months following treatment initiation. We used a structured questionnaire to collect data on demographics, ART regimen, drug formulations and switches, viral suppression, retention, and drug administration challenges. Data were summarized as frequencies and percentages, using SAS ver.9.4.Of 310 children enrolled in the study, 169 (54.5%) were female, and median age at ART initiation was 5.9 years (IQR 1.1\u201311.1). During follow-up, 19 (6.1%) children died, 41 (13.2%) were lost to follow-up and 74 (23.9%) transferred to non-study sites. At baseline, 144 (46.4%) children were receiving efavirenz-based ART regimen, 133 (42.9%) LPV/r, 27 (8.7%) DTG, 5 (1.6%) nevirapine; 1 child had incomplete records. By study end, 143 (46.1%) children were receiving LPV/r-based ART regimen, 109 (35.2%) DTG, and 58 (18.7%) were on efavirenz or nevirapine-based regimen. Of 116 children with viral load results after six months or more on a consistent regimen, viral suppression was seen in 35/53 (66.0%) children on LPV/r, 36/38 (94.7%) children on DTG and 19/24 (79.2%) children on efavirenz.Following optimal ART introduction in Lesotho, most children in the cohort were transitioned and many attained or maintained viral suppression after transition; however, we recommend more robust viral load monitoring and patient tracking to reduce losses and improve outcomes after ART transition. An estimated 1.7 million children are living with HIV worldwide , 3. PrevThe extensive use of NVP for PMTCT and for treatment, has over the decades, led to a build-up of high-level HIV resistance to NNRTI agents \u201310. EarlDolutegravir (DTG), an integrase strand transfer inhibitor (InSTI) was initially approved for use in adults and children 6 years or older who weighed \u2265 20kg. More recently, WHO approved its use in infants and children from 4 weeks of age and a body weight \u2265 3kg . DTG is In Lesotho, the 2016 national ART guidelines were aligned to WHO guidance that recommended use of ABC+3TC+LPV/r or EFV as first-line ART for children, with NVP as an alternative to LPV/r in young children on treatment for tuberculosis or as an alternative to EFV in older children who did not tolerate EFV . In 2019We enrolled and followed a cohort of HIV-positive children and adolescents 0\u201319 years of age who were either ART-na\u00efve or started ART on or after January 1, 2018 and received HIV care and treatment from selected study health facilities in Lesotho. This analysis is limited to data from children and adolescents less than 15 years of age. Cohort data were collected prospectively and retrospectively from March 2020 to May 2022. We used a structured questionnaire to collect quantitative data from study participants every 3 months during regular clinic visits until 24 months of ART follow-up. For participants who initiated ART prior to March 2020, data were collected retrospectively to ensure coverage of a period of 24 months from ART initiation. If the participant\u2019s duration on ART was less than 24 months and the participant was still in care, 3-monthly visit data were collected prospectively until 24-month follow-up data were obtained. Retrospective data were obtained through chart abstraction, while prospective follow-up involved participant and/or caregiver interviews as well as abstraction of relevant information from medical records .The study was carried out in 4 districts where the Elizabeth Glaser Pediatric AIDS Foundation (EGPAF) is implementing a United States Agency for International Development (USAID)\u2014funded HIV program for care and treatment of children and adults living with HIV. Program data were used to identify high-volume health facilities that had, by March 2019, registered at least 30 children on ART. In total, 21 high volume health facilities that included all 7 hospitals within the districts and 14 high-volume health centers were selected. At the health facilities, routine HIV care is provided based on WHO \u201ctreat-all\u201d approach. Trained counselors provide age-appropriate HIV testing, clinicians prescribe and dispense ART based on national guidelines, and return visits for clinic check-up are scheduled every 3 months. Patients who do not return to the clinics for their scheduled appointments are tracked through phone calls on the day of the missed appointment, and if unavailable, a home visit is scheduled in collaboration with village health workers in charge of the patient\u2019s community. Viral load monitoring for children on ART is carried out every 6 months.Following the national policy change on optimized ART regimen in August 2019, health facilities transitioned children (\u2265 20kg) from EFV or NVP-based ART to DTG (50mg). Younger children weighing < 20kg, were transitioned to LPV/r (40/10mg) pellets or granules. Infants on liquid LPV/r formulation were transitioned to LPV/r granules. All ART transitions were in response to policy change and were not based on virologic outcomes.The study participants were drawn from a population of children receiving HIV care and treatment services at the study health facilities. Participants were enrolled if they were less than 19 years of age, were confirmed to be HIV-positive based on age-appropriate WHO-approved HIV tests, and initiated ART on or after January 1, 2018. For this analysis, only a subset of children less than 15 years was included.We consecutively enrolled eligible children during regular clinic visits and interviewed their caregivers to obtain demographic and medical data. We used electronic tablets with built-in structured questionnaires to collect quantitative data on specified study variables. The data were subsequently uploaded to a secure study database for cleaning and analysis. Participant medical records were reviewed to obtain relevant medical history. History and visit data for children retrospectively enrolled because they were no longer in care or had already reached 24 months on ART prior to the time of data collection were abstracted from clinical records. Follow-up data for prospective participants were collected during clinic visits. Data collection was carried out by trained study nurses stationed at the study sites. The nurses conducted caregiver interviews, reviewed patient medical records and extracted data on specified study variables. Caregivers were interviewed together with their children whenever they returned to the clinics for their scheduled visits.Data on participant demographics, ART regimen, drug formulations, and ART switches were collected. In addition, information on comorbidities, viral suppression following ART initiation, retention in care, and adverse events was obtained. Among participants followed prospectively, primary caregivers were interviewed and a structured questionnaire was used to obtain information on challenges experienced by children and their caregivers during ART drug administration. A primary caregiver was defined as any adult who assumed the most responsibility in caring for the health and well-being of the child/adolescent. To assess participant outcomes, optimal ART formulations were considered the exposure variable, and were defined as ART with a backbone of 2 NRTIs plus either DTG or solid formulations of LPV/r. Viral suppression was the outcome of interest and was defined at two levels: < 1000 copies/ml, a threshold routinely used for assessing progress towards the Joint United Nations Program on HIV/AIDS (UNAIDS) programmatic targets; and < 50 copies/ml for viral undetectability as described in the WHO 2021 guidelines , 22, 23.Baseline categorical demographic, social and clinical characteristics were described as frequencies and percentages while continuous variables were summarized as medians with interquartile ranges. Viral suppression and retention outcomes were summarized using proportions. All data were analysed with SAS software version 9.4 .Ethical clearance to conduct the study was obtained from the Lesotho Ministry of Health\u2019s Research Ethics Committee (IRB00009860) and the Advarra Institutional Review Board (IRB) (IRB00000971) in the United States of America. Caregivers provided written informed consent. In addition, verbal assent was obtained from children 12 years or older. A waiver of consent was obtained from the IRB to abstract data from medical records of patients who had been on ART for 24 months or lost to-follow-up at the time of data collection and were enrolled retrospectively. To maintain confidentiality, participants were assigned unique study identification numbers that were used throughout the data collection process and analysis. Before analysis, all data were anonymized and no information linking to participants were collected. Authors did not have access to identifiable data.This research was carried out by the Elizabeth Glaser Pediatric AIDS Foundation in close collaboration with the Lesotho Ministry of Health. Additional information regarding the ethical, cultural, and scientific considerations specific to inclusivity in global research is included in the Supporting Information .A total of 310 children <15 years were enrolled in the study; 236 (76.1%) were enrolled retrospectively (started ART before the period of data collection), while the remainder were prospectively enrolled. The median age at ART initiation was 5.9 years (IQR 1.1\u201311.1), and 144 (46.4%) children were below 5 years of age . At ART During the study period, a total of 248 (80.0%) participants were followed for a median duration of 23.2 months (IQR: 14.7\u201324.3). Sixty-two (20.0%) participants did not return after the baseline visit. Nineteen (6.1%) children died during follow-up, 41 (13.2%) were lost to follow-up and 74 (23.9%) transferred to non-study sites. Of those who died, 17 (89.5%) were under 5 years of age, and 2 (10.5%) were 5\u20139 years of age. Sixteen (84.2%) of the 19 deaths occurred within the first 6 months of ART initiation . Twenty-All children were initiated on ART. Follow-up data were available for the 248 children who returned to the clinics after the enrolment visit. Of these, 147 (59.3%) children never changed ART regimens, 97 (39.1%) had one regimen change, three (1.2%) had two changes within the study period, and one (0.4%) child initially seen at baseline, had only one return visit to the clinic at 15 months and was considered lost to follow-up. Of the children whose ART regimen was not changed, 88 (59.9%) were on LPV/r, 37 (25.2%) were on EFV, 20 (13.6%) were on DTG and 2 (1.4%) were on NVP. Of those who were on LPV/r throughout follow-up, 34 (38.6%) did not switch formulations; 3 on tablets, 26 on syrup, and 5 on pellets.Ninety-three (95.9%) of 97 children with a single regimen change, were transitioned from an NNRTI-based regimen to optimal regimen containing either LPV/r (solid formulation) or DTG. Two children initially on solid  LPV/r were switched to DTG; one child was changed from LPV/r to EFV and the other child switched from NVP to EFV .At the end of study follow-up, 143 (46.1%) children were on LPV/r, 109 (35.2%) were on DTG and 58 (18.7%) were on non-optimal (NNRTI) ART regimen. Of the 143 on LPV/r, 24 (16.8%) were on tablets, 53 (37.1%) were on liquid formulation, 55 (38.5%) were on pellets, and 11 (7.7%) were on granules.A total of 148 children received LPV/r at some point during follow-up. Of these, 90 (60.8%) children remained on a single formulation, 48 (32.4%) had a single change in LPV/r formulation, eight (5.4%) changed LPV/r formulations twice, and two (1.4%) children changed LPV/r formulations three times. Of the 90 children who remained on a single LPV/r formulation, 20 (22.2%) were on tablets, 54 (60%) were on liquid formulation and 16 (17.8%) were on pellets. Fifty three of 54 participants on liquid LPV/r formulation were enrolled retrospectively (treatment initiated before March 2020), with 27 (50%) of them having received LPV/r on a single visit and were thereafter lost to follow-up. Most of the LPV/r formulation changes were seen in the under 5 age group. Forty-one (85.4%) of 48 participants with a single LPV/r formulation change and eight (80%) of ten children with two or more LPV/r formulation changes were switched from liquid to solid formulations .Viral load results were only available for 189 (61.0%) children. To evaluate viral load suppression (VLS) we isolated the analysis to focus on those who had at least 6 months of follow-up data (N = 180). Of these, 58 (32.2%) were children < 5 years of age, 55 (30.6%) were 5\u20139 years and 67 (37.2%) were adolescents 10\u201314 years. When evaluating the final viral load result amongst the 180 who had at least 6 months of follow up data, a total of 148 (82.2%) children attained viral suppression; 119 (66.1%) had undetectable viral loads, and 29 (16.1%) had viral loads between 50\u2013999 copies/ml.When evaluating viral suppression across age groups, we considered those who had been on the same ART regimen for at least 6 months and had a viral load test done at least 6 months after initiation an ART regimen. This was done to best reflect a viral load associated with a regimen of interest; there were 116 children who met this criterion. Among children < 5 years of age who had been on ART for a median time of 13.4 months (IQR: 9.8\u201319.8), 40 (69.0%) children attained viral suppression, of which 25 (43.1%) had undetectable viral load. Among 5-9-year-olds on ART for a median time of 19.9 months (IQR: 16.3\u201323.0), 49 (89.1%) attained viral suppression, with 45 (81.8%) of them having undetectable viral load. The median ART duration for adolescents 10\u201314 years was 17.9 months (IQR: 11.4\u201322.2). Fifty-nine (88.1%) adolescents attained viral suppression, with 49 (73.1%) of them having undetectable viral load.Of these, 53 (45.7%) were on LPV/r, 38 (32.8%) were on DTG, 24 (20.7%) were on EFV and 1 (0.8%) was on NVP. Viral suppression was attained in 35 (66.0%) children on LPV/r, 36 (94.7%) children on DTG, and 19 (79.2%) children on EFV. Of the 35 children on LPV/r who achieved suppression, only 8 (22.9%) did not switch formulations, 24 (68.6%) had one formulation switch , and 3 (8.6%) had two formulation switches . The one child on NVP did not attain viral suppression .Of 53 children who were on LPV/r for \u2265 6 months and had viral load results, viral suppression at the end of follow-up was attained in 8 (80%) of 10 children who transitioned to tablets, 21 (70%) of 30 children transitioned to pellets, 3 (37.5%) of 8 children transitioned to granules and in 3 (60%) of 5 children who were still on liquid formulation at the last visit.Viral load results were available for 29 participants with a sample collected between 3 and 9 months following consecutive DTG ART use  and 19 participants with a sample collected between 9 and 15 months following consecutive DTG ART use . Among children with an approximate 6-month viral load result, 25 (86.2%) attained viral suppression, of which 21 had an undetectable viral load. Viral suppression was attained in 10/11 children who were newly initiated on DTG and in 15/18 children who switched to DTG from another regimen. All 19 children with an approximate 12-month viral load result had an undetectable viral load. Of these children, 7 were newly initiated and 12 switched to DTG from another regimen.Altogether 74 caregivers of prospectively enrolled participants were interviewed on challenges related to ART administration. Ten caregivers reported difficulties in administering medication to their children at one (n = 4) or multiple visits (n = 6). All the difficulties reported were for children < 5 years receiving ABC+3TC+LPV/r. Among the 9 who reported spitting or vomiting after taking LPV/r, 1 was on syrup, 1 was on pellets/granules, 5 were on tablets, and 2 had an unknown formulation. One caregiver reported the difficulty was that drugs had to be administered at an inconvenient time.Our study, conducted in the real-world public health setting of Lesotho, described transition of an ART program for CLHIV, from suboptimal NNRTI-based or liquid LPV/r-based regimens to optimal DTG or solid LPV/r-based formulations that are known to improve participant outcomes. Nearly half of the participants started with an NNRTI-based regimen, and by study end, 81.3% of children were on optimal LPV/r or DTG regimen. Overall, 82.2% of participants attained viral suppression with a notably higher proportion (94.7%) of viral suppression seen among children who transitioned to DTG.Viral resistance to NVP and NNRTIs in general, has been widely known for the last two decades , 24\u201326, Despite the delays, many of the children were able to attain and maintain viral suppression. The high viral suppression seen in children who transitioned to DTG aligns with results from clinical trials and other adult cohorts elsewhere \u201331. We dThe virologic response in children on LPV/r was, however, less impressive. We note that there were marked age differences between patients on DTG and those on LPV/r, making it difficult for any direct comparisons to be made; however, it is worth noting that only two thirds of children on LPV/r attained viral suppression. The majority of children on LPV/r were under 5 years of age and had transitioned from liquid to solid formulations. We cannot fully explain the poor performance of LPV/r; however, difficulties in administration of LPV/r liquid formulation, previously observed in other cohorts , 32, maySome children on LPV/r were started on a liquid formulation, transitioned to solid formulation, and later reverted to liquid formulations. These many changes may cause confusion among caregivers which may result in suboptimal dosing of ART. We did not collect data to explain these frequent changes; however, in settings where temporary stock-outs of optimal formulations occur, it is not uncommon for clinicians to prescribe the available formulation until the appropriate formulation is re-stocked.Changing ART often introduces new challenges since caregivers have to learn to administer the new drugs or formulations and look out for any challenges or side effects that may arise. Among caregivers who were interviewed on drug administration challenges, only a small minority (13.5%) reported difficulties in administering medication. This is reassuring and agrees with findings of a similar study conducted in Zimbabwe that interviewed 74 caregivers . In contOver the 2-year period of follow-up, 134 of 310 participants were lost from the cohort. The majority of these children  relocated or transferred to other non-study clinics for their chronic care; others (n = 41) were lost to follow-up, and some (n = 19) died. Patient transfers from one clinic to another is common and has been documented in other cohorts within the region , 36. In The high mortality seen within the first 6 months of ART, particularly among young children, has been documented by others , 39. It Our study had several limitations. First, we largely relied on extraction of data that were routinely collected for patient care in public health settings, particularly as three-quarters of our cohort were enrolled retrospectively. The analysis was therefore limited to variables available in the routine records and some of the source documents had incomplete data for key study variables. Secondly, our cohort had suboptimal viral load coverage and we did not conduct resistance testing for participants who did not achieve viral suppression. We were therefore, not able to definitively determine whether the persistent viremia was due to poor adherence to medication or resistance to ART. Thirdly, we were unable to determine final outcomes of participants who transferred to non-study facilities. This may have led to an underestimation of mortality and loss to follow-up. Despite these limitations, our study provides evidence that is relevant for design and implementation of an effective ART transition program in similar settings. In addition, the results provide real-world outcomes of pediatric ART optimization and associated challenges such as difficulty in maintaining continuity of care, frequent formulation switches, and inadequate viral load monitoring, that often occur in settings outside of clinical trials.Our study demonstrated that following change of the national ART policy in Lesotho, many children who received suboptimal ART in the public health setting were successfully transitioned to optimal LPV/r solid or DTG-based ART regimen; and many of the children maintained or attained viral suppression after transition. However, inadequate viral load monitoring, frequent formulation changes, particularly for children who were on LPV/r, and poor retention in care were a challenge to the ART program. To adequately monitor patient outcomes after transition to optimal ART regimen, we recommend establishment of a patient care system that includes robust viral load monitoring and follow-up.S1 File(DOCX)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S1 Checklist(DOCX)Click here for additional data file."}
+{"text": "Pulmonary capillary wedge pressure (PCWP) assessment is fundamental for managing dilated cardiomyopathy (DCM) patients. Although cardiovascular magnetic resonance (CMR) has become the gold-standard imaging technique for evaluating cardiac chamber volume and function, PCWP is not routinely assessed with CMR. Therefore, this study aimed to validate the left atrial expansion index (LAEI), a LA reservoir function parameter able to estimate filling pressure with echocardiography, as a novel CMR-measured parameter for non-invasive PCWP estimation in DCM patients.We performed a retrospective, single-center, cross-sectional study. We included electively admitted DCM patients referred to our tertiary center for further diagnostic evaluation that underwent a clinically indicated right heart catheterization (RHC) and CMR within 24\u00a0h. PCWP invasively measured during RHC was used as the reference. LAEI was calculated from CMR-measured LA maximal and minimal volumes as LAEI\u2009=\u2009 ( (LAVmax-LAVmin)/LAVmin)\u2009\u00d7\u2009100.We enrolled 126 patients  randomly divided into derivation (n\u2009=\u200992) and validation (n\u2009=\u200934) cohorts with comparable characteristics. In the derivation cohort, the log-transformed (ln) LAEI showed a strong linear correlation with PCWP  and remained a strong independent PCWP determinant over clinical and conventional CMR parameters. Moreover, lnLAEI accurately identified PCWP\u2009\u2265\u200915\u00a0mmHg , and the optimal cut-off identified (lnLAEI\u2009\u2264\u20093.85) in the derivation cohort discriminated PCWP\u2009\u2265\u200915\u00a0mmHg with 82.4% sensitivity, 88.2% specificity, and 85.3% accuracy in the validation cohort. Finally, the equation PCWP\u2009=\u200952.33- (9.17xlnLAEI) obtained from the derivation cohort predicted PCWP (-0.1\u2009\u00b1\u20095.7\u00a0mmHg) in the validation cohort.In this cohort of DCM patients, CMR-measured LAEI resulted in a novel and useful parameter for non-invasive PCWP evaluation.The online version contains supplementary material available at 10.1186/s12968-023-00977-2. Pulmonary capillary wedge pressure (PCWP) evaluation is fundamental for managing cardiac diseases since PCWP increase is the hemodynamic hallmark of left heart failure syndromes . In clinThe left atrial expansion index (LAEI) is a simple derived parameter describing left atrial compliance through the relative LA volume increase during the LA reservoir phase. Echo-measured LAEI estimated filling pressures in patients with chronic  and acutWe performed a retrospective, single-center, cross-sectional study. We screened DCM patients referred for further diagnostic evaluation to our tertiary Center  from February 2019 to February 2022. We included only the subject who underwent, within 24\u00a0h, clinically indicated RHC and CMR exams. All patients were elective hospitalization, hemodynamically stable, and underwent no therapeutic change between the two exams. We excluded patients with atrial fibrillation (n\u2009=\u20095), patients with MV prosthesis (n\u2009=\u20092), and patients with insufficient CMR image quality related to frequent ventricular ectopic beats such as bigeminy (n\u2009=\u20093).All enrolled patients were included in the \u201cPadua Cardiac Magnetic Resonance Imaging Registry,\u201d this specific cohort had never been published previously; the local ethics committee approved the study, and all patients provided informed consent. The datasets for the current study are available from the corresponding author upon reasonable request.RHC was performed with a Swan-Ganz catheter (SGC) through femoral transvenous access. PCWP values were measured from the pressure\u2013time recordings at the end of a normal expiration by averaging at least three cardiac cycles with the SGC-inflated balloon in the pulmonary capillary wedge position . PCWP\u2009\u2265\u200915\u00a0mmHg was defined as elevated. All patients were imaged using a 1.5T CMR scanner  with an ECG\u2010triggering and phased array coil system, following the standard protocol . Cine im3. Left atrial maximum volume (LAVmax) and minimum volume (LAVmin) were calculated applying the biplane area-length (BAL) method from the LA areas contoured respectively at ES and ED in both long-axis 4Ch and 2Ch views [CMR measurements were performed by an operator blinded to RHC and clinical data using CVi42\u00ae\u00a0software . LV and right ventricular (RV) volumes were measured, excluding papillary muscles, from the endocardial border tracings on short-axis images at end-diastole (ED) and end-systole (ES). LV ejection fraction (EF) and RVEF were calculated from the corresponding volumes with the conventional formula. LV mass was calculated by subtracting endocardial from epicardial LV ED volume tracings and multiplying it by 1.05\u00a0g/cmFurthermore, LV mass and LAVmax were also used for calculating PCWP with the equation proposed by Garg et al. PCWP\u2009=\u20096.1352\u2009+\u2009 0.07204xLAVmax)\u2009+\u2009 (0.02256xLVmass) . In an ixLAVmax\u2009+Inter- and intra-reader variability analyses were performed in 20 randomly selected cases with repeated measurements on the same images by the same reader at least four weeks later and by a second independent reader, blinded to all prior measurements.Continuous variables were summarized as mean\u2009\u00b1\u2009standard deviation and categorical variables as absolute number with percentage (%). Independent samples T-test and Chi-Square analysis were used for subgroups comparison. Linear correlation was assessed with the Pearson correlation coefficient. lnLAEI was derived by log-transformed LAEI. Multivariate linear regression analysis models tested with the F-test the independent and additive predictive role of lnLAEI for PCWP prediction over clinical and other CMR parameters. Receiver operating characteristic (ROC) curves tested lnLAEI diagnostic accuracy for PCWP\u2009\u2265\u200915\u00a0mmHg identification, and the Youden index analysis derived the optimal lnLAEI cut-off. The performance of lnLAEI for elevated PCWP identification was tested and compared against the Garg Eq. with ROC curves analysis in the validation cohort using the De Long method for the area under the curve (AUC) comparisons. The agreement of PCWP\u2009=\u200955.33\u2013 (9.17xlnLAEI) with the invasively measured PCWP was analyzed in the validation cohort using Bland\u2013Altman analysis and compared with the performance of Garg Eq.. Inter- and intra-reader variability was tested with the coefficient of variation (CoV) and the intraclass correlation coefficient (ICC). LA volumes and LAEI measured with SAX and BAL methods were compared in an independent cohort of 25 patients using paired T-test and Pearson correlation coefficient analysis. P-value\u2009<\u20090.05 was considered statistically significant. Statistical analysis was performed using SPSS 26.0  and MedCalc 19.6.1 .The study population comprised 126 DCM patients . Clinical and CMR parameters are summarized in Table LAEI showed a strong linear correlation with PCWP . However, a logarithmic curve better fitted the association between LAEI and PCWP, with a further improvement in lnLAEI linear correlation with PCWP   had a higher heart rate (HR), larger LAVmax, lower systolic blood pressure, LVEF, RVEF, and lnLAEI than the subgroup with PCWP\u2009<\u200915\u00a0mmHg (n\u2009=\u200940) . Moreover, lnLAEI remained the only PCWP independent predictor along with HR in Model 2 . The derived optimal cut-off lnLAEI\u2009\u2264\u20093.85 had 80.8% sensitivity and 97.5% specificity for discriminating PCWP\u2009\u2265\u200915\u00a0mmHg in the derivation cohort Fig.\u00a0.Fig. 2lnlnLAEI confirmed an excellent accuracy for PCWP\u2009\u2265\u200915\u00a0mmHg identification in the validation cohort . Furthermore, when lnLAEI AUC was compared with the performance of the Garg Eq.  for PCWPThe equation PWCP\u2009=\u200952.33- (9.17xlnLAEI) obtained from the derivation cohort was able to predict in the validation cohort invasively measured PCWP without systematic bias and with a better agreement (bias\u2009=\u20090.1\u2009\u00b1\u20095.7\u00a0mmHg) than Garg Eq. (Bias\u2009=\u2009-1.1\u2009\u00b1\u20099.8\u00a0mmHg). Fig.\u00a0.Fig. 4BlLAEI showed good inter- and intra-reader reproducibility Table .Table 5I2, p\u2009<\u20090.001), LAVmin , and slightly lower LAEI values  than the BAL method. Of note, the correlation between the two techniques was excellent for all three parameters   LAEI had a strong logarithmic correlation with PCWP; (ii) lnLAEI was an independent determinant of PCWP and provided added predictive value after accounting for other clinical and CMR PCWP determinants; (iii) lnLAEI\u2009\u2264\u20093.85 cut-off obtained from the derivation cohort had 85.3% accuracy in identifying PCWP\u2009\u2265\u200915\u00a0mmHg in the validation cohort; (iv) PWCP\u2009=\u200952.33- (9.17xlnLAEI) obtained from the derivation cohort was able to predict PCWP (\u2212\u00a00.1\u2009\u00b1\u20095.7\u00a0mmHg) in the validation cohort; (v) LAEI was more accurate than Garg Eq. in discriminating normal vs. elevated PCWP and for PCWP quantitative estimation.CMR is the gold standard imaging technique for quantifying the size, mass, and global and regional LV and RV function and accurately assessing myocardial scar and fibrosis. Therefore nowadays, CMR is the reference imaging technique for cardiomyopathies evaluation . HoweverIn our study, we found that CMR-measured LAEI had a strong logarithmic association with PCWP , as previously found with Echo-measured LAEI , 16. Int2 was only marginally higher (R2\u2009=\u20090.68) than lnLAEI alone (R2\u2009=\u20090.65), underlying the trivial influence of the other parameters on PCWP estimation when lnLAEI was included in the Model. In Model 2, after accounting for clinical and CMR PCWP determinants , lnLAEI remained the strongest independent determinant of PCWP. Interestingly, despite worse MR determined as expected higher PCWP and lower LAEI, the logarithmic correlation between the two parameters remained stable independently of MR severity, similarly to Echo-measured LAEI [Notably, lnLAEI alone explained 65% of the PCWP variance in the derivation cohort in the univariate analysis. In Model 2, Rred LAEI , 16.2\u2009=\u20090.68 vs. lnLAEI: R2\u2009=\u20090.65) with the advantage of providing a practical and user-friendly equation. However, from our findings, the agreement between the lnLAEI equation and invasive PCWP was still modest in some patients, suggesting that the lnLAEI equation should be currently adopted solely as an integrative parameter for further PCWP quantitative insight. Therefore, the dichotomized evaluation of elevated vs. normal PCWP with lnLAEI should currently remain the cornerstone for a reliable non-invasive PCWP assessment with CMR.We found lnLAEI to be an accurate parameter for dichotomizing elevated vs. normal PCWP as lnLAEI\u2009\u2264\u20093.85 was able to identify in the validation cohort PCWP\u2009\u2265\u200915\u00a0mmHg with an accuracy of 85.3% . However, despite the dichotomized approach  being the current fundament for non-invasive PCWP evaluation as in echocardiography , a quantImportantly, we found that in our cohort of DCM patients, the CMR-measured LAEI was more accurate than the Garg Eq. for discriminating normal vs. elevated PCWP (Accuracy 85.3% vs. 61.8% for PCWP\u2009\u2265\u200915\u00a0mmHg identification) and that PWCP\u2009=\u200952.33- (9.17xlnLAEI) was more accurate than Garg Eq. for quantitative estimation of PCWP. We might speculate that the superiority of lnLAEI over Garg Eq. might be explained by the fact that Garg Eq. was based on sole anatomical parameters (LAVmax and LV mass) and derived from a population that included only 6.2% of patients with reduced LVEF.Other LA reservoir function parameters might play a role in PCWP assessment, and a recent study showed that LA longitudinal strain assessed during rest and stress CMR discriminated patients with elevated PCWP and heart failure with preserved ejection fraction . These rCMR-measured LAEI might become a widespread and practical volume-based LA reservoir parameter for non-invasive PCWP evaluation because it could be easily obtained without additional CMR acquisitions other than conventional cine 4-ch and 2-ch long-axis. Moreover, LAEI calculation is not time-consuming since it only requires the additional measurement of LAVmin to LAVmax, which is already measured in most laboratories. Finally, calculating LAEI does not require any dedicated software package and, therefore, could be promptly integrated into the routine clinical activity of every CMR laboratory.This study was a single-center and retrospective study. A selection bias is possible since patients were referred to our tertiary center for further diagnostic assessment. However, we focused our research on a selected cohort of patients, and our findings were strengthened by assessing LAEI over a wide range of PCWP values and by providing internal validation of our results in an independent validation cohort. CMR and RHC exams were not simultaneous. However, the time-lapse was minimal, and the patients were hemodynamically stable and did not undergo therapeutic changes between the exams. All patients included were in sinus rhythm; therefore, the performance of LAEI in patients with atrial fibrillation has not been assessed. In this study, LAEI was calculated with the BAL method. Although the SAX volumetry provided highly concordant LAEI values compared to the BAL method in an independent cohort of 25 patients, the two approaches still provided slightly different LA volumes and LAEI values. Therefore, future studies are needed to assess the interchangeability between the approaches for LAEI calculation and PCWP evaluation and other potential differences due to changes in acquisition protocols .We did not assess LA strain; therefore, a direct comparison of LAEI with LA strain cannot be performed. Finally, future multicentric and prospective studies are needed to confirm our findings for external validation, to compare different methods for LAEI calculation formally, to explore the performance of LAEI measured with CMR in different cardiac diseases  and ethnic groups .In this cohort of DCM patients, CMR-measured LAEI resulted in an accurate parameter for non-invasive dichotomization of normal versus elevated PCWP. Moreover, additional quantitative PCWP insight might be obtained using a simple LAEI-derived equation.Additional file 1: Fig. S1. PCWP and LAEI logarithmic correlation for MR \u2265 moderate and MR < moderate. Table S1. PCWP and LAEI values by MR degrees."}
+{"text": "Monitoring ferroptosis-related miRNAs is crucial for the treatment and prognosis of patients with intracerebral hemorrhage. In this work, a novel hydrophobic paper (h-paper)-based plasmonic substrate was produced by dropping DS Au nanorods with a narrow range of sizes and morphologies onto h-paper. Raman reporter molecules were adsorbed to the array surface, and surface-enhanced Raman scattering spectra at randomly selected points reveal uniform and significant SERS enhancement. Hairpin DNAs labelled with Raman reporters and hybridized with placeholder DNAs were decorated on SERS substrate to fabricate SERS biosensor. Target miRNAs initiated the \u201cinverse Molecular Sentinel\u201d process. During the process, PHs were removed and the conformation of HPs changed toward the hairpin structure, thus eliciting the proximity of Raman reporter to substrate and a stronger SERS signal. The proposed SERS biosensor performs well in terms of stability, reproducibility, and selectivity. The limits of detection of miR-122-5p and miR-140-5p in serum were 4.17 aM and 4.49 aM, respectively. Finally, the fabricated SERS biosensor was applied to detect miR-122-5p and miR-140-5p in ICH patients and healthy subjects, and the results obtained by SERS were consistent with the results from quantitative real-time polymerase chain reaction, revealing the accuracy of the method. This simple, rapid approach offers great potential for the simultaneous detection of miRNAs in practical clinical applications. In the in vitro  and dispin vitro . Intervein vitro . Studiesin vitro , heart fin vitro , traumatin vitro , as wellin vitro . Yin M e-3\u03b2 axis . Zhao HK-3\u03b2 axis . Thus, m-3\u03b2 axis . HoweverSurface-enhanced Raman spectroscopy (SERS) has received much attention in the field of biomarkers analysis. SERS is a physical phenomenon that enhances Raman scattering when a laser excites specific rough noble metal nanoparticles. SERS technology is an analysis method based on this, which can greatly improve the detection sensitivity . The tec6\u2013107, or even achieved as large as 1015\u00a0at \u201chot spots\u201d and allowed the detection of single molecules  is considered to be the most valuable specimen for the identification of ICH biomarkers. However, some brain-derived diseases such as stoke and traumatic brain injury can lead to the disruption of BBB, resulting in the release of relevant biomarkers into the peripheral blood . Detectiolecules .Herein, a novel SERS biosensor based on hydrophobic paper (h-paper)-based plasmonic substrate and iMS was designed for simultaneous detection of miR-122-5p and miR-140-5p associated with ferroptosis following ICH. Firstly, the h-paper was prepared by soaking the filter paper in an alkyl ketene dimer (AKD) solution to convert the hydrophilic hydroxyl groups of the cellulose fibers in the paper into hydrophobic alkyl groups. The h-paper could prevent analytes and nanoparticles from rapidly absorbing, improving uniformity and reproducibility . Then, t4), silver nitrate (AgNO3), sodium borohydride (NaBH4), ascorbic acid (AA), trisodium citrate dihydrate (TSC), cetyltrimethylammonium chloride (CTAC), benzyldimethylammoniumchloride hydrate (BDAC), concentrated nitric acid , hydrochloric acid , sodium hydroxide (NaOH), cyclohexane and absolute ethanol were obtained from Yangzhou Feichang Chemicals Co. Ltd. (China). Phosphate buffer saline (PBS), 5-carboxyfluorescein (5-FAM), Cyanine5 (Cy5) were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. alkyl ketene dimer (AKD) and filter paper were purchased from Younuo Chemicals Inc. . All oligonucleotide sequences in Chloroauric acid tetrahydrate  and CTAC  were sequentially added to deionized water (1.15\u00a0mL), and the solution was stirred magnetically at room temperature for 30\u00a0min. Then trisodium citrate solution  and newly prepared NaBH4 solution  were added successively under vigorous stirring for 2\u00a0min. The reaction vessel was transferred to an oil bath at 85\u00b0C and stirred gently for 4.5\u00a0h, the color gradually changed from brown to wine red, indicating the formation of decahedral Au nanoseeds. The prepared seed solution was removed from the bath and stored at room temperature. Secondly, a growth solution containing BDAC  and HAuCl4  was immersed in a thermostatic bath at 30\u00b0C under constant stirring. 30\u00a0min later, 200\u00a0\u03bcL HCl (1\u00a0M) and 100\u00a0\u03bcL AgNO3 (10\u00a0mM) were added to the mixture, followed by stirring for 1\u00a0min at 450\u00a0rpm. Then, 75\u00a0\u03bcL AA (100\u00a0mM) and 100\u00a0\u03bcL as-prepared decahedral Au nanoseeds solution was introduced and left in a water bath at 30\u00b0C for 2\u00a0h after stirring for 1\u00a0min. After the reaction, the resulting solution was centrifuged at 9,000\u00a0rpm for 12\u00a0min, washed twice with ultrapure water, and dispersed in CTAC solution for further use.DS Au nanorods were prepared by seed-mediated method. Firstly, the preparation of decahedral Au nanoseeds followed the previously reported experimental protocol  with somvia Au-S bonds. The substrate was washed three times with ultrapure water to remove excess HP1 and HP2. Then the substrate was incubated in the solution mixed with PH1  and PH2  at 37\u00b0C for 50\u00a0min to complete the hybridization. Finally, excess unreacted reagent on the above-functionalized substrate was washed off twice with PBS and then dried at room temperature for SERS measurements.At first, the h-paper was performed by soaking the hydrophobic filter paper in a 0.1% AKD dispersion for 10 min, followed by placed in an oven at 100\u00b0C . The fab\u22121 with a spectral resolution of 3\u00a0cm\u22121. All data were analyzed with SPSS Statistics 21.0 . When the p-value was less than 0.05 (p < 0.05), the difference was considered statistically significant.Target miRNAs solution (10\u00a0\u03bcL) with different concentrations was added separately to the aforementioned SERS biosensor and incubated in a humid chamber at 37\u00b0C for 120\u00a0min. Spectra were collected after thoroughly washing substrate with PBS. The laser wavelength was 785\u00a0nm, the power was 5\u00a0mW, and the exposure time was 10\u00a0s. Each final value is the average spectral value obtained by repeating the experiment three times at three different locations to ensure the accuracy and representativeness of the results. The spectra were collected from 800 to 1800\u00a0cmAll Raman spectra were measured with a Renishaw (in Via) Raman microscope spectrometer (785\u00a0nm excitation) with a \u00d750 objective lens. The WiRETM software of Renishaw was used for Raman system operation and data acquisition. To repress the background noises of instrument, smoothing and baseline correction were applied. The UV-visible absorption spectra were obtained from a UNICO 2100\u00a0PC UV-Vis spectrophotometer and processed with Origin Lab software. Transmission electron microscopy (TEM) images of DS Au nanorods were viewed and characterized under a Tecnai 12 transmission electron microscope (Philips) at an accelerating voltage of 60\u00a0kV. Scanning electron microscopy (SEM) images of DS Au nanorods were observed using a field-emission scanning electron microscope  at a 1\u00a0kV accelerating voltage. The high-resolution TEM (HRTEM) was captured with a Tecnai G2 F30 S-Twin TEM (FEI) at 200\u00a0kV.in-situ functionalization of the substrate. Due to the presence of chain sulfhydryl groups, HP1 and HP2 could be efficiently adsorbed on the DS Au nanorod surface. PH1 and PH2 were then linked to the complementary DNA (HP1 or HP2) by hybridization effects. After the above steps, the SERS biosensor can be successfully prepared. Subsequently, after the addition of target miRNAs, the SERS biosensors could capture and hybridize with the target miRNAs, resulting in the detachment of PHs and the formation of PH/RNA heteroduplexes. The remaining HPs reverted to hairpin structure, and the SERS signal gradually increased as the distance between the Raman reporters and the substrate decreased. Based on the above mechanism, the target miRNAs in clinical serum can be quantitatively detected through the change of SERS signal intensity.The SERS assay strategy for miR-122-5p and miR-140-5p detection was shown in \u20132 M, NBA-labeled DS Au nanorods (10\u20136\u00a0M), and NBA-labeled monolayer DS Au nanorod arrays for investigating the enhancement effect of DS Au nanorods. As can be seen from SERS/CSERS)/(IRS/CRS), where ISERS was the SERS signal intensity obtained for the DS Au nanorods at a specific concentration (CSERS), whereas CRS was the concentration of the NBA, which produced a Raman signal IRS under non-SERS conditions. Thus, the EF value of DS Au nanorod was 5.3\u00d7107 when CSERS was set to 10\u20136 M and CRS was 10\u20132 M. Besides, the EF values of the monolayer DS Au nanorod array was calculated and the value is 4.6\u00d7108, which is 1 order of magnitude higher than that of the DS Au nanorods solution, indicating that the DS Au nanorods array can provide significantly enhanced SERS effect.The physical properties such as morphology and size of the as-prepared DS Au nanorods were analyzed using SEM and TEM images. As shown in \u20136\u00a0mol/L) was adsorbed on the surface of SERS substrate, then 20 spots were randomly selected and SERS spectra were performed with the intensity of the characteristic peak at 1,602\u00a0cm\u22121, which were presented in \u22121 was depicted in \u22121 formed by the positively charged nitrogen was applied  and 1,602\u00a0cm\u22121 (Cy5) were used for the quantitative detection of miR-122-5p and miR-140-5p, respectively. As depicted in \u22121 increased linearly as the concentration of miR-140-5p increased, whereas the intensity at 1,133\u00a0cm\u22121 remained constant. The result demonstrated the feasibility of detecting the two miRNAs simultaneously.Determining whether there is a cross-reaction between miR-122-5p and miR-140-5p is the key to the simultaneous analysis of two miRNAs in this experiment. 10 pM of miR-122-5p was mixed with miR-140-5p at concentrations ranging from 10 aM to 10 pM for evaluating cross-reactivity between the two miRNAs. In this work, the characteristic Raman peak intensities at 1,133\u00a0cm\u22121 and 1,602\u00a0cm\u22121 were employed to quantify the Raman intensity of miR-122-5p and miR-140-5p, respectively. Since the assembly time of HP1 and HP2 determined their assembly amount on the SERS substrate surface, it was necessary to optimize the assembly time to ensure the maximum amount. In \u22121 was set as the time function, when the time was less than 60\u00a0min, the intensity of the peak at 1,133\u00a0cm\u22121 increased almost linearly with time. Then, the rising speed gradually slowed down and reached saturation at 130\u00a0min, indicating that the assembly amount of HP1 on the substrate surface reached a maximum, therefore, the optimal assembly time was 130\u00a0min for HP1. Similarly, the intensity of the peak at 1,602\u00a0cm\u22121 attained a steady state at 130\u00a0min , triple base mismatch sequences (MT3), and random sequences were introduced. As described in R2 = 0.9859), where y is the SERS intensity at 1,133\u00a0cm\u22121, and the logarithm of miR-122-5p concentration is x. Similarly, the regression equation for miR-140-5p is y = 1,486.79x-969.87 (R2 = 0.9887). The LOD was calculated using the formula: LOD = 3\u00d7 (\u03c3/S), where \u03c3 is the standard deviation of y-intercepts and S is the slope from the calibration curve. Thus, the calculated LOD for miR122-5p and miR140-5p were 4.17 aM and 4.49 aM, which is much more sensitive than some other highly sensitive detection methods.  were detected using SERS. equation , and theIn conclusion, a rapid, simple, and ultra-sensitive SERS biosensor was constructed for the simultaneous detection of two ferroptosis-related miRNAs in peripheral blood. The SERS biosensor was composed of DS Au nanorods-modified h-paper with HPs, which were hybridized with PHs and labelled with Raman reporters. The SERS substrate exhibited high specificity, good uniformity as well as excellent reproducibility, with LOD of 4.17 aM and 4.49 aM for miR-122-5p and miR-140-5p in serum, respectively. Moreover, by optimizing several key experimental parameters, such as assembly time, hybridization time, incubation temperature, and type of buffer resolution, the best performance of the SERS biosensor platform was achieved. Finally, the SERS biosensor and traditional qRT-PCR were employed to measure the expression of miR-122-5p and miR-140-5p in ICH patients and healthy subjects, and the results revealed that ICH patients had significantly higher levels of miR-122-5p and miR-140-5p than healthy subjects, which was supported by the qRT-PCR technique. Therefore, the proposed SERS biosensor can be used as a tool to detect miRNAs associated with ferroptosis following ICH in clinical samples, with potential clinical applications."}
+{"text": "Cardiac arrest is the most time-critical emergency medical students and junior physicians may face in their personal or professional life. However, many studies have shown that most of them lack the necessary knowledge and skills to efficiently perform resuscitation. This could be related to the fact that advanced cardiovascular resuscitation courses are not always part of the undergraduate medical curriculum.The aim of this study was to describe the development, pilot implementation, and assessment of an advanced cardiovascular resuscitation course designed to enable senior medical students to manage the initial resuscitation phase in case of cardiac arrest.An introductory advanced cardiovascular resuscitation course was developed on the initiative of fifth-year medical students, in collaboration with the prehospital emergency medical service team of the Geneva University Hospitals. The 60 slots available to the 157 members of the fifth-year promotion of the University of Geneva Faculty of Medicine were filled in less than 8 hours. This unexpected success prompted the creation of a first questionnaire, which was sent to all fifth-year students to determine the overall proportion of students interested in attending an advanced cardiovascular resuscitation course. This questionnaire was also used to assess basic life support education and experience among course participants. A postcourse questionnaire was used to gather feedback regarding the course and to assess student confidence regarding the resuscitation skills they had been taught.Out of 157 fifth-year medical students, 73 (46%) completed the first questionnaire. Most thought that the current curriculum did not provide them with enough knowledge and skills regarding resuscitation and 85% (62/73) wished to attend an introductory advanced cardiovascular resuscitation course. All the participants who would have wanted to follow the full Advanced Cardiovascular Life Support course before graduating were set back by its cost . Of the 60 students who had registered for the training sessions, 56 (93%) actually attended. The postcourse questionnaire was completed by 42 (87%) students (out of 48 who had registered on the platform). They unanimously answered that an advanced cardiovascular resuscitation course should be part of the standard curriculum.This study demonstrates the interest of senior medical students in an advanced cardiovascular resuscitation course and their willingness to see such a course integrated as a part of their regular curriculum. Senior medical students about to graduate are sometimes expected to have skills close to those of certified physicians even though their training is still ongoing . During During their 6-year curriculum, medical students at the University of Geneva Faculty of Medicine (UGFM), Switzerland, follow 4 basic life support (BLS) training sessions. Even though the last of these sessions, which takes place during their fifth year, confronts them with more difficult cases requiring a better understanding of the pathophysiology of cardiac arrest (CA), they are not expected to practice skills beyond standard BLS procedures.In 2021, a small group of fifth-year medical students was interested in taking an Advanced Cardiovascular Life Support (ACLS) course since they were concerned about their resuscitation skills. Since this course is both expensive and time-consuming, they contacted the prehospital emergency medical service  of the Geneva University Hospitals to determine if an introductory advanced cardiovascular resuscitation course could be organized free of charge. A total of 60 training slots were provided to the 157 students of the fifth-year promotion, all of which were filled in less than 8 hours. This unexpected enthusiasm prompted the elaboration of a questionnaire to explore fifth-year medical students\u2019 support of the inclusion of an introductory advanced cardiovascular resuscitation course during the undergraduate emergency training program as part of the standard curriculum.The goal of this study was to describe the development, pilot implementation, and assessment of an introductory advanced cardiovascular resuscitation course designed to enable senior medical students to manage the first 10 minutes of resuscitation in case of CA.In accordance with the Swiss federal law on human research, the regional ethics committee  issued a \u201cdeclaration of no objection\u201d (Req-2021-00628) regarding this study .This was a retrospective analysis of data collected prospectively through fully automated web-based questionnaires. Participants were informed that anonymized data would be collected and presented to the UGFM committee for undergraduate education. The students were also informed by email that a publication was considered and had the opportunity to ask questions and express their potential opposition. Methods and results are reported according to the CHERRIES (Checklist for Reporting Results of Internet E-Surveys) guidelines .Information about the course was dispatched to all fifth-year medical students by email on February 18, 2021, and was simultaneously posted on the social network used for the promotion. The fifth-year UGFM promotion included 157 students representing our convenience sample. Students could register for the course by responding to the invitation email on a first come, first served basis. No financial incentive was given to promote participation, and there were no exclusion criteria.A web-based platform was created using the Joomla 3.9 content management system (Open Source Matters) to host the web-based questionnaires, which were created using the Community Surveys 5.5 component . The platform and questionnaires were thoroughly tested by 4 of the authors. The first questionnaire was sent to the participants along with practical information regarding the training sessions on April 1, 2021, and a reminder was sent 3 days later. This questionnaire was divided into 2 sections . The aimTo make sure that participants would reap the highest possible benefit from the course and to facilitate the flow of the practice sessions, they were asked to read a summary of the ACLS guidelines  before aThere were 5 training sessions between April 10 and June 12, 2021, and students were divided into groups accordingly. The course was designed by the SMUR team in collaboration with senior medical students whose pre-existing knowledge was taken into account. Course sessions lasted 5.5 hours and were scheduled during the weekends to reduce the impact on the standard teaching curriculum. Their structure, mostly based on current ACLS guidelines , is detaThree main themes were covered during the simulation sessions: rhythm recognition and management, drug use and timing of drug administration, and specialized care after the return of spontaneous circulation. Nontechnical skills such as leadership and communication were also practiced during these simulations. Regarding airway management procedures, students were taught to prepare and insert an i-gel supraglottic airway device since such devices enhance oxygenation and ventilation and do not require the level of expertise needed to perform endotracheal intubation .To ensure a personalized and efficient training experience, there was a ratio of 1 instructor for 4 medical students. This also helped adhere to the COVID-19 infection prevention guidelines, which were in effect at the time of this study.All instructors were SMUR paramedics certified in BLS training and used to teach advanced resuscitation skills. In Switzerland, paramedics follow a 3-year curriculum and are able to take care autonomously of a wide range of injured or ill patients of all ages. They are allowed to insert intravenous lines, use supraglottic airway devices, and administer a wide variety of drugs including epinephrine, antiarrhythmic agents such as amiodarone, and opiates such as fentanyl . ParamedAn unofficial course completion certificate was given to the students at the end of the course.A few hours after the end of the training session, participants received an email containing a link to the postcourse questionnaire . The goaSince participants had to be registered on the platform to answer this last questionnaire, we were able to send regular reminders to enhance the participation rate.The primary outcome was the proportion of fifth-year students wishing to follow an introductory advanced cardiovascular resuscitation course, regardless of whether they had been able to attend one of the sessions organized for their promotion. Secondary outcomes were the assessment of the motivations and impeding factors to follow such a course. Comments about the usefulness of the pilot course and potential modification, their confidence regarding the skills they learned, and their opinion on its integration into the standard curriculum were also analyzed.P value lower than .05 was considered significant.All data were stored on an encrypted MySQL database (MariaDB 5.5.5) and extracted to a CSV file. Data curation and analysis were performed under Stata . Incomplete questionnaires were not analyzed. The results are presented using descriptive statistics (n [%] and median [IQR]). Normality was assessed graphically, and between-group comparisons were carried out using the chi-square test, except for age for which the Mann-Whitney Wilcoxon test was used. A P<.001) in the subgroup of students who had registered to attend a training session . The characteristics of the respondents who had been able to register were not different from those who had not been able to register . There was no opposition from students regarding the use of their answers for publication. Participation was significantly higher (register .P=.001) in those who did not wish to attend such a course .The proportion of participants who wished to attend an introductory advanced cardiovascular resuscitation course was 85% (62/73). Their main motivation was that they thought it would help them to prepare better for residency . Most students were also interested in improving their resuscitation skills  and in increasing their knowledge . One student  reported an interest in the interprofessional aspect of this course that is, working alongside paramedics. Few of those who wished to attend thought that the current BLS-automatic external defibrillator (AED) curriculum provided them with enough knowledge and skills regarding resuscitation . This proportion was significantly higher (Students who had registered for a practice session had previously attended a median number of 4 (IQR 3-4) BLS-AED courses. Most felt confident or very confident in their BLS-AED abilities , but only a few of them had already performed cardiopulmonary resuscitation in an actual CA . Of the 10 (21%) participants motivated to follow the full ACLS course before graduating, all were set back by the cost of this course .Of the 48 participants who completed the precourse questionnaire, 19 (40%) considered specializing in emergency medicine, intensive care medicine, or anesthesiology.Forty-two of the 48 students who had completed the precourse questionnaire filled the postcourse questionnaire . There was a 93% (56/60) attendance to the practice sessions.All the participants who answered the postcourse questionnaire reported that they liked the course  and that they found its content and structure adequate. All participants thought that the course was either very useful  or useful . They linked their appreciation to the acquisition of new knowledge , to the perceived usefulness of the course , and to the opportunity of practicing resuscitation skills . Nine students wrote free comments. Four of these comments were directly linked to the importance of practicing resuscitation skills. One student commented \u201cwe tend to take for granted too easily the gestures in theory when we notice that it is not the case in practice.\u201d Three students complimented the pedagogy of the instructors: \u201cVery friendly instructors, and very interactive course.\u201d One student acknowledged the interest of including leadership skills training in the course: \u201cVery interesting leadership training.\u201d One student answered that they would feel less helpless if confronted with a CA.Most students answered that the content of the course had completely fulfilled their expectations . The most common suggestion for improvement was to provide an electronic learning (e-learning) module rather than text documents as references prior to the course.After the course, the majority of participants  felt either confident  or very confident  in their ability to apply the skills they had been taught during the course.Finally, all participants thought that such a course should be part of the regular curriculum, with 93% of them answering that it should \u201cabsolutely\u201d be a part of it.This study shows that many fifth-year medical students are highly supportive of the integration of an introductory advanced cardiovascular resuscitation course in their curriculum to feel better prepared for their first professional experience and the responsibilities it implies. The process which led to the creation of this advanced cardiovascular resuscitation course and the results of this study indicate that many students feel unprepared to manage time-critical emergencies such as CA, and ACLS courses are often offered too late during residency. Consequently, while BLS-AED refresher courses need to be held on a regular basis, advanced resuscitation skills should also be taught at least to senior medical students.Conflicting with the results of many previous studies reporting that BLS skills are often lacking among health care students -7, most The disappointing results regarding resuscitation skills reported by many studies are probably linked to skill and information retention. Indeed, it has been shown that BLS-AED skills and knowledge decrease significantly within months after the last training session ,24. The Since the undergraduate medical curriculum is already very dense and demanding, there is significant tension between the importance of teaching more advanced resuscitation skills to senior medical students and the need to continue practicing basic resuscitation maneuvers. Alternative teaching methods could therefore be considered to enhance flexibility and efficiency. In line with one of the comments recorded by a student, including an e-learning module could prove worthwhile. Indeed, interactive e-learning modules and serious games have shown many advantages compared to traditional lectures and their use has been tremendously developed during the COVID-19 pandemic -29. ThesA selection bias cannot be ruled out since the questionnaires were mostly completed by students who had already registered as participants for this course and were therefore probably more interested than some of their colleagues in this particular domain. Therefore, the postcourse confidence may be overestimated, and the lack of a similar question in the precourse questionnaire prevented the assessment of the participants\u2019 confidence. In addition, the design of the first questionnaire, the second part of which could not be filled by the students who had not been able to register for the course may have prevented the acquisition of potentially useful data. Moreover, even though there is considerable overlap between the pregraduate medical curriculum of many universities, we must acknowledge that our convenience sample only consisted of UGFM medical students and that our results might not apply to other universities. Finally, the methods used in this study were hardly ideal. Indeed, given the unforeseen enthusiasm of senior medical students for an advanced cardiovascular resuscitation course, this was not a preplanned study, and the timing of some interventions was far from perfect . This last limitation might have dampened the participation rate.Following our initial results, the UGFM committee for undergraduate education decided to integrate a mandatory blended learning advanced resuscitation course, including an interactive e-learning module, into the pregraduate medical curriculum. The uptake of this course should now be assessed, and its potential shortcomings addressed before an assessment of its actual impact on the knowledge, skills, and confidence of senior medical students can be carried out. Assessing the impact of such a course on confidence through a thorough validated questionnaire would be most relevant according to the theory of planned behavior ,37. FinaThis study demonstrates the interest of senior medical students in an advanced cardiovascular resuscitation course and their willingness to see such a course integrated as part of their regular curriculum. Regardless of the course format, enabling senior medical students to acquire advanced life support knowledge and skills should help them manage more efficiently the time-critical emergencies they will encounter either prior to graduation or during their first years of residency."
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