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+{"text": "PLoS Biology, volume 2, issue 12.In 10.1371/journal.pbio.00203809 beads per g of cells\u201d contains an error. The correct amount of beads is 8 \u00d7 108, i.e., ten times less.In Materials and Methods, the sentence \u201cThe magnetic beads were added to the extract to a ratio of about 8 \u00d710Published February 15, 2005"}
+{"text": "PLoS Computational Biology, vol 2, issue 3:  10.1371/journal.pcbi.0020021In Contributing author Blake Meyers' name and institution should appear:Blake C. Meyers, Department of Plant and Soil Sciences, Delaware Biotechnology Institute, Newark, Delaware, United States of America"}
+{"text": "PLoS Biology, volume 5, issue 3: doi: 10.1371/journal.pbio.0050091In The incorrect image was published with this synopsis. The correct image, along with the caption, appears below."}
+{"text": "DOI: 10.1371/journal.pgen.0010065PLoS Genetics, volume 1, issue 5:In The list of contributing authors does not include Daniel H. Haft, whose name should have appeared between James F. Kolonay and William C. Nelson. He participated in analysis of the data."}
+{"text": "PLoS Medicine, volume 3, issue 8: DOI: 10.1371/journal.pmed.0030330In Reference 13 was mistakenly not cited in the main article. It should have been added as a reference to the sentence: \u201cClaims associated with costs of drug development merit careful scrutiny since they are often used as a rhetorical tool to argue for faster approval times or to justify the high price of pharmaceuticals [13].\u201d"}
+{"text": "PLoS Computational Biology, volume 1, issue 4, DOI: 10.1371/journal.pcbi.0010036. In"}
+{"text": "PLoS Genetics, volume 2, issue 12: doi:10.1371/journal.pgen.0020205In http://bioinfo.lifl.fr/yass/yass.php.In the Materials and Methods, the URL to the Yass online program was incorrectly listed. The correct URL is"}
+{"text": "DOI: 10.1371/journal.pcbi.0020013PLoS Computational Biology, volume 2, issue 2:In http://cssb.biology.gatech.edu/skolnick/files/gpcr/gpcr.html.The URL provided for the GPCR model database in the published article is no longer active. The database is now located at"}
+{"text": "PLoS Genetics, volume 2, issue 7: 10.1371/journal.pgen.0020111In In the Results section, the reference to Figure S7C and S7D is incorrect; the correct reference is to Figure S6D."}
+{"text": "In the Methods section, Equation 2 is incorrect. Please see the correct equation here:"}
+{"text": "In the Methods section, there was an error in the second equation in the fifth rule of the section titled \"Discrete First-Passage-Time Technique.\" The quantity in parentheses is incorrect. It should read 0.5(1-c). For the complete, correct equation, see here:"}
+{"text": "In the hippocampal CA3 network, NMDA receptors are abundant at the recurrent synapses but scarce at the mossy fiber synapses. We generated mutant mice in which NMDA receptors were abolished in hippocampal CA3 pyramidal neurons by postnatal day 14. The histological and cytological organizations of the hippocampal CA3 region were indistinguishable between control and mutant mice. We found that mutant mice lacking NMDA receptors selectively in CA3 pyramidal neurons became more susceptible to kainate-induced seizures. Consistently, mutant mice showed characteristic large EEG spikes associated with multiple unit activities (MUA), suggesting enhanced synchronous firing of CA3 neurons. The electrophysiological balance between fast excitatory and inhibitory synaptic transmission was comparable between control and mutant pyramidal neurons in the hippocampal CA3 region, while the NMDA receptor-slow AHP coupling was diminished in the mutant neurons. In the adult brain, inducible ablation of NMDA receptors in the hippocampal CA3 region by the viral expression vector for Cre recombinase also induced similar large EEG spikes. Furthermore, pharmacological blockade of CA3 NMDA receptors enhanced the susceptibility to kainate-induced seizures. These results raise an intriguing possibility that hippocampal CA3 NMDA receptors may suppress the excitability of the recurrent network as a whole  Hippocampal CA3 pyramidal neurons form abundant recurrent connections with other CA3 neurons in vitro observations raised the hypothesis that the NMDA receptor-mediated LTP contributes to the generation of synchronous network activity. Here, we generated hippocampal CA3 pyramidal neuron-specific NMDA receptor mutant mice on the pure C57BL/6N genetic background. The ablation of hippocampal CA3 NMDA receptors resulted in the enhancement of the susceptibility to kainate-induced seizure and the emergence of characteristic large EEG spikes. We also showed that the virus-mediated ablation of hippocampal CA3 NMDA receptors in the adult mice generated characteristic large EEG spikes and that pharmacological blockade of CA3 NMDA receptors enhanced the susceptibility to kainate-induced seizures. These results raise an intriguing possibility that NMDA receptors may control negatively the excitability of the hippocampal CA3 recurrent network as a whole in vivo.NMDA receptors play key roles in synaptic plasticity and memory GluR\u03b61 gene was isolated by screening a bacterial artificial chromosome (BAC) library prepared from the C57BL/6 strain (Incyte Genomics) with the 2.2 kb-EcoRI fragment from pBKSA\u03b61 EcoRI-XbaI fragment of the BAC clone was used for construction of the targeting vector. The loxP site was inserted into the BamHI site between exon 18 and 19, and the 1.8-kb DNA fragment carrying the loxP sequence and Pgk-1 promoter-driven neomycin phosphotransferase gene (neo) flanked by two Flp recognition target (frt) sites into the SpeI site between exon 20 and 21. Endogenous EcoRI site at the 5\u2032 end of 13.3-kb EcoRI-XbaI genomic fragment was replaced with NotI site and an exogenous EcoRI site was inserted between the second loxP site and neo gene. The targeting vector p\u03b61TV was composed of the 14.8-kb NotI-XbaI fragment, MC1 promoter-driven diphteria toxin gene derived from pMC1DTpA and pBluescript II SK(+) NotI and electroporated into ES cells derived from the C57BL/6N strain EcoRI-digested genomic DNA using 284-bp fragment amplified with primers 5\u2032-ATAGAGAAAGACATGGGGC-3\u2032 and 5\u2032-TGCTACTGTGCAGGAAGTG-3\u2032 from p\u03b61TV, the 0.6 kb PstI fragment from pLFNeo XhoI\u2013EcoRI fragment from the BAC clone as 5\u2032 inner, neo, and 3\u2032 outer probes, respectively. The GluR\u03b61flox allele was also identified by PCR using primers 5\u2032-GCAGTGAGGCTCACACAGGCCTGAAGACTA-3\u2032 and 5\u2032-AGTGAACTCGGATCCTGACCATTGGCCACT-3\u2032. Chimeric mice production and elimination of the neo gene from the genome through Flp/frt-mediated excision were carried out essentially as described Genomic DNA carrying the exon 11 to 22 of the cre gene in the translational initiation site of the GluR\u03b31 gene in frame using ES cells derived from the C57BL/6N strain pgk-1 promoter-driven neo gene flanked by two frt sites cre gene. GluR\u03b61+/flox mice were crossed with GluR\u03b31-Cre mice to yield GluR\u03b31+/cre; GluR\u03b61flox/flox mice. The GluR\u03b31+/cre allele was identified by PCR using primers 5\u2032-AACTGCAGTCTTGCATGCTCTCTGGAGCC-3\u2032, 5\u2032-GGAGCGGAGACACGGGGCAT-3\u2032 and 5\u2032-TTGCCCCTGTTTCACTATCC-3\u2032. Cre recombinase-mediated NMDA receptor ablation is hippocampal CA3 pyramidal neuron-specific in GluR\u03b31+/cre; GluR\u03b61flox/flox mice . Mice were fed rep and cap genes, and the E2A, E4, and VA RNA genes of the adenovirus genome, respectively. HEK293 cells were cotransfected by the calcium phosphate coprecipitation method with the vector plasmid, pAAV-RC, and pHelper. AAV vectors were then harvested and purified by two sequential continuous iodoxale ultracentrifugations. The vector titer was determined by quantitative DNA dot-blot hybridization or quantitative PCR of DNase-I-treated vector stocks. Before administration, AAV vectors were diluted in phosphate-buffered saline to 5\u20138\u00d71010 genome copies/\u00b5l. A glass micropipette was inserted into the hippocampal CA3 region of ketamine-anesthetized mice . Two minutes after the insertion, 1.0 \u00b5l of a virus solution or vehicle was injected at a constant flow rate of 16.6 nl/min, and the glass micropipette was left in this configuration for an additional 2 min, to prevent reflux of the injected material along the injection track, before being slowly retracted. AAV spread 0.5\u20130.7 mm both rostrodorsally and laterally. For every injected animal, the limit of the infected region was verified by immunohistochemistry for Cre recombinase or GluR\u03b61.We employed AAV to deliver Cre recombinase since AAV is safe, non-pathogenic, non-inflammatory and extremely stable stable Immunohistochemistry was done as described 2Cr2O7 (Sigma) and then transferred to a 0.75% solution of AgNO3 (Sigma) for further 4 days. The treated brain was sectioned (100 \u00b5m), dehydrated and mounted on glass slides.Coronal brain sections (2 mm) were immersed for 4 days in a solution composed of 5% glutaraldehyde (Wako) and 2% KAAV-EGFP vector was delivered into the hippocampal CA3 region of ketamine-anesthetized control and mutant mice of 8 weeks old. Fourteen days later, fixed coronal brain sections (150 \u00b5m) were prepared. Neurons were examined with a Leica SP-5 confocal laser scanning microscope. Optical sections were collected at intervals of 0.15 \u00b5m and averaged 16 times using a 100\u00d7objective (N.A. 1.4). The distance between axonal varicosities was measured from 50 \u00b5m-portions of CA3 axons within the CA3 stratum radiatum GluR\u03b31 mRNA density in the CA3 region was directly compared between control and mutant mice. Double in situ hybridization was performed with mixture of [33P]dATP-labeled oligonucleotide probe for GluR\u03b61  and digoxigenin (DIG)-labeled cRNA probe for GAD67  as described Isotopic detection of mRNAs was performed as described Kainate was intraperitoneally administered to mice, and they were monitored for 1 h to determine whether they exhibited seizures with generalized tonic-clonic activity accompanying the loss of postural tone. Mice were then fixed under deep pentobarbital anesthesia for immunohistochemical analysis with the c-Fos antibody (Oncogene) 2 h after kainate administration.2, 1.3 MgSO4, 1 NaH2PO4, 26.2 NaHCO3, and 11 glucose, which was equilibrated with 95% O2/5% CO2. Synaptic responses were evoked via a bipolar stimulating electrode placed in the CA3 stratum radiatum and whole-cell recordings were made from CA3 pyramidal cells using the blind-patch technique. The stimulus strength was set at the beginning of each experiment so that the average amplitude of synaptic responses in the absence of any antagonists is around 200 pA at a holding potential of \u221280 mV. The AMPA receptor-mediated excitatory postsynaptic current (AMPA-EPSC) was isolated by subtracting the synaptic response in the presence of 10 \u00b5M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) from that in its absence. The NMDA receptor-mediated excitatory postsynaptic current (NMDA-EPSC) was recorded at +50 mV in the presence of 10 \u00b5M CNQX and 0.1 mM picrotoxin. The GABAA receptor-mediated inhibitory postsynaptic current (GABAA-IPSC) was recorded at 0 mV in the presence of 10 \u00b5M CNQX and 25 \u00b5M D-2-amino-5-phosphonovaleric acid (D-APV). The stimulus strength was constant throughout each experiment. The slow hyperpolarizing currents induced by high-frequency stimulation  were recorded at \u221220 mV in the presence of 0.1 mM picrotoxin as described previously 3-GTP . For pharmacological experiments, 10 mM BAPTA was added in the pipette solution or potassium methansulfonate in the pipette solution was replaced by cesium methansulfonate. Voltage-clamped responses were recorded with an Axopatch 1D amplifier  and the signal was filtered at 1 kHz, digitized at 2.5 kHz, and stored on a personal computer.Transverse hippocampal slices (400 \u00b5m thick) were superfused with an artificial cerebrospinal fluid (aCSF) containing (in mM): 119 NaCl, 2.5 KCl, 2.5 CaClUrethane-anesthetized mice  were fixed in a stereotaxic head holder (Narishige). For the recording of local field potentials, a tungsten electrode  or a silicon probe  was inserted into the hippocampal CA3 region , the hippocampal CA1 region  or the dentate gyrus . Signals were amplified , band-pass filtered , digitized at 1 kHz through an AD converter , and stored in a computer. Analyses of data were performed offline using LabVIEW  and IGOR (Wave matics) software. Recordings using a glass electrode  were carried out as described 2 (1.4 mM), MgCl2 (0.8 mM), Na2HPO4 (0.8 mM), and NaH2PO4 (0.2 mM). During drug infusions, the mice were restrained lightly in the disposable vinyl jacket  and 0.5 \u00b5l of the drug or aCSF was infused at a rate of 0.2 \u00b5l/min using a microinjection pump . The infusion cannulae  were left in place for a further 1 min to diffuse the drug from the needle tip, and the animal was then returned to its home cage. Kainate was delivered i.p. 20\u201330 min after APV injection.Mice were anesthetized with ketamine  and xylazine , and fixed to a stereotaxic apparatus . Two single guide cannulae  were implanted into the CA3 region of the hippocampus bilaterally , according to an atlas of the mouse brain t-test as appropriate. Statistical significance was set at p<0.05.All behavioral experiments were performed in a blind fashion. Data were expressed as mean\u00b1SEM. Statistical analysis was performed using Fisher's exact probability test, Kolmogorov-Smirnov test, log-rank test and Student GluR\u03b61/NR1 gene specifically in the hippocampal CA3 pyramidal cells by Cre-loxP recombination on the C57BL/6N genetic background. By crossing a target mouse line carrying two loxP sequences flanking exon 19 and 20 of the GluR\u03b61 gene (GluR\u03b61+/flox mice) with a hippocampal CA3 region-dominant Cre mouse line carrying the Cre recombinase gene inserted into the GluR\u03b31/KA-1 gene (GluR\u03b31-Cre mice), we obtained GluR\u03b31+/cre; GluR\u03b61flox/flox mice and GluR\u03b61flox/flox mice  . Immunohistochemical analyses showed that immunoreactivity for GluR\u03b61 protein was present in the CA3 region at P7, though the amount appeared to be decreased (y 1 (P1) . At P7, ant mice . At P21 ant mice . Residuaecreased . Howeverecreased .n\u200a=\u200a4 each; t-test, P\u200a=\u200a0.03). Thus, NMDA receptors were abolished in hippocampal CA3 pyramidal neurons of mutant mice by P21. We used mutant and control mice at P21 to P23 in the following experiments unless otherwise specified.We examined NMDA-EPSCs by whole-cell patch-clamp recordings from the pyramidal cell in the CA3 region of the hippocampus at P21 to P23. NMDA-EPSCs were evoked by stimulating associational/commissural fibers that mainly terminate in the stratum radiatum since NMDA receptors are more abundantly expressed in the stratum radiatum than in the stratum lucidum . In mutain vivo, we tested the kainate sensitivity of mutant mice since the administration of kainate to rodents stimulates initially the CA3 region and then generates seizures P<0.001, Fisher's exact probability test). Mice of both genotypes showed seizures at a higher dosage of kainate (12 mg/kg), but the latency to the onset of seizures was significantly shorter in mutant mice . Neither mutant nor control mice showed seizures after saline-administration. These results suggest that kainate-induced seizure susceptibility was enhanced in mutant mice. Susceptibility to the seizure was comparable between control GluR\u03b61flox/flox mice and GluR\u03b31+/cre; GluR\u03b61+/flox mice, indicating that the insertion of the Cre gene in one allele of GluR\u03b31 locus did not influence the susceptibility.To monitor the excitability of CA3 recurrent circuits in vivo, we employed c-Fos immunohistochemistry. There was little c-Fos immunoreactivity in the hippocampus of both control and mutant mice administrated with saline . In contrast, no significant immunoreactivity was detectable in the hippocampus of kainate-administrated control mice (n\u200a=\u200a3). Kainate at 12 mg/kg induced strong c-Fos immunoreactivity in both control and mutant mice with seizures, while the number of Fos-immunopositive cells in the hippocampus was significantly smaller in mutant mice than in control mice (n\u200a=\u200a20 sections from 5 mice). The cellular imaging of neural activity with c-Fos immunohistochemistry confirmed the enhanced seizure susceptibility of mutant mice.To monitor the neuronal activity e n\u200a=\u200a3, . AdminisUnexpectedly, we found that mutant mice lacking NMDA receptors selectively in CA3 pyramidal neurons became more susceptible to kainate-induced seizures. One obvious possibility is that the ablation of NMDA receptors may disturb the neural wiring of the hippocampal CA3 region, leading to abnormal excitability of the network. We thus examined the histological features of the hippocampal CA3 region in detail. The laminar organization and cellular distribution of the hippocampal CA3 region examined by Nissl staining was indistinguishable between control and mutant mice . Immunosn\u200a=\u200a8; mutant, 17.0\u00b11.1, n\u200a=\u200a9; P\u200a=\u200a0.80; t-test) and the primary , secondary  and tertiary dendrites  between two genotypes   . Consist\u200a=\u200a0.15) .Immunoreactivities for postsynaptic proteins, PSD-95 and GluR\u03b11/GluR1, were comparable in the hippocampal CA3 region between the two genotypes . Distribin vivo. By recording local field potentials in vivo from the hippocampal CA3 region of urethane-anaesthetized mutant mice at the age of postnatal 8 weeks, we found characteristic spikes with large amplitudes (1.5\u20134.0 mV) (n\u200a=\u200a136 from 6 mice) was 0.23\u00b10.02 Hz and the distribution of interspike intervals showed a peak at 4.75 s (Since seizure is produced by synchronous firing of a population of neurons in the brain \u20134.0 mV) . These Et 4.75 s .n\u200a=\u200a8 from 4 mice). Recording from the cortex and hippocampal CA1 region, spikes reversed their polarity in the CA1 stratum oriens. CSD analyses revealed a large sink in the CA1 pyramidal cell layer (n\u200a=\u200a8 from 4 mice). On the other hand, EEG spikes recorded from the dentate gyrus showed neither polarity reversal nor sinks in CSD maps (n\u200a=\u200a8 from 4 mice). These results suggest that characteristic spikes are generated in the pyramidal cell layers of the CA3 and CA1 regions, but not in the dentate gyrus.To investigate the origin of characteristic EEG spikes, we recorded field potentials in various hippocampal regions of mutant mice using a silicon probe with 16 recording sites. Simultaneous recording of a single EEG spike event from the hippocampal CA3 region and surrounding neocortex showed that the amplitude of EEG spikes was largest in the CA3 pyramidal cell layer. EEG spikes reversed their polarity in the CA3 stratum oriens . CurrentFurther analysis revealed that the frequency of MUA in the CA3 pyramidal cell layer was enormously high during spike events . The strin situ hybridization , while the level of GAD67 mRNA was slightly but significantly reduced in the mutant mice (P<0.001). There were no significant differences in hybridization signals of other GluR mRNAs between control and mutant mice.Because either enhanced excitation or reduced inhibition can increase the excitability of hippocampal CA3 network, we examined the mRNA levels of excitatory glutamate receptor (GluR) subunits and glutamic acid decarboxylases (GADs) expressed in the hippocampal CA3 region of the mutant mice by dization , Table 1n\u200a=\u200a32; mutant \u221273.7\u00b11.0 mV, n\u200a=\u200a26, P\u200a=\u200a0.37; input resistance: control, 113.2\u00b15.3 M\u03a9; mutant, 117.7\u00b17.2 M\u03a9, P\u200a=\u200a0.62; membrane capacitance: control, 251.8\u00b19.4 pF; mutant, 250.2\u00b18.0 pF, P\u200a=\u200a0.90). We then compared GABAA-IPSCs in the hippocampal CA3 region, which have been shown to suppress the excitability of the pyramidal cell through postsynaptic inhibition A-IPSCs were measured with the same stimulus strength at 0 mV in the presence of both the non-NMDA receptor antagonist CNQX and the NMDA receptor antagonist D-APV. The ratio of GABAA-IPSCs to AMPA-EPSCs was indistinguishable between the two genotypes   . Thus, t2+-dependent K+ channels 2+-dependent K+ channels in hippocampal CA3 neurons. At a holding potential of \u221220 mV, high-frequency stimulation, which should activate both AMPA receptors and NMDA receptors in normal mice, induced slowly decaying outward currents in the pyramidal cells of control mice (n\u200a=\u200a12). In contrast, such slow outward currents were hardly evoked by the same high-frequency stimulation in mutant mice . The outward currents in control mice were abolished by D-APV , suggesting that NMDA receptors are required for the response. NMDA receptor activation results in influx of Ca2+ into postsynaptic cells, which would activate Ca2+-dependent K+ channels. In fact, inclusion of the Ca2+ chelator BAPTA in the internal solution of patch pipettes diminished the outward currents . The outward currents were also diminished when recorded with a Cs+-based internal solution , suggesting that the currents were mediated by postsynaptic K+ channels. Taken together, the slow kinetics and sensitivities to D-APV, BAPTA and Cs+ of the outward hyperpolarizing currents suggest that the high-frequency stimulation evokes slow AHP currents 2+-activated K+ channels, which are activated by Ca2+ influx through NMDA receptor channels. These results suggest that the NMDA receptor-slow AHP coupling is diminished in the hippocampal CA3 pyramidal neurons of mutant mice, which may result in enhanced excitability of the CA3 recurrent network as a whole. The coupling between NMDA receptors and AHP currents is found in various regions In hippocampal CA1 pyramidal neurons, synaptic excitation is followed by an early GABA-mediated hyperpolarization and late AHP mediated by CaThese results with hippocampal CA3-specific NMDA receptor mutant mice raise an intriguing possibility that MDA receptors suppress the excitability of the CA3 recurrent network as a whole by restricting synchronous firing of CA3 neurons, although the possibility cannot be excluded that the enhanced excitability of the mutant mice might be due to subtle cytoarchitectural abnormalities of CA3 pyramidal neurons. To test the possibility, we then examined the effect of NMDA receptor ablation in the CA3 region of the adult brain on hippocampal network oscillations by employing a virus-mediated gene knockout technique 10) was streotaxically microinjected to the hippocampal CA3 region of GluR\u03b61flox/flox mice at 8\u20139 weeks old. Immunohistochemical analysis revealed that the infection of AAV-Cre was limited to the hippocampal CA3 region and spread within 40\u201370% of the region   (n\u200a=\u200a8 spikes). Thus, the ablation of CA3 NMDA receptors induced by AAV-Cre infection in the adult brain also resulted in the generation of characteristic EEG spikes.Local field potential recording from the CA3 region showed characteristic EEG spikes with large amplitudes in  9 mice) . The frety test) . CSD anan\u200a=\u200a8 each; P\u200a=\u200a0.23, Fisher's exact probability test) . Thus, the focal blockage of CA3 NMDA receptors also enhanced the susceptibility to kainate-induced seizure.We finally examined the seizure susceptibility of wild-type mice by focal injection of a competitive NMDA receptor antagonist, APV. We bilaterally injected 30 mM APV or aCSF into the hippocampal CA3 region of C57BL/6N mice at postnatal 8\u201310 weeks. About 20\u201330 minutes later, the animals were intraperitoneally administrated with the convulsive dose of kainate (30 mg/kg) ty test) . HoweverGluR\u03b61 mRNA was comparable between mutant and control mice at P1 but strongly decreased in mutant mice at P7. The significant expression of GluR\u03b61 protein, though reduced, was found in the CA3 region at P7 but diminished to a negligible level by P14. We found that the mutant mice lacking NMDA receptors in the hippocampal CA3 pyramidal neurons showed enhanced susceptibility to kainate-induced seizures. This observation was rather unexpected since NMDA receptor-mediated LTP was implied to contribute to the generation of synchronous network activity by in vitro studies in vivoin vitroin vivo.Here, we generated hippocampal CA3 pyramidal neuron-specific NMDA receptor mutant mice on the C57BL/6N genetic background. The expression of the in vitroh currents + channels in vivo, although the possibility cannot be excluded that the enhanced excitability of the mutant mice might be due to subtle developmental abnormalities of CA3 pyramidal neurons.It is possible that the ablation of NMDA receptors may disturb the neural wiring of the hippocampal CA3 region, leading to abnormal excitability of the network. It is well known that the NMDA receptor plays a role in the activity-dependent refinement of synaptic connections and neural pattern formation in vivo by restricting synchronous firing of CA3 neurons, although the mechanism remains to be solved. Since slow AHP currents are involved in accommodation of action potential discharge of CA1 pyramidal neurons We thus examined whether the excitability of the CA3 network is enhanced by ablation of NMDA receptors in the adult brain with a virus-mediated gene knockout technique"}
+{"text": "Cross-reactive neutralizing activity (CrNA) is elicited in around 30% of HIV-1 infected individuals and is most likely directed against conserved regions of the envelope glycoprotein complex (Env). We hypothesized that the induction of CrNA may at least to a certain extent depend on the phenotypic characteristics of Env from viruses early in infection.We selected 34 patients from the Amsterdam Cohort Studies (ACS) who had varying levels of CrNA at 2-4 years after seroconversion (SC). We retrospectively generated Env sequences from clonal HIV-1 variants that were isolated between 2 and 14 months after SC and analyzed the length of the variable regions and the number of potential N-linked glycosylation sites (PNGS).For 31 out of 34 patients we observed a correlation between a higher level of CrNA and on one hand a shorter variable region 1 (P=0.04) and on the other hand a increased number of NXS sequons relative to the number of PNGS (P=0.04), which decreases the probability of glycosylation at that site. In contrast, in the 3 patients with the most potent CrNA, defined as elite neutralizers, the viral V1 region was longer with a higher number of NXT sequons relative to the number of PNGS. These viral characteristics are similar to those in patients with low potency of CrNA, rather than to those in patients with higher potency of CrNA in their serum.Our results suggest that in general the development of CrNA in HIV-1 infected patients is associated with a more open structure of the viral Env, mediated by a short V1 loop and a low probability of glycosylation. However, our three elite neutralizers were exceptions to this rule. The identification and understanding of Env characteristics that are involved in the development of CrNA should help the rational design of an effective antibody-based vaccine immunogen."}
+{"text": "The incidence of Alzheimer\u2019s disease increases in people who have had an ischemic episode. Furthermore, APP expression is increased following ischemic or hypoxic conditions, as is the production of the A\u03b2 peptide. To address the question of why APP and A\u03b2 are increased in hypoxic and ischemic conditions we induced an ischemic episode in APP knockout mice (APP\u2212/\u2212) and BACE1 knockout mice (BACE\u2212/\u2212). We find that both APP\u2212/\u2212 and BACE\u2212/\u2212 mice have a dramatically increased risk of mortality as a result of cerebral ischemia. Furthermore, APP knockout mice have reduced cerebral blood flow in response to hypoxia, while wild-type mice maintain or increase cerebral blood flow to the same conditions. The transcription factor, serum response factor (SRF), and calcium-binding molecule, calsequestrin, both involved in vascular regulation, are significantly altered in the brains of APP\u2212/\u2212 mice compared to wild type controls. These results show that APP regulates cerebral blood flow in response to hypoxia, and that it, and its cleavage fragments, are crucial for rapid adaptation to ischemic conditions. Alzheimer\u2019s disease is a progressive neurodegenerative disease that affects the elderly. The disease is thought to occur due to the accumulation of the A\u03b2 peptide in the aging brain. The accumulation of A\u03b2 in soluble oligomers and extracellular plaques initiates a cascade of downstream events, culminating in synaptic and neuronal toxicity APP is produced in most of the tissues in the body, is evolutionarily conserved and has two mammalian APP-like family members, APLP1 and APLP2, suggesting that APP plays important biological roles. There is evidence that APP and its cleavage fragments are involved in a number of crucial cellular functions including cell survival, neurite outgrowth, synaptogenesis, synaptic plasticity, memory, neurogenesis, cell adhesion, and neuroprotection To determine whether APP\u2212/\u2212 and/or its cleavage products are an integral part of a metabolic stress response, we subjected APP\u2212/\u2212 mice to a bilateral common carotid artery occlusion resulting in global ischemia. Strikingly, whereas the control mice survived 48-hours following a global ischemic injury, both the APP\u2212/\u2212 and BACE\u2212/\u2212 mice experienced a mortality rate of around 60%. Additionally, we found that the APP\u2212/\u2212 mice show a dysregulated vascular response to hypoxia and altered proteins associated with vascular changes. Together, our data indicate that APP and/or its cleavage fragments play a necessary role in response to cellular stressors like global ischemic injury.tm1Dbo/J, B6.129-Bace1tm1Pcw/J. All three strains of mice are on the C57BL/6J background.All mouse experiments were performed in accordance with animal protocols approved by the Institutional Animal Care and Use Committee, University of California, Irvine. The animal approval number is 2011-2974. APP\u2212/\u2212, BACE\u2212/\u2212 and their control non-transgenic mice C57BL/6J were obtained from Jackson Laboratories  strain names B6.129S7-AppTo perform a gross check of the vasculature of the Circle of Willis we injected carbon black in a modified protocol 2 and their brains were collected. An additional group of no-injury control APP\u2212/\u2212 and non-transgenic mice were sacrificed and brain, heart, muscle and blood samples were collected.Global cerebral ischemia was induced by transient, bilateral clamping of the common carotid arteries for 12-minutes (two vessel occlusion -2VO) as previously described 25 APP\u2212/\u2212 mice were injected intraperitoneally with phenytoin or Saline as a control  at 30 mg/kg IP in 1\u22361 PBS and DMSO, 1-hour before global ischemia surgery .Nontransgenic (n\u200a=\u200a4) and APP\u2212/\u2212 (n\u200a=\u200a4) mice with exposed skulls were placed in an imaging setup while breathing normal air  under 1% isoflurane anesthesia from a gas mask. Baseline images were taken for 3-minutes before switching the inhaled gas mixture to hypoxic air  for 5-minutes.Using the same setup as for the hypoxia challenge, a second group of 4 C57BL/6J and 4 APP\u2212/\u2212 mice were imaged at baseline for 5-minutes followed by hypercapnic gas  for 7-minutes.Animals were sacrificed using pentobarbital overdose and their brains were collected. All brain samples were cut in half sagittally. Half of the samples were preserved in paraformaldehyde and the other half flash frozen.2Hb) and deoxy- (ctHHb) hemoglobin concentration maps on a pixel-by-pixel basis 2Hb and ctHHb, and tissue oxygen saturation (O2 Sat) was calculated as 100*ctO2Hb/TotHb. The mean flow rate and hemoglobin concentrations were calculated from a region of interest selected over the mouse forebrain as described above.Laser speckle imaging (LSI) and spatial frequency domain imaging (SFDI) were used to study blood-flow dynamics and hemoglobin oxygenation states, respectively, associated with the respiratory challenges. These methods have been previously described in detail Light-level immunohistochemistry was performed with an avitin\u2013vitinerimmunoperoxidase technique  and visualized with diaminobenzidine as previously described Fluorescent immunolabeling was performed using free-floating sections as previously described 2 nitrocellulose membranes, blocked for 1 hour in 5% (v/v) nonfat milk in Tris-buffered saline (pH 7.5) supplemented with 0.2% Tween20. The blots were then probed for the primary antibody of interest overnight, washed 3 times in Tris-buffered saline (pH 7.5) supplemented with 0.2% Tween20 then exposed the the appropriate secondary antibody for one hour at room temperature. The membrane was then washed 3 more times and then developed using Pico Chemiluminescent Substrate  and imaged on a Mini Medical Film Processor . Quantitative densiometric analyses were performed on digitized images of immunoblots with ImageJ. All error bars represent SEM.Western Blotting (N\u200a=\u200a4 control mice and 4 APP\u2212/\u2212 mice) was performed as described previously All data were analyzed by Student\u2019s two-tailed t-tests. Results were considered significant only when p<0.05. Outliers greater than 3x the standard deviation of the mean were removed from statistical analysis. All Western blots were normalized to GAPDH. Error bars indicate standard error of the mean (SEM).A number of studies have found elevated levels of AD-related proteins such as APP and A\u03b2 acutely following cerebral hypoperfusion or hypoxic injuries We examined the brains of the mice to determine if the mortality we observed in the APP\u2212/\u2212 and BACE\u2212/\u2212 mice resulted from a gross increase in cell loss following ischemia. We found no clear differences in cell morphology, measured by H&E stain, or cell death, measured by Fluoro-Jade staining, between the control and knockout mice in the cohort that survived the surgery and were sacrificed 48-hours after injury . These fPrevious studies reported that BACE\u2212/\u2212 mice are more susceptible to seizures and more susceptible to cell loss and acute mortality during pharmacologically-induced seizures To determine if APP\u2212/\u2212 mice had an altered response to vascular insults we exposed APP\u2212/\u2212 and C57BL/6J mice to either hypoxic or hypercapnic conditions while imaging the brain . SpatialBased on the data that APP\u2212/\u2212 mice have altered vascular response to low oxygen environments, we explored the protein levels of Serum Response Factor (SRF), a transcription factor involved in vessel contraction, which is upregulated in APP overexpressing mice and in AD patients We next assessed other SRF target genes in the brains of the APP\u2212/\u2212 and control mice. We did not find any significant differences between levels of any target proteins including the vascular-related Myosin light chain and \u03b1-actin or the immediate early genes phosphorylated CREB and C-fos  G, H.APP, and A\u03b2 production, has been widely reported to be upregulated following a number of metabolic insults and stresses We found that acute mortality was not directly related to tissue damage, as Fluorojade staining was equivalent between APP\u2212/\u2212, BACE\u2212/\u2212 and wild type mice subjected to the bilateral occlusions. Given that all of the mice are on the same C57BL/6J background, it is unlikely that our results are due to vascular differences between the mice. However, it is possible that is that the lack of APP or BACE1 induces structural changes in the brain during development, which we are unaware of, and that these changes render the animals susceptible to ischemia induced mortality. We cannot discount this possibility, but until conditional APP\u2212/\u2212 mice are generated we are unable to rule this out. Of note, and with a similar observation to our study, two groups have published data showing that BACE1\u2212/\u2212 mice were more susceptible to spontaneous and pharmacologically-induced seizures as well as acute mortality as the result of induced seizures in a subset of the animals Using Laser Speckle Imaging and Spatial Frequency Domain Imaging, we examined vascular responses in the APP\u2212/\u2212 and control mice during a hypoxic and hypercapnic insult. We found that during hypercapnia challenges the dilatory and contractile mechanisms were intact. However, performing hypoxia challenges revealed a decrease in blood flow in the APP\u2212/\u2212 mice but not the C57BL/6J mice. This altered vascular response suggests that APP plays a role in the vascular response to hypoxic stress. Additionally, we found alterations in some proteins involved in vessel contraction; however additional experiments are necessary to determine the role of protein alterations in these mice.Our study indicates that APP or an APP cleavage fragment is an integral part of stress response. However, more research is necessary to determine which fragment is involved and what the molecular pathways are underlying the relationship between the lack of APP and/or its fragments and mortality during ischemic stress. Understanding this interaction is of clinical value given the interest in both APP fragments as drug targets for AD, and given the high rate of cerebral hypoperfusion injuries in the elderly population."}
+{"text": "In recent decades, droughts have occurred in the western-to-central United States (US), significantly affecting food production, water supplies, ecosystem health, and the propagation of vector-borne diseases. Previous studies have suggested natural sea surface temperature (SST) forcing in the Pacific as the main driver of precipitation deficits in the US. Here, we show that the aerosol forcing in China, which has been known to alter the regional hydrological cycle in East Asia, may also contribute to reducing the precipitation in the western-to-central US through atmospheric teleconnections across the Pacific. Our model experiments show some indications that both the SST forcing and the increase in regional sulphate forcing in China play a similar role in modulating the western-to-central US precipitation, especially its long-term variation. This result indicates that regional air quality regulations in China have important implications for hydrological cycles in East Asia, as well as in the US. The western-to-central United States (US) has experienced anomalously low precipitation over the past decades , and thi2367Atmospheric aerosols are known to affect regional synoptic meteorology by directly absorbing and scattering solar radiation, a phenomenon referred to as aerosol forcing. The aerosol forcing increase has modified the regional hydrological cycle, such as by enhancing or reducing precipitation in China during certain seasons101112We use the monthly Climate Prediction Center Merged Analysis of Precipitation (CMAP)5In addition, the total precipitation variability from the CMAP observations shows an increasing trend of summer precipitation in southeastern China for 1985\u20132010 . Althoug1710http://www.cesm.ucar.edu/models/cesm1.0/cam/). The first set includes model simulations prescribed with the historical SST over the globe without the Asian SO2 emission for 1985\u20132010 (hereafter referred to as the SST-run). The second set includes model runs prescribed with the climatological monthly mean SST averaged for 1985\u20132010, but with the time-varying SO2 emission in Asia (hereafter referred to as the SO2-run). Finally, we use the historical SST for 1985\u20132010 and the time-varying SO2 emission in Asia in model runs (hereafter referred to as the SST-SO2 run). Each set of experiments is performed with four ensemble members, the ensemble mean of which is presented in this study. A detailed explanation of the model runs and some simulations of the atmospheric circulation and its associated precipitation variability during the summer in the SST-SO2 run is provided in the To further explore the influence of the increase of sulphate aerosol and SST forcing on the observed precipitation variability in southeastern China, we conduct three sets of ensemble model experiments using Community Atmosphere Model version 5.1.1, which consists of the atmospheric component in the Community Earth System Model , respectively. It is evident that the precipitation amounts in both the SST-run , NCAR CAM5 (Community Atmosphere Model version 5) model, and observations. The sulphate aerosol concentrations are obtained from the GEOS-Chem simulation for the period 1985\u20132010, using the assimilated meteorological data from MERRA NASA. GEOS-Chem has been extensively evaluated for aerosol simulations everywhere, including AsiaLinear regression and composite analysis are used to identify the teleconnective effects of sulphate aerosol forcings in the observation data and model outputs.All calculations in this paper are performed for the boreal summer season (June\u2013July\u2013August).http://cola.iges.org).All maps and plots were produced using Grid Analysis and Display System ."}
+{"text": "The use of forearm crutches has been associated with pain and neuropraxia along the ulnar bone. Whilst anatomic grips have improved comfort of crutch walking, to date anatomic forearm cuffs have not been clinically evaluated. The aim of this clinical pilot study was to determine if the use of forearm crutches with anatomic cuffs reduces pain and increases comfort and function in long-term users of forearm crutches during a 4-week period.Prospective study in ten patients suffering from end-stage osteoarthritis of the lower extremity. All participants were long-term users of conventional forearm crutches. Participants used forearm crutches with an anatomically shaped cuff for 4-weeks. General health was assessed using the SF-36, and the crutches were evaluated using a newly developed questionnaire focusing on symptoms along the forearm.p\u2009=\u2009.004) and 3.5 points , respectively, after using the crutches with the new anatomic cuff for 4\u00a0weeks. Comfort and sense of security of crutch use increased by 3.0 points  and 2.4 points . Cross-correlation analysis revealed correlations among items in the same item category and no correlations between items of different item categories of the new questionnaires.Pain and paresthesia along the forearms decreased by 3.3 points : , An anatomically shaped cuff increases comfort of forearm crutches. Further research should confirm long-term clinical improvement.ISRCTN 11135150) on 14/02/2017.This study was registered retrospectively in ISRCTN (TRN:  Because of known demographic changes, the number of patients requiring permanent walking aids will substantially increase in the near future. Today, more than half a million patients in the USA use crutches permanently, mainly because of chronic musculoskeletal or neurological disorders , 2. In tWhile in the USA axillary crutches are predominantly used, conventional forearm crutches are more common in Europe. The advantages of forearm crutches are the absence of pressure on the axilla with potentially associated nerve damage  and theiThe main pressure between the forearm and conventional crutch cuff is located over the ulnar bone during crutch walking . With inTen patients  with different musculoskeletal disorders of the spine or lower extremity  with anatomic hand grip . The length of the crutches were adjusted by the study team to the patients\u2019 hand height during stance with the arms positioned at 20 to 30\u00b0 elbow flexion . PatientWe developed a questionnaire focusing on symptoms along the forearm  with a unipolar 9-point Likert-scale  Fig.\u00a02)2). QuestAll statistical analyses were carried out in GraphPad Prism Version 6 . The data were tested for normality using Shapiro-Wilk tests , 22 and The mean pain score at the forearm decreased by 3.3 points : ; Table\u00a0Discomfort decreased at the forearms by 3.5 points , at the shoulders by 3.6 points , at the hands by 5.4 points  and at the elbows by 3.6 points (95%CI: ; Table\u00a0General crutch comfort significantly increased by 3.4 points (95%CI: ; Table\u00a0We observed a significant increase in physical functioning by 11.0 points (95%CI: 1.8; 20.2]; Table\u00a00.2; TablThe purpose of this clinical pilot study was to determine if the use of forearm crutches with anatomic cuffs reduces pain and increases comfort and function in long-term users of forearm crutches during a 4-week period. The results of this pilot study clearly showed that compared to conventional crutches, pain and discomfort along the forearm significantly improve by the use of crutches with an anatomically shaped cuff. These results suggest that using an anatomically shaped crutch cuff is a promising solution to increasingly frequent clinical reports of pain and discomfort along the ulnar bone using conventional forearm walking aids especially in individuals with chronic walking disability.Anatomic grips in forearm crutches have shown to reduce the load of the wrist, increase comfort and facilitate better control of crutch movements . AlthougThe patient population included in this clinical pilot study was highly heterogenic in terms of age, underlying disorder and other variables. Most participants showed large improvements when using the anatomically shaped cuff despite of the heterogeneity in personal characteristics, and we were unable to identify patient subgroups responding differently to the new anatomic cuff. Nonetheless it is possible that older individuals with presumably less soft tissue who are more likely to develop nerve entrapment symptoms might benefit more from the ulnar protection. Moreover, it is conceivable that younger individuals may also experience and benefit from greater comfort with the anatomic cuff during mid- or short-term use of crutches. It should be noted, however, that correct instruction of crutch walking provided, for instance, by the physiotherapist is critical for preventing overuse symptoms \u201327.This study was a clinical pilot study involving ten patients. The positive effect of the crutch with anatomic cuffs needs to be confirmed in a larger setting although blinding appears difficult because of the obvious differences between the anatomic and conventional cuffs. Eight patients had used conventional crutches with a normal grip prior to and hence also during the study whilst all crutches with anatomic cuff also had anatomic grips. Thus, potential effects of the anatomic grip on symptoms not only at the hands but also at the forearms cannot be excluded. However, changes in scores with the anatomic cuff did not depend on anatomic grip use. Interestingly, the two patients who had previously used anatomic grips also showed an improvement of their symptoms on the hands (Fig.\u00a0To date, evaluating specific aspects of forearm crutches has been difficult. In this study, we used a newly developed questionnaire comprising 17 questions categorized into four item categories. While this questionnaire has not been formally validated, cross-correlations among but not within item categories suggest that the item groups indeed assess the different aspects of pain and comfort of crutch walking investigated in this study. Hence, this questionnaire is very useful for future studies aimed at improving crutch design or for choosing optima crutches for a specific patient.The anatomic cuff for forearm crutches investigated in this study improved comfort and quality of life for patients with long-term crutch use. The significance of the anatomic cuff in reducing structural damage such as skin bruises or nerve entrapment should be investigated in larger trials."}
+{"text": "Background: Reducing vaccine wastage is important. Bacille Calmette-Gu\u00e9rin (BCG) vaccine is produced in vials of 20 infant doses. The reconstituted vaccine is discarded after 4\u20136\u00a0hours. Therefore, to reduce vaccine wastage, a 20-dose vial of BCG is often only opened if at least 10\u201312 infants are present, jeopardising BCG vaccination coverage and timely vaccination. We observed that nurses were not able to withdraw 20 doses from the vials and aimed to quantify how many doses could be obtained from these vials by experienced nurses under real-life circumstances.Methods: At the maternity ward of the national hospital in Guinea-Bissau, since 2002 the same two nurses have been vaccinating all eligible children with BCG before discharge. During a month in 2015, within a randomised trial comparing BCG-Denmark and BCG-Russia, we registered how many doses the nurses were able to withdraw from the two types of vaccine vials.Results: The median number of doses which it was possible to withdraw from the vials was 13 (range 11\u201317): 13 (11\u201316) for BCG-Denmark  and 15 (12\u201317) for BCG-Russia.Conclusions: In real life, experienced nurses could only obtain 13\u201315 doses from the 20-dose vials. Thus, vaccine wastage is much lower than assumed. Adjusting practice to the real-life number of doses would immediately suggest vials should be opened if 7 rather than 10 infants are present. As other studies have indicated that BCG may have beneficial non-specific effects on overall mortality, the potential gain by opening a 20-dose vial even for one child may be considerable. Bacille Calmette-Gu\u00e9rin (BCG) vaccine, the live attenuated vaccine against tuberculosis, is provided as a vial of freeze-dried vaccine together with a vial containing 1\u00a0ml solvent. According to the manufacturers\u2019 specifications, one vial of reconstituted vaccine contains 1\u00a0ml, corresponding to 10 doses for adults and children aged 12\u00a0months and over (0.1\u00a0ml) or 20 doses for infants less than 12\u00a0months (0.05\u00a0ml). The reconstituted vaccine should be discarded after 4\u20136\u00a0hours [Discarding remaining BCG vaccine after completion of a vaccine session, so-called \u2018open vial wastage\u2019, is an important source of wastage. Though the World Health Organization (WHO) states that \u2018it is always recommended to open a vial of vaccine for one infant or a small number of infants\u2019 , nationaOne way to reduce open vial wastage is to increase the number of children vaccinated at each session . Indeed,In order to calculate correct wastage rates, it is important to know the real-life number of doses in a 20-dose vial. In Guinea-Bissau, the Bandim Health Project (BHP) has conducted several randomised controlled trials (RCT) using BCG vaccine. For this purpose, the BHP has implemented BCG vaccination at the maternity ward of the national hospital, and since 2002, the same two skilled BCG vaccinators have vaccinated all neonates eligible for BCG vaccine. We used this set-up to test how many doses two experienced vaccinators could obtain from two different types of 20-dose BCG vaccine preparations: BCG-Denmark and BCG-Russia, both pre-qualified vaccines used interchangeably by UNICEF.In Guinea-Bissau, approximately 6,500 children/year are delivered at the maternity ward of the national hospital.Within an ongoing trial, BCG-Denmark  and BCG-Russia  are compared for their effect on overall morbidity within the first 6\u00a0weeks of life. Healthy neonates delivered at the maternity ward are randomised 1:1 to the two vaccines at discharge, with an average enrolment rate of 14 children per day. In addition, a smaller RCT at the hospital\u2019s neonatal intensive care unit randomises neonates 1:1 to receive BCG-Denmark either at admission or at discharge. The average inclusion rate for this trial is three per day. Furthermore, BCG may be given to infants born at the hospital who did not participate in an RCT .One of the two vaccinators provide all vaccinations on a given day. Vaccines are administered intradermally using a SoloShot Mini auto-disabling 0.05\u00a0ml syringe . After each vaccination, the vaccinator stores the used syringes in a safe container, one for BCG-Denmark and one for BCG-Russia.The present study took place from 12 October\u20139 November 2015. At the end of each vaccination session, the vaccinator counted the used syringes for each vial of BCG opened. Subsequently, the vaccinator drew up any excess vaccine doses left in each vial, using a new 0.05\u00a0ml BCG syringe for each dose, until no further full 0.05\u00a0ml dose could be drawn. For each vial, the vaccinator noted the number of doses used during the vaccination session and the number of doses subsequently drawn from the vial. All procedures were supervised on a daily basis by a BHP staff member.Ethical approval for the BCG trial was obtained from the Guinean Ethical Committee; the Danish ethical committee gave its consultative approval. No permission was necessary for the present study, which did not involve any study participants.With 28 vials of each vaccine, the study would have 95% power to detect if the vaccine vials in fact contained 19 rather than 20 doses, assuming an SD of 1.6 and with a one-sided alpha of 0.05.All statistical analyses were done as linear regression analyses in Stata 12.1.p\u00a0<\u00a00.00001, During 29\u00a0days, the two vaccinators opened 68 BCG vaccine vials, 39 (57%) BCG-Denmark and 29 (43%) BCG-Russia (see Supplementary Table). The median number of doses obtained was 13 (range 11\u201317): 13 (range 11\u201316) for BCG-Denmark and 15 (range 12\u201317) for BCG-Russia. More doses were obtained from the BCG-Russia vials than the BCG-Denmark vials  and 15 (BCG-Russia) doses, respectively.Common sense predicts that it would be implausible to obtain 20 doses of 0.05\u00a0ml each, when combining freeze-dried vaccine substance with 1\u00a0ml solvent, considering the several sources of wastage: first, loss in solvent vial (remaining liquid); second, dead space in needle used to reconstitute the vaccine; third, loss in vial with reconstituted vaccine; fourth, dead space in needle for injection; fifth, loss in de-airing of the syringe before injection . VaccineThe results are very important because using the correct denominator to calculate open vial wastage rates for BCG could help provide realistic wastage targets. With a request to keep wastage for 20-dose vials at 15% , an averThis change in perception would immediately alleviate pressure on health workers, no longer having to restrict BCG vial opening as fiercely, with important consequences for BCG vaccination coverage, and for the mothers, who often come several times in vain before getting their child vaccinated. For instance, in 2010 in rural Guinea-Bissau, only 38% were BCG-vaccinated by 1\u00a0month of age, yet 25% of the BCG-unvaccinated children had been in contact with a health facility; many of their mothers reported having been told to come back another day for vaccination. Had the children been vaccinated at that contact, the BCG vaccination coverage by 1\u00a0month would have soared to 54% rather than 38% [Timely BCG vaccination is particularly important because BCG vaccine provided at birth has been shown in RCTs to reduce neonatal mortality by more than 40% ,6. The gWe found that in real life, experienced nurses could only obtain 13-15 doses from 20-dose BCG vials. Currently, to reduce vaccine wastage, in many low-income countries a BCG vial is not opened for less than 10-12 infants. Adjusting wastage-reducing practice to the real-life number of doses would immediately suggest vials be opened if 7 infants are present. As BCG may have beneficial non-specific effects, the potential gain by opening a vial even for one child may be considerable.Click here for additional data file."}
+{"text": "Overall, high school students were neutral in their concern, although Food and Nutrition students specifically were significantly less concerned (p = 0.002) than high school students overall. University undergraduates were moderately unconcerned about food allergies. Concern was highest in younger students, decreasing between 13 and 18 years of age and plateauing between 19 and 23 years. Among students aged 13\u201318 years, concern was higher among those who worked or volunteered in a daycare and who had previously taken a food preparation course. Among students aged 19\u201323 years, concern was higher among females and those with less advanced cooking abilities. Concern was significantly correlated with perceiving food allergies as a personal threat. This study offers a first exploration of perceived risk of food allergies among this demographic and can guide future, more rigorous assessments. Our objective was to explore the perceived risk of food allergies among students in Ontario, Canada. We analyzed blinding questions  from three existing food safety surveys, given to high school and university undergraduate students ( Food allergies are a growing public health concern in many countries . In CanaYouth and young adults are a unique group in that they are generally growing in autonomy from their parents and increasingly making independent decisions about how to behave with respect to risks; in the realm of food, for example, youth and young adults often make risky food consumption and food handling choices \u201313. ThosIn the province of Ontario, Canada, the transition from elementary school to secondary school through to postsecondary school is important in the context of food allergies. Following the enactment of Sabrina's Law  in 2006,We argue that youth and young adults are important actors in the management of the risks of food allergies; however, little is known about the perception of the risks of food allergies in this group. Towards informing policy responses to food allergies, the goal of this study was to explore perceived risk of food allergies among the youth and young adult students at different academic stages. Specifically, we explored food allergy-related concern among high school and university students in Ontario, Canada, by conducting a secondary analysis of blinding questions from three surveys related to food safety more generally.We analyzed data from three separate surveys , conductOur outcome of interest was students' \u201cstated concern\u201d about food allergies, measured via the question \u201cI am concerned about food allergies\u201d  and analyzed as a continuous measure . We alsot-tests with Bonferroni's adjustment for multiple comparisons. The nine covariate predictors of stated concern were assessed using linear multivariable regression modelling as follows. We first assessed the association between stated concern and each covariate individually, adjusting for survey source as a fixed effect. We then modelled the association between the nine potential predictors and stated concern, by first fitting the full model including all nine covariates and then removing any nonsignificant variables; survey source was retained in all models where possible, as a fixed effect regardless of significance. We then explored potential confounding by reintroducing any excluded variables and assessing whether the sign, significance, or magnitude of any regression coefficients changed meaningfully, retaining any variables for which this was the case. We then assessed all possible two-way interactions, for all significant variables in the final model. Data were analyzed using Stata/SE 14.1 for Mac .Data from the surveys were pooled and analyzed together. Student characteristics and food allergy perceptions were summarized descriptively. Differences in means were tested using p = 0.002) than high school students overall. University undergraduate students were moderately unconcerned  and significantly less concerned than Food and Nutrition students (p = 0.03).Student characteristics, their \u201cstated concern\u201d about food allergies, and their perception that \u201cfood allergies are currently a big threat to my health\u201d are shown, by survey source and overall, in p = 0.001), whereas for students aged 19 and over, age and stated concern were not significantly associated .The associations between individual predictors and stated concern (adjusting for survey source) are shown in Because stated concern varied with age, multivariable analyses of this outcome were stratified over two age groups: \u201c13\u201318 years\u201d  and \u201c19\u201323 years\u201d (which included university students only). Results of the final multivariable model for stated concern for students aged 13\u201318 are shown in Results of the final multivariable model for stated concern for students aged 19\u201323 are shown in p = 0.001). Most students strongly disagreed  or disagreed , whereas 2.1% (11/515) were neutral, 7.2% (37/515) agreed, and 5.4% (28/515) strongly agreed with the statement \u201cfood allergies are currently a big threat to my health.\u201d When examined individually (adjusting for survey source), sex was the only predictor significantly associated with the perception that food allergies are a personal threat, with this perception increased by 0.24 points out of 5 for females versus males (p = 0.047). In all of the multivariable models explored, sex remained the only significant predictor associated with the perception that food allergies were a personal threat, and this relationship did not change when potential confounders and interactions were included . Responses to the personal threat statement were significantly correlated with students' stated concern, among both Food and Nutrition  and university students , and mean stated concern increased linearly with perceiving allergies as a personal threat  to moderately unconcerned (university students). In addition to whether students were in high school or university, we found that older students were significantly less concerned about food allergies than younger students and that the association between other student characteristics and concern differed by age as follows.Here, students' stated concern about food allergies changed with age, with students aged 13 years old reporting the highest, yet still slight, concern about food allergies. Concern then decreased to being slightly unconcerned by the age of 18 and plateaued across ages 19\u201323. Without overstating the significance of this finding, it may be possible that these results reflect changes in feelings of personal vulnerability across adolescence. A previous study found that older adults have higher perceived concerns of food allergies than younger adults [We found that, among students aged 13 to 18 years, concern about food allergies was significantly greater among those who worked or volunteered in a daycare or other location involving children versus those who did not. This elevated concern may be due to increased awareness of, and exposure to, a subpopulation with a disproportionately high risk of food allergies  witHere, among students aged 19\u201324, but not also among those aged 13\u201318, female students reported greater concern about food allergies than did male students. This is consistent with other studies that have identified a higher perceived risk of food allergy among females  as well This study is subject to several limitations. The most significant is that we did not know participants' allergy status and thus could not assess how stated concern related to, or varied by, actual food allergies. Rather, our measure of food allergy concern likely represents a composite of personal food allergy status , as well as the diagnosed and perceived food allergy status of those in the student's network of friends, family members, classmates, and others. Future studies should assess how food allergy concern relates to personal food allergy status and familiarity with food preparation practices, to help further elucidate these relationships. A second main limitation is that our data were cross-sectional; we could not discern the temporal sequence of the various factors we assessed and could not determine whether differences observed by age are due to age, period, or cohort effects. Future studies that follow a cohort of individuals over time are needed to more fully uncover how food allergy concern relates to physical age and birth cohort and life stage effects. Despite these limitations, this study is the first to focus on the perceived concerns of food allergies among high school and university undergraduate students in Canada and offers insight into the unique experiences of this population as they navigate increasingly risky environments with respect to food allergies.This study found that, overall, high school and university undergraduate students in Ontario, Canada, are generally unconcerned about food allergies but that certain types of students, such as younger high school students and those who work or volunteer with children, as well as university students with less well-developed cooking abilities, are more concerned than others. Students who report that food allergies are a personal threat express the most concern. This first exploration can be used to guide future, more rigorous assessments of the perceived risk of food allergies in various related populations."}
+{"text": "This Special Issue on lung diseases is aimed at giving emergent researchers and clinicians an important forum to share their original research and expert reviews on key topics within respiratory diseases. This Special Issue will be of interest to general physicians and respiratory specialist and will equip the reader with up-to-date knowledge on a wide array of lung diseases, including interstitial lung diseases, COPD, and Asthma. Journal of Clinical Medicine. Our objectives are to showcase emergent researchers working at the cutting edge of scientific and clinical advances in the broad field of respiratory diseases.Lung diseases are amongst the leading causes of mortality worldwide. One-sixth of total deaths are attributed to lung disease, accounting for over 9.5 million deaths worldwide. This burden of disease is the prime reason for this timely Special Issue on lung diseases in the Here, we provide the reader with comprehensive reviews that would interest those with general medical and specialist respiratory interests. We have two reviews covering chronic obstructive pulmonary disease (COPD), the leading cause of respiratory mortality worldwide. Dey et al.  discuss Journal of Clinical Medicine.This Special Issue on lung diseases also presents a number of primary research articles spanning all aspects of respiratory diseases showcasing emergent researchers at the forefront of advances in respiratory medicine. We present a variety of themes from novel aspects of lung physiology testing in pulmonary hypertension to the prevalence of fatigue in asthmatic patients and the use of macrolide therapies during hospitalisation of children with childhood disease, amongst other interesting primary research articles, which would be of interest to our growing number of readers of the"}
+{"text": "Translational Respiratory Medicine is a truly international, peer-reviewed journal devoted to the publication of articles on outstanding work with translational potentials between basic research and clinical application to understanding respiratory disease. Translational respiratory medicine will more focus on biomarker identification and validation in pulmonary diseases in combination with clinical informatics, targeted proteomics, bioinformatics, systems medicine, or mathematical science; on different translational strategies of cell-based therapy to clinical application to treat lung diseases; on targeted therapies in combination with personalized medicine; and on distant electronic medicine to monitor a large population of people\u2019s health. Translational Respiratory Medicine is an additional but unique opportunity for scientists and clinicians who work on pulmonary diseases to publish their outstanding findings, initiative results, and critical and perceptive opinions in the journal.Respiratory diseases will become one of the top 3 leading causes of estimated mortality in 2020 and become about one third of total causes of estimated mortality. The journal of  Translational Respiratory Medicine is a truly international, peer-reviewed journal devoted to the publication of articles on outstanding work with translational potentials between basic research and clinical application to understanding respiratory disease. The journal provides a forum for exchange of ideas on potential molecular and cellular mechanisms and novel treatment for respiratory diseases. Translational Respiratory Medicine aims to play an important, critical, and recognized role in the understanding of human respiratory diseases and improvement of the prognosis of patients.The journal of http://www.who.int/evidence/bod. The journal of Translational Respiratory Medicine is an efficient and effective communication channel for publishing articles from leading research scientists and clinicians at the forefront of translational medicine and bridge basic research and clinical application, with a special focus on respiratory diseases. Translational Respiratory Medicine becomes the premier source of information in the field of translational medicine in respiratory diseases and one of the leading platforms to translate basic sciences into clinical use, and clinical imaging into bioinformatics.Respiratory diseases need global attentions and solutions now more than ever, especially from clinical and translational medicine. Respiratory diseases have become the second leading cause of mortality with over 130 major causes according to the WHO Global Burden of Disease 2000 study\u2019s (GBD 2000) estimates of incidence, health state prevalence, severity and duration, and mortality for 17 sub-regions of the world [Translational Respiratory Medicine will promote and accelerate the exchange of bidirectional information on respiratory diseases between basic and clinical researchers, innovators and clinicians, scientists and politicians, and doctors and patients. Translational Respiratory Medicine, as a part of clinical and translational medicine, is intended to optimize new methodologies, foster clinical application of new therapeutic strategies, and ultimately improve the quality of life for patients with respiratory diseases [There is solid evidence that translational research on respiratory diseases has increased greatly. Most current journals related to pulmonology focus on either molecular biology or clinical practices. This means that the most important link between them has been ignored. Translational medicine in respiratory diseases will bring basic research to clinical application and clinical questions to bench. Thus, there is a great need to have a critical and important channel and platform for such communications. diseases . Lung cadiseases , which mdiseases . TranslaTranslational Respiratory Medicine is expected to accelerate and standardize the process from post-validation to authority approval, clinical application to policy and regulation, and individual medicine to public health.Biomarkers have been defined as an important factor in diagnosing the early phase of diseases, monitoring the severities of the disease and sensitivities to therapies, or even predicting the prognosis of patients with lung diseases. An increasing number of biomarkers are being discovered and identified from preclinical research, while a few can be used clinically. For example, network biomarkers and dynamic network biomarkers, a new type of biomarkers with protein-protein interactions, can be monitored and evaluated at different stages and time points during the development of diseases . Recent Increasing evidence from preclinical studies demonstrated that local or systemic administration of external mesenchymal or induced pluripotent stem cells could prevent or treat lung diseases or improve the severity of diseases, even though the exact mechanism remained unclear. For example, stem cell-based therapy has been considered as an attractive new therapeutic approach for acute lung injury. Multiple paracrine factors produced from stem cells were proposed to play critical and necessary therapeutic roles in the maintenance of endothelial and epithelial integrity, down-regulation of both tissue and circulating inflammation, acceleration of tissue repair process, and/or inhibition of bacterial growth . It is aA large number of constantly advancing biotechnologies need to be translated into diagnoses and therapies for respiratory diseases as new applications to benefit patients. Genetic engineering to create tumor-fighting immune cells for cancer therapies has been considered as one of 5 recent breakthrough innovations in biotechnology. The cancer-targeted specific therapy by genetic engineering of the recipient\u2019s immune system could be carried out by transfer of T-cell receptor genes, taken from lymphocytes of a patient who had an immune response to cancer, to lymphocytes from other patients . The feaTranslational Respiratory Medicine will publish papers in all relevant areas designed to further understand the molecular mechanisms underlying organ or cell dysfunction in human pulmonary diseases, dedicated to the discovery and validation of diagnostic and prognostic disease biomarkers, and the identification and validation of novel drug targets, application of tissue genomics, transcriptomics, proteomics and bioinformatics in drug efficacy and toxicity in clinical research.Another topic of translational respiratory medicine is to translate the understanding of molecular mechanisms into personalized medicine and therapies and hospital treatments and manipulations into distant electronic medicine (eMedicine). We create and establish a system of distant eMedicine to closely connect people at risk from respiratory diseases with pulmonologists through the cloudy system. People can reach the pulmonologist and report their symptoms and lung function tests in a real time, without the need to come to the hospital. This is a new aspect of translational respiratory medicine from clinical treatment to social prevention, static outpatient clinic to dynamic and real-time monitoring, and a small group of patients with respiratory diseases to a large population of people with any risk of respiratory disorders or discomfort. Without limits, Translational Respiratory Medicine will refresh the \u201cold\u201d clinical topics, e.g. mechanical ventilation and lung function, and translate clinical challenges and considerations to molecular biologists to innovate and develop new therapies and supports to patients with lung diseases. One way is to identify and validate gene and protein biomarkers from patients with lung diseases for the correlation with lung function measurements, even better to follow up the alternations by time . Or the Translational Respiratory Medicine with Dr Michael Matthay, a close friend, supportive colleague, and valuable mentor for my post-doctoral education. Translational Respiratory Medicine is an additional but unique opportunity for scientists and clinicians who work on pulmonary diseases to publish their outstanding findings, initiative results, and critical and perceptive opinions in the journal. In initiating the journal, we would like to express our special appreciations to all colleagues for their encouragement, support, comments, suggestions and contributions. With the support of our Associate Editors and Editorial Board Members, we have enough reason to believe that Translational Respiratory Medicine will be one of the leading platforms to translate basic sciences associated with respiratory diseases into clinical applications and benefit the healthcare of humans.I am pleased and honored to be co-editor of the journal"}
+{"text": "Deviating from the normal karyotype dramatically changes gene dosage, in turn decreasing the robustness of biological networks. Consequently, aneuploidy is poorly tolerated by normal somatic cells and acts as a barrier to transformation. Paradoxically, however, karyotype heterogeneity drives tumor evolution and the emergence of therapeutic drug resistance. To better understand how cancer cells tolerate aneuploidy, we focused on the p38 stress response kinase. We show here that p38-deficient cells upregulate glycolysis and avoid post-mitotic apoptosis, leading to the emergence of aneuploid subclones. We also show that p38 deficiency upregulates the hypoxia-inducible transcription factor Hif-1\u03b1 and that inhibiting Hif-1\u03b1 restores apoptosis in p38-deficent cells. Because hypoxia and aneuploidy are both barriers to tumor progression, the ability of Hif-1\u03b1 to promote cell survival following chromosome missegregation raises the possibility that aneuploidy tolerance coevolves with adaptation to hypoxia.  \u2022The p38 stress response kinase promotes apoptosis following aneuploidy induction\u2022Aneuploidy-induced metabolic collapse is ameliorated upon inhibition of p38\u2022p38 deficiency upregulates Hif-1\u03b1, buffering aneuploidy-induced metabolic collapse\u2022Aneuploidy tolerance may have coevolved with adaptation to hypoxia Sim\u00f5es-Sousa et\u00a0al. show that chromosome missegregation induces metabolic collapse and apoptosis, mediated by the p38 stress response kinase. Inhibiting p38 elevates Hif-1\u03b1, boosts glycolysis, and limits metabolic collapse, in turn allowing expansion of aneuploid clones. Adapting to hypoxia during tumor development may therefore also permit aneuploidy tolerance. Aneuploidy, a deviation from the normal karyotype, arises following chromosome missegregation during mitosis and meiosis . This leBub1b develop aneuploidy and aging-related phenotypes including cataracts and muscle wasting (BUB1B mutation leads to mosaic variegated aneuploidy (MVA), a rare disorder characterized by progeroid features and early death  has also been implicated in mitotic and post-mitotic responses , with twTP53\u2212/\u2212 cells generated by adeno-associated virus (AAV)-enhanced gene targeting  override, we asked if the canonical p38 pathway was activated following chromosome missegregation. However, SAC override induces a variety of mitotic abnormalities, some of which can lead to DNA damage . Indeed,Having confirmed that sequential inhibition of Cenp-E and Mps1 induces aneuploidy in HCT116 cells, mitotic cells exposed to GSK923295 were isolated by selective detachment, re-plated in AZ3146, harvested at various time points, and then analyzed by immunoblotting to interrogate the p38 pathway. Note that p38 is phosphorylated by upstream kinases MKK3/6, in turn leading to phosphorylation of MK2 and Hsp27 B and S2AMAPK14, which encodes p38\u03b1, the isoform expressed in most cell types  analysis (see below). Beyond this baseline, out of 23 untreated p38\u03b1 null cells, three had a trisomy affecting either chromosome 2 or 13; and out of 22 AZ3146-treated cells, one had a highly deviant karyotype trisomic for six chromosomes and monosomic for another five .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Stephen S. Taylor . HCT116 Flp-In\u2122 T-REx\u2122 derivatives were generated using the Flp-In\u2122 T-REx\u2122 System (Invitrogen) according to manufacturer instructions. HCT116 gelstein . To creaAZ3146 , SB203584 cells were seeded per well in a 24-well plate (Corning) and maintained at 37\u00b0C in a humidified 5% CO2 atmosphere overnight. Transfections using Lipofectamine 2000 was performed according to the manufacturer\u2019s instructions to transfect a pD1301-based plasmid (Horizon Discovery), which expresses Cas9, GFP and a small guide-RNA (sgRNA) targeting the gene of interest. After incubating at 37\u00b0C in a humidified 5% CO2 atmosphere for 48\u00a0hr, transfected cells were sorted by flow cytometry using a BD Influx cell sorter and GFP-positive cells seeded 1 cell per well in 96-well plates (Corning) to generate clonal cell lines which were then screened by immunoblotting to identify desired cell lines.For CRISPR-Cas9-mediated mutagenesis, 5x10TP53 and a pBluescript-based plasmid containing an open reading frame encoding GFP flanked by regions of TP53 generated by PCR amplification of genomic DNA isolated from HCT116 cells using Purelink Genomic DNA Mini kit (Invitrogen). To enrich for targeted clones, 10\u00a0days after transfection, cells were exposed to Nutlin-3 for 24\u00a0hr to stabilize GFP-p53, green fluorescent cells isolated via sorting by flow cytometry, expanded into clones then screened by immunoblotting to identify desired cell lines.A biosensor line expressing GFP-tagged p53 was generated by co-transfecting HCT116 Flp-In T-REx\u2122 cells with the pD1301-AD:153663 plasmid (Horizon Discovery) containing a sgRNA targeting MAPK14\u2212/\u2212, a p38\u03b1 cDNA (Accession#: NM_139012) was amplified by RT-PCR using Superscript III One Step RT-PCR Platinum Taq HiFi (Invitrogen) with RNA template extracted from HCT116 cells using RNeasy Plus Mini kit (QIAGEN). This PCR product was cloned into pcDNA5/FRT/TO (Invitrogen) and transformed into XL1-Blue competent cells. Plasmid DNA was extracted using QIAprep Spin Miniprep Kit (QIAGEN) and co-transfected with pOG44 into GFP-p53 MAPK14\u2212/\u2212 HCT116 Flp-In T-REx\u2122 cells. Following selection in 400\u00a0\u03bcg/ml hygromycin B (Sigma), colonies were pooled and expanded to create an isogenic polyclonal cell line. Expression of p38\u03b1 by the addition of tetracycline hydrochloride (1\u00a0\u03bcg/ml) was confirmed by immunoblotting.To reconstitute p38\u03b1 function in For RNAi-mediated inhibition, cells were plated in flat bottom, low evaporation 24-well plates (Corning) then transfected with a final concentration of 66\u00a0nM of the desired siRNA using DharmaFECT 1 transfection reagent (Dharmacon) in Opti-MEM\u00ae media (Life-Technologies). Knock-down was confirmed by immunoblotting.For DNA content analysis, cells were seeded in 6-well plates (Corning) then treated with small molecule inhibitors  for 24\u00a0hr, fixed in ethanol overnight, treated with RNase A (50\u00a0\u03bcg/ml), stained with propidium iodide (40\u00a0\u03bcg/ml) then analyzed on CyAn (DakoCytomation).Proteins were extracted by boiling cell pellets in sample buffer , resolved by SDS-PAGE, then electroblotted onto Immobilon-P membranes. Following blocking in 5% dried skimmed milk dissolved in TBST , membranes were incubated with primary antibodies overnight at 4\u00b0C. Note, the following monoclonal and polyclonal antibodies were used: Anti-mouse p53 (DO-1), anti-mouse p21 (F-5), anti-mouse Chk1 (G-4) ; anti-mouse phospho-p38\u03b1 (T180/Y182) (R&D Systems), anti-mouse Hif1\u03b1 (Clone 54) (BD Transduction Laboratories), anti-mouse p38\u03b1, anti-rabbit phospho-MAPKAP-K2 (Thr334), anti-rabbit phospho-p53 (Ser 46), anti-rabbit p38 MAPK, anti-rabbit phospho-HSP27 (Ser82), anti-rabbit phospho-MKK3/6 (Ser189/207), anti-rabbit phospho-p44/42 MAPK (ERK1/2), anti-rabbit phospho-SAPK/JNK (T183/Y185), anti-rabbit phospho-Chk1 (Ser 345) ; anti- rabbit ATM [Y170], anti-mouse phospho-ATM (S1981), anti-rabbit DNA-PKcs, anti-rabbit phospho-DNA-PKcs (S2056) (All from Abcam); anti-mouse phospho-KAP1 (S824) (Bethyl Laboratories), anti-rabbit phospho-gamma H2AX (S139) (Novus Bio), anti-sheep Bub3 , anti-sheep Tao1 . Membran5 cells were seeded per well in \u03bcclear\u00ae 96 well plates (Greiner Bio-One) and IncuCyte Kinetic Caspase-3/7 Apoptosis Assay Reagent (Essen BioScience) added. Shortly after adding inhibitors, cells were then imaged using an IncuCyte\u00ae ZOOM (Essen BioScience) equipped with a 20x objective and maintained at 37\u00b0C in a humidified 5% CO2 atmosphere. Phase contrast and fluorescence images (3-4 images per well) were collected every 10-30\u00a0min and IncuCyte\u00ae ZOOM software, used in real-time, measured confluence and fluorescence as a proxy for proliferation and apoptosis, respectively. Apoptosis was quantitated by measuring green fluorescence object count in 3-4 images per well, sampling at least 800 cells. Image sequences were then exported in MPEG-4 format and analyzed manually to generate cell fate profiles. Timing data were imported into Prism 7 (GraphPad) for statistical analysis and presentation. Note that 0\u00a0hr on the fate profiles represents when cells entered mitosis or when imaging started.To measure proliferation, apoptosis induction, and to perform cell fate profiling, 1\u00a0\u00d7 103 cells were seeded per well in Agilent Seahorse 96-well XF Cell Culture microplates and incubated at 37\u00b0C in a humidified 5% CO2 atmosphere overnight. Seahorse XF cartridges were hydrated with Seahorse XF Calibrant and placed in a 0% CO2 incubator for at least 6h. Prior to the assay, DMEM was replaced with XF Base Medium  supplemented with 2\u00a0mM L-glutamine only (glycolysis assay) or a mixture of 2\u00a0mM L-glutamine, 2\u00a0mM sodium pyruvate and 10\u00a0mM D-(+)-glucose (oxidative phosphorylation assay). Compound injections for different stress tests were prepared according to the manufacturer\u2019s instructions (Seahorse XF Cell Mito Stress Test Kit and Seahorse XF Glycolysis Stress Test Kit) and added to the respective ports on the cartridge. Five measurements were made for basal metabolism and three measurements were made for each compound injection. Results were normalized for protein content in each well and assessed by Bradford assay (Bio-Rad). Note that values were derived from three independent experiments.To analyze glycolysis and oxidative phosphorylation, cells were analyzed in a Seahorse XFe96 Analyzer . 1x103 c2 atmosphere for 24\u00a0hr. Cells were treated with 0.66\u00a0\u03bcM nocodazole for 6\u00a0hr, harvested and cell pellets incubated in hypotonic buffer for 20\u00a0min at 37\u00b0C before overnight fixation in methanol:acetic acid (3:1). Samples were dropped onto glass slides and stained with Hoechst 33258 (Sigma). Images of individual metaphase spreads were taken on Zeiss Axiovert 200 with a 100x objective and chromosomes were counted manually.For chromosome counting, cells were seeded in 6-well plates (Corning) and incubated at 37\u00b0C in a humidified 5% CO2 atmosphere overnight. Cells were treated with the inhibitors and washed out after 24\u00a0hr of incubation. Cells were incubated for a further 13\u00a0days to allow colony development and finally fixed in 1% formaldehyde and stained with 0.05% (w/v) crystal violet solution. Colonies were counted using a GelCount (Oxford Optronix) and imaged using a ChemiDoc\u2122 Touch Imaging System (BioRad).For colony formation assays, 500 cells were seeded per well in 6-well plates and incubated at 37\u00b0C in a humidified 5% COSingle G1 nuclei were isolated, sorted, and sequenced as described  Briefly,To prepare chromosome spreads, cells were seeded in 6-well plates (Corning) and incubated for 24\u00a0hrs, treated with 150\u00a0ng/ml nocodazole for 4-8\u00a0hrs, then the entire population harvested by trypsinisation. Cell pellets were incubated for 25\u00a0mins at 37\u00b0C in 0.8% sodium citrate hypotonic buffer followed by overnight fixation in freshly prepared methanol:acetic acid (3:1). Samples in fixative were dropped onto glass slides and air-dried overnight at room temperature and stained using Hoechst 33258 (Sigma). Images of individual metaphase spreads were taken on Zeiss Axiovert 200 with a 100x objective and chromosomes manually counted. Slides were then experimenter-blinded and hybridized with the M-FISH probe kit 24XCyte (Zeiss MetaSystems) following the manufacturer instructions, then analyzed using an Olympus BX60 microscope for epifluorescence equipped with a Sensys CCD camera . Images were collected and analyzed using the Genus Cytovision software (Leica). A minimum of 25 metaphases were karyotyped for each cell line/condition.4 cells per well in FluoroBrite DMEM media (Life Technologies) plus 1\u00a0\u03bcg/ml tetracycline hydrochloride and incubated at 37\u00b0C in a humidified 5% CO2 atmosphere for 24\u00a0hrs. GSK923295 and AZ3146 inhibitors were added at 100\u00a0nM and 0.5\u00a0\u03bcM respectively, and time-lapse microscopy was performed on a manual microscope  equipped with an automated stage  and an environmental control chamber (Solent Scientific), which maintained the cells at 37\u00b0C in a humidified stream of 5% CO2. Imaging was performed using a 40x Plan NEOFLUAR objective. Shutters, filter wheels, and point visiting were driven by MetaMorph software . Images were taken using an Evolve delta camera (Photometrics).HCT116 Flp-In T-REx\u2122 GFP-H2B cells were seeded in \u03bcclear\u00ae 96 well plates (Greiner Bio-One) at 1x10\u2217p\u00a0< 0.05, \u2217\u2217p\u00a0< 0.01, \u2217\u2217\u2217p\u00a0< 0.001, \u2217\u2217\u2217\u2217p\u00a0< 0.0001, ns: p > 0.05. Details of statistical analyses are described in the Figure\u00a0legends. Values on apoptosis and proliferation line graphs show the mean and SD or SEM from three technical replicates. Lines on scatterplots show mean and interquartile ranges. Box-and-whisker plots show the median, interquartile ranges, and the full range. To determine EACR and OCR, five measurements were made for basal metabolism and three measurements were made for each compound injection . Results were normalized for protein content in each well and assessed by Bradford assay (Bio-Rad). The values shown in Prism 7 (GraphPad) was used for statistical analysis, where The accession number for the raw unaligned single-cell sequencing reads reported in this paper is European Nucleotide Archive (ENA): PRJEB27319."}
+{"text": "P < .05) with mixed clade infection in 2. Nonvaricella-associated VZV encephalitides may be caused by simultaneous viral reactivation from multiple neurons.Cerebrospinal fluid varicella zoster virus (VZV) from encephalitis (8) was significantly more diverse than vesicular fluid (24) ( Varicella zoster virus (VZV) may cause encephalitis, both with and without rash. Here we investigate whether viruses recovered from the central nervous system  differ genetically from those recovered from non-CNS samples.Enrichment-based deep sequencing of 45 VZV genomes from cerebral spinal fluid (CSF), plasma, bronchoalveolar lavage (BAL), and vesicles was carried out with samples collected from 34 patients with and without VZV infection of the CNS.Viral sequences from multiple sites in the same patient were identical at the consensus level. Virus from vesicle fluid and CSF in cases of meningitis showed low-level diversity. By contrast, plasma, BAL, and encephalitis had higher numbers of variant alleles. Two CSF-encephalitis samples had high genetic diversity, with variant frequency patterns typical of mixed infections with different clades.Low viral genetic diversity in vesicle fluid is compatible with previous observations that VZV skin lesions arise from single or low numbers of virions. A similar result was observed in VZV from cases of VZV meningitis, a generally self-limiting infection. CSF from cases of encephalitis had higher diversity with evidence for mixed clade infections in 2 cases. We hypothesize that reactivation from multiple neurons may contribute to the pathogenesis of VZV encephalitis. Primary infection with varicella zoster virus (VZV), an alphaherpesvirus, causes chickenpox (varicella). Following latency in the neurons of peripheral sensory ganglia, VZV reactivates in 30% to cause herpes zoster (shingles). Around 50% of herpes zoster cases occur in subjects with reduced cell-mediated immunity . In commStudies of equid herpesvirus1  have reported that a naturally occurring point mutation in the viral DNA polymerase significantly increases the risk of encephalitis . Here weThe samples  used in http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Read pairs were aligned against the VZV reference strain Dumas (NC_001348) using BBMap (http://sourceforge.net/projects/bbmap/) while duplicate read pairs were removed using MarkDuplicates (http://broadinstitute.github.io/picard) and local realignment performed using IndelRealigner [https://github.com/genome/bam-readcount) and the fpfilter function in VarScan2 [Sequence libraries were constructed from 200 ng of starting DNA, derived from original GOSH DNA extracts, supplemented with human DNA  where necessary. DNA was sheared on a Covaris E220 . Multiplexed libraries were run on Illumina MiSeq or NextSeq sequencers. Paired-end sequence data were demultiplexed and sequence reads trimmed to remove low quality 3\u2032 bases and adapter sequences using TrimGalore , defined as the average number of nucleotide differences between reads at a site  . Strandt test was used to compare numbers of variant alleles present in samples grouped according to pathology, sample type, and immune status. The Kolmogorov-Smirnov (nonparametric) test was used to discern whether variant allele frequency distributions differed between vesicle and nonvesicle populations.The Student Forty-five diagnostic clinical samples, obtained from a total of 34 patients with VZV-related conditions, were sequenced following enrichment for VZV DNA , 13. ForA consensus sequence was generated for each sample and aligned with 12 VZV genomes, representative of 6 different clades obtained from Genbank . A phyloWe next generated estimates of within (intra-) sample nucleotide (population) diversity . Our mett test, P < .001) as it was between vesicular and encephalitis samples (Student t test P < .05).For subsequent analyses, variant alleles were filtered to flag and remove artefactual variant alleles generated by sequencing/mapping errors. Plots of the frequencies of filtered variant alleles and their positions along the genome are shown for vesicle  and nonvP < .001) compared to vesicular fluid  or immune status . All bar 1 of the CSFs were from immunocompetent patients either with CNS disease alone or with concomitant herpes zoster and there were too few of the other sample types for statistical analysis.Nonvesicular samples also showed a different distribution of variant allele frequencies  and 3 (majority) viruses . For CSFThese results are compatible with previous conclusions that individual skin vesicles are typically founded by 1 to 3 virions , a signiP < .05). Viral diversity was not related to viral load, which we have previously shown can be inferred in relatively acellular fluids such as CSF from the percentage of on-target reads [By contrast, although numbers were small, we observed significantly higher diversity in CSF from cases of encephalitis, with 7 out of 8 CSF samples from these patients having greater than the median numbers of variants, as compared with vesicular fluid and CSF samples from patients with meningitis CSFs 8\u201311) (et reads . Insteadet reads . How oftet reads . Reinfecet reads . We and  (et readet reads . RecombiOther encephalitis samples such as CSF7, with high variant numbers at low frequencies, could represent reactivation of the same clade-virus from multiple sites, giving a similar allelic frequency profile as the pooled patient D vesicle alleles . In contTwo other samples with higher viral diversity, PLAS2 and BAL1, both came from patients with profound immunosuppression. We have previously shown the presence of a mixed vOka founder population in endotracheal aspirates from a case of vOka pneumonitis in an immunocompromised child, presumably from the multiple foci of infection in the lungs. Vesicular fluid from the same child was, as expected, derived from a single virion . ExtrapoIn summary, using genetic studies we have shown that, like the VZV vOka vaccine strain, vesicles arising during VZV wild-type infections are likely to be founded by few virions, a major bottleneck event that results in restricted genome diversity within vesicles as the founder VZV population expands . By contThe Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Supplementary Figures and LegendsClick here for additional data file.Supplementary TablesClick here for additional data file."}
+{"text": "Litseacubeba (Lour.) Pers.) (Lauraceae) is an important industrial crop as an ingredient in cosmetics, pesticides, food additives and potential biofuels. These properties are attributed to monoterpenes and sesquiterpenes. However, there is still no integrated model describing differentially expressed genes (DEGs) involved in terpenoid biosynthesis during the fruit development of L. cubeba. Here, we performed digital gene expression (DGE) using the Illumina NGS platform to evaluated changes in gene expression during fruit development in L. cubeba. DGE generated expression data for approximately 19354 genes. Fruit at 60 days after flowering (DAF) served as the control, and a total of 415, 1255, 449 and 811 up-regulated genes and 505, 1351, 1823 and 1850 down-regulated genes were identified at 75, 90, 105 and 135 DAF, respectively. Pathway analysis revealed 26 genes involved in terpenoid biosynthesis pathways. Three DEGs had continued increasing or declining trends during the fruit development. The quantitative real-time PCR (qRT-PCR) results of five differentially expressed genes were consistent with those obtained from Illumina sequencing. These results provide a comprehensive molecular biology background for research on fruit development, and information that should aid in metabolic engineering to increase the yields of L. cubeba essential oil.Mountain pepper ( Litseacubeba (Lour.) Persoon) is a fast-growing and aromatic plant of the Lauraceae family, indigenous to Eastern Asia. L. cubeba has a diversity of uses. The fruit and bark are commonly used as traditional medicines for the treatment of stomach aches, common cold, inflammation, and coronary heart disease. The aromatic essential oil extracted from the fruits and roots, which possesses an intensely lemon-like odor, has been widely used as a raw material for cosmetics, pesticides, food additives and biodiesel fuel [L.cubeba are attributed to the main components in the fruit oils, monoterpenes and sesquiterpenes, the most dominant component of which is citral, which constitutes more than 80% of the chemical content of the essential oils. Considerable research efforts related to the oil biosynthesis in fruits are urgently needed, and it is believed to be a promising avenue for increasing the content of chemicals of interest.Mountain pepper (sel fuel . Meanwhisel fuel . These uRaphanus sativus) [Arabidopsis [Brassica juncea [Hevea brasiliensis [Antirrhinum majus) [d-xylulose 5-phosphate reductoisomerase (DXR) have been identified in tomato [Citral belongs to the terpenoids. Terpenoids, represented mainly by isoprene C5), monoterpenes C10), sesquiterpenes (C15), diterpenes (C20), are the largest category of plant secondary metabolites. They play important roles in plant development, the interactions of plants with the environment, and reproduction through attraction of pollinators and seed disseminators . The bio, monotersativus) . 3-Hydrobidopsis , Brassica juncea  and Heveiliensis . Three g, sesquitn tomato .L. cubeba. Chang reported the isolation and functional characterization of three monoterpene synthase (mono-TPS) genes in L. cubeba [L. cubeba, but functional characterization of the corresponding enzymes was not studied [L. cubeba. Significant improvements in oil accumulation must be accompanied by changes in the expression of the terpenoids biosynthetic genes during fruit development. Identification of these genes and their regulatory pathways would provide not only new genetic information to understand the development of L. cubeba fruit, but also to control gene expression to alter oil accumulation.However, there are few studies about the enzymes involved in terpenoid biosynthesis in . cubeba . cDNA en studied . At the L. cubeba has been carried out using the Illumina platform [L. cubeba.In recent years, the rapid development of next-generation sequencing (NGS) technologies has offered new cost-efficient high-throughput approaches for global measurements of gene expression of model or non-model species in recent years ,15,16. Dplatform  aiming tL. cubeba fruit development, which should provide an invaluable resource for the identification genes involved in terpenoid biosynthesis, and promote a systematic understanding of the molecular mechanisms of oil production of L.cubeba in the future. All of the genes identified in our research also provide much information to improve the oil contents and quality of terpenoids in L. cubeba breeding.In this study, we employed DGE tag profiling using the Illumina NGS platform to analyze the gene expression profiles of five different developmental stages of fruit  and to identify differentially expressed genes. DGE generated over eight million reads per sample, which produced expression data for approximately 19354 genes. Twenty six candidate genes were selected using pathway analysis and characterized as those responsible for terpenoid biosynthesis. Our results provide a comprehensive understanding of DGE patterns and expression levels during L. cubeba, we analysed the concentration of citral at different stages (60 to 165 DAF)  and \u03b2-citral  are the major compounds in 165 DAF) A. To expanalyzed B. The reFive libraries were constructed, and sequence analyses were conducted. Approximately 34.19 million raw reads were generated. Date evaluation of the GC percentage of the clean reads in the five DGE libraries was performed . The GC L. cubeba. The average percentage of reads mapped to the reference transcriptome was 89.16% for the five development periods, including mismatching and empty position. Furthermore, after excluding mismatching and empty position, the average percentage of reads mapped to the reference transcriptome was 63.85%. Using the number of reads per kilobase transcriptome per million mapped reads (RPKM) approach, the expression level of each gene was measured by reading the sequencing depth and gene length. The results showed that mRNAs transcribed from the major types of genes were represented by low RPKM values, and only a small proportion of genes was highly expressed  increased proportionally to the total number of clean reads, sequencing data saturation analysis was performed. L. cubeba fruit, the differentially expressed genes (DEGs) between the two samples were identified using statistical criteria (|log2 (fold-change)| \u2265 1 and p-value \u2264 0.01). Fruit at 60 days after flowering (DAF) served as the control, a total of 920, 2606, 2271 and 2661 genes were differentially expressed at 75, 90, 105 and 135 DAF, respectively. There were 415, 1255, 449 and 811 up-regulated genes and 505, 1351, 1823 and 1850 down-regulated genes identified at 75, 90, 105 and 135 DAF, respectively. The down-regulated genes were more abundant than the up-regulated genes. A Venn diagram showing all of the DEGs is shown in To identify genes that were differentially expressed during the development of In our research, DEGs were classified into three functional categories: cellular components, molecular functions, or biological processes . In the L. cubeba fruit. The results showed that 14 subsets of the biological processes category and 20 subsets of the molecular functions category were found to be involved in terpenoid biosynthesis. Furthermore, 75 DEGs were identified to be involved with terpenoid biosynthesis. Within the biological ontology category, the DEGs involved in terpenoid biosynthesis could be further classified into two categories: precursor biosynthetic processes  and biosynthetic processes of diverse terpenoids . In the molecular function ontology category, 35 DEGs were identified (nine of which were up-regulated and 26 down-regulated)  .KEGG pathway enrichment analysis of the five DEG libraries was performed. All annotated genes were mapped to terms in the KEGG database to search for significantly enriched genes involved in metabolic or signal transduction pathways. A total of 3384 DEGs were mapped to 262 KEGG pathways, including the terpenoid backbone biosynthesis pathways, the ubiquinone and other terpenoid-quinone biosynthesis pathways, the carotenoid biosynthesis pathways, the steroid biosynthesis pathways, the prenyltransferases pathways, the diterpenoid biosynthesis pathways, the limonene and pinene degradation pathways and the geraniol degradation pathways. The 447 DEGs identified in the 60 DAF vs. 75 DAF contrast group were assigned to 120 KEGG pathways. Furthermore, the 1138 DEGs identified in 60 DAF vs. 90 DAF contrast group were mapped to 181 KEGG pathways, the 1320 DEGs identified in the 60 DAF vs. 105 DAF contrast group were mapped to 186 KEGG pathways, and the 1092 DEGs identified in the 60 DAF vs. 135 DAF contrast group were mapped to 180 KEGG pathways. From all those pathways, the terpenoid backbone biosynthesis pathway was selected for further analysis. A total of 26 DEGs were identified in this pathway , 14 of w2-transformed RPKM values of the four stages to the 60 DAF RPKM values generated using Multiple Array Viewer  during the fruit development was performed with the correlated expression profiles. The genes were clustered according to the similarity of their expression patterns. The heatmap of the ratio of the normalized logy Viewer . Up-reguTo examine the results obtained from the DGE analysis, five genes were selected for qRT-PCR . The genL. cubeba fruit development were examined using DGE profiling technology. A total of 8459 DEGs were identified across four respective comparisons of developmental stages. Among the identified DEGs, 26 DEGs were associated with terpenoid biosynthesis pathway as identified using pathway analysis. The average percentage of reads mapped to the reference transcriptome was 63.85%. The lack genome sequence date and incomplete transcriptome reads of L. cubeba explain the occurrence of the unmapped tags.In this study, the DEGs associated with terpenoid biosynthetic processes during Arabidopsis thaliana, Astragalus membranaceus, and Tropaeolum majus [Among those differentially expressed genes, more DEGs were down-regulated than up-regulated, and a similar phenomenon was observed in the DEGs involved in terpenoid backbone biosynthesis pathways 24 vs. 2). The fact suggested that these genes may be the negatively controlled genes in terpenoid biosynthesis. However, it didn\u2019t mean lower expression of these genes leaded to lower volatile constituent contents. In other words, the expression levels of terpenoid biosynthetic genes did not correspond with the storage of volatile constituents, since the terpenoids biosynthetic pathway is a complex pathway. Most of the key enzymes involved in this pathway were encoded by multiple genes with different expression patterns and subcellular localizations . Anotherum majus ,24,25. vs. 2. TThe biosynthesis pathways of all terpenoids include three main processes, the synthesis of the C5 precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the synthesis of the immediate diphosphate precursors, and the formation of the diverse terpenoids. A large group of enzymes play a key role in volatile terpene synthesis ,5. In thL. cubebe essential oil was citral (60\u201380%), which is an isomeric mixture of geranial and neral [Ocimum basilicum cv. Sweet Dani, and found that GEDH activity levels were the highest in young leaves which have more glands per unit area [Zingiber officinale. The expression levels of ZoGeDH in various ginger plant tissues were in accordance with the accumulation of geranial [Perilla [2 (fold-changed)| < 1), the RPKMs of g36695, which KEGG annotation identified as GEDH, were increased from 60 DAF to 90 DAF, and then decreased until 135 DAF. This trend was inconsistent with the accumulation of citral. It is believed that GEDH expression is decreased during the maturation of fruit and the citral is synthesized during the young stage of fruit development and stored during maturation in L. cubeba. This inconsistent phenomenon was also found in Zingiber officinale and Citrusunshiu. In old rhizomes of Zingiber officinale, the loss of ZoGeDH expression was concomitant with the accumulation of abundant geranial [Citrus unshiu, the expression of four clones, CitMTSE1, CitMTS3, CitMTS61, and CitMTS62 coding for d-limonene synthase, \u03b3-terpinene synthase, and \u03b2-pinene synthase, respectively, appeared mainly in the peel at an early stage of fruit development, whereas they decreased or disappeared in later stages of development [The most predominant chemical constituent in nd neral . Geraniand neral , and cannd neral ,29. GEDHnit area . The autgeranial . In addi[Perilla . In our geranial . In Citrelopment .d-limonene, geraniol (the content were above 1%), were decreased from 60 DAF to 135 DAF  and artemisinic aldehyde, was a result of higher expression of the amorpha-4,11-diene synthase (ADS) and artemisinic aldehyde \u039411-13 reductase (DBR2) genes [However, the expression level of other genes were consistent with the content. The expression of several compounds, including camphene, caryophyllene, \u03b1-pinene, \u03b2-pinene, \u03b2-myrcene,  135 DAF , which w 135 DAF . The phephyllene . The hig2) genes .L. cubebe fruit development, and most of them were down-regulated. For example, g 5372 (for GPPS) was up-regulated compared to 60 and 75 DAF, but then down-regulated compared to 60 vs. 90 DAF, 60 vs. 105 DAF, and 60 vs. 135 DAF contrast groups. Additionally, g 4493 and g 21893 (for FPPS) and g 50359 and g 57414 (for GGPPS) were down-regulated in four respective groups. These results suggested that the terpenoid biosynthesis might ocurr during the young stage of fruit development and the products stored during maturation. However, the changes in there DEGs were not consistent during the fruit development, which suggests that terpenoid biosynthesis is a complex pathway. For example, GPP exists in both the homodimeric and heterodimeric forms in both angiosperms and gymnosperms [GPPS. In Salvia miltiorrhiza, GPPS.LSU is less tissue specific compared with that of GPPS.SSU and the two types have different gene organization [In the second phase of terpenoid biosynthesis, IPP and DMAPP are used to produce the immediate precursors of monoterpenes, sesquiterpenes and diterpenes, which are namely geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranyl geranyl diphosphate (GGPP) by geranyl diphosphate synthase (GPPS), farnesyl diphosphate synthase (FPPS), and geranyl geranyl diphosphate synthase (GGPPS), and the immediate precursors of monoterpenes, sesquiterpenes and diterpenes, respectively. In our research, these three enzymes were found to be differentially expressed during nosperms , and twod-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (MCT), 4-(cytidine 50-diphospho)-2-C-methyl-d-erythritol kinase (CMK), 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (MDS), (E)-4-hydroxy-3-methyl-but-2-enyldiphosphate synthase (HDS), (E)-4-hydroxy-3-methyl-but-2-enyl diphosphatereductase (HDR). Studies have suggested that these enzymes could affect the yield of essential oil [Fourteen enzymes are involved in the first phase of terpenoid biosynthesis, namely AACT HMGS, HMGR, mevalonate kinase (MVK), phosphomevalonate kinase (PMK), and mevalonate diphosphate decarboxylase (MVD), isopentenyl diphosphateisomerase (IDI), 1-deoxy-tial oil ,39. TherL. cubeba.The terpenoid biosynthetic pathways involve the cooperation of multiple genes. Therefore, it is difficult to increase oil content by overexpressing a single gene. However, in our research, large amount of DEGs involved in the terpenoids biosynthetic pathway were found, which provides a basis to identify key regulatory processes affecting oil accumulation and further molecular genetics and improvement of L. cubeba were collected in 2014 at Fuyang Forest Park, Hangzhou City, Zhejiang Province , China. We observed the development process of L. cubeba fruit from flowering until fruit maturation from May-August, 2014. Fruits without peduncle were hand-collected at 60 days after flowering (DAF) (the immature stage), and then every 15 days to full maturity, which covered a total range of 105 days. The fruits at every stage were consistently collected from three trees for three replicates. The fruits at every stage were divided into two parts. One part was used to measure the oil contents. The other part was flash frozen in liquid nitrogen and stored at \u221280 \u00b0C until mRNA extraction, sequencing and q-RT PCR validation.Fruits of 2SO4) and stored at 4 \u00b0C until analysis. The essential oil compositions were analysed using gas chromatography\u2013mass spectrometry (GC-MS) methodology. GC-MS was performed on an Agilent 6890N gas chromatograph  equipped with an Agilent 5975B Mass Spectrometer using an HP-5MS fused silica capillary column . The temperature program of 50 \u00b0C for 2 min was increased to 120 \u00b0C for 2 min at a rate of 3 \u00b0C/min, then to 250 \u00b0C for 2 min at 15 \u00b0C/min. The carrier gas was N2 at a flow rate of 1 mL/min. The injected volume was 1.0 \u03bcL (1:10 in Et2O) and the injector temperature was 220 \u00b0C. Mass spectrometer conditions were as follows: GC-MS interface temperature, 250 \u00b0C; ion source temperature, 230 \u00b0C; quadrupole temperature, 150 \u00b0C; ionisation mode, EI; and ionisation energy, 70 eV. Compounds were identified by comparing their mass spectra with the mass spectra obtained from an MS database (NIST 08). The MS identifications were confirmed by comparing the GC retention times of the analysed samples with those from pure standards. The identification was confirmed by comparing the retention indices (RI) of the samples with those reported in the literature [To extract the essential oil, fruit hand-collected at 60, 75, 90, 105, 120, 135, 150 and 165 DAF were air-dried and subjected to hydrodistillation using a Clevenger-type apparatus for essential oil. The volatile distilled oils were dried over anhydrous sodium sulphate . The yield and quality of RNA samples was determined using a NanoDrop2000 spectrophotometer . Approximately 6 \u00b5g of total RNA was used for cDNA library construction. The cDNA library was constructed by using a SuperScriptIII 1st Strand cDNA Synthesis Kits . Briefly, after mRNA was isolated and purified using Oligo (dT) magnetic beads, it was fragmented using fragmentation buffer. The first-strand cDNA was synthesized using a random hexamers with mRNA as the template. Then the second-strand cDNA was synthesized by adding buffer, dNTPs, RNase H and DNA polymerase I. Double-stranded cDNA was purified using a MinElute PCR Purification Kit , and then repaired by adding EB buffer. The A-tails were ligated to the 3\u2019 ends, and the sequenced joints were added. Agarose gel electrophoresis was used for fragment size selection, and a cDNA library was constructed using PCR amplification. The quality of library was assessed using the Agilent 2100 Bioanalyzer . Finally, the library was constructed and sequenced on the Illumina HiSeq\u2122 2000 sequencing system.http://www.ncbi.nlm.nih.gov/sra/?term=). Then the clean data was retained and mapped to the reference transcriptome of L. cubeba [http://genome.ucsc.edu/cgi-bin/hgBlat) [L. cubeba was downloaded directly from the NCBI website (http://www.ncbi.nlm.nih.gov/). The accession number of the transcriptome is SRA080286. The read numbers mapped to each gene was counted using HTSeq v0.5.4p3 (http://www-huber.embl.de/users/anders/HTSeq/). Then, the expression abundance of the genes was calculated by the number of reads per kilobase of exon model per million mapped reads (RPKM) [http://telethon.bio.unipd.it/bioinfo/IDEG6/ [2 (fold-changed)| \u2265 1.Raw data (raw reads) in the FASTQ format were transformed into clean data after data-processing: removal of the low quality reads, adaptors and reads containing ploy-N. Then the clean data were used for the subsequent analysis. The quality parameters of clean data including the length, number of reads, and GC-content, were used for the data evaluation. The raw data in the FASTQ format was deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database (. cubeba  using BL/hgBlat) ,43. The s (RPKM) . Differeo/IDEG6/ . The falTo annotate, classify, and functionally map the differentially expressed genes, these genes were analysed in the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database using Blast2GO  and BLASTX  programs, respectively. Clustering analysis of differentially expressed genes was conducted using the Multiple Array Viewer (MAV) software . The heat maps and corresponding HCL tree were constructed using the Pearson correlation method with average linkage. UBC) gene served as an internal control gene [http://frodo.wi.mit.edu/primer3/) to amplify products approximately 120\u2013200 bp long. qRT-PCR with the SYBR Premix Ex Taq\u2122 Kit (TliRNaseH Plus) (2X)  was carried on 7300 Real Time PCR System . According to the manufacturer\u2019s protocol, PCR reactions were run in a 20 \u00b5L volumes containing 10 \u00b5L of 2\u00d7 SYBR\u00ae Premix Ex Taq\u2122, 0.4 \u00b5l of each primer, 0.4 \u00b5L of 50 \u00d7 ROX reference dye, 2 \u00b5L of diluted cDNA and 6.8 \u00b5l of sterile distilled water. The cycling conditions were 30 s at 95 \u00b0C followed by 40 cycles of 5 s at 95 \u00b0C and 31 s at 60 \u00b0C. Melting curves after 40 PCR cycles were carried out by heating from 60 \u00b0C to 95 \u00b0C to verify the specificity of each amplicon. Each cDNA was analysed three times. The results were normalized to the expression level of the UBC. The relative expression levels of genes were calculated using E\u2212\u0394\u0394CT (gene/UBC) [To verify the data obtained by Illumina sequencing, qRT-PCR was performed on five genes involved in terpenoid synthesis, and the ubiquitin-conjugating enzyme E2 (rol gene . The RNAene/UBC) .L. cubeba fruit development. Gene ontology analyses identified 75 DEGs associated with terpenoid biosynthesis in L. cubeba. Pathway analysis revealed 26 DEGs associated with terpenoid biosynthesis pathways in L. cubeba. Our results provide a comprehensive understanding of DEG transcription patterns and expression levels during L. cubeba fruit development, particularly the process of oil accumulation. The DEG transcription patterns should provide an invaluable resource for the identification of genes involved in terpenoids biosynthesis in L. cubeba, and should promote the systematically understanding of the molecular mechanisms of oil production of L. cubeba in the future.In this study, a total 8459 DEGs were identified across four respective comparisons of developmental stages during"}
+{"text": "Context: Temporal lobe epilepsy (TLE) is resistant to antiepileptic drugs (AEDs) and is associated with cognitive impairment. The modern Chinese medicine, compound Danshen dripping pills (CDDP), is clinically effective in treating epilepsy and improving cognitive impairment.Objective: This study evaluated the protective effects of CDDP alone and in combination with carbamazepine (CBZ) on kainic acid-induced TLE and cognitive impairment in rats.Materials and methods: Sprague\u2013Dawley rats were randomly divided into five groups: control (sham operated), model, CDDP, CBZ and combined. A TLE model was then created via bilateral intrahippocampal injection of 0.35\u2009\u03bcg kainic acid (KA). Rats received CDDP (85\u2009mg/kg), CBZ (100\u2009mg/kg) or combined (85\u2009mg/kg CDDP +100\u2009mg/kg CBZ) via intragastric administration for 90\u2009d, respectively. Seizure intensity, apoptosis and glial cell line-derived neurotrophic factor (GDNF) were measured. Furthermore, the improvement in cognitive impairment and hippocampal neuronal damage was evaluated.Results: CDDP combined with CBZ significantly decreased seizure severity and frequency (p\u2009<\u20090.05) and ameliorated cognitive impairment (p\u2009<\u20090.05). The model group showed a significant reduction of neurons and Bcl-2/Bax expression in the hippocampus CA3 area (p\u2009<\u20090.01), the combined groups significantly reversed these change (p\u2009<\u20090.01). GDNF expression in the combined groups showed a clear increase over the model group (p\u2009<\u20090.05).Conclusion: These findings support the use of CDDP as an adjuvant drug for the treatment of TLE and cognitive deficit. Its mechanism might be related to an anti-apoptosis effect and up-regulation of GDNF. Epilepsy is a terrible chronic neurologic disease of brain dysfunction that is characterized by an enduring state of spontaneous recurrent seizures (SRS); it affects approximately 1\u20132% of the world\u2019s population  (known as \u201cdanshen\u201d in Chinese), Panax notoginseng (Burk.) F.H. Chen  (known as \u201csanqi\u201d in Chinese) and borneol are a modern Chinese medicine preparation based on TCM theory and modern preparation technologies. With its multiple targets, components and effects, CDDP is now used extensively in China to prevent and treat diseases including angina pectoris, hyperlipidemia, and coronary heart disease  is primarily used as a complementary and alternative medical approach in the treatment of epilepsy. A combination of TCM and Western medicine may increase the curative effect and decrease the recurrence rate and adverse reactions to Western medicine. These combination therapies may offer a new way to treat TLE. Compound Danshen dripping pills (CDDP), consisting of Hippocampal neuronal loss is usually a feature of human TLE  rats weighing 240\u2009\u00b1\u200920\u2009g were purchased from the Laboratory Animal Service Centre of Lanzhou University  2013-0002). They were housed at a controlled temperature (22\u2009\u00b1\u20093\u2009\u00b0C) and relative humidity (40%) with a 12\u2009h light/dark cycle. All animal experimental procedures were performed in accordance with the animal care guidelines of the National Institutes for Health and approved by the Animal Ethics Committee of Lanzhou University Second Hospital.n\u2009=\u200914): control (sham operated), model, CDDP, CBZ and combined. Then rats in model, CDDP, CBZ and combined groups were selected to create a TLE rat model by intrahippocampal injection of KA. After the rats were weighed, they were anesthetized with 10% chloral hydrate by intraperitoneal injection (350\u2009mg/kg) and mounted on a stereotaxic instrument . The scalps of the rats were incised to expose the skull surface, and holes about 1.0\u2009mm in diameter were drilled with an incisor bar on each lateral ventricle according to the following coordinates: AP\u2009=\u20090.9\u2009mm anterior to the bregma, ML\u2009=\u2009\u00b1\u20092.0\u2009mm from the midline and DV\u2009=\u20095.6\u2009mm below the dura. Then, 0.35\u2009\u03bcL of KA  was injected into each hole with a micro-syringe. The needle was left in place for at least 5\u2009min. Bone wax was then used to seal the holes and suture the scalp. Rats in the control group were selected to create a sham-operated model by intrahippocampal injection of an equal volume of physiologic saline solution using the same method.Seventy male SD rats were randomly divided into five groups  and CBZ (maximum daily dose 20\u2009mg/kg) for adults are equivalent to CDDP (85.05\u2009mg/kg/d) and CBZ (126\u2009mg/kg/d) for rats. Based on the relevant literature and clinical practice, we chose to give daily doses of CDDP at 85\u2009mg/kg and CBZ at 100\u2009mg/kg by oral administration. The CDDP  and CBZ  were ground and dissolved in normal saline solution before use. An HPLC fingerprint of CDDP was shown in After intrahippocampal injection of KA, the rats in each group underwent continuous gavage with the corresponding drug once per day for 90\u2009d. The rats in the control and model groups received physiologic saline solution at 2.5\u2009mL/kg body weight. The rats in the CDDP group underwent gavage with CDDP at 2.5\u2009mL/kg body weight . The rats in the CBZ group received CBZ at 2.5\u2009mL/kg body weight . The rats in the CDDP\u2013CBZ combined group underwent gavage with CDDP at 2.5\u2009mL/kg body weight and CBZ at 2.5\u2009mL/kg body weight. The experimental process is shown in The rats\u2019 postoperative behaviour was observed over a 9\u2009h period (from 09:00 to 18:00). The Racine stages Racine  used to The MWM test Morris  was usedEach test had two parts: a place navigation test and a spatial probe trial. The place navigation test was performed four times a day for four consecutive days of training. The test started by placing a rat at the border of a randomly chosen quadrant other than the SW quadrant and facing the wall of the pool. The test finished when the rat escaped onto the hidden platform and the escape latency was then recorded. Any rat that failed to find the platform within 120\u2009s was guided to the platform and kept there for 15\u2009s; its escape latency was recorded as 120\u2009s. Probe trials without a platform were performed on day 5. The trials allowed the rats to swim freely from the NE quadrant. The platform-crossing frequency and time spent in the NE quadrant were recorded.The rats underwent transcardial perfusion with physiologic saline solution followed by 4% paraformaldehyde for fixation after deep anaesthesia, and their brains were quickly removed and post-fixed over 24\u2009h at 4\u2009\u00b0C with 4% paraformaldehyde and transferred into 10%, 20% and finally 30% sucrose solution . A bicinchoninic acid kit  was used to determine the concentration of the protein. Equal amounts of protein were separated by SDS-polyacrylamide gels, and the protein was transferred onto polyvinylidene fluoride membranes  at 100\u2009mA for 75\u2009min at 4\u2009\u00b0C. Blocking was done with 5% skim milk in Tris-buffered saline solution containing 0.05% Tween 20 (TBST) for 1.5\u2009h at room temperature. The membranes were then incubated overnight at 4\u2009\u00b0C with mouse anti-\u03b2-actin , anti-GDNF , anti-Bcl-2  or anti-Bax  antibodies. The membranes were then incubated with a goat anti-rabbit antibody conjugated to a horseradish peroxidase secondary antibody  for 1.5\u2009h at room temperature. After washing with TBST (3\u2009\u00d7\u20097\u2009min), bands were detected with an enhanced chemiluminescence detection system . Densitometric analysis was performed using Bio-Rad\u2019s Quantity One software. To determine the relative band density ratio, all values were normalized against \u03b2-actin.p value of less than 0.05 was considered to indicate statistical significance.SPSS 17.0 software  was used for statistical analysis. One-way repeated-measures analysis of variance was used for statistical comparisons; Tukey\u2019s test was used for comparisons of two groups. The measurement data were expressed as the mean\u2009\u00b1\u2009SEM (standard error of the mean). In MWM tests, one-way repeated-measures analysis of variance followed by Tukey\u2019s test was used to evaluate differences in the escape latencies among the groups. A Seizures induced by intrahippocampal KA injection were divided into three stages according to behavioural characteristics: acute phase, incubation period and chronic phase. The acute phase occurred within 24\u2009h of intrahippocampal injection of KA. From 30\u2009min to 2\u2009h after the injections, all rats that had received KA developed status epilepticus, which was characterized by continuous clonic convulsions associated with intermittent rolling and falling, a type of seizure that can cause death in severe cases. After that, the frequency and degree of epilepsy seizures gradually declined and the duration of each epileptic seizure reduced. Finally, the seizures gradually stopped and the rats returned to normal activity. Incubation periods occurred at 2\u201314\u2009d after surgery. Chronic phases occurred after the incubation period. In this stage, the rats developed SRS and displayed a different Racine stage.p\u2009<\u20090.05). Although the degree and the frequency of epileptic seizures in the CBZ and CDDP groups were slightly lower than those in the model group, the results were not statistically significant. Thus, the results show that CDDP in combination with CBZ can effectively control epilepsy.To assess the effect of CDDP and its combination with CBZ on the degree of seizure, rats in each group underwent behavioural assessments for 1\u2009week at 90\u2009d after surgery . The salp\u2009<\u20090.05). The combined group also had longer escape latencies than the control group on the second (p\u2009<\u20090.05) day, whereas the CDDP and combined groups showed significantly quicker escape latencies during the training sessions than those recorded for the model group (p\u2009<\u20090.05). In the spatial probe trials (p\u2009<\u20090.05), whereas the frequency of crossing was significantly higher in the CDDP (p\u2009<\u20090.01) and combined groups (p\u2009<\u20090.05) than in the model group. The model and CBZ groups spent significantly less time in the target quadrant than the control group did (p\u2009<\u20090.05), whereas the CDDP and combined groups significantly improved their performance compared with the model group (p\u2009<\u20090.05). In addition, the CDDP group spent significantly more time in the target quadrant than the CBZ group did (p\u2009<\u20090.05). These results indicate that CDDP alone and in combination with CBZ could markedly improve learning and memory.We used the MWM test to assess the effect of CDDP and its combination with CBZ on cognitive impairment at 90\u2009d after surgery. In the place navigation test , all groe trials , the frep\u2009<\u20090.01) (p\u2009<\u20090.01). Furthermore, the number of surviving neurons in the combined group was significantly greater than that in the CDDP (p\u2009<\u20090.01) and CBZ (p\u2009<\u20090.05) groups. The results show that CBZ and CDDP combined with CBZ clearly reversed the loss of hippocampal neurons in the CA3 area induced by KA, with the combined group showing the most improvement.Hippocampal neuronal damage was assessed by Nissl staining at 90\u2009d after surgery. The number of neurons in the hippocampus CA3 area was counted. We found that KA induced neuronal cell loss in this area. The combined group showed no obvious neuronal loss when compared with the control group, whereas the other groups showed extensive loss (\u2009<\u20090.01) . The CBZp\u2009<\u20090.05), whereas the expression of GDNF was significantly greater in the combined groups than in the model group (p\u2009<\u20090.05). The expression of Blc-2/Bax in all groups was lower than in the control group (p\u2009<\u20090.05). Compared with the model group, the combined treatment significantly suppressed the KA-induced decrease of Blc-2/Bax expression (p\u2009<\u20090.05). These results indicate that CDDP in combination with CBZ can remarkably up-regulate the expression of GDNF protein and the level of Bcl-2/Bax in the hippocampal CA3 region of KA-induced rats.GDNF and Bcl-2/Bax expression in the hippocampal CA3 area was evaluated by western blotting at 90\u2009d after surgery. As shown in Rats that are given intracerebral or systemic kainic acid (KA) develop neuropathological changes such as neurodegeneration, severe status epilepticus (SE), neuronal cell death and memory loss and experience chronic, spontaneous, recurrent seizures (Contractor et\u00a0al. TLE is often associated with cognitive dysfunction (Tavakoli et\u00a0al. A pathological characteristic of TLE is progressive damage to and pronounced neuronal loss in the hippocampus Kapur . CognitiGDNF is widespread in the rat and human central nervous system and is also present in the hippocampus (Schmidt-Kastner et\u00a0al. In conclusion, our findings support the use of CDDP as an adjuvant drug for the treatment of TLE and cognitive deficit. Combined administration of CDDP with CBZ was more effective for control of epileptogenesis, protection against cognitive impairment and inhibition of hippocampal neuronal loss in a model of KA-induced TLE. Its mechanism might be related to anti-apoptosis and up-regulation of GDNF expression level. This combination, if applicable to individual clinical therapy, may provide a potential novel therapeutic option to enhance the treatment of patients with TLE."}
+{"text": "There is a debate over whether actions that resist devaluation  are primarily habit- or goal-directed. The incentive habit account of compulsive actions has received support from behavioral paradigms and brain imaging. In addition, the self-reported Creature of Habit Scale (COHS) has been proposed to capture inter-individual differences in habitual tendencies. It is subdivided into two dimensions: routine and automaticity. We first considered a French version of this questionnaire for validation, based on a sample of 386 undergraduates. The relationship between two dimensions of habit and the risk of substance use disorder and impulsive personality traits was also investigated. COHS has good psychometric properties with both features of habits positively associated with an Obsessive-Compulsive Inventory score. Besides, the propensity to rely more on routines was associated with lower levels of alcohol abuse and nicotine use, suggesting that some degree of routine might act as a protective factor against substance use. In contrast, a high automaticity score was associated with an increased risk of harmful alcohol use. These results demonstrate that the COHS is a valid measure of habitual tendencies and represents a useful tool for capturing inter-individual variations in drug use problems in undergraduates. A constantly changing environment requires behavioral and decisional adjustments. The repetition of reinforced actions performed within the same context results in behavior triggered automatically in that environment , while the second sample consisted of 286 students from the Universit\u00e9 Libre de Bruxelles . In exchange for their participation, they received course credits. The answers of 21 participants were removed due to their low involvement in the questionnaires. All participants gave informed consent to be part of the experiment. The experiment was approved by the C.H.U. Brugmann Ethics Committee (n\u00b0 OM 026) and was performed according to the Declaration of Helsinki.After giving their informed consent and some demographic information (age and number of succeeded years from 12 years old), each participant completed the 27 COHS questions on the online LimeSurvey platform. In addition, they complete several validated French version of other questionnaires: harmful alcohol was measured with the Alcohol-Use Disorder Test  contains 16 items, the second (\u201cAutomaticity\u201d) 11 items. To assess whether our data fit the same 2-factor solution, a principal component analysis with a varimax rotation was first performed on the first sample. Items with a rotated factor loading lower than 0.35 have been removed. Subsequently, a confirmatory factor analysis was performed on the second sample. All the analyses were made with R studio (v1.1.146), the Lavaan package Rosseel, , and IBMt-tests  were used to compare COHS scores between (1) participants with an AUDIT score up to 12 (non-dependent drinkers) vs. those with an AUDIT score of 13 and higher . Bartlett's sphericity test, which tests the overall significance of all the correlations within the correlation matrix, was significant for a 2-factor solution  with good sampling adequacy (KMO = 0.66). The rotated component matrix . The MI showed that four pairs of items were too redundant . We analyzed the pair and decided to remove the pair with the lowest factor load , because they were semantically very similar. The rotated factor loads of the remaining variables with the subscales were significant (p < 0.001). The construct validity measures were all on their respective cut-off score  and were close to those obtained by Ersche et al. , close those initially observed by Ersche et al. .The descriptive scores for each variable are presented in r = 0.25, p < 0.001).Correlations were performed between each subscale, AUDIT score and clinical profile see . As was X2 = 4.9, p = 0.03), and alcohol  than males. The AUDIT score significantly correlated with the routine  scores, but not with the automaticity score (p > 0.05). Routine score was significantly lower in dependent alcohol drinkers than in non-dependent drinkers  and lower in nicotine smokers than in non-smokers . The automaticity score was significantly higher in dependent drinkers than in non-dependent drinkers  and did not significantly differ according to nicotine consumption (p > 0.05). The mean scores for each group are shown in In addition, females reported being significantly less exposed to nicotine  and to estimate the association between the tendency to act routinely and/or automatically and substance use  among undergraduates. First, COHS has good psychometric properties with two distinct features of habits, namely routine behaviors and automatic responses, both of which were associated with an Obsessive-Compulsive Inventory score (OCIR)  can be found at: The studies involving human participants were reviewed and approved by C.H.U. Brugmann Ethics Committee (n\u00b0 OM 026). The patients/participants provided their written informed consent to participate in this study.FW: conceptualization, methodology, formal analysis, investigation, data curation, writing\u2014original draft, and visualization. AC: conceptualization, methodology, investigation, writing\u2014review, and editing. MS, NJ, CF-H, and CK: writing\u2014review and editing. XN: conceptualization, writing\u2014original draft, writing\u2014review and editing, and supervision. 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": "Malignancies of alimentary tract include esophageal carcinoma (ESCA), stomach adenocarcinoma (STAD), colon adenocarcinoma (COAD), and rectum adenocarcinoma (READ). Despite of their similarities in cancer development and progression, there are numerous researches concentrating on single tumor but relatively little on their common mechanisms. Our study explored the transcriptomic data of digestive tract cancers from The Cancer Genome Atlas database, yielding their common differentially expressed genes including 1,700 mRNAs, 29 miRNAs, and 362 long non-coding RNAs (lncRNAs). There were 12 mRNAs, 5 miRNAs, and 16 lncRNAs in the core competitive endogenous RNAs network by RNA-RNA interactions, highlighting the prognostic nodes of SERPINE1, hsa-mir-145, and SNHG1. In addition, the weighted gene co-expression network analysis (WGCNA) illustrated 20 gene modules associated with clinical traits. By taking intersections of modules related to the same trait, we got 67 common genes shared by ESCA and READ and screened 5 hub genes, including ADCY6, CXCL3, NPBWR1, TAS2R38, and PTGDR2. In conclusion, the present study found that SERPINE1/has-mir-145/SNHG1 axis acted as promising targets and the hub genes reasoned the similarity between ESCA and READ, which revealed the homogeneous tumorigenicity of digestive tract cancers at the transcriptome level and led to further comprehension and therapeutics for digestive tract cancers. The incidence and mortality of alimentary tract malignancies have been high in the world, seriously endangering public health and human life . For insAn important reasons why digestive tract cancers contribute to numerous deaths is the lack of suitable and effective diagnosis . BesidesWith the development of high-throughput sequencing and omics profiling in life sciences, there are increasing reliable methods and constantly updated databases , 6. The https://gdc-portal.nci.nih.gov/). The mRNA expression data of ESCA included a total of 171 tumor samples consisting of 160 tumor samples and 11 normal samples. The mRNA expression data of STAD included a total of 407 samples consisting of 375 tumor samples and 32 normal samples. The mRNA expression data of COAD included a total of 177 tumor samples consisting of 167 tumor samples and 10 normal samples. The mRNA expression data of READ included a total of 521 samples consisting of 480 tumor samples and 41 normal samples. The miRNA expression data of ESCA included a total of 198 samples consisting of 185 tumor samples and 13 normal esophageal samples. The miRNA expression data of STAD included a total of 491 samples consisting of 446 tumor samples and 45 normal samples. The miRNA expression data of COAD included a total of 458 tumor samples consisting of 450 tumor samples and 8 normal samples. The miRNA expression data of READ included a total of 84 samples consisting of 83 tumor samples and 1 normal sample. The sequencing data were all publicly available and no ethical issues were involved. The Gencode database (https://www.gencodegenes.org/human/) was used to distinguish and annotate the mRNA and lncRNA from the RNA-seq. LncRNA and mRNA were extracted by Perl to make lncRNA and mRNA data matrixes. The edgeR package in Bioconductor was used to screen the differentially expressed miRNAs, differentially expressed lncRNAs and differentially expressed mRNAs . The hallmarks gene sets were downloaded from Molecular Signature database (https://www.gsea-msigdb.org/gsea/msigdb). And p-value <0.05 and FDR <0.05 were set as the cutoff criterion to screen the prominent hallmarks.For the differentially expressed mRNAs of four digestive tract cancers, Gene set enrichment analysis (GSEA) was conducted to investigate the hallmarks of the four digestive tract cancers by online tool WebGestalt (https://david.ncifcrf.gov/) online tool , including Targetscan database, miRDB database and miRTarBase database, aiming to predict target mRNA of different expressed miRNA and took the intersection between the predicted mRNA and common differentially expressed mRNA. We got the adjusted relationships between the miRNA and mRNA. We used the miRcode database (http://www.mircode.org/) to obtain the relationships of lncRNA-miRNA and took the intersection between the predicted lncRNA and common differentially expressed lncRNA. Thus, the visual ceRNA network was constructed by cytoscape software  was used to show co-expression relationships between lncRNA and mRNA. Besides, the ImmLnc database (http://bio-bigdata.hrbmu.edu.cn/ImmLnc/) was used to show the Immune-related pathways.For the lncRNA of highest connectivity, the GEPIA2 database (https://string-db.org/) was used to construct the PPI network and cytoscape MCODE plug-in was used to screen the hub genes (https://metascape.org/) and David database (https://david.ncifcrf.gov/) were used to perform the GO and KEGG pathways enrichment analysis to detect the potential biological functions of common genes  was used to analyze the correlation between hub genes expression and clinical traits. For the methylation analysis, We explored the association between hub genes\u2019 expression levels and their methylation status by Disease Meth version 2.0 (http://bio-bigdata.hrbmu.edu.cn/diseasemeth/) and MethSurv (https://biit.cs.ut.ee/methsurv/). Besides, the online database TIMER2.0 (http://timer.cistrome.org/) was used to verify that the above key nodes in ceRNA network and hub genes were the common features in alimentary tract malignancies.The \u201cWGCNA\u201d package in R software was used to construct gene co-expression network and identify the co-expression genes modules . For fouub genes . And Meton genes , 12. ForIn total, 3,999 mRNAs were identified as differentially expressed (DE) mRNAs in ESCA, including 1,776 up-regulated and 2,223 down-regulated mRNAs  was used to show the hallmarks of different cancers . The resTo gain insight into the biological functions of four tumors in common, GO and KEGG pathways analysis , 12 wereThe Competing endogenous RNAs (ceRNAs) regulate RNA transcripts by competitively binding with shared miRNAs . AccordiThe correlations between ceRNAs and the survival outcomes of patients were analyzed to validate the prognostic value. The results showed serpin peptidase inhibitor (SERPINE1), hsa-mir-145 and small nucleolar RNA host gene 1 (SNHG1) were significantly correlated with overall survival  showed that SERPINE1, SNHG1, ADCY6, CXCL3, ADCY6, CXCL3, NPBWR1, TAS2R38, and PTGDR2 were mainly differentially expressed in alimentary tract malignancies , a G protein-coupled receptor, implicates in the biological process of energy homeostasis, neuroendocrine function and modulating inflammatory pain , 44. AndFor the other hub genes, ADCY6 encodes a member of the adenylate cyclase protein family, and it is required for the synthesis of cAMP . In our CXCL3 is also known as CINC-2 alpha (Cytokine-induced neutrophil Chemoattractant) and is a number of signaling pathways such as ERK1/2 MAPK, by activating CXCR2 receptor \u201355. TherIn conclusion, by constructing ceRNA network and using WGCNA analysis, we find a potential regulatory axis and several significant gene modules in alimentary tract cancers. For the clinical treatment of these malignancies, the axis SERPINE1/hsa-mir-145/SNHG1 could serve as prognostic biomarkers and potential therapeutic targets. The hub gene ADCY6, CXCL3, NPBWR1, TAS2R38, and PTGDR2 highly influence the clinical traits of age, histology types, neoplasm histologic grade, and overall survival in ESCA and READ, leading to their similarity in these clinical traits. Further study is guaranteed for sure, aiming to identify the therapeutic value of SERPINE1/hsa-mir-145/SNHG1 axis and fully reveal the hub genes contribution to the clinical outcomes.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/KY and D-BJ developed the original idea. KY, D-BJ, and L-HZ designed the whole study. Y-CL, J-QS, Z-XZ, and J-YZ ran the most of data processing. Y-CL, H-KZ, and Y-CF did data visualization. Z-HL and Z-CL helped to access the public data. S-YY, X-YZ, YL, and Y-JS reviewed the manuscript and raised suggestions. Y-CL and D-BJ drafted the manuscript. D-BJ and J-QS revised the writings. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China (No. 81772763 and No. 82073154) and Key Research and Development Program of Shaanxi Province (No. 2020SF-200).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": "Textured sockets were compared to an Original Squirt-Shape (OSS) socket and a smooth thermoformed socket. Sockets were fitted with a mock residual limb and bi-axially loaded to 350 N compression with simultaneous rotation  using a custom rotation assembly attached to a uniaxial hydraulic material testing system. There was a statistically significant three-way interaction between suspension, angle and texture (p < 0.0005). Torques between textured and reference sockets, for all rotation angles and both suspension conditions, were significantly different (p < 0.0005). Using newly developed testing protocols, it was demonstrated that some texture patterns significantly increased torque  in the transverse plane compared to both OSS and smooth sockets, especially for passive suction. Rotation testing of sockets may provide insight into socket design to improve suspension in the transverse plane.Creating a secure and comfortable linkage between the residual limb and prosthetic socket in persons with lower limb amputation is a critical factor for successful rehabilitation, including ambulation and other activities of daily living. Unwanted rotation within the socket can be a clinical problem for prosthesis users. One way of addressing issues experienced with transverse plane control of the socket may be through increased friction interface forces. It has been proposed that friction at the residual limb/socket interface may be increased by adding texture to interface components. Three-dimensional (3D) printing may be used to fabricate sockets with texture patterns added to the inner socket surface. Hence, the aim of this study was to investigate the effects of socket texturing on transverse plane rotation of the socket on a mock residual limb under two suspension conditions: passive suction and active vacuum. To conduct this study, we developed a mechanical testing protocol as no standardized tests currently exist to assess prosthetic sockets. Sockets with 14 different texture patterns were fabricated using the Squirt-Shape Creating a secure and comfortable linkage between the residual limb and prosthetic socket in persons with lower limb amputation is a critical factor for successful rehabilitation, including ambulation and other activities of daily living. A secure linkage between the residual limb and socket requires some friction forces acting at the interface between them  to diminThe generation of sufficient interface forces to control relative motion without causing other problems continues to be a clinical challenge both in the longitudinal/axial direction as well as the transverse plane. Reducing pistoning \u20139 and imRotational control of the prosthetic socket on the residual limb is needed during both stance and swing phases of walking. In swing phase, transverse plane gait deviations known as whips can occur due to poor suspension . In stan\u2122 3D printer . Squirt-Shape sockets with slight horizontal striations/texturing have been in clinical use for over a decade.An alternative way of addressing issues experienced with transverse plane control of the socket may be through increased friction interface forces. It has been proposed that friction at the residual limb/socket interface can be increased by adding texture to interface components \u201321. ThrePrevious studies , 21 repoMechanical testing has the potential to offer insights regarding interface designs aimed at reducing relative motion between the socket and residual limb, yet no guidelines exist for how this may be accomplished. Previous studies modified the International Organization for Standardization (ISO) 10328 standard  testing Given the above, the aim of this project was to investigate the effects of socket texturing on transverse plane rotation of the socket on a mock residual limb under two suspension conditions: passive suction and active vacuum. In order to conduct this study, we adapted a rack and pinion design by Edwards and Troy  and deve\u2122 3D Printer . A selection of seven novel texture patterns were programmed and 3D printed into the socket wall. Texture patterns included simple designs such as horizontal and vertical lines, horizontal and vertical rectangles, as well as more complicated designs such as checkered, hemisphere and half-hemisphere [Total Surface Bearing prosthetic sockets were fabricated using polypropylene copolymer pellets  and a Squirt-Shapemisphere . Two socmisphere .During socket fabrication, our goal was to obtain a similar fit between the sockets with different textures and a mock residual limb. This was achieved by ensuring that there was resistance when sliding the mock residual limb into the socket while obtaining distal end contact. The volume of each socket was globally altered in the computer-aided design (CAD) software to obtain these characteristics. Estimates of socket volume from the digital CAD files indicate that heavy dense sockets had a greater socket volume (range: 1.16%\u20134.85%) when compared to the light sparse sockets.Additionally, two reference sockets were fabricated to be used for comparison with novel textured sockets. An OSS socket was fabricated using the same 3D printer and the same polypropylene copolymer material as the novel textured sockets. The OSS socket had horizontal striations approximately 1.2 mm in depth and 0.75 mm in thickness , 23. A sRotational testing was conducted using a uniaxial hydraulic material testing system . Assembly and fixtures needed to conduct testing were designed using CAD modeling software . The transformation of linear actuation of a uniaxial material testing system into rotational movement has been described previously for torsional testing of human cadaveric proximal tibiae using a rack and pinion design . SimilarThe custom rotation assembly consisted of a custom external loading frame constructed of 6061 aluminum stock , high-load steel gear rack  length, 16 pitch, face width 3/4\" (19.05 mm), height 3/4\" (19.05 mm), McMaster-Carr, Elmhurst, IL) and high-load steel pinion gear , face width 3/4\" (19.05 mm), McMaster-Carr, Elmhurst, IL), steel tapered-roller bearings (3/4\" (19.05 mm) shaft diameter, trade No. 09067, 1-15/16\" (49.21 mm) OD, McMaster-Carr Elmhurst, IL), one-piece steel thrust ball bearing (3/4\" (19.05 mm) OD, open, McMaster-Carr, Elmhurst, IL), flange-mount ball transfer (heavy duty 1/2\" (12.70 mm) steel ball, steel housing, 100 lb (45.36 kg) capacity, McMaster-Carr, Elmhurst, IL), 34mm wide guide rail for ball bearing carriage , spring plunger  with compression spring , hole diameter 0.720\" (18.29 mm), wire diameter 0.148\" (3.76 mm), Lee Spring, Brooklyn, NY), hardened 4140 alloy steel rod (3/4\" (19.05 mm) diameter x 2 feet (0.61 m), McMaster-Carr, Elmhurst, IL), and additional custom and stock fixtures, shaft collars, bolts, screws and washers  necessary to position the socket and mock residual limb onto the external loading frame.T (Nm) was calculated using the force F (N) measured by the load cell and the moment arm r, which was the pitch radius (0.0635 m) of the pinion gear:The pinion gear and gear rack assembly rotation was mounted inside the aluminum housing using steel tapered-roller bearings and a steel rod and it was connected via a threaded rod matching the hole of the main load cell . The pisf calculated based on pilot testing (1.60 N\u20141.99 N) was considered negligible compared to the measured loads, hence the torque calculations were simplified to:Friction An additional compression load cell  with load button cell design, FUTEK Advanced Sensor Technology, Inc., Irvine, CA) was attached at the distal end of the socket\u2013mock residual limb rotation assembly  to measu\u2122, WillowWood, Mt. Sterling, OH) connected to the manifold. During testing the pump was set to 20 inHg (67.73 kPa).A 3 mm liner and one nylon stocking were donned over the mock residual limb before it was seated inside the socket, which had an elevated vacuum attachment plate with manifold with air fitting  attached to the distal end. A sealing sleeve  was donned over the exterior of the socket and sealed proximally to the liner above the socket trim lines. Distally, electrical tape was used to seal the sleeve to the socket given that more aggressive texturing caused some leaking of air from between the liner and socket. It is not uncommon in clinical practice for tape to be used to ensure that the distal end of the sleeve seals well to the exterior of the socket. Two suspension conditions were tested: 1) passive suction, which was created by attaching a one-way valve to the manifold, and 2) active vacuum, which was created using an electronic vacuum pump  resembles partial body weight on one limb of a 72 kg person and was determined during pilot testing to be the maximum that could be applied. Higher compression loads resulted in large, potentially damaging offset moments to the Instron. Tuning of the Instron for displacement control was performed according to manufacturer instructions . Tuning To assess frictionless movement of our rotational testing set-up, we conducted pilot rotation tests using the same testing protocol with the socket mounted but without the mock residual limb attached. Ten trials per rotation angle were performed. To assess repeatability, tests were repeated using the same method, user, environmental conditions, and calibrated load cells. Average friction of the set-up was recorded as 1.60 (\u00b10.72) N, 1.79 (\u00b10.81) N and 1.99 (\u00b10.82) N for rotation angles 2.5\u00b0, 5\u00b0 and 7.5\u00b0, respectively. Hence, the set-up was deemed nearly frictionless and with little variability.Each socket was subjected to six trials consisting of ten cycles at each of three rotation angles in the following order: 2.5\u00b0, 5\u00b0 and 7.5\u00b0. These values were informed by previous studies that reported 8\u00b0 of external rotation of the shank over the foot and 3\u00b0 to 8.5\u00b0 relative internal-external rotation between the pelvis and socket during the stance phase of walking of persons with a transfemoral prosthesis  and rotaDespite pre-compression of the socket and mock residual limb, additional settling of the mock residual limb inside the socket occurred during the first trial, hence only five trials of data from each socket were included in the final analysis. We avoided testing the same liner and the same mock residual limb more than once per day in order to avoid any unwanted motion that may occur due to flow characteristics of the elastomeric materials used in the mock residual limb and liner , 37.Torques between the socket and mock residual limb and the corresponding rotation angles were recorded and analyzed using custom MATLAB  code. Statistical analyses were performed using SPSS version 25  with torque as the dependent variable having three rotation angles . We assessed the torque of 16 different sockets: 2 reference sockets (smooth thermoformed and OSS sockets) and 14 novel textured sockets. Each socket was tested six times in rotation under two suspension conditions: passive suction suspension using a one-way valve (OV) and active vacuum suspension (VAC). Three independent variables were analyzed: texture (a between socket measure), and suspension  and rotation angle .A three-way mixed analysis of variance \u201cbetween-within-within\u201d (ANOVA BWW) was conducted first. Torque data were not normally distributed as indicated by the Shapiro\u2013Wilk test. Hence, we normalized torque data using a two-step rank procedure . We procSignificant interactions and main effects among all the independent variables were found, hence we performed additional Welch one-way ANOVAs to assess our hypotheses. Games-Howell post-hoc analysis for pairwise comparisons between novel and reference textures was conducted, analyzing six torques (three at each rotation angle for each suspension condition). Critical alpha was initially set at 0.05, then corrected to a Bonferroni-adjusted alpha level of 0.003125.Figs at 2.5\u00b0 rotation angle: OV: 10.387 to 16.060 Nm and VAC: 10.894 to 16.645 Nm;at 5\u00b0 rotation angle: OV: 17.693 to 26.055 Nm and VAC: 20.960 to 28.545 Nm; andat 7.5\u00b0 rotation angle: OV: 20.473 to 37.266 Nm and VAC: 24.801 to 43.030 Nm;There was a statistically significant three-way interaction between suspension, rotation angle and texture, F = 353.280, p < 0.0005, partial \u03b72 = .883, \u03b5 = .804. There was also a statistically significant (p < 0.0005) simple two-way interaction between suspension and rotation angle for all texture types, and a statistically significant (p < 0.0005) simple-simple main effect of suspension on all measured torques for all rotation angles. More detailed statistical results are summarized in the Torques in the transverse plane between texture and reference sockets for all rotation angles and both suspension conditions were significantly different at p < 0.0005. Specifically:for 2.5\u00b0 rotation angle: OV: Welch\u2019s F = 1366.253; VAC: Welch\u2019s F = 3232.694;for 5\u00b0 rotation angle: OV: Welch\u2019s F = 1956.274; VAC: Welch\u2019s F = 1977.798; andfor 7.5\u00b0 rotation angle: OV: Welch\u2019s F = 1727.067; VAC: Welch\u2019s F = 2246.391.For passive suction across all rotation angles, greater torques were observed for 10 of 14 novel textured sockets when compared to the smooth socket, and 11 of 14 when compared to the OSS socket. When active vacuum was applied, fewer novel textured sockets produced increased torques compared to the two reference sockets .For the smallest rotation angle of 2.5\u00b0 with passive suction, 9 of 14 novel textured sockets had significantly greater torques than the smooth socket. The same 9 novel textured sockets and the smooth socket had significantly greater torques than the OSS socket. Additionally, 5 of 14 novel textured sockets and the OSS socket had significantly smaller torques than the smooth socket and 3 of 14 novel textured sockets had significantly smaller torques than the OSS socket. With active vacuum, 4 of 14 novel textured sockets had significantly greater torques than both the smooth and OSS sockets. Additionally, 9 of 14 novel textured sockets had significantly smaller torques than the smooth socket and 10 of 14 novel textured sockets had significantly smaller torques than the OSS socket.For the next rotation angle of 5\u00b0 with passive suction, 9 of 14 novel textured sockets had significantly greater torques than the smooth socket. Ten of 14 novel textured sockets and the smooth socket had significantly greater torques than the OSS socket. Additionally, 4 of 14 novel textured sockets had significantly smaller torques than both the smooth and OSS sockets. With active vacuum, 2 of 14 novel textured sockets had significantly greater torques than the smooth socket. Three of 14 novel textured sockets and the smooth socket had significantly greater torques than the OSS socket. Additionally, 12 of 14 novel textured sockets and the OSS socket had significantly smaller torques than the smooth socket and 11 of 14 novel textured sockets had significantly smaller torques than the OSS socket.For the largest rotation angle of 7.5\u00b0 N with passive suction, 7 of 14 novel textured sockets had significantly greater torques than the smooth socket. Nine of 14 novel textured sockets and the smooth socket had significantly greater torques than the OSS socket. Additionally, 6 of 14 novel textured sockets and the OSS socket had significantly smaller torques than the smooth socket and 5 of 14 novel textured sockets had significantly smaller torques than the OSS socket. With active vacuum, 2 of 14 novel textured sockets had significantly greater torques than the smooth socket and 3 of 14 novel textured sockets and the smooth socket had significantly greater torques than the OSS socket. Additionally, 12 of 14 novel textured sockets and the OSS socket had significantly smaller torques than the smooth socket and 10 of 14 novel textured sockets had significantly smaller torques than the OSS socket.The aim of this study was to investigate the effect of socket texturing on transverse plane rotation of the socket on a mock residual limb under two suspension conditions: passive suction and active vacuum. Rotational testing demonstrated that socket texturing influences socket suspension in the transverse plane. Our hypothesis that novel textures would increase torques in the transverse plane compared to smooth and OSS sockets was partially supported. While most of the textures demonstrated increased torques in the transverse plane compared to the smooth and OSS sockets in the passive suction condition, fewer novel textures demonstrated increased torques compared to the smooth and OSS sockets under active vacuum. Our hypothesis that active vacuum would provide increased torque for a given rotation angle compared to passive suction was supported. Increased torques were observed under active vacuum for almost all of the sockets compared to passive suction at all rotation angles.Our results suggest that there was no difference in torque between aggressive texture patterns and/or those with primarily vertical pattern orientation when compared to more subtle texture patterns and/or those with primarily horizontal pattern orientation. On the contrary, two of the light and sparse sockets with texture patterns that had a primarily horizontal orientation\u2013Horizontal Rectangle LS and Horizontal Line LS\u2013resulted in the highest torque at 7.5\u00b0 rotation angle under active vacuum. It is possible that this may be in part because the mock residual limb was insufficiently compliant to fully engage the height of the aggressive textures. Furthermore, based on the socket volume estimated from digital CAD files, socket volume was greater for the heavy dense sockets than the light sparse sockets in order to fit onto the mock residual limb, and this may similarly have contributed to less engagement with the aggressive textures. Unfortunately, our exploration of texture patterns was limited by the subjective rank order applied to texture orientation and the combination of factors compared. Future studies would benefit from a more systematic assessment and less subjective categorization of different texture dimensions and orientations.New methods were developed to assess socket suspension in the transverse plane  given that standardized tests are not available. The rotation testing protocol we developed to assess socket suspension in the transverse plane has merit based on the collected data. Firstly, changes in torque scaled systematically with the rotation angles applied such that the lowest rotation angle resulted in the least amount of torque and the highest rotation angle resulted in the most amount of torque. Additionally, the results demonstrated little variability in torque across the 10 cycles measured within each rotation angle.Our rotation testing protocol resembles partial body weight on one limb of a 72 kg person, hence, for ease of comparison to previously reported studies, we have normalized torque data by a mass of 72 kg. Normalized torque collected under passive suction ranged from 0.148 \u00b10.013 to 0.229 \u00b10.007 Nm/kg for 2.5\u00b0 rotation angle, from 0.253 \u00b1 0.014 to 0.372 \u00b1 0.008 Nm/kg for 5\u00b0 rotation angle, and from 0.292 \u00b1 0.026 to 0.532 \u00b1 0.021 Nm/kg for 7.5\u00b0 rotation angle. Torque collected under 20 inHg (67.73 kPa) active vacuum ranged from 0.156 \u00b10.012 to 0.238 \u00b1 0.007 Nm/kg for 2.5\u00b0 rotation angle, from 0.299 \u00b1 0.015.84 to 0.408 \u00b1 0.010 Nm/kg for 5\u00b0 rotation angle, and from 0.354 \u00b1 0.036 to 0.615 \u00b1 0.024 Nm/kg for 7.5\u00b0 rotation angle.Our normalized torque results for 2.5\u00b0 rotation angle under passive suction are comparable to transverse plane moments during walking reported in persons with transtibial amputation , 15, 39.This study focused on socket rotation around a mock transtibial residual limb. While transverse plane rotation of the socket can be an issue for persons with transtibial amputation, causing discomfort, pain and/or soft tissue damage, issues with socket rotation around the residual limb are more prevalent in persons with transfemoral amputation  given thin vivo. It remains to be demonstrated if the more promising textures identified in this study  could be worn comfortably by actual prosthesis users and whether or not they would increase liner wear and tear.The results of our study suggest that texturing may be used to improve socket suspension in the transverse plane and reduce unwanted socket rotation compared to the smooth thermoformed and OSS sockets especially under passive suction. The greater torques produced by the novel textured sockets reflects higher resistance to unwanted rotation compared to the reference sockets, thus improving socket suspension in the transverse plane. Generally, resistance against unwanted rotation was improved to a lesser degree under active vacuum compared to passive suction. For example, 10 out of 14 textures outperformed the smooth socket under passive suction and 4 out of 14 textures outperformed the smooth socket under active vacuum. Similarly, under passive suction 11 out of 14 textures outperformed the OSS socket and 4 out of 14 textures outperformed the OSS socket under active vacuum. Our results suggest that texturing of a socket with passive suction improves suspension in the transverse plane producing torque that is comparable to a smooth socket with active vacuum. Texturing of the socket was expected to increase friction between the socket/liner and residual limb and we observed increases in the resistance against unwanted rotation with certain textures, resulting in increased torque. What remains unknown is whether such texturing is helpful or not in vivo when muscles contract. With respect to the test set-up and protocol we developed, it was limited to bi-axial loading, where compression and rotation were applied simultaneously. Even though bi-axial testing is more complex than the uniaxial testing typically used when evaluating sockets [Limitations of this study include both the extent to which the mock residual limb resembled that of a human residual limb and the extent to which the testing protocol simulates the range of loading conditions a prosthesis user would experience every day. With respect to the mock residual limb, the outer shell was made from urethane of 20A shore hardness, which is higher than the shore hardness reported for skin and subcutaneous tissues , 43. A h sockets \u201331 it do\u2122 3D Printer. Different texture patterns or applying texture in a different manner (e.g. to flexible inner sockets or liners) might improve suspension in the transverse plane to a greater extent than what was evident in this study and deserve further attention.The results of this study apply only to the particular texture patterns and dimensions that we 3D printed into rigid polypropylene copolymer sockets using the Squirt-Shape\u2122 3D Printer to add texture to polypropylene copolymer sockets significantly increased torque in the transverse plane compared to both a smooth and Original Squirt-Shape socket especially for passive suction. Increased torque relates to higher resistance against unwanted rotation, thus improving socket suspension in the transverse plane.Mechanical testing protocols were developed to investigate the effect of socket texturing on transverse plane rotation of the socket on a mock residual limb. Results indicated that using the Squirt-ShapeS1 Appendix(PDF)Click here for additional data file."}
+{"text": "COVID-19 has spread globally to now be considered a pandemic by the World Health Organisation. Initially patients appeared to have a respiratory limited disease but there are now increasing reports of multiple organ involvement including renal disease in association with COVID-19. We studied the development and outcomes of acute kidney injury (AKI) in patients with COVID-19, in a large multicultural city hospital trust in the UK, to better understand the role renal disease has in the disease process.This was a retrospective review using electronic records and laboratory data of adult patients admitted to the four Manchester University Foundation Trust Hospitals between March 10 and April 30 2020 with a diagnosis of COVID-19. Records were reviewed for baseline characteristics, medications, comorbidities, social deprivation index, observations, biochemistry and outcomes including mortality, admission to critical care, mechanical ventilation and the need for renal replacement therapy.There were 1032 patients included in the study of whom 210 (20.3%) had AKI in association with the diagnosis of COVID-19. The overall mortality with AKI was considerably higher at 52.4% compared to 26.3% without AKI . More patients with AKI required escalation to critical care . Following admission to critical care those with AKI were more likely to die  and more likely to require mechanical ventilation .We have shown that the development of AKI is associated with dramatically worse outcomes for patients, in both mortality and the requirement for critical care. Patients with COVID-19 presenting with, or at risk of AKI should be closely monitored and appropriately managed to prevent any decline in renal function, given the significant risk of deterioration and death. In December 2019, the first cases of a cluster of pneumonia of unknown cause was described in Wuhan, China, later identified as a novel coronavirus\u2014SARS Coronavirus 2 (SARS-Cov-2) . The cliSeveral further studies have focused attention on AKI in critically and non-critically ill patients with COVID-19. In March 2020, Cheng at al reported on a prospective cohort of 701 COVID-19 cases in Tongji Hospital, Wuhan, China and found that 43.9% of these patients had proteinuria during hospitalisation with AKI occurring in 5.1% . AKI staAs the incidence of COVID-19 rose across European countries and the USA, reports showed that AKI was a more predominant feature of the condition than seen in initial reports from China. In April 2020, Richardson et al. described the presenting characteristics, co-morbidities and outcomes of 5700 patients admitted with COVID-19 to New York Hospitals in the USA, and of the 2626 discharged dead or alive . This paThe COVID-19 pandemic extended to the UK in March 2020, with peak infection rates seen between early and mid-April 2020 . Our HosAt the height of the COVID-19 pandemic locally, the trust admitted cumulatively over 1000 patients between March and May 2020. This paper sets out to further understand the epidemiology, clinical characteristics and outcomes of hospitalized patients with COVID-19 disease who developed AKI. To our knowledge, this study is the largest cohort of hospitalized COVID-19 and AKI patients, including critically ill patients in ICU stetting\u2019s, and ward-based patients in United Kingdom or Europe.This was a retrospective review using electronic records and laboratory data of adult (age >18 years) patients admitted to Manchester University Foundation Trust Hospitals between March 1 and April 30 2020 and had a confirmed clinical and/or virologic diagnosis of COVID-19. After consultation with the National Health Service Research Ethics Committee (NHS REC) and the Trust Research and Development department , the study was exempt from requiring a specific ethical approval and therefore waived, as it was considered a retrospective audit and considered a service evaluation. Given this, no consent was required or obtained. Data for this study was obtained from four different hospitals using the electronic health records such as Patient administration service (PAS) for hospital admissions and co-morbidities, ICE and Sunquest ICE for laboratory data and Patientrack\u2122 by Alcidion for observations data. All data was manually checked to exclude duplicate values. All patients\u2019 information and data was anonymised for analysis and reporting.Manchester University NHS Foundation Trust (MFT) Hospitals is the leading provider of tertiary and specialist healthcare services in Greater Manchester. It comprises 10 hospitals/managed clinical services across six separate sites, providing a wide range of services from comprehensive local general hospital care through to highly specialised regional and national services. The trust is the main provider of hospital care to approximately 750,000 people in Manchester and Trafford and the single biggest provider of specialised services in the North West of England. It is also the lead provider for a significant number of specialised services including Breast Care, Vascular, Cardiac, Renal, Renal Transplant Respiratory, Urology, Paediatrics, Women\u2019s Services, Ophthalmology and Genomic Medicine. It is also the main teaching hospital affiliated to the University of Manchester.Data for this study was obtained from four of the trust\u2019s hospitals that cared for adult inpatients with COVID-19 infection . All adult patients (age \u2265 18 years), including the current inpatients and any new admissions from 10th of March to 30th of April 2020 (51 days), who tested positive by polymerase chain reaction testing of a nasopharyngeal or lower respiratory tract sample for COVID-19, were eligible. Patients who were transferred between hospitals within our own trust were treated as one hospital episode and the first admission nearest to the COVID 19 result date was considered. Day cases and all known Haemodialysis patients were excluded. Records were abstracted for demographic and co-morbidity data. Postal area codes were used to link to government calculated Deprivation Indices . The patOnce the cohort was identified, laboratory data was matched for confirmation of AKI defined according to Kidney Disease: Improving Global Outcomes (KDIGO)  criteriaBlood results for all the patients were collected from data collectable tables derived from the trusts clinical laboratory reporting system (ICE\u00ae and SUNQEST ICE\u00ae). Two sets of blood results data were analysed and defined as baseline and peak results. Baseline results were the results on day 0 (day of the first positive COVID sample) and if no blood tests on that day then the sample nearest to the day 0 result (no greater than 7 days earlier were used). The second set of blood test results (peak results) were taken up to 30 days from the COVID positive date, where the highest and lowest ranges of the selected blood results were identified. If there is no repeat sample blood test, then these patients were excluded for the peak blood results analysis.Review of medication history was undertaken to check if the cohort patient was prescribed an angiotensin-converting enzyme (ACE) inhibitor and/or an angiotensin receptor blocker (ARB). This was collected manually by reviewing discharge summaries and General Practitioner records.Patientrack\u2122 by Alcidion is an electronic clinical observation system which is used to collect the observations at the bedside of patients across MFT. Observation data , respiratory rate, oxygen saturations and early warning score (EWS)) for the patient cohort was collected for 72 hours around the time of COVID diagnosis . The nearest set of observations to COVID-19 positive date were considered.Comorbidities based on ICD-10 codes obtained from patient electronic records as described above. Definitions of comorbidities and Charlson comorbidity score calculated as described by Quan et al. and Charlson et al. respectively, and using R Comorbidity package \u201326.The Raw data required to reproduce these findings cannot be shared due to patient privacy, legal and ethical reasons. However, the processed data that support the findings of this study are available from the corresponding author upon reasonable request.Categorical variables presented as number and percentage. Normality assessed using quantile-quantile (QQ) plots and Shapiro-Wilk normality test. Non-parametric continuous variables presented as median and interquartile range. Risk factors impacting on the development of AKI was assessed with the use of logistic regression. Multivariable models adjusted for demographics and variables showing significance at univariable analysis. Model 1 included vital signs as the amalgamated Early Warning Score  (EWS) atFrom March 10, 2020 to April 30, 2020, 11,721 patients were admitted to the four hospitals, and 1166 of these had a diagnosis of COVID-19. After excluding the 35 dialysis patients and 99 day-cases, there were a total of 1032 COVID-19 positive patients included in the analysis; 20.3% (n = 210) of these had a diagnosis of AKI, and 79.7% (n = 822) patients never developed AKI. The median age at diagnosis for the overall cohort was 71 years (IQR 56\u201383), with 55.1% (n = 569) males and 44.9% (n = 463) females. There were significant differences between the ethnicity of the two cohorts although the majority of patients admitted were White British, with 71.3% (n = 586) in the non-AKI cohort and 66.5% (n = 139) in the AKI cohort. There were more Black patients in the AKI cohort with 14.8% (n = 31), compared to the non-AKI cohort with 7.7% (n = 63); p-value 0.012. There were also significantly more males in the AKI group compared to the non-AKI group, p-value 0.022.There were more patients on RAAS inhibition in the AKI group with 29.5% (n = 62), compared to the non-AKI group with 20.7% . Patients in the AKI groups were more likely to have had a previous diagnosis of renal disease compared to those in the non-AKI group . Diabetes with complications was significantly higher in the AKI cohort with 8.5% (n = 15) patients compared to 2.8% (n = 21) in the non-AKI cohort, p-value 0.001, but no difference between the two groups in patients with diabetes without complications .AKI occurred in 210 patients (20.3%). At the time of initial diagnosis of their AKI then 58% (n = 123) were in stage 1, 18% (n = 37) stage 2 and 24% (n = 50) were in stage 3 AKI At peak AKI stage then 36% (n = 75) had stage 1, 20% (n = 43) stage 2 and 44% (n = 92) had stage 3 AKI. AKI worsened in 31% (n = 65) of the patients. 59% (n = 123) of patients had admission AKI and 42% (n = 87) patients developed AKI post admission. The median time to the initial AKI from the date of COVID-19 positivity was -1.00 days (IQR -1.00\u20132.00). 52% (n = 110) of patients were in AKI during the 72 hours of COVID-19 positive date  and 24% (n = 50) of patients developed AKI post 24hrs of COVID-19 positive date.The total number of patients requiring renal replacement therapy (RRT) was 32 (15.2%), and this was provided for 24 patients by continuous RRT alone, seven patients receiving both continuous RRT and haemodialysis and one patient needing haemodialysis only.Of the 32 patients needing RRT 65.6% (n = 21) died, two patients needed long term haemodialysis and the rest recovering their kidney function (n = 8).At baseline, patients with a higher serum Sodium , Urea  and Creatinine  were more likely to have AKI compared to those in the non-AKI cohort. There was no statistical difference in the lymphocyte titre between the groups  and it did not appear to be a risk factor for developing AKI; OR 1.02, 95% CI 0.88\u20131.16, p = 0.719. Patients in the AKI cohort had a higher baseline CRP, with a median of 104.00nmol/L (IQR 51.00\u2013196.00) compared to 59.00nmol/L (IQR 23.00\u2013119.25), and was a significant risk factor for the development of AKI; OR 1.01, 95% CI 1.00\u20131.01, p<0.001. Patients with a previous diagnosis of Renal disease were more likely to develop AKI , as were those with Diabetes with complications . Tables Multiple logistic regression analysis shows that baseline serum sodium , CRP , male sex  and a previous diagnosis of Renal disease  remain statistically significant for the development of AKI in association with COVID-19 after adjusting for variables. Overall, 31.6% (n = 326) patients died following a diagnosis and admission for COVID-19. Overall mortality was significantly higher in the AKI group with 52.4% (n = 110) compared with 26.3% (n = 216) in the non-AKI group . Patients with AKI were more likely to be admitted to critical care  and more likely to be ventilated once there . They were also more likely to die, both in the critical care unit  and overall .For the whole study population, AKI is an independent risk factor for mortality with an HR 1.48  and it remains significant after adjusting for multiple variables with a HR 1.82 . Figs For patients admitted to critical care, AKI is a statistically significant risk factor for death with a HR 2.05  and remains statistically significant at multivariable regression analysis with a HR 2.89 . Figs In the group of 100 AKI patients who survived, 82% (n = 82) of the patients recovered their kidney function, and only two patients remained dialysis dependent. Recovery was based on a creatinine level up to 50% above their baseline . The meaThere was no difference in overall mortality from either the initial stage of AKI or the peak AKI stage; HR 0.96, 95% CI 0.76\u20131.20, p-value 0.707 and HR 0.91, 95% CI 0.74\u20131.13, p-value 0.406 respectively. Our study found a high incidence of AKI in patients hospitalised with COVID-19 infection (20.3%), with the mortality in patients with AKI practically double that of patients without AKI (52.4% versus 26.3%). When the current COVID-19 pandemic emerged, initial reports suggested a respiratory limited condition with little multi-organ involvement. As the disease has progressed around the world the picture is evolving, and it is now evident that the disease results in extra-respiratory organ involvement, with significant prothrombotic disorders, neurological problems and cytokine storm patterns, as well as AKI, and all of these can have a dramatic impact on patient outcomes , 30\u201332.We have analysed the outcomes of all patients admitted to a large teaching hospital in a diverse city in the UK. The patient cohort, although majority white, has a significant proportion of minority ethnic groups and a diverse cross section of social deprivation making it an ideal environment to study the disease across a range of demographics. ICNARC data showed greater than half of our ICU admissions came from social deprivation index scale 4 and 5 .Fortunately, the vast majority of patients who contract COVID-19 appear to have a minor illness with little or no sequelae. Despite this, due to the vast numbers worldwide and its seeming predisposition in older, more comorbid patient groups, this has resulted in a significant cohort requiring hospitalisation which is placing a high burden on healthcare systems worldwide .Many previous studies, including from our trust , have deIn those hospitalised, the disease has a high mortality and morbidity as in our cohort of more than one thousand patients. Of the 1032 patients, 31.6% died following the diagnosis of COVID-19, compared to 14% deaths in all sectors across the UK as of 31st May 2020.What is apparent from our study is the dramatic effect having an acute kidney injury has on the patients\u2019 disease progression. In almost every metric studied, AKI results in significantly worse outcomes. Patients with AKI in association with COVID-19 are more likely to die (52.4% vs 26.3%) and more likely to be admitted to ICU (34.8% vs 11.2%). Once in ICU they were more likely to die in ICU (52.1% vs 22.8%), to die at any point following admission (54.8% vs 25.0%) and to require mechanical ventilation (86.3% vs 66.3%). Even adjusting for all other parameters, AKI remains a significant factor of overall mortality  and following admission to ICU . The mortality rates of ICU patients with AKI and COVID 19 are similar to those found in other European centres , 35.Conversely, overall mortality in our cohort with AKI (52.4%) is higher than in that described by Hirsch et al. in New York City (36.6%) and in Wuhan, China (33.7% in patients with raised baseline creatinine) , 16. WhiThere were a number of independent baseline characteristic risk factors associated with the development of AKI such as being male or black and being on RAAS inhibition similar to findings by Hirsch et al.  but they did not find these drugs were related to greater AKI risk. Patients also had a higher serum sodium and haemoglobin, higher urea and creatinine, and a higher CRP, along with lower blood oxygen saturations and temperature. Once adjusted for all parameters, being male, having a higher baseline serum sodium and CRP as well as a previous diagnosis of Renal disease were all still significantly associated with AKI but RAAS inhibition was not.Despite the poorer outlook for patients with AKI in the context of COVID-19 in regards to mortality, it seems if a patient survives they do appear to have a much higher chance of recovering their renal function (82%) compared to the 57% recovery in a study by Chan et al. . This maSimilar to previous reports, the majority of our patients were found to have AKI on admission or within a day of diagnosis , with feGiven the novel nature of the disease, the exact pathophysiology of AKI in COVID-19 has not been elicited fully as yet. It is, however, highly likely to be a multifactorial process involving any combination of, and not limited to, sepsis, nephrotoxins, hypovolaemia and hypotension, along with a background of renal disease. This is along with emerging data for more direct renal damage from endothelial injury, thrombotic events and glomerulopathy , 38\u201342. There are a number of limitations associated with this study in that it is a retrospective study with all the inherent bias associated with this. It has not been possible to determine the cause of death for patients aside from that patients died whilst being treated for COVID-19. It has also not been possible to say that AKI in of itself was a direct cause of mortality. It may be that the existence of AKI in a patient with COVID-19 is a surrogate marker for the severity of disease, as is already accepted in other diseases such as sepsis. The fact that baseline biochemistry such as CRP, and vital signs such as temperature, respiration rate and oxygen saturations were worse in patients with AKI does add some credence to this. Renal recovery was assessed based on serum creatinine. In the cohort of patients showing serological renal recovery there may be a subset who have still taken a significant \u201crenal hit\u201d as lower creatinine levels occur in patients recovering from an acute and/or prolonged illness. This is in part due to a loss of muscle bulk and poor dietary intake. We could not assess this which may then overestimate the true renal function. Given this was a retrospective study, there were also multiple parameters that were unable to be studied due to the amount of missing data, including weight and body mass index, along with biochemistry such as Ferritin, Troponin T, Lactate Dehydrogenase, D-Dimer, Procalcitonin, Creatinine Kinase, pro-B-type natriuretic peptide, and urinalysis.Despite the limitations, this study, across a diverse large population in the UK, shows that the development of AKI, no matter how severe, is associated with dramatically worse outcomes for patients in terms of mortality and the requirement of critical care admission and organ support. Patients presenting with AKI and those at risk of AKI, with suspected COVID-19 should be closely monitored given the risk of deterioration and death. With the findings of this study future strategies to deal with COVID-19 should include systems and processes for rapid detection, prevention and proactively management acute kidney injury. Future prospectively designed studies on COVID-19 should consider management strategies that minimise the potential for AKI.S1 TableAKI\u2013Acute Kidney Injury; BAME\u2013Black, Asian and Minority Ethnic; RAASi\u2013Renin-angiotensin-aldosterone-system inhibitors; IMD\u2013Index of multiple deprivation; CCI\u2013Charlson Comorbidity index; COPD\u2013Chronic Obstructive Pulmonary Disease.(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 File(PDF)Click here for additional data file."}
+{"text": "Acute kidney injury (AKI) is expected to occur commonly in patients with chronic hepatitis C. In addition, AKI may affect the survival of patients with chronic hepatitis C. However, few studies are available on this topic. We aimed to evaluate the incidence of AKI in patients with chronic hepatitis C and investigate the factors related to overall mortality.Between January 2005 and December 2018, 1252 patients with chronic hepatitis C virus (HCV) infection, defined as persistent HCV RNA for at least 6\u2009months, were retrospectively enrolled at two centers. Of them, 1008, 123, and 121 patients had chronic hepatitis (CH), compensated cirrhosis (Com-LC), and decompensated cirrhosis (Decom-LC) or hepatocellular carcinoma (HCC) at entry, respectively. Factors associated with AKI and overall mortality were evaluated using the Cox proportional regression model. The Kaplan-Meier survival curves for the development of AKI and overall mortality were generated.P\u2009<\u20090.001). In multivariate Cox regression analysis, AKI  remained an independent risk factor for overall mortality.Over a mean follow-up period of 5.2\u2009years, 285 patients developed AKI, with an incidence rate of 4.35 per 100 person-years. The incidence of AKI increased gradually with progression of chronic hepatitis C: CH (3.32 per 100 person-years), Com-LC (5.86 per 100 person-years), and Decom-LC or HCC (17.28 per 100 person-years). The patients without AKI showed better survival rates at 14\u2009years than the patients with AKI (94.2% vs. 26.3%, AKI is common in patients with chronic HCV infection and is associated with significant overall mortality. Therefore, clinicians should carefully monitor the occurrence of AKI, which is an important predictor of mortality in patients with chronic hepatitis C.The online version contains supplementary material available at 10.1186/s12879-021-05991-2. Renal impairment occurs in approximately 20\u201350% of hospitalized patients with cirrhosis \u20134. AcuteThe presence of hepatitis C virus (HCV) infection increases not only the risk of AKI development, but also of chronic kidney disease development and prognosis , 11. AKIWe established a retrospective, longitudinal HCV cohort study in Korea to investigate the incidence of AKI in patients with chronic hepatitis C. The aims of the study were to elucidate the incidence of AKI in patients with chronic hepatitis C according to the natural course of liver disease and to investigate the factors related to overall mortality.n\u2009=\u2009390); (2) prior kidney transplant or end-stage renal disease (n\u2009=\u200923); (3) <\u200918\u2009years of age (n\u2009=\u20092); (4) seropositivity for hepatitis B virus surface antigen (n\u2009=\u200970); or (5) seropositivity for the human immunodeficiency virus (HIV) (n\u2009=\u20096). The remaining 1252 patients with chronic hepatitis C infection were finally included in the analysis. The laboratory test results and comorbidities including diabetes and hypertension were extracted from the electronic medical records. The patients\u2019 medical histories were invesgated to identify age, gender, alcohol consumption, and antiviral therapy. This study was approved by the Institutional Review Boards of two centers, Gyeongsang National University Changwon Hospital and Gyeongsang National University Hospital.Between January 2005 and December 2018, a retrospective cohort of the natural history of liver disease progression was established in 1743 consecutive patients. The patients had a detectable HCV RNA of chronic hepatitis C diagnosed at two centers. Patients with at least two or more creatinine measurements obtained more than 3\u2009months apart were included. Of these patients, 491 met any of the following exclusion criteria: (1) a follow-up period of less than 6\u2009months without mortality  and direct-acting agents (since 2014) was administered to patients in whom it was indicated according to the clinical decisions of the treating physicians. In our study, sustained virologic response (SVR) was defined as a serum HCV RNA test below limit of detection performed at least 12\u2009weeks after the end of treatment.Liver cirrhosis was determined as (1) presence of clinical sign of portal hypertension manifested as ascites, varices, or hepatic encephalopathy or (2) compatible imaging findings accompanied by thrombocytopenia . Hepatic decompensation was defined by the presence of hepatic encephalopathy, ascites, hepatorenal syndrome, or documented gastro-esophageal variceal bleeding. Chronic hepatitis C infection was classified as (1) CH, (2) compensated cirrhosis (Com-LC), and (3) Decom-LC or HCC according to liver disease progression.AKI was defined according to the International Club of Ascites (ICA)-AKI criteria for patients with cirrhosis and to the Kidney Disease Improving Global Outcomes criteria for patients with CH , 14. It The AKI stages for severity were classified as follows:  AKI stagSubjects with AKI were classified into 4 types according to the cause of kidney impairment: (1) pre-renal AKI, (2) hepatorenal syndrome (HRS)-AKI, (3) intrinsic-renal AKI, and (4) post-renal AKI. The diagnostic criteria of HRS-type AKI were defined using the ICA criteria as previously reported .U test was performed to analyze quantitative variables. Fisher\u2019s exact and Pearson\u2019s chi-square tests were conducted to analyze qualitative data. The incidence rate of AKI per 100 person-years was calculated by dividing the number of newly developed AKI event cases by the person-years of follow-up. In our study, the time to event was calculated from the date of enrollment to the study to the date of AKI occurrence, death, or last observation or to December 31, 2019, in each diagnosis group at entry. Survival curves for the development of AKI and overall mortality were calculated using the Kaplan-Meier method and compared with log-rank test. Univariate and multivariate analyses were conducted using the Cox proportional regression model to identify potential factors associated with AKI and overall mortality. To assess the risk factors associated with AKI, variables including age, gender, presence of hypertension and diabetes, alcohol consumption, HCV genotype, diagnosis at entry , extrahepatic malignancy, SVR, HCV RNA, albumin, bilirubin, platelet, INR, Child-Pugh score, and MELD score were used in Cox univariate regression. Moreover, to assess the risk factors associated with overall mortality, Cox univariate analysis was performed by adding the presence of AKI to the existing variables for AKI. The significant variables (P\u2009<\u20090.05) by univariate analysis were subjected to Cox multivariate analysis. The risk was expressed as hazard ratio (HR) and 95% confidence interval (CI). A two-sided P value of <\u20090.05 was considered statistically significant for all analyses.Statistical analyses were performed using PASW software . The Mann-Whitney A total of 1252 patients with chronic hepatitis C were followed up for a mean of 5.2\u2009years. Baseline characteristics are shown in Table\u00a0Of the 1252 patients, 285 (22.8%) developed AKI. Patients with AKI were older than those without AKI. The proportions of male sex, hypertension, diabetes, and extrahepatic malignancy were higher in patients with AKI than in those without AKI. The risk of AKI incidence gradually increased as chronic hepatitis progressed to advanced liver disease.A total of 149 (52.3%), 80 (28.1%), and 56 (19.6%) of the 285 patients had initial AKI stage 1, stage 2, and stage 3, respectively. During hospitalization, 121 (42.5%) patients had a peak AKI stage 1, 76 (26.7%) patients had a peak AKI stage 2, and 88 (30.9%) patients had a peak AKI stage 3. A resolution of AKI occurred in 77.7, 53.9, and 31.8% of the patients with peak AKI stages 1, 2, and 3, respectively  pre-renal AKI in 127 (44.6%) patients, (2) HRS-AKI in 44 (15.4%) patients, (3) intrinsic-renal AKI in 108 (37.9%) patients, and (4) post-renal AKI in 6 (2.1%) patients.P\u2009<\u20090.001) and with Com-LC  than in patients with CH  , age , hypertension , diabetes mellitus , platelet count , Model For End-Stage Liver Disease (MELD) score at entry , Child-Pugh score at entry , SVR , extrahepatic malignancy , Com-LC at entry , and Decom-LC or HCC at entry . In the final multivariate analysis for the development of AKI, the risk factors included age , hypertension , diabetes mellitus , MELD score at entry , Child-Pugh score at entry , SVR , extrahepatic malignancy , and Decom-LC or HCC at entry  . Among the 606 patients who achieved SVR, the incidence rate of AKI in patients with SVR before enrollment, patients treated with interferon and ribavirin, and patients treated with direct-acting agents was 7.7, 8.9, and 7.3%, respectively.In our study, among the 400 patients treated with IFN, 10 (2.5%) developed AKI, and of the 347 patients treated with DAA, 7 (2.0%) developed AKI. Therefore, only a small number of patients who received antiviral therapy developed AKI. However, patients who achieved SVR through antiviral therapy had reduced risks of AKI. Of the 748 patients who started antiviral treatment after enrollment, 400 patients received pegylated interferon alpha and ribavirin treatment, with 291 patients (72.8%) achieving SVR, and 329 patients received direct-acting agents, with 302 patients (91.8%) achieving SVR . In addition, 13 patients achieved SVR before study enrollment (Supp Table n\u2009=\u200957), HCC (n\u2009=\u200925), extrahepatic malignancy (n\u2009=\u200923), sepsis (n\u2009=\u200923), and others (n\u2009=\u20099). The cumulative survival rate at 14\u2009years was higher for patients without AKI (94.2%) than for patients with AKI  . The causes of mortality in patients with chronic hepatitis C were hepatic failure (P\u2009=\u20090.007), age , platelet count , MELD score at entry , Child-Pugh score at entry , SVR , extrahepatic malignancy , Com-LC at entry , Decom-LC or HCC at entry , and AKI . The factors associated with overall mortality in the final multivariate analysis included SVR , extrahepatic malignancy , Decom-LC or HCC at entry , and AKI  , intrinsic-renal AKI (P\u2009<\u20090.001), and postrenal-AKI (P\u2009<\u20090.001)  and AKI stage 3 (P\u2009<\u20090.001) . Patients without AKI had a better survival rate than patients with AKI. In multivariate analysis, AKI remained an independent risk factor for overall mortality (HR 6.66).Few data are available on the incidence of AKI in patients with chronic hepatitis C ; previouWe found an increasing incidence of AKI according to chronic HCV infection progression from CH to Com-LC, Decom-LC, or HCC Figs.\u00a0 and 2. TThe most common causes of AKI were prerenal-AKI (44.6%), followed by intrinsic-AKI (37.9%), HRS-AKI 15.4%), and postrenal-AKI (2.1%). Mechanisms that explain the development of AKI in chronic hepatitis C include factors such as chronic impairment of kidney, poor liver function, ascites, low arterial pressure, low serum sodium, shock, high leukocyte, and use of antiviral agent 5.4%, and, 18. We We identified that AKI was also associated with a significant increase in the overall mortality. The peculiar result of our study was the approximately 5.6-fold increase (HR 6.66) in the risk of overall mortality in chronic HCV-infected patients with AKI compared with that in patients without AKI. A previous study by Nadkarni et al. using a nationally representative database reported that dialysis-requiring AKI is associated with a significant increase in in-hospital mortality among HCV-positive adults . HoweverThis study has a few limitations. First, its retrospective design may limit the collection of factors related to AKI and mortality. Second, the irregular timing of laboratory assessment in each patient may cause missing AKI not found in this analysis . Despite these limitations, the strength of this study is that it is the largest study about AKI in patients with chronic HCV infection to date and that it showed the effect of AKI on mortality. Moreover, our study describes renal outcome in patients with chronic HCV infection in both outpatient and inpatient settings.Based on the findings of this large longitudinal cohort study, we found that the occurrence of AKI has a significant effect on mortality in the natural history of patients with chronic HCV infection. Our observation suggests that AKI events are common in the natural history of chronic hepatitis C, and that those with AKI should be monitored closely.Additional file 1: Supp. Table 1. Treatment outcome (n=1252).Additional file 2: Supp. Figure 1. Course of acute kidney injury (n=285).Additional file 3: Supp. Figure 2. Overall survival according to etiologies of acute kidney injury (n=285).Additional file 4: Supp. Figure 3. Overall survival according to stage of acute kidney injury (n\u2009=\u2009285)."}
+{"text": "The World Health Organization (WHO) has named the virus as 2019 novel coronavirus on January 12, 2020, and has declared a public health emergency globally on January 30, 2020. The epidemic started in Wuhan, China, in December of 2019 and quickly spread to over 200 countries. COVID-19 can cause multiple organ injuries . Among them, acute kidney injury (AKI) is a critical complication due to its high incidence and mortality rate. So, it is essential to evaluate AKI in COVID-19 patients during this pandemic state. The aim of this work is to detect the occurrence of AKI in hospitalized COVID-19 patients. So, a retrospective study was conducted on hospitalized adult patients >\u200918\u2009years old with confirmed SARS-CoV-2 infection admitted to the Abo Teeg Hospital at Assiut City, Egypt, from May 1, 2020, to July 1, 2020. All data were collected from medical records, patients\u2019 follow-up, and charts. Data were verified, coded by the researcher, and analyzed using IBM-SPSS 21.0.Eighty-six COVID-19 patients were admitted to Abo Teeg Hospital in Assiut City, Egypt, between May and July 2020. Thirty-eight patients (33%) were of the male gender. Mean age was 58.07 \u00b1 17.9, and 61 patients developed AKI. 32.8% of the AKI group were a stage I severity (increase in serum creatinine by 0.3\u2009mg/dl within 48\u2009h), 21.3% of them presented by stage II (2\u20132.9 times increase in serum creatinine), and 45.9% were in stage III (3 times or more increase in serum creatinine). The overall hospital mortality for the patients admitted to ICU with AKI was 6.7% (11/61), compared to 1% (4/25) in those without AKI.Hospitalized patients with COVID-19 had a higher risk of AKI, and we recommended that those patients should be evaluated after discharge for the development of CKD. There was no determination on the animal species carrying the nCoV. The preliminary studies showed that the nCoV is closely related to the coronavirus isolated from bats, postulating the theory of possible transmission from bats to humans [The 2019 novel coronavirus disease (COVID-19) is a newly defined serious infectious disease caused by the SARS-CoV-2 virus. The World Health Organization (WHO) has named the virus as 2019 novel coronavirus (2019-nCoV) on January 12, 2020, and has declared a public health emergency globally on January 30, 2020 . Among them, acute kidney injury (AKI) is a critical complication due to its high incidence and mortality rate . AKI in A retrospective observational study was conducted on adult patients admitted to the Abo Teeg Hospital, Assiut, Egypt, with laboratory and radiologically confirmed SARS-CoV-2 infection between May 1, 2020, and July 1, 2020, under the supervision of our medical staff of Al-Azhar University Hospital, Assiut Branch. Patients with known end-stage kidney disease (ESKD) before admission and patients who were hospitalized for <\u200948\u2009h were excluded.2 with MDRD  equation in patients without known chronic kidney disease [KDIGO  criteria were used to define AKI according to both urinary output and serum creatinine as follows: stage I, an increase in serum creatinine by 0.3\u2009mg/dl within 48\u2009h or 1.5\u20131.9 times increase in serum creatinine from baseline or urinary output <\u20090.5\u2009ml/kg/h for 6\u201312\u2009h within 7\u2009days; stage II, 2.9 times increase in serum creatinine or urinary output <\u20090.5\u2009ml/kg/h for \u2265 12\u2009h within 7\u2009days; and stage III, 3 times or more increase in serum creatinine or to \u2265 4.0\u2009mg/dl or urinary output <\u20090.3\u2009ml/kg/h for \u2265 24\u2009h or anuria for \u2265 12\u2009h within 7\u2009days. Baseline creatinine was defined as the best value in the 3 preceding months, if unavailable as the lowest value during the hospital stay or was back-calculated based on a glomerular filtration rate of 60\u2009ml/min/1.73 mAll data were collected from medical records, patients\u2019 follow-up, and charts. Baseline patients\u2019 characteristics were collected Table , includit test analysis was carried out to compare the means of normally distributed data, while the Mann-Whitney U test was calculated to test the median differences of the data that do not follow a normal distribution. The Kaplan-Meier curve was used to estimate the median survival time. The log-rank test was used to compare survival curves between the categories of the explanatory variables. A significant P value was considered when it is less than 0.05 [Data were verified, coded by the researcher, and analyzed using IBM-SPSS 21.0 . For descriptive statistics, means, standard deviations, medians, and ranges were calculated. For the test of significances, chi-square/Fisher\u2019s exact test was calculated to compare the frequencies among groups. For continuous variables, independent han 0.05 .n = 36, 41.9%), diabetes mellitus , and chronic kidney disease  were observed, in addition to ischemic heart disease  and chronic liver disease . As regards vital signs, the AKI group had a lower PO2 (80.5 (28\u2013224)) and SO2 (95 (46\u2013100)) than the non-AKI group with no significant differences as regards blood pressure, blood PH, and PCO2  were of the male gender. 8.2% of those patients developed acute COVID-19 symptoms (8% in the non-AKI group), 37.7% developed gradual onset (36% in the non-AKI group), and lastly 45.1% developed symptoms suddenly (56% in the non-AKI group). Within 7\u2009days of admission, 61 patients developed AKI and meet the KDIGO criteria. The AKI group had a higher grade of fever than the non-AKI group with no significant differences in other symptoms such as cough, dyspnea, and loss of smell , compared to 1% 4/25) in those without AKI. Survival probability is lower in patients with AKI. The Kaplan-Meier survival curves for patients with and without AKI were shown\u00a0in Fig. /25 in th/61, compThe present study has limitations. First, because this is a retrospective and observational study, we cannot make a causal relationship between exposures and AKI where renal biopsy may be needed to prove the direct link between AKI and COVID-19 and explain such role of comorbidities. Second, we cannot generalize our findings in the outpatient AKI settings because non-hospitalized patients were not part of the present study.This study concluded that acute kidney injury is a serious feature in critically ill hospitalized patients with COVID-19. It is more common in patients with comorbidities such as hypertension and diabetes in addition to the development of AKI which is associated with increased severity of illness, prolonged duration of hospitalization, and increased mortality. So, we recommended that hospitalized patients with COVID-19 should be evaluated for development of acute renal injury and should be regularly tested for renal functions after discharge to monitor those patients for the progression to CKD."}
+{"text": "Background: Acute kidney injury (AKI) complicating cardiogenic shock is associated with increased mortality. We hypothesize that renal replacement therapy (RRT)\u00a0improves survival in cardiogenic shock supported by Impella-CP  complicated by AKI.Methods: A retrospective chart review identified 34 patients on Impella-CP for cardiogenic shock between January 2015 and December 2017. AKI was defined as an increase in serum creatinine\u22650.3 mg/dL from baseline. Three groups were analyzed: AKI plus RRT, AKI minus RRT, and no AKI. Pre-existing dialysis patients were excluded. The only indication for RRT was AKI not responding to diuretics. Thirty-day mortality was analyzed.Results: There were 13 patients with no AKI, 9 with AKI plus RRT groups, and 12 with AKI minus RRT. Thirty-day mortality was similar between no AKI and AKI plus RRT groups  2.25 ]. Thirty-day mortality was higher in AKI minus RRT group compared to the no AKI group .Conclusion: In cardiogenic shock patients on Impella-CP, AKI minus RRT is associated with a higher 30-day mortality compared to patients without AKI and/or patients with AKI plus RRT. Short-term mortality may improve in cardiogenic shock patients with AKI who are treated with RRT. Despite advances in technology for the treatment of cardiogenic shock, mortality has not dramatically improved .\u00a0CardiogOur cohort was a single-center retrospective study including all patients on Impella-CP for cardiogenic shock admitted to Albany Medical Center between January 2015 and December 2017. Data were obtained by a retrospective review of the electronic medical record. Cardiogenic shock was defined as elevated serum lactate (>2.0 mmol/L) and hypotension requiring inotrope/vasopressors to maintain a mean arterial blood pressure above 65 mmHg. Eligible patients were classified based on AKI at presentation (increase in serum creatinine>0.3 mg/dL from baseline); those with AKI were further categorized by RRT. This led to three groups: no AKI, AKI minus RRT, and AKI plus RRT. RRT included hemodialysis or continuous RRT.The exclusion criteria included pre-existing hemodialysis-dependent patients, unknown baseline renal function, or patients lost to follow-up within 30 days. The chi-square test was utilized to compare 30-day mortality of AKI plus RRT and no AKI groups as well as AKI minus RRT and no AKI groups. Continuous variables (lab parameters at presentation) were compared between groups using ANOVA. Type 1 error was prespecified at less than or equal to 5%. The protocol was approved by the Institutional Review Board at Albany Medical Center.Between January 2015 and December 2017, 34 patients with cardiogenic shock on Impella-CP met the inclusion criteria for our study. There were 13 patients with no AKI, 9 had AKI plus RRT, and 12 had AKI minus RRT. The only indication for RRT was AKI not responding to diuretics (oliguria or anuria). Baseline characteristics and lab parameters at presentation are included in Tables AKI was associated with higher 30-day mortality compared to patients with no AKI in our cardiogenic shock cohort  6.16 ]. Thirty-day mortality was similar in patients with no AKI and AKI plus RRT . Patients with AKI minus RRT had a higher 30-day mortality than patients with no AKI . Outpatient use of calcium channel blockers and hemoglobin greater than 11 g/dL were associated with decreased likelihood of AKI at presentation (Table 1/21 vs. AKI in severe system illness and particularly cardiogenic shock is associated with poor outcomes ,8.\u00a0AKI iOur single-center retrospective study evaluated outcomes of cardiogenic shock in patients on Impella-CP complicated by AKI with and without RRT.\u00a0We found that patients with AKI had a similar 30-day mortality when receiving RRT compared to patients without AKI at presentation.It seems intuitive that early recognition of AKI in cardiogenic shock and aggressive treatment with RRT may improve outcomes, but data are conflicting. Aside from cardiogenic shock populations, the Impella-CP device is shown to be protective against AKI in patients undergoing high-risk\u00a0percutaneous coronary intervention\u00a0. In a siThe limitations of our study are acknowledged: small sample size, retrospective examination, single-center review, and wide confidence intervals.AKI is an independent predictor of mortality in cardiogenic shock. Based on our single-center, retrospective comparison of cardiogenic shock supported by Impella-CP, 30-day mortality was similar between patients with no AKI and those who received RRT for AKI. Early initiation of RRT for AKI coupled with improved left ventricular unloading with Impella-CP may offset the detrimental effects of AKI in cardiogenic shock. Our study provides preliminary evidence to warrant further investigation on early RRT for AKI in cardiogenic patients supported with Impella-CP."}
+{"text": "Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the associated Coronavirus Disease 2019 (COVID-19) is a public health emergency. Acute kidney injury (AKI) is a common complication in hospitalized patients with COVID-19 although mechanisms underlying AKI are yet unclear. There may be a direct effect of SARS-CoV-2 virus on the kidney; however, there is currently no data linking SARS-CoV-2 viral load (VL) to AKI. We explored the association of SARS-CoV-2 VL at admission to AKI in a large diverse cohort of hospitalized patients with COVID-19.th and May 19th, 2020 with SARS-CoV-2 in a large academic healthcare system in New York City  with available VL at admission quantified by real-time RT-PCR. We extracted clinical and outcome data from our institutional electronic health records (EHRs). AKI was defined by KDIGO guidelines. We fit a Fine-Gray competing risks model (with death as a competing risk) using demographics, comorbidities, admission severity scores, and log10 transformed VL as covariates and generated adjusted hazard ratios (aHR) and 95% Confidence Intervals (CIs). VL was associated with an increased risk of AKI  with a 4% increased hazard for each log10 VL change. Patients with a viral load in the top 50th percentile had an increased adjusted hazard of 1.27  for AKI as compared to those in the bottom 50th percentile.We included patients hospitalized between March 13VL is weakly but significantly associated with in-hospital AKI after adjusting for confounders. This may indicate the role of VL in COVID-19 associated AKI. This data may inform future studies to discover the mechanistic basis of COVID-19 associated AKI. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of patients worldwide . SARS-CoAcute kidney injury (AKI) is now emerging as a common complication in hospitalized patients with COVID-19. Up to 40% of hospitalized patients with COVID-19 develop AKI, and it is linked to substantially higher morbidity and mortality , 6. In aSeveral mechanisms, including direct viral kidney invasion and cytokine storm have been linked to increased SARS-CoV-2 viral load (VL) phenomenon; however, there is currently no consensus . We haveWe included patients hospitalized between March 13th 2020 and May 19th 2020 with SARS-CoV-2 confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) of nasopharyngeal swab samples measured by the Roche cobas\u00ae 6800. Viral load of patients who tested positive was quantified using an assay developed by the Mount Sinai Health System clinical laboratory, All patients were admitted from five hospitals in the Mount Sinai Health System in New York City.th 2020. We extracted this data from the EPIC CLARITY database using custom SQL code. Our EHR database consists of several tables that replicate the backend of our clinical EHR server. Briefly these tables include diagnoses, medication administration, problem list, patient demographics, and procedure codes. All data exists in structured fields with the exception of clinical notes. Individual terms from clinical notes were mapped to SNOMED concepts using a natural language processing engine. History of clinical comorbidities were determined using ICD-9/10 diagnostic codes  with outcomes determined on or before the data freeze date of June 5ic codes . As per ous work . If patious work . SpecifiRealtime RT-PCR was performed with the Quantifast Pathogen RT-PCR kit (Qiagen) in a Lightcycler 480 II (Roche) using the N2 primer from the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel. All extraction and RT-PCR were performed in a CLIA certified laboratory in the Mount Sinai Health System. The limit of detection, defined as the concentration at which at least 95% of replicates test positive, was determined to be 5 genome copies/mL. Viral load was quantified using the delta Ct method by plotting a standard curve fitting a linear regression model with Ct values for known concentrations of viral RNA.Details of statistical analysis and results are provided in a STROBE checklist .10 transformed VL to minimize skewness. Time-to-AKI was determined as the difference between time of AKI onset and VL measurement. Individuals who remained hospitalized at the time of data freeze were censored. We fit a Fine Gray competing risks model [10 transformed viral load as covariates. We performed a time-to-AKI analysis using a competing risks model where death was treated as a competing risk since individuals who died in their hospital course may have died of causes unrelated to AKI. Since these individuals might have developed AKI if they had not died, including death as a competing risk accounts for this group of patients. We associated viral load with maximal AKI stage in a multivariate regression model. All statistical analysis was performed using R version 3.6.3 [We logks model  with ageon 3.6.3 . For uniThis research has been approved by the Institutional Review Board (IRB) of the Icahn School of Medicine at Mount Sinai. All data were patient level data and not anonymized. The IRB waived requirement for informed consent.10 mean VL at admission , were significantly older , more frequently Black , and had a higher prevalence of history of diabetes  and chronic kidney disease  as compared to patients who did not develop AKI  for AKI as compared to those in the bottom 50th percentile  with a 4% hazard increment for each logrcentile . We thenWe then performed a sensitivity analysis by adjusting for inflammatory markers at the time of admission in addition to the above covariates. Individuals who developed AKI had a significantly greater C-reactive protein, procalcitonin, D-dimer, and ferritin and significantly lower lymphocyte count than controls . After aIn a large, diverse group of patients with COVID-19, we report a significant adjusted association between admission VL and risk of in-hospital AKI. The association between VL and AKI was weak but significant and persisted after adjustment for both chronic comorbidities and admission severity of illness.Although AKI has emerged as a common complication during the hospital course of COVID-19 , 6, 8, tWe add to this literature by showing that admission VL independently associates with an increased risk of AKI in a competing risks framework. Importantly, we adjusted our analysis for the history of CKD since individuals with CKD may both have increased VL and be more predisposed to AKI. Since it is unknown whether individuals who died of other causes may have developed AKI if they had not died, it is necessary to account for death as an alternate outcome that precludes an individual from ever developing AKI. To account for this, we utilized a competing risks model with death as a competing risk. In this manner, our analysis partially accounts for the potential confounding of non-AKI related death.Higher VL at admission may represent more severe disease and a strong proinflammatory state. However, we find that even after adjusting for inflammatory markers at the time of admission, VL is still significantly associated with risk of AKI. Thus, although an inflammatory milieu may contribute to AKI, VL has an independent role in driving AKI through an alternative mechanism.ACE2 receptor expression in the kidney, allowing for greater viral renal tropism. Recent work has shown that VL correlates with lung disease severity [ACE2 receptor expression, viral load may be an indicator for renal tropism. However, correlation with autopsy specimens is required to investigate this hypothesis. Finally, it is possible that high VL leads to a cytokine release syndrome, contributing to organ injury [Our work sheds light into the role of VL at admission and the risk of AKI. Recent work suggests that individuals with high VL at admission have an increased risk of death . Our resseverity , likely n injury . These hOur work should be interpreted in the light of certain limitations. First, VL may be influenced by certain medications such as corticosteroids administered before hospitalization. Second, although we adjusted for potential confounding, there may be unmeasured confounders that may have influenced our results. Third, since individuals presented to the hospital at different timepoints in their disease course, the VL measurements included in our study may or may not represent the peak VL for each individual and do not represent the VL at time of initial infection. Each patient\u2019s true peak VL may correlate better with degree of kidney injury. Additionally, after resolution of AKI, whether VL decreases as viral particles are cleared remains an unanswered question. Large prospective studies with serial measurement of VL to determine peak VL are needed. Our cohort was selected to include all patients who were hospitalized with a positive COVID-19 test without strict inclusion and exclusion criteria to limit selection bias. However, since our cohort only included patients from New York City where resources were limited during the peak of the COVID pandemic, these results may not be generalizable and will need to be validated in other cohorts. Additionally, controls in our dataset were defined as all individuals who did not develop AKI and thus may have had other comorbidities or hospital complications.In summary, we demonstrate an independent, significant association with admission VL and in-hospital AKI. This work may open new avenues to understanding the pathophysiological and mechanistic basis of COVID-19 associated AKI.S1 TableIndividual diagnosis codes were extracted from each patient\u2019s electronic health record. Patients were identified as having a specific comorbidity if they had at least one of the corresponding diagnosis codes in their record.(CSV)Click here for additional data file.S2 Table(DOCX)Click here for additional data file."}
+{"text": "IntroductionCoronavirus disease 2019 (COVID-19) primarily affects the pulmonary system and presents itself as shortness of breath, fever, and cough. However, it may affect other systems as well, including the nervous system. This study aimed to determine the frequency of neurological symptoms in COVID-19 patients and its association with the severity of the disease.MethodsThis cross-sectional study was conducted at a public sector tertiary care teaching hospital in Karachi, Pakistan, from April to July 2020. All patients with positive polymerase chain reaction (PCR) tests were included, except those with pre-existing neurological and psychiatric conditions.ResultsThe most common neurological symptom was dizziness (17.5%), followed by headache (15.7%). Three (2.6%) patients had a stroke. Nine (7.8%) participants had a taste impairment, and another nine (7.8%) had a smell impairment. There was no significant difference in the frequency of neurological symptoms when severe and non-severe disease was compared.ConclusionNeurological symptoms are frequent in COVID-19. Care should be taken to identify them early. COVID-19 should be suspected in patients presenting with neurological abnormalities and should be included in the differential diagnosis to prevent further virus transmission. Coronavirus disease 2019 (COVID-19) is an infectious disease caused by a new strain of coronavirus; its first case was reported\u00a0in December 2019 in Wuhan, China ,4. Mao eAngiotensin-converting enzyme 2 (ACE2) was identified as the functional receptor for\u00a0severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in January 2020. ACE2 receptors are present in various organs, including the nervous system . The expThis study will try to determine the frequency of neurological manifestations in COVID-19 and its association with COVID-19 disease severity. The study aims to help in the diagnosis of\u00a0COVID-19 patients presenting with non-conventional symptoms.This cross-sectional study was conducted at a public sector tertiary care teaching hospital in Karachi, Pakistan, from April to July 2020. All patients with positive oropharyngeal or nasopharyngeal swabs polymerase chain reaction (PCR) tests for SARS-CoV-2 were included. Patients with prior neurological and psychiatric diseases were excluded from the study. The hospital's ethics committee approved the study protocol, and all patients provided oral informed consent.A self-structured survey was designed for COVID-19 patients, which recorded various neurological manifestations such as headache, altered sense of smell, altered sense of taste, nausea, dizziness, seizures, cerebrovascular event, and skeletal muscle injury. Acute cerebrovascular disease was diagnosed with the help of clinical symptoms and head CT. Clinical symptoms were used to diagnose seizures. Skeletal muscle injury was diagnosed with the help of laboratory values (serum kinase level greater than 200 U/L) and skeletal muscle pain . PakistaData were processed and analyzed using SPSS Statistics for Windows, version 22.0 . Quantitative variables were summarized as mean and standard deviation (SD), and qualitative variables as frequency and percentage. Qualitative variables were cross-tabulated with disease severity and compared with chi-square tests. A p-value of less than 0.05 indicated that the difference in neurological manifestation between mild to moderate and severe disease was significant enough to discard the null hypothesis.A total of 114 patients was selected for the study. The mean age of the participants was 51 \u00b1 14 years. There were 49 (42.9%) participants who were less than 50 years of age and 65 (57.1%) participants older than 50 years. There were 52 (45.6%) females and 62 (54.4%) males. There was no significant difference in severity when patients were stratified for age and gender; 28 (24.5%) patients had neurological manifestations. The most common neurological symptom was dizziness (17.5%) followed by headache (15.7%). Nine (7.8%) participants had taste impairment, and another nine (7.8%) had a smell impairment. There was no significant difference in the frequency of neurological symptoms when severe and non-severe disease was compared  was present in three patients in our cohort. Various factors are responsible for stroke in patients with COVID-19. Viral infection of endothelial cells, which may damage blood vessels, may increase the risk of stroke. Viral infection of vascular endothelial cells accompanied by damage to the vasculature can predispose to infarct ,15. SARSIn this study, 12 10.5%) participants had skeletal muscle injury. Muscle injury has been reported by various studies ,10. Musc.5% partiIn this study, nine (7.8%) participants reported altered taste. One possible mechanism for altered taste is that salivary glands, as they have\u00a0(ACE2) receptors, may be an early target of SARS-CoV-2 infection. This infection of the salivary gland may decrease saliva flow, which may be responsible for altered taste in COVID-19 . Nine (7The present study had several limitations. Firstly, all our data were collected from one institute. Also, long-term implications of neurological symptoms were not noted, and not all tests were performed in all participants included in the study as they were self-isolated at home after the initial diagnosis.Our study, along with other studies, has shown that neurological manifestations are not uncommon in COVID-19. All patients presenting with respiratory and neurological signs and symptoms should be tested for COVID-19 to avoid delay in diagnosing the condition. A multidisciplinary approach is needed to manage\u00a0COVID-19 patients presenting with neurological symptoms.In our study, neurological manifestations among patients were not uncommon. Patients commonly reported dizziness, headache, impaired consciousness, skeletal muscle injury, and altered senses. Patients presenting with neurological manifestation should undergo testing for COVID-19 during this pandemic. Clinicians and researchers should make a joint effort to gather more data related to neurological manifestations in COVID-19 and, if deemed necessary, changes in diagnostic guidelines should be made."}
+{"text": "IntroductionAdministering intravenous thrombolytic therapy within 60 minutes on arrival in any healthcare facility is challenging, especially when done by Emergency Medicine Physicians (EMP) via telemedicine in centres without onsite neurology cover. Prior quality improvement interventions have improved median Door-to-Needle (DTN) timings in our centre; however, it still falls short of the DTN target of 60 minutes.\u00a0MethodsVarious quality improvement interventions were implemented over four months by a multi-disciplinary telestroke workgroup led by EMPs to improve DTN timings for patients presenting with acute ischaemic strokes. A retrospective observational study was conducted to review if these interventions resulted in an improvement in DTN timings while keeping the rates of stroke mimics given thrombolytic therapy, haemorrhagic conversions and 30-day mortality rates low.\u00a0ResultsA total of 279 patients were evaluated. Median DTN timings significantly improved from 71.0 minutes pre-intervention to 62.0 minutes post-intervention (p=0.012). Correspondingly, the proportion of patients with DTN \u2264 60 minutes increased from 31.7% pre-intervention to 47.0% post-intervention, giving an odds ratio of 1.91 . There were no significant differences found in the rates of stroke mimics, haemorrhagic conversions and 30-day mortality pre and post-intervention.ConclusionThe implementation of EMP led to systemic quality improvement interventions is associated with improved DTN timings without compromising clinical quality outcome measures like haemorrhagic conversion rates and 30-day mortality rates. EMPs, with a broad knowledge base and familiarity, interacting with various specialities and co-ordinating care, are uniquely suited in this role to drive change. More work in the public health sector would also have to be done to improve the population\u2019s response to acute stroke symptoms. In our current day and age, administering intravenous thrombolytic therapy for acute ischaemic stroke in eligible patients presenting to the Emergency Department (ED) in hopes of improving neurological outcomes is considered standard of care\u00a0. As per BackgroundThis study was conducted at an urban Emergency Department (ED) with an average ED daily attendance of 420 patients, in a 1,000-bed acute regional hospital without onsite neurology cover where a hub-and-spoke model of telestroke was developed. All screened eligible patients presenting with acute stroke symptoms to the ED (spoke) would undergo an immediate Computer Tomography (CT) scan of the brain before a video-assisted evaluation by off-site Neurologists at a national specialist centre (hub). This web-based video conferencing process was facilitated by using the KrixiCareTM program, which allowed a real-time audio-visual link between both centres and CT images transfer. The entire telestroke process is reliant on the Neurologist, ED staff and Radiology staff to work together efficiently and quickly to achieve the goal of a Door-to-Needle (DTN) timing of 60 minutes or less. As part of prior quality improvement initiatives, the telestroke process had been subdivided into five phases for the ease of analysis and to aid in implementing specific interventions\u00a0,11, withPrior quality improvement initiatives had seen the median DTN timings drop from an initial of 92.7min to 86min\u00a0\u00a0and a suObjectiveThe objective of this study was to evaluate if the various quality improvement interventions implemented from June to September 2016 resulted in an improvement in the DTN timings for patients presenting with acute ischaemic stroke to the ED; while at the same time, ensuring that clinical standards were maintained by keeping the rates of stroke mimics given thrombolytic therapy, haemorrhagic conversions and 30-day mortality rates low.This retrospective observational study was approved by the Centralised Institutional Review Board (CIRB) of the study institution and consent waiver was approved as all data collected were anonymised. All patients aged \u226521 years old who were administered thrombolytic therapy for an acute ischaemic stroke in the ED from January 2015 to December 2017 were included in this study. Patients who were not in the thrombolytic window, had contraindications to thrombolytic therapy, were not offered thrombolytic therapy by the neurologist or declined thrombolytic therapy were excluded from the study. Trained research assistants extracted required data from patient electronic medical records and phone operator call logs. As the series of quality improvement interventions were implemented gradually over time from June to September 2016, the Pre-intervention period would be defined as from January 2015 to August 2016 and the Post-intervention period would be from September 2016 to December 2017. However, during the study CIRB renewal in November 2017, the study consent waiver was no longer approved, resulting in excluding the data collected from November to December 2017 to comply with the new CIRB conditions. Thus, the Post-intervention period would run from September 2016 to October 2017 instead.This study\u2019s primary outcome measure was the median DTN timings, including the breakdown of timings for each phase, and the proportion of patients achieving the DTN target of 60 minutes. Secondary outcome measures include rates of intracranial haemorrhagic conversion post thrombolysis, rates of stroke mimics given thrombolytic therapy, 30-day mortality rates and Modified Rankin Score (MRS) on discharge from hospital.Statistical analysis was performed with SPSS statistical software, version 19.0 . Categorical data were presented as frequency (percentage). Numeric data were presented as mean (standard deviation) for parametric distribution and median (interquartile range) for non-parametric distribution. The differences in characteristics between pre-and post-intervention were examined using Chi-Square test or Fisher\u2019s Exact test for categorical variables, two-sample t-test or Mann Whitney U-test continuous variables, where appropriate. Odds Ratio (OR) was reported where appropriate. A two-tailed, p-value of <0.05 was considered statistically significant.Interventions implementedMonthly stroke audits, where an in-depth analysis of factors contributing to delays in each of the phases, were carried out by an EMP led multi-disciplinary workgroup to review cases from the previous month that did not meet DTN targets and new measures  as shown in Table\u00a0The intracranial haemorrhagic conversion rates post thrombolytic therapy pre-, and post-intervention remained almost similar and had a calculated odds ratio of 1.05 (95% CI 0.57 - 1.94). The numbers of stroke mimics also remained almost similar to pre-and post-intervention with a calculated odds ratio of 0.96 (95% CI 0.36 - 2.56). Both secondary outcomes were found not to be statistically significant (refer to Table\u00a0The systemic quality improvement interventions described have helped significantly reduce DTN timings, as shown in the results above. Although the ultimate goal of all eligible patients receiving thrombolytic therapy within 60 minutes was not achieved, a significant improvement in the proportion of patients achieving this goal from 31.7% to 41% was shown after the implementation of our methods. In truth, telestroke involves extra hurdles and multiple stakeholders like ED, radiology, neurology and even IT support to work closely together. Thus, it is conceivable that DTN timings would be longer than that for direct stroke activations where the in-house neurology team takes over direct management of the acute stroke patient. Other telestroke centres have also reported comparable DTN timings of 61-106 minutes\u00a0,12-15, fThe intention of dividing into five phases, as described above is to break down the large, complicated telestroke process into five small time capsules that are more easily understood and analysed. Within each of the five phases, the telestroke workgroup led by EMPs and comprising of all the vital stakeholders - EMPs and ED Nurses, Neurologists, Radiologists, Radiographers and IT support - brainstormed and came up with quality improvement interventions to smoothen the telestroke process out (refer to Figure\u00a0Comparing with other described quality improvement interventions in the literature\u00a0-18, mostLooking at the analysis of the timings obtained for the different phases, the results show a significant increase in the timings obtained for Phase 1, which describes the period starting from patient ED arrival to a stroke standby being called (refer to Figure\u00a0On the other hand, Phase 5, which describes the period from neurology offer of thrombolytic therapy to the administration of thrombolytic therapy showed the most improvement - from a median of 21.5 minutes pre-intervention to 15.0 minutes post-intervention (refer to Table\u00a0The secondary outcome measures, the haemorrhagic conversion rates, stroke mimic rates, MRS scores and 30-day mortality rates have remained largely similar pre and post-intervention despite significant improvement in DTN timings and the proportion of patients achieving target DTN timings. This finding was unexpected, as improved clinical outcomes were expected to improve DTN timings ,4,16 sigLimitationsAs a retrospective observational study, our results are inherently affected by the accuracy of available documented records and recall bias. Also, given its retrospective nature, our study would not establish any causal relationships from the interventions done. Any confounders and biases would also not be able to be controlled. However, as the focus of this study was to share our experience of identifying areas for improvement and systemic quality improvement interventions implemented helped improve DTN, we feel that the above limitations do not detract too much from the original goals of the study.Also, specific clinical outcomes like 30-day NIHSS scores, three-month MRS scores and discharge destinations were not captured. Our primary focus was on the impact these new systemic quality improvement interventions had on DTN timings. The sample size was also not pre-calculated to detect significant differences in mortality rates. Lastly, our study is a single-centre study and the interventions and results described may not be as applicable to other patient populations and settings.In conclusion, the implementation of systemic quality improvement initiatives as detailed above with the buy-in of all the stakeholders involved is shown to be associated with improved DTN timings for patients receiving thrombolytic therapy for acute ischaemic stroke. Our median DTN timing post-intervention at 62min, though not the gold standard, came very close to the ideal target of 60 minutes recommended by AHA/ASA. Although telestroke quality improvement interventions have improved DTN timings, clinical quality outcome measures like the haemorrhagic conversion rates and 30-day mortality rates have remained largely stable. EMPs, with a broad knowledge base and familiarity, interacting with various specialities and coordinating care, are uniquely suited in this role to drive change. More work in the public health sector would have to be done to improve the population's response to acute stroke symptoms."}
+{"text": "TFS) and time to maximum number of elasmobranchs observed (tMaxN) were examined. We found that TFS occurred earlier for species groups with high occurrence, and on BRUVS with high elasmobranch abundance, yet longer soak times were not essential to observe rarer species. Our models indicated an optimum of 95% of both sighting event types  was recorded within 63\u201377 minutes, and a soak time of 60 minutes recorded 78\u201394% of the elasmobranch sighting events recorded (78\u201394% of TFS events and 82\u201390% of tMaxN events), when species rarity and abundance on BRUVS was accounted for. Our study shows that deployments of ~ 77 minutes are optimal for recording all species we observed, although 60 minutes soak time effectively samples the majority of elasmobranch species in shallow coral reef habitats using BRUVS.Effective sampling of marine communities is essential to provide robust estimates of species richness and abundance. Baited Remote Underwater Video Stations (BRUVS) are a useful tool in assessment of fish assemblages, but research on the optimal sampling period required to record common and rare elasmobranch species is limited. An appropriate \u2018soak time\u2019 (time elapsed between settlement of the BRUVS on the seabed and when it is hauled off the seabed) requires consideration, since longer soak times may be required to record species rare in occurrence, or sightings in areas of generally low elasmobranch abundance. We analysed 5352 BRUVS deployments with a range of soak times across 21 countries in the Coral Triangle and Pacific Ocean, to determine the optimal soak time required for sampling reef-associated elasmobranchs, considering species rarity, and community abundance at each site. Species were categorised into 4 \u2018rarity\u2019 groups (very rare to common), by their relative occurrence in the dataset, defined simply by the proportion of BRUVS on which they occurred. Individual BRUVS were categorised into 3 \u2018abundance\u2019 groups (low to high) by overall relative elasmobranch abundance, defined as total number of all elasmobranchs sighted per unit of sampling effort. The effects of BRUVS soak times, and levels of rarity and abundance groupings, on the time to first sighting ( The abundance of elasmobranchs can be highly variable , 2 and wThere are a variety of fishery-independent methods available for estimating the abundance of elasmobranchs . Two of One of the main benefits of the baited video technique is that it removes the need for skilled observers in the field, yet is easily standardised for application to a variety of locations and depths. UVC requires a scuba diver highly skilled in fish identification to conduct each survey and there is no visual record of the sample. The presence of video footage permits visualisation of the seascapes in the field of view and species identifications in an archive that can be interrogated repeatedly for a variety of research questions . Due to The status of reef-associated elasmobranchs has been the subject of numerous studies which have produced varying perspectives about their conservation status and the efficacy of their management , 5, 22. The application of BRUVS provides an opportunity to sample a wide array of depths and habitat types using multiple units simultaneously  and to survey large areas more efficiently than UVC surveys. A large number of BRUVS can be deployed in quick succession to sample a broader area than is surveyed in a UVC transect. The amount of time a BRUVS is deployed on the seabed (\u2018soak time\u2019) is constrained by the travelling time between sampling sites, the handling time in setting and retrieving the units, camera storage capacity and battery life. A common presumption is that longer soak times would be needed in areas of low abundance (e.g. ), and thCarcharodon carcharias, since their mean time to first sighting was 148 minutes for a maximum of one individual at any time [Although sharks and rays are easily attracted to bait, it is unclear how they interact with BRUVS units. It could be assumed that in areas of high abundance BRUVS are likely to be placed in proximity to local sharks. In these instances individuals could be expected to appear in the BRUVS field of view early in the deployment, and accumulate to high numbers. In contrast, it could also be assumed that in areas of low overall elasmobranch abundance where individuals are sparsely distributed, or for particular species that are rarely encountered in the region, that likelihood of deploying a BRUVS in their proximity is low and hence arrival times would be longer (e.g. ). For exany time . While 1https://globalfinprint.org/) has conducted BRUVS deployments in coral reef ecosystems around the world to estimate the relative abundance of reef-associated elasmobranchs. Countries within the Coral Triangle and Pacific Ocean comprise a wide diversity and abundance of elasmobranchs, with species varying in rarity (i.e. occurrence on BRUVS), and sites ranging from low to high overall abundance . We examined this data to identify the optimal soak time required to capture the events of the time to first sighting (TFS) and time to maximum number of elasmobranchs observed (tMaxN), compared with the commonly used soak time of 60 minutes. The soak time required to effectively capture these sighting events was assessed under varying levels (categories) of: (1) species rarity: the occurrence of each species on the individual BRUVS in the dataset, grouped into categories of species rarity; and (2) overall elasmobranch abundance on individual BRUVS. These findings provide a basis and recommendations for future research using BRUVS to help define the occurrence and relative abundance of tropical, reef-associated elasmobranchs.The Global FinPrint project (https://www.gopro.com) within a housing overlooking a bait bag positioned 1.5 m from the camera (Our study included data from 5352 baited remote underwater video station (BRUVS) deployments of the Global FinPrint project in 21 countries. Deployments were conducted in coral reef habitats in depths from 1\u201370 m. Each BRUVS consisted of a lightweight aluminium frame fitted with a video camera  recorded for each species, and MaxN updated if the number increased at a later point in the video.Data comprised standardised legacy data and records from videos analysed using the FinPrint Annotator (v.1.1.44.0) which were examined by two readers, with reader discrepancies, outliers and a subset of images reviewed by a master annotator to ensure accuracy in species identification. Elasmobranchs were identified to species, and the maximum number of individuals of each species seen together in one frame (\u2018bundance ), was reTFS); MaxN; and \u2018time to MaxN\u2019 (tMaxN). The \u2018soak time\u2019 was measured as the minutes elapsed between the start time and the instant at which the BRUVS was hauled off the seabed (the end time). For instances where bait was entirely removed from a BRUVS, the time at which this occurred was classified as the end time and marked completion of the soak, for standardisation among BRUVS. Elasmobranch observations beyond the end time were not included in analyses. The \u2018time to first sighting\u2019 (TFS) was the time elapsed between the start of the sampling period and the first record of a particular species in the field of view. The \u2018time to MaxN\u2019 (tMaxN) for each species was the time elapsed between the start of the sampling period and the MaxN event, representing the time at which maximum relative abundance was observed. These metrics have been reviewed [TFS data; and tMaxN data, which includes an additional 1337 BRUVS, for which only MaxN for each species was recorded , being MaxN values summed across species on individual BRUVS divided by the soak time, multiplied by 60 to provide an hourly rate. SPUE was used to assign three categories of overall elasmobranch abundance on individual BRUVS: \u2018low\u2019 (SPUE \u2264 1); \u2018medium\u2019  and \u2018high\u2019 (SPUE > 3). Records for each species were assigned to four levels of \u2018occurrence\u2019 (\u2018rarity\u2019) based on the number of BRUVS samples on which they were recorded in the TFS dataset  was used to estimate the fraction of observations of TFS and tMaxN that were less than or equal to a specified value (using the ecdf function in R). The ECDF plots were used to find the 75th and 95th percentiles of TFS and tMaxN by species occurrence (4 levels of rarity), and by abundance on individual BRUVS (3 levels based on SPUE).Where cumulative frequency shows the frequency of an event in an interval, the empirical cumulative density function ; overall elasmobranch abundance (3 levels based on SPUE per BRUVS); and soak time. We identified the optimal soak times, and considered the commonly used soak time of 60 minutes as sufficient if ABTs indicated the majority of events occurred within 60 minutes, i.e. for all categories of abundance and species rarity. ABTs use the benefits of both regression techniques and classification by machine learning to derive a single, optimal tree by cross-validation  werion see , 34). Th. ThTFS aion  to 0.001 (very slow) were tested with tree sizes of 1000 and 5000.BRUVS were excluded from the ABT analyses if soak time > 126 , due to scarcity of cases with very long soaks beyond this time. Responses were transformed by 4TFS was available for 2349 BRUVS and data were obtained for tMaxN from 3384 BRUVS , blacktip reef shark (C. melanopterus), and whitetip reef shark (Triaenodon obesus) (Taenura lymma), maskrays (Neotrygon spp.), eagle rays (Aetobatus spp.), and the Galapagos shark   of the 2349 BRUVS where TFS information was available, 1382 of the 3384 BRUVS with TMaxN data  of 2989 individuals. The larger tMaxN dataset (MaxN = 1 for most species and most BRUVS (e.g. Three species comprised the \u2018common\u2019 category of species rarity based on occurrence: grey reef shark ( obesus) . The \u2018unagensis) . Most ota 40.8%; , and had dataset  showed sUVS e.g. .TFS and TMaxN event observations for each minute were calculated and represented as a proportion of the total observations per level of rarity. Boxplots of the event proportions observed per minute for 20 minute time bins showed no evidence that rarer species were being sighted later in videos, or that the highest proportions peaked later than those of the more common categories  groups  on BRUVS where higher overall abundance was recorded, compared to BRUVS with medium and low SPUE, particularly within the first 60 minutes  groups . At leas minutes . Thus, abundance , linked w curves .TFS (n = 3652 on 2314 BRUVS) and tMaxN (n = 5393 on 3347 BRUVS) for 47 species were modelled as responses to the predictors soak time, species rarity and abundance groups for ABT analyses. Less than ~ 10% of soak times ended before ~ 60 minutes, and less than 10% of soaks were longer than ~ 100 minutes . The relative prediction error of 0.85, equated to an R-squared of only 15.3%, and the relative influence of soak was 58.9%, with a generally increasing relationship between soak time and mean predicted TFS (species and BRUVS pooled)  . The rel minutes .TFS for a given soak time was observed faster than for BRUVS where SPUE of elasmobranchs were low to medium , with a high relative prediction error (0.94), equating to a very small R-squared of 6.3%. The model for tMaxN with soak time alone had a marginally lower R-squared of 6.1%. The relative influence soak time was 80.1%, with relative influence of 12.3% and 7.6% for rarity and abundance groups respectively , maximisensu , 39, 40,sensu . All fousensu , 42\u201346).MaxN, times to first arrival and times to MaxN values for Hawaiian bottom fish assemblages, found soak times of 60 minutes were considerably more powerful for detecting statistical differences in sessile \u2018macropiscivores\u2019 and \u2018generalist macropiscivores\u2019 [MaxN and mean species-specific MaxN did not differ among soak times of 30, 60 and 90 minutes in estuarine habitats, but \u2018precision\u2019 rose with increasing sampling time for small fish on seagrass and sandy substrata [Our ability to test the effect of varying soak times on these metrics for elasmobranchs extends research on the dynamics of visits to baited cameras by teleosts. Soak times for teleost community studies typically range from 30 to 90 minutes  and are civores\u2019 . Yet eitcivores\u2019 . Mean spubstrata . Precisiubstrata . TherefoCarcharodon carcharias) ranged from 15 to 299 minutes with a mean of 148 \u00b1 15 minutes [Somniosus microcephalus) was found between 118 to 280 minutes [MaxN, accounting for only 9% and 8% of the total predicted variation in TFS and tMaxN, respectively. This result likely reflects the high potential for sighting reef-associated elasmobranchs on BRUVS deployed in clear water reef habitats. There were obviously other major factors not analysed here that governed elasmobranch dynamics, such as micro-habitat type in the field of view, reef type and fishing pressure. These factors are important in other studies of reef-associated elasmobranch abundance [Studies using BRUVS to specifically identify and count elasmobranchs have used soak times of 60 minutes , 90 minu minutes , and meabundance .MaxN was observed differed little between abundance groups, but for sites characterised by high abundance (SPUE > 3 elasmobranchs), TFS occurred earlier. This was obvious within the first 60 minutes (94% of events observed), particularly for deployments where elasmobranchs were viewed immediately on settlement of the BRUVS on the seabed. The trend in TFS is in accordance with a presumption that arrival times to BRUVS will be faster at sites inhabited by more elasmobranchs. For example, mean arrival times of S. microcephalus was found to occur within the first half of long (up to 10 hour) soak times with a significant negative exponential relationship between first arrival times and total individuals sighted [Carcharhinus amblyrhynchos) on BRUVS was not accompanied by any significant change in TFS or tMaxN in the field of view [Our analyses showed a slight trend for delay in events for a given soak time at sites where overall elasmobranch abundance was classified as \u2018low\u2019 or \u2018medium\u2019. The times  sighted . In cont of view . Our comTFS and tMaxN) in the Global FinPrint datasets occurred, on average, within ~ 46 minutes in ABT models for all abundance groups, and the mean, global TFS and tMaxN occurred at 30.6 and 33.5 minutes across the variety of soak times recorded. The ECDFs indicated that approximately 83\u201394% of all TFS and 85\u201390% of all tMaxN events occurred by 60 minutes, and 95% by 77 minutes of soak time, for all abundance groups. Abundance groups also indicated that while first sightings of species occurred faster on BRUVS where elasmobranch SPUE was highest, ABT models predicted that all observations were observed within the first 60 minutes. These analyses imply that regardless of BRUVS-specific relative abundance, soak times of 60\u201377 minutes effectively survey elasmobranch assemblages of associated with shallow, coral reefs in the Pacific and Coral Triangle in daylight hours. These results may also apply to other coral reef systems, as the rates of elasmobranch sightings (SPUE) in our abundance groups are comparable to other studies, including the Tubbataha no-take Marine Reserve in the Philippines which was identified as a \u201chotspot\u201d for elasmobranchs [Events  rarely move over large distances and tend to have fairly consistent activity spaces on the scale of several kilometres [Galeocerdo cuvier) may have species-specific habitat preferences, broader movements, or nocturnal foraging patterns that reduce their incidental interaction with the BRUVS field of view (e.g. [C. brevipinna), white shark (Carcharodon carcharias), thresher shark (Alopias spp.) and the reticulate whipray , amongst many other singletons, could be considered transients. While a ~ 77 minute soak time may be more optimal than 60 minutes for research solely targeting one of these rarer transients in this region, increased replication of 60 minute BRUVS or complementary sampling via other approaches such as 360 degree cameras [We found no evidence that rarer species were sighted only on the longest video records or that counts of their maximum number peaked later than those of more abundant species. Species groups classified as \u2018very rare\u2019 showed a delay of only ~10 minutes in the timing of ely (see ). Most rlometres , 52\u201355, ew e.g. , 57). Th. ThtMaxN cameras , 59 woulMaxN values were only 1 individual for most species on most BRUVS. This may be because separate visits of single, different, elasmobranchs in the field of view are not recognised in the conservatism of the MaxN metric [MaxN suggests that reef-associated elasmobranchs are not typically found in large groups and/or very high local abundances. Most reef elasmobranchs are not known to aggregate as readily as some pelagic or inshore species . Lo. LoMaxN es  in the field of view of BRUVS. Unless specifically investigating a single very rare transient species in areas with few elasmobranchs, greater benefit is likely gained through increased replication of 60 minute BRUVS than extending soak times. These metrics are fundamental to addressing questions about the status of populations when using the BRUVS technique, and we provide a baseline for the areas within the regions sampled. We recommend pilot studies compare a range of sampling durations using these methods to gain an understanding of completeness of the biodiversity inventories assessed.We have concluded that a sampling time of 63\u201377 minutes is optimal for sighting individuals for defining relative abundance of rare to common species, at sites with varying levels of elasmobranch abundance, on shallow tropical reefs across a broad geographic area. Extending deployments and video analysis beyond 60 minutes could have undesirable logistical consequences and questionable benefits, and may not be necessary since 60 minutes appears sufficient to capture the majority of sighting events. Understanding the effectiveness of the commonly used 60 minute soak time is important for robust comparisons among tropical locations, especially for rare or uncommon species. We recommend that researchers routinely record all events relating to timing of first arrival and subsequent timing of increase in S1 TablePercentage of BRUVS where elasmobranchs were recorded is shown.(PDF)Click here for additional data file.S1 Fig(PNG)Click here for additional data file."}
+{"text": "In the past decade, bacteria\u2010based cancer immunotherapy has attracted much attention in the academic circle due to its unique mechanism and abundant applications in triggering the host anti\u2010tumor immunity. One advantage of bacteria lies in their capability in targeting tumors and preferentially colonizing the core area of the tumor. Because bacteria are abundant in pathogen\u2010associated molecular patterns that can effectively activate the immune cells even in the tumor immunosuppressive microenvironment, they are capable of enhancing the specific immune recognition and elimination of tumor cells. More attractively, during the rapid development of synthetic biology, using gene technology to enable bacteria to be an efficient producer of immunotherapeutic agents has led to many creative immunotherapy paradigms. The combination of bacteria and nanomaterials also displays infinite imagination in the multifunctional endowment for cancer immunotherapy. The current progress report summarizes the recent advances in bacteria\u2010based cancer immunotherapy with specific foci on the applications of naive bacteria\u2010, engineered bacteria\u2010, and bacterial components\u2010based cancer immunotherapy, and at the same time discusses future directions in this field of research based on the present developments. Bacteria\u2010based cancer immunotherapy has shown significant potential in the realm of tumor therapy, mainly because of bacterial specific targeting and preferential colonization of tumors, the abundant natural antigen library of bacteria, and the great engineerability. These high\u2010quality properties of bacteria have been and are being researched extensively, allowing the discovering of best immunotherapies. TIME undergoes dynamic changes along with the rapid growth of tumor, acting as a hotbed for tumor's rapid proliferation and invasion and a helper for tumor immune escape. In fact, it is the tumor's hypoxic environment that provides a solid foundation for this hotbed. The oxygen concentration in the tumor tissue is only 7\u201328\u00a0mm Hg (1\u20134%), while it is 40\u201360\u00a0mm Hg (5\u20138%) for the normal tissue. Such a hypoxic environment not only poses a huge challenge to tumor radiotherapy and chemotherapy but also directly facilitates the formation of tumor immunosuppressive microenvironment. Therefore, the extreme hypoxia of the tumor and the immunosuppressive microenvironment are two major features of malignant tumors, which can be regarded as the key for developing tumor treatments.The ongoing exploration of the tumor microenvironment (TME) in recent years has gradually unveiled a fact that the tumor immunosuppressive microenvironment is another big hurdle that needs to be overcome in cancer treatment besides those already known adverse factors that severely limit the therapeutic effect like the tumor interstitial pressure, hypoxia, nutritional deprivation, inadequate blood perfusion, immature lymphatic system, etc.1 The  Although the delivery efficiency of nanoparticles can be improved in this way, its total enrichment in tumor remains at a relatively low level. Compared to inactive nanoparticles, bacteria have a variety of complex physiological functions , making them good therapeutic agents or vectors that have potential applications in cancer therapy. As early as the late nineteenth century, William Coley injected heat\u2010killed streptococcal organisms in conjunction of Serratia marcescens (\u201cColey's Toxins\u201d) into tumor patients to treat their sarcomas and other malignancies, and observed tumor ablation. Since then, William Coley was regarded as a trailblazer in bacteria\u2010based cancer immunotherapy. Afterward, researchers found that certain obligate anaerobes and facultative anaerobes could preferentially colonize the hypoxic/necrotic zone inside the solid tumor after systemic administration. Although the mechanism behind bacteria's efficiency in targeting tumors has not yet found a consensus on the answer, it is agreed that bacteria's tumor\u2010targeting ability is inseparable from the hypoxia property of tumor based on the analysis of the distribution of different strains in the body. Clarifications on this issue have been provided by many researchers. Neil S. Forbes reported that more than 10\u00a0000\u2010fold of Salmonella typhimurium (S. typhimurium) could be accumulated in tumors compared with other organs after one week of systemic injection. Several mechanisms were proposed to explain the excellent specificity: a) chaotic vasculature trapped bacteria in the tumor; b) attraction to specific TME; c) preferential replication in TME. Although a pool of studies has claimed to see a great tumor\u2010colonization of bacteria after intravenous administration, the initial phase of bacteria escape from vessels to tumor site still remains a mystery. Sara Leschner observed a dramatic increase in tumor necrosis factor\u2010\u03b1 (TNF\u2010\u03b1) in blood after the intravenous injection of Salmonella enterica serovar typhimurium in an ectopic transplantable tumor model, inducing the disruption of vessels in tumors. As a result, bacteria were flushed into the tumor with the influx of blood and were trapped inside ever since. More convincingly, authors neutralized TNF\u2010\u03b1 in the serum of tumor\u2010bearing mice and observed retardation of blood influx together with a delay of bacterial tumor\u2010colonization. Therefore, TNF\u2010\u03b1 and the induced hemorrhage inside the tumor play a major part at the early stage of bacterial invasion of the tumor.As an alternative idea, some researchers focused on the tumor hypoxia\u2010tropic nanoparticles to realize a preferential engagement in hypoxic tumor tissues.5 AlthFigure\u00a0 For example, the components of bacteria including peptidoglycan, lipopolysaccharide (LPS), lipoteichoic acid (LTA), flagellum, DNA, RNA, etc., could be identified by pattern recognition receptors (PRRs) on dendritic cells (DCs), macrophages, and neutrophils, which subsequently triggered the corresponding immune response. Among them, LPS, found in the outer membrane of Gram\u2010negative bacteria, is a typical strong immunogenic microbial\u2010associated molecular patterns (MAMPs), which is mainly combined with Toll\u2010like receptors (TLRs) on the immune cell membranes. The increase of LPS would cause the overexpression of interleukin\u20106 (IL\u20106), activation of nuclear factor kappa B (NF\u2010\u03baB) signaling and TLRs pathway, and phosphorylation of signal transducer and activator of transcription 3 (STAT3). These pathways could promote the maturation of DCs and the proliferation of immune cells, thereby enhancing anti\u2010tumor immunity. Similarly, bacterial flagellum would activate TLR5\u2010mediated innate immune response. For Gram\u2010positive bacteria without LPS and flagellum, such as Bifidobacterium, it could activate macrophages, natural killer (NK) cells, DCs and B lymphocytes by peptidoglycan, extracellular polysaccharides and DNA. Then, these effector cells could be promoted to produce immune active substances, such as IL\u20101, IL\u20106, IL\u201012, TNF\u2010\u03b1, IFN\u2010\u03b3, and nitric oxide (NO), and finally facilitate the anti\u2010tumor effect. Therefore, it seems that bacteria and cancer immunotherapy will inevitably be combined . The hypoxic microenvironment in tumor and the nutrients released from the dead tumor cells facilitate the massive proliferation of anaerobic bacteria. Moreover, the immunosuppressive TME prevents the immune system from clearing the bacteria in the tumor at the early stage. Then, these multiplied bacteria will activate the host's immune system, causing a large amount of immune cells to infiltrate within tumors.In the late 19th century, the anti\u2010tumor effect of Coley's toxins made people realize for the first time the huge potential of bacteria in tumor treatment.19 Alt+) oxidase and elevated intracellular Ca2+ amount. Both of the two mechanisms can form reactive oxygen species (ROS), which is a highly cytotoxic free radical. ROS can initiate the immunogenic death of tumor cells, then activate CD8+ T cells to eliminate residual tumor cells, and finally prevent metastases. Meanwhile, Listeria can infect bone marrow\u2010derived suppressor cells (MDSCs) at the tumor site, causing a significant quantity decrease in MDSC and subsequently transforming the immunosuppressive microenvironment to an immunostimulatory status. Moreover, IL\u201012 can be produced upon the transformation of the remaining infected MDSCs into immunostimulatory phenotype, which can improve the T cells\u2019 and NK cells\u2019 response.It is worth noting that the underlying mechanisms in the immune system for bacteria and host to interact with each other depend on bacterial strains and tumor models. For example, Listeria infection can directly kill tumor cells through the activation of nicotinamide adenine dinucleotide phosphate  from polymorphonuclear neutrophils. Studies have reported that matrix metalloproteinase 8 (MMP\u20108) played a vital role in this release process. Moreover, the early expansion of Clostridium in solid tumors could lead to intratumoral infiltration of granulocytes and macrophages, and the increased secretion of chemokines would further trigger the adaptive immunity and recruit immune cells  to the tumor site.Salmonella is another type of bacteria that has been widely studied in the field of cancer therapy; its interaction with the immune system has been clarified as well. After infecting tumor cells, Salmonella directly destroys tumor cells either by initiating autophagy pathway or inducing apoptosis. Furthermore, pathogen\u2010associated molecular patterns (PAMPs) such as flagellin and LPS released from Salmonella can be captured and recognized by antigen\u2010presenting cells (APCs). Flagellin can promote the maturation of APCs and up\u2010regulate pro\u2010inflammatory cytokines  and co\u2010stimulatory molecules  by binding and activating TLR5 and Nod\u2010like receptors (NLRs) on APCs. These inflammatory mediators can subsequently stimulate the secretion of interferon\u2010gamma (IFN\u2010\u03b3) and the T helper type 1 (Th1) cell\u2010mediated immune response. In this process, macrophages and DCs secrete pro\u2010inflammatory IL\u20101\u03b2 and TNF\u2010\u03b1 under the stimulation of LPS\u2010induced TLR4 signaling and tumor cell debris. Flagellin has been found to be capable of activating NK cells to produce IFN\u2010\u03b3 through a TLR\u2010independent pathway involving IL\u201018 and Myeloid differentiation factor 88 (MyD88), and the produced IFN\u2010\u03b3 will lower the frequency of CD4+ CD25+ regulatory T cells (Tregs) in the TME. Studies have shown that Salmonella infection can lead to the up\u2010regulation of connexin 43 (Cx43) in human and murine melanoma cells  was tested on 24 patients with metastatic melanoma and one patient with metastatic renal carcinoma. Patients received 106 to 109 cfu m\u2212\u00b2 of VNP20009 through systematic administration and then were evaluated for dose\u2010related toxicities, selective replication within tumors, and anti\u2010tumor effects. The maximum\u2010tolerated dose was determined as 3.0\u2009 \u00d7 \u2009108 cfu m\u2212\u00b2. And some tumor colonization was observed at the highest tolerated dose. Although dose\u2010related increase of several proinflammatory cytokines  was observed, no patients experienced objective tumor regression. The clinical results suggest that additional methods were required to enhance the efficacy and reduce the toxicity. With the advancement of synthetic biology, bacteria have been engineered for attenuation to ensure safety. After decades of rapid development, this transformative approach not only enables researchers to focus on the attenuation but also adopts a variety of engineering methods to develop intelligent bacteria for a wide range of applications.Although native anaerobic and facultative anaerobic bacteria are excellent at colonization in tumor and killing tumor cells through immune cells\u2019 infiltration, the single functionality and ineffective therapeutic action seriously limit their development and application. Besides, some toxic and side effects have been observed after systemic injection of native bacteria.39 Wit2.22.2.1Immunotherapeutic agents include monoclonal antibodies, chemokines, and cytokines, etc. Due to their large molecular weight and good water solubility, it is particularly difficult even for nano carriers to deal with the targeted delivery of these proteins. The rapid development of synthetic biology enables researchers to creatively develop engineered bacteria that produce and express multiple immunotherapeutics. These bacteria can efficiently and safely express immunotherapeutic agents in the local tumor, thereby triggering a strong anti\u2010tumor immune response. This kind of immune stimulation usually results from the joint action of two aspects. One is that the expressed immunotherapeutic agent specifically targets a certain part of the immune system, while the other is that the bacteria supplement the immune stimulation through PRRs, thereby generating maximum anti\u2010tumor immunity. Another benefit of this method is to reduce the off\u2010target of the therapeutic agent, hence reducing the toxic side effects.Escherichia coli (E. coli) strain to release nanobody antagonist of CD47 (CD47nb), which is an anti\u2010phagocytic receptor highly expressed in some human cancer types. The E. coli strain used in this work comprised a synchronized lysis circuit (SLC) in which lyse bacteria released its therapeutic products once reaching a critical density  via transforming the high\u2010copy plasmid carrying programmed death\u2010ligand 1 (PD\u2010L1) nanobody and cytotoxic T\u2010lymphocyte\u2010associated protein 4 (CTLA\u20104) nanobody sequences to locally and controllably express PD\u2010L1 and CTLA\u20104 antagonists  by activating MyD88 after dimerization. In the last step, it activates NF\u2010\u03baB and regulates cells from cellular transcription. Therefore, flagellin can also be used as an immune adjuvant, mainly based on the ability to activate DCs and non\u2010intrinsic immune cells . The interaction of flagellin and TLR5 results in the expression of varied proinflammatory cytokines, NO, H2O2, chemokines, and host defense proteins. It is revealed that engineered Salmonella in the TME exerts a strong anti\u2010tumor efficacy through the activation of the TLR4 and TLR5 pathways. The engineered flagellin\u2010secreting Salmonella has a greater inhibitory effect on tumors than free Salmonella, indicating that TLR4 and TLR5 pathways have a synergistic effect on tumor growth inhibition. Moreover flagellin is also closely related to tumor immunosuppressive microenvironment. The inhibitory signals produced from tumor cells in the TME effectively prevent the surveillance of the immune system. MDSCs can recruit regulatory T cells and inhibit the activation of NK cells and effector T cells. Intratumoral injection of flagellin can effectively reduce the number of MDSCs in the tumor. In TME, tumor\u2010associated M2\u2010like macrophages promote tumor growth by inhibiting DCs maturation, down\u2010regulating the expression of major histocompatibility complex (MHC) molecules, and recruiting regulatory T cells. Interestingly, it has been proved that flagellin can reverse the tumor immunosuppressive microenvironment into immune\u2010responsive TIME by the polarization of M2\u2010like macrophages to M1\u2010type macrophages. Jin Hai Zheng et\u00a0al. reported that an engineered attenuated S. typhimurium strain could successfully suppress the tumor growth and metastasis in murine colon and melanoma models through a secretion of Vibrio vulnificus flagellin B (FlaB) .50 As  STING can be activated by binding to cyclic dinucleotides (CDNs) and initiating a potent pro\u2010inflammatory cytokines production, including type I IFNs. Numerous studies have shown that STING agonists exhibit potent anti\u2010tumor activity, opening the way for developing STING\u2010based cancer immunotherapy. However, although the STING agonists can activate the STING pathway to trigger an anti\u2010tumor immune response in cancer treatment, it still has some shortcomings, such as off\u2010target effects and toxicity. Therefore, the use of synthetic biology technology to engineer bacteria to produce and release STING agonists after colonization on tumor sites has become a preferred solution. Also, as a good source of immune stimulation through PRRs, bacteria can activate complementary immune pathways to provide better curative effects. In a recent report, Daniel S. Leventhal and co\u2010workers engineered a strain of E. coli Nissle (referred to as SYNB1891) to express STING\u2010agonist cyclic diAMP (CDA) . These results confirmed the great potential of SYNB1891 in triggering an anti\u2010tumor immune response. Moreover, two distinct murine tumor models (B16F10 and A20) and two different genetic backgrounds mice (C57BL/6 and BALB/c) were carried out and verified that SYNB1891 could trigger effective anti\u2010tumor immunity and immunological memory.Like TLRs, as an important part of the innate immune system, the stimulator of interferon genes (STING) pathway has received elevated attention since its discovery.58 STIS. typhimurium to express LIGHT, which is a TNF\u2010family cytokine that has been demonstrated to generate promising anti\u2010tumor activity. In several mouse tumor models, authors observed satisfactory tumor suppression with no significant toxicity. And the TME analysis demonstrated that the efficacy was associated with the massive inflammatory cell infiltration into tumors including the elevated frequency of DCs. Based on this novel strategy, authors further armed attenuated S. typhimurium with the ability to synthesize IL\u201018, which is an IFN\u2010\u03b3\u2010inducing factor with multiple functions related to the proliferation and activation of immune cells. In preclinical mouse cancer models, the endowment of IL\u201018\u2010producing ability greatly increased S. typhimurium\u2019s anti\u2010tumor activity. The mechanism was associated with increased frequency of tumor infiltrating CD3+/CD4+ T cells and DX5+ NK cells. Meanwhile, several immunostimulatory and inflammatory cytokines were brought into play in tumor suppression, including GM\u2010CSF, IL\u20101\u03b2, and TNF\u03b1. These preclinical findings show that cytokine\u2010producing bacteria could elicit strong immune response with no side effects, suggesting a novel strategy for bacteria\u2010based cancer immunotherapy.Cytokines are widely studied due to their intrinsic functions to promote the activation and proliferation of immune cells to achieve anti\u2010tumor effects. However, systemic administration of cytokines causes serious side effects, limiting its clinical application. Among different routes to deliver cytokines to the tumor site, engineered tumor\u2010targeting bacteria to express cytokines have multiple advantages such as high\u2010level specificity but less side effects. Markus Loeffler and co\u2010workers engineered attenuated activity.62 In 2.2.2 Nanomaterials can achieve a variety of functions in cancer therapy due to different synthesis methods and encapsulated drugs. Therefore, compared with the genetic modification of bacteria to secrete therapeutic agents, it seems more direct to integrate nanomaterials with other diverse functions in the outer membrane of bacteria. The biggest benefit brought by this is the easy preparation and the low cost; however, there still exist some shortcomings.In addition to the use of genetic technology to engineer bacteria, the integration of bacteria and nanomaterials for multifunctional synergistic therapy is considered to be a promising treatment.40c Na To prevent the early detachment of nanomaterials in the body fluid environment and the loss of the expected versatility on the collaborative treatment platform, a strong connection between the nanomaterials and bacteria is particularly important. Therefore, researchers mostly use stable amide bonds to connect the two bodies to prevent functional materials from falling off in advance. Furthermore, the proper function should be selected to integrate with the ontology of bacteria to produce a synergy rather than antagonism. The response to external stimuli allows the integrator to act on its orders and achieve a controlled treatment. Light irradiation, magnetic field, and ultrasonic are the commonly used externally applied stimuli. Among them, light irradiation is the most\u2010widely used and mature stimulus in tumor treatment.First of all, bacteria can target tumors partly because the outer membrane of bacteria is rich in outer membrane proteins, some of which are used to sense the surrounding environment and perform chemotactic movement of certain specific molecular signals. When integrating nanomaterials into the outer membrane of bacteria, the chemotactic function of the bacteria will be inevitably affected if a dense and continuous shell is formed to completely enclose the bacteria, thereby weakening the targeting ability of bacteria. Another consideration lies in the strength of the connection between nanomaterials and bacterial membranes. The engineered bacteria need to face a complex physiological environment, such as high\u2010speed blood flow, high salinity, a variety of metal ions, and abundant fibrin.65 To  Applying engineering methods to integrate phototherapy nanoparticles with bacteria can effectively achieve precise tumor targeting and powerful primary tumor ablation. Moreover, the sequent anti\u2010tumor immunity could achieve multi\u2010functional combination therapy to maximize the therapeutic effect of tumors. However, although the tumor's hypoxia microenvironment provides an excellent condition for bacterial intrinsic tumor targeting ability, it greatly limits the therapeutic effect of PDT. The photosynthetic bacteria were previously applied for cardiovascular disease because of its highly efficient oxygen generation. The light\u2010controllable growth and photosynthesis of bacteria could relieve tumor hypoxia for enhanced immunogenic PDT. Lanlan Liu et\u00a0al. engineered photosynthetic bacteria  by integrating photosensitizer\u2010encapsulated nanoparticles on the bacterial surface via amide bonds  that involved the up\u2010regulated expression of calreticulin (CRT) on the cell surface. The CRT exposure on the membrane of tumor cells can promote the activation and maturation of DCs. Interestingly, not only CRT but also ROS produced by PDT could accelerate the maturation of DCs. In addition to producing oxygen locally, Syne as immunogenic bacteria can trigger moderate immune activation through upregulating the percentage of MHC II and increasing the production of IL\u201012p40. Meanwhile, improved tumor hypoxia promotes the infiltration of effector T cells to the tumor since T cells tend to avoid hypoxic areas and head to neighboring normoxic regions in TME. Photosynthesis\u2010boosted immunogenic PDT shows a great ability to ameliorate the TIME by elevating the percentage of immune effector cells such as CD8+ T cells, CD4+ T cells, and NK cells, and decreasing the number of immunosuppressive cells such as Tregs, MDSCs, and M2\u2010like tumor\u2010associated macrophages (TAM) which are important for suppressing anti\u2010tumor immunity.Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), has gained increasing attention due to its precision and non\u2010invasion.66 App Due to the good biocompatibility and biodegradability, photosensitizer polydopamine (pDA) is a promising candidate for PTT. To achieve the combination of hypoxic tumor targeting of bacteria and photothermal behavior of pDA, researchers have integrated pDA into the surface of anaerobe Salmonella strain VNP20009 to make it a thicker artificial outer membrane  to block programmed cell death protein 1 (PD\u20101). AUNP\u201012 has been proven to perform well on the antagonism of PD\u20101 by subcutaneous administration and showed a safe toxicological profile. Although peptide antagonists have better tumor penetration properties, short\u2010term retention in tumor tissues limits their application. Therefore, the authors applied subcutaneous injection of AUNP\u201012\u2010loaded phospholipid\u2010based phase separation gel (PPSG) near the tumor to achieve long\u2010term sustained release of the antagonist. With gel sustained\u2010release system, the immune checkpoint inhibitor could stay in the tumor tissue for up to 42 days, enhancing the anti\u2010tumor effect of triple combination therapy. Compared with the VNP20009 treatment alone, the amount of TNF\u2010\u03b1 at the tumor in the triple therapy group was 7.9\u00d7 that of the bacterial treatment group. Moreover, every element in the triple therapy contributed to the immune activation of the tumor. With the release of tumor\u2010associated antigens, the maturation of DCs, the production of proinflammatory cytokines, the recruitment and functioning of T cells, and the promotion of NK cells, the triple therapy including pDA engineered bacteria and immune checkpoint blockade achieved full\u2010stage promotion of anti\u2010tumor immunity and exhibited effective suppression of advanced melanoma.Some researchers also tried to engineer the bacteria with PTT photosensitizer. PTT, as a non\u2010invasive cancer treatment, can cause strong tumor ablation and simultaneously induce heat shock proteins (HSP) produced by tumor cells, which is a family of proteins with moderate immunostimulant function.74 DueIn addition to genetic engineering, the hybridization of bacteria and nanomaterials is another strategy to functionalize bacteria. The appropriate modification of nanomaterials on the surface of live bacteria would not affect the intrinsic tumor\u2010targeting ability of bacteria. The additional functions  achieved by the introduction of nanomaterials have a synergistic effect on bacterial therapy and enhance the therapeutic effect. Usually, such combination therapy can simultaneously enhance several aspects of anti\u2010tumor immune activation, leading to the reversal of the tumor immunosuppressive microenvironment and maximizing anti\u2010tumor immunity. Although the combination of phototherapy with bacteria\u2010based cancer immunotherapy has made great achievements, the intrinsic drawbacks of phototherapy including the insufficient light penetration depth and the potential phototoxicity to the skin need to be considered in future studies.2.32.3.1 OMVs mainly consist of cellular components of the bacterial periplasm and the outer membrane, including membrane lipids, proteins, LPS, peptidoglycans (PG), and virulence factors. Some intracellular components may also be wrapped, such as some intracellular proteins, DNA, RNA, ions, metabolites, signaling molecules, and enzymes. The mechanism of OMVs production remains unclear. There are currently three hypotheses widely recognized: 1) the accumulation of phospholipid in the outer leaflet of the bacterial outer membrane and regulation by VacJ/Yrb ATP\u2010binding cassette transport system for most Gram\u2010negative bacteria result in the OMVs formation; 2) the cross\u2010linking of the outer membrane and the PG layer. The cell wall of Gram\u2010negative bacteria is composed of outer membrane and PG, while lipoprotein crosslinks them with covalent bonds to maintain the integrity of the envelope structure. When decomposition or abnormal synthesis of the PG layer happens, a small part of the outer membrane would dissociate from the PG layer and protrude out of the cell to form OMVs; 3) the accumulation of the misfolded proteins and abnormal envelope components in the periplasm would promote the formation of OMVs. These unexpected substances may reduce the integrity of the envelope, thereby separating the PG layer and the outer membrane layer. As a result, the buds are extruded outward to form OMVs. OMVs containing a large number of MAMPs  can interact with host PRRs to induce innate immune responses. Owing to the abundant natural adjuvant components in OMVs, systemic injection of OMVs or siRNA\u2010packaged OMVs from a mutant E. coli strain has been reported to up\u2010regulate the production ofTNF\u2010\u03b1, IL\u20106, IFN\u2010\u03b3, and anti\u2010tumor cytokines CXCL10, which are closely related to the anti\u2010tumor immunity. Among a variety of MAMPs, the LPS stands out like a star. It is composed of lipid A, core polysaccharide and O\u2010antigen. O\u2010antigen is a kind of polysaccharide exposed on the surface of the bacterial outer membrane, which is an epitope of bacterial cell antigens. Lipid A is regarded as the active biological center of LPS; it can cause strong inflammation and regulate immune response such as stimulating immune cells to produce antibodies against multiple antigens. However, current studies have shown that excess LPS would cause immunosuppressive reactions, and blocking lipid A can remove endotoxin activity and reduce immunosuppression. OMVs as immune adjuvants need to appropriately reduce the toxicity of LPS, that is, knocking out the activity of lipid A through genetic modification to achieve the purpose of attenuation. Oh Youn Kim et\u00a0al. knocked out the msbB gene encoding endotoxin in E. coli to avoid the immunosuppression caused by lipid A, and found that the use of G\u2010bacteria OMVs in mouse colon cancer model could inhibit tumor growth. The outer membrane protein of vesicles stimulated NK cells and T cells to produce INF\u2010\u03b3, which in turn has growth inhibitory effect on tumor. In some cases, naturally\u2010produced OMVs were used directly as carriers for drug delivery. For example, immunomodulatory molecules, photosensitizers, and chemo drugs can be loaded in OMVs and then transported to tumor, achieving a combination of immunotherapy and chemotherapy/phototherapy. Qi Chen et\u00a0al. coated DSPE\u2010PEG\u2010RGD\u2010 hybridized bacterial OMVs on drug\u2010loaded polymer micelles to produce an innovative nanomedicine for effective cancer immunotherapy and metastasis prevention , further realizing remarkable inhibition of pulmonary metastasis. Therefore, in order to enrich the functions of OMVs and achieve more effective tumor suppression, two schemes have been generally adopted. The first solution is to hybridize some functionalized lipid polymers or other biological membranes to OMVs to achieve new functions or to improve their inherent performance. The second method is to take advantage of the high loading capacity of OMVs to realize the anti\u2010tumor immune activation of other treatment methods and OMVs per se by delivering therapeutic agents  to the tumor site. Synergistic therapy could further improve the anti\u2010tumor efficacy. In a recent report, Qi Chen et\u00a0al. designed and constructed a hybrid eukaryotic\u2010prokaryotic nanoplatform by fusing melanoma cytomembrane vesicles with attenuated Salmonella OMVs  have plenty of specific antigens on the surface that can be recognized by APCs and benefit its maturation, which in turn can trigger the anti\u2010tumor immune response. Furthermore, the homologous targeting ability of CCMVs allows more vesicles to reach the tumor site, thus triggering a stronger immune response. In order to inherit the excellent characteristics of CCMVs and OMVs, the authors designed a fusion eukaryotic\u2010prokaryotic vesicle (EPV) which exhibited great potential to suppress the tumor growth based on DCs immune activation and the CTL\u2010derived tumor\u2010specific immunity. Due to the limited tumor\u2010eradication capacity of monoimmunotherapy, the combination with other therapeutics would be more effective. The authors made the best use of EPV's great loadability to enclose a photothermal agent inside the EPV, achieving thermal ablation of tumor mass. The immunogenic death of tumor cells caused by thermal ablation can provide supplementary antigens to the immune system and further improve the anti\u2010tumor immune response.Outer\u2010membrane vesicles (OMVs) are nano\u2010sized spherical vesicles produced by Gram\u2010negative bacteria.78 OMVFigure\u00a0 This approach would trigger the following four sequential steps: to produce tumor\u2010specific antigens by radiation therapy; to capture cancer neoantigens and enhance the uptake of antigens in DCs by BNP; to initiate the anti\u2010tumor T\u2010cell response; and finally to activate both innate and adaptive immunity. Bacterial membranes are abundant in PAMPs such as TLR agonists that initiate innate immunity and DCs activation. The maleimide group modification on bacterial membranes endows a better capturability for ICD\u2010induced tumor\u2010specific antigens, thus strengthening the activation of anti\u2010tumor immunity. In murine melanoma or neuroblastoma, the results showed that combining BNP with radiation therapy could lead to a remarkable activation of DCs and effector T cells, significant tumor regression, and anti\u2010tumor immune memory, indicating that BNP has a great ability to facilitate in situ immune identification of a radiated tumor with a potent immune activation.The superiority of membrane vehicles is not just reflected in their strong carrying capacity; the membrane can see more functions available by simply carrying out some basic modifications. For example, Ravi B. Patel and colleagues developed a bacterial membrane\u2010coated nanoparticle (BNP) consisting of an immunostimulator PC7A/CpG polyplex core coated with maleimide groups\u2010modified bacterial membrane. BNP was used as an in situ vaccine in combination with radiation therapy for cancer treatment Figure\u00a013.[91 Owing to the strong chemotactic and phagocytic function, neutrophils can quickly cross the blood vessel composed of endothelial cells (ECs) when pathogens cause infection, subsequently follow the concentration gradient of chemokines to arrive the non\u2010vascular area and finally migrate to the inflamed area, thus clearing the pathogens through phagocytosis and intracellular killing. One characteristic of tumors is inflammation, which makes neutrophils actively chemotax to tumors. Therefore, neutrophils possess natural advantages as a carrier for nanoparticles or drugs. Due to the short life cycle of neutrophils, they are extremely difficult to be engineered in vitro and injected back into the blood. An alternative solution can be the use of neutrophils and pathogens with high affinity loaded nanoparticles in the blood circulation. As a bacterial product, OMVs completely inherit the protein on the surface of the bacterial membrane. Naturally, it is also a pathogen that can be recognized by neutrophils. Min Li and colleagues designed pathogen mimicking nano\u2010pathogenoids (NPNs) containing PAMPs by cloaking NPs with OMVs, which can be recognized by PRRs on neutrophils  and macrophage\u2010inflammatory protein\u20102 (MIP\u20102), NPNs could be carried to migrate to tumor. Then, under the stimulation of inflammation in tumor, neutrophils would release the loaded nanoparticles that were internalized by the surrounding tumor cells to kill the tumor cells. Generally speaking, OMVs are more like an efficient carrier for cancer therapeutic agents. Indeed, it showed a wide range of application scenarios in loading anticancer agents. On the other hand, more novel applications may be developed by exploiting the potential of OMVs to interact with the human immune system due to the rich and completely inherited bacterial outer membrane information on the surface of OMVs. It is promising that these applications may exert the full potential of OMVs and spawn more effective tumor immunotherapeutics.Apart from reaching the tumor through enhanced permeability and retention (EPR) effect, OMVs can also hitchhike the immune cells to transport them to tumor by immune response. For instance, neutrophils are important natural immune cells in mammals. On the frontline of host defender, neutrophils sense and ingest pathogens by recognizing PAMPs, followed by initiating the innate immune response to prevent the invasion of pathogenic microorganisms.92 Owi2.3.2 The anti\u2010tumor bacterial toxins can be divided into two categories: conjugated on the surface antigen of tumor cells or conjugated on the ligand. Due to the high expression of specific antigens on the surface of tumor cells, bacterial toxins that specifically target these antigens, such as Diphtheria toxin (DT) and Clostridium perfringens enterotoxin (CPE), and Pseudomonas exotoxin (PE) can be used for targeting and killing cancer cells. Among them, DT has been widely used in tumor treatment both in mice and humans because of its relatively desiable anti\u2010tumor effect. This may benefit from its strong cytotoxicity or the simultaneously induced anti\u2010tumor immunity. Silvio Buzzi and colleagues employed a non\u2010toxic cross\u2010reacting material 197 (CRM197) to treat a group of cancer patients. CRM197 is a nontoxic mutant of DT that possesses similar immunological properties as DT. Similar to DT, CRM197 targets heparin\u2010binding epidermal growth factor (HB\u2010EGF), which is commonly overexpressed in cancer cells. The authors found that subcutaneous injection of CRM197 caused inflammatory\u2010immunological reactions, triggering biological anti\u2010tumor response. Through the experimental results, the authors speculated that neutrophils and TNF\u2010\u03b1 were involved in the anti\u2010tumor process. Thus, bacterial toxins not only show a great lethal effect on cancer cells but also initiate anti\u2010tumor immunity. The fusion proteins composed of bacterial toxins and targeting antibody fragments are also called immunotoxins. The fused targeting antibody fragment can target cancer cells, leading to a more potent lethal effect of bacterial toxin fragment on target cells. Immunotoxins that contain bacterial toxins which achieve potent cytotoxicity by blocking protein translation have been proved to be effective in treating some hematological malignancies. In a previous study, Ontak, a fusion protein composed of DT and anti\u2010IL\u20102, was studied for treating chronic lymphocytic leukaemia (CLL) based on the overexpression of high\u2010affinity IL\u20102 receptors on CLL cells, and satisfactory results were achieved. Similar to chemotherapy, repeated administration is necessary for immunotoxins treatment to maintain the optimal cell lethal concentration. However, the retreatment is limited to its immunogenetics, which is also known as the formation of anti\u2010drug antibodies (ADA). After treatment with immunotoxin, many patients will have a rapid immune response and the formation of ADA, which will neutralize the efficacy of immunotoxins and prohibit multiple administrations. To solve this problem, some researchers have tried to combine immunotoxin with chemo drugs or modify bacterial toxins to evade recognition of the immune system. These methods have achieved effective immunogenetic reduction. A more direct and well\u2010accepted method is to delete or mutate T cell epitopes by rational design of recombinant proteins to achieve immunogenetic reduction. Ronit Mazor et\u00a0al. reported a novel immunotoxin consisting of disulfide stabilized Fv of anti\u2010Tac antibody and PE38 with nine point mutations both in domains II and III. Moreover, they demostrated that domain II is essential to CD25\u2010mediated cell killing, which is different from CD22\u2010mediated internalization. Compared with LMB\u20102 (anti\u2010Tac(Fv)\u2010PE38), the newly constructed immunotoxin LMB\u2010142 shows both a strong cytotoxic activity in vitro and a five\u2010time lower nonspecific toxicity in mice.Bacterial toxins are a class of highly toxic proteins that are produced and released by bacteria with certain functions. Bacterial toxins have been proven to be a tool for cancer treatment due to high toxicity.95 The As the vanguard of the immunosuppressive microenvironment, Tregs promote the growth of tumors. This has prompted researchers to develop many immunotherapies for Tregs, including their function suppression and consumption. One method of depleting Tregs is to reposition bacterial toxins to take advantage of their efficient cytotoxicity to kill Tregs directly. This approach involves replacing the natural binding domain of bacterial toxins with known ligands of Treg receptors, transferring the killing mechanism of toxins to the cells rich in Treg receptors, thereby eliminating them instead. Benefits include the alleviation of immunosuppression of TME and the minimization of the toxicity associated with bacterial toxins to non\u2010target cells.In addition to using immunotoxins to treat T\u2010cell malignancies, Tregs depleting fusion protein toxins create a paradigm shift in cancer immunotherapy for other solid tumors as well. Tregs are an important population of T cells, which are regarded as the brake of the effector T cell\u2010mediated immune response. Besides, Tregs play important roles in immune tolerance, prevention of autoimmune diseases, and suppression of anti\u2010tumor immunity.103 As Studies have shown that the abundant CD25 on Tregs can consume IL\u20102 in the local environment, while the lack of cytokines will cause the apoptosis of activated effector T cells. Therefore, CD25 can be selected as an ideal targeting site. Laurene S. Cheung and co\u2010workers developed a new generation of IL\u20102 receptor\u2010targeted diphtheria fusion toxin, which has a good anti\u2010tumor effect related to the reduction of Tregs. In fact, this new\u2010generation fusion toxin has also seen a good synergy effect together with anti\u2010PD\u20101 to treat melanoma. The risk of vascular leakage and production issues  limit the clinical application of denileukin diftitox (Ontak), which is a fusion protein composed of the bacterial toxin DT and anti\u2010IL\u20102. The authors reported a production method that employed Corynebacterium diphtheriae to directly replicate biologically\u2010active and fully\u2010folded s\u2010DAB\u2010IL\u20102 as a monomer into the culture medium. Furthermore, the authors prepared a more advanced fusion protein s\u2010DAB\u2010IL\u20102(V6A) with a single amino acid mutation (V6A). Compared with s\u2010DAB\u2010IL\u20102, V6A reduced vascular leak in vitro by 50\u00d7 and lethality in mice by 3.7\u00d7. In mouse melanoma model, significant suppression in tumor growth was observed for both s\u2010DAB\u2010IL\u20102(V6A) monotherapy and the combination therapy with anti\u2010PD\u20101. The authors analyzed and confirmed that the excellent therapeutic effect was related to the depletion of Tregs and the enhancement of effector T cells.The high expression of Foxp3 in Tregs leads to a high\u2010level CD25 expression on the surface of Tregs, forming a heterotrimeric high\u2010affinity IL\u20102 receptors.105 StAlthough bacterial toxins have been widely studied on imposing the great toxicity on tumor cells for directly killing, it is also an effective method for fusion with Tregs targeted proteins for Tregs depletion and anti\u2010tumor response enhancement. More hopefully, the successful combination with immune checkpoint blockade makes it possible for bacterial toxins to be applied in cancer immunotherapy.2.3.3 They can survive in oxygen\u2010rich tissues for a long time without germination. When encountering a suitable environment, such as the hypoxic/necrotic area of the tumor core, spores will germinate and multiply. There is no severe hypoxic environment in normal human tissues, which means that spores will not show toxicity to normal human organs. Researchers have tried to inject Clostridium histolyticum spore suspension into the tumor and observed effective suppression of transplanted mouse sarcomas without obvious systemic toxicity. Not only limited to intratumoral injection, some researchers observed the death of mice due to tetanus within 48 h after intravascular injection of Clostridium spores in tumor\u2010bearing mice. The healthy mice with the same treatment were asymptomatic for 40 days. This confirms that spores exhibit tumor\u2010specific germination even with vascular administration. Clostridium novyi (C. novyi) has been widely investigated because of its extreme sensitivity to oxygen and its excellent mobility owing to the numerous peritrichous flagella. These two characteristics lead to tumor enrichment of C. novyi even with only a small amount of spore germination. Considering the toxicity, the major systemic toxin (\u03b1\u2010toxin) gene of C. novyi was removed, and a new attenuated C. novyi\u2010NT was created which has a better prospect for application because of its lower systemic toxicity. Nishant Agrawal et\u00a0al. observed that systemic injection of C.novyi\u2010NT spores in fully immune tumor\u2010bearing mice could achieve tumor regression and have long\u2010term effects  and tissue inhibitor of metalloproteinases 1 (TIMP\u20101) to attract tumoral infiltration of various immune cells. At the very beginning, it was mainly a neutrophil response, but led to the participation of monocytes subsequently. The inflammatory response can inhibit the spread of bacterial infections and provide a second layer of control besides the first layer provided by an anaerobic environment. Inflammation may also directly cause tumor cells\u2019 destruction through the production of ROS, proteases and other enzymes. Moreover, inflamation induces an effective cellular immune response, which continues to destroy the remained tumor cells that have not been killed by the bacteria. The authors observed that 30% of tumor\u2010bearing mice were cured, which is a quite gratifying result. In a later report, injection of C. novyi\u2010NT spores into the naturally occurring tumors on dogs induced a strong immune response. The authors observed that intratumoral administration of C. novyi\u2010NT spores resulted in enhanced phagocytosis and increased NK cell\u2010like function. Intravenous injection of C. novyi\u2010NT spores will lead to LPS\u2010triggered TNF\u2010\u03b1 production, LTA\u2010triggered IL\u201010 production, and also increasing of NK cell\u2010like function. This indicates that the administration of C. novyi\u2010NT spores can induce long\u2010term changes in immune cell function. In another report, John T. Heaps and colleagues injected engineered Clostridial spores into blood vessels to effectively suppress and cure human colon carcinoma in the mouse xenograft model. The engineered spores can germinate and be activated after reaching the hypoxic necrosis area of the tumor core, and then release the prodrug\u2010converting enzyme (PCE) that can convert non\u2010toxic prodrug molecule into a strong cytotoxic form at the tumor site, a process further causing tumor cells into death.As a dormant form of bacteria, spores are highly resistant.108 Th3 Since both bacteria and their components display inherent stimulation to the host immune system, thereby producing a positive anti\u2010tumor immune response, the bacteria\u2010based tumor immunotherapy has developed rapidly. From bacteria attenuation, transformation engineering, and individual component extraction and application to synergistic therapy, researchers have tried a variety of solutions to obtain the optimal efficacy. The latest research also shows that bacteria play a vital role in both the occurrence and development of tumors. Although the triangle interactions between bacteria, tumors, and the immune system are not yet clear, it is hopeful that with the deepening of research, researchers can make the best use of the great adaptability of bacteria to regulate the interaction of the other two to achieve better efficacy.Further clarifications on the interaction between tumor and the human immune system has unveiled that immunotherapy is one of the most promising approaches to cure cancer.113 Si Due to the specific colonization of bacteria in the tumor, the chance of immunotherapeutics reaching the target is greatly improved, thus reducing the repeated administration of free drugs and reducing the possible side effects. However, bacteria are complex therapeutic agents with vitality rather than precise formulations. The targeting effect of bacteria is variable, and there may be differences in efficacy between patients with different tumors during treatment. Besides, the level of immunotherapeutics expressed by bacteria may vary. Too low expression levels may result in poor efficacy, while excessive expression levels may cause autoimmune diseases. The long\u2010term stable production of bacteria in the host is very important. Moreover, some uncontrollable mutations may bring toxicity to patients in the process of bacteria proliferation. Fortunately, with the rapid development of synthetic biology, solutions to these potential problems may be available and worth of exploration. Researchers can precisely control the production and release behavior of immunotherapeutics by controlling gene copy number, promoter strength, bacterial metabolic rate, and initial bacteria injection dose. For patients with different tumors and at different stages, a personalized plan is preferred by adjusting the expression intensity of the therapeutics to yield the optimized treatment effect.Current engineered bacteria exhibit new functions that enable them to locally produce and release immune checkpoint blockers or other immunotherapeutics in tumors.61 Due Therefore, the immune cells infiltrated in the tumor may have spatially distributed heterogeneity, resulting in an unsatisfactory anti\u2010tumor effect. Some other tumor treatment methods (such as radiotherapy) have a better therapeutic effect on areas where the outer layer of the tumor has good oxygen perfusion. The combination of these therapies focusing on different areas may produce better treatment results.Although the administration of bacteria into the body can cause a variety of immune cell responses to produce anti\u2010tumor efficacy, most of the bacteria are colonized in the core necrosis area of the tumor with less distribution in the outermost area of the tumor.115 ThLactobacillus and Bifidobacterium etc.) to pathogenic bacteria. Meanwhile, most probiotics are less virulent than pathogenic bacteria, even compared with attenuated strains, which completely eliminates the worry of potential toxicity on patients.Some extracted components of bacteria are used as immunostimulators. Although they can effectively elicit anti\u2010tumor immune activation, their single action site in the entire anti\u2010tumor immune response link extremely limits the maximum efficacy. Despite that the increased dose or rounds of injection can better suppress tumor growth, some new problems come out. One of the problems is that the extraction of certain components of bacteria with high purity is a very time\u2010consuming task and may limit large\u2010scale applications. Another problem is that patients may not accept multiple injections as easily as oral administration. Therefore, replacement of oral dosage forms will help patients better accept multiple administrations. As a promising treatment approach, oral administration has attracted increasingly more attention in recent years not only because of the most natural and easiest administration, but also its self\u2010assistance that eliminates the requirement for nursing. However, it still remains a challenge as the complex physiological conditions  in gastrointestinal tract may digest or deactivate drugs or bacteria before they reach the action site. In order to avoid these problems, the therapeutics is usually wrapped in some protective materials and made into capsules to improve the utilization rate and also achieve sustained release. As another alternative and creative strategy, some researchers took full advantage of bioengineering technology to retrofit orally\u2010taken bacteria to serve as \u201cliving factory\u201d to produce \u201cproducts\u201d in the gut, which may play beneficial roles in microbiome\u2010induced anti\u2010tumor immunity. Different from tumor\u2010targeting bacteria, orally\u2010taken bacteria prefer probiotics (such as bacteria.116 MeThe complexity of bacteria as a living body determines the difficulties and risks of transforming them into weapons to fight against tumor. However, complexity is precisely the most fascinating part of bacteria, which enables scientist to fine\u2010tune the various functions of different strains to realize anti\u2010tumor activities unachievable via other therapies. This complexity is also reflected in the interactions between bacteria and the human immune system. On top of that, with the in\u2010depth understanding of their relationship, bacteria\u2010based cancer immunotherapy will provide a steady stream of power to fight against cancer.The authors declare no conflict of interest."}
+{"text": "The 0.125, 0.25, 0.5, 1, 2, and 3 mg t-PA, 20,000 U, and 40,000 U urokinase (u-PA) were taken for the corresponding blood clot for dissolution test. The blood clot volume and dissolution volume was measured at 0, 30, 60, 90, 120, and 150 min.P < 0.05). Without intervention, hematocrit (HCT) was correlated with blood clot volume and the correlation decreased with time. The 30, 60, and 90 min dissolution curves of each group showed an upward trend (P < 0.05), and the dissolution curves tended to be flat at 120 min and 150 min. The dissolution volume of.125 mg/3 ml, 0.25 mg/3 ml, 0.5 mg/3 ml rt-PA, 20,000 U, 40,000 U u-PA was higher than that of 1, 2, 3 mg/ml rt-PA (P < 0.05). The dissolution volume of.125 mg/3 ml, 0.25 mg/3 ml, 0.5 mg/3 ml rt-PA was not significantly different from 20,000 and 40,000 U u-PA (P > 0.05). Gender differences did not affect the effects of the above drugs.Without intervention, the blood clot volume of men was higher than that of women at 0, 30, 60, and 90 min (In vitro, low-concentration rt-PA has a better dissolution effect, and it shows a time-dependent effect, reaching the highest effect in 90 min. Hypertensive intracerebral hemorrhage has a high incidence, a long treatment period, and a poor prognosis, which causes a large burden on the family and society \u20133. At prin vitro was prepared, and different doses of rt-PA intervention were given to observe the relationship between rt-PA and dissolution effect. This study aimed to provide theoretical support to further optimize the treatment of hypertensive intracerebral hemorrhage.The main component of rt-PA is glycoprotein which can bind to fibrin through lysine residues and activate plasminogen bound to fibrin to turn it into plasmin, thereby dissolving blood clots or thrombus. Rt -PA is a third-generation thrombolytic drug with the characteristics of safety, efficiency, and convenience . HoweverThirty-five patients with hypertensive cerebral hemorrhage diagnosed from 2020-2-6 to 2020-6-30 in the Department of Neurosurgery of Rudong Hospital Affiliated with Nantong University and Haimen District People's Hospital were selected as the research objects. Inclusion criteria: (1) meet the diagnostic criteria for hypertensive intracerebral hemorrhage; (2) the hematoma was located in the basal ganglia, thalamus, or brain lobe; (3) consciousness disorder, hemiplegia, aphasia, limb numbness, and other neurological disorders; (4) the time from onset to hospitalization did not exceed 48 h; (5) normal cardiopulmonary function, normal blood coagulation function. Exclusion criteria: (1) age <18 years; (2) cerebral hemorrhage caused by other causes; (3) those with a history of surgery within one month; (4) abnormal blood coagulation function; (5) The time from onset to hospitalization exceeded 48 h; (6) oral anticoagulants; (7) drug users; (8) pregnant women; (9) severe heart, lung, kidney, and liver function abnormalities. Among the 35 patients with hypertensive intracerebral hemorrhage, 27 were males and 8 were females, ranging in age from 28 to 81 years old, with an average of 64.14 \u00b1 13.00 years old. The average systolic blood pressure at admission was 171.34 \u00b1 25.24 mmHg, and the average diastolic blood pressure was 96.91 \u00b1 11.66 mmHg. There were 5 cases with a history of stroke; 3 cases with a history of diabetes; 13 cases with a history of hypertension were clearly stated, only 4 cases with a history of regular use of antihypertensive drugs; 2 cases with a history of smoking; 4 cases with a history of drinking. The studies involving human participants were reviewed and approved by the Ethics Committee of Rudong County People's Hospital and Haimen District People's Hospital. The patient's family provided written informed consent to participate in this study.A total of 40 ml of arterial blood was drawn from each patient and randomly injected into 10 blood collection tubes (4 ml/tube), centrifuged, then the serum was aspirated to prepare the blood clot model, and divided into the standard group, control group, 0.125 mg/3ml, 0.25 mg/3ml, 0.5 mg/3ml, 1 mg/3ml, 2 mg/3ml, 3 mg/3ml rt-PA groups, 20,000 U, and 40,000 U urokinase (u-PA) groups. No intervention in the standard group for checking blood clot volume, the control group was added with 3 ml saline, then 0.125 mg/3 ml, 0.25 mg/3 ml, 0.5 mg/3 ml, 1 mg/3 ml, 2 mg/3 ml, 3 mg/3 ml rt-PA groups were added with the corresponding dose of the rt-PA , and the 20,000 and 40,000 U u-PA groups were added with the corresponding dose of the u-PA . Then, the tube was placed in a 37\u00b0C electric-heated thermostatic water bath , and the corresponding dissolved volume or blood clot volume was measured 30, 60, 90, 120, and 150 min after the intervention with a pipette gun .P < 0.05 was considered the difference to be statistically significant.The SPSS 21.0 data software  package was used for the statistical analysis of the data obtained in this study. The measurement data obtained in the study was verified by the Shapiro-Wilk test to conform to the normal distribution, expressed by mean\u00b1SD, and the independent-test or ANOVA was used for comparison between groups. The relationship between blood clot volume and the blood test indexes of patients was analyzed with Spearman correlation analysis. P < 0.05), and the difference was not statistically significant at 120 min, 150 min. There was no statistically significant difference between the subgroups of other clinical factors at any time point. The results were shown in The blood clot volume of males was bigger than that of females at 0, 30, and 90 min (P < 0.05). There was no correlation between other relevant blood test indexes and blood clot volume (P > 0.05). The results were shown in Hematocrit (HCT) was correlated with blood clot volume and the correlation decreased with time (P < 0.05). The dissolution volume at 120 and 150 min in the 20,000 and 40,000 U u-PA groups was similar to that at 90 min. Although the dissolution volume of the other groups continued to increase at 120 min, there was no statistically significant difference compared with 90 min (P > 0.05). The dissolution volume at 150 min in all groups was similar to that at 120 min . Among them, the dissolution volume of 0.125 mg/3 ml, 0.25 mg/3ml, and.5 mg/3 ml rt-PA was not significantly different from that of 20,000 and 40,000 U u-PA (P > 0.05). The dissolution volume of.25 mg/3 ml rt-PA was slightly higher than that of.125 mg/3 ml and.5 mg/3 ml rt-PA, but the difference was not statistically significant (P > 0.05). The dissolution volume of.125 mg/3 ml, 0.25 mg/3 ml, 0.5 mg/3 ml rt-PA, 20,000 U, 40,000 U u-PA was higher than that of 1 mg/3 ml, 2 mg/3 ml, and 3 mg/3 ml rt-PA (P < 0.05). The dissolution volume of 1 mg/ml rt-PA was higher than the dissolution volume of 2 mg/3 ml and 3 mg/3 ml rt-PA (P < 0.05). There was no significant difference in the dissolution volume of 2 mg/3 ml and 3 mg/3 ml rt-PA (P > 0.05) (The dissolution volume of different doses of rt-PA and u-PA was higher than that of the control group ( > 0.05) ; Table 3 > 0.05) .in vitro to explore the effect of fibrinolytic drugs on the hematoma. Because hypertensive cerebral hemorrhage intracranial hematoma is formed by arterial blood coagulation after arterial rupture, the experimental blood clot model was closer to the clinic, and the results were more credible. The method of constructing the blood clot model was scientific and reliable  for the publication of any potentially identifiable images or data included in this article.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by the Jiangsu Provincial Science and Technology Program (BRA2019206) and the fifth phase of Project 333 scientific research in Jiangsu Province in 2019 (BRA2019206).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Vascular endothelial cells in adults are composed of about 10-60 trillion cells, weighing about 1 kg and having a body surface area of 1~7 m2  discoverThis Special Issue, entitled \u201cEndothelial Dysfunction: From Pathophysiology to Novel Therapeutic Approaches\u201d, is focused on the pathophysiology of endothelial dysfunction, new biomarkers for endothelial dysfunction related to cardiovascular disorders or tumors, and novel therapeutic approaches for endothelial dysfunctions. This Special Issue includes 13 review articles and 6 research papers, in which several novel biomarkers or target proteins associated with endothelial dysfunction are described. New concepts, such as new biomarkers, therapeutic targets, and treatment technologies for endothelial dysfunction are, included.In this short editorial, I would like to highlight the paper that introduced a new concept related to endothelial cell dysfunction. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is an essential multifunctional protein. In 2013, the secretion of APE1/Ref-1 into the cultured medium in response to hyperacetylation was first reported . Lee et Nannelli G et al. focused on the role of the detoxifying enzyme aldehyde dehydrogenase 2 (ALDH2) in the maintenance of endothelial function . ALDH2 iThere is a new technique for treating endothelial cell dysfunction. Red and near-infrared photobiomodulation is a technology that uses light of various wavelengths to inhibit inflammation, angiogenesis, and promote blood vessel function. Although such long-wavelength light treatment technology requires extensive randomized clinical trials, it has been partially used in clinical practice . RegularBiomedicines.Finally, scientific efforts for endothelium dysfunction will contribute to the development of therapeutic and preventive substances that inhibit endothelial damage in cardiovascular diseases. With the worldwide spread of COVID-19, many researchers are working to develop specific inhibitors to inhibit vascular endothelial dysfunction. We would like to thank the many scientists who participated in peer reviews, as well as the managing editors of this Special Issue of"}
+{"text": "Androgen deprivation therapy (ADT) is the effective treating prostate cancer but is often accompanied by cancer treatment-induced bone loss (CTIBL), which impairs the patient\u2019s quality of life. In patients with nonmetastatic castration-sensitive prostate cancer (M0CSPC) who already have osteoporosis before starting ADT, appropriate bone-modifying agent intervention must be performed in parallel, as the patient has a high risk of future fracture. However, little is known about therapeutic interventions aimed at preventing the progression of CTIBL and new fractures. The present study explored the effect of once-yearly zoledronic acid 5\u2009mg (ZOL 5\u2009mg) on bone mineral density (BMD) and new vertebral fractures (VFs) in M0CSPC patients with coexisting osteoporosis before starting ADT.n\u2009=\u200926; control group, n\u2009=\u200916). The association of the ZOL 5\u2009mg with changes in the BMD from baseline to 12\u2009months and the incidence of VFs were evaluated.We conducted a retrospective, multi-institutional, cohort study involving 42 M0CSPC patients with osteoporosis who had undergone ADT with/without a single intravenous infusion of ZOL 5\u2009mg at the start of ADT  and significantly decreased the TRACP-5b  at 12\u2009months after starting ADT. Incident VFs were identified in 19.0% of all patients at 12\u2009months after starting ADT. After adjusting for the age, BMI, and changes in the LS-BMD, ZOL 5\u2009mg was not significantly associated with incident VFs .Prevalent VFs were found in 47.6% of all patients at baseline. ZOL 5\u2009mg significantly increased the lumbar spine BMD (LS-BMD) (mean rate of change: +\u20094.02%, ZOL 5\u2009mg significantly increased the LS-BMD 12\u2009months after starting ADT, and our short-term results showed that ZOL 5\u2009mg was not significantly correlated with the suppression of incident vertebral fractures. Androgen deprivation therapy (ADT), which forms the core of prostate cancer treatment, reduces the overall bone strength by decreasing the bone mineral density (BMD) and consequently the bone quality , 2. OsteThe Japanese management manual for CTIBL caused by ADT does not specify a first-line drug for the treatment of osteoporosis, only suggesting good treatment outcomes for zoledronic acid (ZOL) and denosumab . Little Given the above, the present study clarified the effects of once-yearly ZOL 5\u2009mg on BMD and the development of new vertebral fractures in Japanese men with M0CSPC who had osteoporosis before starting ADT.n\u2009=\u200926) and an osteoporosis group that did not receive ZOL 5\u2009mg .In this retrospective multi-institutional cohort study, from January 2016 to December 2017, we retrospectively evaluated 108 patients who were diagnosed with prostate cancer and were newly started on ADT for \u226512\u2009months at Koto Hospital  and Jikei University Hospital . The inclusion criteria were that the patient had osteoporosis that required treatment prior to starting ADT, with the following definition: 1) there was a proximal femoral or vertebral fracture, and/or 2) the BMD T score  before starting ADT was \u2264\u2009\u2212\u20092.5 . The excAt the time this study was conducted, all patients diagnosed with osteoporosis based on the latest DXA images underwent BMA intervention or orthopedic consultation by the attending physician.The study\u2019s retrospective protocol was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Koto Hospital  and The Jikei University School of Medicine (32\u2013123(10199), approved on Sep. 29, 2020).The primary outcome evaluated in this study was the occurrence of incident vertebral fracture. A spine surgeon (M.T.) and a radiologist (K.W.) examined the chest-to-pelvis sagittal images of CT or X-ray photographs performed before and 12\u2009months after the start of ADT. The anterior, central and posterior heights of each vertebral body from Th1 to L5 were measured. Vertebral fracture was diagnosed if at least 1 of these 3 measurements was reduced by more than 20% compared to the height of the nearest uncompressed vertebral body . VertebrFurthermore, clinical data, including the age, body height, weight, body mass index (BMI), serum creatinine levels, estimated glomerular filtration rate (eGFR), tartrate-resistant acid phosphatase 5b (TRACP-5b), prostate-specific antigen (PSA), and medical history , were also collected. ZOL 5\u2009mg is a renal excretion-type drug, and proximal tubular disorders and acute renal failure have been reported as side effects of unknown frequency . The mect-test or the chi-square test. The paired t-test was used to compare continuous variables between 2 paired groups before and after 12\u2009months of ADT. A logistic regression analysis was used to assess the effect of ZOL 5\u2009mg on incident vertebral fractures. P values of <\u20090.05 were considered to indicate statistical significance. All statistical analyses were performed using the JMP\u00ae 14 software program .Differences in clinical parameters between the ZOL 5\u2009mg group and the control group were determined by Student\u2019s The baseline characteristics of 42 patients with M0CSPC who had osteoporosis are shown in Table\u00a02) at 12\u2009months after ADT was significantly higher than the baseline value (1.06\u2009\u00b1\u20090.21\u2009g/cm2) (p\u2009<\u20090.0001), and no significant change in the FN-BMD was observed. The average rate of change in the LS-BMD with ZOL 5\u2009mg in combination with ADT was +\u20094.02%. In the control group, the LS-BMD (1.03\u2009\u00b1\u20090.16\u2009g/cm2) and FN-BMD (0.76\u2009\u00b1\u20090.09\u2009g/cm2) after 12\u2009months of ADT were significantly lower  than the baseline values . On comparing the rate of change between the two groups, the percentage changes in the LS-BMD and FN-BMD of the ZOL 5\u2009mg group were significantly higher than those of the control group .Table\u00a0p\u2009<\u20090.0001) was observed in the ZOL5 mg group, while a significant increase  was observed in the control group. No significant changes were observed in the serum creatinine levels or eGFR in either group. On comparing the change rates of each marker between the two groups, the ZOL 5\u2009mg group had a significantly lower percentage change in TRACP-5b than the control group (p\u2009<\u20090.0001). The serum creatinine levels and percentage change in the eGFR were not significantly different between the two groups.Regarding TRACP-5b, a significant decrease compared with the baseline .Incident vertebral fractures were identified in 8 of 42 patients at 12\u2009months after the start of ADT . The effect of ZOL 5\u2009mg in combination with ADT on incident vertebral fractures was evaluated by a logistic regression analysis Table\u00a0. The logIn this study, once-yearly ZOL 5\u2009mg significantly increased the LS-BMD in M0CSPC patients with osteoporosis who were introduced to ADT for the first time and significantly decreased the levels of the bone resorption marker TRACP-5b. The mean rate of change in the LS-BMD for 12\u2009months with ZOL 5\u2009mg in combination with ADT was +\u20094.02% from baseline, a result that was nearly consistent with the results of an RCT performed in general men with osteoporosis treated with once-yearly ZOL 5\u2009mg . In contUnder present circumstances, urologists may not be very concerned about osteoporosis. In fact, it has been reported that too few ADT-introduced prostate cancer patients are being screened for BMD and that many patients in need of treatment for osteoporosis remain untreated , 14. Whep\u00a0=\u20090.7774), suggesting the potential efficacy of ZOL 5\u2009mg for bone management in M0CSPC patients with osteoporosis. Further studies on the association between once-yearly ZOL 5\u2009mg administration to M0CSPC patients with osteoporosis and the risk of new fractures are required.The CTIBL management manual in Japan does not specify a first-line osteoporosis drug for cancer patients undergoing hormone depletion treatment, only suggesting good treatment results with zoledronic acid and denosumab . There hIn general, one of the issues with drug therapy in osteoporosis patients is the need to improve the treatment continuation rate. A cohort study of 40,002 people treated for osteoporosis in Pennsylvania, USA, reported that 45.2% were not taking the drug as prescribed at 1\u2009year after the start of treatment, and 52.1% had dropped out by 5\u2009years later . As in oSeveral limitations associated with the present study warrant mention. First, this was a retrospective observational study, and the assessed sample size and 12-month observation period were not sufficient for evaluating the association between ZOL 5\u2009mg and fracture risks. In this short observation period, there were not many cases in which incident vertebral fractures were confirmed. To validate the new fracture suppression effect, an RCT in which once-yearly ZOL 5\u2009mg is administered for a longer period of time with a continuous evaluation should be conducted. Second, all M0CSPC patients included in this study were Japanese. The plasma levels of insulin-like growth factor-I (IGF-I) and IGF-binding protein-3, daily physical activity and the BMI, which can affect bone metabolism and fracture development, have been reported to differ among races \u201334. The In summary, once-yearly ZOL 5\u2009mg significantly increased the LS-BMD at 12\u2009months in M0CSPC patients with osteoporosis who were introduced to ADT for the first time and significantly reduced the level of TRACP-5b, a bone resorption marker. The results of this study, only 12\u2009months in duration, showed that ZOL 5\u2009mg did not correlate significantly with the suppression of incident vertebral fractures. Further studies are needed to elucidate the correlation between ZOL 5\u2009mg and the risk of incident vertebral fractures with long-term follow-up."}
+{"text": "Continuous peripheral nerve catheters (PNCs) have been shown to provide superior postoperative analgesia, decrease opioid consumption, and improve patient satisfaction compared with single injection techniques. In order to achieve success and reliability, accurate catheter positioning is an essential element of PNC placement. An agitated solution of normal saline, D5W, or a local anesthetic solution can be produced by the introduction of air to the injectate, creating air bubbles that can enhance ultrasonographic visualization and possibly improve block success.Eighty-three patients were enrolled. Ultrasound-guided continuous popliteal sciatic nerve blocks were performed by positioning the tip of a Tuohy needle between the tibial and common peroneal branches of the sciatic nerve and threading a catheter. An agitated local anesthetic solution was injected through the catheter, viewed with color Doppler ultrasound and video recorded. A peripheral block score  was calculated based upon the motor and sensory testing at 10, 20, and 30\u2009min after block completion. The color Doppler agitation coverage pattern for the branches of the sciatic nerve was graded as follows: complete (>\u200950%), partial , or none (0%).p <\u20090.01] and block onset was faster (p = 0.03). The block success was higher in groups with partial or complete coverage of the branches of the sciatic nerve vs no coverage .The degree of nerve blockade at 30\u2009min as judged by median  peripheral block score was significant for partial or complete color Doppler coverage of the sciatic nerve injectate compared to no coverage [3  vs 8 ; Injection of an agitated solution through a popliteal sciatic perineural catheter is predictive of accurate catheter placement when partial or complete coverage of the sciatic nerve branches is visualized with color Doppler ultrasound.NCT01591603 Continuous peripheral nerve catheters (PNCs) have been shown to provide superior postoperative analgesia, decrease opioid consumption, and improve patient satisfaction compared with single injection techniques , with the first author as the principal investigator. After obtaining IRB approval, 83 ASA physical statuses I to III adult patients scheduled for foot and ankle surgery were enrolled in a prospective study conducted at Vanderbilt University Medical Center and the Nashville Surgical Center. Written, informed consent was obtained from each participating patient. Patients with any of the following were excluded from the study: <\u200918\u2009years old, body mass index (BMI) >\u200935, pregnancy, known lower extremity peripheral neuropathy, planned amputations, polytrauma patients, local anesthetic allergy, chronic pain , and patients in whom communication was a problem. Patients, in-room anesthesia providers, surgeons, and recovery room nurses were blinded to the agitation coverage pattern. All data extraction was completed by the first author and all data was de-identified for statistical analysis.The study was registered at The popliteal sciatic nerve blocks were performed in a pre-operative holding area primarily by senior level anesthesia residents and regional anesthesia fellows who were supervised by a regional anesthesiologist. A regional anesthesiologist was defined as a board-certified attending anesthesiologist on faculty at Vanderbilt University who spent the majority of their clinical time in the administration of regional anesthesia. The patient was placed in a supine position with routine monitors and provided supplemental oxygen. Midazolam and fentanyl or ketamine were administered and titrated to produce light sedation. After the operative leg was placed in a leg holder, the distal thigh and knee were sterilely prepped circumferentially and draped. Mask and sterile gloves were worn.An ultrasound machine  and ultrasound transducer  were used in each case. After the ultrasound transducer was draped with a sterile sleeve cover, it was placed against the popliteal fossa of the knee. A transverse (short axis) view of both branches of sciatic nerve  was obtained that produced an image of the two branches at the bifurcation point , tibial (plantar foot), superficial peroneal , and deep peroneal (first web space of foot). The following motor nerves were tested: tibial (plantar flexion), deep peroneal (dorsiflexion), and superficial peroneal (foot eversion). A 3-point scale was used to determine sensory or motor function: 2 = normal , 1 = partial function , and 0 = no function (no sensation/no motor). At the discretion of the operating room anesthesiologist, the block was used as the primary anesthetic or a general anesthetic was administered. Postoperatively, if the catheter was determined to be ineffective, it was adjusted or replaced for optimal patient care.Data collected for each patient included gender; age; body mass index (BMI); American Society of Anesthesiologists (ASA) classification; diabetes mellitus; sedation medication; pain medication administered before, during, and after the surgical procedure; sciatic nerve visualization; local anesthetic injected; color Doppler video; Tuohy depth; catheter depth; sensory and motor groups blocked at 10, 20, and 30\u2009min; surgical procedure; surgical region (hindfoot vs forefoot); length of surgery; and pain score . Agitation coverage scoring was done for tibial branch, common peroneal branch, and the combination of the two branches. Adobe Premiere Pro CC 2018  was utilized to capture the best image before and after agitation. Adobe Photoshop CC 2018  image analysis tool was then used to calculate the percentage of area covered by the agitated solution for the tibial branch, common peroneal branch, and the combination of the two branches. The agitation coverage pattern was graded as follows: complete (>\u200950% coverage), partial , or none (0% coverage).The peripheral block score was computed by summing the sensory and motor values, which ranged from 0 to 14. A lower peripheral block score was associated with greater blockade. Block success was judged by postoperative recovery room pain score (\u2264 3) or no need to treat pain with intra-operative or postoperative pain medications.Patient demographics and ultrasound characteristics were compared between agitation coverage groups using Kruskal-Wallis test and Pearson\u2019s Chi-squared test for continuous and categorical variables, respectively. Continuous variables were summarized with the median (10th and 90th percentile) and categorical variables were summarized with percentages.For the primary analysis, proportional odds models were used to examine the association between the peripheral block score at 30\u2009min and the agitation coverage pattern for the tibial branch, common peroneal branch, and the combination of the branches. Since the agitation coverage pattern was not randomized and possibly influenced by confounding variables, an unadjusted and adjusted propensity score analysis was performed. The propensity model included patient gender, age, BMI, ASA classification, diabetes mellitus, and surgical region. In a secondary analysis, the onset of popliteal sciatic nerve blockade was compared between agitation groups by fitting proportional odds models. Robust sandwich standard error estimates were used to test whether there was a time-related decrease in peripheral block score (block onset difference) between agitation coverage groups. This analysis also was done with an unadjusted and adjusted propensity score.All analyses were performed using R version 3.2.1, and two-sided significance levels of 5% were used to define statistical significance.Eighty-one of 83 patients were included in the study. Two patients were excluded because there was no video record displaying agitation coverage. Ten of the 81 patients did not have a complete sensory and motor evaluation, which was due to insufficient time to complete the evaluation before the patient was transferred to the operating room. There were no significant differences among the groups with respect to gender, age, BMI, ASA classification, diabetes mellitus, surgical region, catheter depth, or sciatic nerve visualization (Table p <\u20090.01] (Table p = 0.03). The overall block success rate was significantly higher in groups that demonstrated sciatic nerve branch agitation coverage compared to no coverage .The median  peripheral block score at 30\u2009min was significantly lower (greater blockade) in patients that had partial or complete sciatic nerve branch agitation coverage compared to no coverage  Table  and blocp <\u20090.01; Table p = 0.17). Compared to no coverage, partial or complete coverage of the combination of the tibial and common peroneal nerves was associated with a 14.53  fold increase in the adjusted odds ratio of obtaining greater blockade.The propensity score adjusted odds ratio of obtaining greater blockade due to partial or complete tibial branch coverage compared to no coverage was 6.46 , 6 nonfunctioning catheters were replaced, 3 from the no coverage group, 2 from the partial coverage group, and 1 from the complete coverage group. On catheter day 2, all catheters were working except 3 catheters that had become dislodged, 2 from the partial coverage group and 1 from the complete coverage group. There was no significance associated with the rate of dislodgement between the agitation groups.As a method to suggest adequate PNC placement, an agitated solution with Doppler visualization is a promising adjunct to traditional and sometimes challenging confirmation methods such as catheter visualization and spread visualization without Doppler. In this study, we demonstrated that the degree of color Doppler ultrasound enhancement of sciatic nerve area covered by an agitated injectate was associated with faster onset and higher block success for a popliteal sciatic PNC. In addition, tibial branch coverage compared to common peroneal branch coverage showed a higher correlation with greater blockade. The tibial branch of the sciatic nerve is larger than the common peroneal branch; therefore, better coverage of the tibial branch may have led to easier subsequent spread of local anesthetic to the smaller common peroneal branch. Ninety-six percent (68/71) of the catheters that had partial or complete coverage were clinically successful. Seventy percent (7/10) of catheters that did not show agitation coverage were still clinically effective. Therefore, positive agitation coverage correlated well with successful catheter placement (high sensitivity), but zero agitation coverage did not necessarily correlate with catheter failure (low specificity). In this study, the catheter was not allowed to be moved after initial placement, but in routine clinical practice a perineural catheter could be adjusted to improve agitation coverage. There were no adverse clinical events associated with the injection of the agitated solution during the study.The \u201cDhir showed a high success rate and patient satisfaction with perineural catheter placement using an agitated solution and color Doppler ultrasound; however, correlation was not made with the agitation coverage pattern (Dhir & Ganapathy, This study is not without its limitations. First, we acknowledge the small sample size. We designed this study as a pilot study with a primary goal being feasibility. Secondly, because this study was a feasibility study, we excluded classes 3 and 4 obesity as interpretation of ultrasound is usually more difficult in larger patients. However, we included class 1 obesity as we felt this would strike a balance between generalizability and feasibility. Future studies with larger sample sizes should incorporate individuals with class 2 and higher obesity levels. Many of the blocks that failed to show the agitated injectate around the sciatic nerve still resulted in successful blockade. The mechanism for this is unclear but might be related to proximal or distal migration of the catheter tip during placement. Though anesthesiologists were asked to find the location closest to the catheter prior to injecting the agitated local anesthetic, it is possible that positioning of the ultrasound probe affected the proportion of agitated injectate seen around the nerves. Finally, technologic advancements such as three dimensional ultrasound (Feinglass et al., In conclusion, we demonstrate that injection of an agitated solution through a popliteal sciatic PNC assisted in confirmation of accurate catheter placement and successful blockade when partial or complete coverage of the sciatic nerve or its branches were visualized by color Doppler ultrasound. The presence of partial or complete coverage of the branches of the sciatic nerve was associated improved propensity-adjusted odds of obtaining greater blockade. However, the lack of agitation visualization was not specific for a failed catheter position. We believe an agitated solution can be a useful tool to validate the position of a peripheral nerve catheter placed under ultrasound guidance."}
+{"text": "In the title compound, the benzimidazole ring system is inclined to the the benzene ring by 78.04\u2005(10)\u00b0. The crystal structure features O\u2014H\u22efN and C\u2014H\u22efO hydrogen bonding and C\u2014H\u22ef\u03c0 and \u03c0\u2013\u03c0 inter\u00adactions. 15H12N2O2, the benzimidazole ring system is inclined to the benzene ring by 78.04\u2005(10)\u00b0. The crystal structure features O\u2014H\u22efN and C\u2014H\u22efO hydrogen bonding and C\u2014H\u22ef\u03c0 and \u03c0\u2013\u03c0 inter\u00adactions, which were investigated using Hirshfeld surface analysis.In the title compound, C The principal weak inter\u00adactions are clearly visible. The surface coverage corresponding to O\u2014H\u22efN and C\u2014H\u22efO inter\u00adactions are 9% and 11.8%, respectively. The dark-red spot indicates significant hydrogen bonding.A Hirshfeld surface analysis was performed and the two-dimensional fingerprint plots generated (McKinnon b), C\u22efH/H\u22efC  and N\u22efH/H\u22efN 9%  inter\u00adactions. The contributions of inter\u00adactions such as C\u22efC 4.8% are negligible.The two-dimensional finger plots are given in Fig.\u00a049% Fig.\u00a04d inter\u00ad2, ZnO and other metal oxide nano-materials, which are electro-conducting in themselves  of organic mol\u00adecule is determined by a Tauc plot from the absorption spectra . The band gap energy, Eg = 4.6\u2005eV, of the title compound is very large  were mixed in a round-bottom flask in aceto\u00adnitrile  under an inert atmosphere. The mixture was then allowed to stirred for 60\u2005min at 363\u2005K then treated with 2-(bromo\u00admeth\u00adyl) benzo\u00adnitrile , and the resulting solution refluxed for 24\u2005h. After completion of this step, the solution was allowed to cool to room temperature and the mixture was poured slowly onto ice\u2013water (100\u2005ml) under constant stirring. A greenish muddy crystalline precipitate was obtained and it was left to stand at 293\u2005K for two days. After two days, a crystalline powder of 2-[(1H-benzo[d]imidazol-1-yl)meth\u00adyl]benzo\u00adnitrile was obtained  and dry KH-benzo[d]imidazol-1-yl)meth\u00adyl]benzo\u00adnitrile, 2\u2005g being mixed with 20 equimolar of potassium hydroxide  in water. The solution was refluxed at 373\u2005K for 36\u2005h, the resultant solution was then allowed to cool at room temperature and then poured onto ice\u2013water, and after that acidified using 6\u2005N HCl for protonation. The protonated solution was kept for slow evaporation. After two weeks, pale-yellow cubic crystals were obtained in good yield, which were suitable for data collection. The reaction scheme is shown in Fig.\u00a06The title compound was synthesized by hydrolysis of 2-[ I. DOI: 10.1107/S2056989021006435/ex2044Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021006435/ex2044Isup3.cmlSupporting information file. DOI: 2091351CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:"}
+{"text": "Enterobacter xiangfangensis is an emerging antibiotic-resistant bacterial pathogen from the Enterobacter genus and has the ability to acquire resistance to multiple antibiotic classes. Currently, there is no effective vaccine against Enterobacter species. In this study, a chimeric vaccine is designed comprising different epitopes screened from E. xiangfangensis proteomes using immunoinformatic and bioinformatic approaches. In the first phase, six fully sequenced proteomes were investigated by bacterial pan-genome analysis, which revealed that the pathogen consists of 21,996 core proteins, 3785 non-redundant proteins and 18,211 redundant proteins. The non-redundant proteins were considered for the vaccine target prioritization phase where different vaccine filters were applied. By doing so, two proteins; ferrichrome porin (FhuA) and peptidoglycan-associated lipoprotein  were shortlisted for epitope prediction. Based on properties of antigenicity, allergenicity, water solubility and DRB*0101 binding ability, three epitopes  were used in multi-epitope vaccine designing. The designed vaccine construct was analyzed in a docking study with immune cell receptors, which predicted the vaccine\u2019s proper binding with said receptors. Molecular dynamics analysis revealed that the vaccine demonstrated stable binding dynamics, and binding free energy calculations further validated the docking results. In conclusion, these in silico results may help experimentalists in developing a vaccine against E. xiangfangensis in specific and Enterobacter in general.Antibiotic resistance is a global public health threat and is associated with high mortality due to antibiotics\u2019 inability to treat bacterial infections.  Escherichia coli, Salmonella typhi, Staphylococcus aureus, Clostridium burdile and Enterobacter species have been seen to demonstrate resistance to several classes of antibiotics [Antimicrobial resistance is a widespread public health problem; it affects the treatment of bacterial diseases, increases the hospital stay of patients and is linked to a higher rate of human mortality ,2. Antibibiotics . The misibiotics ,6. Recomibiotics ,7. New aibiotics ,9.Enterobacter xiangfangensis is a Gram-negative, motile, and 0.8\u20131 \u00d7 1\u20131.5 \u00b5m size bacteria [E. xiangfangensis is involved in multiple hospital-acquired infections and shows high resistance to broad-spectrum antibiotics [Enterobacter cloacae complex (ECC), no appropriate vaccine is available, which makes the situation worse globally [Neisseria meningitidis serogroup B. Before that, there was no effective vaccine for N. meningitidis [E. xiangfangensis to support vaccine research against the targeted bacteria.bacteria ,11. E. xibiotics . It is aglobally . Vaccineglobally . Genome-ngitidis . Once thE. xiangfangensis is presented in The overall flow diagram followed for the in silico design of a multi-epitope vaccine against E. xiangfangensis from the National Center for Biotechnology Information (NCBI) database  followed by bacterial pan-genome analysis (BPGA). BPGA provides the core proteome of the pathogen, which is vital for broad-spectrum vaccine development [The study began with the retrieval of the complete proteome of elopment . In addielopment ,17. Aftehttp://weizhong-lab.ucsd.edu/cd-hit/, accessed on 15 March 2022) [https://www.psort.org/psortb/, accessed on 15 March 2022) [http://www.mgc.ac.cn/VFs/, accessed on 15 March 2022. The selection criteria for virulence proteins were that the proteins must have >100 bit score and >30% sequence identities [Duplicated genes are paralogous genes and are mostly not required for vaccine development . Hence, ch 2022) . Surfacech 2022) . The cytentities . Virulenentities .https://services.healthtech.dtu.dk/service.php?TMHMM-2.0, accessed on 16 March 2022, and proteins having more than 1 transmembrane helix were discarded [http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html, accessed on 17 March 2022 [https://www.ddg-pharmfac.net/AllerTOP/, accessed on 18 March 2022 [https://pepcalc.com/peptide-solubility-calculator.php, accessed on 19 March 2022. Physicochemical property analysis was performed using ProtParam Expassy at https://web.expasy.org/protparam/, accessed on 20 March 2022 [Lactobacillus rhamnosus (taxid: 47715), L. johnsonii (taxid: 33959) and L. casei (taxid: 1582) to discard host homologous and probiotic proteins to avoid autoimmune responses and accidental inhibition of probiotic bacteria, respectively [https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 22 March 2022 [Transmembrane helix analysis was performed using TMHMM-2.0 available at iscarded ,25. Protiscarded . Antigenrch 2022 . A thresrch 2022 . The allrch 2022 . Differerch 2022 . The proectively . This warch 2022 .https://www.iedb.org/, accessed on 22 March 2022 [https://webs.iiitd.edu.in/raghava/abcpred/ABC_submission.html, accessed on 23 March 2022), which is an artificial neural network based B-cell prediction tool. T-cell epitopes were predicted using B-cell epitopes as input [https://services.healthtech.dtu.dk/service.php?NetMHC-4.0, accessed on 25 March 2022). Further, common epitopes were prioritized based on percentile score. A lower percentile score indicates a stronger binder. The antigenicity, allergenicity, water solubility and toxicity of good binders were evaluated using VaxiJen [https://webs.iiitd.edu.in/raghava/toxinpred/algo.php, accessed on 28 March 2022), respectively. Additionally, the selected epitopes were also assessed for HLA-DRB1*0101 binding, and good HLA-DRB1*0101 binders were selected while the rest of the non-antigenic, probable allergic and poorly water-soluble epitopes were discarded [In the epitope selection and prioritization phase, linear B-cell epitopes were predicted using Bepipred Linear Epitope Prediction 2.0 on the IEDB web server at rch 2022 ,34. The as input . The T-c VaxiJen , Allerto VaxiJen , Innovagiscarded .In multi-epitope designing and processing, a multi-epitope vaccine was constructed and then processed ,38. In thttp://scratch.proteomics.ics.uci.edu/, accessed on 1 April 2022) [https://galaxy.seoklab.org/, accessed on 2 April 2022) [The 3D structure of the designed vaccine construct was modeled using the online Scratch Protein Predictor tool (il 2022) . Ab initil 2022) .http://cptweb.cpt.wayne.edu/DbD2/, accessed on 3 April 2022) [http://www.jcat.de/, accessed on 15 April 2022) [https://www.snapgene.com/, accessed on 15 April 2022).Disulfide engineering was performed to retain the stability of the designed vaccine construct using Designed 2.0 (il 2022) . The disil 2022) , and thehttps://bio.tools/pdbsum_generate, accessed on 16 April 2022) [https://protein-sol.manchester.ac.uk/, accessed on 16 April 2022) [https://prosa.services.came.sbg.ac.at/prosa.php, accessed on 16 April 2022) [http://tools.iedb.org/population/, accessed on 16 April 2022. During the analysis, the final set of epitopes and their respective best binding alleles were used. The tool predicts the percentage of individuals who are likely to respond to the given set of epitopes with known HLA background. The calculation was performed by considering both class I and class II combined.The secondary structure of the designed vaccine was generated using the pdbsum generate tool (il 2022) . The seril 2022)  and Prosil 2022) . Moreoveil 2022) . This waMolecular docking is an in silico approach where non-covalent interactions of the molecules such as proteins and ligands are predicted . For docMolecular dynamics simulation is a computer-based approach that is mainly used to investigate the physical movement of docked complexes . The ABMTo check the antibody and different immune responses of the host to the vaccine, an online C-ImmSim server was utilized . The calIn this research study, total of six fully sequenced proteomes, namely (i) ASM80740v4, (ii) ASM81422v1, (iii) ASM396479v2, (iv) ASM399975v1, (v) ASM1493169v1 and (vi) ASM172978v1, were retrieved from the NCBI database. In the retrieval phase, several filters were applied , humans were considered as hosts and incomplete proteomes were discarded. The BPGA pipeline was then utilized to extract core proteomes from pathogen complete proteomes. The BPGA results revealed that the pathogen consists of 21,996 core proteins, while the CD-HIT analysis revealed that the pathogen core proteins contain 3785 non-redundant proteins and 18,211 redundant proteins. The core proteins are regarded as good vaccine targets due to their major role in bacterial essential pathways and functionality . The nonAmong the above-filtered sequences, no unstable proteins were predicted. Furthermore, no significant hits against human and probiotic bacteria were found, which ensures that autoimmune reactions will not be generated if the proteins are used in subunit vaccine designing . SolubilIn the epitope mapping phase, two proteins: ferrichrome porin (FhuA) and peptidoglycan-associated lipoprotein  were subjected to B-cell epitope prediction phase. From FhuA, eight B-cell epitopes were predicted, while from Pal, four epitopes were predicted, as tabulated in 50 value (nM) less than 100 nM. The 3D structure was predicted using Scratch Protein Predictor, as shown in Physicochemical property analysis predicted 168 amino acids for the vaccine. The vaccine\u2019s molecular weight is 18.16 kDa, while the theoretical pI is 9.21. The estimated half-life is 30 h. The instability index (II) is computed to be 31.78; this classifies the protein as stable. The aliphatic index is 78.57 and the grand average of hydropathicity (GRAVY) is \u22120.277 for the final vaccine construct. The final vaccine construct also revealed an overall prediction for the protective antigen of 0.6534. The vaccine is a non-allergen, has good water solubility and has a good DRB*0101 binding score of an ICThe primary sequence of the vaccine is given in To retain the rigidity of the vaccine and remove structure errors from the vaccine structure, refinement was performed, and the results are mentioned in Escherichia coli K12 strain [In in silico codon optimization, the vaccine was converted to the DNA sequence \u201cATGATCAAACTGAAATTTGGCGTCTTCTTCACCGTCCTGCTGTCTTCTGCTTACGCTCACGGTACCCCGCAGAACATCACCGACCTGTGCGCTGAATACCACAACACCCAGATCTACACCCTGAACGACAAAATCTTCTCTTACACCGAATCTCTGGCTGGTAAACGTGAAATGGCTATCATCACCTTCAAAAACGGTGCTATCTTCCAGGTTGAAGTTCCGGGTTCTCAGCACATCGACTCTCAGAAAAAAGCTATCGAACGTATGAAAGACACCCTGCGTATCGCTTACCTGACCGAAGCTAAAGTTGAAAAACTGTGCGTTTGGAACAACAAAACCCCGCACGCTATCGCTGCTATCTCTATGGCTAACGAAGCTGCTGCTAAAGGTCCGGCTCCGACCATCGCTGCTAAACGTGGTCCGGGTCCGGGTGCTACCAAAACCGACACCCCGATCGAAAAAGGTCCGGGTCCGGGTCGTAACAACGGTACCACCGCTGAAATC\u201d and then inserted into the pET28a (+) vector as shown by red color after 6x histidine. The primary nucleotide sequence of vaccine is provided in 2 strain .In population coverage analysis, the vaccine construct was checked for world and country-wise coverage. According to IEDB population coverage, the designed vaccine construct molecule was able to cover a worldwide population of 99.74%, while the highest countrywide populations are in China (97.83%), India (97.35%) and Pakistan (97.13%). The vaccine construct has the ability to provide immunity against the pathogen in 100% of the population of Sweden, as shown in Molecular docking analysis was performed to check the binding affinity of the vaccine construct to immune cell receptors MHC-I, MHC-II and TLR-4. For docking purposes, Cluspro2.0 was utilized ; in eachMDS analysis is an in silico approach for evaluating the dynamic behavior of docked molecules. The simulation analysis consists of (i) root mean square deviation (RMSD)  and (ii)The \u201cC-IMMSIM\u201d server was utilized to check the immunogenic profile of the model vaccine for a period of about 35 days. The server predicted primary as well as secondary immune responses to the vaccine in the form of different types of antibodies. Additionally, the combination of IgM and IgG was also seen in the higher titers, followed by \u201cIgG1 + IgG2\u201d and IgM, as shown in E. xiangfangensis and other members of the genus Enterobacter, though several are under clinical investigation. In the present study, a chimeric multi-epitope vaccine construct was designed to tackle E. xiangfangensis infections by considering all sequenced strains of the said pathogen. Two proteins, FhuA and Pal, were shortlisted as good vaccine candidates and harbored both B- and T-cells epitopes. The predicted epitopes were checked for antigenicity, allergenicity, water solubility and toxicity, and only three epitopes were predicted as probable antigen, non-allergen, non-toxic, DRB*0101 binders with good water solubility. The designed vaccine construct was subjected to molecular docking study with immune cell receptors, which predicted that the designed vaccine has the ability to interact with said immune cell receptors and can evoke both humoral and cellular immunity as demonstrated by C-immune simulation. Additionally, the molecular dynamics simulation analysis revealed that the intermolecular interactions between vaccine construct and immune cell receptors are quite stable. Regardless of the promising results, experimental validations are still needed to determine the real immune protection ability of the vaccine. Currently, no FDA-approved vaccines are available against"}
+{"text": "Outbreaks of monkeypox virus infections have imposed major health concerns worldwide, with high morbidity threats to children and immunocompromised adults. Although repurposed drugs and vaccines are being used to curb the disease, the evolving traits of the virus, exhibiting considerable genetic dynamicity, challenge the limits of a targeted treatment. A pan-genome-based reverse vaccinology approach can provide fast and efficient solutions to resolve persistent inconveniences in experimental vaccine design during an outbreak-exigency. The approach encompassed screening of available monkeypox whole genomes (n = 910) to identify viral targets. From 102 screened viral targets, viral proteins L5L, A28, and L5 were finalized based on their location, solubility, and antigenicity. The potential T-cell and B-cell epitopes were extracted from the proteins using immunoinformatics tools and algorithms. Multiple vaccine constructs were designed by combining the epitopes. Based on immunological properties, chemical stability, and structural quality, a novel multi-epitopic vaccine construct, V4, was finalized. Flexible-docking and coarse-dynamics simulation portrayed that the V4 had high binding affinity towards human HLA-proteins  with low conformational fluctuations (<1 \u00c5). Thus, the vaccine construct (V4) may act as an efficient vaccine to induce immunity against monkeypox, which encourages experimental validation and similar approaches against emerging viral infections.  The global burden of monkeypox cases as a result of the 2022 outbreak is estimated to be ~73,500 (6.6% increase in October 2022) across 109 countries around the world, imposing high to moderate threats in most parts of the world [The monkeypox virus (MPXV), a double-stranded DNA (dsDNA) virus of the Orthopoxvirus genus, Chordopoxvirinae subfamily and Poxviridae family, is the etiological agent of monkeypox, a zoonotic illness having high morbidity threats in children and immunocompromised people . Historihttps://www.ncbi.nlm.nih.gov/labs/virus (accessed on 20 September 2022)). In total, we have retrieved 2798 genomes of MPXV from the NCBI database. The genomes that were flagged as complete were screened and subsequently we manually curated those datasets to exclude duplicates. The genomes of MPXV with complete assembly (n = 910) were retrieved and downloaded in FASTA format (https://github.com/tseemann/prokka (accessed on 20 September 2022)) [https://github.com/sanger-pathogens/Roary (accessed on 21 September 2022 )) [All complete genomes (n = 910) of MPXV were reannotated de novo with Prokka v1.14.6 (r 2022))  for gene 2022 ))  with a 1 2022 )) . The ann 2022 ))  to generhttp://csbio.sjtu.edu.cn/bioinf/virus-multi/ (accessed on 23 September 2022)). The knowledge of subcellular localization of viral proteins plays an important role in designing antiviral drugs [http://www.ddg-pharmfac.net/vaxijen/ (accessed on 23 September 2022)) and ANTIGENpro (http://scratch.proteomics.ics.uci.edu/ (accessed on 23 September 2022)). The VaxiJen server classifies a protein as an antigen based on auto cross-covariance transformation of protein sequences into uniform vectors of principal amino acid properties [http://scratch.proteomics.ics.uci.edu/ (accessed on 23 September 2022)), a sequence- based prediction method, was used to identify the propensity of a protein to be soluble upon overexpression [An intersection operation was performed to obtain the core genes in all the subjected genomes. Then, the non-homologous sequences to the human host proteome were filtered out using BLASTp with an E-value > 0.005 . This sial drugs . The viroperties , whereasoperties . Both thpression . The proThe shortlisted proteins were submitted to multiple tools/servers to predict the potential MHC class-I epitopes, MHC class-II T-cell epitopes and B-cell epitopes, which induce good immune responses after binding .http://crdd.osdd.net/raghava/propred1/ (accessed on 26 September 2022)), MHC-I binding predictions (http://tools.iedb.org/mhci/ (accessed on 26 September 2022)) and class-I immunogenicity (http://tools.iedb.org/immunogenicity/ (accessed on 26 September 2022)). The latter two are from the Immune Database (IEDB) analysis resource. The ProPred1 is a matrix-based web server, used to find the binding regions and proteasome cleavage sites in the given protein sequence for 47 MHC class-I alleles. The server utilizes matrices obtained from the BIMAS server to identify promiscuous MHC binders [The MHC class-I binding regions in the antigenic proteins were predicted by using three different platforms: ProPred1 ( binders  that bin binders . The pep binders . The pep binders  with a chttp://tools.iedb.org/mhcii/ (accessed on 26 September 2022)). The tool incorporates nine prediction methods: 1. IEDB recommended, 2. consensus method, 3. combinatorial library, 4. NN-align \u22122.3, 5. NN-align 2.2, 6. SMM-align, 7. Sturniolo, 8. NetMHCIIpan \u22123.1, and 9. NetMHCIIpan \u22123.2 [http://tools.iedb.org/CD4episcore/ (accessed on 26 September 2022)). The IEDB recommended a combined method to predict the MHC class-II binding peptides, and this method combines the predictions from 7-allele and immunogenicity method. The peptides can be considered immunogenic when their percentile rank is <66 [The prediction of peptide binding to MHC class-II molecules was performed by IEDB MHC-II binding predictions ) [The shortlisted MHC class-I/II epitopes were double-checked by clustering MHC-restricted alleles based on their binding specificity using MHCcluster v2.0 (r 2022)) . The serhttps://services.healthtech.dtu.dk/service.php?BepiPred-2.0 (accessed on 27 September 2022)) uses a random forest algorithm trained on the epitopes annotated from antigen\u2013antibody structures [http://crdd.osdd.net/raghava/abcpred/ (accessed on 27 September 2022)) uses ANN method of static length patterns to predict B-cell epitope in an antigenic sequence [The epitopes must be recognized by B-lymphocytes to initiate humoral immunity, which can eliminate the foreign bodies by secreting antibodies against antigens exposed in humans. The linear B-cell epitopes were identified by employing two tools: BepiPred v2.0 and ABCPred. BepiPred v2.0  and PADRE (Pan HLA-DR reactive epitope) sequences to the shortlisted epitopes . Later, https://web.expasy.org/protparam/ (accessed on 27 September 2022)). The tool gives information on a protein\u2019s molecular weight, instability index and grand average of hydropathicity (GRAVY) [https://ddg-pharmfac.net/AllergenFP/ (accessed on 27 September 2022)) and VaxiJen v2.0 [The physio-chemical and functional properties of the vaccine constructs were evaluated by the Expasy ProtParam tool ( (GRAVY) . AdditioJen v2.0  were empJen v2.0 . Based on the physicochemical and immunological suitability, the multi-epitope vaccine constructs were finalized and subjected to modelling and structural validation for further analyses.https://swissmodel.expasy.org/ (accessed on 27 September 2022)), Robetta (https://robetta.bakerlab.org/ (accessed on 27 September 2022)) and Modeller were used for this purpose [http://molprobity.biochem.duke.edu/index.php (accessed on 30 September 2022)) and Procheck (https://saves.mbi.ucla.edu/ (accessed on 30 September 2022)) [2 order parameter values of the vaccine constructs, the DynaMine online interface (https://bio2byte.be/dynamine/ (accessed on 1 October 2022)) calculated the relative residue-level backbone dynamics. Based on constrained atomic bond vector orientations, S2 values represented experimental NMR chemical shifts. S2 values of >0.8 depict rigid conformations, while lower values indicate flexibility [https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html (accessed on 1 October 2022)) was used to determine the peptides\u2019 secondary structural patterns [The 3-D structure of the finalized peptide vaccine candidates was modeled as per our previous studies combining homology and ab initio approach ,46. Swis purpose ,48. By dr 2022)) ,50,51. Uxibility . SOPMA wpatterns . The moshttps://galaxy.seoklab.org/ (accessed on 1 October 2022)) by reducing clash scores, poor rotamers, percentage Ramachandran outliers and minimizing side-chain rotamers [The RCSB-Protein Data Bank was used to retrieve the target proteins 1H15 (HLA-DR B501:01), 2FSE (HLA-DR B101:01), 1A6A (HLA-DR B103:01), 2SEB (HLADRB1 04:01), 3C5J (HLA-DRB302:02), 2Q6W (HLA-DR B3 01:01) and 2Z65 (Human-Toll-Like receptor-4). Swiss-PDB viewer (SPDBV) was used to screen these six unique HLA alleles and toll-like receptor (TLR) alleles for missing residues and undesired hetero-atoms . The prorotamers . Finallyrotamers ,56,57.https://bioinfo3d.cs.tau.ac.il/PatchDock/ (accessed on 3 October 2022)) [https://bioinfo3d.cs.tau.ac.il/FireDock/ (accessed on 3 October 2022)) extension (rapid interaction refinement in molecular docking) was adopted, which provided the top 10 best solutions for final refinements based on global binding energy, atomic contact energies and intermolecular bonding contributors. The PatchDock docked complexes were therefore improved and re-scored [The vaccine construct was finally tested for interactions with six different HLA alleles and TLR-4 using the PatchDock web interface (r 2022)) . PatchDoe-scored . The gloe-scored ,61.http://biocomp.chem.uw.edu.pl/CABSflex2 (accessed on 4 October 2022)) was employed to calculate the residue-level fluctuations of the target-vaccine complexes. The Root Mean Square Fluctuation (RMSF) trajectories, which were produced based on default restraint parameters, were used to determine the residue-level propensity, signifying the most advantageous conformation. Using an all-atom force-field-based MD simulation, the consensus protein variations in an aqueous environment were determined . The default mode was selected, with minimum and maximum conformational distances of 3.8 and 8.0, respectively, and gap = 3 (minimum distance between the previous and next amino acid in the chain to be restrained). The relative atomic fluctuations were plotted to validate the most stable docked complex.The CABSflex interface , were antigenic and treated as vaccine candidates for further analysis. The results obtained from VaxiJen v2.0, ANTIGENpro and SolPro are given in The MHC class-I T-cell epitopes were identified by subjecting the three antigenic protein sequences to ProPred1. It resulted in the identification of 61, 70 and 557 epitopes in L5L, A28 and L5 proteins, respectively. In addition to ProPred1, we submitted antigenic protein sequences in IEDB. From IEDB, a total of 26,331 epitopes were found. After considering of cut-off IC50 values < 50 nM, 18, 27 and 39 unique epitopic sequences were identified in L5L, A28 and L5 proteins, respectively. In order to increase the efficacy of our vaccine constructs, we chose the epitopes commonly (n = 42) presented in proteins from both tools and tested for their immunogenicity. The IEDB immunogenicity analysis revealed, out of 42, only 23 epitopes as most immunogenic epitopes. Additionally, these 23 epitopes were evaluated for their toxicity and antigenicity, and their scores are provided in The MHC class-II T-cell epitopes were identified by the IEDB server, which generated a total of 23,779 epitopes in three shortlisted proteins. With percentile rank < 0.2 and IC50 < 50 nM, the L5L, A28, and L5 proteins contained unique 4, 33 and 67 MHC class-II T-cell epitopes, respectively. These were considered as good binders to MHC class-II molecules. Subsequently, we checked immunogenicity for these good binders by CD4+ T-cell immunogenicity prediction tool and observed that only five epitopic sequences presented in the L5 protein were immunogenic with percentile rank cut-off of <66. The immunogenic MHC class-II T-cell epitopic sequences in the other two proteins, L5L and A28, were absent. Moreover, the five epitopic sequences in the L5 protein were non-toxic and antigenic, as predicted by ToxinPred and VaxiJen v2.0 tool. Hence, they were considered for our vaccine construction. The immunogenicity, antigenicity, and toxicity of the finalized MHC class-II T-cell epitopes are given in MHC class-I and MHC class-II alleles were further investigated based on their IC50 value. All the finalized epitopic sequences (12 MHC class-I T-cell epitopes and 5 MHC class-II T-cell epitopes) were independently evaluated for their MHC interaction analysis using MHCcluster v2.0. The epitopic sequences were clustered by interaction with HLA, and the interacted alleles were shown as a heat map . In the Along with cellular immunity, humoral immunity is also responsible for eliminating the pathogen from the host. Our study used BepiPred v2.0 and ABCPred to predict the B-cell epitopes. From ABCPred, 10, 14 and 9 B-cell epitopic sequences were found in L5L, A28 and L5 proteins, respectively. In addition, the resultant epitopes were further scrutinized based on the BepiPred v2.0 . In the The overlapping and distinctive epitopes were chosen for vaccine construction from the shortlisted MHC class-I, MHC class-II T-cell epitopes, and linear B-cell epitopes .Hence, the overlapping epitopes would have the ability to induce both innate and humoral immunity. This study used \u03b2-defensin, heparin-binding haemagglutinin (HBHA), HBHA conserved and L7/L12 ribosomal proteins as the adjuvants. The epitopes were placed in various combinations after adjuvant. The linkers EAAK, GPGPG, and RVRR were used to separate the adjuvants, PADRE sequence, and epitopic sequences, respectively. The EAAK linkers were added in the N and C terminals of the adjuvants. These linkers are immunogenicity boosters. The PADRE sequence was added in order to increase the efficacy of the vaccine by stimulating CD4+ cells. Finally, His Tag was added to the C-terminal to form the final vaccine construct. The classical form of vaccine constructs is shown in The designed vaccine constructs were then assessed for their properties to be considered as an effective vaccine. The allergenicity, antigenicity and physio-chemical properties of 16 vaccine constructs are given in Foremost, the vaccine constructs were tested for allergenicity, and it was found that all our vaccine constructs were non-allergen. Upon checking for their antigenicity, it was observed that V6, V10 and V14 were non-antigenic in nature. Thus, we excluded these vaccine constructs from our study. The physio-chemical properties were calculated for the remaining vaccine constructs by the ProtParam tool. All vaccine constructs showed high solubility, and their molecular weight ranged from 26\u201339 kDa, with pI score of 9.11\u201310.40. The GRAVY value ranging between \u22120.2 and \u22120.6 can elicit a good immunogenic response. While comparing the stability among vaccine constructs, it was noted that only V4, V8, V12, and V16 were more stable compared to other vaccine constructs. Thus, we finalized these four predicted vaccine constructs for our study and further evaluated them through structural parameters. A combined approach comprising homology and ab initio approaches was used to model the vaccine constructs, followed by structural validations. The final model or the most suitable vaccine construct, i.e., V4, was chosen based on the highest Ramachandran favored region (96.06%) and overall quality factor (99.05). V4 possessed marginally lower backbone rigidity than V8, V12 and V16. The relative structural properties of the models are given in 2-scores depicting backbone flexibility also showed V4 having marginally lesser rigidity (0.783) than the other vaccine constructs (0.785) in the multi-epitopic domain. The residue-wise S2-scores are given in The average SThe docked complexes of V4 depicted its selective affinity towards different HLA-alleles and TLR-4. According to the lowest global binding energy, V4 possessed the highest affinity towards two HLA-alleles, 1H15  and 2FSE  besides TLR-4 or 2Z65 . The same was contributed by attractive van der Waal forces, atomic contact energies and hydrogen bonds . The strong hydrophobic interaction between 26 amino acid residues of the V4 with 29 residues of the \u03b1 and \u03b2-chains of the target 1H15, besides four hydrogen bonds  and one salt-bridge (Lys196), contributed to its superior binding profiles, as given in The relative dynamics of V4 interacting with the HLA-alleles and TLR-4 validated the docking profiles. It was observed that RMSF derived from the coarse-grained simulations suggested a relative low difference in average fluctuations (<1 \u00c5) among the docked complexes. However, the average fluctuations of V4 while interacting with 1H15 (0.875) and 2FSE (0.964) were the lowest as compared to the rest, which further validated their low binding energy (from docking) and stable conformation. The relative average RMSF values are illustrated in MPXV is a dsDNA orthopoxvirus causing emerging and re-emerging outbreaks in various parts of the world, with increasing mortality rates in children and immunocompromised people ,3. The dSince immunoinformatics approaches are used as popular first-line vaccine design techniques, there have been a few notable studies on the same approach adding to the quest to design suitable multi-epitopic MPXV vaccines ,63,64,65Immunogenicity can be induced by coupling B-cell and T-cell epitopes. The humoral immune response elicited by B-lymphocytes comprises pathogen-specific antibodies that can effectively combat viral loads by neutralizing the antigenic determinants. On the other hand, cytotoxic T-cells (CTLs) activated by MHC class-I epitopes can engender long-lasting protection against virus-infected cells ,67,68. ICellular immunity plays an influential role against viral infections, activating CD4+ T-cells, CD8+ T-cells and B-cells . Outer-m2-score < 0.8) of V4 over the others was favorable for it to establish transient interactions with its HLA-targets [The set of 16 vaccine constructs were prioritized out of various combinations based on immunological complementarity and chemical stability. The finalized vaccine constructs V4, V8, V12 and V16) were validated with docking and dynamics analyses. All the finalized vaccine constructs have L7/L12 ribosomal proteins as adjuvants, which are more stable than other vaccine constructs with \u03b2-defensin, HBHA and HBHA conserved proteins. Firstly, the chosen constructs were modelled using a combined extended modelling approach to resolve ambiguities concerning low-compatibility (much less sequence identity/coverage) modelling templates. The most suitable construct, i.e., V4, represented high-quality structural profiles and backbone dynamics to be considered for further analyses. The relatively low backbone rigidity  to encourage extensive in vitro/in vivo research and development.Emerging and re-emerging MPVX infections have threatened the healthcare sector due to evolving genomic traits and a lack of targeted therapy. The present study endeavored to screen potential vaccine targets through extensive pangenome analyses followed by epitope identification and designing suitable multi-epitopic vaccine constructs. Immunologically compatible and safe vaccine constructs established high affinity and stability towards interacting with human HLA-proteins and TLR-4. A potent vaccine construct was finally designed to encourage future experimental evaluations."}
+{"text": "Background and Objectives:\u00a0Citrobacter freundii (C. freundii) is an emerging and opportunistic Gram-negative bacteria of the human gastrointestinal tract associated with nosocomial and severe respiratory tract infections. It has also been associated with pneumonia, bloodstream, and urinary tract infections. Intrinsic and adaptive virulence characteristics of C. freundii have become a significant source of diarrheal infections and food poisoning among immune-compromised patients and newborns. Impulsive usage of antibiotics and these adaptive virulence characteristics has modulated the C. freundii into multidrug-resistant (MDR) bacteria. Conventional approaches are futile against MDR C. freundii. Materials and Methods: The current study exploits the modern computational-based vaccine design approach to treat infections related to MDR C. freundii. A whole proteome of C. freundii (strain: CWH001) was retrieved to screen pathogenic and nonhomologous proteins. Six proteins were shortlisted for the selection of putative epitopes for vaccine construct. Highly antigenic, nonallergen, and nontoxic eleven B-cell, HTL, and TCL epitopes were selected for mRNA- and peptide-based multi-epitope vaccine construct. Secondary and tertiary structures of the multi-epitope vaccine (MEVC) were designed, refined, and validated. Results: Evaluation of population coverage of MHC-I and MHC-II alleles were 72% and 90%, respectively. Docking MEVC with TLR-3 receptor with the binding affinity of 21.46  occurred through the mmGBSA process. Further validations include codon optimization with an enhanced CAI value of 0.95 and GC content of about 51%. Immune stimulation and molecular dynamic simulation ensure the antibody production upon antigen interaction with the host and stability of the MEVC construct, respectively. Conclusions: These interpretations propose a new strategy to combat MDR C. freundii. Further, in vivo and in vitro trials of this vaccine will be valuable in combating MDR pathogens. Citrobacter spp. are emerging and opportunistic dwellers of the human gastrointestinal tract, inhabitants of animals\u2019 intestinal tracts, wastewater, and soil. Citrobacter freundii is a Gram-negative, facultatively anaerobic, nonsporulating, and rod-shaped bacillus bacterium that belongs to the family Enterobacteriaceae [Citrobacter freundii is the most infectious species of Citrobacter spp. It participates in an array of nosocomial contagious diseases, including bloodstream infections (BSIs), respiratory tract infections (RTIs), biliary tract infections (BTI), and urinary tract infections (UTIs) [freundii have also been reported to have intrinsic and adaptive virulence characteristics that cause diarrheal infections and food poisoning in the gastrointestinal tract of human beings, generally found in neonates and immunosuppressed patients [eriaceae . Citrobas (UTIs) . It has s (UTIs) . Certainpatients .Citrobacter freundii has acquired resistance against multiple antibiotics, including extended-spectrum \u03b2-lactamase (ESBL), carbapenemase, and cephalosporins, and there are also strains of quinolone-resistant Citrobacter freundii. It has become a significant challenge for healthcare departments as most cases are associated with hospital-acquired infections, perpetuating hospitalization and immortality in patients relying on antibiotics [Imprudent applications of antibiotics invoke several resistance mechanisms, prompting multidrug resistance (MDR) among various bacteria, especially Gram-negative bacteria ,6. Multiibiotics .Multidrug resistance of this opportunistic bacterium reduces the effectiveness of antibiotics so that novel antibiotics face resistance opposition strains of bacteria, consequently complicating the treatment of infected individuals. Immunotherapeutic and novel therapeutic approaches are considered to mitigate the emerging resistance mechanism in bacteria as there is no proper antibiotic to treat this infection-causing bacteria . FormerlCitrobacter freundii-related proteins assessed through proteome subtraction. Multiple computational-based bioinformatics tools were applied to predict PTO predicter T lymphocytes (HTL) and cytotoxic T lymphocytes (CTL) epitopes from the subtractive proteome. Putative epitopes were shortlisted based on outer membrane, pathogenicity, antigenicity, allergenicity, and immunogenicity. Highly antigen, nonallergen, and nontoxic peptide- and mRNA-based vaccines were constructed. Furthermore, the secondary and tertiary structures of the vaccine were predicted, codon-optimized, and validated through immune simulation and molecular dynamic simulation. These interpretations will assist researchers in developing precise and effective peptide- and mRNA-based vaccines against multidrug-resistant Citrobacter freundii.The current study aims to design a novel peptide- and mRNA-based vaccine by evaluating Citrobacter freundii (strain: CWH001) proteome through UniProt ID: UP000229029 (https://www.uniprot.org/) (accessed on 15 January 2022) [The Universal Protein Resource (UniProt) was accessed for the subtraction of ry 2022)  and chrory 2022) . The flohttp://cello.life.nctu.edu.tw/cello2go/) (accessed on 15 January 2022), a web server for the subcellular localization of proteins, was applied to screen pathogenic proteins based on their molecular localization function [CELLO (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) (accessed on 17 January 2022) [https://www.ddg-pharmfac.net/AllerTOP/) (accessed on 17 January 2022) [The CD-HIT suite (weizhongli-lab.org) a database for clustering protein sequences, was applied for screening nonconcurrent and contrasting protein sequences with default parameters of 0.8 and 80% as cut-off value and identity percentage, respectively . Vaxijenry 2022) . PathogeTo evaluate whether screened proteins were embedded in the cell membrane, we ran the TMHMM v.2.0 web server  . The hidhttp://tools.iedb.org/bcell/) (accessed on 19 January 2022) [http://tools.iedb.org/main/tcell/) (accessed on 19 January 2022), where ANN 4.0 method was performed, sorting predicted epitopes by IC50 value, for the prediction of CTL epitopes [http://tools.iedb.org/main/tcell/) (accessed on 20 January 2022) [http://tools.iedb.org/population/) (accessed on 20 January 2022) to recognize the interaction between MHC-I and MHC-II and their respective T-cell lymphocyte epitopes [Putative B-cell or antibody epitopes, helper T-cell lymphocytes (HTL), and cytotoxic T-cell lymphocytes (CTL) were predicted utilizing different online servers. Immune Epitope Database (IEDB), an online server, was performed under the Bepipred linear epitope prediction 2.0 method to predict linear B-cell epitopes  [The best putative epitopes were selected to design an mRNA-based vaccine against d GGGGS)  along wid GGGGS) .The construction of a peptide-based vaccine was similar to the mRNA vaccine construct, except that adjuvants such as \u03b2-defensin epitopes were joined together through EAAK, AAY, GPGPG, and KK linker, and the \u03b2-defensin adjuvant. B-cell, cytotoxic T-cell, and helper T-cell epitopes from six shortlisted proteins were linked through EAAK, AAY, GPGPG, and KK linkers and adjuvants .https://web.expasy.org/protparam/) (accessed on 22 January 2022) [http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html, accessed on 22 January 2022) and Aller TOPv 2.0 . Toxicity analysis was performed using the ToxinPred webserver  [https://loschmidt.chemi.muni.cz/soluprot/ accessed on 24 January 2022) [Physiochemical properties of MEVC were predicted by applying the Expassy Protpram tool (ry 2022) . Antigenry 2022) , and forry 2022)  was utilhttp://bioinf.cs.ucl.ac.uk/psipred/, accessed on 28 January 2022), was run to predict the secondary structure of the vaccine construct under default parameters [https://galaxy.seoklab.org/, accessed on 1 February 2022) [https://saves.mbi.ucla.edu/, accessed on 1 February 2022), which were applied for the stereochemistry analysis using the Ramachandran plot [https://prospro SAvices.came.sbg.ac.at/prosa.php, accessed on 1 February 2022) [PSIPRED, an online server (rameters . RoseTTAry 2022) , PROCHECry 2022) , and ERRry 2022)  servers ran plot , and atory 2022) , which vhttps://cluspro.bu.edu/login.php, accessed on 4 February 2022) [http://cadd.zju.edu.cn/hawkdock/, accessed on 4 February 2022) [Molecular docking of vaccine constructs and human ligand TLR-3 (2A0Z), retrieved from RCSB PDB, was performed using protein\u2013protein docking server Cluspro 2.0 (ry 2022) . To intery 2022)  was run http://www.jcat.de/Literature.jsp, accessed on 6 February 2022) [E. coli as reference host. Moreover, the optimized sequence was run in SnapGene software  for restriction site generation; DNA fragments and two restriction sites were inserted as desired vector for cloned plasmid formation [The JCat tool (ry 2022)  was performation .https://www.iac.rm.cnr.it/~filippo/c-immsim/index.html, accessed on 7 February 2022), was utilized to evaluate immune response generated in the form of antibodies against desired constructed antigens [C-ImmSim, a web server ImmSim  was used for the molecular dynamic simulation analysis to describe the physical properties of atoms and molecules of the vaccine concerning its stability [To ensure the stability of the vaccine construct-receptor (MEVC-TLR-3), the Imod server  to design MEVC as shown in Citrobacter freundii, highly antigenic and nonallergen epitopes were selected. In the mRNA-based vaccine construct, two epitopes of B-cell from five different protein sequences were selected, making up a total of 10 epitopes of each immune cell; and one epitope of B-cell was selected from a single protein sequence, thus making up a total of 11 B-cell epitopes. Likewise, there were 11 HTL epitopes and 11 TCL epitopes from six different protein sequences. These epitopes were joined through linkers  with an additional signal peptide, five m7G Cap, Kozak sequence, and five untranslated regions (UTR) along with the addition of stop codon followed by poly-A tail for the construction of the mRNA vaccine as described in For the construction of an mRNA-based vaccine against emerging MDR Citrobacter freundii, 11 HTL, 11 B-cell, and 11 TCL epitopes from six different protein sequences were selected and linked through linkers along with adjuvants, and constructed a peptide-based MEVC with a length of 414 amino acids analyzed for antigenicity, toxicity, allergenicity, solubility, and physiochemical properties, as shown in Similar to the case with designing a peptide-based vaccine against emerging MDR The tertiary structure of the vaccine construct from Rosetta was visualized in PyMOL, as shown in E. coli, and then formed DNA fragments exploited in the pET28a-MH6 vector for cloning, as illustrated in Computational cloning was performed through the codon optimization tool, JCat, which improved the DNA sequence of the vaccine construct with the enhanced value of CAI 0.96 and GC content of about 51.3. The optimized vaccine constructs, due to enhanced codon sequence, provided better results when subjected to protein expression in Molecular dynamics simulations interpret results in B-factor mobility, and atomic index graph predicts the vaccine construct reliability in the environment. The simulation uses the I-Mods web server, as shown in Citrobacter freundii is the most infectious species of Citrobacter spp., which efficiently play a role in an array of nosocomial and community-acquired infections [Citrobacter freundii impede the activity of broad-spectrum antibiotics, thereby emerging as highly opportunistic multidrug-resistant Citrobacter freundii. MDR Citrobacter freundii has become challenging in regard to treating its infections, so there is a need for a new immunoinformatics-approach-based therapy to treat such infections [fections . Inherenfections . This apfections ,44.Citrobacter freundii to treat its infections effectively. A whole proteome was retrieved, and pathogenic nonhomologous proteins were screened. These screened proteins were further analyzed to exclude overlapping proteins, and transmembrane helices analysis was performed to predict signal transduction across the cell membrane [This study aimed to construct a vaccine against the emerging multidrug-resistant membrane . Immune membrane . Multi-emembrane ,47,48.Molecular docking analyses were performed between the vaccine construct human toll-like receptor 2A0Z docked results, with the lowest binding affinity selected as it shows a bond strength between receptor and vaccine. Further, validations of vaccine construct were carried out to predict the best interpretations, stability, and immune-level interaction following the protocol from a previous study . InitialCitrobacter freundii, this vaccine could be a potential therapeutic candidate to combat various nosocomial and community-acquired infections.The primary objective of creating a vaccination was to induce long-term memory. B- and T-cell activation must coincide with accomplishing this production. The host will be prepared to produce a rapid and efficient defense against future infections. However, the vaccine\u2019s success relies heavily on strategically deploying a subset of antigens called epitopes. As a result, it is crucial to locate the epitope that may trigger the merging of both cells to develop an effective vaccination. In addition to IL-4 and IFN-, the HTL can also produce IL-10. Human T-lymphotropic (HTL) epitopes are shown on antigen-presenting cells (APCs), and lymphocytes can release chemokines that play critical roles in the immune system\u2019s defense against these microorganisms. After successfully eliminating infection, all immune cells, except memory cells, perish. Recognizing the antigenic epitopes is accomplished by the B-cell membrane-bound immunoglobin receptor. This way, the epitopes are induced and processed before being presented to T-cells via MHC class-II molecules. T-cell receptors (TCR) are specific for recognizing them. Because of this, B-cells undergo a differentiation process into plasma cells, which then generate antibodies to fight off invading memory and foreign cells. Vaccines effectively eradicate infections or diseases for the long term with minimal chances of resistance compared to other therapeutic measures such as antibiotics. The vaccine can produce enduring immunity in the host through acquired immunity. Against Citrobacter freundii. Therefore, an immunoinformatics-approach-based mRNA vaccine was designed from selected putative epitopes. Physiochemical properties evaluation and immune simulation demonstrated the potency of the vaccine construct. The vaccine design elicits the anticipated physiochemical and immune responses. The immunization was shown to produce an immune response that was consistent with our goals in the immunological simulation. This framework should be considered as a potential candidate for in vitro and in vivo research in contraindications to Citrobacter freundii, with numerous serological tests performed to validate the trigger of reaction on demand.A potential therapeutic strategy is needed to restrict the MDR"}
+{"text": "Burkholderia pseudomallei, a gram-negative soil-dwelling bacterium, is primarily considered a causative agent of melioidosis infection in both animals and humans. Despite the severity of the disease, there is currently no licensed vaccine on the market. The development of an effective vaccine against B. pseudomallei could help prevent the spread of infection. The purpose of this study was to develop a multi-epitope-based vaccine against B. pseudomallei using advanced bacterial pan-genome analysis. A total of four proteins were prioritized for epitope prediction by using multiple subtractive proteomics filters. Following that, a multi-epitopes based chimeric vaccine construct was modeled and joined with an adjuvant to improve the potency of the designed vaccine construct. The structure of the construct was predicted and analyzed for flexibility. A population coverage analysis was performed to evaluate the broad-spectrum applicability of B. pseudomallei. The computed combined world population coverage was 99.74%. Molecular docking analysis was applied further to evaluate the binding efficacy of the designed vaccine construct with the human toll-like receptors-5 (TLR-5). Furthermore, the dynamic behavior and stability of the docked complexes were investigated using molecular dynamics simulation, and the binding free energy determined for Vaccine-TLR-5 was delta total \u2212168.3588. The docking result revealed that the vaccine construct may elicit a suitable immunological response within the host body. Hence, we believe that the designed in-silico vaccine could be helpful for experimentalists in the formulation of a highly effective vaccine for B. pseudomallei. Burkholderia pseudomallei, a member of the family Burkholderiaceae, is a rod-shaped, gram-negative, motile, and multitrichous flagella bacterium typically of 1\u20135 \u03bcm long and diameter of 0.5\u20131.0 \u03bcm  construct has many advantages over the single peptide-based vaccine. MEVC elicits both humoral and cellular immune responses because it comprises both B and B-cell derived T-cell epitopes , 17. OthB. pseudomallei were obtained from the National Center for Biotechnology Information (NCBI)   followedn (NCBI) . CD-HIT n (NCBI)  was usedhttps://services.healthtech.dtu.dk/service.php?TMHMM-2.0). Physiochemical properties like molecular weight, theoretical index, instability index, and GRAVY (hydrophilicity check) for proteins with transmembrane helices < 2 were checked by using the Protparam tool (https://web.expasy.org/protparam/). The molecular weight of the proteins should be <100 kDa as they can be easily purified from the cell, and their instability index should be <45. Further, antigenicity and allergenicity of the proteins were checked via Vaxijen v2.0 . Epitopes with IC50 <100 nM were considered good binders and further scrutinized for antigenicity, allergenicity, toxicity, and solubility. Only epitopes that were antigens, non-allergen, non-toxic, and soluble were used to design a multi-epitope vaccine construct.All the protein sequences meeting the criteria of being a potential candidate and shortlisted from all the checks were then used for epitope mapping utilizing the Immune Epitope Database and Analysis Resource (IEDB) . The proPopulation coverage analysis of the designed vaccine was evaluated using the Population coverage tool of IEDB. The alleles which cover most of the world population were used for the analysis.Shortlisted epitopes were linked together to design a vaccine construct. GPGPG linkers were used to link epitope to epitope while EAAAK linker was used to connect epitopes peptide with an adjuvant. The adjuvant molecule was used to boost vaccine immune responses. Here, we used four different adjuvants resulting in four different constructs which were further analyzed for physiochemical properties, antigenicity, allergenicity, adhesion probability, and secondary structures. Construct with Cholera toxin-B (CTB) adjuvant met the criteria to be considered best amongst the four. MALP-2 (GNNDESNISFKEK) was added to the construct, EAAAK linker was used to link the last epitope and MALP2 sequence. MALP-2 (macrophage activating lipoprotein) is a toll-like receptor (TLR) agonist mostly used to treat certain infections and also as an adjuvant for a vaccine. Physiochemical properties like molecular weight, hydrophilicity, theoretical, and instability index of the final vaccine construct were evaluated by the ProtParam tool . We predhttp://bioinf.uab.es/aggrescan/).The cabs-flex analysis is a computational approach to investigating the structural flexibility of the protein. The multi-epitopes peptide vaccine was subjected to coarse-grind simulations using Cabs-flex 2.0 . The anaDisulfide by Design 2.0 was used to perform disulfide engineering of the vaccine model. It is usually done to identify unstable residues being mutated into cysteine residues narrating the induction of disulfide bonds making the structure stable .in-silico cloning via Snapgene (in silico cloning was done in vector pET-28a (+).Java Codon Adaptation Tool (JCat)  was usedSnapgene . The in A computational immune simulation is an approach to analyzing the vaccine construct for its immunogenicity and ability to induce immunity. This analysis was done through the C-ImmSim webserver . The webhttp://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html) and Discovery Studio v2021.Toll-like receptor-5 (TLR-5) and vaccine model (ligand) were subjected to molecular docking using an online docking tool ClusPro 2.0 . Severalvia (Assisted Model Building with Energy Refinement tool) AMBER 20 software. The simulation was conducted for 50 ns to understand the dynamic behavior in an aqueous solution. The force field \u201cff14SB\u201d was used to generate parameters for both the vaccine and the TLR5, followed by incorporation of complex into TIP3P water box by maintaining 12 \u00c5 padding distance. The Na+ ions were added to neutralize the system. The carbon alpha atoms, non-heavy atoms, hydrogen atoms, and solvation box energies were minimized for 500, 1,000, 300, and 1,000 steps, respectively. Langevin dynamics were applied to maintain the system's temperature by the execution of system heating to 300 K for 20ps. The SHAKE algorithm was applied to constraint hydrogen bonds. The production run trajectories were analyzed by using the AMBER CPPTRAJ module. Binding free energies were predicted using the MMPBSA.py module .Molecular dynamics simulation was performed for the docked complex of the vaccine model and TLR5 B. pseudomallei to get core proteome . The protein sequences with a sequence identity of more than 30% and a bit score >100 were considered the best vaccine targets. Virulent proteins numbered 17 in the extracellular group, 18 in the periplasmic group, and 12 in the outer membrane group. The sequences were further analyzed for the presence of transmembrane helices that should be 0 or 1. The number of transmembrane helices was 0 for extracellular proteins, except for one. In the periplasmic group, only one had transmembrane helices out of 18 proteins while the remaining were lacking the transmembrane helices. Out of the 12 outer membrane proteins, only three  had transmembrane helices, while the rest had 0. In all three groups, the proteins with no transmembrane helices were subjected to physicochemical evaluation. The parameters evaluated were molecular weight, theoretical index, GRAVY (Grand average of hydropathy), and instability index. Molecular weight was <100 kDa, while the GRAVY calculated was negative and the proteins having an instability index >40 were considered unstable proteins. The instability index of 15 extracellular proteins, 13 periplasmic, and nine outer membrane proteins were stable and within the range of 40. All these stable proteins were subjected to antigenicity (threshold 0.5), allergenicity, solubility, and adhesion stability (threshold 0.5) checks. All the non-antigenic and allergen proteins were discarded including eight extracellular, four periplasmic, and two outer membrane proteins. Similarly, the poorly soluble and the proteins having adhesion probability <0.5 were discarded, and only antigenic, non-allergens, soluble, and having adhesion probability were shortlisted as potential vaccine candidates. Four proteins were filtered out of which three  were from the extracellular group and only 1 (core/3042/1/Org1_Gene5247) was a periplasmic protein as listed in In epitope mapping, T-cell epitopes were predicted from B-cell epitopes for eliciting both humoral and cellular immune responses. Using the shortlisted proteins as input sequences, the B-cell derived T-cell epitopes were predicted by IEDB. The B-cell epitope prediction tool of IEDB was used to predict potential peptides acting as B-cell epitopes. Peptides having a value \u22650.5 (threshold) were considered as B-cell epitopes. Thirteen peptides were predicted as B-cell epitopes. A total of 78 T-cell MHC class II binding epitopes were predicted from the B-cell epitopes. These epitopes were then used to predict the MHC class I binding epitopes on T-cells, and 120 of these epitopes with lengths of 9 or 10 residues were selected based on their percentile rank. The compete reference set of alleles was selected as a parameter for MHC class II and I binding epitopes prediction.The predicted epitopes were prioritized using different parameters. First, they were subjected to MHCPred to select epitopes with IC50 values <100 nM to confirm the epitopes as good binders. With the help of this analysis, 64 epitopes were chosen and further examined by VaxiJen for antigenicity; only 52 epitopes were antigenic, of which 24 were non-allergen. All the epitopes were non-toxins. Five epitopes were predicted by Innovagen to be poorly soluble and were deleted while overlapping ones were also removed. Finally, 10 epitopes listed in Population coverage analysis by IEDB was performed for the shortlisted epitopes to estimate their binding probability to MHC molecules covering the world population . World pvia the EAAAK linker at the end presumed to be involved in enhancing the antigenicity of the construct. The CTB vaccine construct along with MALP 2 was again evaluated for physiochemical properties thus having a molecular weight of 29.01 kDa, theoretical index of 8.92, \u22120.65 GRAVY, and stable with a 23.28 instability index. It was non-allergen and antigen with antigenicity of 1.0059.The 10 shortlisted epitopes that were antigenic, non-allergen, non-toxic, and soluble were considered as potential epitopes to be a part of the vaccine construct. Epitopes were chosen using GPGPG linkers. We designed four constructs using four different adjuvants like TLR-4 agonist, B-defensin, Cholera B toxin (CTB), and 50 s ribosomal adjuvant. The rationale for using different adjuvant molecules was to check which adjuvants show the best compatibility with the designed vaccine molecule and generate strong and protective immune responses. The designed constructs were then evaluated for their physiochemical properties, antigenicity, allergenicity, adhesion probability, and prediction of secondary structure on four parameters . The conThe finalized vaccine construct was modeled into a 3D structure  using thAGGRESCAN analysis was performed before a cabs-flex analysis  colored red in We used a C-ImmSim online webserver for the computational immune simulation demonstrating the immune responses expected against the designed multi-epitopes vaccine. Production of immunoglobulins and interleukins proved the effectiveness of the vaccine. B. pseudomallei , root-mean-square fluctuation (RMSF), and radius of gyration (RoG) were computed for the system. RMSD analysis during simulation revealed that the complex reaches 7\u00c5 at 10.5 ns, however, this is due to the presence of loops. The graph became stable and showed maximum stability toward the end of simulation time as presented in B. pseudomallei is a gram-negative bacterium that causes melioidosis and can be lethal if not adequately treated. It can sometimes act as a facultative intracellular pathogen, emphasizing the pathogenesis of the infection (B. pseudomallei. Immunoinformatic approaches for vaccine designing can develop effective vaccines in less time and can provoke both innate and humoral immune responses (B. pseudomallei strains and subjected to various filters resulting in potential vaccine candidates. This was done via a subtractive proteomics approach in which desired proteins were filtered (Acinetobacter baumannii (Enterobacter hormaechei (Enterobacter xiangfangensis (nfection . Long meesponses . The preesponses . Core prfiltered , 43. Thefiltered , 43. Thefiltered . The virfiltered . These pfiltered . Predictaumannii , Enterobrmaechei , and Entangensis  etc. Thevia the EAAAK linker. The purpose of utilizing linkers is to facilitate the separation of epitopes and adjuvants while avoiding overlaps (The vaccine construct was designed by linking the epitopes with GPGPG linkers and joining the epitope to the adjuvant overlaps , 47. Fouoverlaps . CTB is overlaps . The vacoverlaps , 50. Theoverlaps , 50. Stroverlaps  explaineoverlaps . The maioverlaps . To genein-silico host immune simulation revealed that the chimeric vaccine can induce a proper immune response in the host body. The immune response was observed in the form of different antibodies and cytokines. According to our findings, the proposed vaccine is promising and capable of eliciting a proper immune response against B. pseudomallei.In this study, we performed a docking analysis to assess the vaccine's binding potency with TLR-5. The server generates 10 complexes with different binding energy scores. From the docking analysis, the designed vaccine can interact with TLR-5 molecules and can provoke proper immune reactions. For long-term immunity, the docking stability of the docked complexes must be maintained. To evaluate the dynamic movement of the docked complex, a molecular dynamics simulation approach was performed. The results of B. pseudomallei. There is no licensed vaccination to protect against B. pseudomallei infection. Immunoinformatics, bioinformatics, and reverse vaccinology were employed to speed up vaccine target discovery while also lowering costs and saving time. In the current research, a multi-epitopes-based vaccine was designed that may produce innate and adaptive immunity against B. pseudomallei using a variety of bioinformatics, immunoinformatic, and reverse vaccinology methodologies. For the above-said purpose, the first proteins were shortlisted from pathogen core proteins and utilized for probable antigenic, non-allergic, non-toxic, and good water-soluble epitopes prediction. Several analyses demonstrate that the developed vaccine model is capable of inducing a proper innate and adaptive immune response against the target pathogen. However, wet laboratory confirmation and validations are strongly advised to validate in-silico findings. Although the in-silico results are encouraging, additional efforts are required, such as the use of more accurate servers/tools to validate the results.Antibiotic abuse in animals, humans, and agriculture has resulted in the emergence of antibiotic-resistant bacterial infections, which has significantly raised hospitalization, community mobility, and mortality rates. Melioidosis, a condition that can be fatal, is caused by Publicly available datasets were analyzed in this study. This data can be found here: NCBI GenBank.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Plesiomonas shigelloides is one of the etiological agents of intestinal infection in humans. It is a gram-negative rod-shaped bacterium that is highly resistant to several classes of antibiotics, and no licensed vaccine against the aforementioned pathogen is available. Hence, substantial efforts are required to screen protective antigens from the pathogen whole genome that can be subjected easily to experimental evaluations. Here, we employed a reverse vaccinology (RV) approach to design a multi-antigenic epitopes based vaccine against P. shigelloides. The complete genomes of P. shigelloides were retrieved from the National Center for Biotechnological Information (NCBI) that on average consist of 5226 proteins. The complete proteomes were subjected to different subtractive proteomics filters, and in the results of that analysis, out of total proteins, 2399 were revealed as non-redundant and 2827 as redundant proteins. The non-redundant proteins were further checked for subcellular localization analysis, in which three were localized in the extracellular matrix, eight were outer membrane, and 13 were found in the periplasmic membrane. All surface localized proteins were found to be virulent. Out of a total of 24 virulent proteins, three proteins (flagellar hook protein (FlgE), hypothetical protein, and TonB-dependent hemoglobin/transferrin/lactoferrin family receptor protein) were considered as potential vaccine targets and subjected to epitopes prediction. The predicted epitopes were further examined for antigenicity, toxicity, and solubility. A total of 10 epitopes were selected . The selected epitopes were linked with each other via specific GPGPG linkers in order to design a multi-epitopes vaccine construct, and linked with cholera toxin B subunit adjuvant to make the designed vaccine construct more efficient in terms of antigenicity. The 3D structure of the vaccine construct was modeled ab initio as no appropriate template was available. Furthermore, molecular docking was carried out to check the interaction affinity of the designed vaccine with major histocompatibility complex (MHC-)I (PDB ID: 1L1Y), MHC-II (1KG0), and toll-like receptor 4 ((TLR-4) (PDB: 4G8A). Molecular dynamic simulation was applied to evaluate the dynamic behavior of vaccine-receptor complexes. Lastly, the binding free energies of the vaccine with receptors were estimated by using MMPB/GBSA methods. All of the aforementioned analyses concluded that the designed vaccine molecule as a good candidate to be used in experimental studies to disclose its immune protective efficacy in animal models.The swift emergence of antibiotic resistance (AR) in bacterial pathogens to make themselves adaptable to changing environments has become an alarming health issue. To prevent AR infection, many ways can be accomplished such as by decreasing the misuse of antibiotics in human and animal medicine. Among these AR bacterial species,  With advancements in genomics, many significant advancements in vaccinology took place. Along with this, shotgun sequencing and bioinformatics tool/servers have aided in predicting antigens localized on pathogens\u2019 surfaces for successful vaccine development [meningococcal serogroup B vaccine (4CMenB) [The elevated use of antibiotics in treating human and animal infections as well as in agriculture prompts bacteria to evolve antibiotic resistance, which has contributed to significant economic losses and elevated mortality and morbidity . AR is aelopment . Reverseelopment . RV has (4CMenB) . Compare(4CMenB) .Plesiomonas shigelloides with the objective of predicting probable antigenic epitopes from the core genome of P. shigelloides and to design a multi-epitope vaccine (MEV) against the pathogen. SP in genomics and proteomics is a technique to search proteomes of bacterial pathogens in quest of vaccine candidates [Herein, we employed two approaches; subtractive proteomics (SP) and RV to present a best vaccine model against ndidates ,14. The P. shigelloides belongs to the family of Enterobacteriaceae and mainly causes diarrhea and gastrointestinal infections [P. shigelloides as well as fast-tracking the vaccine development process. Also, as the vaccine design is based on proteins that form the core genome of the pathogen, the vaccine is likely to provide cross-protection against all sequenced strains of the bacteria.fections . Morpholfections . These bfections . Severalfections ,19,20. Afections . The proP. shigelloides is schematically demonstrated in The flow chart for designing a chimeric vaccine for P. shigelloides strain were retrieved from the National Center for Biotechnology Information (NCBI) and subjected to SP. The proteins which are present in the pathogen core proteome, host non-similar, and are essential for survival were selected [A total of two proteomes of selected . In thisselected .The initial step in the pre-screening phase was to screen proteins; that show sequence conservation among complete sequenced genomes of the pathogen , host noThe genome was processed using the bacterial pan-genome analysis (BPGA) tool. The core proteins were the primary targets for vaccine development. In the process, redundant proteins, resulting in the redundancy of biochemical function, were not selected . TherefoPathogen non-redundant core proteins were identified by using the CD-HIT server. Proteins showing 60% sequence homology were removed . Using BThe resulting core and non-redundant proteome were then analyzed for extracellular, periplasmic, and outer membrane region proteins (OMPs), respectively . For achLactobacillus rhamnosus (taxid: 47715), L. casei (taxid: 1582), and L. johnsonii (taxid: 33959). Only proteins that showed a bit score >100, and E-value threshold value <0.005 were selected.The secretome and exoproteome of the bacteria were further filtered to get virulence factor through BLASTp against the virulence factor database (VFDB) . ProteinUsing Vaxijen 2.0, the antigenicity of proteins was analyzed , and preThe selected proteins were subjected to the epitopes prediction phase and analyzed via the Immune Epitope Database (IEDB) . These e50 values are less than 100 nM for the DRB*0101 gene were subjected to the next steps. The pathogenicity of epitopes was determined by Virulentpred [Through the MHcPred server, a DRB*0101 binding analysis was performed and only those epitopes whose IClentpred , and antlentpred .The GPGPG linkers were used to join the predicted B-cell derived T-cell epitopes. Finally, the designed epitopes peptide was joined at the N-terminal of the B subunit of cholera toxin adjuvant using an EAAAK linker . The finhttps://www.snapgene.com/ (accessed on 1 June 2022).For achieving a stable vaccine, a disulfide engineering using Design 2.0 was performed and then reverse translated to DNA sequence through JCat server . Lastly,Molecular docking was carried out for the vaccine with different human immune receptors . For preAdditionally, the designed vaccine was revealed for its dynamics in 200-ns of computer simulations, including system making, pre-processing, and production steps using Assistant model construction with Energy Refinement (AMBER) 20 . Using aThrough the MMPBSA.py module, the MMPBSA binding free energy of vaccine-TLR4 was estimated using the AMBER 20 package . OverallThrough a computer immune simulation server, C-Immune Server, host immune reactions (immunogenicity) against the vaccine construct were characterized . The posP. shigelloides were retrieved from NCBI. The MS-17-188 is a multi-drug resistant strain recovered from catfish in 2017. This strain is responsible for gastrointestinal tract infections and is resistant to chloramphenicol, doxycycline, gentamicin, tetracycline, oxytetracycline, sulfamethoxazole-trimethoprim, novobiocin, florfenicol, streptomycin, azithromycin, and spectinomycin. The NCTC10360 is isolated from patients with diarrhea and shows resistance to many antibiotics. All the retrieved data were in fasta format and the proteome data of the strains were subjected to further study. The size and GC content of each genome is tabulated in In the current study, a total two of completely sequenced genomes of The two proteomes were processed in the BPGA tool and the core sequences of the bacteria were extracted. The core proteome consists of dispensable proteins and strain-specific proteins. The proteomic data of strains consist of 5226 total proteins. The proteins contain pathogen core proteome, host non-similarity, and essential proteins. A CD-HIT analysis revealed 2399 non-redundant proteins and 2827 redundant proteins [For the prediction of ex-proteome and secretome, a subcellular localization strategy was applied to get surface proteins that have the qualities of invasion, adherence, and proliferation. The PSORTb tool was used to check the localization of proteins . The majThe selected proteins were further analyzed to achieve virulence factors and other targets using the virulence factor database (VFDB) . As viruThe selected proteins were subjected to physicochemical properties . DiffereProbiotic bacteria are those organisms that produce different enzymes to stop the growth and survival of harmful microbes. They produce different vitamins such as biotin, vitamin K2 and are also a key factor for the secretion of antibodies and regulatory cells that stimulate the host immune response. The proteins were aligned with the reference human proteins, and we selected those proteins that showed non-homology with the human genome to avoid an autoimmune response. In this study, only three protein sequences have shown homology with human proteins and 10 protein sequences have shown similarity with the normal flora of the host. The three proteins were found to be antigenic, non-allergenic and showed an adhesion probability value greater than 0.6, indicating that they are good vaccine candidates. An antigenic value greater than 0.4 refers to the strong ability of the proteins to induce immune responses compared to those with a value less than 0.4. Similarly, a protein can be classified as an adhesive if its value is higher than 0.5 . Among t50 values <100 nM for the DRB*0101 gene. DRB*0101 belongs to human leukocyte antigen II and is present in most human populations. After this, the epitopes were analyzed for allergenicity, solubility, and toxicity analysis. All of the predicted B and T-cell epitopes with percentile rank IC50 predicted scores are mentioned in The proteins were first utilized to predict B-cell epitopes in which those B-cell epitopes were selected showing scores greater than 0.8 . Only thIn these analyses, allergic and non-antigenic epitopes were removed, and the remaining probable antigenic and non-allergic epitopes were selected for further analysis. The solubility was determined via InvivoGen and ToxinPred for toxicity to avoid the toxic effect of all the toxic epitopes. Ten epitopes were found to be soluble and non-toxic and were considered for the designing of multi-epitopes as mentioned in The multi-epitopes based vaccine construct consisted of different epitopes rather than a single epitope in order to generate strong and protective immune responses. The multi-epitopes vaccine was designed by linking all top 10 screen epitopes with each other with the help of GPGPG linkers. Furthermore, the designed vaccine was also linked with Cholera Toxin B subunit adjuvant via an EAAAK linker to enhance the efficacy of immune response , which hUsing a 3Dpro SCRATCH predictor tool, the tertiary structure of the multi-epitopes construct was modeled as shown in To avoid the breakdown of the designed vaccine weak regions, disulfide engineering was done to stabilize the bonding between residues having unfavorable energy . DisulfiE. coli vector and calculate it with the aid of a codon adaptation index (CAI) and its GC percentage values. The designed vaccine was then expressed into the pET-28a (+) vector through SnapGene, as represented in After the above process, through the use of the Java Codon Adaptation Tool (JCat), the sequence of the designed vaccine construct was first reverse translated to DNA sequence to get the maximum level of expression of vaccine in the Blind docking was performed for the prediction of interaction between the vaccine with MHC-I (PDB ID: 1L1Y), MHC-II (1KG0), and TLR-4 (PDB: 4G8A) with the help of PATCHDOCK  and the \u22121), attractive van der Waals (0 kJ\u00b7mol\u22121), repulsive VdW (0 kJ\u00b7mol\u22121), atomic contact energy (ACE) (0 kJ\u00b7mol\u22121) and hydrogen bonding (0 kJ\u00b7mol\u22121). In the case of MHC-II solution, number 8 has the lowest global energy (3.11 kJ\u00b7mol\u22121), attractive van der Waals (\u22124.44 kJ\u00b7mol\u22121), repulsive van der Waals (1.75 kJ\u00b7mol\u22121), ACE (1.71 kJ\u00b7mol\u22121) and hydrogen bond energy (0. kJ\u00b7mol\u22121). In case of TLR-4, solution number 4 was selected as having the lowest global energy (\u221215.33 kJ\u00b7mol\u22121), attractive van der Waals (\u221222.6), repulsive VdW (7.55), ACE) (2.29 and hydrogen bonding (\u22122.62 kJ\u00b7mol\u22121) were found more stable due to lower global energy and selected for high structure configuration studies through UCSF Chimera 1.13.1 [The FIREDOCK  server ra 1.13.1 . DockingPeptide antigen processing and presentation to immune cells by MHC molecules is crucial for the adaptive immune response. Before antigen processing and presentation, the foreign peptide antigen is required to interact with different types of immune cells to generate an appropriate immune response. These intermolecular interactions of MHC-I, MHC-II and TLR-4 are critical to deciphering residues important from a vaccine recognition perspective. The model vaccine construct showed strong interactions with several key amino acid residues of MHC-I, MHC-II and TLR-4 immune cells receptor molecules as find out in UCSF chimera and tabulated in Molecular dynamic simulation is a computer simulation process for the analysis of the dynamic behavior of macromolecules. Molecular dynamics simulations of docked complexes were performed for 200 nanoseconds (ns) to evaluate the structural stability of the systems. The simulations were carried out using the AMBER20 simulation package . The anaThrough the MMPBSA.py module, the MMPBSA/MM/GBSA binding free energies of the vaccine- receptor was estimated . Only 10c (cytotoxic killer T-cell), macrophages (M\u03c6), natural killer cells, and dendritic and epithelial cells is shown in The immunogenic efficacy of the final vaccine construct was evaluated by performing in silico immune simulations with the help of the C-immSim server 10.1 for 350 days [P. shigelloides is one of the AR bacterial species and shows resistance to several classes of commercially available antibiotics such as azithromycin, penicillin, doxycycline, and erythromycin. P. shigelloides is a group of opportunistic gram-negative, motile, and rod-shaped pathogens belonging to the Enterobacteriaceae family. It causes many infections, including diarrhea, gastrointestinal infection, CNS abnormalities, neonatal sepsis and vision problems. P. shigelloides isolates from hospital patients are reported to show high resistance to antibiotics leading to high mortality and morbidity rates (https://doi.org/10.3389/fmicb.2018.03077 (accessed on 24 September 2022)).AR is the outcome of the bacterial evolution process to make itself resistant to antibiotics. To prevent infection caused by AR pathogens, vaccination is an alternative approach to generate a proper immunological response against specific organisms . P. shigP. shigelloides to lower the burden of AR [https://doi.org/10.1038/s41598-022-12651-1 (accessed on 24 September 2022)), Onchocerca volvulus (https://doi.org/10.3389/fitd.2022.1046522 (accessed on 24 September 2022)), and Listeria monocytogenes (https://doi.org/10.1038/s41467-022-33721-y (accessed on 24 September 2022)). Traditional vaccinology is a failure for pathogens that are unable to be cultured or grown in vitro. As compared to conventional RV, pan-genomic reverse vaccinology (PGRV) is more effective as it screens highly conserved targets than strain specific ones. For example, the genome of Streptococcus agalactiae determined four protective antigens identified with the help of the PGRV approach [P. shigelloides.Hence, in this study, we designed an in silico vaccine model against en of AR . The genen of AR . RV is aen of AR . By usinen of AR . The metA good vaccine candidate has the following properties: it should be antigenic, immunogenic, non-homologous, non-allergic, and is located on the pathogen surface region. All of these properties are literature-based and highly desirable to design a chimeric vaccine against a specific pathogen. Immune cell epitopes prediction, analysis and processing of potential and safe antigens, population coverage and conservation analysis, toxicity prediction of the antigens, allergenicity evaluation, docking and simulation approaches, and binding energies estimations are steps used in computational vaccinology . In the Three different types of subcellular localized proteins: (i) flagellar hook protein FlgE (ii) hypothetical protein; and (iii) hemoglobin/transferrin/lactoferrin family receptor) were selected due to non-allergic, probable antigenic, and non-similar with human and microbiota. The shortlisted proteins were utilized for B and T-cell epitopes prediction . The preP. shigelloides.In the multi-epitopes designing phase, all the shortlisted epitopes were linked by GPGPG linkers to design a multi-epitopes vaccine construct. The designed vaccine construct was further linked with Cholera Toxin B subunit adjuvant via EAAAK linker for making the designed vaccine more efficient. The tertiary structure of the vaccine construct was modeled and refined to maintain structural stability. The construct was subjected to blinding docking to check the binding interaction between the construct and MHC-I, MHC-II, and TLR-4, and to examine the immune responses. We found solution 10 in MHC-I, 8 in MHC-II, and 4 in TLR-4 more stable because of lower global energy. The behavior of molecules within the host cell was achieved in the molecular dynamic phase and binding energies of a construct with receptor were estimated. The integrated SP and RV approach successfully identified an antigenic epitope for the design of a chimeric vaccine to boost up the host immune response against Clinically, the designed vaccine may be quite useful as the vaccine contains core epitopes and thus could provide broad spectrum immune protection against all strains of the pathogen. Also, the vaccine is safe as it is non-allergic and non-toxic. Some limitations of the study need to be overcome in future studies. First, the need for an experimental evaluation to get the best combination epitopes in the vaccine construct for maximum level of immune responses must be done. Second, the refinement of MHC molecules epitopes prediction algorithms is under way. Lastly, the real immune protection of the vaccine required extensive in vivo and in vitro testing.P. shigelloides, which is one of the most troublesome human pathogens and is highly resistant to several antibiotics. This is worrisome in addition to the absence of a licensed vaccine against the pathogen. By using core, non-homology, non-redundant proteins, we designed a vaccine consisting of non-allergic, antigenic, non-toxic, and soluble epitopes. The epitopes were joined to each other by GPGPG linkers and linked with cholera toxin B subunit adjuvant with the help of another EAAAK linker to enhance the potency of the designed vaccine. The vaccine candidate was used as a comprehensive immune system inducer and the strongest candidates were prioritized in future vaccine development efforts to prevent future P. shigelloides disease outbreaks. We believe that the product of this work is a designed peptide vaccine for researchers to investigate its immune protection ability in vivo, and the findings of the study will increase the vaccine antigens library against P. shigelloides as well as fast-track the vaccine development process. Furthermore, as the vaccine design is based on proteins that form the core genome of the pathogen, the vaccine is likely to provide cross-protection against all sequenced strains of the bacteria.In the current study, we employed RV, SP, and immunoinformatics approaches to design a multi-antigenic epitope- based vaccine against"}
+{"text": "STAT3 (STAT3-HIES). Patients suffering from HIES show an increased susceptibility to Staphylococcus aureus (S. aureus) including skin abscesses and pulmonary infections. To assess if the underlying immune defect of STAT3-HIES patients influences the resistance patterns, pathogenicity factors or strain types of S. aureus. We characterized eleven S. aureus strains isolated from STAT3-HIES patients (n\u2009=\u20094) by whole genome sequencing (WGS) to determine presence of resistance and virulence genes. Additionally, we used multi-locus sequence typing (MLST) and protein A (spa) typing to classify these isolates. Bacterial isolates collected from this cohort of STAT3-HIES patients were identified as common spa types in Germany. Only one of the isolates was classified as methicillin-resistant S. aureus (MRSA). For one STAT3 patient WGS illustrated that infection and colonization occurred with different S. aureus isolates rather than one particular clone. The identified S. aureus carriage profile on a molecular level suggests that S. aureus strain type in STAT3-HIES patients is determined by local epidemiology rather than the underlying immune defect highlighting the importance of microbiological assessment prior to antibiotic treatment.Hyper-IgE syndromes (HIES) are a group of inborn errors of immunity (IEI) caused by monogenic defects such as in the gene  Staphylococcus aureus (S. aureus) frequently colonizes skin and mucosa, it is also a major pathogen causing harmful infections such as abscesses, pneumonia, and septicemia [S. aureus is the leading cause for surgical site infections and especially methicillin-resistant S. aureus (MRSA) infections are associated with significant costs for health care systems [While pticemia . In fact systems .STAT3 (STAT3-HIES) [S. aureus infections [S. aureus can also cause infections in immunocompetent individuals; the predisposition of STAT3-HIES patients towards this pathogen, however, offers further insights into the immune defense against S. aureus. STAT3-HIES patients show impaired Th17 cell function and diminished memory B cell development [S. aureus toxin despite chronic colonization [S. aureus, STAT3-HIES patients also suffer from reduced epithelial immunity towards this pathogen [Hyper-IgE syndromes (HIES) are a group of inborn errors of immunity (IEI), of which the most frequent form is caused by monogenic defects in the gene T3-HIES) . STAT3-Hfections \u20136. Severfections \u20139. S. auelopment \u201313. Addinization . Importapathogen . STAT3-Hpathogen , 17. NevS. aureus colonization and infection are key symptoms of STAT3-HIES and patients frequently require antibiotic prophylaxis or therapy, little is known about the molecular features of S. aureus in these patients. Moreover, it is unclear if the immunodeficiency of STAT3-HIES patients selects S. aureus strains with a specific \u201cgenetic fingerprint\u201d. A recent study used multi-locus sequence typing (MLST) and protein A (spa) typing to analyze the genetic background of 13 S. aureus isolates collected from STAT3-HIES patients in the USA [S. aureus carriage profile of STAT3-HIES patients in Germany to optimize antibiotic treatment using bacterial whole genome sequencing (WGS) and to investigate virulence and resistance genes in these isolates.Although  the USA . Here, mSTAT3 mutation, were unrelated, and presented with the characteristic clinical findings of their genetically confirmed diagnosis at inclusion of study  isolates collected from skin lesions (n\u2009=\u20095) and one clinical isolate from a lymph node abscess  following manufacturer\u2019s instructions. Isolates were sequenced using Illumina technology and Nextera XT version 2 chemistry, with a 250-bp paired-end protocol on a MiSeq sequencer . Quality trimming of fastq files  and de novo assembly using SKESA [S. aureus cgMLST scheme passed quality control; otherwise, sequencing was repeated. Target gene sets for virulence factors, resistance, and toxin genes as well as spa type were analyzed using the SeqSphere\u2009+\u2009software [ng SKESA  were perng SKESA . Only gesoftware , 22. Presoftware .Table 2TS. aureus isolates were collected either as screening isolates  or from different skin sites. One clinical isolate (P1.2) was isolated from a lymph node abscess.Patients included in our study were between 5 and 53\u00a0years of age at enrollment. All four patients had a history of pulmonary and skin infections Table\u00a0. During spa type. The collected S. aureus isolates were predominantly ST5 or ST582 and the most frequent spa types were t179 and t084 (Table\u00a0pvl), staphylococcal enterotoxins Q (seq), or staphylococcal enterotoxins K (sek) gene  and e Tables\u00a0. These t the USA .Table 3TS. aureus isolates collected from STAT3-HIES patients was analyzed by cgMLST. cgMLST combines \u201cclassical\u201d typing approaches to classify bacteria with the extensive genetic data sets obtained by WGS [S. aureus up to 1861 conserved target genes are compared to discriminate between isolates achieving a sufficient resolution for epidemiological and surveillance studies [S. aureus isolates were isolated from skin lesions at different sites yet at the same time point (P3.1\u2009\u2212\u2009P3.5). WGS showed that these isolates belonged to the same ST (ST582) and spa type (t084) but differed by up to 6 alleles or 6 single nucleotide polymorphisms (SNPs) in their core genome  was not genetically related to the clones colonizing the patient at other sites and differed from colonizing isolates in 1401 alleles or 9382 SNPs , yet showed significant variation in their core genome. Strains P1.1 and P1.3 were both isolated from a nose/throat screening swab but were collected 8\u00a0years apart and differed by 30 alleles or 31 SNPs in their core genome. Taken together the analyses of these bacterial isolates indicate that STAT3-HIES patients are carrying several genetically diverse S. aureus isolates at a time.Next, the genetic relatedness of d by WGS . For S.  studies . The isoome Fig.\u00a0. In anotNPs Fig.\u00a0. The colS. aureus to cause soft tissue infection and inflammation with the activity of pathogenicity factors and bacterial toxin genes [S. aureus isolates of STAT3-HIES patients for genes of the accessory regulator (agr), hemolysin genes (hl), and genes encoding for enterotoxins, adhesion molecules and the immune evasion complex (IEC)  was present in all isolates. Two colonizing isolates showed a stop codon in the hl gene (P1.3 and P1.4). One colonizing isolate lacked the hemolysin \u03b4 (hld) gene (P3.4). The IEC consists of the genes coding for staphylokinase (sak), staphylococcal complement inhibitor (scn), chemotaxis inhibitory protein (chp), and staphylococcal enterotoxins A (sea). IEC genes are well-known pathogenicity factors that aid S. aureus to bypass the human immune response by counteracting key steps in innate immunity such as complement activation and chemotaxis. Interestingly, three of the eleven isolates collected from STAT3-HIES patients contained all four IEC genes . As STAT3-HIES patients show reduced Th17 and B cell responses towards S. aureus, bacterial pathogenicity factors that impair innate immunity might result in a more pronounced effect in these patients. However, long-term follow-up studies are needed to address this research question.Hemolysin \u03b1 (S. aureus clones in STAT3-HIES patients remain largely unknown. The MLST ST and spa types of the eleven S. aureus isolates collected during this study differed significantly from the typing results described in a previous report from the USA [spa types are common in Germany [22). The ST and spa type results imply that the local epidemiology is the main factor determining which S. aureus strains colonize STAT3-HIES patients. Although all patients enrolled in our study have received long-time antibiotic prophylaxis and therapy, MRSA was not prevalent in our patient cohort (Table\u00a0S. aureus bacteria isolated from patients in the community setting are MRSA [S. aureus isolates collected from STAT3-HIES patients. In our study, we analyzed the genetic relatedness of colonizing S. aureus isolates from several STAT3-HIES patients based on WGS data. Here, we demonstrated that STAT3-HIES patients were colonized with genetically diverse S. aureus clones at a time. Similar observations have been made in a study analyzing S. aureus isolates collected from eczematic lesions of atopic eczema patients by WGS. In this publication, a broad genetic diversity of bacterial isolates was detected suggesting that clonal expansion of a bacterial population takes place during a disease flare [Presently, the molecular characteristics and genetic variability of  the USA . Howeverare MRSA . Furtherse flare .S. aureus isolates generated as part of this study offer a comprehensive characterization of multiple virulence factors. An improved understanding of pathogenicity factors may pave the way for new therapeutic strategies for STAT3-HIES patients. Currently, long-acting monoclonal antibodies that are capable to neutralize S. aureus toxins in the respiratory tract are under development to treat MRSA lung infections [S. aureus toxins, the potential of these monoclonal antibodies as treatment for severe S. aureus lung infections could be evaluated [The WGS data of fections , 32. As valuated , 33.S. aureus isolates collected from STAT3-HIES patients, including genetic relatedness of isolates, methicillin resistance and presence of virulence factors and demonstrates the benefits of using molecular approaches to study host\u2013pathogen dynamics in IEI.Taken together, our study provides a detailed view into the molecular characteristics of the"}
+{"text": "Short tandem repeats on the Y chromosome (Y-STRs), characterized by paternal inheritance, are valuable in forensic practice. Notably, the potential application of Y-STRs in pedigrees should be drawn upon, especially in China\u2019s surname-concentrated natural villages. The study focused on 50 Y-STRs, including 13 rapidly mutating (RM) Y-STRs that largely constitute the current Y-STR commercial kits, and determined the differences in these Y-STRs between branches in a large pedigree and the discriminatory power of these haplotypes in different units for male relatives. As indicated in the results, 14 inconsistencies were observed at 9 Y-STRs between 10 father-son pairs. In addition, these 50 Y-STR haplotypes discriminated 10 out of 47 father-son pairs, 106 of 148 cousin pairs, 70 of 119 uncle-nephew pairs, 17 of 39 brother pairs, and 14 out of 33 grandfather-grandson pairs in a large pedigree. The RM Y-STR set is able to differentiate close male relatives in a large pedigree. With the genetic characteristics of paternal inheritance and lack of recombination, Y-chromosomal short tandem repeats (Y-STRs) are extremely useful tools in forensic practice . In partPlenty of China\u2019s natural villages are surname-concentrated, in which thousands of residents with the same surname share paternal genetic ancestry and are grouped into numerous branches. A village has been reported where the residents have a single surname for dozens of generations. If a crime occurs in these places and the criminal happens to be a member of the patrilineal line, the power of conventionally used Y-STRs is insufficient to exclude numerous patrilineal relatives of the suspect. It would be time consuming to collect samples from thousands of male suspects. Recently, 13 rapidly mutating (RM) Y-STRs have been identified , and theThis study focused on 50 Y-STRs including RM STRs, found in the most common Y-STR commercial kits, to observe the variation in Y-STRs in a large pedigree, which will provide an advantage compared with simple mutation investigation.The samples were 53 male individuals from the four-generation pedigree, and they belonged to five branches designated A, B, C, D and E, as presented in Their bloodstains were collected on FTA Cards . DNA was extracted by using Chelex 100 in a total volume of 100\u2009\u03bcL without subsequent quantitation of the DNA amount . Writtenhttp://www.genome.ucsc.edu/cgi-bin/hgPcr).Thirty-seven conventional Y-STRs  and 13 RM Y-STRs  were involved. Among them, one had \u201cI\u201d and \u201cII\u201d parts (i.e. DYS389) and two had \u201ca\u201d and \u201cb\u201d parts (i.e. DYS526 and DYF403S1). With the primer sequences attained from published literature, their exact map positions were retrieved adopting the UCSC in silico PCR tool  , the AGCPCR products were separated by multicapillary electrophoresis on the ABI Prism 3130 Genetic Analyzer . The fragment sizes were analysed using Gene-Mapper v.3.2. Allele designations were determined by comparing the sample fragments with those of allelic ladders from the kits or a ladder of RM Y-STRs made in our laboratory. The nomenclature adopted was that of the latest recommendations from the DNA Commission of the International Society of Forensic Genetics. RM Y-STR markers were named following the allele designation method adopted by Ballantyne [For the Database Y24, Y SUPP, and RM set, 9948 was used as control DNA, while 007 was employed for Y-filer\u00ae Plus.The mutation events of 50 Y-STRs were observed using father-son pairs, and the mutant allele was evaluated for its potential application to distinguish among different branches in a pedigree. The 50 Y-STR markers were selected to constitute different units, including the minimum YHRD marker set, the various commercial kits , and the 13 RM Y-STR sets. For all 50 Y-STRs and each unit, the value was ascertained for differentiating the pairs of father-son, uncle-nephew, brothers, cousins, and grandfather-grandson.Supplementary Figure 1. Seven of them are multi-copy systems  and are associated with the palindromic sequences on the Y chromosome, especially in band q11.23. Two copies for DYS526 were observed, which did not comply with the number of physical locations along the Y chromosome per Y-STR predicted using the UCSC PCR tool. This was because the forward primer of DYS526 binds to two distinct areas, with a primer pair generating DYS526a and DYS526b as PCR products. In this regard, this phenomenon needs to be noted so that two or more peaks at this locus are not automatically interpreted as originating from a mixture of males. In addition, several STR loci, such as DYF403S1, are not simple sequence repeats [The physical locations of the 50 Y-STR markers on the Y chromosome involved in this work are presented in  repeats , and the repeats .\u22122), which has been found to be highly mutated in our previous mutation analysis on 100 father-son pairs [The genotypes of 50 Y-STRs from 53 individuals were successfully obtained. The overlapping Y-STR loci covered in different kits showed consistent genotypes, which indicated that these kits showed good compatibility. For the majority of Y-STRs, 41 genotypes remained consistent for all individuals from the four-generation pedigree. These Y-STRs could be considered characteristic surname markers. Fourteen inconsistencies were observed at the remaining nine Y-STRs and six of them were RM. All inconsistencies were one-repeat changes due to the occurrence of mutation events, as shown in on pairs .There are a maximum of two Y-STR inconsistencies observed within the four father-son pairs. Our observation agrees with the recommended exclusion criteria of the minimum of three Y-STRs in paternity testing . NaturalAlthough autosomal STRs should be investigated first for differentiating close patrilineal relatives, Y-STRs, especially RM ones may be prioritized when analyzing male/female mixed samples. Provided that two close patrilineal relatives were involved in a suspected gang rape case, the Y-STR haplotype could be used to differentiate them. These 50 Y-STR haplotypes differentiated 10 out of 47 father-son pairs, marking a differentiation rate of 21.3%; moreover, they could differentiate 106 out of 148 cousin pairs, 70 out of 119 uncle-nephew pairs, 17 out of 39 brother pairs, and 14 out of 33 grandfather-grandson pairs. The differentiation rate rises in line with the degree of kinship. The distant relatives indicate considerable meiosis and a higher probability of mutation events, which can occur at each meiosis independently. In summary, the 50 Y-STR haplotype allowed 56.2% of these relative pairs to be separated. For other units that are part of the Y-STRs among 50 units, the number of pairs with inconsistencies and differentiation rates between two related men for different units are listed in In conclusion, for male/female mixed samples, the question remains of whether the sample is from the perpetrator or whether different but related men were involved. The subsequent analysis of the RM Y-STRs will provide further evidence for the exclusion of close male relatives."}
+{"text": "The Korean government implemented a value incentive program providing incentives to providers based on C-section rates, with the rates being publicized. The program ended in 2014 after the administration decided that the effects of the incentive program were limited. In this report, we analyzed changes in C-section rates with the value incentive program.The analysis used claim data from Korea\u2019s National Health Insurance. The study period (2011\u20132016) was divided into two phases: before and after the program. This study included 95 providers that were tertiary or general hospitals having more than 200 deliveries per year during the study period. The dependent variable was the risk-adjusted C-section rate. Independent variables included time and hospital characteristics such as hospital type, district, and ownership. Interrupted time series analysis was performed to analyze the data.P < .05), was statistically significant, as was the trend after intervention, at 0.21% (P < .0001). The slope showed an increase after the intervention to 0.25% per medical institution, which was contrary to the trend of the preintervention decline (negative slope).Our results showed that risk-adjusted C-section rates increased immediately after the end of the incentive program for C-sections. The immediate effect of intervention, a change of 1.73% (Risk-adjusted C-section rates increased immediately after the discontinuation of a value incentive program. Tertiary hospitals showed greater increases in C-section rates than general hospitals after the intervention. WHO also indicated that the negative consequences of cesarean delivery must be limited, especially if C-section rates exceed 7%.2 C-section rates above the appropriate level not only increase the prevalence and risk of death for mothers and newborns but also contribute to the inefficient use of medical resources.3According to the 2017 Organisation for Economic Co-operation and Development (OECD) Health Statistics, the number of C-sections per 1000 births in Korea was 452, which was the second highest after Turkey (531 cases) and 190 cases higher than the average of OECD member countries (264 cases). The number of C-sections per 1000 births in Korea was higher than that for women in the Asia\u2013Pacific region, Japan, Taiwan, or the United States, who have similar obstetric conditions as Korean women. The World Health Organization (WHO) recommends maintaining C-section rates at 10\u201315%,4 Currently, only monitoring services have been implemented since 2016.Therefore, the Korean government implemented a Value Incentive Program in January 2011 to assess the quality of medical services and provide incentives to hospitals to protect maternal health and optimize C-section rates, ultimately reducing health insurance finances. According to the results of the quality assessment of medical services, the providers were incentivized by adding 1%\u20135% of the fee to higher-grade institutions and quality improvement institutions, and by applying 1%\u20135% of the fee reduction to medical institutions below the standard. However, the Value Incentive Program was terminated in January 2014 as the attainment of a certain level of medical quality and the introduction of the Korean Diagnosis-Related Group (KDRG) had diminished the influence of policy.5By disclosing assessment results, the Korean Value Incentive Program forced hospitals to manage quality assessment indicators. However, the rating information is no longer available on the Health Insurance Review and Assessment Service (HIRA) website, a quasi-governmental organization that reviews National Health Insurance (NHI) program claims in South Korea. Furthermore, the C-section rate may be increasing since the program was eliminated.7 Jang et al found that the repeated public releases decreased the cesarean section rate (by 0.81 %).8 According to Hong et al, it was confirmed that most clinical indicators  which are subject to incentives have risen since the implementation of the Korean Value Incentive Program.9 However, no one has explored the relationship between the discontinuation of the Korean Value Incentive Program and the subsequent C-section rates.Previous studies have explored the effects of publicizing assessment results on C-section rates. Ko et al confirmed the average change of cesarean rate decreased by 10.2% after the public disclosure of information and the fluctuation also decreased.The purpose of this study was to analyze how hospitals responded to terminating the Korean Value Incentive Program for cesarean delivery. Specifically, this study calculated the risk-adjusted C-section rates for each hospital and analyzed whether they changed after the Korean Value Incentive Program ended.Data from 72 months (January 2011 to December 2016) were analyzed, with the period divided into two segments: 36 months before and 36 months after January 2014. The study data included the delivery mode, maternal age, region, and health at the birth of women of childbearing age (16\u201349 years) in tertiary hospitals and general hospitals with more than 200 deliveries per year. Data were retrieved from the customized database of the NHI program.10Delivery was defined according to the ICD-10 code , HIRA procedure code , and HIRA code for Diagnosis Related Groups (DRGs) .12. The risk-adjusted C-section rates were calculated by dividing the crude rates of C-section in hospitals by the predicted rates of C-section in those hospitals, multiplied by the crude C-section rates of all patients. The predicted C-section rates in hospitals were calculated by dividing the sum of the predicted probability of C-section for a patient by the total number of deliveries.13The dependent variable was monthly C-section rates in hospitals, which was adjusted for risk differences among patients from different hospitals. The risk adjustment model was established based on the selected risk factors 14 The hospital type was coded as \u201cTertiary\u201d = 1 and \u201cGeneral\u201d = 0; the administrative district as \u201cMetropolitan\u201d = 1 and \u201cProvince\u201d = 0; and ownership type as \u201cPrivate\u201d = 1 and \u201cPublic\u201d = 0.Time and hospital characteristics were used as independent variables. The \u201ctime\u201d variable was coded as 1 to 72 to represent the monthly time intervals from January 2011 to January 2016. The Korean Value Incentive Program ended in January 2014. The policy intervention was dummy coded: intervention type = 0 for the 36 months from January 2011 to December 2013, and intervention = 1 for the 36 months from January 2014 to December 2016. The \u201ctime after policy\u201d variable represented time since the policy was discontinued. This variable was coded as 0 for times before the policy intervention and by month in numerical order starting with 1 after the policy intervention. The model includes hospital characteristics known to affect the C-section rates.13 The risk-adjustment model was developed in the following order.10 First, a univariate analysis was performed to compare the distribution of 188,094 cases of vaginal delivery and 176,258 cases of cesarean section of 364,352 claims for each of the 17 risk factors. Second, selected risk factors were included in multivariate analysis, and the final model was developed based on their statistical significance and regression coefficients. Using this approach, 13 factors were selected as factors for the risk-adjusted model, excluding 4 factors .A risk-adjusted model was established with 17 risk factors selected based on a previous report.16 Details related to autocorrelation were mentioned in Appendix 3, Supplementary Digital Content, http://links.lww.com/MD/G988. GEE is used to estimate the causal model of panel data and is an analytical method that can address time-series data that are difficult to handle in Generalized Linear Model (GLM).17 The GEE analysis method is used in medical and health studies with repeated measurements where a phenomenon is commonly evaluated using more than one outcome variable.18Because the analysis used time-series data, an interrupted time series analysis was performed using a generalized estimated equation (GEE) considering the correlation that has been repeatedly measured.16 The regression model for the interrupted time series (ITS) analysis used in this study was as follows.19Data in this study included 36 months of panel data for which the correlation of error terms within a group could be determined; that is, the GEE was used to consider the autocorrelation of the error terms within each group.Y is the monthly C-section rate by a hospital, time is the monthly time flow from 2011 to 2016, intervention is the endpoint of the policy (January 2014), time after the intervention is the monthly time flow after the end of the policy, X = is the hospital characteristics, and e is the error term. The regression coefficient for the \u201cintervention\u201d variable represents the extent of change in the dependent variable at the time of the policy intervention; thus, the immediate effect of the policy can be seen. The regression coefficient for the \u201ctime after policy\u201d variable allows us to assess the long-term effects of the policy through trends in the dependent variables after policy intervention.Where This study used SAS 9.4 to identify the risk-adjusted C-section rates and to perform ITS analysis.http://links.lww.com/MD/G988)).All 13 risk factors in the model showed a significant association with C-section delivery, with odds ratios >1.0. The odds ratio was highest for breech malpresentation (56.03), followed by placenta previa (55.15). Diabetes had the lowest odds ratio, 1.10. When all 13 risk factors were used in the risk-adjustment model, the C-statistic was 0.91 and the Hosmer\u2013Lemeshow Chi-square statistic (4472.80) was significant .The monthly average risk-adjusted C-section rates from 2011 to 2016 are shown in Figure C-section rates increased after the intervention  in hospitals of all types. The rate of change was 14.49% and 14.42% for tertiary and general hospitals, respectively (Table \u03b22), a change of 1.73% (P < .05), was statistically significant, as was the trend after intervention (\u03b23), at 0.21% (P < .001),. The slope (Table The trend before intervention \u03b2 and the P < .05). Before the intervention, a tendency toward a decrease, that is, a drop of 0.08%, was noted, and the immediate effect after the intervention was a relative increase of 4.32%. After the intervention, the rate gradually increased, by 0.27%. In tertiary hospitals, the risk-adjusted C-section rate tended to decrease before the intervention and then increased steadily immediately after the intervention.In tertiary hospitals, the trends before intervention  and the accompanying information disclosure. These findings are consistent with previous studies showing that C-section rates changed due to the publication of assessment results and previous studies showing that policy interventions affect the behavior of medical institutions.26 They may fear the way of normal delivery or the physical damage caused by vaginal delivery. Lee et al (2004) questioned 505 Korean women and found that women\u2019s attitudes were not related to cesarean section surgery.27 They proposed that the preferences of healthcare practitioners and the healthcare system were the main factors in determining the mode of delivery. The study period (from 2011 to 2016) was not long enough to cause changes in the patients\u2019 perspectives on the mode of delivery. The main cause of C-section rate changes will have come from the changes in hospital behavior.The C-section rates could be affected by the mother\u2019s preferences for the mode of delivery.According to OECD health statistics, the C-section rate in Korea in 2017 was 45.2%, far exceeding the average rate of other OECD countries. Thus, policy intervention is required. Since interventions addressing C-section rates have been discontinued at this time, management incentives are required for quality assessment.First, to resume quality assessment, a risk-adjustment model must be developed that can be applied to the DRG payment system, which is one of the reasons that quality assessment for cesarean delivery was terminated. This model must determine whether the assigned KDRG fully reflects the patient\u2019s clinical status.28 Transition from a performance payment system to an absolute evaluation method will likely be accepted as a means of acknowledging the doctors\u2019 expertise rather than as a mechanism of control over the provider.29 Previous studies have shown that the absolute evaluation method was more effective than the relative evaluation method.31Second, an improved evaluation method is required. In Korea, pay-for-performance (P4P) programs adopt a relative evaluation method based on a ranking of hospitals. A large number of medical institutions oppose the system because of the uncertainty it raises about the potential increases or decreases in payments based on these evaluations.33 A previous study showed that when the rates were publicized through mass media outlets, there was a reduction in C-section rates relative to other periods.6Third, while publicizing assessment results is important, the public will need to easily access and understand the information. In addition, an effective disclosure strategy for the assessment results must be used when selecting medical providers.This study is meaningful in that it is an empirical study that evaluated the effect of the end of discontinuation of the Korean Value Incentive Program on the quality level of medical services by calculating the index which is evaluated using the risk-adjusted cesarean delivery rate as a dependent variable. In addition, to compensate for the short analysis period, which was suggested as a limitation in previous studies, and the impact could not be confirmed due to the lack of a control group, a total of 6 years were analyzed. However, since this study is not an analysis including a control group, there is a limit to estimating the pure effect of the policy only for the Korean Value Incentive Program. In particular, it is difficult to affirm the effect of the performance of the incentive system or information disclosure only, because the incentive system was carried out in conjunction with the disclosure of the evaluation results.16 that the coefficients calculated by pooling individual estimates using inverse variance weights and the coefficients calculated through average estimates for each hospital unit yielded consistent results. Therefore, it would be better to consider these in further studies.Another limitation of the study is that the uncertainty that does occur by using the risk-adjusted C-section rates was not considered. This study was performed according to previous studiesThis study showed that C-section rates have steadily increased since the discontinuation of the Korean Value Incentive Program and the associated disclosure of assessment results. This represents a return to the selective behavior of physicians on delivery from before the implementation of the program due to the absence of financial and social incentives.To manage the increasing C-section rates, new policy intervention may be implemented. However, rather than applying for the previous assessment program, it should be modified to reflect environmental changes, such as the adoption of KDRG and the use (and publicizing) of a new evaluation method. Successful implementation of the next-generation evaluation program will require discussion with diverse stakeholders in the medical community and will require a reasonable compensation system.This study investigated how hospitals responded to terminating the Korean Value Incentive Program for cesarean delivery.Risk-adjusted C-section rates increased immediately after the discontinuation? of value incentive program.Tertiary hospitals showed greater increases in C-section rates than general hospitals after the intervention.http://www.textcheck.com/certificate/Xvf6L2. This study was prepared by partially revising and supplementing the first author's master's thesis.The authors would like to sincerely thank the institute of health and welfare, Yonsei University who allowed us to partake in this study to share their expert knowledge on the Korean Value incentive program. The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see PYH and LKS designed the study. KJH performed the literature review and interpretation for data analysis. PYH and LKS analyzed the data. PYH and LKS wrote the draft. ALL authors read and approved the final manuscript."}
+{"text": "Selection in patients with functional mitral regurgitation (MR) to identify responders to interventions is challenging. In these patients, frailty might be used as a\u00a0multidimensional parameter to summarize the resilience to stressors. Our objective was to evaluate frailty as a\u00a0predictor of outcome in patients with moderate to severe secondary MR.We conducted a\u00a0single-center retrospective observational cohort study and included 239 patients with moderate to severe secondary MR aged 65\u00a0years or older between 2014 and 2020. Echocardiography was performed at baseline; frailty was evaluated using the clinical frailty scale (CFS). The combined primary endpoint was hospitalization for heart failure and all-cause mortality.p\u202f<\u20090.001), 1\u2011year mortality  even after adjustment for EuroSCORE2. The CFS was predictive for the combined endpoint  and outperformed EuroSCORE2 . In sensitivity analyses, we found that frailty was associated with adverse outcomes at least in trend in all subgroups.A\u00a0total of 53% (127) of all patients were classified as CFS 4 (living with mild frailty) or higher. Frail patients had a\u00a0higher risk for the combined endpoint (hazard ratio, HR 3.70, 95% confidence interval, CI 2.12\u20136.47; For older, medically treated patients with moderate to severe secondary mitral regurgitation, frailty is an independent predictor for the occurrence of death and heart failure-related readmission within 1 year and outperformed the EuroSCORE2. Frailty should be assessed routinely in patients with heart failure to guide clinical decision making for mitral valve interventions or conservative treatment. Valvular heart disease (VHD) affects 50% of the population aged 65\u00a0years and older, with a\u00a0general increase in age and higher prevalence in men and is also a\u00a0common reason for heart failure (HF) in this population . Given iThe aim of our study was to evaluate frailty as an outcome predictor in a\u00a0real-world population with moderate to severe secondary MR to support clinical decision making for mitral valve intervention.We conducted a\u00a0single-center retrospective observational cohort study from 1 January 2014\u201330 september 2020. The study was approved by the local ethics committee (1170/2020). Our hospital electronic health care records were searched for patient cases with ICD-10 codes related to mitral regurgitation (MR). Complete clinical, echocardiographic and pharmacologic data were obtained from the digital fever chart by means of comprehensive chart review. Only adults aged 65\u00a0years and older were analyzed. Further inclusion criteria were any-cause hospitalization and in-hospital performed echocardiography with a\u00a0diagnosis of moderate to severe MR. Individuals with primary MR and patients who were referred to surgery or mitral valve repair were excluded. The diagnosis primary or secondary MR was made according to European Society of Cardiology guidelines for echocardiography . All patThe combined primary endpoint was hospitalization due to heart failure and all-cause death. To reduce confounding effects due to competing risks, we decided to shorten the observation period to a\u00a0follow-up of 12\u00a0months. Frailty was assessed from the nursing records using the revised version (2.0) of the CFS . As a\u00a0bi2-test was used to calculate differences between groups. Survival and hospitalization were visualized by means of Kaplan Meier plots in Fig.\u00a0p-value of <\u202f0.05 was considered statistically significant. As not all parameters were available for all categories, patients had to be excluded for the subgroup analyses. For this reason, not all patient numbers add up to 100% (see tables). Stata\u00a017 was used for all statistical computations .Baseline characteristics were expressed as median with interquartile range (IQR) for continuous variables in parentheses. Differences between groups were calculated using U\u2011test. Categorial variables, were expressed as frequencies and percentages, and \u03c7p\u202f=\u20090.75), frail patients were older . Echocardiographic parameters like left ventricular ejection fraction, left atrial size and the presence of aortic stenosis did not differ significantly  and poor mobility . They required higher levels of assistance, whether from family members, home care or nursing homes . The rate of polypharmacy was high in both groups .Table\u00a0p\u202f=\u20090.001) as well as the combined endpoint at 1 year   as well as 1\u2011year mortality . Frailty remained associated with higher risk for the combined endpoint  and 1\u2011year mortality  after adjustment for EuroSCORE2. The CFS was predictive for the combined endpoint  and outperformed EuroSCORE2 .Fig.\u00a0p\u202f=\u20090.03). The categorical frailty scale was associated with the combined endpoint in univariate and multivariable modelling  better than EuroSCORE2 \u00a0frail patients had a\u00a0significantly higher rate of reaching the combined primary endpoint (death and readmission due to heart failure). 2)\u00a0In multivariable analysis, frailty was an independent predictor of the occurrence of the primary endpoints. 3)\u00a0For that purpose, the CFS was a\u00a0better prognostic tool than the EuroSCORE\u00a0II. 4)\u00a0Overall mortality was low in both groups.Metze et\u00a0al. showed that frail patients undergoing percutaneous mitral valve repair face a twofold increase in hazard of death or heart failure-related readmission in long-term outcomes compared to nonfrail patients. Frailty as assessed using the Fried criteria is in this study an independent predictor of adverse outcomes after adjustment for logistic EuroSCORE . Our stuAccording to expectations, mortality as well as the readmission rate are both significantly higher in the frailty group; however, it has to be mentioned that despite numerous readmissions, overall mortality is still low in both groups. These findings are consistent with previous trials in patients with conservatively and interventionally treated secondary MR . NeverthRecent findings show that other predictive variables for increased mortality, regardless of intervention, are older age, high Charlson comorbidity index, renal dysfunction, anemia, elevated right atrial pressure (estimated by echocardiography), admission for heart failure, and lack of mitral valve intervention , 30. In Furthermore, in univariate analysis, CFS resulted in a\u00a0higher AUC compared to EuroSCORE2. The poorer performance of the EuroSCORE\u00a0II might be seen in the light of the fact that this score was designed for the eligibility of cardiac surgery in mainly younger patients. Nevertheless, there does not seem to be a\u00a0more appropriate risk score for outcome prediction in this sample. According to recent studies, our findings strengthen the presumption that the clinical judgement of patients by means of CFS is a\u00a0better evaluation tool than the measurement of various clinical parameters and assessment of comorbidities and also predicts the outcome . This suAn important limitation is that this is a\u00a0single-center study with a\u00a0small cohort of patients, which reflects common patients in non-tertiary care centers. The inclusion was not focused on candidacy for intervention.Furthermore, all variables were obtained retrospectively and frailty was assessed using nursing documentation. There might be a\u00a0selection bias, given the fact that patients were recruited via ICD-10 diagnoses only. The recording of every single diagnosis in our digital fever chart is conducted by doctors in charge; however, the accuracy of these recordings and the consecutive completeness of our data cannot be guaranteed. For adjustment, we used EuroSCORE2, in awareness of the fact that this score is not validated in this setting. Nevertheless, EuroSCORE2 is routinely assessed and commonly used for cardiac risk evaluation as it has a\u00a0high degree of familiarity.No objective frailty assessment method was used, e.g. 5\u2011minute walk test, grip strength, etc. Frailty was a\u00a0subjective determination using the CFS. No additional frailty assessments were used, as the CFS is reliable and comparable to more complex methods, such as Frieds frailty phenotype in identifying frailty . We did For older, medically treated patients with moderate to severe secondary mitral regurgitation, frailty is an independent predictor for the occurrence of death and heart failure-related readmission within 1\u00a0year. In univariate analysis, CFS resulted in a\u00a0higher AUC compared to EuroSCORE2. We therefore believe that at least in an older medically treated population, reflected in our study cohort, CFS is more useful than EuroSCORE2. This is supported by our own clinical experience that CFS is easier to implement in everyday clinical practice compared to EuroSCORE2. CFS should therefore be assessed routinely in patients with heart failure to guide clinical decision making for mitral valve interventions or conservative treatment."}
+{"text": "Antimicrobial resistance (AMR) is a threat to public health. Addressing unnecessary antibiotic use provides an opportunity to reduce antibiotic consumption and to slow AMR. Understanding people\u2019s beliefs is important for informing antimicrobial stewardship (AMS) initiatives. Within New Zealand, health inequities exist between M\u0101ori and non-M\u0101ori; however, no research has examined M\u0101ori beliefs about antibiotics and AMR. The aim of this study was to explore the experiences related to antibiotic use of M\u0101ori in New Zealand. In-depth, semi-structured interviews were conducted with 30 M\u0101ori adults recruited from primary care to explore the experiences, perceptions and beliefs that M\u0101ori have about antibiotics, and about AMR. Overall, 30 M\u0101ori adults  participated. Three themes emerged: systemic-, social-, and individual-related factors. From these themes, seven subthemes explained the factors that influenced antibiotic use and their perceptions of AMR in M\u0101ori: general practitioner (GP) times and ratios, effect of colonisation, lack of knowledge and information, access and poverty barriers, relationship with health professionals, illness perceptions, treatment beliefs and Whaakaro (thoughts), and beliefs pertaining to natural (rongo\u0101) and Western medicine. Participants identified potential solutions to improve antibiotic use such as cultural support and involving Te Ao M\u0101ori; recognising these can inform future AMS initiatives. Antibiotic resistance has become a threat to public health at individual, community and national levels . In New High rates of antibiotic dispensing for M\u0101ori, in large part driven by higher rates of infectious disease, contribute to the development of antibiotic resistance. Addressing unnecessary antibiotic use provides an important opportunity to reduce antibiotic consumption ,11. One Understanding people\u2019s beliefs and perceptions about antibiotics is important for informing antimicrobial stewardship initiatives, as perceptions can have a significant influence on the patient-health professional interaction and can impact antibiotic use ,17. GeneDiffering worldviews on health can influence treatment and healthcare expectations in individuals, and health professionals\u2019 perceptions and behaviours are influenced by these expectations, potentially contributing to suboptimal or unnecessary antibiotic prescribing ,15. A siThis paper thus aims to explore the experiences, perceptions and beliefs that M\u0101ori have about antibiotics and the use of antibiotics in regard to acute upper respiratory tract symptoms, and of antimicrobial resistance, using a qualitative research approach. A total of 30 participants participated in the study; 21 participants were interviewed one-on-one and the remainder were interviewed in four focus groups with one group of three and three groups of two using a semi-structured interview guide . Twenty-Nine participants reported living with a long-term illness, which included breast cancer, heart disease, rheumatoid arthritis and diabetes . Eleven Three overall themes influenced the use of antibiotics; these themes related to Systemic, Social and Individual factors. From these overall themes, seven subthemes emerged to explain the factors that influenced antibiotic use and resistance. Additionally, one specific theme related to potential solutions was raised by participants to improve antibiotic use .Most of the participants were living with chronic health conditions and, due to limited consultation time with the GP, additional problems including infections were often not able to be addressed in the consultation. This was recognised as a potential factor that could lead to suboptimal use of antibiotics in M\u0101ori, \u201c\u2026because the thing today is, you\u2019re only allowed 15 minutes to try and get everything in, that was another thing with the (GP). Oh, what was it, I think it was my hearing, and something else, and when I went in there to see him and I tried [to] ask him about my hearing and about this other thing and he said \u2018well, it\u2019s one or the other,\u2019\u201d\u2014P23, F, 66 years old.Participants also reported that there was not enough consultation time with the GP to give a full narrative of all health symptoms that they were experiencing, thus impacting their understanding of their health and the role of medication and treatment, as \u201cthere\u2019s not enough [time], I guess, when the time is confined to 15 minutes. You can\u2019t put everything into 15 minutes, yeah,\u201d\u2014P9, F, 69 years old. Along with time, participants identified lack of GP availability as a contributing factor to why they could not access the care they need. One participant said that \u201c\u2026you\u2019ve got to be quick to get that spot to see a doctor. There [are] not enough doctors here,\u201d\u2014P5, F, age unknown. Meanwhile, another highlighted the mismatch between GP availability and need for help: \u201c\u2026because when I need to see a doctor, she\u2019s not available or she\u2019s somewhere else,\u201d\u2014P9, F, 63 years old.The negative impacts of colonisation for M\u0101ori were both explicitly and implicitly discussed by participants. Many recognized the \u2018downstream\u2019 effects of colonisation because of how colonisation underpins the systems and structures in society that participants perceived provided advantage to NZ European ways over M\u0101ori values. Consequently, participants described experiencing disadvantaged socioeconomic and health status, which in turn undermines socioeconomic opportunities and resources such as health literacy, eventually resulting in increased risk for infections and subsequently, antibiotic overuse.Many participants reported that the removal of traditional Rongo\u0101  knowledge had an impact on their healthcare utilisation and perceptions of medicine, as \u201c\u2026our M\u0101ori-ism [sic] needs to be brought into our thing.I don\u2019t know why people don\u2019t know anything about it, you know, like medicine for the kids, the cream that they get. Yeah it\u2019s good, but our medicineis just\u2026 better,\u201d\u2014P19, M, age unknown.Participants also reported the effects of urbanization of M\u0101ori communities, as a result of land-loss, resulted in a loss of culture and identity over time: \u201cI was bought up on the marae\u2026 It was a good life. My mother spoke fluent M\u0101ori, more so my mum then my dad\u2026 The government at that time change(d); there was a generational change, so their house was built \u2026so we moved from the marae up there,\u201d\u2014P7, F, age unknown. This culture shift impacted their health-seeking behaviour and how they integrated into the health and societal systems.Commonly, participants were not able to distinguish the difference between a bacterial and viral infection. Participants said they did not understand what an antibiotic was before an explanation was presented by their health provider and some reported having antibiotics for viral infections. Participants reported having had limited or unmemorable past exposure to information regarding antibiotics or that they \u201c\u2026can\u2019t remember. It\u2019s been a while. I heard [information about antibiotics] before, but I can\u2019t remember,\u201d\u2014P22, M, 63 years old.Information regarding medications such as antibiotics was perceived to be scarce. Participants reported having fears of taking antibiotics due to the lack of understanding and information regarding what they are and what they do to the body: \u201cI get scared if I don\u2019t know the medication. See but don\u2019t even explain that what it is. It\u2019s just an antibiotic, what is that, what is the medication doing for you, so I\u2019m just more like Augmentin. I know what, I don\u2019t know exactly what Augmentin is, but I know what it does for me. It kills what needs to be, the bacteria inside myself,\u201d\u2014P11, M, 46 years old. In terms of information sources, only a few participants reported receiving information about antibiotics from their GP. Similar to the concerns reported in Theme 1, this may be due to issues of access to the GP, and time availability during the consultation. Furthermore, some participants reported that they were not able to understand information provided by their GP, as \u201cthey do write in medical language and not everyone is medical and then you ask the pharmacy and they\u2019re only reading off what the bottle says or on the paper,\u201d\u2014P16, F, age unknown. Many participants reported using the internet or friends and wh\u0101nau as sources of information about antibiotics: \u201cI ask for the name of [the medicine] and I googled [sic],\u201d\u2014P27, F, 44 years old.Many participants indicated a lack of knowledge in terms of when to use antibiotics and what the purpose of the medication is for, stating that antibiotic use is required for more serious illness, for example when \u201c[the illness is] really bad. That\u2019s the only time I will take an antibiotic,\u201d\u2014P23, F, 66 years old. Participants described the \u2018severity\u2019 of how bad the infection is based on their experience of symptoms: \u201cI guess it depends really on how mucus-y [sic] you are or whoever is,\u201d\u2014P5, F, age unknown.Poverty and other barriers to accessing timely and quality healthcare influenced medication provision and use for the participants. Nearly all participants described having trouble accessing GP appointments due to cost, which also led to barriers in accessing antibiotics for the correct use. The cost of an appointment was highlighted as a concern for most of the participants. Many did not have the finances to seek healthcare (NZD$17 for adults) or the antibiotics (NZD$5 per medicine) when these were prescribed, as \u201c\u2026it costs money and it still gotta [sic] come back again for thrush, so it\u2019s a waste of time coming here to get you [antibiotics] if I have to come back and get you [thrush medication],\u201d\u2014P6 \u2014F, age unknown. Due to the cost of a GP appointment, many participants commented on the incurred debt that sat at the clinic. This debt caused them to worry that they may be turned away from necessary care and produced emotional strain on participants: \u201cI did not want to come in this morning. I had a bit of an argument with my partner. I was telling her that, you know, that I might get here and they might say \u2018no,\u2019 because of the money I owed them\u2026 So yeah, that\u2019s all I was afraid of coming in,\u201d\u2014P1, M, age unknown.Poverty forced participants to prioritise expenditure on items other than their healthcare, which delayed access to timely care; for example, one participant had to weigh up the options of spending money on food or medication: \u201cI\u2019ll get them [medication], and it was like $2 or $5 and sometimes I didn\u2019t have the finance, and when I did have the finance it was $5 food or $5 tablet,\u201d\u2014P26, F, 45 years old. Several participants reported the issues they faced when suffering from poverty, in which the cost of healthcare in general prohibited them from accessing timely care, as there were other expenses they needed to pay: \u201cI\u2019m asking for a food grant. I had to pay extra in parking. I get $4 after everything is paid and that\u2019s what comes from work and income and that\u2019s all we get and there are four little girls and I\u2019ve got two teenagers at home as well. But after the rent is paid, the power is paid, that\u2019s it, $4. Then the daycare bill, school fees, it\u2019s like \u201coh please give me a break,\u201d\u2014P2, F, 39 years old.Travel was also a barrier for participants to get necessary and timely healthcare. Some participants relied on wh\u0101nau to get to the GP clinic while others used other modes of transport such as buses, which were associated with additional costs or time-delays. Others reported having to travel long distances to get the help they required to treat their symptoms: \u201cthat\u2019s a half an hour, three-quarters of an hour drive [to go to the doctor]\u2026 sitting there in the car holding yourself up,\u201d\u2014P19, M, age unknown.One participant highlighted a cycle of poverty and crime. The participant reported that due to poverty and debt, they had to find the financial assistance to pay for their medication by other means, such as crime and drugs, to be able to access healthcare: \u201c\u2026that\u2019s usually what the crime is coming from you know. They can\u2019t get the medicine, so they get the other drug and they\u2019re in debt, so they have to do crime to pay it back and you know, it\u2019s a cycle,\u201d\u2014P1, M, age unknown.Many participants reported having a good relationship and rapport with their GP and that they adhered to the advice of their GP. In certain cases, this supported access to necessary medication in general and information, with one participant describing feeling culturally safe to share information during their consultation: \u201cI just think they\u2019re very culturally safe as [I] feel [they] welcome me and my wh\u0101nau,\u201d\u2014P28, F, 29 years old.Many also reported they took the advice of their GP, because the doctor is the health professional and was more knowledgeable about health in general than the participants themselves, as the GP \u201cseems to be really good, I try to be honest with them \u2018cause [sic] at the end of the day they know more than I know, and I need their help,\u201d\u2014P1, M, age unknown. However, one participant acknowledged it \u201ccomes down to, you know, a personal choice, you go to the doctors to see them because\u2026 when you\u2019re like concerned and you want to get a second opinion from your GP,\u201d\u2014P25, F, 49 years old.A few participants reported having negative experiences with their GP, which led to loss of trust with their GP, as \u201cthere was a bit of controversy there\u2026 It has taken me a long time after that doctor [for]\u2026 my trust and faith in the hospital, in the doctor [to come back]\u2026\u201d\u2014P7, F, age unknown. Other participants reported there were times when participants did not feel heard by their healthcare provider \u201cbecause [the GP] had already put things in place without listening\u2026 and I know he doesn\u2019t advocate antibiotics, but this particular time moko [my grandchild] needed it,\u201d\u2014P9, F, 63 years old.Participant illness perceptions, treatment beliefs and whaakaro were important factors that emerged from the interviews. There was variability between participants in their beliefs about antibiotics. Some participants described being happy to receive antibiotics as they believed this would be the best or only treatment for them to get better, providing \u201ca bit of a relief that I\u2019m going to be fixed and feeling better and I can do what I want, you know,\u201d\u2014P19, M, age unknown. Participants stated that they believed getting antibiotics was a necessity, that \u201cit is a must now\u2026 especially for your children. If your children need it, give it. It\u2019s worse if they suffer,\u201d\u2014P5, F, age unknown. Participants discussed other positive treatment beliefs and emotions associated with getting antibiotics and the help that they believed they needed\u2014including feeling \u201creally happy, grateful, thankful. Yeah, I was so happy that someone had helped me,\u201d\u2014P1, M, age unknown. In contrast, participants also discussed that antibiotics were more harmful than useful, expressing concerns about the effect of antibiotics on the body: \u201c[antibiotics are] more harmful than anything else. Nah, I just don\u2019t like it at all,\u201d\u2014P6, F, age unknown. Some also believed that \u201cif you take too much, you could damage your [body] system more,\u201d\u2014P26, F, 45 years old. There were concerns about adverse effects caused by antibiotic use that influenced participants\u2019 ideas about antibiotic use. This led to avoidance of antibiotic use because of participants\u2019 previous negative experiences, including side effects and misdiagnosis. In one example, the \u201cdoctors have prescribed my moko [kids]\u2026 with the wrong antibiotic and the side effects [occurred],\u201d\u2014P17, F, age unknown. In some cases, this prevented participants from accessing the antibiotics, \u201cbecause whatever is in it gives me bad thrush\u2026 so it\u2019s a waste of time coming here to get you [antibiotics] if I have to come back and get you [thrush medication],\u201d\u2014P6, F, age unknown. Other participants were adamant that they did not want antibiotics at all unless it was the last resort: \u201cI try not to take antibiotics unless I really have to,\u201d\u2014P25, F, 49 years old.Participants highlighted the importance of incorporating natural medicine as part of their healing process with western medicine. Participants perceived alternative healing as aligning with their spiritual and physical well-being: \u201cI take alternate healing. Sometimes the wairua needs to be aligned as well and I always believe that because my father was a healer, so he did healing with his hands,\u201d\u2014P9, F, 63 years old. This was particularly important for some as they were raised with natural medicine, which has influenced participants\u2019 perceptions of medication used today: \u201cwhen I got brought up, my grandmother\u2019s like, some of the medicines she just didn\u2019t agree, so she would go out and pick these leaves and just like\u2026 yeah, and it worked,\u201d\u2014P16, F, age unknown. Many participants discussed preferring natural medicine such as rongo\u0101, M\u0101ori medicine, over pharmaceuticals like antibiotics, perceiving that \u201crongo\u0101 is better than anything,\u201d\u2014P3, F, 20 years old, and that \u201crongo\u0101 would work better,\u201d\u2014P6, F, age unknown. In contrast, a few participants reported not having exposure to rongo\u0101 or not having exposure to the information regarding rongo\u0101, stating that they have \u201cnever used it, but \u2026have recommended it because [they] have been told about it \u2026 but\u2026 never used it \u2026 because [they] don\u2019t know where [they] can get anything like that,\u201d\u2014P27, F, 44 years old. Almost all participants provided suggestions to improve antibiotic use. Suggestions centred on better communication about antibiotics, with a focus on improving people\u2019s knowledge about antibiotics. Participants felt that GPs \u201cshould give out more info[rmation] to our people [M\u0101ori] or to whoever, give out more information and explain to them that antibiotics that they\u2019re giving to them,\u201d\u2014P6, F, age unknown. There were suggestions about disseminating information using pamphlets and presentations with accessible health information as an engaging way to deliver messages about antibiotics, to \u201c\u2026have more info[rmation] about [antibiotics], even with pamphlets, in the health thing, and all that. They got all the you need to stop smoking\u2014you need to do this, medication wise. They need pamphlets for the actual medication they\u2019re giving out\u2026 Yeah, sort of do like a presentation thing\u2026 on the marae,\u201d\u2014P26, F, 45 years. Other participants reinforced the importance of considering how the message was delivered, for example, some participants suggested the marae as a good venue to share information when learning about antibiotics as the \u201cmarae has always been a place, as a kid, that we always learnt a lot on our maraes\u201d and \u201cit\u2019s an open place for k\u014drero, for everything,\u201d\u2014P25, F, 49 years old. Having conversations face-to-face with health professionals was thought to important to increase antibiotic knowledge: \u201c\u2026a lot of verbal info[rmation], like I hate reading, like give me that, I\u2019ll go home and read it but I hate reading\u2026 Sitting down and having a conversation about it\u2026 and you telling me directly the good and the bad\u2026 Well kanohi ki te kanohi \u2026 is the best, we need to sit down and talk. Never mind about all this paper trail thingy,\u201d\u2014P23, F, 66, Waikato/Ng\u0101puhi. This was supported by the view from some participants who recognized that engagement with their GP was an important aspect in being able to have good knowledge in relation to antibiotics, which can help address any illness or treatment misconceptions. Some believed that their GP needed to engage better with them and their wh\u0101nau to improve understanding about antibiotics, as \u201c\u2026health literacy is important. I think that\u2026 they need to know more, and health professionals need to know how to engage and educate you. Know the consumer, wh\u0101nau, so that they have an understanding of what antibiotics actually do,\u201d\u2014P28, F, 28 years old.Participants emphasised the importance of cultural support and involving Te Ao M\u0101ori into learning about antibiotics, for example having \u201cmore cultural support to engage people with antibiotics\u2026 Understanding the medicines and the interactions. Using M\u0101ori Reo and Tikanga as way to help,\u201d\u2014P5, F, age unknown. A few participants highlighted that this is a \u2018P\u0101keha world\u2019 involving antibiotics, and suggested using Te Ao M\u0101ori to help patients understand, as \u201c\u2026you can\u2019t put a group of M\u0101ori in a room of P\u0101keha who are assessing that k\u014drero\u2026 So, it\u2019s someone who can view both worlds and yeah, I just reckon that\u2019s exactly what I think because you\u2019ve got to learn both worlds to understand them,\u201d\u2014P28, F, 29 years old. A few participants also commented on prevention of infectious disease to avoid using antibiotics in the first place, that the focus of education should be \u201cmore healthy living and that can avoid you from having to come on to see the GP [for infections] if we can live a more healthier life,\u201d\u2014P24, M, 49 years old.This is the first study to explore M\u0101ori beliefs about antibiotics and their use for cold or flu symptoms. We adopted a qualitative research approach to allow exploration of the experiences and perceptions of M\u0101ori in-depth. We identified systemic, social and individual factors that influenced antibiotic use for M\u0101ori. Personal beliefs were influenced by the wider health systems and infrastructures within NZ, which also impacted on the social issues that people experienced. Most participants appeared to have low expectations for antibiotic treatment for cold and flu symptoms; many expressed that they did not want to have unnecessary antibiotics or reported experiencing adverse effects with previous treatment.From the themes identified from this study, there are several key factors that could potentially be modifiable or targeted in an intervention to improve antibiotic use. First, knowledge relating to infections  and their management (symptomatic care or antibiotics) appeared to be lacking in this participant group. Participants described seeking information or clarification from other sources immediately after their GP consultation, using Google or wh\u0101nau, which reflects a clear need for better, more accessible information in this population. As provision of information in way that is tailored to an individual\u2019s health literacy level reflects the response of the health service or provider, and not simply the knowledge or capacity of the person seeking care, these findings suggest that health professionals need to develop and implement more effective resources for M\u0101ori who present with infections that may or may not require antibiotics 8, 10). Participants described a hierarchy during the consultation with the doctor, where the doctor\u2019s advice was seen as an important \u2018direction\u2019 even if they did not understand the information about antibiotics. People felt that it was not safe to ask questions or question treatment decisions. Unfortunately for M\u0101ori in NZ, there is both under-prescribing of necessary treatments and over-prescribing of inappropriate medications , creatin0. ParticWe identified issues of access and poverty that play a big factor in healthcare. Any relationship between the participants and their doctors has potential to be hindered by access and poverty issues. Participants described a cycle of M\u0101ori being unable to access care due to poverty and lack of transport, health professionals not seeing or understanding these issues and behaving in ways that caused misinformation or mistrust\u2014for example, communicating information in a way that participants felt like they had not been listened to, and people\u2019s beliefs about antibiotics being further undermined. This cycle needs to be broken. Any campaigns focusing on the education in M\u0101ori communities need to be done in a culturally safe way . ProgramParticipants highlighted a key point about their preference not to take antibiotics or at least for health providers to consider incorporating traditional medicine or alternative M\u0101ori traditional remedies as part of their treatment plan. This was reinforced by participants\u2019 negative experiences and past adverse effects with antibiotics, which impacted their preference to seek alternative remedies (rongo\u0101). Ancestral knowledge was lost because of colonisation with unequal treatment of M\u0101ori within societal institutions displacing many cultural traditions such as tohunga . The TohThese findings have important implications for antimicrobial stewardship initiatives in NZ. Participants suggested solutions to support and promote optimal antibiotic use in M\u0101ori. Developing a framework that addresses the downstream determinants of health such as information and patient experience with antibiotics would be key. Targeting this area would improve health knowledge and work to improve health literacy . It woulThere is a tension that exists with antibacterial use and the inequities that are seen in both infection and prescribing rates between M\u0101ori and non-M\u0101ori. Care is required to balance appropriate antibiotic use with the risks of resistance that can arise from overuse. Nationally, the drive to reduce antibiotic use to reduce the risk for antimicrobial resistance in NZ is countered within a context of M\u0101ori missing out on appropriate and necessary antibiotics relative to health need ,27. ThisA potential intervention could be one informed by constitutional reform, for example, one that challenges colonisation and acknowledges the power imbalance between M\u0101ori and P\u0101keha . ConstitThere were several strengths to the research. Interviews used in this research provided an opportunity for open discussion with M\u0101ori in a comfortable setting. The study focused on M\u0101ori participants and an area of healthcare that has not been studied in NZ before, filling a knowledge gap. The qualitative nature of the research allowed a wide variety of ideas, opinions and beliefs to be expressed and provided a crucial starting point to address inequities regarding antibiotic prescribing in NZ. Limitations to the study included sampling and methodological issues. Whilst a sample size of 30 is generally sufficient in qualitative research to ensure data richness and to reach data saturation . Our stuParticipants were recruited from a general practice with a high M\u0101ori patient population . Papakura Marae is located in Papkura, a south-eastern suburb of T\u0101maki Makaurau, approximately 32 kilometres south of the Auckland central business district. The Papakura Local Board area has a population of 45,633, with 24% of residents being 15 years and under (compared to 20.9% across Auckland) and the highest number of M\u0101ori (28.1%) within its Local Board boundary (compared to 10.7% across Auckland) . ParticiPatients identified as suitable for inclusion and who consented to find out more information about the study were then introduced by the health worker to the interviewer (KH). The study was explained to them by the interviewer and when patients agreed to participate, verbal and written informed consent was obtained from all participants prior to the interview. All participants had sufficient time to discuss with wh\u0101nau  to ensure consistency of coding. All themes were second-coded and checked by another member of the research team (AC). There are many factors influencing antibiotic use within M\u0101ori communities, at a system, societal and individual level. Importantly, the study highlighted key socioeconomic determinants for M\u0101ori that impact their use of antibiotics, and their experiences of the health system, including what to do when having an infection and how to receive appropriate treatment. These socio-economic inequities for Maori can be traced to historical and contemporary colonisation and attempts to address these upstream factors are likely to require political transformation to a system in which Maori leadership is supported. Considerations need to be made to consider Te Ao M\u0101ori in healthcare when prescribing antibiotics, and as lack of recognition of this can lead to negative downstream effects in health for M\u0101ori, such as increase in infections and substandard antibiotic use. M\u0101ori face reduced consultation times, leading to less rapport with health professional, decreased health literacy regarding antibiotic use and an increase in infection rates."}
+{"text": "W*, is used as the failure criterion. The effect of the hardener composition, the testing temperature, and the value of the phase angle \u03b2 determining the proportion of normal and shear stresses at the adhesive interface on the threshold value W* is detected. W*(\u03b2) diagrams (fracture loci) are plotted and analytically described logarithmic functions. They can be used to make strength calculations for adhesive joints in structures and metal-polymer composites.The regularities of the effect of a complex stress state on the strength of an AlMg5/epoxy adhesive joint are experimentally studied at \u221250 and +23 \u00b0C in tension+shear and compression+shear tests with different normal-to-shear stress ratios. The tests use modified Arcan specimens and Brazil-nut-sandwich specimens, with the lateral faces of the adhesive layer having a shape of a mushroom-like \u201cridge\u201d aimed at reducing stress concentration at the specimen edges. An original computational model of a selected microvolume including the interface together with the adjacent substrate and adhesive layers is used to process the experimental results. The attainment of the threshold value of strain energy density in the selected microvolume,  Using hybrid metal-polymer assemblies is a promising trend in developing composite materials and honeycomb and laminated load-bearing structures combining high strength and small weight ,2,3. TheThe cohesive strength of adhesive materials, as well as the overall rigidity of structures, can be increased by using reinforcing elements, e.g., glass or carbon tapes and fabrics, hard dispersoids, etc. However, the ability of metal-polymer assemblies to resist delamination-type failure is determined exclusively by the adhesive strength of the metal\u2013binder interface.Cold-curing adhesives are generally used to form an adhesive interface under conditions when it is difficult or technically impossible to use heating to cure thermosetting adhesives. Like other structural materials, an adhesive used as a binder in a metal-polymer composite must provide interfacial strength sufficient for its reliable operation in a structure. This is especially important for structures designed for Arctic regions, where low environmental temperatures aggravate the effect of mechanical loads.Composite materials and structures are currently designed mainly by means of engineering analysis systems involving global strength calculations of structural components and their assemblies, which are generally in a complex stress-strain state when being used. Reference databases included in engineering analysis software  containing characteristics of the ultimate properties of structural materials lack information of polymer adhesives. Engineering documents generally contain information only on the ultimate shear  strength of adhesive joints and only at room temperature. Also note that there is currently no fully formed idea of what criteria should be taken to estimate the strength of adhesive joints and what characteristics of the stress state should be used for this purpose. Engineering calculations often use force criteria based on the application of conventional theories of the strength of solids, as well as criteria that were specialized for solving problems of adhesive strength G is generally used as the constitutive parameter of CZMs, which is calculated as the work spent on the formation of a free surface unit during crack propagation [Gc and identification of models by traction\u2013separation diagrams, various tests are performed under conditions of cleavage (GcI), shear (GcII), and transverse shear (GcIII), including those regulated by ASTM D5528. The use of CZMs enables one to solve important problems of preserving the survivability of an adhesive joint in the case of the presence or formation of cracks in it. An important step in predicting the failure of adhesive interfaces was the development of cohesive zone models (CZM), according to which crack propagation is preceded by the development of material damage in front of the crack tip, and crack extension occurs when damage reaches a critical level. CZMs were focused on in a special issue of the Journal of Engineering Fracture Mechanics 70 (2003) and in later reviews ,11,12. Epagation . The modWhen an adhesive assembly is designed, it is important to determine the level of acceptable mechanical loads, above which initial discontinuities may start to appear at the interface. With further loading, these discontinuities cause the appearance and propagation of cracks and eventually the fracture of the assembly. An effective indication of this can be the ultimate value of the work/energy spent by external forces on the formation of local discontinuities at the most heavily loaded and dangerous points. The criteria based on strain energy take into account all the stress and strain components; therefore, they are more suitable as the criteria of initial fracture than stress and strain criteria. Besides, as distinct from energy release rate, this quantity is cumulative, and this provides a principal possibility to take into account the effect of the history of deformation conditions on the failure of an adhesive assembly. Strain energy density averaged over the adhesive volume being deformed was used as the failure criterion in . The autGc as dependent on the value of the phase angle \u03c8 characterizing the ratio of shear \u03c3s and normal \u03c3n stresses in front of the crack tip [The ultimate strain energy of an adhesive assembly depends on the stress state; however, few results of experimental studies of these dependences are found in the scientific and technical literature. The majority of them deal with rack tip ,16,17,\u03c8s*~\u03c3n*) is also common [The representation of the threshold characteristics of adhesive assemblies in tension+shear and compression+shear tests as diagrams in the coordinates  was studied. The alloy containing 5.2% Mg, 0.5% Mn, 0.3% Si, and 0.2% Zn is used to produce structural members. Adhesive compositions were made on the basis of ED-20 epoxy (bisphenol A diglycidyl ether)  with an epoxy number of 21.1%. Modified Arcan specimens  with gluMetal specimens were made from a hot-extruded bar with a diameter of 20 mm . The contact surfaces of the specimens were then machined by milling in order to obtain the required roughness and to remove the oxide film. A roughness Ra of 0.4 \u03bcm on the contact surfaces of the plates after machining and abrasion was determined by means of a Wyko NT1100 optical surface profiler . In order to create adhesive joints, three versions of epoxy compositions were used, differing in the use of curing agents or hardeners . The composition of the materials is shown in Investigations with an Eplexor 100 N dynamic mechanical analyzer  were performed with the aim of determining the elastic properties of epoxy compositions, which were necessary for simulating the stress-strain state of adhesive joints during testing. For this purpose, cylindrical specimens with a diameter of 4 \u00b1 0.1 mm and a height of 6 \u00b1 0.2 mm were made by turning from 10 mm diameter bars of epoxy compositions obtained by casting into Teflon molds. The tests were conducted with the use of the time sweep software option of the device, which allows one to change the temperature in the test chamber at a constant specified static load. The temperature conditions of specimen heating/cooling were automatically controlled by the executive control systems of the device. The specimens were cooled by liquid nitrogen vapors and heated by the heating elements. In order to measure the dynamic complex elastic modulus during the tests under a static compressive load of 100 N, sinusoidal mechanical vibrations with a frequency of 1 Hz and an amplitude of 5 N were additionally applied to the specimen. The specimens were first cooled in the test chamber to a temperature of \u221270 \u00b0C, held for 15 min to equalize the temperature of the specimens, and then the temperature in the test chamber was raised at a rate of 1\u00b0/min until it reached +100 \u00b0C.E* was calculated by the formulaThe parameters  were recorded in the function of the test time into the built-in controller of the device. The measurement results were processed by means of Eplexor 9 software of the device. The complex modulus E* is represented as a complex operator,E\u2032 = E* cos \u03b4 is the elastic modulus (storage modulus), E\u2033 = E* sin\u03b4 is the loss modulus, and \u03b4 is the lag angle between the strain change and the stress change under sinusoidal loading conditions (the loss angle). The real part of the complex operator (3) characterizes the elastic properties of the material, and the imaginary part characterizes the viscous ones. The higher the value tan \u03b4 = E\u2033/E\u2032, the greater the manifestation of the viscous properties of the material. The temperature dependences of E* are graphically shown in The complex modulus Tg = 57 \u00b0C for A1, 59 \u00b0C for A2, and 62 \u00b0C for A3 were determined according to ASTM D7028 at the points of intersection of the tangents to the curves of the temperature dependence of E\u2033 and tan(\u03b4). The values of the glass transition temperature Modified Arcan specimens were tested in a Zwick/Roell Z2.5 testing machine  in a KTKh-20 climate chamber  keeping the test temperature constant within \u00b11 \u00b0C by means of a two-circuit cooler, with Freon as the refrigerant, and heat-producing electric heating elements.kn = \u03c3n/n= 0.58, and the value of the shear stress concentration factor in shear tests sk = \u03c3s/s= 1.02 . Under these conditions, the appearance of edge delamination cracks is hardly probable. The existence of a ridge has an insignificant effect on the deformation force since its thickness is small. The ridge can be easily formed with a shaped scraper removing excess adhesive from the lateral surface of the inserts to be bonded. Gained in [In order to increase the uniformity of stresses at the contact surfaces of Arcan specimens, many researchers make the specimen edges beak-shaped ,28,29,30ained in , the posArcan specimens with bonded inserts a were plP during the testing was measured with an accuracy of \u00b11% by means of a strain-measuring system incorporated in the testing machine unit, and the change in the dimension of the adhesive layer in the direction of loading was measured by means of an Epsilon attached extensometer with an accuracy of \u00b11 \u03bcm. Three Arcan specimens were tested for each test temperature and each angle \u03b1, i.e., 30 specimens for each adhesive composition.The specimens to be tested were fixed in the testing machine grips housed in the climate chamber. After reaching the specified temperature in the chamber, they were held for 30 min until the specimen temperature was equal to the temperature in the climate chamber. The specimens were tensioned at a rate of 1 mm/min to the fracture of the adhesive joint. The load \u03b1 between the direction of compression and the interface plane varied from 4\u00b0 to 21\u00b0.For specimen preparation, 20 mm diameter bars were cut into disks with a thickness of 20 \u00b1 0.2 mm. The disks were then cut into bars in the meridional plane and abrased to a roughness Ra of 0.4 \u03bcm on the contact surfaces, which was checked with a Veeco Wyko NT1100 non-contact profiler. The prepared halves were bonded by the epoxy compositions, with shaping the lateral surface of the adhesive layer as a mushroom-like ridge, as was described for the Arcan specimens. The appearance of the specimens and the loading condition are shown in Since compression+shear testing requires a greater force than is allowed by the Zwick/Roell Z2.5 testing machine, the Brazil-nut-sandwich specimens were tested in an Instron 8801 universal testing machine  with a maximum load of 100 kN, in a climate chamber included in the testing machine delivery set. Cooling in the chamber was performed by passing liquid nitrogen vapors, and heating was performed by electric heat-producing elements. The temperature was automatically controlled with an accuracy of \u00b12 degrees by means of the standard software of the machine on the basis of the measurement data of the chromel-alumel thermocouples. All in all, 25 to 30 Brazil-nut-sandwich specimens were tested for each adhesive composition.n and shear \u03c3s stresses can be used to evaluate the stress state at fracture [P is the load at bond failure, and S is the bond area.It follows from the simulation results  that thefracture ,\u03c3s= PW* of strain energy density in some selected microvolume, including the interface, is used as the criterion determining the condition for the local fracture of the adhesive joint. The selected microvolume shown in h2 belongs to the substrate, and the part of thickness h1 belongs to the adhesive. It is assumed that the reinforcing filler of the adhesive layer is not in the selected microvolume, and it does not participate in the adhesive interaction with the metal constituent of the composite. This assumption seems to be fairly logical since the adhesive interaction occurs at the interface between the adhesive and the substrate. The microvolume is in equilibrium, and on its opposite faces there are equal stresses \u03c3ij in the local coordinate system . The z\u2019-axis is directed along the normal to the interface, and the x\u2019- and y\u2019-axes lie in its plane.The attainment of the threshold value ij and the corresponding strains \u03b5ij are determined from solving the problem of mechanics on finding the stress-strain state of bonded solids under external loading. In the case under study, the selected microvolume is considered to be in the plane stress state under the action of normal and shear stresses determined by Equation (4). Since the loading diagrams of the specimens being tested were straight lines up to fracture, it was assumed that fracture occurs within the elastic strain of the adhesive.The stresses \u03c3W* and its component at the fracture of the joint is determined asWn*, Ws* are the normal and shear components of strain energy density; The threshold strain energy density in the selected microvolume E1, \u03bd1, G1 and E2, \u03bd2, G2 are the values of the normal elastic modulus, Poisson\u2019s coefficient, and shear modulus of the binder and the substrate, respectively. In the calculations, the values of the elastic characteristics E2 = 71 GPa, \u03bd2 = 0.3, G2 = 27 GPa for AlMg5 were set according to the reference data [The effective values of the elastic characteristics can be calculated by the known formulas for unidirectional composites ,32,(6)hnce data .h1 and h2 in the selected microvolume are introduced mathematical abstractions; therefore, they are physically undefined. It is only their ratio that can be physically meaningful. It characterizes the involvement of both materials in adhesive interaction. Suppose, in the first approximation, that this ratio is inversely proportional to the values of the normal elastic moduli of the adhesive and the substrate, i.e.,The formula represented by Equation (6) cannot be used directly for calculations since the values of the layer thicknesses h1 and h2, for calculating the effective values of Then, simple transformations of Equation (6) result in the following formulas, without E* can be considered equal to normal Young\u2019s elastic modulus, i.e., E1 = E*. Using the data from It follows from dynamic mechanical analysis that the material of the adhesives was in the glassy state during the tests; therefore, during deformation in the elastic region, W* on the phase angle \u03b2 calculated by the formulaTo make a quantitative evaluation of the effect of the stress state on the strength of the adhesive joint, we use the dependence of W* is seen to increase as the values of the phase angle \u03b2 (taking into account the sign) decreases. This dependence reflects the effect of normal stresses on the cleavage failure of adhesive joints as follows: when \u03b2 > 0, the interface is affected by external tensile normal stresses inducing cleavage; when \u03b2 < 0, there are compressive normal stresses hindering cleavage; \u03b2 = 0 corresponds to the stress state of simple shear, when there is no action of normal stresses. For all the joints, W* is higher under shear than under cleavage. Note that, as distinct from the use of the phase angle calculated by Equation (1), the fracture loci have no discontinuities when \u03b2 is used to characterize the stress state. The rate of increase of W* is much higher in the region of compressive normal stresses (\u03b2 < 0) than in the region of tensile normal stresses (\u03b2 > 0). The comparison of the obtained diagrams shows that, at room temperature, the adhesive joint with A2 (PAPA hardener) has the highest fracture energy consumption  throughout the stress range studied, and the adhesive joint with A1 (PEPA hardener) has the lowest one. The situation is indefinite at \u221250 \u00b0C, namely: when \u03b2 > 0, the values of W* are a little higher for the joint with A2, but when \u03b2 < 0, the threshold strain energy density of the joint with A3 (DETA hardener) rapidly increases; therefore, using DETA as the hardener for adhesive assemblies operating at low temperatures is preferable.W*(\u03b2) for each adhesive joint more obvious, let us study the curves in W* for the joint with A2 at \u221250 \u00b0C is higher when \u03b2 > 0 than at +23 \u00b0C. Under shear (\u03b2 = 0), the values of W* are identical at both temperatures, and at \u03b2 < 0 and T = \u221250 \u00b0C, the values of W* are lower than at +23 \u00b0C. For the joint with A3, the trend of the effect of test temperature depending on the stress state is opposite to that for A2; namely, W* is higher at +23 \u00b0C when \u03b2 > 0, and it is lower at \u221250 \u00b0C when \u03b2 < 0. For the joint with A1, the values of W* are higher at \u221250 \u00b0C in the entire range of \u03b2.To make the estimate of the effect of test temperature on W*(\u03b2) can be used as a criterion for the nucleation of cleavage fracture of adhesive joints in structures and metal-polymer composites. In this case, the used strength of the joint \u03a8 and its residual strength margin \u03a8res can be quantitatively evaluated as follows:W is the local strain energy density in the most stressed (dangerous) place of the adhesive joint at a given time.The dependence res can be conveniently used in design and checking calculations of adhesive assemblies. Note that the selection of the form of assumption (7) will have no effect on the values of \u03a8 and \u03a8res provided that the effective elastic characteristics of the selected microvolumes will be determined with the use of the same versions of assumption (7) in the determination of the experimental dependence W*(\u03b2) and in the calculation of W values for the same composition used in the structural component.The parameters \u03a8 and \u03a8W*(\u03b2) are approximated by the following function:c1, c2, c3 are empiric coefficients, whose values for the adhesive assemblies under study are shown in The dependences The approximation was made in Statistica v.8 software by experimental data processing with the application of the least square method and the quasi-Newton procedure. It follows from The regularities of the effect of the stress state type under conditions of low and room temperatures on threshold strain energy density for AlMg5/epoxy adhesive joints with polyethylenepolyamine, polyamidepolyamine, and diethylenetriamine curing agents have been studied in experiments on the tension of modified Arcan specimens (tension+shear) and compression of Brazil-nut-sandwich specimens (compression+shear). W*, should be used as the failure criterion for adhesive joints. This value depends on the test temperature and the magnitude of the phase angle \u03b2 determining the normal-to-shear stress ratio at the adhesive bond interface. A computational model of a selected microvolume including the interface together with the adjacent substrate and adhesive layers has been used to process the experimental results. The dependence W*(\u03b2) has been described by an exponential equation, and the numerical values of the approximation coefficients have been found.It has been proposed that the attainment of the threshold value of strain energy density in a selected microvolume, W*(\u03b2) yields the following conclusion: at a temperature of +23 \u00b0C, the adhesive joint with the hardener A2 (polyamidepolyamine) has the highest W* in the whole range of the stress state; at a test temperature of \u221250 \u00b0C, in the region of compressive normal stresses (\u03b2 < 0), the highest strength is shown by the adhesive joint with the hardener A2, while the joint with the hardener A3 (diethylenetriamine) has the highest strength in the region of tensile normal stresses (\u03b2 > 0).The analysis of the fracture loci W*(\u03b2) by an exponential equation and to determine the numerical values of the approximation coefficients. The obtained dependences can be used for calculating the strength of adhesive joints in structures and metal-polymer composites.The results of statistical analysis have enabled us to describe the dependence"}
+{"text": "In the published article, there was an error in affiliation \u201c1\u201d. Instead of \u201cTianjin People's Hospital\u201d, it should be \u201cTianjin Union Medical Center\u201d.Due to the change of the name of our institution from \u201cTianjin People's Hospital\u201d to \u201cTianjin Union Medical Center\u201d, we propose to correct the original institution name.The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated."}
+{"text": "The efficacy and selectivity of the synthesized derivatives 1) against four P2Y receptors i.e., t-P2Y1, h-P2Y2, h-P2Y4, and r-P2Y6Rs was carried out by calcium mobilization assay. The results revealed that except 1b, 1d, 1l, 1m, 1o, 1u, 1v, 1w, and 1y, rest of the synthesized derivatives exhibited moderate to excellent inhibitory potential against P2Y1 receptors. Among the potent antagonists, derivative 1h depicted the maximum inhibition of P2Y1 receptor in calcium signalling assay, with an IC50 value of 0.19 \u00b1 0.04\u00a0\u00b5M. The potential of inhibition was validated by computational investigations where bonding and non-bonding interactions between ligand and targeted receptor further strengthen the study. The best identified derivative 1h revealed the same binding mechanism as that of already reported selective antagonist of P2Y1 receptor i.e (1-(2- (2-tert-butyl-phenoxy) pyridin-3-yl)-3\u20134-(trifluoromethoxy) phenylurea but the newly synthesized derivative exhibited better solubility profile. Hence, this derivative can be used as lead candidate for the synthesis of more potential antagonist with much better solubility profile and medicinal importance.The P2Y receptors are responsible for the regulation of various physiological processes including neurotransmission and inflammatory responses. These receptors are also considered as novel potential therapeutic targets for prevention and treatment of thrombosis, neurological disorders, pain, cardiac diseases and cancer. Previously, number of P2Y receptor antagonists has been investigated but they are less potent and non-selective with poor solubility profile. Herein, we present the synthesis of new class of benzimidazole derived sulfonylureas ( Different cellular activities are controlled by extracellular nucleotides through P2X which is ionotropic and P2Y which is G protein-coupled receptor i.e., endocrine and exocrine secretion, neurotransmission, immune cell regulation, proliferation, differentiation and intracellular signalling . Eight tP2YRs are expressed in almost all human tissues and exerts various biological functions . Over-ex+2] which is useful for the screening of agonist and antagonist of purinergic receptors by performing calcium flux assay in P2Y transfected 1321N1 astrocytoma cell line (The N-terminus which is extracellular of P2Y receptor has many glycosylation sites (N-linked), and it has seven transmembrane spanning motifs that help in the creation of the ligand pocket . Additioell line . Cell liell line .Epithelial cells, macrophages, neutrophils and cardiomyocytes expressed P2Y2 and P2Y4Rs. These receptors are also expressed in some organs such as the heart, kidneys, brain, liver and spleen . P2Y2 anXenopus . In order to confirm the in silico interaction between target and ligand, in silico studies were conducted.P2Y1 receptors have been discovered and reported in humans, rats, mice, cows, chickens, turkeys, and Xenopus . P2Y1 reXenopus . CompareXenopus . The P2Yal cells . BPTU  and were used without any additional purification. Using capillary tubes (open ended) and the Gallenkamp melting point equipment, melting points of synthetic substances were calculated. On a BRUKER spectrometer, NMR spectroscopy (Proton and carbon NMR) was carried out in both DMSO and chloroform. FTIR (Agilent Technologies\u2019 Cary 630) spectrophotometer was employed to conduct IR spectrum research. LCMS Agilent 1,200/6,210 (TOF) was used for the HRMS analysis, and the electrospray ionisation (ESI) or electron ionisation  methods were used to get the HRMS data.2a-h) Sulfonyl chlorides (0.2\u00a0mmol) in 10\u00a0mL of methanol 10\u00a0mL. For 2\u00a0hours, the mixture of reaction was allowed to stir continuously at rt. TLC verified that reaction had finished. Then this reaction mixture was dried by evaporating it under decreased pressure. Product was then extracted with the 15\u00a0mL each of H2O and 15\u00a0mL CH3COOC2H5. To extract the compounds as solid-products, layer of Ethyl acetate was isolated, and washed with saturated saline thrice with 15\u00a0mL and at the end product was dried out with anhydrous Na2SO4, and further and dried up by evaporation  was added to suitable (poration .5a-f (1\u00a0mmol) and Triethylamine (2\u00a0mL) to 0\u00b0C by using ice-bath. Thereto, slowly emulsifying Phenyl chloroformate 6  solution in a THF (5\u00a0mL) which is anhydrous was then added to it. For a further 2hrs, this mixture remained continued for stirring at RT. After this, the mixture of reaction was vacuum evaporated, 30\u00a0mL of Ethyl acetate was used for extracting the product, and then washed away with 30\u00a0mL of saline. Using anhydrous Na2SO4, the organic upper layer was isolated and dried. It was then filtered and evaporated for dryness. Without additional purification, these intermediates were employed in the last stage after being verified by TLC.Anhydrous Tetrahydrofuran (5\u00a0mL) was used and chilled a combination of compound 4a-h (0.1\u00a0mmol), anhydrous Sodium hydride  and 7a-f (0.12\u00a0mmol) intermediates were combined and allowed for stirring in dry Dimethylformamide (5\u00a0mL) at RT for a whole day. Then the mixture of reaction was washed away with 20\u00a0mL of H2O after the reaction was finished, and the upper organic portion was then isolated, recovered with the anhydrous Na2SO4, filtrated, and allowed for evaporation until it get dried. Recrystallization was used to purify the residual residue.Compounds 1H NMR (DMSO-d6 - 250\u00a0MHz) \u03b4 (ppm) 10.81 , 10.11 , 7.85 , 7.54 , 7.38 , 7.37 . 13C NMR  \u03b4 168.7 (C=O), 167.9 (C-OH), 148.4 (C-NH2), 133.8 (C-NO2), 132.9, 130.2, 125.2 (C-Cl), 122.0, 121.3, 111.8, 104.7; HRMS (ESI-TOF) m/z: calcd [M+H]+ for C14H9Cl2N5O6S, 444.1270; found, 444.1265.Yield: 65%; off-white powder; mp: 107\u2013110\u00b0C; FTIR : 2,939 (N\u2013H), 3,450 (OH), 1,613 (C=O), 1H NMR (DMSO-d6 -250\u00a0MHz) \u03b4 10.76 , 7.54 , 7.51 , 6.87 , 6.83 , 1.95 .13C NMR  \u03b4 168.7 (C=O), 159.2 (C-NH2), 156.5, 150.5, 140.5, 138.0, 127.0, 126.9, 121.9, 115.3, 112.7, 111.2 (Ar-Cs), 20.0 (C-CH3); HRMS (ESI-TOF) m/z: calcd [M+H]+ for C15H13N5O5S, 375.0637; found, 375.0636.Yield: 62%; light brown solid; mp: 113\u2013115\u00b0C; FTIR : 3,429 (N\u2013H), 1,633 (C=O), 1,104 (S=O), 1H NMR (DMSO-d6 -250\u00a0MHz) \u03b4 10.68 , 8.86 , 8.09 , 8.05 , 7.47 , 7.43 , 1.94 .13C NMR  \u03b4 167.9 (C=O), 147.6, 144.8, 144.2, 132.1, 127.2, 123.2, 121.1, 20.1(C-CH3); HRMS (ESI-TOF) m/z: calcd [M+H]+ for C15H12N6O7S, 420.0488; found, 420.0488.Yield: 77%; off-white powder; mp: 117\u2013119\u00b0C; FTIR : 3,262 (N\u2013H), 1,633 (C=O), 1,034 (S=O), 1H NMR (DMSO-d6 -250\u00a0MHz) \u03b4 9.91 , 7.78 , 7.64 , 7.61 \u2013 7.58 , 7.39 , 7.34 , 7.13 . 13C NMR  \u03b4 167.9 (C=O), 146.9 (C-NH2), 146.9 (C-N), 144.3, 144.2, 139.8, 136.1, 128.2, 121.1, 118.6, 116.1, 112.7(Ar-Cs); HRMS (ESI-TOF) m/z: calcd [M+H]+ for C14H10N6O7S, 406.0331; found, 406.0333.Yield: 70%; off-white powder; mp: 114\u2013116\u00b0C; FTIR : 3,436 (N\u2013H), 1,643 (C=O), 1,460 (N-O), 1,110 (S=O), 1H NMR (DMSO-d6\u2013300\u00a0MHz) \u03b4 10.10 , 8.86 , 7.84 , 7.56 , 7.37 , 7.28 . 13C NMR  \u03b4 168.7 (C=O), 167.9 (C-OH), 148.4 (C-NH2), 132.9 (C-S), 130.2, 128.9, 125.2(C-Cl), 124.0, 122.0, 121.3, 121.0, 118.2, 114.1; HRMS (ESI-TOF) m/z: calcd [M+H]+ for C14H10Cl2N4O4S, 399.9799; found, 399.9796.Yield: 60%; brown solid; mp: 115\u2013117\u00b0C; FTIR : 3,345 (O-H), 2,947 (N\u2013H), 1,660 (C=O), 1,043 (S=O), 1H NMR (DMSO-d6 -300\u00a0MHz) \u03b4 9.89 , 8.86 , 8.21 , 8.19 , 7.85 , 7.82 , 7.25 .13C NMR  \u03b4 167.9 (C=O), 154.2(C-NH2), 147.2 (C-N), 130.7, 129.1, 127.7, 126.8, 124.0, 121.5, 117.5, 110.8 (Ar-Cs); HRMS (ESI-TOF) m/z: calcd [M+H]+ for C14H11ClN4O3S, 350.0240; found, 350.0243.Yield: 65%; off-white powder; mp: 110\u2013112\u00b0C; FTIR : 3,429 (N\u2013H), 1,645 (C=O), 1,128(S=O), 1H NMR (DMSO-d6 - 300\u00a0MHz) \u03b4 9.91 , 8.87 , 8.51 , 8.38 , 7.66  7.28 , 7.12 , 2.65 .13C NMR  \u03b4 168.9 (C=O), 168.4 (C-NH2), 148.1 (C-NO2), 146.9, 145.3, 139.0, 134.4, 132.6, 127.7, 124.7, 123.7, 121.6 (Ar-Cs), 20.9 (CH3).); HRMS (ESI-TOF) m/z: calcd [M+H]+ for C15H13N5O5S, 375.0637; found, 375.0638.Yield: 70%; light yellow solid; mp: 183\u2013184\u00b0C; FTIR : 3,436 (N\u2013H), 1,502 (C=O), 1,048 (S=O), 1H NMR (DMSO-d6 -300\u00a0MHz) \u03b4 9.92 , 8.85 , 8.37 , 8.21 , 8.04 , 7.81 , 7.14 . 13C NMR  \u03b4 154.1 (C=O), 149.8 (C-NH2), 147.2 (C-OH), 144.9, 129.3, 129.1, 129.1, 128.6, 126.9, 126.8, 124.2, 124.0, 117.9, 115.6 (Ar-Cs); HRMS (ESI-TOF) m/z: calcd [M+H]+ for C14H9Cl3N4O4S, 433.9410; found, 433.9411.Yield: 66%; off-white solid; mp: 124\u2013126\u00b0C; FTIR : 3,303 (OH), 2,975 (N\u2013H), 1,650 (C=O), 1,027 (S=O), 887 (C-Cl) 1H NMR (DMSO-d6 -250\u00a0MHz) \u03b4 9.21 , 7.57 , 7.39 , 7.35 , 7.29 ; 13C NMR  \u03b4 167.9 (C=O), 154.2 (C-NH2), 147.2 (C-N), 130.70, 129.1, 127.7, 126.8, 124.0, 121.5, 117.5, 110.8 (Ar-Cs); HRMS (ESI-TOF) m/z: calcd [M + H]+ for C14H10Cl2N4O3S, 383.9850; found, 383.9852.Yield: 61%; light brown solid; mp: 129\u2013131\u00b0C; FTIR : 3,429 (N\u2013H), 1,502 (C=O), 1,062 (S=O), 889 (C-Cl), 1H NMR (DMSO-d6- 300\u00a0MHz 3) \u03b4 10.79 , 8.06 , 7.43 , 7.25 , 7.08 , 7.06 , 3.12 . 13C NMR  \u03b4 168.9 (C=O), 168.4 (C-NH2), 148.1 (C-NO2), 146.9, 145.3, 139.0, 134.4, 132.6, 127.7, 124.7, 123.7, 121.6 (Ar-Cs), 20.9 (CH3); HRMS (ESI-TOF) m/z: calcd [M + H]+ for C15H12ClN5O5S, 409.0247; found, 409.0248.Yield: 72%; off-white solid; mp: 144\u2013146\u00b0C; FTIR : 3,443 (N\u2013H), 1,504 (C=O), 1,032 (S=O), 871 (C-Cl), 1H NMR (DMSO-d6-300\u00a0MHz) \u03b4 9.88 , 8.85 , 8.37 , 8.21 , 8.18 , 8.02 , 7.85 .13C NMR  \u03b4 167.9 (C=O), 159.0 (C-NH2), 154.2 (C-NO2), 147.2, 139.2, 129.6, 129.1, 126.8, 124.0, 123.3 (Ar-Cs); HRMS (ESI-TOF) m/z: calcd [M+H]+ for C14H10ClN5O5S, 395.0091; found, 395.0091.Yield: 75%; light brown powder; mp: 183\u2013184\u00b0C; FTIR : 3,359 (N\u2013H), 1,502 (C=O), 1,062 (S=O), 881 (C-Cl), 1H NMR (DMSO-d6-250\u00a0MHz) \u03b4 9.42 , 8.10 , 7.81 , 7.69 , 7.60 , 7.41-7.30 . 13C NMR  \u03b4 150.9 (C=O), 143.3, 142.4, 142.3, 138.6, 130.1, 128.9, 128.8, 126.6, 122.3, 121.3, 119.9, 115.0 (Ar-Cs); HRMS (ESI-TOF) m/z: calculated [M+H]+ for C26H19N3O3S, 453.1147; found, 453.1148.Yield: 81%; off-white solid; mp: 133\u2013135\u00b0C; FTIR : 3,429 (N\u2013H), 1,502 (C=O), 1,062 (S=O), 1H NMR (DMSO-d6-250\u00a0MHz) \u03b4 10.29 , 8.32 \u2013 8.30 ), 8.10 , 7.91 , 7.71 , 7.50-7.30 , 7.2 .13C NMR  \u03b4 150.0 (C=O), 136.1, 131.2, 130.7, 130.1, 129.5, 129.1, 128.2, 127.7, 127.4, 127.1, 123.6, 121.3, 115.0, 114.5 (Ar-Cs); HRMS (ESI-TOF) m/z: calcd [M+H]+ for C20H15N3O3S, 377.0834; found, 377.0835.Yield: 75%; light brown solid; mp: 154\u2013156\u00b0C; FTIR : 3,443 (N\u2013H), 1,627 (C=O), 1,096 (S=O), 1H NMR (DMSO-d6 -250\u00a0MHz) \u03b4 9.41 , 8.10-8.00 , 7.90-7.81 , 7.70 \u2013 7.50 , 7.30-7.21 .13C NMR  \u03b4 150.8 (C=O), 147.1, 142.9, 142.3, 136.4, 132.9, 130.1, 129.5, 129.3, 129.0, 128.9, 127.6, 127.4, 126.6, 122.4, 121.3, 121.2, 115.1, 114.8 (Ar-Cs); HRMS (ESI-TOF) m/z: calcd [M+H]+ for C20H14ClN3O3S, 411.0444; found, 411.0445.Yield: 78%; off-white solid; mp: 158\u2013160\u00b0C; FTIR : 3,436 (N\u2013H), 1,641 (C=O), 1,062 (S=O), 706 (C-Cl); 1H NMR  \u03b4 13.05 , 8.22-8.19 , 7.69 \u2013 7.64 , 7.57 , 7.55 , 7.53 , 7.51 , 7.48 , 7.46 , 7.21 .13C NMR  \u03b4 161.5 (C=O), 151.1, 150.6, 143.7, 135.0, 130.1, 128.8, 127.6, 127.0, 126.4, 122.4, 121.2, 118.7, 113.9, 112.6, 111.3 (Ar-Cs); HRMS (ESI-TOF) m/z: calcd [M+H]+ for C20H14N4O5S, 422.0684; found, 422.0685.Yield: 80%; white powder; mp: 162\u2013164\u00b0C; FTIR : 3,323 (N\u2013H), 1,688 (C=O), 1,487 (N-O), 1,026 (S=O), 1H NMR (DMSO-d6-500\u00a0MHz) \u03b4 8.22 , 7.90-7.81 , 7.60 , 7.51 , 7.20-7.10 , 3.80 . 13C NMR  \u03b4 167.4 (C=O), 159.7, 143.1, 141.8, 136.6, 132.1, 127.5, 120.8, 115.1, 114.4, 113.2 (Ar-Cs), 56.0 (OCH3).); HRMS (ESI-TOF) m/z: calcd [M+H]+ for C15H12ClN3O4S, 365.0237; found, 365.0238.Yield: 80%; off-white solid; mp: 160\u2013162\u00b0C; FTIR : 3,401 (N\u2013H), 1,627 (C=O), 1,026 (S=O), 835 (C-Cl), 1H NMR (DMSO-d6-300\u00a0MHz -) \u03b4 8.10 , 7.80-7.71 , 7.49-7.40 , 7.28-7.25 . 13C NMR  \u03b4 166.9 (C=O), 142.8, 141.5, 135.4, 131.6, 130.7, 130.1, 128.2, 126.0, 120.2, 114.6 (Ar-Cs); HRMS (ESI-TOF) m/z: calcd [M+H]+ for C14H10ClN3O3S, 335.0131; found, 335.0133.Yield: 77%; white solid; mp: 179\u2013181\u00b0C; FTIR : 3,457 (N\u2013H), 1,627 (C=O), 1,161 (S=O), 831 (C-Cl), 1H NMR (DMSO-d6-500\u00a0MHz) \u03b4 8.10 , 7.93 , 7.88 , 7.86 , 7.74 , 7.73-7.70 , 7.64-7.62 .13C NMR  \u03b4 167.4 (C=O), 144.1, 143.4, 142.5, 139.1, 132.2, 132.1, 129.4, 128.8, 127.5, 126.7, 126.4, 120.3, 115.1(Ar-Cs); HRMS (ESI-TOF) m/z: calcd [M+H]+ for C20H14ClN3O3S, 411.0444; found, 411.0443.Yield: 73%; brown solid; mp: 172\u2013174\u00b0C; FTIR : 3,387 (N\u2013H), 1,620 (C=O), 1,161 (S=O), 847 (C-Cl), 1H NMR (DMSO-d6-500\u00a0MHz) \u03b4 8.10 , 7.75 , 7.60 , 7.40 . 13C NMR  \u03b4 167.4 (C=O), 147.7, 144.5, 142.8, 132.0, 129.5, 128.1, 127.9, 120.1, 115.1 (Ar-Cs); HRMS (ESI-TOF) m/z: calcd [M+H]+ for C14H9Cl2N3O3S, 368.0956; found, 368.0955.Yield:84%; light brown solid; mp: 180\u2013182\u00b0C; FTIR : 3,436 (N\u2013H), 1,627 (C=O), 1,160 (S=O), 824 (C-Cl),1H NMR (DMSO-d6-500\u00a0MHz) \u03b4 8.20 , 7.72-7.67 , 7.60-7.59 , 7.13 , 6.92 .13C NMR  \u03b4 167.4 (C=O), 148.7, 145.8, 142.3, 132.1,- 129.1, 128.5, 126.0, 118.0, 115.1 (Ar-Cs); HRMS (ESI-TOF) m/z: calcd [M+H]+ for C14H9ClN4O5S, 379.9982; found, 379.9984.Yield: 75%; light brown solid; mp: 166\u2013168\u00b0C; FTIR : 3,436 (N\u2013H), 1,627 (C=O), 1,160 (S=O), 842 (C-Cl),1H NMR (Chloroform-d-500\u00a0MHz) \u03b4 8.03 , 7.58 , 7.27 , 7.27 , 7.26 , 3.80 , 3.76 .13C NMR  \u03b4 158.2 (C=O), 156.5, 145.5, 132.0, 128.9, 128.5, 126.1, 123.9, 122.8, 119.9, 109.5, 109.3, 52.4 (O-CH3), 50.9 (O-CH3).); HRMS (ESI-TOF) m/z: calcd [M+H]+ for C16H15N3O5S, 361.0732; found, 361.0734.Yield: 60%; light brown solid; mp: 177\u2013179\u00b0C; FTIR : 3,408 (N\u2013H), 1,627 (C=O), 1,096 (S=O),1H NMR (Chloroform-d-500\u00a0MHz) \u03b4 7.78 , 7.64 , 7.38 , 7.36 , 7.32 , 1.62 . 13C NMR  \u03b4 158.3 (C=O), 156.2, 145.5, 132.1, 128.2, 128.5, 126.1, 123.9, 122.8, 119.9, 109.5, 109.3, 50.91(O-CH3); HRMS (ESI-TOF) m/z: calcd [M+H]+ for C15H12ClN3O4S, 365.0237; found, 365.0239.Yield: 65%; light brown solid; mp: 152\u2013154\u00b0C; FTIR : 3,387 (N\u2013H), 1,627 (C=O), 1,160 (S=O), 841 (C-Cl),1H NMR (DMSO-d6-300\u00a0MHz) \u03b4 10.75 , 7.70 , 7.68 , 7.52 , 7.30 , 6.89 , 6.67  2.80 . 13C NMR  \u03b4 169.0 (C=O), 167.9, 159.2, 156.9, 140.8, 139.5, 136.3, 127.0, 125.5, 112.7, 20.3 (CH3); HRMS (ESI-TOF) m/z: calcd [M+H]+ for C15H14N4O3S, 330.0786; found, 330.0785.Yield: 70%; pale yellow powder; mp: 165\u2013167\u00b0C; FTIR : 3,390 (N\u2013H), 1,650 (C=O), 1,029 (S=O), 880 (C-Cl),1H NMR (DMSO-d6-250\u00a0MHz) \u03b4 9.91  8.87 , 7.75 , 7.52 , 7.35 , 7.15 , 6.95 , 1.85 . 13C NMR  \u03b4 167.9 (C=O), 138.3, 132.3, 131.8, 130.5, 129.5, 127.7, 127.7, 126.8, 121.5, 113.5, 20.3 (CH3); HRMS (ESI-TOF) m/z: calcd [M+H]+ for C15H13ClN4O3S, 364.0396; found, 364.0394.Yield: 73%; off-white solid; mp: 188\u2013190\u00b0C; FTIR : 3,422 (N\u2013H), 1,502 (C=O), 1,029 (S=O), 1H NMR (DMSO-d6-500\u00a0MHz) \u03b4 8.02 , 7.88 , 7.77 \u2013 7.71 , 7.71 \u2013 7.63 , 7.57 \u2013 7.45 , 7.39 \u2013 7.25 , 7.19 , 7.12 \u2013 7.05 , 6.95 , 6.84 . 13C NMR  \u03b4 155.9, 155.8, 155.7, 154.1, 152.4, 149.0, 139.5, 137.3, 134.4, 131.2, 129.5, 128.2, 121.2, 120.0, 119.4, 114.6, 101.9. HRMS (ESI-TOF) m/z: calcd [M+H]+ for C20H16N4O3S, 392.0943; found, 392.0944.Yield: 70%; off-white solid; mp: 176\u2013178\u00b0C; FTIR : 3,380 (N\u2013H), 1,627 (C=O), 1,128 (S=O), 2 incubator which was maintained at (5%) and 37\u00b0C temperature until 90% of the cells were confluent. Throughout the experiment, cells were periodically checked for the confluency and strictly watched for the microbial contamination.Dr. Gary A. Weisman provided human-1321N1 astrocytoma cells that had been transfected by the tP2Y1R, hP2Y2R, hP2Y4R, and rP2Y6R and untransfected 1321N1 astrocytoma cells . The DMEM Dulbecco\u2019s Modified Eagle -Medium (Gibco) was added in flasks to cultivate the cell line. 10% of the Fetal bovine serum was also added to the media together with 500\u00a0\u03bcg/mL of Geneticin (G418) and 1% of the Pen/Strep. The cells in T-75 flask were then cultured and incubated in a CO2 (5%) incubator. Next to this, the prepared culture media was changed to media which was free from FBS, the test compounds were added and the incubation process was repeated. After incubation of 24\u00a0h, the serum free media from the wells was discarded, and 100\u00a0\u03bcL of the reagent i.e., MTT reagent was pipetted. Plate was then additionally incubated at 37\u00b0C for 4\u00a0h. Utilizing a microplate reader, absorbance was determined at 570\u00a0nm and 630\u00a0nm for calculation of cell viability   .Real-time PCR was used to assess the efficacies of targeted genes . For this purpose, extraction of RNA was done by using reagent Trizol which was bought from (Invitrogen-Thermo Fisher Scientific) from stable transfected cell lines. According to the manufacturer instructions (Thermo Scientific), 2\u00a0\u03bcg mRNA of each desired gene (quantified by LVis plate) was occupied for the synthesis of cDNA using Reverse Transcription Kit. Program settings for the thermocycler were 25\u00b0C for time period of 5 min, 42\u00b0C for time period of 70 min, 70\u00b0C temp for 5\u00a0min, and infinity at 8\u00b0C. For RT-PCR, a master mixture which contains 1.5\u00a0\u03bcL = cDNA, 0.25\u00a0\u03bcL = reverse primer, 0.25\u00a0\u03bcL = forward primer, 10\u00a0\u03bcL = SYBR Green, and 8\u00a0\u03bcL from RNAse-free water was utilised, yielding a master volume of around 20\u00a0\u03bcL for each sample  were cultured in T-75 flask. When cells reached the confluency of 80%\u201390%, they were washed with PBS and then Trypsin was added. After trypsinization for 3 min, 20,000 cells/well were allowed to seed in a black wall and 96 well clear bottom plate which was coated with Poly-D-Lysine and allowed to incubate for 24\u00a0h in 37\u00b0C, 5% CO2 incubator. After incubation, 200\u00a0\u03bcL culture media was changed with 100\u00a0\u03bcL HBSS containing Fura-2 AM which is calcium chelating dye and allowed the plate to incubate for 45\u00a0min in dark at temp of 37\u00b0C in incubator which is maintained with carbon dioxide (5% CO2). After incubation for 45 min, synthesized molecules were added to HBSS washed cells in each well of 96 well plate and re-incubated for 30\u00a0min at 37\u00b0C. In next step, injection of the endogenous ligand such as ADP through the injector was followed by excitation and emission measurements of Fura-2 AM at 380/520\u00a0nm and 340/520. Final volume of the assay per well 200\u00a0\u03bcL. IC50 values were computed for the compounds displaying more than 50% of activity using Prism 5.0 software  by using analysis i.e., nonlinear regression  microplate reader with two injectors was used to measure the Cagression .In eppendorf tubes, over-saturated chemical solutions were prepared by mixing synthesized compound with PBS buffer (pH 7.4). To confirm compound saturation, suspensions were rapidly vortexed for 1 min, sonicated at ambient temperature for 10 min, and then incubated with overnight shaking. After that, compound suspensions were centrifuged at 15,000 RPM for 20\u00a0min. Supernatants were cautiously taken out of the tubes and filtered once more using 0.22\u00a0m syringe filters. By using UV-vis absorption spectrometry with standard curves and an already-determined absorption extinction coefficient in the same buffer, the concentrations of the drug in the supernatants were identified.in vitro results of the sulfonylurea derivatives of benzimidazole (1a-y) towards P2Y receptor, docking was conducted. Crystal structure of P2Y1R with PDB ID = 4XNV was used for this study (https://www.rcsb.org/). Targeted proteins were prepared by using software  with default parameters, the energy minimization and amino acid residues sequence correction was done through quick preparation, after that active amino acid residues were selected, and then docking was performed by applying dummies atoms. The ligand structure were sketched by using Chemdraw 15.0 and the energy minimization were also done in 3D Chem draw after selection of prepared ligand then run docking of top 20 poses. Using Discovery Studio Visualizer DS, the best poses with low binding affinity and highest binding affinity were chosen and examined  ammonium hydroxide was done. In second step, interaction of benzimidazole and phenyl chloroformate with triethylamine (TEA) as catalyst produced the intermediates of carbamate. The twenty-five target sulfonylurea products in good to excellent yield were synthesized in last and final step by reacting the synthesized sulfonamides (4a-h) and carbamates (7a-f) for 15h with sodium hydride as catalyst.Sulfonylurea analogues were synthesized in a three steps of reaction as depicted in 1a-y) such as colour and melting points were determined. Structure elucidation of compounds was done through FTIR, 13C NMR, 1H NMR and HRMS. In spectra of FTIR, absorbance bands for different functional groups appeared at appropriate ranges confirmed the synthesis of various analogues. Stretching frequency around 3,300 and 3,400 cm-1 relate to N-H while the stretching of carbonyl (C=O) appeared around 1,680 and 1,500\u00a0cm-1 confirmed the functionalities. In case of 1H NMR, the characteristic broad signals between 7.30ppm and 10.81ppm were observed for N-H respectively. Peaks for the protons of benzimidazole ring were detected in a range of 6.65\u20138.80\u00a0ppm. In 13C-NMR, all of the synthesized molecules display characteristic signals at \u03b4 168.78, 167.92 for carbonyl carbon.Physical properties of synthesized compounds   assay. Among the anti-cancer drugs cisplatin is used as standard for positive control. The results are shown in 2+ flow functional assay was used to further establish P2YR expression. Here, Ca2+ influx through these gates was activated by using ADP (500\u00a0nM) for t-P2Y1, ATP (500\u00a0nM) for h-P2Y2, UTP (500\u00a0nM) for h-P2Y4, and UDP (800\u00a0nM) in the case of r-P2Y6 pyridin-3-yl)-3-4-(trifluoromethoxy) phenylurea having IC50 value of 0.28 \u00b1 0.24 \u00b5M, reported earlier. The results are reported as inhibition rate at . The structure activity relationship for these compounds is summarized in All the synthesized compounds were evaluated for inhibition rate and IC1b, 1d, 1l, 1m, 1o, 1u, 1v, 1w and 1y depicted less than 50% inhibition of all the targeted receptors while derivative 1a, 1e, and 1h were found potent derivatives exhibited strong inhibition of P2Y1 receptor. All these three derivatives have same R\u2032 and R\u2034 but different R\u2033group. Compound 1h exhibited more potential as compared to 1a and 1e with an IC50 values of 0.19 \u00b1 0.04, 0.37 \u00b1 0.02 and 0.34 \u00b1 0.01\u00a0\u00b5M  at para position on ring B and no substitution at meta position on ring B resulted in the decreased potency of about five times relatively to the derivative 1h. The inhibition curve of these potent inhibitor showed in 1u and 1v. The compound 1v having 4-Cl group did not show any effect but better %inhibition result as compared to 1u, only because of the presence of electronegative substitution. Similarly when R\u2034 is replaced by Cl group like in 1p, 1q, 1r, 1s and 1t resulted in the different response. Among these derivatives the compounds, the derivative 1s having R\u2032 as 4-Cl substitution exhibited excellent inhibition of all the selected receptors non-selectively but found more potent towards P2Y1. Here, the substitution with the methoxy group resulted in the loss of activity towards all the targeted receptors except P2Y1 just because of presence of Cl irrespective of the position. An interesting behaviour was observed when the structure activity relationship of those derivatives was studied having phenyl ring at position R\u2034 i.e., derivative 1l, 1m, 1n and 1o, it was observed that only 1n exhibited some inhibitory potential towards P2Y1 and P2Y6 but did not show any response towards P2Y2 and P2Y4 receptor. This derivative showed inhibition only because of the presence of 4-Cl substitution (at R\u2032).The structure activity relationship was studied based on a variety of structural modification among different derivatives which contain benzimidazole nucleus and sulphonyl urea linkage. Among all the synthesized derivatives, compound  0.01\u00a0\u00b5M . The detectively . The act3) i.e., compounds 1b and 1d at benzene sulphonyl moiety showed no antagonistic effect against P2Y1, 2, 4 and P2Y6Rs. But the attachment of nitro group alone and together with alkyl group at benzene sulphonyl moiety  showed inhibition against P2Y1 receptor with IC50 value of 12.42 \u03bcM, 6.04 \u03bcM, 11.18 \u03bcM and 13.44\u00a0\u03bcM respectively which revealed that nitro group is favourable for antagonistic activity against P2Y1 receptor. The presence of bulky groups such as (-C6H5 and -OCH3) showed no favourable activity. Therefore, it can be suggested that introduction of electronegative Cl group resulted in the improved inhibition of the targeted receptors however, the position 3 and position 5 resulted in the improved inhibition of P2Y1 receptor.Conclusively, the SAR study of these compounds presented that existence of groups which are electron withdrawing i.e., chloro (Cl) substituent at m-position on benzene sulfonylurea moiety reserved the activity towards P2Y1 receptor suggested that chloro substitution was favorable at benzene sulphonyl moiety. While compounds with an alkyl moiety  bundles and is in contact with the phospholipid bilayer of the receptor. BPTU is a non-nucleotide antagonist of P2Y1R belonging to aryl urea derivatives while our synthesized compounds belong to the same family showing similar characteristic and behaviour. In case of compound 1h, NH of sulphonyl urea linkage, Oxygen of SO2 and free NH2 group of the benzimidazole ring show H-bonding with Leu102, Ala106 and Gln127 respectively. Chloro (-Cl) at meta position of benzimidazole ring show \u03c0-alkyl interaction, with Leu102 and chloro (-Cl) groups attached at meta position of phenyl ring showed \u03c0-alkyl interaction with Phe119 and Met123 respectively. Similarly, compound 1a, 1e and 1f showed same binding mode of interaction as compare with the 1h due to structural similarity among them. Some extra H-bonding interaction were observed in our synthesized compounds such as Gln127and Ala106 which are not present in the standard antagonist.1h) of series (1a-y) in aqueous solution using UV absorption spectroscopy, respectively , thus suggesting that only one H-bond with the backbone of L102 is needed to bind at the P2Y1R.The leading interaction that were observed in standard antagonist (BPTU) were also observed in our synthesized compounds. There are a few interactions found to be conserved on P2Y1R-ligands 1a-y) was synthesized as P2Y1 receptor antagonists in this study.Over-expression of P2Y1 receptors have been reported to be involved in pathophysiology of various disorders including inflammation, thrombosis, pain, osteoporosis, bladder abnormalities as well as neurological dysfunctions. There are limited antagonists of P2Y1 receptors available. Therefore, there is a need to synthesize new class of potent P2Y1 receptor antagonists with improved pharmacokinetic properties. Based on the presence of urea group in BPTU and suramin, sulphonylurea in Elinogrel, carboxamide group in SAR 216471 hydrochloride and benzimidazole nucleus in KCB77033 of previously reported compounds, shown in 1a-y).Cell viability assay was performed to evaluate the cytotoxic nature of the effective compounds and lower the possible effects of calcium influx that might be produced by cell toxicity producing false positive results. None of the sulfonylurea derivatives were identified as cell toxic, when the synthesised molecules were investigated with dose up to 50\u00a0\u03bcM. As a result, it was believed that any inhibitory impact seen by calcium influx assays was unlikely to be caused by the cytotoxicity of synthesized series  ester of Fura-2 fluorescence dye was injected into stably transfected P2YRs- 1321N1 astrocytoma cells. Four P2YR subtypes i.e., turkey P2Y1R, human P2Y2R and P2Y4R, and rat P2Y6 receptors were used in this study. Gq-protein coupled receptors, such as P2Y receptor upon ligand activation., ATP, ADP, UDP and UTP causes abrupt rise in the intracellular Ca+2 concentration as a result of the release of the fluorescence light by the Fura-2 AM dye. Synthetic substances that act as P2Y receptor antagonists prevent the increase in the intensity of the fluorescence which is measured via a fluorescent microplate reader. Among all the synthesized derivatives, compound 1a, 1e and 1h were found potent derivatives exhibited strong inhibition of P2Y1 receptor. Compound 1h exhibited more potential as compared to 1a and 1e with an IC50 values of 0.19 \u00b1 0.04, 0.37 \u00b1 0.02 and 0.34 \u00b1 0.01\u00a0\u00b5M. The presence of electronegative Cl group resulted in the improved inhibition of the targeted P2Y1 receptor. The detailed structure activity relationship was described in detail in result section.The effectiveness of the synthesised compounds were assessed through fluorescently based calcium mobilization assay which follow the standard methodology i.e., the Ca1h provides a way to evaluate their functional profile in vivo. In silico studies of the potent compounds were also performed which support in vitro results. Compound 1h showed considerable interaction comparable with BPTU at the allosteric site which is located on the outer hydrophobic surface of the transmembrane (TM) bundles of the receptor.The improved aqueous solubility of compound 1h with 3,5-diCl, 2-OH substitution showed the highest potency with an IC50 value of 0.19\u00a0\u03bcM against the P2Y1 receptor, which is comparable to the non-selective inhibitor suramin. Additionally, compounds 1a, 1e, and 1f showed excellent inhibition towards P2Y1 receptor. Compound 1h also depicted inhibitory potential against P2Y2 and P2Y4 receptors but was inactive against P2Y6. The aqueous solubility of compound 1h was significantly higher than the reported BPTU antagonist, indicating its potential for in vivo functional activity. Furthermore, computational studies revealed that compounds 1a, 1e, and 1h formed complexes with P2Y1R-ligands that interacted with amino acid residues Leu102, Ala106, Phe119, and Met123. These results suggest that the P2Y1 receptor may bind with these ligands. Overall, the synthesized benzimidazole-based sulfonylurea derivatives represent a promising group of compounds for further development as P2Y receptor subtype inhibitors.All the synthesized compounds based on benzimidazole and sulfonylurea were synthesized and characterized through spectroscopic techniques and evaluated for their inhibitory potential against P2Y receptor subtypes. The synthesized compounds exhibited less cytotoxicity compared to the reference compound, and most of them displayed good to excellent inhibitory activity. Compound"}
+{"text": "Serum thyroglobulin (Tg) is a cornerstone on the follow up of differentiated thyroid cancer (DTC) and is widely used for diagnostic and prognostic purposes . Some faIn addition to a well-performed neck ultrasound, the ultrasound guided fine needle aspiration biopsy (FNAB) should be performed in every suspicious lymph node in order to confirm malignancy. As these biopsies still carry up to 10% of false negative results  recommenMartins-Costa and cols. , assesseI131 uptake, however, only 57% had positive cytology. When Tg-FNAB was < 10 ng/mL, only 10% had confirm metastatic disease and FNAB was able to identify only 2%. In group 2, when Tg-FNAB was above 10 ng/mL, also all patients had confirmed metastatic disease and positive cytology but when less than 10 ng/mL, around 45% had confirmed metastatic disease by histology or I131 uptake but only 25% had positive cytology. In both groups there were a high frequency of non-diagnostic cytology FNABs when Tg-FNAB was less than 10 ng/mL, showing that neither Tg-FNAB nor cytology were able to identify those cases of lymph node metastasis.Patients were divided into two groups: group 1 (203 FNAB samples): serum negative TgAB and group 2 (29 FNAB samples): serum positive TgAB. In the first group, all patients with Tg-FNAB > 10 ng/mL had metastatic disease confirm by histology or The authors nicely showed that Tg-FNAB is a useful tool to diagnose lymph node metastasis as they proved that, when above 10 ng/mL, all patients had confirmed metastatic disease, independently on the Tg-AB serum status. Thus, if Tg-FNAB is high in a suspicious lymph node, is likely that will be confirmed as metastatic disease. Interestingly, Tg-FNAB was even more accurate than cytology. That information reinforces the recommendation that Tg-FNAB should be measured when suspicious lymph nodes are biopsied in order to increase sensitivity.Conversely, when Tg-FNAB was lower, the accuracy was not as good but neither was cytology.Even more important, in the present study, the authors also showed that the same phenomenon occurs in serum TgAB positive and negative patients, suggesting that the presence of TgAB in the serum does not interfere significantly in Tg-FNAB measurement. This is an ongoing discussion in the literature. Shin and cols., studied 239 lymph nodes from 201 PTC patients and found that the presence of serum TgAB in fact interfered with Tg-FNAB measurement  and simi"}
+{"text": "The Renal Activity Index for Lupus (RAIL) consists of urine protein assessment of neutrophil gelatinase\u2013associated lipocalin, kidney injury molecule-1, monocyte chemotactic protein 1, adiponectin, hemopexin, and ceruloplasmin, which non-invasively identifies lupus nephritis (LN). We aimed to delineate RAIL scores with inactive versus active LN and changes over time with response to LN induction therapy.There were 128 pediatric patients with systemic lupus erythematosus (SLE) and age-matched healthy controls recruited in a prospective case control study, with kidney biopsy confirmation of LN. Laboratory and clinical information was recorded and urine collected at diagnosis and end of induction and during maintenance therapy. Response to therapy was assessed by repeat kidney biopsy or laboratory parameters. Urine was assayed for RAIL biomarkers and the RAIL score calculated.p\u2009<\u20090.0017). pRAIL scores significantly decreased with complete LN remission by 1.07\u2009\u00b1\u20091.7 (p\u2009=\u20090.03). Pediatric RAIL (pRAIL) scores from 128 children and young adults with SLE (with/without LN: 70/38) including 25 during LN induction therapy, differentiated clinically active LN from inactive LN or without LN, and controls (all The RAIL biomarkers differentiate LN patients based on activity of kidney disease, with decreases of\u2009\u2265\u20091 in pRAIL scores indicating complete response to induction therapy. Significantly lower RAIL scores in healthy controls and in SLE patients without known LN raise the possibility of subclinical kidney disease.The online version contains supplementary material available at 10.1007/s00467-023-05888-z. Kidney involvement with systemic lupus erythematosus (SLE), or lupus nephritis (LN), continues to account for sizable morbidity, mortality, and kidney failure . An estiTo provide accurate non-invasive means of monitoring LN disease activity and response to LN therapy, we discovered and validated a urine biomarker panel consisting of 6 urine proteins: neutrophil gelatinase\u2013associated lipocalin (NGAL), monocyte chemoattractant protein-1 (MCP-1), kidney injury molecule-1 (KIM-1), ceruloplasmin, adiponectin, and hemopexin. Renal Activity Index for Lupus (RAIL) scores, with or without standardization of urine creatinine levels, are calculated from the urine concentrations of urine proteins based on patient age. There is a pediatric RAIL (pRAIL) algorithm and one for adults (aRAIL). A standardized pRAIL score of 0.39 or higher correctly identifies 92% of histologically proven, active LN in patients aged\u2009<\u200924\u00a0years , 12. LikWe hypothesized that the improvement in RAIL score will detect those who respond to induction therapy versus those who do not. We evaluated RAIL scores based on the clinical activity of LN and compared RAIL scores to those in healthy controls. In this study, we aimed to delineate changes in RAIL scores during induction therapy of LN.We performed a prospective case control study. With approval of local institutional review boards of the participating academic centers at Cincinnati Children\u2019s Hospital (CCHMC), Children\u2019s Hospital of Philadelphia, Washington University School of Medicine, and Baylor College of Medicine, clinical data and urine samples of patients with cSLE were included in this study. Study participants were pediatric patients diagnosed with SLE by the 1997 or the 2019 American College of Rheumatology (ACR) classification criteria , 16, irrSLE patients were classified as having LN based on the result of a kidney biopsy . PatientBesides demographics, information needed to score the SLE Disease Activity Index (SLEDAI) and the renal domain score of the SLEDAI  was extracted . This inUrine collected was spun at 2200\u2009\u00d7\u2009g for 10\u00a0min and stored at\u2009\u2212\u200980\u00a0\u00b0C for no longer than 5\u00a0years. Human NGAL and human urine creatinine were measured on a Roche Cobas c 311 clinical chemistry analyzer using a commercially available assay . NGAL had a lower limit of detection of 9.8\u00a0ng/mL while creatinine had a lower limit of detection of 1.1\u00a0mg/dL. The remaining biomarkers were measured via ELISA, all done in duplicate. Human urinary KIM-1  had a mean minimal detectable dose of 0.009\u00a0ng/mL. Human MCP-1 , diluted 1:1, had a mean minimal detectable dose of 1.7\u00a0pg/mL. Human adiponectin , diluted 1:5, had a mean minimal detectable dose of 0.246\u00a0ng/mL. Human ceruloplasmin , diluted 1:50, had a mean minimal detectable dose of 0.085\u00a0ng/mL. Human hemopexin , diluted 1:20, had a mean minimal detectable dose of 4.2\u00a0ng/mL. KIM-1 and MCP-1 used a four-parameter logistic curve to fit the standard curve. Adiponectin, ceruloplasmin, and hemopexin used a log/log curve to fit the standard curve. Analyte concentrations that were above or below the limits of detection were imputed by 50% of the level of lower limit of detection and 50% over the upper limit of detection, respectively.and/or hematuria\u2009\u2265\u20095 RBC/HPF and/or eGFR\u2009<\u200990\u00a0mL/min/1.73\u00a0m2 [plus GFR\u2009>\u200990\u00a0mL/min/1.73 m2plus inactive urine sediment (no hematuria) [plus at least stable eGFR compared to LN diagnosis. Those patients with LN who failed to achieve a partial or complete response to induction therapy were considered non-responders.There is no consensus how to define active LN based on the NIH-AI scores; scores from 2 to 6 have been proposed previously \u201330. For /1.73\u00a0m2 . Accordimaturia) . Partialt-test (mean\u2009\u00b1\u2009SD) or ANOVA test (mean\u2009\u00b1\u2009SD). The Wilcoxon test  or Kruskal\u2013Wallis test  was performed to compare group differences for continuous variables for patients with LN (active or inactive LN), SLE patients without known LN (no-LN), and age-matched healthy controls. Matching controls were recruited to address potential sources of bias. Study size was determined by prior studies using biomarkers. Fisher\u2019s exact or chi-square tests, as appropriate, were performed for testing of group differences of categorical variables. Correlation was established via Pearson\u2019s correlation coefficient, with r of 0.1\u20130.39 representing weak correlation, 0.4\u20130.69 representing moderate correlation, 0.7\u20130.89 representing strong correlation, and greater than 0.9 representing very strong correlation [t-test was performed in evaluating patient changes of pRAIL scores from the start to the end of induction therapy for LN patients classified as LN responders  during induction therapy, but not for non-responders. Two-sided p-values\u2009<\u20090.05 were considered statistically significant.In patient-level variables, descriptive analyses are reported and compared by Student\u2019s relation . BaselinPrior to use in the RAIL algorithm, biomarker concentrations were natural-log transformed. The pRAIL algorithm was used only for pediatric and young adult cSLE patients up to age 23\u00a0years at enrollment, as previously published , 14. TheA total of 128 pediatric or young adult patients  were included for the analyses of changes of pRAIL scores. The study duration was from June 2019 to December 2021. Among 70 lupus nephritis patients, 32 patients were undergoing induction therapy for LN, of which 25 met our criteria for active LN, while the remaining 7 did not due to NIH-AI scores\u2009<\u20094.p\u2009<\u20090.0001). Most pediatric and young adult LN patients had proliferative LN with a median NIH-AI score of 7  and NIH-CI score of 1  on kidney biopsy, respectively.Table p\u2009<\u20090.03). The pRAIL scores differed between the active LN group and other groups but not between the inactive LN group versus the non-LN group, irrespective of whether pRAIL scores were standardized by urine creatinine or not.The pRAIL scores significantly differed by LN status  and by healthy controls. Table r value of 0.14. Considering the subset of active LN patients with available NIH-AI scores, pRAIL scores were moderately  and UPCR weakly  correlated with NIH-AI scores as shown in Fig.\u00a0There was only a weak correlation between pRAIL scores and UPCR, with an n\u2009=\u200914) and oral mycophenolate mofetil (n\u2009=\u200911). There were 22 responders to induction therapy, of whom 15 patients met criteria for complete response, 7 patients were partial responders, and 3 were non-responders to induction therapy. Distribution of age at baseline, gender, race, ethnicity, and ISN/RPS classes were all similar across groups. Proliferative nephritis was evenly distributed across response status. Of the 17 patients with class III or IV LN, 2 patients (66.67%) were non-responders, 5 patients (71%) were partial responders, and 10 patients (66.67%) were complete responders. Finally, baseline pRAIL scores were similar between partial and complete responders  and therefore excluded, and 25 patients who had active LN at the start of induction therapy. Immunosuppressive therapy for active LN included intravenous cyclophosphamide (p\u2009=\u20090.03). For partial responders, there was no change of the pRAIL score during induction therapy .Among the three non-responders, standardized and non-standardized pRAIL scores numerically increased during induction therapy see Fig.\u00a0. Conversp\u2009=\u20090.0007). Overall, mean\u2009\u00b1\u2009SD absolute pRAIL scores of complete responders verified by repeat kidney biopsy decreased by 1.08\u2009\u00b1\u20091.13, and standardized pRAIL scores by 0.43\u2009\u00b1\u20090.31, respectively. The average UPCR pre-therapy was 4.56\u2009\u00b1\u20094.74 and the average UPCR post-biopsy was 1.29\u2009\u00b1\u20092.04. As shown in Fig.\u00a0r\u2009=\u20090.46 and 0.37, respectively), while UPCR had no correlation with NIH-AI at either time point, with r values of 0 and 0.1, respectively.Ten patients underwent a repeat kidney biopsy upon completion of induction therapy, including nine with complete renal response and 1 non-responder. The mean\u2009\u00b1\u2009SD of NIH-AI scores decreased from 9.89\u2009\u00b1\u20093.92 to 1.1\u2009\u00b1\u20091.45 among complete responders during induction therapy , we were unable to show statistically significant decreases in standardized pRAIL scores in this subgroup.We had previously shown that both absolute and standardized pRAIL scores were similarly well suited to discriminate moderate to highly active LN (NIH-AI\u2009>\u200910) from LN with lesser degrees of inflammation as seen on kidney biopsy , 14. UsiAnother reason for the small changes in pRAIL scores in partial LN responders may be the choice of the response definition selected for this study, which was based on a prior consensus formation exercise in children with LN . NonethePatients who achieve partial LN response have better outcomes than non-responders, but have worse outcomes than complete LN responders . If confThe degree of histological activity on kidney biopsy at the time of LN diagnosis or on repeat biopsy is an established prognostic factor for the longer-term outcomes of LN , 39, 41.In this study, levels of urine biomarkers and RAIL scores were lower in age-matched healthy controls compared to SLE patients, irrespective of kidney involvement. Lower levels of kidney biomarkers and RAIL scores could be considered a reflection of common, subclinical kidney inflammation in SLE patients, even those without abnormal urinalysis.There are several limitations to this study. First, we only had access to patients with available repeat kidney biopsy at the end of induction therapy, requiring reliance on other definitions of active/inactive LN. This was an observational study, so we relied on data from real clinical encounters; thus, study results may be biased due to missing data and informational bias. There were a small number of non-responders, limiting the analyses that could be performed in the induction group. However, the observations in this study were consistent with those of our prior validation studies of the RAIL , 13, 14.In conclusion, we provide evidence for the value of a composite urinary biomarker score and RAIL to support the severity of LN and its response to induction therapy.Graphical Abstract (PPTX 54 KB)Below is the link to the electronic supplementary material."
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